WO2018049248A1 - Virus oncolytique équipé de molécules d'engagement bispécifiques - Google Patents

Virus oncolytique équipé de molécules d'engagement bispécifiques Download PDF

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WO2018049248A1
WO2018049248A1 PCT/US2017/050803 US2017050803W WO2018049248A1 WO 2018049248 A1 WO2018049248 A1 WO 2018049248A1 US 2017050803 W US2017050803 W US 2017050803W WO 2018049248 A1 WO2018049248 A1 WO 2018049248A1
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antigen
binding domain
binding
seq
amino acid
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PCT/US2017/050803
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Xiaotong Song
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Icellhealth Consulting Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to oncolytic viruses, anti-EpCAM antibodies, and methods of use thereof.
  • Oncolytic viruses are promising agents for cancer treatment, due to their ability to infect, replicate in, and lyse tumor cells and spread through tumor cells in successive rounds of replication (Russell et al., Nat Biotechnol. 2012, 30:658-670; Kelly and Russell, Mol Ther. 2007, 15:651-659). Their major mode of action is to lyse tumor cells, which may induce antigen-specific T-cell responses to target metastatic diseases, even if the OVs are delivered locally. OV has been tested in both preclinical models and clinical trials, but complete clinical responses have only been rarely observed, highlighting the need for further improvement of OV therapy.
  • T-cell immunotherapy has the ability to control tumor growth and prolong survival in cancer patients.
  • tumor-specific T-cell responses are hard to achieve and sustain, likely due to the limitations of various immune escape mechanisms of tumor cells (Shafer-Weaver et al., Adv Exp Med Biol.2007, 601:357– 368; Shafer-Weaver et al., J Immunol.2009, 183:4848–4852).
  • Bispecific engager molecules such as those comprising a T-cell surface molecule-binding domain and a tumor antigen- binding domain, have provided a way to engage T cells to tumor cells and shown some clinical success, such as killing tumor cells in patients with non-Hodgkin's lymphomas and B-cell precursor acute lymphoblastic leukemia (Bargou et al., Science.2008, 321:974-977; Topp et al., J Clin Oncol. 2011, 29:2493-2498; Nagorsen et al., Leuk Lymphoma.2009, 50:886-891).
  • bispecific engager molecules such as bispecific T-cell engagers
  • have short half-life, which require continuous infusion (Hammond et al., Cancer Res.2007, 67:3927-3935; Lutterbuese et al., Proc Natl Acad Sci USA.2010, 107:12605-12610; Friedrich et al., Mol Cancer Ther.2012, 11:2664–2673; Choi et al., Proc Natl Acad Sci USA. 2013, 110:270–275).
  • the inability for bispecific T-cell engagers to actively accumulate at tumor sites lead to systemic adverse effects such as within the central nervous system.
  • the present application provides oncolytic virus encoding low-affinity bispecific engager molecule, compositions (such as pharmaceutical compositions), and methods of use thereof.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M, such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M.
  • OV oncolytic virus
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 -4 M to about 10 -9 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 -4 M to about 10 -8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 -6 M to about 1 ⁇ 10 -8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -7 M.
  • the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3.
  • the tumor antigen is EpCAM, FAP, or EGFR.
  • the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell.
  • the effector cell is a T lymphocyte, such as a cytotoxic T lymphocyte.
  • the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. In some embodiments, the cell surface molecule is CD3.
  • the first and/or second antigen-binding domain is a single chain variable fragment (scFv).
  • the first antigen-binding domain and the second antigen-binding domain are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain.
  • the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus, Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, and Newcastle disease virus (NDV).
  • the OV is a vaccinia virus.
  • the VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR).
  • the vaccinia virus is a WR strain.
  • the VV comprises double deletion of thymidine kinase (TK) gene and vaccinia virus growth factor (VGF) gene.
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator.
  • the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.
  • the immune checkpoint modulator is an immune checkpoint inhibitor.
  • the immune checkpoint modulator is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4.
  • the immune checkpoint modulator is an inhibitor of PD-1.
  • the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule, such as anti-PD-1 antibody.
  • the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule.
  • the immune checkpoint modulator is a ligand that binds to PD-L1, PD-L2, HHLA-2, CD47, or CXCR4.
  • the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin.
  • the immune checkpoint modulator is a TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin.
  • the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules.
  • the immune checkpoint modulator is an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin.
  • the Fc fragment is an IgG4 Fc.
  • the OV further comprises a third nucleic acid encoding a cytokine, such as GM-CSF.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter.
  • the second nucleic acid encoding the immune checkpoint modulator is operably linked to a promoter.
  • the promoter is a late promoter.
  • the promoter is a VV promoter.
  • the promoter is a VV late promoter, such as F17R.
  • a pharmaceutical composition comprising any of the OVs described above, and a pharmaceutical acceptable carrier.
  • a pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific engager molecules described above), and a second OV comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a pharmaceutical acceptable carrier.
  • a pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific engager molecules described above), and a second OV comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a pharmaceutical acceptable carrier.
  • a pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen- binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific engager molecules described above), a second OV comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a third OV comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a pharmaceutical acceptable carrier.
  • Another aspect of the present application provides a method of treating a cancer in an individual, comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific engager molecules described above), and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a second pharmaceutical acceptable carrier.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific engager molecules described above), and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a second pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific engager molecules described above), and a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a second pharmaceutical acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a third pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV comprising a first nucleic
  • the effective amount is about 10 5 to about 10 13 pfu, such as about 10 9 pfu.
  • the pharmaceutical composition is administered systemically, such as intravenously. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally.
  • the cancer is a solid tumor, such as colorectal cancer, lung cancer, liver cancer, skin cancer (such as melanoma), brain cancer (such as glioblastoma), and breast cancer.
  • the method of treating cancer described herein further comprises administering to the individual an additional cancer therapy, such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. In some embodiments, the individual is a human.
  • Another aspect of the present application provides constructs specifically recognizing EpCAM. Also provided are methods of preparation, pharmaceutical compositions and uses thereof.
  • FIG.1 depicts an exemplary rationale for TEA-VV.
  • FIG. 2 depicts an exemplary scheme of expression cassettes encoding EpCAM- scFv-CD3-scFv (EpCAM-TEA-VV) or GFP (GFP-VV), all under control of the late promoter F17R.
  • EpCAM-TEA-VV EpCAM-TEA-VV
  • GFP-VV GFP-VV
  • FIG. 3 depicts the SDS-PAGE of EpCAM-TE (EpCAM-CD3) secreted by tumor cells after EpCAM-TEA-VV infection.
  • EpCAM-TE is the positive EpCAM-bispecific scFv control produced in 293-T cells.
  • FIGS. 4A and 4B depict successful binding of EpCAM T-cell engager (EpCAM- TE) secreted by EpCAM-TEA-VV infected cells to EpCAM on different human tumor cells, by FACS assay (FIG. 4A) or Western blot (FIG. 4B). Actin expression in corresponding human tumor cells was used as a control in the Western blot assay (FIG.4B).
  • FIG. 5 depicts comparable replicative capacity of EpCAM-TEA-VV and GFP-VV in infected cells CV-1, SK-BR-3 and HT-29. The respective viral titers were measured at 0, 24, 48, and 72 hours post-infection.
  • FIG. 6 depicts comparable tumor lytic ability induced by EpCAM-TEA-VV and GFP-VV in Huh7, SK-BR-3, and HT-29 tumor cell lines.
  • Huh7, SK-BR-3 and HT-29 cells were infected with EpCAM-TEA-VV or GFP-VV at MOI of 0, 0.01, 0.1, 1, and 5 (x-axis), and cell viability was analyzed using MTS assay (y-axis).
  • FIG. 7 depicts the capability of EpCAM-TEA-VV to activate human PBMCs.
  • PBMCs were either untreated, or mixed with GFP-VV or EpCAM-TEA-VV infected tumors. Subsequently, the level of CD69+ cells was analyzed by FACS (gated for CD4+ or CD8+ cell population), which is indicative of the level of PBMC activation.
  • FIGS. 8A-8B depict the ability of EpCAM-TEA-VV to induce tumor lysis in the presence of human PBMCs.
  • HT-29-GFP and SK-BR-3-GFP cell lines were mock-infected or infected with HN3-TEA-VV or EpCAM-TEA-VV, and co-cultured with human PBMC for 48 hours before cell viability analysis.
  • FIG. 8A depicts the FACS plot of viable, GFP positive tumor cells.
  • FIG. 8B depicts immunofluorescence analysis of viable, GFP positive tumor cells.
  • FIGS. 9A-9B depict the ability of EpCAM-TEA-VV to induce bystander tumor lysis in the presence of human PBMC.
  • HT-29 and SK-BR-3 cell lines were mock-infected or infected with HN3-TEA-VV or EpCAM-TEA-VV, and cell culture media was harvested after 48 hours of culture.
  • Non-infected HT-29-GFP or SK-BR-3-GFP cells were co-cultured with PBMC in the presence of the respective media collected, for 48 hours.
  • FIG. 9A depicts the FACS plot of viable, GFP positive tumor cells, indicating effective bystander killing of non- infected HT-29-GFP and SK-BR-3-GFP cells by EpCAM-TEA-VV in the presence of PBMC, but not by GFP-VV or HN3-TEA-VV.
  • FIG. 9B depicts immunofluorescence analysis of viable, GFP positive tumor cells, indicating effective bystander killing of non-infected HT- 29-GFP and SK-BR-3-GFP cells by EpCAM-TEA-VV in the presence of PBMC, but not by HN3-TEA-VV.
  • FIGS. 10A-10C depict the ability of EpCAM-TEA-VV to inhibit HT-29 tumor growth in vivo.
  • 4 ⁇ 10 6 HT-29 cells were inoculated subcutaneously into the right flank of NSG mice, followed by i.p. injection of 1 ⁇ 10 8 pfu of EpCAM-TEA-VV, GFP-VV or no VV (PBS) on day 5, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 7.
  • FIG. 10A depicts the tumor volume over time in mock, human PBMC only, VV only, or VV/human PBMC-treated mice.
  • FIG.10B shows the representative excised tumors from mock, human PBMC only, VV only, or VV/human PBMC-treated mice after 24 days.
  • FIG. 10C shows the survival curve of mock, human PBMC only, VV only, or VV/human PBMC- treated mice.
  • FIG. 11 depicts the ability of EpCAM-TEA-VV to inhibit SK-BR-3 tumor growth in vivo.
  • SK-BR-3 cells were mixed with or without PBMC and inoculated s.c. into the right flank of NSG mice, followed by i.p injection of EpCAM-TEA-VV, HN3-TEA-VV, or PBS at day 0.
  • SK-BR-3 tumor cells infected with EpCAM-TEA-VV in the presence of human PBMC showed significant inhibition of SK-BR-3 tumor size growth in vivo.
  • FIG. 12A depicts an exemplary scheme of expression cassettes encoding FAP-scFv- human CD3-scFv (hFAP-TEA-VV), GPC3-scFv-human CD3-scFv (hGPC3-TEA-VV), or GFP (GFP-VV), all under control of the late promoter F17R.
  • the YFP-GFP selection marker was removed after confirming the expression of hFAP-TE, GPC3-TE, or GFP.
  • FIG. 12B depicts the SDS-PAGE of hFAP-TE secreted by tumor cells after hFAP-TEA-VV infection. The left-most lane is the protein ladder.
  • FIG. 12C depicts quantification of hFAP-TE purified from SK-BR3 and HT-29 cells infected with hFAP-TEA-VV at MOI of 0.1, 1, and 5 after 24 hours culturing.
  • FIG. 13 shows comparable replicative capacity of hFAP-TEA-VV, GPC3-TEA-VV, or VSC20 in infected tumor cells CV-1, SK-BR-3 and HT-29.
  • the respective viral titers were measured at 0, 24, 48, and 72 hours post-infection.
  • FIG. 14 depicts comparable tumor lytic ability induced by hFAP-TEA-VV, GPC3- TEA-VV, and parental VSC20-VV in U87, SK-BR-3, and HT-29 tumor cell lines.
  • U87, SK- BR-3 and HT-29 cells were infected with VV at MOI of 0, 0.01, 0.1, and 1 (x-axis), and cell viability was analyzed using MTS assay (y-axis).
  • FIGS. 15A-15B show tumor lytic ability induced by hFAP-TEA-VV in the presence of PBMC.
  • U87 tumor cells were infected with hFAP-TEA-VV or GPC3-TEA-VV. Mock transduced cells were used as a control. Transduced cells were co-cultured with PBMC for 72 hours.
  • FIG. 15A depicts FACS plots of viable, GFP positive tumor cells (upper panel), and tumor cells exhibiting apoptotic markers Annexin-V and propidium iodide (PI) (lower panel).
  • FIG. 15B demonstrates FACS quantification of GFP signal (upper panel) and cell viability demonstrated by MTS assay (lower panel), indicating tumor lytic activity induced by hFAP- TEA-VV in the presence of PBMC.
  • FIGS. 16A-16B demonstrate the ability of hFAP-TEA-VV to activate human PBMCs in vitro.
  • PBMCs were either mock-treated, or mixed with hFAP-TEA-VV or GPC3- TEA-VV infected U87 tumors for 72 hours.
  • FIG. 16A depicts the FACS plots showing levels of CD69+ cells (gated for CD4+ or CD8+ cell population), which is illustrative of the level of PBMC activation.
  • FIG. 16A depicts the FACS plots showing levels of CD69+ cells (gated for CD4+ or CD8+ cell population), which is illustrative of the level of PBMC activation.
  • 16B depicts levels of cytokines IFN ⁇ and IL2 collected from the supernatants of PBMC co-cultured with medium, GPC3-TEA-VV infected tumor cells, or hFAP-TEA-VV infected tumor cells.
  • the cytokine levels were determined by ELISA.
  • FIGS. 17A-17E demonstrate bystander killing of uninfected tumor cells by hFAP- TEA-VV infected tumor cells.
  • FIG. 17A shows an exemplary scheme whereby the culture media from U87 tumor cells infected with VV was used for co-culturing uninfected FAP+ tumor cells and PBMC for 48 hours.
  • FIG. 17B depicts measurement of cell viability of FAP+ U87-GFP cells by immunofluorescence and FACS detection of GFP.
  • FIG. 17C depicts quantitation of FACS analysis.
  • FIG. 17D depicts MTS assay of cells co-cultured at various PBMC:U87 ratios from an infection MOI of 1.
  • FIG. 17E depicts MTS assay from cells co- cultured at PBMC:U87 ratio of 5:1 at MOI of 0, 0.01, 1, 5, and 10.
  • FIGS. 18A-18C demonstrate the ability of hFAP-TEA-VV to inhibit SK-BR-3 tumor growth in vivo.
  • FIG.18A depicts an exemplary scheme of the establishment of the SK- BR-3 tumor model. NSG mice were inoculated subcutaneously into the right flank at day 0 with 4 ⁇ 10 6 SK-BR-3 cells, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV or GPC3- TEA-VV or no VV (PBS) into the right flank tumor on day 8, and i.v. implantation of 2 ⁇ 10 7 non-activated human PBMC cells on day 10.
  • FIG. 18B depicts volume of tumors measured by caliper.
  • FIG.18C depicts weight of tumors excised from SK-BR-3 mice.
  • FIGS. 19A-19B demonstrate the ability of hFAP-TEA-VV to inhibit HT-29 tumor growth in vivo.
  • Mice were inoculated subcutaneously into the right flank at day 0 with 4 ⁇ 10 6 HT-29 cells, followed by injection of 1 ⁇ 10 8 pfu of FAP-TEA-VV or GFP-VV or no VV (PBS) into the right flank tumor on day 8, and i.v. implantation of 2 ⁇ 10 7 non-activated human PBMC cells on day 10.
  • FIG.19A depicts volume of tumors from HT-29 mice measured by caliper.
  • FIG.19B shows monitoring of HT-29 mouse survival.
  • FIGS. 20A-20D demonstrate a correlation between stromal destruction and hFAP- TEA-VV virus spread in SK-BR-3 tumor environment.
  • Biopsies of right flanks of SK-BR-3 mice treated with GPC3-TEA-VV, hFAP-TEA-VV, and no virus (PBS) with or without PBMC implantation were obtained.
  • FIG. 20A depicts FACS analysis of biopsies followed by staining of anti-mFAP-APC and mCD45-FITC.
  • FIG 20B depicts qRT-PCR measurement of mouse FAP mRNA in biopsied tumors.
  • FIG. 20C depicts immunofluorescence of the stromal marker ⁇ -SMA. DAPI was employed to visualize nuclei.
  • FIG. 20D depicts higher viral spread capacity of hFAP-TEA-VV in the presence of PBMC in biopsied tumors measured by plaque assays.
  • FIGS. 21A-21B demonstrate stromal destruction by hFAP-TEA-VV in the HT-29 tumor environment.
  • Biopsies of right flanks of HT-29 mice treated with GFP-VV, hFAP- TEA-VV, and no virus (PBS) in the presence of PBMC were obtained.
  • a biopsy from HT-29 tumor injected with hFAP-TEA-VV without implantation of PBMC was served as a control.
  • FIG. 21A depicts FACS analysis of biopsies followed by staining of mFAP.
  • FIG. 21B depicts immunofluorescence of the stromal marker ⁇ -SMA. DAPI was employed to visualize nuclei.
  • FIGS. 22A-22D demonstrate the ability of hFAP-TEA-VV to activate T cells in the SK-BR-3 tumor model.
  • Biopsies of right flanks of SK-BR-3 mice treated with GPC3-TEA- VV/PBMC, hFAP-TEA-VV/PBMC, no virus (PBS)/PBMC and hFAP-TEA-VV alone were obtained at day 15.
  • FIG.22A depicts FACS analysis of biopsies followed by staining of anti- hCD3-FITC.
  • FIG. 22B depicts immunofluorescence of the T cell marker anti-human CD3 at day 21. DAPI was employed to visualize nuclei.
  • FIGS. 22C-22D depict levels of cytokines IFN ⁇ and IL2 collected from biopsies determined by qRT-PCR.
  • FIGS. 23A-23B demonstrate the ability of hFAP-TEA-VV to facilitate T cell infiltration and activate T cells in the HT-29 tumor model.
  • Biopsies of right flanks of HT-29 mice treated with hFAP-TEA-VV/PBMC, GFP-VV/PBMC or PBMC alone were obtained.
  • FIG. 23A depicts FACS analysis of biopsies followed by staining of anti-hCD3-FITC.
  • FIG. 23B depicts FACS analysis of biopsies followed by staining of anti-hCD8-FITC and hIFN ⁇ .
  • FIG. 24 shows distribution of VV in HT-29 tumor model following intratumor injection of hFAP-TEA-VV.
  • Blood and urine samples were collected from hFAP-TEA-VV- treated HT-29 mice at 20 minutes, 5 days, and 10 days following injection of hFAP-TEA-VV, and VV distribution was analyzed by quantitative PCR of VV genome.
  • FIGS. 25A-25C depict toxicity of hFAP-TEA-VV in HT-29 mice following treatment with hFAP-TEA-VV/PBMC, GFP-VV/PBMC, PBS alone, PBMC alone, or hFAP- TEA-VV alone.
  • FIG. 25A depicts measurement of mouse body weight over the course of 24 days.
  • FIG. 25B depicts measurement of bone marrow cellularity at day 21 after treatment.
  • FIG. 25C depicts measurement of quadriceps mass at day 21 after treatment.
  • FIG. 26A depicts an exemplary scheme of expression cassettes encoding mouse FAP-scFv-mouse CD3-scFv (mFAP-TEA-VV), EphA2-scFv-mouse CD3-scFv (mEphA2- TEA-VV) or GFP (GFP-VV), all under control of the late promoter F17R.
  • the YFP-GFP selection marker was removed after confirming the expression of mFAP-TE, mEphA2-TE, or GFP.
  • 26B depicts the SDS-PAGE of proteins secreted by tumor cells after infection with mFAP-TEA-VV, mEphA2-TEA-VV, or GFP-VV.
  • the left-most lane is the protein ladder and EphA2-CD3 protein was used as a control.
  • FIGS. 27A-27D depict comparable tumor lytic ability induced by mFAP-TEA-VV (mCD3-FAP), mEphA2-TEA-VV (mCD3-EphA2), mGM-CSF-VV (mGM-CSF), and GFP- VV (GFP) in tumor cell lines.
  • B16, GL261, MC38, and CT26 cell lines were transduced with VV at MOI of 0, 0.01, 0.1, 1, 5, and 10.
  • Cell viability was determined at 48 hours post- infection by MTS assay.
  • FIG. 28 demonstrates the ability of mFAP-TEA-VV to enhance tumor lytic ability in the presence of PBMC.
  • FAP positive GL261-GFP cells were transduced with mFAP-TEA- VV or mEphA2-TEA-VV at MOI of 1 or 0.1. Mock transduced cells were used as a control (Medium). Cells were co-cultured with fresh murine splenocytes C57BL/6 for 24 or 48 hours. Cell viability was then assessed by FACS against GFP signal.
  • FIGS. 29A-29C demonstrate bystander killing of uninfected tumor cells by mFAP- TEA-VV infected cells.
  • FIG. 29A shows an exemplary scheme of bystander killing test, whereby culture media from MC38 or GL261 tumor cells infected with VV were applied to uninfected FAP+ tumor cells in the presence of T cells.
  • FIG. 29B depicts measurement of cell viability of uninfected MC38-GFP and GL261-GFP cells co-cultured with T cells in the presence of culture media from corresponding tumor cells infected with GFP-VV, mEphA2- TEA-VV, or mFAP-TEA-VV. The quantification was carried out by MTS.
  • FIG. 29C depicts FACS analysis against GFP signal in uninfected MC38-GFP and GL261-GFP cells co- cultured with T cells in the presence of culture media from corresponding tumor cells infected with GFP-VV, mEphA2-TEA-VV, or mFAP-TEA-VV, indicating bystander killing induced by mFAP-TEA-VV.
  • FIGS. 30A-30C demonstrate the ability of mFAP-TEA-VV to inhibit B16 tumor growth in vivo.
  • FIG. 30A depicts an exemplary scheme of the B16 s.c. (subcutaneous) tumor model. 1 ⁇ 10 5 B16 cells were implanted subcutaneously into the right flank of C57/BL6 mice at day 0, followed by implantation of 5 ⁇ 10 4 B16 cells in the left flank on day 4. Mice were injected with 1 ⁇ 10 8 pfu of mFAP-TEA-VV, mEphA2-TEA-VV, mGM-CSF-VV, or PBS (control) on day 7, 10, 13, and 16.
  • FIG. 30B depicts volume of tumors measured by caliper in the right flank over the course of 22 days.
  • FIG. 30C depicts volume of tumors measured by caliper in the left flank over the course of 15 days.
  • FIGS. 31A-31D demonstrate the ability of mFAP-TEA-VV to inhibit B16 tumor growth in vivo.
  • FIG. 31A depicts an exemplary scheme of the construction of the B16 i.v. (intravenous) tumor model. 2 ⁇ 10 5 B16 cells were injected intravenously into C57/BL6 mice at day 0, followed by i.v. injection of 1 ⁇ 10 8 pfu of mFAP-TEA-VV, mEphA2-TEA-VV, mGM-CSF-VV, or PBS on day 1 and 3.
  • FIG. 31B shows representative excised lung tumors from VV-treated mice after 15 days.
  • FIG. 31C shows the mean tumor nodules in lung of VV- treated mice.
  • FIG. 31D shows the survival curve of VV-treated mice over the course of 42 days post B16 inoculation.
  • FIGS. 32A-32D depict a correlation between stroma destruction and mFAP-TEA- VV virus spread in the B16 tumor environment. Biopsies of right flanks of mFAP-TEA-VV, mEphA2-TEA-VV, mGM-CSF-VV, or PBS treated mice were obtained.
  • FIG. 32A depicts immunofluorescence with anti-mFAP showing stroma staining. DAPI was employed to visualize nuclei.
  • FIG. 32B depicts quantification of mFAP+ immunostained cells.
  • FIG. 32C depicts replicative capacity of mFAP-TEA-VV, mEphA2-TEA-VV, mGM-CSF-VV in biopsied tumors measured by plaque assays.
  • FIG. 32D depicts the correlation between tumor stroma cell destruction and virus spread ability.
  • FIG. 33 demonstrates the ability of mFAP-TEA-VV to increase tumor infiltration by T cell.
  • Biopsies of right flanks of B16 tumor bearing C57 mice treated with mGM-CSF- VV, mFAP-TEA-VV, no virus (PBS) and mEphA2-TEA-VV were obtained and analyzed by immunofluorescence with anti-CD3. DAPI was employed to visualize nuclei.
  • FIG. 34 demonstrated the ability of mFAP-TEA-VV to enhance anti-tumor T cell response. Splenocytes of the VV treated mice were harvested for ELISPOT assay.
  • FIG. 35 depicts higher EpCAM expression in MCF7 cells, and lower EpCAM expression in Huh7 cells, as measured by FACS.
  • MCF7 and Huh7 cells were stained with PE-anti-EpCAM.
  • FIG. 36 depicts the binding affinity measured by FACS of low affinity EpCAM-TE (EpCAM lo -TE) and high affinity EpCAM-TE (EpCAM hi -TE) to MCF7 (EpCAM hi expression) and Huh7 (EpCAM lo expression) cells.
  • FIG. 37A depicts potent tumor killing activity of both EpCAM lo -TE and EpCAM hi - TE in MCF7-GFP cells expressing high-level EpCAM co-cultured with PBMC, as measured by FACS against GFP signal.
  • FIG. 37B upper panels depict potent tumor lytic activity of EpCAM hi -TE, but less potent tumor killing activity of EpCAM lo -TE, in Huh7-GFP cells expressing low-level EpCAM co-cultured with PBMC.
  • FIG. 37B lower panels depict potent T cell infiltration induced by EpCAM hi -TE, but less potent T cell infiltration induced by EpCAM lo -TE, in Huh7-GFP cells expressing low-level EpCAM co-cultured with PBMC.
  • FIG. 38 depicts lower EGFR expression in MCF7 cells, and higher EGFR expression in MDA-MB-468 cells, as measured by FACS.
  • MCF7 and MDA-MB-468 cells were stained with PE-anti-EGFR.
  • FIG. 39 depicts potent tumor lytic activity induced by EGFR hi -528-TE, and less potent tumor lytic activity induced by EGFR lo -C10-TE, in MCF7-GFP cells with low EGFR expression co-cultured with PBMC, as measured by GFP immunofluorescence (upper panels), light picture (middle panels), and apoptosis marker Annexin-V by FACS (bottom panels).
  • FIG. 40 depicts potent tumor lytic activity induced by EGFR hi -528-TE, and less potent tumor lytic activity induced by EGFR lo -C10-TE, in MCF7-GFP cells with low EGFR expression co-cultured with PBMC, as measured by GFP immunofluorescence(upper panels), light picture (middle panels), and GFP and hCD45 (T cell activation marker) by FACS (bottom panels).
  • FIG. 41 depicts potent tumor lytic activity induced by EGFR hi -528-TE and EGFR lo - C10-TE in MDA-MB-468-GFP cells with high EGFR expression co-cultured with PBMC, as measured by GFP immunofluorescence (upper panels), and GFP and hCD3 (indicative of T cell infiltration) by FACS (bottom panels).
  • FIG. 42 depicts exemplary schemes of expression cassette PD1-Ig-VV encoding PD1 (extracellular domain)-IgG4-Fc, expression cassette FAP-TEA-VV (FAP-CD3-VV) encoding low-affinity bispecific engager molecule FAP-TE (FAP-CD3), and expression cassette PD1-Ig-FAP-TEA-VV (PD1-Ig-FAP-CD3-VV) co-encoding low affinity FAP-TE (FAP-CD3) and PD1-IgG4-Fc, all under control of the late promoter F17R.
  • the YFP-GFP selection marker was removed after confirming the expression of PD1-Ig, FAP-CD3, or FAP- CD3 and PD1-Ig.
  • the present invention provides a low-affinity T-cell engager-armed oncolytic virus (TEA-OV) as a new strategy to activate the immune system and achieve greater antitumor activity, especially for solid tumors.
  • TAA-OV T-cell engager-armed oncolytic virus
  • the present invention is based on the finding that oncolytic viruses expressing low-affinity bispecific engager molecules (hereinafter also referred to as“bispecific molecule”,“engager molecule”, or“engager”) are still effective in killing tumor cells, despite of the low-affinity of the bispecific engager molecules to tumor antigens and/or effector cell surface molecules.
  • the oncolytic viruses expressing low-affinity bispecific engager molecules described herein exhibit lower toxicity as compared to OVs expressing bispecific engager molecules with targeting moieties showing higher affinity, which may lead to on-target toxicity against normal tissues expressing low amounts of targeted tumor-associated antigens (TAAs).
  • TAAs tumor-associated antigens
  • the oncolytic viruses expressing low-affinity bispecific engager molecules described herein are capable of killing infected tumor cells, as well as bystander tumor cells not infected by OVs in the presence of T cells.
  • one aspect of the present invention provides an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen, such as epithelial cell adhesion molecule (EpCAM), fibroblast activation protein- ⁇ (FAP), or epidermal growth factor receptor (EGFR), and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell, such as CD3 on T lymphocytes, wherein the KD of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), hereinafter referred to as“low- affinity bispecific engager molecule”, “low-affinity bispecific
  • the present application also provides bispecific engager molecules (such as any of the bispecific molecules described herein). These bispecific engager molecules can be incorporated into oncolytic viruses, OV vectors, or can be provided in isolated forms. [0067] Also provided are compositions (such as pharmaceutical compositions), kits and articles of manufacture comprising the oncolytic virus expressing the low-affinity bispecific engager molecules, and methods of treating cancer using the oncolytic virus expressing the low-affinity bispecific engager molecules described herein.
  • novel anti-EpCAM antibodies such as EpCAM-T-cell engager, and uses thereof.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • Treatment is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment.
  • the term“prevent,” and similar words such as“prevented,”“preventing” etc. indicate an approach for preventing, inhibiting, or reducing the likelihood of the recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of the symptoms of a disease or condition. As used herein,“prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to recurrence of the disease or condition.
  • “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a method that“delays” development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals.
  • Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.
  • CAT Scan computerized axial tomography
  • MRI Magnetic Resonance Imaging
  • abdominal ultrasound clotting tests
  • clotting tests arteriography
  • biopsy biopsy.
  • cancer progression may be initially undetectable and includes occurrence, recurrence, and onset.
  • an effective amount refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation.
  • an effective amount is an amount sufficient to delay development.
  • an effective amount is an amount sufficient to prevent or delay recurrence.
  • An effective amount can be administered in one or more administrations.
  • the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • an“individual” or a“subject” refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.
  • bispecific T-cell engagers or“BiTEs” are used herein interchangeably to refer to an antibody or fragment thereof that has polyepitopic specificity, with one specificity directed to a T-cell surface molecule.
  • immune checkpoint inhibitor refers to a molecule that totally or partially reduces, inhibits or interferes with one or more inhibitory immune checkpoint molecules that may inhibit T-cell activation and function.
  • an“activator of a stimulatory immune checkpoint molecule” refers to a molecule that stimulates, activates, or increases the intensity of an immune response mediated by stimulatory immune checkpoint molecules.
  • An“isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • transfected or“transformed” or“transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or“transformed” or“transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • Adjuvant setting refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgery resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of development of the disease.
  • Treatment or administration in the“adjuvant setting” refers to a subsequent mode of treatment.
  • the degree of risk e.g., when an individual in the adjuvant setting is considered as“high risk” or“low risk” depends upon several factors, most usually the extent of disease when first treated.
  • Neoadjuvant setting refers to a clinical setting in which the method is carried out before the primary/definitive therapy.
  • Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of“X”.
  • reference to“not” a value or parameter generally means and describes“other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
  • the present invention provides an oncolytic virus (OV; such as vaccinia virus, “VV”) comprising a nucleic acid encoding a bispecific molecule (such as bispecific T-cell engager) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • VV oncolytic virus
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to (e.g., the same, or about 1-2 times of the K D of the binding between the first antigen-binding domain and the tumor antigen), or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the oncolytic virus is a VV.
  • the OV is a Western Reserve (WR) strain VV.
  • the OV comprises double deletion of thymidine kinase (TK) gene and vaccinia virus growth factor (VGF) gene.
  • the first and/or second antigen-binding domain is a scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen- binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • the oncolytic virus expressing low-affinity bispecific engager molecule described herein can: 1) facilitate T-cell tumor infiltration and T-cell activation at tumor sites, 2) effectively lyse tumor cells that are infected or not infected by the bispecific engager-armed OV (bystander killing); 3) minimize on-target toxicity against normal tissues expressing low amounts of targeted tumor-associated antigens; and 4) minimize systemic adverse events (e.g. flulike symptoms and CNS effects) and increase tumor site bispecific engager molecule concentration by delivering and sustaining bispecific engager molecules selectively within the tumors.
  • systemic adverse events e.g. flulike symptoms and CNS effects
  • the K D of the binding between the second antigen-binding domain of the bispecific engager molecule and the cell surface molecule is about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the KD of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 2-10 times of the K D of the binding between the first antigen-binding domain
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain.
  • the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • the K D of the binding between the second antigen-binding domain of the bispecific engager molecule and the cell surface molecule is the same as, or about 1-2 times of the K D of the binding between the first antigen-binding domain and the tumor antigen (hereinafter refer to as“similar to” or“similar binding affinity”).
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule can be about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, or about 1 to about 1.1 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is the same as the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to the K D of the binding between the first antigen-binding domain and the tumor
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain.
  • the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • Tumor antigens can be a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
  • TAA or TSA is expressed on a cell of a solid tumor.
  • Tumor antigens include, but are not limited to, EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3).
  • the tumor antigen is EpCAM.
  • the tumor antigen is FAP.
  • the tumor antigen is EGFR.
  • Effector cells include, but are not limited to T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, NKT-cell, or the like.
  • the effector cell is a T lymphocyte.
  • the effector cell is a cytotoxic T lymphocyte.
  • Cell surface molecules on an effector cell include, but are not limited to CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, NKG2D, or the like.
  • the cell surface molecule is CD3.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen- binding domain and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the KD of the binding between the first antigen-binding domain and EpCAM.
  • an oncolytic virus such as VV
  • VV an oncolytic virus
  • a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes)
  • the K D of the binding between the first antigen-binding domain and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and FAP.
  • an oncolytic virus such as VV
  • VV an oncolytic virus
  • a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes)
  • the K D of the binding between the first antigen-binding domain and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and EGFR.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain.
  • the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen-binding domain.
  • the first antigen-binding domain is C- terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • the low-affinity bispecific molecule described herein can be of any format.
  • the first antigen-binding domain is a scFv.
  • the second antigen-binding domain is a scFv.
  • both the first and second antigen- binding domains are scFvs.
  • the first antigen-binding domain and the second antigen binding domain are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen-binding domain.
  • the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR), and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first antigen-binding domain is a scFv.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR), and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen- binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the second antigen-binding domain is a scFv.
  • VV oncolytic virus
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first scFv and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • an effector cell such as CD3 on T lymphocytes
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain e.g., scFv
  • the second antigen-binding domain e.g., scFv
  • the first antigen- binding domain e.g., scFv
  • the second antigen-binding domain e.g., scFv
  • the K D of the binding between the second antigen-binding domain (e.g., scFv) and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain (e.g., scFv) and the tumor antigen.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an immune checkpoint modulator such as immune checkpoint inhibitor
  • a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first scFv and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second scFv specifically recognizing a cell surface molecule on an effector cell
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first scFv and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C-terminal to the second scFv.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second scFv specifically recognizing a cell surface molecule on an effector cell
  • the K D of the binding between the second scFv and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first scFv and the tumor antigen.
  • the first scFv and the second scFv are connected by a linker.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an immune checkpoint modulator such as immune checkpoint inhibitor
  • a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • VV oncolytic virus
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C-terminal to the second scFv.
  • VV oncolytic virus
  • the K D of the binding between the second scFv and CD3 is similar to or about 2-10 times of the K D of the binding between the first scFv and EpCAM.
  • the first scFv and the second scFv are connected by a linker.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • an immune checkpoint modulator such as immune checkpoint inhibitor
  • a third nucleic acid encoding a cytokine (such as GM- CSF).
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10- 5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • VV oncolytic virus
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C- terminal to the second scFv.
  • VV oncolytic virus
  • the K D of the binding between the second scFv and CD3 is similar to or about 2-10 times of the K D of the binding between the first scFv and FAP.
  • the first scFv and the second scFv are connected by a linker.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an immune checkpoint modulator such as immune checkpoint inhibitor
  • a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • VV oncolytic virus
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C-terminal to the second scFv.
  • VV oncolytic virus
  • the K D of the binding between the second scFv and CD3 is similar to or about 2-10 times of the K D of the binding between the first scFv and EGFR.
  • the first scFv and the second scFv are connected by a linker.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • an immune checkpoint modulator such as immune checkpoint inhibitor
  • a third nucleic acid encoding a cytokine (such as GM- CSF).
  • Exemplary oncolytic virus include without limitation vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus (AdV), herpes simplex virus (HSV, such as HSV1 and HSV2), reovirus, myxoma virus (MYXV), poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, Newcastle disease virus (NDV), or the like.
  • the OV is a VV.
  • the OV is a Western Reserve (WR) strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • an VV comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • the VV is a WR strain. In some embodiments, the VV comprises double deletion of TK and VGF genes. In some embodiments, the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen. In some embodiments, the first and/or second antigen-binding domain is a scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain.
  • the first antigen-binding domain is C- terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C-terminal to the second scFv.
  • the K D of the binding between the second scFv and CD3 is similar to or about 2-10 times of the K D of the binding between the first scFv and EpCAM.
  • the first scFv and the second scFv are connected by a linker.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C-terminal to the second scFv.
  • the K D of the binding between the second scFv and CD3 is similar to or about 2-10 times of the K D of the binding between the first scFv and FAP.
  • the first and second scFvs are connected by a linker.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is N-terminal to the second scFv.
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first scFv is C-terminal to the second scFv.
  • the K D of the binding between the second scFv and CD3 is similar to or about 2-10 times of the K D of the binding between the first scFv and EGFR.
  • the first and second scFvs are connected by a linker.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • the oncolytic virus (such as VV) encoding the low-affinity bispecific molecule described herein further comprises a second nucleic acid encoding an immune checkpoint modulator.
  • the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.
  • the immune checkpoint modulator is an immune checkpoint inhibitor, such as an inhibitor of PD- 1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4.
  • the immune checkpoint modulator is an inhibitor of PD-1.
  • the immune checkpoint modulator is an antibody against an immune checkpoint molecule, such as an anti-PD-1 antibody.
  • the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule, such as PD-L1/PD-L2.
  • the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).
  • the immune checkpoint modulator is a ligand that binds to HHLA2.
  • the immune checkpoint modulator is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.
  • the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4.
  • the immune checkpoint modulator comprises an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as an immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as an immune checkpoint inhibitor, e.g.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the OV is a VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK gene and VGF genes.
  • the first and/or second antigen-binding domain is a scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen- binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain. In some embodiments, the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • a promoter such as a late promoter, e.g. F17R
  • the second nucleic acid encoding the immune checkpoint modulator is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine, such as GM-CSF.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and EpCAM is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen- binding domain and EpCAM.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen- binding domain is a scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen- binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R). In some embodiments, the second nucleic acid encoding the immune checkpoint modulator is operably linked to a promoter (such as a late promoter, e.g. F17R). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a cytokine such as GM-CSF
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and FAP is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen- binding domain and FAP.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen- binding domain is a scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen- binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R). In some embodiments, the second nucleic acid encoding the immune checkpoint modulator is operably linked to a promoter (such as a late promoter, e.g. F17R). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a cytokine such as GM-CSF
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and EGFR is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the oncolytic virus further comprises a nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen- binding domain and EGFR.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen- binding domain is a scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen- binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R). In some embodiments, the second nucleic acid encoding the immune checkpoint modulator is operably linked to a promoter (such as a late promoter, e.g. F17R). In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a cytokine such as GM-CSF
  • nucleic acids encoding the low-affinity bispecific molecule, the immune checkpoint modulator, and/or the cytokine described herein can be operably linked to a promoter. In some embodiments, at least two of the nucleic acids encoding the low-affinity bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the low- affinity bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to the same promoter.
  • the promoter is a late promoter. In some embodiments, the promoter is a VV promoter. In some embodiments, the promoter is a vaccinia virus late promoter. In some embodiments, the promoter is F17R.
  • an OV (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first antigen-binding domain comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20.
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • the first antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 33.
  • the second antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 45.
  • the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 55.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and EpCAM.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen- binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen- binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the first scFv comprises a heavy chain variable region (VH) comprising (1) a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR
  • VH heavy chain variable region
  • the first and second scFvs are connected by a linker.
  • the T lymphocyte is a human T lymphocyte.
  • the T lymphocyte is a mouse T lymphocyte.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the VV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the VV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an OV (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first antigen-binding domain comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; and (3) a HVR-H3 comprising the
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising: a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 83.
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • the first antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 36.
  • the second antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 45.
  • the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 58.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and EpCAM.
  • the OV is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the first scFv comprises a heavy chain variable region (VH) comprising (1) a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 76; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; and a light chain variable region (VL) comprising (1)
  • the first and second scFvs are connected by a linker.
  • the T lymphocyte is a human T lymphocyte.
  • the T lymphocyte is a mouse T lymphocyte.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the VV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the VV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an OV (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first antigen-binding domain comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (3) a HVR-H3 comprising the amino acid
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising: a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 32.
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • the first antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 49.
  • the second antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 45.
  • the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 59.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and FAP.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the first scFv comprises a heavy chain variable region (VH) comprising (1) a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 25; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain variable region (VL) comprising (1) a HVR
  • VH heavy chain variable region
  • the first and second scFvs are connected by a linker.
  • the T lymphocyte is a human T lymphocyte.
  • the T lymphocyte is a mouse T lymphocyte.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the VV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the VV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an OV comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the first antigen- binding domain comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 84; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID
  • VH heavy chain variable region
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 90, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 91.
  • the second antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • the first antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 50.
  • the second antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 45.
  • the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 74.
  • the K D of the binding between the second antigen-binding domain and CD3 is similar to or about 2-10 times of the K D of the binding between the first antigen- binding domain and EGFR.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK and VGF genes.
  • the first and/or second antigen- binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen- binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule is operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a VV comprising a nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the first scFv comprises a heavy chain variable region (VH) comprising (1) a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 84; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 86; and a light chain variable region (VL) comprising (1)
  • VH heavy chain variable region
  • the first and second scFvs are connected by a linker.
  • the T lymphocyte is a human T lymphocyte.
  • the T lymphocyte is a mouse T lymphocyte.
  • the VV is a WR strain.
  • the VV comprises double deletion of TK and VGF genes.
  • the VV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor).
  • the VV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).
  • Oncolytic viruses are capable of selective replication in dividing cells (e.g. cancer cell) while leaving non dividing cells (e.g. normal cells) unharmed. As the infected dividing cells are destroyed by lysis, they release new infectious virus particles to infect the surrounding dividing cells.
  • Cancer cells are ideal hosts for many viruses because they have the antiviral interferon pathway inactivated or have mutated tumor suppressor genes that enable viral replication to proceed unhindered (Chernajovsky et al., 2006, British Med. J. 332: 170-2).
  • Exemplary oncolytic virus include without limitation vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus (AdV), herpes simplex virus (HSV, such as HSV1 and HSV2), reovirus, myxoma virus (MYXV), poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, Newcastle disease virus (NDV), or the like (see, e.g. , Kirn et al., Nat. Med. 7:781 (2001); Coffey et al., Science 282:1332 (1998); Lorence et al., Cancer Res. 54:6017 (1994); and Peng et al., Blood 98:2002 (2001)).
  • VV vaccinia virus
  • SVV Seneca Valley virus
  • AdV adenovirus
  • HSV herpes simplex
  • Oncolytic viruses may utilize DNA or RNA as their genetic material.
  • Oncolytic DNA viruses may have capsid symmetry that is icosahedral or complex. Icosahedral oncolytic DNA viruses may be naked or comprise an envelope. Families of oncolytic DNA viruses include the Adenoviridae (for example, Adenovirus, having a genome size of 36-38kb), Herpesviridae (for example, HSV1, having a genome size of 120-200 kb) and Poxviridae (for example, Vaccinia virus and myxoma virus, having a genome size of 130-280 kb).
  • Oncolytic RNA viruses include those having icosahedral or helical capsid symmetry.
  • Icosahedral oncolytic viruses are naked without envelope and include Reoviridae (for example, Reovirus, having a genome of 22-27 kb) and Picornaviridae (for example, Poliovirus, having a genome size of 7.2-8.4 kb).
  • Helical oncolytic RNA viruses are enveloped and include Rhabdoviridae (for example, VSV, having genome size of 13-16 kb) and Paramyxoviridae (for example MV and NDV, having genome sizes of 16-20 kb).
  • the oncolytic virus is a vaccinia virus (VV).
  • VV vaccinia virus
  • the VV can be Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, or Western Reserve (WR) strain.
  • VVA vaccinia virus
  • WR Western Reserve
  • the VV is a WR strain.
  • the oncolytic vaccinia virus of the present invention is modified by altering for one or more viral gene(s).
  • Said modification(s) preferably lead(s) to the synthesis of a defective protein unable to ensure the activity of the protein produced under normal conditions by the unmodified gene (or lack of synthesis).
  • Modifications encompass deletion, mutation and/or substitution of one or more nucleotide(s) (contiguous or not) within the viral gene or its regulatory elements. Modification(s) can be made in a number of ways known to those skilled in the art using conventional recombinant techniques. Exemplary modifications are disclosed in the literature with a specific preference for those altering viral genes involved in DNA metabolism, host virulence, IFN pathway (see e.g.
  • the oncolytic virus comprises an inactivating mutation in a thymidine kinase (TK) gene to produce a negative TK phenotype.
  • TK thymidine kinase
  • the TK gene of the OV is deleted.
  • the TK enzyme is involved in the synthesis of deoxyribonucleotides. TK is needed for viral replication in normal cells as these cells have generally low concentration of nucleotides whereas it is dispensable in dividing cells which contain high nucleotide concentration. Therefore TK deletion limits virus replication significantly in resting cells allowing efficient virus replication to occur only in actively dividing cells (e.g. cancer cells).
  • VGF for VV growth factor
  • VGF is a secreted protein which is expressed early after cell infection and its function seems important for virus spread in normal cells. Replication of VGF deleted vaccinia viruses is highly attenuated in resting (non-cancer) cells.
  • the VV of the present invention does not express functional vaccinia growth factor (VGF).
  • the oncolytic virus of the present invention is a vaccinia virus defective for both TK and VGF activities. The effects of TK and VGF deletions have been shown to be synergistic.
  • an oncolytic VV comprising the nucleic acid encoding the low-affinity bispecific molecule described herein, wherein the oncolytic VV is a WR strain, and wherein the oncolytic VV comprises double deletion of TK and VGF genes.
  • the present invention also relates to an oncolytic viral vector comprising the nucleic acid molecule described in the present disclosure.
  • viral vector is used according to its art-recognized meaning. It refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged into a viral vector particle. The viral vector particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo.
  • Oncolytic viral vectors include, but are not limited to, vaccinia virus (VV) vectors, Seneca Valley virus (SVV) vectors, adenovirus (AdV) vectors, herpes simplex virus vectors (e.g. HSV1 vector), reovirus vectors, myxoma virus (MYXV) vectors, poliovirus vectors, vesicular stomatitis virus (VSV) vectors, measles virus (MV) vectors, lentiviral vectors, retrovirus vectors, morbillivirus vectors, influenza virus vectors, Sinbis virus vectors, and Newcastle disease virus (NDV) vectors.
  • VV vaccinia virus
  • SVV Seneca Valley virus
  • AdV adenovirus
  • MYXV myxoma virus
  • poliovirus vectors vesicular stomatitis virus
  • MV measles virus
  • lentiviral vectors lentiviral vectors
  • the oncolytic viral vector is a VV vector.
  • VV vector encoding the low-affinity bispecific molecule described in the present disclosure.
  • the present application in some embodiments also provides low-binding affinity engager molecules.
  • These low-binding affinity engager molecules can be provided in a composition (such as a pharmaceutical composition) or expressed in any of the oncolytic viruses or oncolytic viral vectors discussed herein.
  • Binding affinity can be indicated by K D , K off , K on , or K a .
  • K off is intended to refer to the off rate constant for dissociation of an antibody (or antigen-binding domain) from the antibody/antigen complex, as determined from a kinetic selection set up.
  • K on is intended to refer to the on rate constant for association of an antibody (or antigen-binding domain) to the antigen to form the antibody/antigen complex.
  • equilibrium dissociation constant“K D ” or “K d ”, as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding domains present in a solution of antibody molecules at equilibrium, and is equal to K off /K on .
  • the measurement of K D presupposes that all binding agents are in solution.
  • the affinity constant, K a is the inverse of the dissociation constant, K D .
  • the dissociation constant (K D ) is used as an indicator showing affinity of antibodies to antigens.
  • K D dissociation constant
  • Scatchard method using antibodies marked with a variety of marker agents, as well as by using BiacoreX (made by Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user's manual and experiment operation method attached with the kit.
  • BiacoreX made by Amersham Biosciences
  • the K D value that can be derived using these methods is expressed in units of M (Mols).
  • An antibody or antigen- binding fragment thereof that specifically binds to a target may have a dissociation constant (K D ) of, for example, ⁇ 10 -4 M, ⁇ 10 -5 M, ⁇ 10 -6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 -9 M, ⁇ 10 -10 M, or ⁇ 10 -11 M.
  • K D dissociation constant
  • an antibody or antigen-binding fragment thereof that specifically binds to a target with a dissociation constant (K D ) of about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M) is referred to as of“low-affinity” or“low-binding affinity”.
  • Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen in the bispecific engager molecule described herein is about 1 ⁇ 10 ⁇ 5 M to about 0.1 ⁇ 10 ⁇ 5 M, about 1 ⁇ 10 ⁇ 5 M to about 0.5 ⁇ 10 ⁇ 5 M, about 0.5 ⁇ 10 ⁇ 5 M to about 0.1 ⁇ 10 ⁇ 5 M, about 1 ⁇ 10 ⁇ 6 M to about 0.1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 6 M to about 0.5 ⁇ 10 ⁇ 6 M, about 0.5 ⁇ 10 ⁇ 6 M to about 0.1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 7 M, about 1 ⁇ 10 ⁇ 7 M to about 0.5 ⁇ 10 ⁇ 7 M, about 0.5 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 7 M, about 1 ⁇ 10 ⁇ 8 M to about 0.1 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 8 M to about 0.5 ⁇ 10 ⁇ 8 M, or about
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 5 M to about 5 ⁇ 10 ⁇ 9 M, about 10 ⁇ 5 M to about 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 6 M to about 5 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 6 M to about 5 ⁇ 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 7 M to about 5 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 7 M to about 5 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 7
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 9 M. In some embodiments, the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 8 M. In some embodiments, the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 8 M. In some embodiments, the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M.
  • the KD of the binding between the second antigen-binding domain and the cell surface molecule is more than the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule can be about 1-10 times, about 10-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-60 times, about 60-70 times, about 70-80 times, about 80-90 times, about 90-100 times, about 100-500 times, or about 500-1000 times of the K D of the binding between the first antigen- binding domain and the tumor antigen.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1-15 times, about 1-14 times, about 1-13 times, about 1-12 times, about 1-11 times, about 1-10 times, about 1- 9.5 times, about 1-9 times, about 1-8.5 times, about 1-8 times, about 1-7.5 times, about 1-7 times, about 1-6.5 times, about 1-6 times, about 1-5.5 times, about 1-5 times, about 1-4.5 times, about 1-4 times, about 1-3.5 times, about 1-3 times, about 1-2.5 times, about 1-2 times, about 2-10 times, about 2-9 times, about 2-8 times, about 2-7 times, about 2-6 times, about 2- 5 times, about 2-4 times, about 2-3 times, about 2-2.5 times, about 3-10 times, about 3-9 times, about 3-8 times, about 3-7 times, about 3-6 times, about 3-5 times, about 3-4 times, about 3-3.5 times, about 4-10 times, about 4-9 times, about 4-8 times, about 4-7 times, about 4-6 times, about 4-5 times, about 4-4.5
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, or about 10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 2-10 times of the K D of the binding between the first antigen- binding domain and the tumor antigen.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the KD of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 4 M to about 0.1 ⁇ 10 ⁇ 4 M, about 1 ⁇ 10 ⁇ 4 M to about 0.5 ⁇ 10 ⁇ 4 M, about 0.5 ⁇ 10 ⁇ 4 M to about 0.1 ⁇ 10 ⁇ 4 M, about 1 ⁇ 10 ⁇ 5 M to about 0.1 ⁇ 10 ⁇ 5 M, about 1 ⁇ 10 ⁇ 5 M to about 0.5 ⁇ 10 ⁇ 5 M, about 0.5 ⁇ 10 ⁇ 5 M to about 0.1 ⁇ 10 ⁇ 5 M, about 1 ⁇ 10 ⁇ 6 M to about 0.1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 6 M to about 0.5 ⁇ 10 ⁇ 6 M, about 0.5 ⁇ 10 ⁇ 6 M to about 0.1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 7 M, about 1 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 7 M, about
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 2 ⁇ 10 ⁇ 9 M, about 10 ⁇ 4 M to about 5 ⁇ 10 ⁇ 9 M, about 10 ⁇ 4 M to about 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 4 M to about 2 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 4 M to about 5 ⁇ 10 ⁇ 8 M, about 10 ⁇ 4 M to about 10 ⁇ 7 M, about 5 ⁇ 10 ⁇ 5 M to about 2 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 5 M to about 5 ⁇ 10 ⁇ 9 M, about 5 ⁇ 10 ⁇ 5 M to about 1 ⁇ 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 5 M to about 2 ⁇ 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 5 M to about 5 ⁇ 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 5 M to about 5 ⁇ 10 ⁇ 8 M, about 5 ⁇ 10 ⁇ 5 M to about 1 ⁇ 10 ⁇ 7 M, about 1 ⁇ 10 ⁇ 5 M to about
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 10 ⁇ 8 M. In some embodiments, the K D of the binding between the second antigen- binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 10 ⁇ 7 M. In some embodiments, the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 7 M. In some embodiments, the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 2 ⁇ 10 ⁇ 9 M. In some embodiments, the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 2 ⁇ 10 ⁇ 8 M. In some embodiments, the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 2 ⁇ 10 ⁇ 8 M. In some embodiments, the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 5 M to about 0.1 ⁇ 10 ⁇ 5 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is 1 ⁇ 10 ⁇ 4 M to about 0.2 ⁇ 10 ⁇ 5 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 5 M to about 0.5 ⁇ 10 ⁇ 5 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is 1 ⁇ 10 ⁇ 4 M to about 1 ⁇ 10 ⁇ 5 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 0.5 ⁇ 10 ⁇ 5 M to about 0.1 ⁇ 10 ⁇ 5 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 0.5 ⁇ 10 ⁇ 4 M to about 0.2 ⁇ 10 ⁇ 5 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 6 M to about 0.1 ⁇ 10 ⁇ 6 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 5 M to about 0.2 ⁇ 10 ⁇ 6 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 6 M to about 0.5 ⁇ 10 ⁇ 6 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 5 M to about 1 ⁇ 10 ⁇ 6 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 0.5 ⁇ 10 ⁇ 6 M to about 0.1 ⁇ 10 ⁇ 6 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 0.5 ⁇ 10 ⁇ 5 M to about 0.2 ⁇ 10 ⁇ 6 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 6 M to about 0.2 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 7 M to about 0.5 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 0.5 ⁇ 10 ⁇ 7 M to about 0.1 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the second antigen- binding domain and the cell surface molecule is about 0.5 ⁇ 10 ⁇ 6 M to about 0.2 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 8 M to about 0.1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 7 M to about 0.2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 8 M to about 0.5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 0.5 ⁇ 10 ⁇ 8 M to about 0.1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 0.5 ⁇ 10 ⁇ 7 M to about 0.2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 4 M to about 2 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 5 M to about 5 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 4 M to about 1 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 4 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 5 M to about 2 ⁇ 10 ⁇ 9 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 6 M to about 5 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 5 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 5 M to about 2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 5 M to about 2 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 6 M to about 5 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 5 M to about 1 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 5 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 2 ⁇ 10 ⁇ 9 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 5 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 2 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 6 M to about 2 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 7 M to about 5 ⁇ 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 6 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 1 ⁇ 10 ⁇ 7 M to about 5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 1 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 9 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 9 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 9 ⁇ 10 ⁇ 8 M to about 5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 9 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 8 ⁇ 10 ⁇ 8 M to about 5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 8 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 7 ⁇ 10 ⁇ 8 M to about 5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 7 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 6 ⁇ 10 ⁇ 8 M to about 5 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 6 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 9 ⁇ 10 ⁇ 8 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 9 ⁇ 10 ⁇ 7 M to about 4 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 8 ⁇ 10 ⁇ 8 M to about 3 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 8 ⁇ 10 ⁇ 7 M to about 6 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 7 ⁇ 10 ⁇ 8 M to about 4 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 7 ⁇ 10 ⁇ 7 M to about 8 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 8 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 8 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 7 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 7 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 6 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 6 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 4 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 4 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 3 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 3 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 2 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 2 ⁇ 10 ⁇ 7 M to about 2 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 9 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 10 ⁇ 8 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 ⁇ 5 M to about 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 ⁇ 4 M to about 10 ⁇ 7 M
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen- binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 6 M to about 1 ⁇ 10 ⁇ 7 M.
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 ⁇ 8 M to about 1 ⁇ 10 ⁇ 8 M
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 ⁇ 7 M to about 1 ⁇ 10 ⁇ 7 M.
  • the KD of the binding between the second antigen-binding domain of the bispecific engager molecule and the cell surface molecule is the same as, or about 1-2 times of the K D of the binding between the first antigen-binding domain and the tumor antigen (hereinafter refer to as“similar to” or“similar binding affinity”).
  • the binding between the second antigen-binding domain and the cell surface molecule can be about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, or about 1 to about 1.1 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is the same as the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • a cell surface molecule on an effector cell such as CD3 on a T lymphocyte
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M, and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 -4 to about 10 -9 M.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • a cell surface molecule on an effector cell such as CD3 on a T lymphocyte
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen- binding domain and the tumor antigen is about 10 -5 to about 10 -8 M, and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 10 -4 to about 10 -8 M.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • a cell surface molecule on an effector cell such as CD3 on a T lymphocyte
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -7 to about 1 ⁇ 10 -8 M, and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 -6 to about 1 ⁇ 10 -8 M.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • a cell surface molecule on an effector cell such as CD3 on a T lymphocyte
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M, and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 -7 to about 1 ⁇ 10 -8 M.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • a cell surface molecule on an effector cell such as CD3 on a T lymphocyte
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M, and wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • a cell surface molecule on an effector cell such as CD3 on a T lymphocyte
  • the antigen specifically recognized by the first antigen- binding domain of the engager molecule is a tumor-associated antigen (TAA) or a tumor- specific antigen (TSA).
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • the TAA or TSA is expressed on a cancer cell.
  • the TAA or TSA is expressed on a blood cancer cell.
  • the TAA or TSA is expressed on a cell of a solid tumor.
  • solid tumor cancer include, by way of non-limiting example, a glioblastoma, a non-small cell lung cancer, a lung cancer other than a non-small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, liver cancer, colorectal cancer, stomach cancer, a cancer of the spleen, skin cancer (such as melanoma), a brain cancer other than a glioblastoma, a kidney cancer, a thyroid cancer, head and neck tumors, bladder cancer, esophageal cancer, or the like.
  • a glioblastoma a non-small cell lung cancer
  • a lung cancer other than a non-small cell lung cancer breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, liver cancer, colorectal cancer, stomach cancer, a cancer of the spleen, skin cancer (such as melanoma), a brain cancer other than a glioblastoma, a kidney cancer, a thyroid cancer, head
  • the TAA or TSA is one or more of, e.g., an scFv on the engager is specific for one or more of EphA2, HER2, GD2, Glypican-3, 5T4, 8H9, ⁇ v ⁇ 6 integrin, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR, Folate Receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, Mesothelin
  • the antigen specifically recognized by the first antigen- binding domain of the engager molecule is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3.
  • the tumor antigen is EpCAM, FAP, or EGFR.
  • the antigen specifically recognized by the first antigen- binding domain of the engager molecule is Epithelial cell adhesion molecule (EpCAM, CD326), also known as 17-1A, ESA, AUA1, EGP40, etc., which is a 40 kDa transmembrane glycoprotein composed of 314 amino acid.
  • EpCAM is specifically expressed in various types of epithelial cells, and major types of human malignancies. For example, EpCAM is highly expressed in colon cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, breast cancer and ovarian cancer.
  • the antigen specifically recognized by the first antigen- binding domain of the engager molecule is fibroblast activation protein (FAP).
  • FAP fibroblast activation protein
  • Fibroblasts are connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules. Fibroblasts in the tumor-stroma (i.e., tumor supporting tissue) synthesize FAP, a type II transmembrane protein that functions as a serine protease. FAP is selectively overexpressed in over 90% of stromal fibroblasts associated with colon, breast and lung carcinomas.
  • FAP is also expressed in some tumor cells, such as human malignant gliomas cell line U87 and murine lewis lung cancer cell line LL2 (Kraman et al., Science 330: 827-830 (2010)). Overexpression of FAP reportedly leads to promotion of tumor growth and increases in metastatic potential, whereas treatment with anti-FAP antibodies inhibits tumor growth.
  • the antigen specifically recognized by the first antigen- binding domain of the engager molecule is Epidermal growth factor receptor (EGFR).
  • EGFR is a member of the ErbB family of closely related receptors including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4).
  • Activation of EGFR leads to receptor tyrosine kinase activation and a series of downstream signaling events that mediate cellular proliferation, motility, adhesion, invasion, and resistance to chemotherapy as well as inhibition of apoptosis, processes that are crucial to the continual proliferation and survival of cancer cells.
  • Expression of the EGFR is associated with poor prognosis in a number of tumor types including stomach, colon, urinary bladder, breast, prostate, endometrium, kidney and brain (e.g., glioma).
  • EphA2 is referred to as EPH receptor A2 (ephrin type-A receptor 2; EPHA2; ARCC2; CTPA; CTPP1; or ECK), which is a protein that in humans is encoded by the EPHA2 gene in the ephrin receptor subfamily of the protein-tyro sine kinase family.
  • Receptors in this subfamily generally comprise a single kinase domain and an extracellular region comprising a Cys-rich domain and 2 fibronectin type III repeats; embodiments of the antibodies of the disclosure may target any of these domains.
  • EphA2 encodes a protein that binds ephrin-A ligands.
  • An exemplary human EphA2 nucleic sequence is in GenBank® Accession No. NM_004431, and an exemplary human EphA2 polypeptide sequence is in GenBank® Accession No. NP_004422, both of which sequences are incorporated herein in their entirety.
  • HER2 is referred to as human Epidermal Growth Factor Receptor 2 (Neu, ErbB-2, CD340, or pi 85), which is a protein that in humans is encoded by the ERBB2 gene in the epidermal growth factor receptor (EFR/ErbB) family.
  • HER2 contains an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules.
  • GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans. GD2 is present and concentrated on cell surfaces, with the two hydrocarbon chains of the ceramide moiety embedded in the plasma membrane and the oligosaccharides located on the extracellular surface, where they present points of recognition for extracellular molecules or surfaces of neighboring cells.
  • Glypican-3 is an oncofetal antigen re-expressed in a high frequency of neoplastic hepatocytes.
  • the GPC3 gene encodes a 70-kDa precursor core protein, which can be cleaved by furin to generate a 40-kDa amino (N) terminal protein and a 30-kDa membrane-bound carboxyl (C) terminal protein.
  • the C-terminus is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor.
  • Vascular endothelial growth factor receptor 2 (VEGFR2, KDR3) is a VEGF receptor, which is one of the most potent and specific positive regulators of angiogenesis.
  • VEGFR2 is highly expressed in tumor associated endothelial cells and contributes to tumor growth, invasion and metastasis (Dias et al., J Clin Invest.106(4):511-521, 2000; Santos et al., Blood 103(10):3883-3889, 2004; St. Croix et al., Science 289:1197-1202, 2000).
  • VEGFR2 is also expressed on the surface of several tumor cells including: B cell lymphoma and leukemia, multiple myeloma, urothelial bladder cancer, breast cancer, and lung cancer, among others (El-Obeid et al., Leuk Res. 28(2): 133-137, 2004; Kumar et al., Leukemia 17(10):2025-2031, 2003; Gakiopoulou-Givalou et al., Histopathology 43(3):272-279, 2003; Kranz et al., Int J Cancer 84(3):293-298, 1999; Decaussin et al., J Pathol.188(4):369-377, 1999).
  • the relatively high level of expression on tumor cells relative to normal vascular endothelial cells suggests that VEGFR2 is a suitable target of tumor therapy.
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the KD of the binding between the first antigen-binding domain and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the KD of the binding between the first antigen-binding domain and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte), wherein the KD of the binding between the first antigen-binding domain and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • Exemplary effector cell includes without limitation a T lymphocyte, a B lymphocyte, a natural killer (NK) cell, a dendritic cell (DC), a macrophage, a monocyte, a neutrophil, an NKT-cell, or the like.
  • the effector cell is a T lymphocyte.
  • the effector cell is a cytotoxic T lymphocyte.
  • the effector cell is allogenic.
  • the effector cell is autologous.
  • a cell surface molecule on an effector cell of the present invention is a molecule found on the external cell wall or plasma membrane of a specific cell type or a limited number of cell types.
  • cell surface molecules include, but are not limited to, membrane proteins such as receptors, transporters, ion channels, proton pumps, and G protein-coupled receptors; extracellular matrix molecules such as adhesion molecules (e.g., integrins, cadherins, selectins, or NCAMS); see, e.g., U.S. Pat. No. 7,556,928, which is incorporated herein by reference in its entirety.
  • Cell surface molecules on an effector cell include but not limited to CD3, CD4, CD5, CD8, CD16, CD27, CD28, CD40, CD64, CD89, CD134, CD137, CD278, NKp46, NKp30, NKG2D, and an invariant TCR.
  • the cell surface molecule-binding domain of an engager molecule can provide activation to immune cells.
  • immune cells have different cell surface molecules.
  • CD3 is a cell surface molecule on T-cells
  • CD16, NKG2D, or NKp30 are cell surface molecules on NK cells
  • CD3 or an invariant TCR are the cell surface molecules on NKT-cells.
  • Engager molecules that activate T-cells may therefore have a different cell surface molecule-binding domain than engager molecules that activate NK cells.
  • the activation molecule is one or more of CD3, e.g., CD3 ⁇ , CD3 ⁇ or CD3 ⁇ ; or CD27, CD28, CD40, CD134, CD137, and CD278.
  • the cell surface molecule is CD16, NKG2D, or NKp30, or wherein the immune cell is a NKT-cell, the cell surface molecule is CD3 or an invariant TCR.
  • CD3 comprises three different polypeptide chains ( ⁇ , ⁇ and ⁇ chains), is an antigen expressed by T cells.
  • the three CD3 polypeptide chains associate with the T-cell receptor (TCR) and the ⁇ -chain to form the TCR complex, which has the function of activating signaling cascades in T cells.
  • TCR T-cell receptor
  • the CD3 specific antibody OKT3 is the first monoclonal antibody approved for human therapeutic use, and is clinically used as an immunomodulator for the treatment of allogenic transplant rejections.
  • the second antigen-binding domain of the bispecific molecule described herein specifically recognizes CD3 on a human T lymphocyte.
  • the VH and VL regions of the CD3 specific domain are derived from an antibody specifically recognizing human CD3, wherein the binding affinity of the antibody and CD3 is about 10 -4 M to about 10 -9 M (such as about 10 -4 M to about 10 -8 M, about 5 ⁇ 10 -6 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M).
  • the VH and VL regions of the human CD3 specific domain are derived from Blinatumomab (Blincyto®, CD19-CD3 bispecific antibody).
  • VH and VL regions derived from antibodies/antibody derivatives and the like are capable of specifically recognizing the human CD3- ⁇ chain or human CD3- ⁇ chain.
  • the second antigen-binding domain of the bispecific molecule specifically binds to an epitope within the human CD3- ⁇ chain.
  • the second antigen-binding domain of the bispecific molecule described herein specifically recognizes CD3 on a mouse T lymphocyte.
  • the VH and VL regions of the CD3 specific domain are derived from an antibody specifically recognizing mouse CD3, wherein the binding affinity of the antibody and CD3 is about 10 -4 M to about 10 -9 M (such as about 10 -4 M to about 10 -8 M, about 5 ⁇ 10 -6 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M).
  • VH and VL regions are derived from antibodies/antibody derivatives and the like are capable of specifically recognizing the mouse CD3- ⁇ chain or mouse CD3- ⁇ chain.
  • the second antigen-binding domain of the bispecific molecule specifically binds to an epitope within the mouse CD3- ⁇ chain or mouse CD3- ⁇ chain.
  • the TCR complex is an octomeric complex of variable TCR ⁇ and ⁇ chains with three dimeric signaling modules CD3 ⁇ / ⁇ , CD3 ⁇ / ⁇ and CD3 ⁇ / ⁇ or ⁇ / ⁇ .
  • the engager molecule described herein targets CD3 ⁇ with a scFv
  • CD3 ⁇ targets CD3 ⁇ with a scFv
  • targeting other CD3 molecules, especially CD3 ⁇ , or the TCR ⁇ and ⁇ chains, with a specific scFv is also encompassed in the disclosure.
  • targeting molecules that are not part of the TCR complex for example, CD27, CD28, CD40, CD134, CD137, and CD278) is encompassed in the disclosure.
  • the bispecific molecule described herein comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on a T lymphocytes, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the T lymphocyte is a human T lymphocyte.
  • the T lymphocyte is a mouse T lymphocyte.
  • the T lymphocyte is a cytotoxic T lymphocyte.
  • the engager molecule described herein can be of any format known in the art.
  • Such engager molecules generally comprise a tumor antigen-binding domain and an effector cell surface molecule-binding domain.
  • the engager molecule's antigen-binding domain may be designed so as to bind to one or more tumor antigens present on target cells, while engager molecule's effector cell surface molecule-binding domain may be designed so as to bind to one or more cell surface molecules present on effector cells, for example, T lymphocytes.
  • the effector cell kills the tumor cells.
  • the engager is bipartite (e.g., comprising one tumor antigen- binding domain and one effector cell surface molecule-binding domain that may optionally be joined by a linker), or may be tripartite or multipartite (e.g., comprising one or more tumor antigen-binding domains and/or one or more effector cell surface molecule-binding domains, or other domains, including one or more co-stimulatory domains and/or one or more dimerization, trimerization or multimerization domains).
  • bipartite e.g., comprising one tumor antigen- binding domain and one effector cell surface molecule-binding domain that may optionally be joined by a linker
  • tripartite or multipartite e.g., comprising one or more tumor antigen-binding domains and/or one or more effector cell surface molecule-binding domains, or other domains, including one or more co-stimulatory domains and/or one or more dimerization, trimerization or
  • the engager is bispecific (e.g., comprising one tumor antigen-binding domain and one effector cell surface molecule-binding domain that may optionally be joined by a linker, wherein the tumor antigen and cell surface molecule are different).
  • Engagers may also be multispecific (e.g., comprising one tumor antigen-binding domain and two effector cell surface molecule-binding domains that may optionally be joined by a linker, wherein the tumor antigen and two cell surface molecules are all different).
  • the engager molecule can be in any format known in the art (see, e.g., Weidle et al., Cancer Genomics Proteomics, 10(1):1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015; Stamova et al., Antibodies, 1(2):172-198, 2012).
  • the engager may be a format class of “IgG-derived molecules” comprising Fc regions.
  • the engager molecule can be in the format of, but are not limited to, Common LC (light chain), DAF (dual acting Fab), which comprises evolved Fvs with dual specificity), CrossMab, IgG-dsscFv2 (disulfide-stabilized scFv2), DVD (dual variable domain), IgG-dsFv (disulfide-stabilized Fv), processed IgG-dsFv, IgG-scFab (single chain Fab), scFab-dsscFv, or Fv2-Fc.
  • Common LC light chain
  • DAF dual acting Fab
  • CrossMab crossMab
  • IgG-dsscFv2 disulfide-stabilized scFv2
  • DVD dual variable domain
  • IgG-dsFv disulfide-stabilized Fv
  • processed IgG-dsFv IgG-scFab (single chain Fab), scFab
  • Knobs-into-holes technologies can be used for heterodimerization of different H- chains in, for example, common LC, CrossMab, IgG-dsF, IgG-scFab or Fv2-Fc.
  • the engager may also be an“Fc-less bispecific” format class, which usually comprises individual scFvs of Fabs of different specificities fused together via linkers.
  • the engager can be in the format of, but are not limited to, Fab-scFv2, Fab-scFv, scFv-scFv (such as BiTE ® ), diabody, scBsDb (single-chain bispecific diabody), DART (dual-affinity retargeting molecule), TandAb (tetravalent tandem antibody), scBsTaFv (single-chain bispecific tandem variable domain), DNL-F(ab) 3 (dock-and-lock trivalent Fab), scFv-HSA-scFv (scFv-human serum albumin-scFv), or bssdAb (bispecific single-domain antibody).
  • Bi- or trivalent Fab-Fv or Fab-Fv2 formats are generated by fusion of VH-CH1 and/or L chains to scFvs.
  • scFv-scFv molecules (such as BiTE ® ) are generated by fusions of scFvs of different specificities.
  • the linker peptide length can be modulated so that VH and VL correctly pair, such as in Diabodies, DARTs, and TandAbs. These molecules can be further stabilized by interchain disulfide bonds (e.g. in DART, or between VH and VL of antibodies comprising scFvs).
  • the engager molecule may also be a format class of an antibody mimetics, which are small engineered proteins comprising antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and comprise a single polypeptide.
  • Exemplary engagers in the format class of antibody mimetics can be a Designed ankyrin repeat protein (DARPin; comprising 3-5 fully synthetic ankyrin repeats flanked by N- and C-terminal Cap domains), an avidity multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target), or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized).
  • DARPin Designed ankyrin repeat protein
  • avimer a high-affinity protein comprising multiple A domains, each domain with low affinity for a target
  • an Anticalin based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized.
  • the engager molecule to be employed in accordance with the disclosure can be chemically modified derivative of any of the aforementioned engager formats, or it may comprise ligands, peptides, or combinations thereof.
  • the engager molecule to be employed in accordance with the disclosure can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) (e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination.
  • modification(s) e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation
  • the bispecific engager molecule of the present invention is a bispecific single chain Fv (scFv).
  • a scFv in general contains a VH and VL domain connected by a linker peptide.
  • the secretable engager is composed of a signal peptide (to allow for secretion) from cells, followed by 2 scFvs connected by linker peptides (Lx, Ly, Lz).
  • Linkers may be of a length and sequence sufficient to ensure that each of the first and second domains can, independently from one another, retain their differential binding specificities.
  • Bispecific single chain molecules are known in the art and are described in WO 99/54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loffler, Blood, (2000), 95, 6, 2098-2103; and Bruhl, J. Immunol., (2001), 166, 2420-2426.
  • an exemplary molecular format of the disclosure provides an oncolytic virus comprising a nucleic acid encoding a polypeptide comprising a signal peptide followed by two scFvs, wherein the first scFv specifically recognizes a tumor antigen (such as EpCAM, FAP, or EGFR), and the second scFv specifically recognizes a cell surface molecule on an effector cell (such as CD3 on T lymphocytes).
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a cell surface molecule on an effector cell such as CD3 on T lymphocytes.
  • Each scFv comprises one V H and one V L region.
  • Bispecific scFv may be tandem bi-scFv or diabody.
  • Bispecific scFvs can be arranged in different formats: V H ⁇ -Lx-V L ⁇ -Ly-V H ⁇ -Lz-V L ⁇ , V L ⁇ -Lx-V H ⁇ -Ly-V H ⁇ - Lz-V L ⁇ , V L ⁇ -Lx-V H ⁇ -Ly-V L ⁇ -Lz-V H ⁇ , V H ⁇ -Lx-V L ⁇ -Ly-V L ⁇ -Lz-V H ⁇ , V H ⁇ -Lx-V L ⁇ -Ly-V H ⁇ - Lz-V L ⁇ , V L ⁇ -Lx-V L ⁇ -Ly-V H ⁇ -Lz-V H ⁇ , V H ⁇ -Lx-V H ⁇ -Ly-V L ⁇ -Lz-V H ⁇ , V H ⁇ -Lx-V H ⁇ -Ly-V L ⁇ -Lz-V H ⁇ , V H ⁇ -L
  • the linkers can be peptide linkers of any length.
  • the peptide linker between VH and VL of an antigen-binding domain (such as scFv) is from 1 amino acids to 20 amino acids long, from 2 amino acids to 19 amino acids long, from 3 amino acids to 18 amino acids long, from 4 amino acids to 17 amino acids long, from 5 amino acids to 17 amino acids long, from 6 amino acids to 17 amino acids long, from 7 amino acids to 18 amino acids long, from 8 amino acids to 17 amino acids long, from 9 amino acids to 17 amino acids long, from 10 amino acids to 17 amino acids long, from 11 amino acids to 16 amino acids long, from 12 amino acids to 17 amino acids long, from 13 amino acids to 16 amino acids long, from 14 amino acids to 16 amino acids long, or from 14 amino acids to 15 amino acids long.
  • the peptide linker between VH and VL of an antigen-binding domain is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In some embodiments, the peptide linker between VH and VL of an antigen-binding domain (such as scFv) is 14 or 15 amino acids long. In some embodiments, the peptide linker between the first and second antigen-binding domains (such as scFv) is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In some embodiments, the peptide linker between the first and second antigen- binding domains (such as scFv) is 5 amino acids long.
  • the peptide linker does not comprise any polymerization activity.
  • the characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80).
  • a particularly preferred amino acid in context of the“peptide linker” is Gly.
  • peptide linkers that also do not promote any secondary structures are preferred.
  • the linkage of the domains to each other can be provided by, e.g., genetic engineering.
  • the peptide linker can be a stable linker, which is not cleavable by protease, especially by Matrix metalloproteinases (MMPs).
  • MMPs Matrix metalloproteinases
  • the linker can also be a flexible linker.
  • exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n and (GGGS) n , where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
  • bispecific antibody molecule can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired bispecific antibody molecule structure.
  • the VH and VL domains of the first antigen-binding domain are linked together by a linker of sufficient length to enable the domains to fold in such a way as to permit binding to the tumor antigen, such as EpCAM, FAP, or EGFR.
  • a linker may comprise, for example, the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 46), or GGGGSGGGGSGGSA (SEQ ID NO: 47).
  • the VH domain and VL domains of the second antigen- binding domain are linked together by a linker of sufficient length to enable the domains to fold in such a way as to permit binding to cell surface molecule on an effector cell, such as CD3 on T lymphocytes.
  • a linker may comprise, for example, the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 46), or GGGGSGGGGSGGSA (SEQ ID NO: 47).
  • the first and second antigen-binding domains are linked together by a linker of sufficient length to enable the domains to fold in such a way as to permit binding both to the tumor antigen (such as EpCAM, FAP, or EGFR) and to the cell surface molecule on effector cells (such as CD3 on T lymphocytes).
  • a linker may comprise, for example, the amino acid sequence of GGGGS (SEQ ID NO: 48).
  • the engager additionally comprises one or more other domains, e.g., one or more of a cytokine, a costimulatory domain, a domain that inhibits negative regulatory molecules of T-cell activation, or a combination thereof.
  • the cytokine is IL-15, IL-2, and/or IL-7.
  • the co- stimulatory domain is CD27, CD80, CD83, CD86, CD134, or CD137.
  • the domain that inhibits negative regulatory molecules of T-cell activation is polypeptide that inhibits PD- 1, PD-L1, CTLA4, or B7-H4.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the first scFv specifically recognizing EpCAM comprises a heavy chain variable region (VH) comprising (1) a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first scFv specifically recognizing EpCAM comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first scFv specifically recognizing EpCAM comprises an amino acid sequence of SEQ ID NO: 33.
  • the first scFv specifically recognizing EpCAM comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first scFv specifically recognizing EpCAM comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 83.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first scFv specifically recognizing EpCAM comprises an amino acid sequence of SEQ ID NO: 36.
  • the VH and VL of the first scFv specifically recognizing EpCAM are connected by a linker comprising the amino acid sequence of SEQ ID NO: 46.
  • the second scFv specifically recognizing CD3 on T lymphocytes comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second scFv specifically recognizing CD3 on T lymphocytes comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • VH and VL of the second scFv specifically recognizing CD3 on T lymphocytes are connected by a linker comprising the amino acid sequence of SEQ ID NO: 46.
  • the second scFv specifically recognizing CD3 comprises the amino acid sequence of SEQ ID NO: 45.
  • the first and second scFvs are connected by a linker comprising the amino acid sequence of SEQ ID NO: 48.
  • the first scFv is at N-terminal to the second scFv. In some embodiments, the first scFv is at C-terminal to the second scFv.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 55.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EpCAM is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 58.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on human T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the first scFv specifically recognizing FAP comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first scFv specifically recognizing FAP comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 32.
  • VH and VL of the first scFv specifically recognizing FAP are connected by a linker comprising the amino acid sequence of SEQ ID NO: 47.
  • the first scFv specifically recognizing FAP comprises an amino acid sequence of SEQ ID NO: 49.
  • the second scFv specifically recognizing CD3 on T lymphocytes comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second scFv specifically recognizing CD3 on T lymphocytes comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • VH and VL of the second scFv specifically recognizing CD3 on T lymphocytes are connected by a linker comprising the amino acid sequence of SEQ ID NO: 46.
  • the second scFv specifically recognizing human CD3 comprises an amino acid sequence of SEQ ID NO: 45.
  • the first and second scFvs are connected by a linker comprising the amino acid sequence of SEQ ID NO: 48.
  • the first scFv is at N-terminal to the second scFv. In some embodiments, the first scFv is at C-terminal to the second scFv.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on human T lymphocytes, wherein the K D of the binding between the first scFv and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 59.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M).
  • the first scFv specifically recognizing EGFR comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 84; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 86; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 87; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 89.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first scFv specifically recognizing EGFR comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 90, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 91.
  • VH and VL of the first scFv specifically recognizing EGFR are connected by a linker comprising the amino acid sequence of SEQ ID NO: 46.
  • the first scFv specifically recognizing EGFR comprises an amino acid sequence of SEQ ID NO: 50.
  • the second scFv specifically recognizing CD3 on T lymphocytes comprises a heavy chain variable region (VH) comprising (1) a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 37; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 40; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second scFv specifically recognizing CD3 on T lymphocytes comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44.
  • VH and VL of the second scFv specifically recognizing CD3 on T lymphocytes are connected by a linker comprising the amino acid sequence of SEQ ID NO: 46.
  • the second scFv specifically recognizing human CD3 comprises an amino acid sequence of SEQ ID NO: 45.
  • the first and second scFvs are connected by a linker comprising the amino acid sequence of SEQ ID NO: 48.
  • the first scFv is at N- terminal to the second scFv. In some embodiments, the first scFv is at C-terminal to the second scFv.
  • the bispecific molecule described herein comprises a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the K D of the binding between the first scFv and EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and wherein the bispecific molecule comprises an amino acid sequence of SEQ ID NO: 74.
  • the oncolytic virus encoding the low-affinity bispecific molecule of the present invention further comprises a second nucleic acid encoding an immune checkpoint modulator.
  • Immune checkpoints are molecules in the immune system that either turn up (stimulatory molecules) or turn down a signal (inhibitory molecules). Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Stimulatory checkpoint molecules include, but are not limited to, CD27, CD40, OX40, GITR and CD137, which belong to tumor necrosis factor (TNF) receptor superfamily, as well as CD28 and ICOS, which belong to the B7-CD28 superfamily.
  • TNF tumor necrosis factor
  • Inhibitory checkpoint molecules include, but are not limited to, program death 1 (PD-1), Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), Lymphocyte Activation Gene-3 (LAG-3), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3, HAVCR2), V- domain Ig suppressor of T cell activation (VISTA, B7-H5), B7-H3, B7-H4 (VTCN1), HHLA2 (B7-H7), B and T Lymphocyte Attenuator (BTLA), Indoleamine 2,3-dioxygenase (IDO), Killer-cell Immunoglobulin-like Receptor (KIR), adenosine A2A receptor (A2AR), T cell immunoreceptor with Ig and ITIM domains (TIGIT), 2B4 (CD244) and ligands thereof.
  • PD-1 program death 1
  • CTL-4 Cytotoxic T-Lymphocyte-Associated protein 4
  • LAG-3 Lymph
  • checkpoint proteins have been studied extensively, such as CTLA-4 and its ligands CD80 (B7-1) and CD86, and PD-1 with its ligands PD-L1 (B7-H1, CD274) and PD- L2 (B7-DC, CD273) (See, for example, Pardoll, Nature Reviews Cancer 12: 252-264 (2012)).
  • Immune checkpoint modulators can be immune checkpoint inhibitors (inhibitors of inhibitory immune checkpoint molecules) or activators of stimulatory immune checkpoint molecules.
  • Immune checkpoint inhibitors are of particular interest in the present invention, such as inhibitors of PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-DC, CD273), LAG-3, TIM-3 (HAVCR2), BTLA, CTLA-4, TIGIT, VISTA (B7-H5), B7-H4 (VTCN1), CD160 (BY55), HHLA2 (B7-H7), CXCR4, 2B4 (CD244), CD73, B7-1 (CD80), B7-H3 (CD276), KIR, or IDO.
  • the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.
  • the activator of a stimulatory immune checkpoint molecule is a natural or engineered ligand of a stimulatory immune checkpoint molecule, including, for example, ligands of OX40 (e.g., OX40L), ligands of CD28 (e.g., CD80, CD86), ligands of ICOS (e.g., B7RP1), ligands of 4-1BB (e.g., 4-1BBL, Ultra 4-1BBL), ligands of CD27 (e.g., CD70), ligands of CD40 (e.g., CD40L), and ligands of TCR (e.g., MHC class I or class II molecules, IMCgp100).
  • OX40 e.g., OX40L
  • CD28 e.g., CD80, CD86
  • ICOS e.g., B7RP1
  • the activator of a stimulatory immune checkpoint molecule is a secreted protein.
  • the activator of a stimulatory immune checkpoint molecule is an antibody (such as an agonist antibody), such as anti-CD28, anti-OX40, anti-ICOS, anti-GITR, anti-4- 1BB, anti-CD27, anti-CD40, anti-CD3, and anti-HVEM.
  • the immune checkpoint modulator is an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor targets T cells.
  • the immune checkpoint inhibitor targets tumor cells. For example, in some cases, tumor cells can turn off activated T cells, when they attach to specific T-cell receptors. However, immune checkpoint inhibitors may prevent tumor cells from attaching to T cells so that T cells stay activated (see, for example, Howard West, JAMA Oncol. 1(1):115 (2015)).
  • the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM-3 (e.g., Galectin-9), ligands of A2A Receptor (e.g., adenosine, Regadenoson), ligands of LAG-3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod, NLG919), ligands of HHLA2 (e.g., TMIGD2), ligands of CTLA-4 (
  • the immune checkpoint inhibitor is secreted.
  • the immune checkpoint inhibitor is an antibody (such as antagonist antibody) that targets an inhibitory immune checkpoint protein, including but not limited to, anti-CTLA-4, anti-TIM-3, anti-LAG-3, anti-KIR, anti-PD-1, anti-PD-L1, anti-CD73, anti-B7-H3, anti-CD47, anti-BTLA, anti-VISTA, anti-A2AR, anti-B7-1, anti-B7- H4, anti-CD52, anti-IL-10, anti-IL-35, and anti-TGF- ⁇ .
  • an inhibitory immune checkpoint protein including but not limited to, anti-CTLA-4, anti-TIM-3, anti-LAG-3, anti-KIR, anti-PD-1, anti-PD-L1, anti-CD73, anti-B7-H3, anti-CD47, anti-BTLA, anti-VISTA, anti-A2AR, anti-B7-1, anti-B7- H4, anti-CD52, anti-IL-10, anti-IL-35
  • the immune checkpoint inhibitor is an inhibitor of an inhibitory checkpoint molecule selected from the group consisting of PD-1, PD-L1, LAG-3, TIM-3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, and VISTA.
  • the immune checkpoint modulator is an inhibitor of PD-1.
  • the immune checkpoint modulator is an antibody specifically recognizing PD-1.
  • the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule.
  • the immune checkpoint modulator inhibitor is a ligand that binds to PD-L1 and/or PD-L2.
  • the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin.
  • the Fc fragment is an IgG4 Fc.
  • the immune checkpoint modulator is a ligand that binds to HHLA2.
  • the immune checkpoint modulator is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.
  • the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4.
  • the immune checkpoint modulator comprises an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.
  • PD-1 is a part of the B7/CD28 family of co-stimulatory molecules that regulate T- cell activation and tolerance, and thus antagonistic anti-PD-1 antibodies can be useful for overcoming tolerance.
  • PD-1 has been defined as a receptor for B7-4.
  • B7-4 can inhibit immune cell activation upon binding to an inhibitory receptor on an immune cell. Engagement of the PD-1/PD-L1 pathway results in inhibition of T-cell effector function, cytokine secretion and proliferation. (Turnis et al., OncoImmunology 1(7):1172-1174, 2012).
  • High levels of PD-1 are associated with exhausted or chronically stimulated T cells.
  • increased PD-1 expression correlates with reduced survival in cancer patients.
  • Agents for down modulating PD-1, B7-4, and the interaction between B7-4 and PD-1 inhibitory signal in an immune cell can result in enhancement of the immune response.
  • PD-L1 (Programmed cell death-ligand 1) is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1).
  • PD-L1 serves as a ligand for PD-1 to play a major role in suppressing the immune system during particular events such as pregnancy, tissue allographs, autoimmune disease and other disease states such as hepatitis and cancer.
  • the formation of PD-1 receptor/PD-L1 ligand complex transmits an inhibitory signal which reduces the proliferation of CD8+ T cells at the lymph nodes.
  • PD-L2 (Programmed cell death 1 ligand 2) is also known as B7-DC.
  • PD-L2 serves as a ligand for PD-1.
  • PD-L2 and its inhibitor can be used as a substitute for PD-L1 and its inhibitor respectively.
  • LAG-3 (Lymphocyte Activating Gene-3, CD223) works to suppress an immune response by action to Tregs, as well as direct effects on CD8+ T cells (Huang et al., 2004, Immunity.21(4):503-13; Grosso et al., 2007, J Clin Invest. 117(11):3383-92).
  • TIM-3 Lymphocyte Activating Gene-3
  • TIM-3 (HAVCR2) is an immune checkpoint molecule, which has been associated with the inhibition of lymphocyte activity and in some cases induction of lymphocyte anergy (Pardoll D. Nature Reviews 2012 April Vol. 12: 252).
  • TIM-3 is a receptor for galectin 9 (GAL9), which is up-regulated in various types of cancers, including breast cancers.
  • GAL9 galectin 9
  • TIM- 3 has been identified as another important inhibitory receptor expressed by exhausted CD8+ T cells. In mouse models of cancer, it has been shown that the most dysfunctional tumor- infiltrating CD8+ T cells actually co-express PD-1 and TIM-3.
  • HHLA2 human endogenous retrovirus-H long terminal repeat-associating protein 2, B7-H7
  • B7-H7 human endogenous retrovirus-H long terminal repeat-associating protein 2, B7-H7
  • B7-H7 a retrovirus-H long terminal repeat-associating protein 2
  • It is a membrane protein with three Ig-like domains (IgV-IgC-IgV) in its extracellular region.
  • the expression of HHLA2 is not inducible on T cells, which is different from PD-L1 (B7-H1).
  • HHLA2 receptors can be found on a various immune cells, including T cells, B cells, monocytes, and dendritic cells (DC).
  • DC dendritic cells
  • CD28H CD28H, TMIGD2
  • Ig superfamily member Ig superfamily member with an extracellular IgV-like domain, a transmembrane region, and a cytoplasmic tail
  • Janakiram, M. et al. Expression, clinical significance, and receptor identification of the newest B7 family member HHLA2 protein. Clin Cancer Res 2015; 21:2359-66).
  • HHLA2 was found to over-express in many human cancers such as cancers from breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus (Janakiram, M., et al., Clin Cancer Res 2015, 21:2359-66), and has been demonstrated to inhibit CD4 and CD8 proliferation and cytokine production in vitro (Zhao R. et al., Proc Natl Acad Sci U S A.2013;110:9879–9884).
  • BTLA B and T Lymphocyte Attenuator, CD272
  • CD272 is a member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and PD-1.
  • BTLA has one IgV domain in extracellular domain, a transmembrane domain and an intracellular domain.
  • HVEM herpes virus entry mediator
  • TNFR tumor-necrosis factor receptor
  • CD160 (BY55) is a multimeric glycosylphosphatidylinositol-anchored lymphocyte surface receptor which expression is mostly restricted to the highly cytotoxic CD56 dim CD16 + peripheral blood subset in human. CD160 is also expressed in human by most of TCR ⁇ cells, a subset of TCR ⁇ CD8 bright+ T cells and almost all intestinal intraepithelial lymphocytes (iIELs) (Ma ⁇ za et al. J Exp Med 1993; 178:1121-6; Anumanthan et al. J Immunol. 1998, 161:2780-90).
  • iIELs intestinal intraepithelial lymphocytes
  • CD160 binds to CD160 on circulating NK lymphocytes, and that their interaction triggers their cytotoxic activity and cytokine production (Le Bouteiller at al. PNAS 2002; 99(26):16963-8).
  • the cell surface expression of CD160 is down-modulated by NK cell activation mediated by cytokines including IL-2 and IL-15.
  • cytokines including IL-2 and IL-15.
  • the activation of an immune response leads to the release of a soluble form of CD160 from cells expressing CD160 such as NK cells, T cells, mast cells, or activated endothelial cells.
  • This soluble form of CD160 can then bind to classical and non-classical MHC class I molecules and CD1 molecules, resulting in the inhibition of the cytotoxic CD8+ T cells activity, of the CD160-mediated NK cell activity, and of TCR ⁇ and NKT functions, as was shown in WO2008009711.
  • 2B4 (CD244) belongs to the CD2 family molecules and is expressed on all NK, ⁇ , and memory CD8 + ( ⁇ ) T cells. 2B4 mediated interaction between NK-cell and target cells is thought to modulate NK-cell cytolytic activity. The non-MHC restricted cell killing mediated by 2B4 also applies to subsets of T cells.2B4 becomes engaged upon binding its high-affinity ligand, CD48. The 2B4-L isoform is believed to be a negative immune regulator of immune cells.
  • CD73 or ecto-5'-nucleotidase (5'-NT) is ubiquitously expressed in a number of tissues. This protein is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) linkage, has ecto-enzyme activity, and plays a role in signal transduction.
  • GPI glycosylphosphatidylinositol
  • the primary function of CD73 is the conversion of extracellular nucleotides (e.g., 5'-AMP), to which cells are generally impermeable, to their corresponding nucleosides (e.g., adenosine), which can readily enter most cells.
  • CD73 production of adenosine by the dephosphorylation of AMP has been shown to regulate adenosine receptor engagement in many tissues, indicating that adenosine functions in cytoprotection, cell growth, angiogenesis and immunosuppression, and also plays a role in tumorigenesis.
  • Extracellular adenosine accumulates in cancerous tissues and constitutes an important mechanism of tumor immune escape.
  • tumor-derived adenosine profoundly inhibits infiltrating effector T cells through adenylyl cyclase-activating A2A receptors.
  • CD73 expression has been reported in a range of tumor cells, including leukemia, lymphoma, bladder cancer, colorectal cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer and breast cancer. Elevated CD73 expression has also been associated with tumor invasiveness, metastasis, and reduced patient survival time. CD73 generates an immunosuppressed environment, characterized by increased adenosine levels, which promote the development and progression of cancer. Notably, CD73 expression has been associated with a pro- metastatic phenotype in melanoma and breast cancer.
  • CD73-/- mice are protected from transplanted and spontaneous tumors (Stagg et al., 2010, Cancer Res.71:2892-2900).
  • CTLA-4 is an immune checkpoint molecule, which is up-regulated on activated T- cells.
  • An anti-CTLA-4 mAb can block the interaction of CTLA-4 with CD80/86 and switch off the mechanism of immune suppression and enable continuous stimulation of T-cells by DCs.
  • B7-H4 (VTCN1) is a Type I transmembrane protein and is a member of the B7 superfamily of proteins that provides co-signal in conjunction with a T-cell receptor antigenic signal. B7-H4 is expressed by tumor cells and tumor-associated macrophages. It is a negative regulator of T-cell function - binding to T-cells inhibits their growth, cytokine secretion and cytotoxicity, thus B7-H4 plays a role in tumor escape. The receptor for B7-H4 is unknown and unidentified.
  • B7-H4 expression in tumor tissues was observed in various types of human cancers such as breast (Tringler et al, 2005, Clin Cancer Res 11 (5):1842-8), ovarian (Kryczek et al, 2006, J Exp Med 203(4):871-81), pancreatic, lung (Choi et al, 2003, J Immunol 171(9):4650- 4; Sun et al, 2006, Lung Cancer 53(2):143-51) melanoma (Quandt et al, 2011, Clin Cancer Res 17(10):3100-11) and renal cell carcinoma (Jung et al, 2011, Korean J Urol 52(2):90-5; Krambeck et al, 2006, Proc Natl Acad Sci USA 103(27):10391-6).
  • Human B7-H4 is a 282 amino acid protein (including the amino-terminal signal sequence), of which -227 amino acids are predicted to be in the extracellular space following cleavage of the amino-terminal signal sequence.
  • B7-H4 comprises an Ig-like V-domain, an Ig- like C domain, a transmembrane domain and a short cytoplasmic tail.
  • TIGIT T cell immunereceptor with Ig and ITIM domains
  • TIGIT is an immunomodulatory receptor expressed primarily on activated T cells and NK cells.
  • TIGIT is also known as VSIG9, VSTM3, and WUCAM. Its structure shows one extracellular immunoglobulin domain, a type I transmembrane region and two ITIM motifs.
  • TIGIT forms part of a co- stimulatory network that consists of positive (CD226) and negative (TIGIT) immunomodulatory receptors on T cells, and ligands expressed on APCs (CD 155 and CD112).
  • TIGIT immunoreceptor tyrosine-based inhibition motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • ligation of TIGIT by receptor-ligands CD 155 and CD112 expressed by tumor cells or tumor-associated macrophages may contribute to the suppression of TCR-signaling and T cell activation, which is essential for mounting effective anti-tumor immunity.
  • an antagonist antibody specific for TIGIT could inhibit the CD 155 and CD112 induced suppression of T cell responses and enhance anti-tumor immunity.
  • V-domain Immunoglobulin Suppressor of T cell Activation bears limited homology to PD-L1, but does not belong to the B7 family due to its unique structure
  • VISTA has a single IgV domain with 3 additional cysteine residues. Its cytoplasmic tail does not contain any signaling motifs.
  • VISTA is a checkpoint regulator that negatively suppresses immune responses. See Wang et al.,“VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses,” J. Exp. Med., 208(3) 577-92 (2011).
  • VISTA is exclusively expressed within the hematopoietic compartment, with very high levels of expression on CD11b high myeloid cells, and lower expression levels on CD4+ and CD8+ T cells, and Tregs.
  • a soluble VISTA-Ig fusion protein or VISTA expressed on APCs acts as a ligand to suppress CD4+ and CD8+ T cell proliferation and cytokine production, via an unidentified receptor independent of PD-1.
  • An anti-VISTA mAb 13F3 reversed VISTA-mediated T cell suppression in vitro and suppressed tumor growth in multiple murine tumor models by enhancing the anti-tumor T cell responses.
  • VISTA over- expression on tumor cells impaired protective anti-tumor immunity in vaccinated hosts.
  • VISTA KO mice develop an inflammatory phenotype, which points towards a loss of peripheral tolerance.
  • U.S. Pat. Nos. 8,236,304 and 8,231,872 Published International Applications WO/2011/120013 and WO/2006/116181, U.S. Published Application Nos. 2008/0287358, 2011/0027278, and 2012/0195894, and U.S. Provisional Patent Application See Nos. 60/674,567, filed Apr. 25, 2005, 61/663,431, filed Jun. 22, 2012, Ser. No. 61/663,969, filed Jun. 25, 2012, 61/390,434, filed Oct. 6, 2010, 61/436,379, filed Jan. 26, 2011, and 61/449,882, filed Mar.
  • VISTA is a novel, functionally non-redundant, central negative regulator of immunity, whose expression is primarily myeloid-restricted.
  • HHLA2 is widely expressed in many human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, esophagus and hematological malignancies of leukemia and lymphoma.
  • HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and is an attractive target for human cancer therapy.
  • TMIGD2 has been identified as a receptor for HHLA2. Blocking of HHLA2/TMIGD2 could be an effective strategy for cancer immunotherapy.
  • the immune checkpoint modulator described herein is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).
  • CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRP ⁇ ). Interference with the CD47-SIRP ⁇ interaction could enhance anti-tumor immune responses.
  • SIRP ⁇ receptor signal regulatory protein alpha
  • the chemokine CXCL12 and its receptor CXCR4 are expressed widely in human cancers, including ovarian cancer, in which they are associated with disease progression at the levels of tumor cell proliferation, invasion, and angiogenesis.
  • CXCL12 produced by tumor tissue and surrounding stroma stimulates VEGF-mediated angiogenesis and the recruitment of endothelial progenitor cells from the bone marrow.
  • CXCL12 has also been shown to recruit suppressive CD11b+Gr1+ myeloid cells and pDCs at tumor sites, and induce intratumoral T regulatory cells (Tregs) localization, which impede immune mechanisms of tumor destruction. Therefore, modulation of the CXCL12/CXCR4 axis could impact multiple aspects of tumor pathogenesis including immune dysregulation.
  • Several CXCR4 antagonists have demonstrated antitumor efficacy in preclinical models and have been evaluated in early clinical trials.
  • the immune checkpoint modulator described herein is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4.
  • the immune checkpoint modulator comprises an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.
  • the immune checkpoint modulators contemplated herein are proteins or peptides.
  • the immune checkpoint modulator comprises a single polypeptide chain.
  • the immune checkpoint modulator comprises more than one (such as any of 2, 3, 4, or more) polypeptide chains.
  • the polypeptide chain(s) of the immune checkpoint modulator may be of any length, such as at least about any of 10, 20, 50, 100, 200, 300, 500, or more amino acids long.
  • the nucleic acid sequences encoding the polypeptide chains may be operably linked to the same promoter or to different promoters.
  • the immune checkpoint modulator is a secreted protein.
  • the immune checkpoint modulator is an antibody.
  • Native antibodies such as monoclonal antibodies, are immunoglobulin molecules that are immunologically reactive with a particular antigen.
  • the antibody is an agonistic antibody.
  • the antibody is an antagonistic antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a full-length antibody.
  • the antibody is an antigen-binding fragment selected from the group consisting of V H , V L , V NAR , V H H, Fab, Fab’, F(ab’) 2 , Fv, minibody, scFv, sc(Fv) 2 , tribody, tetrabody, scFv-scFv (such as BiTE ® ), minibody, scFv-Fc, triabody, and other antigen-binding subsequences of the full length antibody or engineered combinations thereof.
  • the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
  • the antibody is a monovalent antibody.
  • the antibody is a multivalent antibody, such as a divalent antibody or a tetravalent antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a single domain antibody (sdAb). In some embodiments, the antibody is a heavy chain-only antibody, such as a camelid antibody or a derivative thereof. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the antibody is a scFv. In some embodiments, the antibody is a fusion protein comprising an antibody fragment (such as an Fc-containing fusion protein, e.g. PD-1 extracellular domain- Fc fusion protein) or any other functional variants or derivatives of a full-length antibody.
  • an antibody fragment such as an Fc-containing fusion protein, e.g. PD-1 extracellular domain- Fc fusion protein
  • the immune checkpoint modulator is an antibody comprising a heavy chain and a light chain.
  • the heavy chain comprises a V H domain.
  • the heavy chain further comprises one or more constant domains, such as C H 1, C H 2, C H 3, or any combination thereof.
  • the light chain comprises a V L domain.
  • the light chain further comprises a constant domain, such as C L .
  • the heavy chain and the light chain are connected to each other via a plurality of disulfide bonds.
  • the antibody comprises an Fc, such as an Fc fragment of the human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody does not comprise an Fc fragment.
  • the immune checkpoint modulator is a scFv.
  • the oncolytic virus expressing the low-affinity bispecific molecule described herein may further express any number (such as any of 1, 2, 3, 4, 5, 6, or more) of immune checkpoint modulators.
  • the oncolytic virus comprises a nucleic acid encoding a single immune checkpoint modulator.
  • the oncolytic virus comprises one or more nucleic acids encoding at least two immune checkpoint modulators.
  • the nucleic acids encoding the at least two immune checkpoint modulators are operably linked to the same promoter.
  • the nucleic acids encoding the at least two immune checkpoint modulators are operably linked to different promoters.
  • nucleic acids encoding the immune checkpoint modulator(s) and the bispecific molecule of the present invention are operably linked to the same promoter. In some embodiments, the nucleic acids encoding the immune checkpoint modulator(s) and the bispecific molecule of the present invention are operably linked to different promoters.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • a second antigen-binding domain such as scFv
  • the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule.
  • the immune checkpoint modulator is an anti-PD-1 antibody (such as scFv). In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand that binds to PD-L1 and/or PD-L2. In some embodiments, the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator is a ligand that binds to HHLA2.
  • an anti-PD-1 antibody such as scFv
  • the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand that binds to PD-L1 and/or PD-L2. In some embodiments, the immune checkpoint modulator is an extracellular domain of PD-1 fused to
  • the immune checkpoint modulator is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).
  • the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4.
  • the immune checkpoint modulator comprises an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).
  • the nucleic acid encoding the bispecific molecule and/or the second nucleic acid encoding the immune checkpoint modulator is operably linked to a promoter (such as late promoter, e.g., F17R).
  • a promoter such as late promoter, e.g., F17R
  • the nucleic acid encoding the bispecific molecule and the second nucleic acid encoding the immune checkpoint modulator are operably linked to the same promoter.
  • the nucleic acid encoding the bispecific molecule and the second nucleic acid encoding the immune checkpoint modulator are operably linked to different promoters.
  • the oncolytic virus (such as VV) comprising a nucleic acid encoding a low-affinity bispecific molecule described herein further comprises a second nucleic acid encoding a cytokine (such as GM-CSF).
  • the oncolytic virus (such as VV) comprising a nucleic acid encoding a low-affinity bispecific molecule described herein further comprises a second nucleic acid encoding an immune checkpoint modulator, and a third nucleic acid encoding a cytokine (such as GM-CSF).
  • cytokine or“cytokines” as used herein refers to the general class of biological molecules, which affect cells of the immune system.
  • the definition is meant to include, but is not limited to, those biological molecules that act locally or may circulate in the blood, and which, when used in the present invention serve to regulate or modulate an individual’s immune response to cancer.
  • interferons such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇
  • all interleukins e.g., IL-1 to IL-29, in particular, IL-1,
  • GM-CSF is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts that functions as a cytokine.
  • GM-CSF induces activation, proliferation, and differentiation of a variety of immunologically active cell populations, thereby facilitating the development of both humoral and cellular-mediated immunity (Warren and Weiner, 2000).
  • the cytokine is GM-CSF.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion
  • an immune checkpoint modulator such as anti-PD-1 antibody, PD-1
  • the cytokine is GM-CSF.
  • the nucleic acid encoding the low-affinity bispecific molecule, and/or the second nucleic acid encoding the immune checkpoint modulator, and/or the third nucleic acid encoding the cytokine is operably linked to a promoter (such as late promoter, e.g., F17R).
  • a promoter such as late promoter, e.g., F17R.
  • at least two of the nucleic acids encoding the low-affinity bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to the same promoter.
  • all of the nucleic acids encoding the low-affinity bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the low- affinity bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to different promoters. Regulatory sequence
  • regulatory sequences may be added to the OV nucleic acid molecules comprised in the disclosure.
  • Such regulatory sequences are known to the artisan and may include a promoter, additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), stability (e.g. introns and non-coding 5’ and 3’ sequences), translation (e.g. an initiator Met, tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.), and insertion site for introducing an insert into the viral vector.
  • the regulatory sequences are promoters, transcriptional enhancers and/or sequences that allow for proper expression of the low-affinity bispecific molecule, the immune checkpoint modulator, and the cytokine of the disclosure may be employed.
  • control sequence refers to DNA sequences that are necessary to affect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoters, ribosomal binding sites, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors.
  • control sequence is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.
  • control sequence“operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence“operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is preferably used.
  • a“promoter”, a promoter region or a promoter element or regulatory region or regulatory element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked.
  • the promoter region includes specific sequences that are involved in RNA polymerase recognition, binding and transcription initiation.
  • the promoter includes sequences that modulate recognition, binding and transcription initiation activity of RNA polymerase (i.e., binding of one or more transcription factors). These sequences can be cis acting or can be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, can be constitutive or regulated. Regulated promoters can be inducible or environmentally responsive (e.g.
  • the present invention also relates to an oncolytic viral vector comprising the nucleic acid molecules described in the present disclosure (e.g., nucleic acids encoding low-affinity bispecific engager molecules, immune checkpoint modulators, or cytokines).
  • the term“viral vector” is used according to its art-recognized meaning. It refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged into a viral vector particle. The viral vector particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo.
  • the oncolytic viral vector is a VV vector.
  • the present invention therefor relates to a VV vector comprising the nucleic acid molecules described herein.
  • the thymidine kinase gene of the vaccinia virus vector may have been deleted.
  • the vaccinia virus vector may have a mutation in a gene encoding vaccinia virus growth factor.
  • the oncolytic viral vector is a lentiviral vector. Lentiviral vectors are commercially available, including from Clontech (Mountain View, Calif.) or GeneCopoeia (Rockville, Md.), for example.
  • “Expression vector” is a construct that can be used to transform a selected host and provides for expression of a coding sequence in the selected host.
  • Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors.
  • Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript.
  • regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV- promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements that are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the recited nucleic acid sequence and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous nucleic acid sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA, (Raum et al., Cancer Immunol Immunother (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL).
  • the choice of the regulatory sequences can depend on such factors as the nucleic acid molecule itself, the virus into which it is inserted, the host cell or subject, the level of expression desired, etc.
  • the promoter is of special importance. In the context of the invention, it can be constitutive directing expression of the nucleic acid molecule in many types of host cells or specific to certain host cells (e.g. tumor-specific regulatory sequences) or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc.) or according to the phase of a viral cycle (e.g. late or early).
  • the expression control sequences are eukaryotic promoter systems in vectors capable of transforming of transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purification of the polypeptide of the disclosure may follow.
  • Promoters can be native or heterologous (with respect to the oncolytic virus described herein). Any suitable promoters, including synthetic and naturally-occurring and modified promoters, can be used.
  • a native promoter is a promoter that is endogenous to the virus and is unmodified with respect to its nucleotide sequence and its position in the viral genome as compared to a wild-type virus.
  • a synthetic promoter is a heterologous promoter that has a nucleotide sequence that is not found in nature.
  • a synthetic promoter can be a nucleic acid molecule that has a synthetic sequence or a sequence derived from a native promoter or portion thereof.
  • a synthetic promoter can also be a hybrid promoter composed of different elements derived from different native promoters. See, e.g. US9005602 for exemplary vaccinia virus synthetic promoters.
  • Viral promoters can include, but are not limited to, VV promoter, poxvirus promoter, adenovirus late promoter, Cowpox ATI promoter, or T7 promoter.
  • the promoter may be a vaccinia virus promoter, a synthetic promoter, a promoter that directs transcription during at least the early phase of infection, a promoter that directs transcription during at least the intermediate phase of infection, a promoter that directs transcription during early/late phase of infection, or a promoter that directs transcription during at least the late phase of infection.
  • Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters.
  • Promoters suitable for constitutive expression in mammalian cells include but are not limited to the cytomegalovirus (CMV) immediate early promoter (US 5,168,062), the RSV promoter, the adenovirus major late promoter, the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpes simplex virus (HSV)-l and the T7 polymerase promoter (WO98/10088).
  • Vaccinia virus promoters are particularly adapted for expression in oncolytic poxviruses.
  • Representative examples include without limitation the vaccinia 7.5K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, pll, pB2R, pA35R and K1L promoters, as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8) as well as early/late chimeric promoters.
  • Promoters suitable for oncolytic measles viruses include without limitation any promoter directing expression of measles transcription units (Brandler and Tangy, 2008, CIMID 31: 271).
  • Inducible promoters belong to the category of regulated promoters.
  • the inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the host cell, or the physiological state of the host cell, an inducer (i.e., an inducing agent), or a combination thereof.
  • Appropriate promoters for expression can be tested in vitro (e.g. in a suitable cultured cell line) or in vivo (e.g. in a suitable animal model or in the subject).
  • the encoded immune checkpoint modulator(s) comprise(s) an antibody and especially a mAb
  • suitable promoters for expressing the heavy component of said immune checkpoint modulator comprise CMV, SV and vaccinia virus pH5R, F17R and pllK7.5 promoters
  • suitable promoters for expressing the light component of said immune checkpoint modulator comprise PGK, beta-actin and vaccinia virus p7.5K, F17R and pA35R promoters.
  • Promoters can be replaced by stronger or weaker promoters, where replacement results in a change in the attenuation of the virus.
  • replacement of a promoter with a stronger promoter refers to removing a promoter from a genome and replacing it with a promoter that effects an increased the level of transcription initiation relative to the promoter that is replaced.
  • a stronger promoter has an improved ability to bind polymerase complexes relative to the promoter that is replaced.
  • an open reading frame that is operably linked to the stronger promoter has a higher level of gene expression.
  • replacement of a promoter with a weaker promoter refers to removing a promoter from a genome and replacing it with a promoter that decreases the level of transcription initiation relative to the promoter that is replaced.
  • a weaker promoter has a lessened ability to bind polymerase complexes relative to the promoter that is replaced.
  • an open reading frame that is operably linked to the weaker promoter has a lower level of gene expression.
  • the viruses can exhibit differences in characteristics, such as attenuation, as a result of using a stronger promoter versus a weaker promoter.
  • vaccinia virus synthetic early/late and late promoters are relatively strong promoters, whereas vaccinia synthetic early, P7.5k early/late, P7.5k early, and P28 late promoters are relatively weaker promoters (see e.g., Chakrabarti et al. (1997) BioTechniques 23 (6) 1094-1097).
  • the promoter is a vaccinia virus promoter.
  • Exemplary vaccinia viral promoters for use in the present invention can include, but are not limited to, P 7.5k , P 11k , P SE , P SEL , P SL , H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b or K1 promoters.
  • Exemplary vaccinia early, intermediate and late stage promoters include, for example, vaccinia P 7.5k early/late promoter, vaccinia P EL early/late promoter, vaccinia P 13 early promoter, vaccinia P 11k late promoter and vaccinia promoters listed elsewhere herein.
  • Exemplary synthetic promoters include, for example, P SE synthetic early promoter, P SEL synthetic early/late promoter, P SL synthetic late promoter, vaccinia synthetic promoters listed elsewhere herein (Patel et al., Proc. Natl. Acad. Sci.
  • the promoter directs transcription during at least the late phase of infection (such as F17R promoter) is employed.
  • the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P 7.5k early/late promoter, P EL early/late promoter, P 11k late promoter, P SEL synthetic early/late promoter, and P SL synthetic late promoter.
  • the late vaccinia viral promoter F17R is only activated after VV infection in tumor cells, thus tumor selective expression of transgene from VV will be further enhanced by the use of F17R promoter.
  • the late expression of the bispecific molecule of the present invention will also allow for sufficient viral replication before T-cell activation and mediated tumor lysis.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the nucleic acid encoding the bispecific molecule is operably linked to a promoter.
  • a first antigen-binding domain such as scFv
  • tumor antigen such as EpCAM, FAP, or EGFR
  • the promoter is a late promoter.
  • the oncolytic virus is a vaccinia virus (VV).
  • the promoter is a vaccinia virus promoter.
  • the promoter is a vaccinia virus late promoter.
  • the promoter is F17R.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the OV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD
  • an immune checkpoint modulator such as anti-PD-1 antibody,
  • the nucleic acid encoding the bispecific molecule and the second nucleic acid encoding the immune checkpoint modulator are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the bispecific molecule and the second nucleic acid encoding the immune checkpoint modulator are operably linked to different promoters.
  • the promoter is a late promoter. In some embodiments, the oncolytic virus is a VV. In some embodiments, the promoter is a vaccinia virus promoter. In some embodiments, the promoter is a vaccinia virus late promoter. In some embodiments, the promoter is F17R.
  • an oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), wherein the OV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-F
  • an immune checkpoint modulator such as anti
  • the nucleic acid encoding the bispecific molecule, the second nucleic acid encoding the immune checkpoint modulator, and the third nucleic acid encoding the cytokine are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the bispecific molecule, the second nucleic acid encoding the immune checkpoint modulator, and the third nucleic acid encoding the cytokine are operably linked to different promoters. In some embodiments, the promoter is a late promoter. In some embodiments, the oncolytic virus is a VV. In some embodiments, the promoter is a vaccinia virus promoter. In some embodiments, the promoter is a vaccinia virus late promoter. In some embodiments, the promoter is F17R.
  • Additional regulatory elements may include transcriptional as well as translational enhancers.
  • the above-described oncolytic viral vectors of the disclosure comprise a selectable and/or scorable marker.
  • Selectable marker genes useful for the selection of transformed cells are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera- Estrella, EMBO J.
  • hygro which confers resistance to hygromycin
  • Additional selectable genes namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci.
  • mannose-6-phosphate isomerase which allows cells to utilize mannose
  • ODC ornithine decarboxylase
  • DFMO ornithine decarboxylase
  • deaminase from Aspergillus terreus confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem.59 (1995), 2336-2338).
  • Useful scorable markers are also known to those skilled in the art and are commercially available.
  • said marker is a gene encoding luciferase (Giacomin, Pl. Sci.116 (1996), 59-72; Scikantha, J. Bact.178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47), YFP, or ⁇ -glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907).
  • Scorable markers are particularly useful for simple and rapid screening of cells, tissues and organisms containing a recited vector.
  • a signal peptide may be included for facilitating secretion from the infected cell.
  • the signal peptide is typically inserted at the N-terminus of the protein immediately after the Met initiator.
  • the choice of signal peptides is wide and is accessible to persons skilled in the art.
  • trans-membrane domain is typically inserted at the C-terminus of the protein just before or at close proximity of the STOP codon.
  • a vast variety of trans-membrane domains are available in the art (see e.g. WO99/03885).
  • the OV (such as VV) encoding the low-affinity bispecific molecule described herein further comprises a signal peptide fused at N-terminal of the bispecific molecule.
  • the signal peptide comprises an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 53.
  • a peptide tag (typically a short peptide sequence able to be recognized by available antisera or compounds) may also be added for following expression, trafficking or purification of the encoded gene product.
  • a vast variety of tag peptides can be used in the context of the invention including, without limitation, PK tag, FLAG octapeptide, MYC tag, HIS tag (usually a stretch of 4 to 10 histidine residues) and e- tag (US 6,686,152).
  • the tag peptide(s) may be independently positioned at the N-terminus of the protein or alternatively at its C-terminus or alternatively internally or at any of these positions when several tags are employed.
  • Tag peptides can be detected by immunodetection assays using anti-tag antibodies.
  • the glycosylation can be altered so as to increase biological activity of the encoded gene product (e.g. to increase). Such modifications can be accomplished, for example, by mutating one or more residues within the site(s) of glycosylation. Altered glycosylation patterns may increase the ADCC ability of antibodies and/or their affinity for their target.
  • the described nucleic acid molecule or vector that is introduced in the host may either integrate into the genome of the host or it may be maintained extrachromosomally.
  • the host can be any eukaryotic cell or prokaryotic cell. It is particularly envisaged that the recited host may be a mammalian cell, such as human or mice cell. Particularly preferred host cells comprise CV-1, BS-C-1, HuTK-143B, BHK-21, CEF, CHO cells, COS cells, myeloma cell lines like SP2/0 or NS/0 cells. III. Pharmaceutical compositions and methods of treating cancer Pharmaceutical compositions
  • compositions comprising any one of the oncolytic virus (such as VV) comprising the nucleic acid encoding a low-affinity bispecific molecule described herein, a viral vector comprising the nucleic acid encoding a low-affinity bispecific molecule described herein, and optionally a pharmaceutically acceptable carrier.
  • the oncolytic virus (such as VV) encoding the low-affinity bispecific molecule further comprises a second nucleic acid encoding an immune checkpoint modulator (such as an immune checkpoint inhibitor, e.g.
  • the oncolytic virus (or oncolytic viral vector) encoding the low-affinity bispecific molecule further comprises a second nucleic acid encoding an immune checkpoint modulator (such as an immune checkpoint inhibitor), and a third nucleic acid encoding a cytokine (such as GM-CSF).
  • an immune checkpoint modulator such as an immune checkpoint inhibitor
  • a third nucleic acid encoding a cytokine (such as GM-CSF).
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a first oncolytic virus (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the low-affinity bispecific molecules described herein), and a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and optionally a pharmaceutical acceptable carrier.
  • a first oncolytic virus such as VV, or viral vector
  • a first nucleic acid comprising a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one
  • a pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the low-affinity bispecific molecules described herein), and a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described herein), and optionally a pharmaceutical acceptable carrier.
  • a first OV such as VV, or viral vector
  • a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the low-affinity bispecific molecules described herein)
  • a pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding bispecific molecule comprising a first antigen- binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the low-affinity bispecific molecules described herein), a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a third OV (such as VV, or viral vector) comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described herein), and optionally a pharmaceutical acceptable carrier.
  • a first OV such as VV, or viral vector
  • a first nucleic acid encoding bispecific molecule comprising a first antigen- binding domain specifically recognizing a tumor antigen and
  • a pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • a first OV such as VV, or viral vector
  • a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10- 8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • a pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding a cytokine (e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier.
  • a cytokine e.g. GM-CSF
  • a pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • a first OV such as VV, or viral vector
  • a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • extracellular domain of PD-1 fused to IgG4 Fc extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to IgG4 Fc
  • a third OV such as VV, or viral vector
  • a cytokine e.g., GM- CSF
  • optionally a pharmaceutical acceptable carrier optionally a pharmaceutical acceptable carrier.
  • the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to (e.g., the same, or about 1-2 times of the K D of the binding between the first antigen-binding domain and the tumor antigen), or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • the oncolytic virus is a VV.
  • the OV is a WR strain VV.
  • the OV comprises double deletion of TK gene and VGF gene.
  • the first and/or second antigen-binding domain is a scFv.
  • the first and second antigen-binding domains are connected by a linker.
  • the first antigen-binding domain is N-terminal to the second antigen- binding domain.
  • the first antigen-binding domain is C-terminal to the second antigen-binding domain.
  • the nucleic acid encoding the bispecific molecule, immune checkpoint modulator, and/or cytokine are operably linked to a promoter (such as a late promoter, e.g. F17R).
  • the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.
  • the immune checkpoint modulator is an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4.
  • the immune checkpoint modulator is an inhibitor of PD-1.
  • the immune checkpoint modulator is an antibody against an immune checkpoint molecule, such as an anti-PD-1 antibody.
  • the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule, such as PD-L1/PD-L2.
  • the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator is a ligand that binds to HHLA2. In some embodiments, the immune checkpoint modulator is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc. In some embodiments, the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • compositions comprising such carriers can be formulated by well- known conventional methods.
  • the solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength.
  • Representative examples include sterile water, physiological saline (e.g. sodium chloride), Ringer's solution, glucose, trehalose or saccharose solutions, Hank's solution, and other aqueous physiologically balanced salt solutions (see, for example, the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).
  • compositions of the disclosure may be administered locally (such as intratumorally) or systematically. Administration will generally be parenteral, e.g., intravenous; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the present disclosure might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.
  • proteinaceous carriers like, e.g., serum albumin or immunoglobulin, preferably of human origin.
  • virus formulation are available in the art either in frozen, liquid form or lyophilized form (e.g. WO98/02522, WO01/66137, WO03/053463, WO2007/056847 and WO2008/114021, etc).
  • Solid (e.g. dry powdered or lyophilized) compositions can be obtained by a process involving vacuum drying and freeze-drying (see e.g.
  • the pharmaceutical composition of the disclosure might comprise, in addition to the bispecific engager molecule (and/or immune checkpoint modulator, cytokine) or nucleic acid molecules or vectors encoding the same (as described in this disclosure), further biologically active agents, depending on the intended use of the pharmaceutical composition.
  • the oncolytic virus pharmaceutical composition is suitably buffered for human use.
  • Suitable buffers include without limitation phosphate buffer (e.g. PBS), bicarbonate buffer and/or Tris buffer capable of maintaining a physiological or slightly basic pH (e.g. from approximately pH 7 to approximately pH 9).
  • the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
  • the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.
  • One aspect of the present application relates to methods of treating or ameliorating of a cancerous (including tumorous) disease comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g.
  • an oncolytic virus such as VV
  • VV an oncolytic virus
  • a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a pharmaceutical acceptable carrier.
  • the individual is a cancer patients or patent susceptible to cancer or suspected of having cancer.
  • the present invention contemplates, in part, viruses, protein constructs (such as low-affinity bispecific molecule), nucleic acid molecules and/or vectors that can be administered either alone or in any combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient.
  • viruses protein constructs (such as low-affinity bispecific molecule), nucleic acid molecules and/or vectors that can be administered either alone or in any combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient.
  • suitable pharmaceutical carriers and excipients that are well known in the art.
  • the compositions prepared according to the disclosure can be used for the treatment or delaying of onset or worsening of cancer.
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen- binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g.
  • an oncolytic virus such as VV
  • VV an oncolytic virus
  • a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen- binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effect
  • the K D of the binding between the first antigen- binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a pharmaceutical acceptable carrier.
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti- PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain- Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator and a third nucleic acid encoding a cytokine (such as GM- CSF).
  • the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting proliferation of cancer cells; (3) inducing redistribution of peripheral T cells; (4) inducing immune response in a tumor; (5) reducing tumor size; (6) alleviating one or more symptoms in an individual having cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) prolonging time to cancer progression; (10) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer; (11) inducing stromal destruction or killing tumor stromal cells in a tumor; (12) promoting oncolytic virus spread through tumors; (13) facilitating T cell infiltration in tumors, and (14) reducing incidence or burden of preexisting tumor metastasis (such as metasta
  • the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the OV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size.
  • the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis.
  • the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months.
  • the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, (such as any one of the bispecific molecules described herein), a second OV (such as VV, viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a pharmaceutical acceptable carrier.
  • a first OV such as VV, viral vector
  • a second OV such as VV, viral vector
  • an immune checkpoint modulator such as any one of the immune checkpoint modulators described herein
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described herein), a second OV (such as VV, viral vector) comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described herein), and a pharmaceutical acceptable carrier.
  • a first OV such as VV, viral vector
  • a second OV such as VV, viral vector
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described herein), a second OV (such as VV, viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), a third OV (such as VV, viral vector) comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described herein), and a pharmaceutical acceptable carrier.
  • a first OV such as VV, viral vector
  • a first nucleic acid encoding a bispecific molecule comprising
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described herein), and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (such as VV, viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a second pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain specifically recognizing a tumor antigen and
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described herein), and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (such as VV, viral vector) comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described herein), and a second pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain specifically recognizing a tumor antigen and a
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (such as VV, viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described herein), and a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV (such as VV, viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a second pharmaceutical acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV (such as VV, viral vector) comprising a third nucleic acid encoding a cytokine (such as any one of the cyto
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a pharmaceutical composition
  • a pharmaceutical composition comprising a first oncolytic virus (e.g. VV) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g.
  • a first oncolytic virus e.g. VV
  • a first nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g. scFv) specifically recognizing
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and a second OV (e.g., VV) comprising a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first oncolytic virus (e.g. VV) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g.
  • EpCAM, FAP, or EGFR EpCAM, FAP, or EGFR
  • a second antigen-binding domain e.g. scFv
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M)
  • a second OV e.g., VV
  • a second nucleic acid encoding a cytokine
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • a first OV such as VV, or viral vector
  • a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • a third OV (such as VV, or viral vector) comprising a third nucleic acid encoding a cytokine (e.g., GM- CSF), and optionally a pharmaceutical acceptable carrier.
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g.
  • VV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g. EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g.
  • a first antigen-binding domain e.g. scFv
  • tumor antigen e.g. EpCAM, FAP, or EGFR
  • a second antigen-binding domain e.g. scFv
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (such as VV) comprising a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first oncolytic virus (e.g. VV) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (e.g.
  • EpCAM, FAP, or EGFR EpCAM, FAP, or EGFR
  • a second antigen-binding domain e.g. scFv
  • the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M)
  • a first pharmaceutical acceptable carrier and an effective amount of a second pharmaceutical composition comprising a second nucleic acid encoding a cytokine (e.g.
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • a first pharmaceutical composition comprising a first OV (such as VV, or viral vector) comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR) and a second antigen-binding domain (e.g.
  • a tumor antigen such as EpCAM, FAP, or EGFR
  • scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV (such as VV, or viral vector) comprising a second nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor, e.g.
  • an immune checkpoint modulator such as immune checkpoint inhibitor, e.g.
  • a third pharmaceutical composition comprising a third OV (such as VV, or viral vector) comprising a third nucleic acid encoding a cytokine (e.g., GM-CSF), and optionally a third pharmaceutical acceptable carrier.
  • a third OV such as VV, or viral vector
  • the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting proliferation of cancer cells; (3) inducing redistribution of peripheral T cells; (4) inducing immune response in a tumor; (5) reducing tumor size; (6) alleviating one or more symptoms in an individual having cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) prolonging time to cancer progression; (10) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer; (11) inducing stromal destruction or killing tumor stromal cells in a tumor (e.g., when a FAP-CD3 T-cell engager (FAP-TE) is expressed); (12) promoting oncolytic virus spread through tumors (e.g., when a FAP-CD3 T-cell engager is expressed); (13) facilitating T-cell infiltration in tumors (e.g., when a FAP-CD3 T-cell engager is expressed), and (14)
  • the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the OV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size.
  • the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis.
  • the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months.
  • the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.
  • a method of inducing stromal destruction or killing tumor stromal cells in a tumor in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g.
  • scFv specifically recognizing FAP and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a pharmaceutical acceptable carrier.
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • an immune checkpoint modulator such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein.
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator and/or a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a method of inducing stromal destruction or killing tumor stromal cells in a tumor in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV encoding a bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and optionally a second pharmaceutically acceptable carrier (and optionally a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein), and optionally a third pharmaceutically acceptable carrier).
  • a first pharmaceutical composition comprising a first OV encoding a bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and optionally a
  • a method of inducing stromal destruction or killing tumor stromal cells in a tumor in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV encoding a low-affinity bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and/or a third OV encoding a cytokine (e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier.
  • a pharmaceutical composition comprising a first OV encoding a low-affinity bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and/or a third OV encoding a
  • the stromal destruction rate or stromal cell killing rate in a tumor mediated by the pharmaceutical composition described herein can be at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
  • the method further comprises administering to the individual an effective amount of another pharmaceutical composition comprising a second oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen that is not FAP (e.g. EpCAM or EGFR) and a second antigen-binding domain (e.g.
  • a second oncolytic virus such as VV
  • VV second oncolytic virus
  • scFv specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the non-FAP tumor antigen (e.g. EpCAM or EGFR) is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally another pharmaceutical acceptable carrier.
  • an effector cell e.g. CD3 on T lymphocytes
  • the K D of the binding between the first antigen-binding domain and the non-FAP tumor antigen e.g. EpCAM or EGFR
  • the K D of the binding between the first antigen-binding domain and the non-FAP tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to
  • the OV encoding the non-FAP bispecific molecules further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • the OV encoding the non-FAP bispecific molecules further comprises a second nucleic acid encoding an immune checkpoint modulator and a third nucleic acid encoding a cytokine (such as GM-CSF).
  • the FAP-T cell engager armed vaccinia virus enhances the antitumor activity of the non-FAP-TEA-VV, such as EpCAM-TEA-VV or EGFR-TEA-VV.
  • a method of promoting oncolytic virus spread through tumors in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing FAP and a second antigen- binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g.
  • an oncolytic virus such as VV
  • an oncolytic virus such as VV
  • the K D of the binding between the first antigen- binding domain and FAP is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a pharmaceutical acceptable carrier.
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator and/or a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a method of promoting oncolytic virus spread through tumors in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV encoding a bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and optionally a second pharmaceutically acceptable carrier (and optionally a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein), and optionally a third pharmaceutical
  • a method of promoting oncolytic virus spread through tumors in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV encoding a low-affinity bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and/or a third OV encoding a cytokine (e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier.
  • a pharmaceutical composition comprising a first OV encoding a low-affinity bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and/or a third OV encoding a cytokine (e.g
  • the method further comprises administering to the individual an effective amount of another pharmaceutical composition comprising a second oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen that is not FAP (e.g. EpCAM or EGFR) and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain and the non-FAP tumor antigen (e.g.
  • a second oncolytic virus such as VV
  • VV second oncolytic virus
  • EpCAM or EGFR is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally another pharmaceutical acceptable carrier.
  • the OV encoding the non-FAP bispecific molecules further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • the OV encoding the non-FAP bispecific molecules further comprises a second nucleic acid encoding an immune checkpoint modulator and a third nucleic acid encoding a cytokine (such as GM- CSF).
  • a cytokine such as GM- CSF
  • the FAP-T cell engager armed vaccinia virus enhances the spread of the non-FAP-TEA-VV of the another pharmaceutical composition, such as EpCAM-TEA-VV or EGFR-TEA-VV.
  • a method of increasing T cell tumor infiltration in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing FAP and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g.
  • an oncolytic virus such as VV
  • the K D of the binding between the first antigen-binding domain and FAP is about 10 -5 to about 10 -9 M (such as about 10 -5 to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally a pharmaceutical acceptable carrier.
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • the OV further comprises a second nucleic acid encoding an immune checkpoint modulator and/or a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a method of increasing T cell tumor infiltration in an individual comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV encoding a bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and optionally a second pharmaceutically acceptable carrier (and optionally a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein), and optionally a third pharmaceutically acceptable carrier).
  • a first pharmaceutical composition comprising a first OV encoding
  • a method of increasing T cell tumor infiltration in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV encoding a low-affinity bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and/or a third OV encoding a cytokine (e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier.
  • a pharmaceutical composition comprising a first OV encoding a low-affinity bispecific molecule (such as any one of the low-affinity bispecific molecules described herein, e.g, FAP-TE), and a second OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and/or a third OV encoding a cytokine (e.g. GM-
  • the method further comprises administering to the individual an effective amount of another pharmaceutical composition comprising a second oncolytic virus (such as VV) comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing the a non-FAP tumor antigen (e.g. EpCAM or EGFR), and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), wherein the K D of the binding between the first antigen-binding domain (e.g.
  • VV second oncolytic virus
  • the non-FAP tumor antigen is about 10 -5 M to about 10 -9 M (such as about 10 -5 M to about 10 -8 M, about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M, or about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M), and optionally another pharmaceutical acceptable carrier.
  • the OV encoding the non-FAP bispecific molecules further comprises a second nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRP ⁇ and a CXCL12 fragment-Fc fusion protein).
  • the OV encoding the non-FAP bispecific molecules further comprises a second nucleic acid encoding an immune checkpoint modulator and a third nucleic acid encoding a cytokine (such as GM-CSF).
  • a cytokine such as GM-CSF.
  • the FAP-T cell engager armed vaccinia virus enhances the infiltration of T cells to tumor cells infected with non-FAP-TEA-VV and/or to bystander tumor cells not infected with TEA-VV.
  • the methods described herein are suitable for treating a variety of cancers, including both solid cancer and liquid cancer.
  • the methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission.
  • the methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (such as T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting (i.e., the method may be carried out before the primary/definitive therapy).
  • the method is used to treat an individual who has previously been treated.
  • the cancer has been refractory to prior therapy.
  • the method is used to treat an individual who has not previously been treated.
  • cancers examples include, but are not limited to, blood cancer, lung cancer (e.g., a non-small cell lung cancer (NSCLC), a lung cancer other than a NSCLC), breast cancer, prostate cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma (HCC)), colon cancer, colorectal cancer, stomach cancer, cancer of the small intestine, spleen cancer, skin cancer (e.g., melanoma), brain cancer (e.g., glioblastoma, a brain cancer other than a glioblastoma), head and neck cancer, cancers of the genitourinary tract (e.g.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • colon cancer colorectal cancer
  • stomach cancer cancer of the small intestine
  • spleen cancer skin cancer (e.g., melanoma)
  • brain cancer e.g., glioblastoma, a brain cancer
  • ovarian cancer endometrial cancer, cervical cancer), kidney cancer, thyroid cancer, gall bladder cancer, cancers of the bile duct, esophagus cancer, cancer of the salivary glands and cancer of the thyroid gland.
  • Hematological malignancies include, but not limited to, leukemia, lymphoma, multiple myeloma, and myelodysplastic syndromes, are treated with methods and compositions of the disclosure.
  • the cancer is EpCAM-, FAP-, or EGFR-positive, for example.
  • the cancer is positive for, e.g., displays on its cell surface, any of the tumor associated antigens or tumor specific antigens listed herein.
  • the administration of the pharmaceutical composition(s) comprising the OV described herein is useful for all stages and types of cancer, including for minimal residual disease, early solid tumor, advanced solid tumor and/or metastatic solid tumor.
  • the method described herein is suitable for treating cancers that overexpress EpCAM on the surface of the cancer cells, such as EpCAM-positive solid cancers.
  • the cancer cells in the individual may express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of EpCAM compared to normal cells.
  • EpCAM-positive solid cancer can be a carcinoma or adenocarcinoma.
  • EpCAM-positive solid cancer include, but are not limited to, small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer (such as melanoma), renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.
  • the method described herein is suitable for treating cancers that overexpress FAP on tumor cells or tumor stromal fibroblasts, such as FAP-positive solid cancers.
  • the tumor stromal fibroblasts in the individual express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of FAP compared to normal fibroblasts.
  • FAP-positive solid cancer can be a carcinoma or adenocarcinoma.
  • the FAP-positive solid cancer is selected from the group consisting of colorectal cancer, breast cancer, brain cancer, lung cancer, and skin cancer (such as melanoma).
  • the method described herein is suitable for treating cancers that overexpress EGFR on the surface of the cancer cells, such as EGFR-positive solid cancers.
  • the cancer cells in the individual (such as a human) express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of EGFR compared to normal cells.
  • EGFR-positive solid cancer can be carcinoma or adenocarcinoma.
  • EGFR- mediated cancers include, but are not limited to, glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retina cancer, skin cancer, liver cancer, genital-urinary cancer, and bladder cancer.
  • the method described herein is suitable for treating a colorectal cancer, such as adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, leiomysarcoma, melanoma, or squamous cell carcinoma.
  • a colorectal cancer such as adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, leiomysarcoma, melanoma, or squamous cell carcinoma.
  • the method described herein is suitable for treating a lung cancer, such as a NSCLC, or a lung cancer other than NSCLC.
  • NSCLC include, but are not limited to, large-cell carcinoma, adenocarcinoma, neuroendocrine lung tumors, and squamous cell carcinoma.
  • the method described herein is suitable for treating a liver cancer, such as liver cell carcinoma, fibrolamellar variants of hepatocellular carcinoma, or mixed hepatocellular cholangiocarcinoma.
  • a skin cancer such as melanoma.
  • melanoma examples include, but are not limited to, superficial spreading melanoma, lentigo maligna melanoma, nodular melanoma, mucosal melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, or acral lentiginous melanoma.
  • the method described herein is suitable for treating a breast cancer, such as early stage breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting.
  • a breast cancer such as early stage breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting.
  • the breast cancer is fibroadenoma, or intraductal papilloma.
  • the breast cancer is HER2 positive or HER2 negative.
  • the breast cancer is a triple negative breast cancer.
  • the method described herein is suitable for treating a brain cancer, such as glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma).
  • a brain cancer such as glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma).
  • the number of viruses (such as VV) employed in an effective amount of the pharmaceutical composition described herein will depend upon a number of circumstances, such as the purpose for the introduction, the particular type and stage of cancer being treated, the protocol to be used, for example, the number and route of administrations, the stability of the viruses, the activity of the encoded low-affinity bispecific molecules, and the like.
  • the dosage regimen of the pharmaceutical composition comprising the OV described herein will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • that effective amount of the pharmaceutical composition comprising the OV described herein is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the pharmaceutical composition is administered to the individual.
  • the oncolytic virus in the effective amount of the pharmaceutical composition described herein is from about 10 5 to about 10 13 pfu, including for example any of about 10 5 to about 10 12 pfu, about 10 6 to about 10 13 pfu, about 10 6 to about 10 12 pfu, about 10 7 to about 10 13 pfu, about 10 7 to about 10 12 pfu, about 10 7 to about 10 11 pfu, about 10 7 to about 10 10 pfu, about 10 7 to about 10 9 pfu, about 10 8 to about 10 13 pfu, about 10 8 to about 10 12 pfu, about 10 8 to about 10 11 pfu, about 10 8 to about 10 10 pfu, about 10 8 to about 10 9 pfu, about 10 9 to about 10 13 pfu, about 10 9 to about 10 12 pfu, about 10 9 to about 10 12 pfu, about 10 9 to about 10 11 pfu, about 10 9 to about 10 10 pfu, about 10 9
  • the oncolytic virus in the effective amount of the pharmaceutical composition described herein is about 10 13 pfu, about 10 12 pfu, about 10 11 pfu, about 10 10 pfu, about 10 9 pfu, about 10 8 pfu, about 10 7 pfu, about 10 6 pfu, or about 10 5 pfu. In some embodiments, the oncolytic virus in the effective amount of the pharmaceutical composition described herein is about 10 5 to about 10 13 pfu. In some embodiments, the oncolytic virus in the effective amount of the pharmaceutical composition described herein is about 10 7 to about 10 9 pfu.
  • the oncolytic virus in the effective amount of the pharmaceutical composition described herein is about 10 9 pfu.
  • the pharmaceutical composition is administered for a single time (e.g. bolus injection). In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times). If multiple administrations, they may be performed by the same or different routes and may take place at the same site or at alternative sites.
  • the pharmaceutical composition may be administered twice per week, 3 times per week, 4 times per week, 5 times per week, daily, daily without break, once per week, weekly without break, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year.
  • the interval between administrations can be about any one of 24h to 48h, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 month to 3 months, 3 months to 6 months, or 6 months to a year.
  • Intervals can also be irregular (e.g. following tumor progression). In some embodiments, there is no break in the dosing schedule.
  • the pharmaceutical composition comprising the oncolytic virus described herein may be administered once or several time (e.g. 2, 3, 4, 5, 6, 7 or 8 times, etc.) at a dose within the range of from about 10 5 pfu to about l0 13 pfu (such as from about 10 7 pfu to about l0 9 pfu, or about l0 9 pfu).
  • the time interval between each administration can vary from approximately 1 day to approximately 8 weeks, from approximately 2 days to approximately 6 weeks, from approximately 3 days to approximately 4 weeks, from approximately 1 week to approximately 3 weeks, or every two weeks.
  • the pharmaceutical composition comprising the oncolytic virus (such as oncolytic VV) described herein is administered 2 to 5 times (e.g. 3 times) intravenously or intratumorally in the range of about 10 5 pfu to about l0 13 pfu (such as from about 10 7 pfu to about l0 9 pfu, or about l0 9 pfu) at approximately 1 or 2 weeks interval.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the pharmaceutical composition comprising the OV described herein may be suitable for a variety of modes of administration, including for example systemic or localized administration.
  • the pharmaceutical composition is administered parenterally, transdermally (into the dermis), intraluminally, intra-arterially (into an artery), intramuscularly (into muscle), intrathecally or intravenously.
  • the pharmaceutical composition is administered subcutaneously (under the skin).
  • the pharmaceutical composition is administered intravenously.
  • the pharmaceutical composition comprising the OV described herein is administered to the individual via infusion or injection.
  • the pharmaceutical composition is directly injected to tumor sites (intratumorally -- into tumor or at its close proximity).
  • compositions comprising oncolytic viral vectors encoding the bispecific molecule described herein and a pharmaceutically acceptable carrier are also encompassed in the present invention.
  • Oncolytic viral vectors may be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of the active agent(s) in the subject.
  • the individual to be treated is a mammal.
  • mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cattle and the like.
  • the individual is a human.
  • the present application also provides novel constructs comprising antibodies, antibody fragments, or antigen-binding fragments thereof that specifically recognizes EpCAM (hereinafter also referred to as“anti-EpCAM antibody”,“anti-EpCAM antibody fragment” or“EpCAM binding domain”), including scFv-scFv that comprises an antigen-binding fragment derived from the anti-EpCAM antibody.
  • EpCAM antibody hereinafter also referred to as“anti-EpCAM antibody”,“anti-EpCAM antibody fragment” or“EpCAM binding domain”
  • scFv-scFv that comprises an antigen-binding fragment derived from the anti-EpCAM antibody.
  • antibody is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen-binding sites, while IgA antibodies comprise from 2-5 of the basic 4- chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N- terminus, a variable domain (V H ) followed by three constant domains (C H ) for each of the ⁇ and ⁇ chains and four C H domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (V L ) followed by a constant domain at its other end.
  • the V L is aligned with the V H and the C L is aligned with the first constant domain of the heavy chain (C H 1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a V H and V L together forms a single antigen- binding site.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in the C H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • A“human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
  • recombinant human antibodies have variable and constant regions in a rearranged form.
  • the recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation.
  • the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (hereinafter defined) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non- human primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit or non- human primate having the desired specificity, affinity, and/or capacity.
  • framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc.
  • the number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as“class-switched antibodies”.
  • Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81 (1984) 6851- 6855; US Patent Nos. 5,202,238 and 5,204,244.
  • the term“monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.
  • phage-display technologies see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
  • full-length antibody “intact antibody” or“whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment.
  • full-length 4-chain antibodies include those with heavy and light chains including an Fc region.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more effector functions.
  • An“antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single- chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called“Fab” fragments, and a residual“Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab') 2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C H 1 domain including one or more cysteines from the antibody hinge region. F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the C H 1, C H 2 and C H 3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
  • The“light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“ ⁇ ”), based on the amino acid sequences of their constant domains.
  • diabodies refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med.9:129-134 (2003).
  • The“Fc” fragment comprises the carboxy-terminal portions of both H chains held together by di-sulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • FcR Fc receptors
  • “Fv” is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as“sFv” or“scFv” are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
  • scFvs are known in the art, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
  • The“variable region” or“variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody.
  • the variable domains of the heavy chain and light chain may be referred to as“V H ” and“V L ”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains.
  • HVRs hypervariable regions
  • the more highly conserved portions of variable domains are called the framework regions (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • HVR hypervariable region
  • HV hypervariable region
  • 4-chain antibodies comprise six HVRs; three in the V H (H1, H2, H3), and three in the VL (L1, L2, L3).
  • H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • HVR delineations are in use and are encompassed herein.
  • the Kabat “Complementarity Determining Regions” (or“CDRs”) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901- 917 (1987)).
  • the AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • the “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below in Table 1.
  • HVRs may comprise“extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the V L and 26-35 (H1), 50-65 or 49-65 (H2) and 93- 102, 94-102, or 95-102 (H3) in the V H .
  • the variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.
  • variable-domain residue-numbering as in Kabat or“amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • “Framework” or“FR” residues are those variable-domain residues other than the HVR residues as herein defined.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)- FR4.
  • the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra.
  • The“EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
  • the term“binds”,“specifically binds to”,“specifically recognizes” or“is specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that binds to, or specifically recognizes, or specifically binds to a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • multispecific refers to an antibody having polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules).
  • bispecific refers to an antibody capable of specifically binding to two different epitopes on one biological molecule, or capable of specifically binding to epitopes on two different biological molecules.
  • the order in which the antigens bound by a bispecific antibody listed is arbitrary. That is, for example, the terms “anti-CD3/EpCAM,” “anti-EpCAM/CD3,” “EpCAM ⁇ CD3,”“CD3 ⁇ EpCAM,”“CD3-EpCAM,”“EpCAM-CD3,” and“EpCAM-TE” may be used interchangeably to refer to bispecific antibodies that specifically bind to both CD3 and EpCAM.
  • Percent (%) amino acid sequence identity and“homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising one, two or three HVRs from SEQ ID NO: 19, and/or a light chain variable region (VL) comprising one, two or three HVRs from SEQ ID NO: 20.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 19, and/or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 20.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising: a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 19, and/or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising one, two or three HVRs from SEQ ID NO: 21, and/or a light chain variable region (VL) comprising one, two or three HVRs from SEQ ID NO: 22.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 21, and/or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 22.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising: a HVR- H1 comprising the amino acid sequence of SEQ ID NO: 7; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 21, and/or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising one, two or three HVRs from SEQ ID NO: 23, and/or a light chain variable region (VL) comprising one, two or three HVRs from SEQ ID NO: 24.
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 23, and/or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 24.
  • the anti-EpCAM antibody or antigen-binding domain comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO: 23, and/or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or
  • the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 23, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 24.
  • the anti-EpCAM antibody is a full-length antibody.
  • the full-length anti-EpCAM antibody comprises an Fc sequence from an immunoglobulin, such as IgA, IgD, IgE, IgG, and IgM.
  • the full-length anti-EpCAM antibody comprises an Fc sequence of IgG, such as any of IgG1, IgG2, IgG3, or IgG4.
  • the full-length anti-EpCAM antibody comprises an Fc sequence of a human immunoglobulin.
  • the full-length anti-EpCAM antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • isolated antibodies or antigen-binding fragments which compete with any of the anti-EpCAM antibodies (or antigen-binding fragment thereof) described herein for binding with EpCAM.
  • an isolated antibody or an antigen-binding fragment thereof which binds to the same epitope as any of the anti-EpCAM antibodies (or antigen-binding fragments thereof) described herein.
  • Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.
  • ELISA enzyme linked immunosorbent assay
  • a monoclonal antibody has the same specificity as a monoclonal antibody of the invention (e.g., the anti-EpCAM antibody comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 20, or the anti-EpCAM antibody comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 22, or the anti-EpCAM antibody comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 24) by ascertaining whether the former prevents the latter from binding to EpCAM (e.g., prevents at least about any of 80%, 85%, 90%, 95% of the binding).
  • the two monoclonal antibodies bind to the same, or a closely related, epitope.
  • An alternative method for determining whether a monoclonal antibody has the specificity of monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with soluble EpCAM protein (with which it is normally reactive), and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind EpCAM. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention.
  • the anti-EpCAM antibody is a monoclonal antibody, such as a monovalent antibody.
  • the anti-EpCAM antigen-binding fragment is in the form of a Fab, Fab’, a F(ab’) 2 , single-chain Fv (scFv), an Fv fragment, a diabody, or a linear antibody.
  • the anti-EpCAM antibody or antigen-binding fragment thereof is a scFv.
  • an anti-EpCAM scFv comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-EpCAM scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-EpCAM scFv comprises a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • an anti-EpCAM scFv comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti- EpCAM scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-EpCAM scFv comprises a VL comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 34.
  • an anti-EpCAM scFv comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti- EpCAM scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-EpCAM scFv comprises a VL comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 35.
  • the anti-EpCAM antibody is a multispecific antibody that binds to EpCAM, and also binds one or more other targets and optionally inhibits their function.
  • Multispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for two or more different antigens (e.g., bispecific antibodies have binding specificities for at least two antigens).
  • one of the binding specificities can be for the EpCAM protein, the other one can be for any other antigen (e.g., non-EpCAM protein, or a different epitope on EpCAM).
  • the other antigen is a cell surface protein, receptor, or receptor subunit.
  • the cell surface protein can be CD3, such as CD3 epsilon.
  • a bispecific antibody that specifically recognizes both EpCAM and, e.g. a cell surface molecule on an effector cell (such as CD3 on T lymphocytes).
  • the effector cell can be a T lymphocyte, a B lymphocyte, a natural killer (NK) cell, a dendritic cell (DC), a macrophage, a monocyte, a neutrophil, an NKT-cell, or the like.
  • the effector cell is a T lymphocyte.
  • the effector cell is a cytotoxic T lymphocyte.
  • Cell surface molecules include, but are not limited to, CD3, CD4, CD5, CD8, CD16, CD27, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, NKp30, NKG2D, and an invariant TCR.
  • the multi-specific anti-EpCAM antibody is, for example, a diabody (Db), a single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a di-diabody, a tandem scFv, a tandem di- scFv (e.g., a bispecific T cell engager), a tandem tri-scFv, a tri(a)body, a bispecific Fab 2 , a di- miniantibody, a tetrabody, a scFv-Fc-scFv fusion, a dual-affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, an IgG-scFab, an scFab-ds-scFv, an Fv2-
  • fusion proteins, conjugates, or isolated cells comprising any of the anti-EpCAM antibodies or antigen-binding fragments thereof described above.
  • Nucleic acids, oncolytic virus, or vectors (e.g., viral vector) encoding the anti-EpCAM antibodies or antigen-binding fragments thereof described above are also contemplated.
  • a bispecific molecule comprising a first antigen-binding fragment (e.g., scFv) specifically recognizing EpCAM and a second antigen- binding fragment (e.g., scFv), wherein the anti-EpCAM antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti- EpCAM antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 19, and/or a VL comprising the amino acid sequence of SEQ ID NO: 20.
  • the anti-EpCAM antigen-binding fragment is a scFv, such as a scFv comprising the amino acid sequence of SEQ ID NO: 33.
  • the second antigen-binding fragment specifically recognizes CD3 (e.g., human or mouse CD3).
  • the second antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 43, and/or a VL comprising the amino acid sequence of SEQ ID NO: 44.
  • the second antigen-binding fragment is a scFv, such as a scFv comprising the amino acid sequence of SEQ ID NO: 45.
  • the first scFv and the second scFv are connected by a linker, such as a linker comprising the amino acid sequence of SEQ ID NO: 48.
  • the anti-EpCAM antigen-binding fragment is N-terminal to the second antigen-binding fragment.
  • the anti-EpCAM antigen-binding fragment is C-terminal to the second antigen-binding fragment.
  • the bispecific molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 55.
  • a bispecific molecule comprising a first antigen-binding fragment (e.g., scFv) specifically recognizing EpCAM and a second antigen- binding fragment (e.g., scFv), wherein the anti-EpCAM antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti- EpCAM antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 21, and/or a VL comprising the amino acid sequence of SEQ ID NO: 22.
  • the anti-EpCAM antigen-binding fragment is a scFv, such as a scFv comprising the amino acid sequence of SEQ ID NO: 34.
  • the second antigen-binding fragment specifically recognizes CD3 (e.g., human or mouse CD3).
  • the second antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 43, and/or a VL comprising the amino acid sequence of SEQ ID NO: 44.
  • the second antigen-binding fragment is a scFv, such as a scFv comprising the amino acid sequence of SEQ ID NO: 45.
  • the first scFv and the second scFv are connected by a linker, such as a linker comprising the amino acid sequence of SEQ ID NO: 48.
  • the anti-EpCAM antigen-binding fragment is N-terminal to the second antigen-binding fragment.
  • the anti-EpCAM antigen-binding fragment is C-terminal to the second antigen-binding fragment.
  • the bispecific molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 56.
  • a bispecific molecule comprising a first antigen-binding fragment (e.g., scFv) specifically recognizing EpCAM and a second antigen- binding fragment (e.g., scFv), wherein the anti-EpCAM antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-EpCAM antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 23, and/or a VL comprising the amino acid sequence of SEQ ID NO: 24.
  • the anti-EpCAM antigen-binding fragment is a scFv, such as a scFv comprising the amino acid sequence of SEQ ID NO: 35.
  • the second antigen-binding fragment specifically recognizes CD3 (e.g., human or mouse CD3).
  • the second antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 43, and/or a VL comprising the amino acid sequence of SEQ ID NO: 44.
  • the second antigen-binding fragment is a scFv, such as a scFv comprising the amino acid sequence of SEQ ID NO: 45.
  • the first scFv and the second scFv are connected by a linker, such as a linker comprising the amino acid sequence of SEQ ID NO: 48.
  • the anti-EpCAM antigen-binding fragment is N-terminal to the second antigen-binding fragment.
  • the anti-EpCAM antigen-binding fragment is C-terminal to the second antigen-binding fragment.
  • the bispecific molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 57.
  • isolated nucleic acid encoding the anti-EpCAM antibodies or antigen-binding fragments thereof OV (or oncolytic viral vector) comprising the nucleic acid encoding the anti-EpCAM antibodies or antigen-binding fragments thereof, isolated cells expressing the anti-EpCAM antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising any of the anti-EpCAM antibodies or antigen-binding fragments thereof (or OV and vectors encoding thereof, host cells expressing thereof), methods of treating cancers in an individual using such pharmaceutical compositions.
  • the pharmaceutical composition is administered to the individual to be treated intravenously.
  • the pharmaceutical composition is administered to the individual to be treated intratumorally.
  • the individual to be treated is a human.
  • the anti-EpCAM antibodies or antigen-binding fragments thereof described herein can be used in a variety of therapeutic and diagnostic methods. Further provided are methods of treating cancer in an individual (such as human), comprising administering an effective amount of the anti-EpCAM antibody or antigen-binding fragment thereof described herein or pharmaceutical compositions comprising thereof to the individual.
  • compositions comprising any of the anti-EpCAM antibodies or antigen- binding fragments thereof (or OV and vectors encoding thereof, host cells expressing thereof) can be used alone or in combination with other agents in treating a disease characterized by abnormal EpCAM expression, including, but not limited to, small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer (such as melanoma), renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.
  • the antibodies provided herein can also be used for detecting EpCAM protein in patients or patient samples.
  • Screening of monoclonal antibodies of the invention can be also carried out, e.g., by measuring EpCAM-mediated signaling, and determining whether the test monoclonal antibody is able to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM-mediated signaling.
  • These assays can include competitive binding assays. Additionally, these assays can measure a biologic readout.
  • Human monoclonal antibodies can be also prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983.
  • Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).
  • EpCAM antibodies of the invention are monoclonal antibodies. Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM-mediated cell signaling are generated, e.g., by immunizing an animal with membrane bound and/or soluble EpCAM, such as, for example, human EpCAM or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding EpCAM such that EpCAM is expressed and associated with the surface of the transfected cells. Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen-binding domain sequences for binding to EpCAM.
  • This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e.,“phage displayed library”). Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to EpCAM.
  • Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco’s Modified Eagle’s Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibodies).
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as Chinese hamster ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, simian COS cells, PER.C6®, NS0 cells, SP2/0, YB2/0, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody, or can be substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody.
  • Monoclonal antibodies of the invention include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • An anti-EpCAM antibody can be generated using any procedures known in the art. For example, anti-EpCAM antibodies can be identified using a modified RIMMS (Repetitive Immunization Multiple Sites) immunization strategy in mice and subsequent hybridoma generation. In other, alternative methods, an anti-EpCAM antibody is developed, for example, using phage-display methods using antibodies containing only human sequences. Such approaches are well-known in the art, e.g., in WO92/01047 and U.S. Pat. No.
  • an anti-EpCAM antibody can be produced by a process wherein at least one step of the process includes immunizing a transgenic, non-human animal with human EpCAM protein. In this approach, some of the endogenous heavy and/or kappa light chain loci of this xenogenic non-human animal have been disabled and are incapable of the rearrangement required to generate genes encoding immunoglobulins in response to an antigen.
  • At least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal.
  • the human loci rearrange to provide genes encoding human variable regions immunospecific for the antigen.
  • the xenomouse produces B-cells that secrete fully human immunoglobulins.
  • VH genes one or more VH genes, one or more D H genes, one or more J H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal.
  • a“minilocus” approach in which an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus.
  • VH genes one or more VH genes, one or more D H genes, one or more J H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal.
  • D H genes one or more D H genes
  • J H genes one or more J H genes
  • a mu constant region preferably a gamma constant region
  • WO 92/03918 WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and related family members.
  • HAMA Human anti-mouse antibody
  • HACA human anti- chimeric antibody
  • Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)).
  • mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
  • the cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest.
  • the DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences.
  • the sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effecter functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.
  • Antibody fragments such as Fv, F(ab’) 2 and Fab may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage.
  • a truncated gene is designed.
  • a chimeric gene encoding a portion of the F(ab’) 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • Consensus sequences of H, L, and J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.
  • C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.
  • Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like.
  • a convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed.
  • splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions.
  • the resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)).
  • retroviral LTRs e.g., SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)).
  • native Ig promoters and the like may be used.
  • human antibodies or antibodies from other species can be generated through display type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art.
  • display type technologies including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art.
  • antibodies can be generated to EpCAM expressing cells, soluble forms of EpCAM, epitopes or peptides thereof, and expression libraries thereto (see e.g., U.S. Patent No. 5,703,057) which can thereafter be screened as described above for the activities described herein.
  • the anti-EpCAM antibodies of the invention can be expressed by a vector containing a DNA segment encoding the single chain antibody described herein.
  • Vectors can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc.
  • Vectors include chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, etc.
  • the vectors can be chromosomal, non-chromosomal or synthetic.
  • Retroviral vectors include moloney murine leukemia viruses.
  • DNA viral vectors are preferred.
  • These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad.
  • HSV herpes simplex I virus
  • Pox viral vectors introduce genes into the cellular cytoplasm.
  • Avipox virus vectors result in only a short term expression of the nucleic acid.
  • Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells.
  • the adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors.
  • Vaccinia virus vectors are capable to multiply in a large number of different types of cells. The particular vector chosen will depend upon the target cell and the condition being treated.
  • the introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPO 4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.
  • the vector can be employed to target essentially any desired target cell.
  • stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location.
  • the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System.
  • icv intracerebroventricular
  • a method based on bulk flow, termed convection has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell (see Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)).
  • Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.
  • These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of EpCAM in a sample.
  • the EpCAM antibody can also be used to bind and disrupt EpCAM-mediated signaling.
  • Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of Fabexpression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab’) 2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F (ab’)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • the invention also includes F v , Fab, Fab’ and F(ab’) 2 anti-EpCAM fragments, single chain EpCAM antibodies, single domain antibodies (e.g., nanobodies or VHHs), multispecific (such as bispecific) anti-EpCAM antibodies, and heteroconjugate anti-EpCAM antibodies.
  • bispecific antibodies Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co- transfected into a suitable host organism.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory“cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab’-TNB derivatives is then reconverted to the Fab’-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab’ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab’) 2 molecule.
  • Each Fab’ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • This method can also be utilized for the production of antibody homodimers.
  • The“diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light- chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • scFv single-chain Fv
  • Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al., J. Immunol.147:60 (1991).
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980), and for treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • the antibody of the invention can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders associated with aberrant EpCAM signaling.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)).
  • ADCC complement-mediated cell killing and antibody-dependent cellular cytotoxicity
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a radioactive isotope (i.e., a radioconjugate), or a label for detecting the target antigen (such as EpCAM) in patient samples or in vivo by imaging (e.g., radioisotope, fluorescent dye and enzyme).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a radioactive isotope (i.e., a radioconjugate),
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026).
  • Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities.
  • This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.
  • the preferred binding is, however, covalent binding.
  • Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules.
  • representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
  • Preferred linkers are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker.
  • MBS M-maleimidobenzoyl-N- hydroxysuccinimide ester
  • linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem.
  • linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties.
  • sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.
  • Disulfide linkages are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
  • Sulfo-NHS in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • the anti-EpCAM antibody or antigen-binding fragment thereof can be used for making chimeric antigen receptor (CAR) comprising: a) an extracellular domain comprising any of the anti-EpCAM antibodies or antigen-binding fragments described herein; and b) an intracellular signaling domain.
  • a transmembrane domain may be present between the extracellular domain and the intracellular domain.
  • intracellular signaling domains for use in the anti-EpCAM CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Any method for producing a CAR may be used herein.
  • TCR T cell receptor
  • the anti-EpCAM antibody or antigen-binding fragment thereof can be used for making recombinant T cell receptor (TCR) comprising an extracellular domain comprising any of the anti-EpCAM antibodies or antigen-binding fragments described herein.
  • TCR T cell receptor
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • PEG-PE PEG- derivatized phosphatidylethanolamine
  • compositions comprising the anti-EpCAM antibodies (or antigen-binding fragment thereof) descried herein, and optionally a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions comprising nucleic acids, OV, or vectors (such as viral vector) encoding the anti-EpCAM antibodies (or antigen-binding fragment thereof), or host cells expressing the anti-EpCAM antibodies (or antigen-binding fragment thereof) are also contemplated.
  • a method of treating cancer in an individual comprising administering to the individual an effective amount of the pharmaceutical composition comprising the anti-EpCAM antibodies (or antigen- binding fragment thereof) descried herein, and optionally a pharmaceutically acceptable carrier.
  • the treatment effects may include, but are not limited to, killing cancer cells; inhibiting proliferation of cancer cells; inducing redistribution of peripheral T cells; inducing immune response in a tumor; reducing tumor size; inhibiting tumor metastasis; reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); prolonging survival of an individual; prolonging time to cancer progression; preventing, inhibiting, or reducing the likelihood of the recurrence of a caner, etc.
  • the pharmaceutical composition comprising the anti-EpCAM antibodies (or antigen-binding fragment thereof) described herein is administered to the individual intravenously or intratumorally.
  • the individual to be treated is a mammal.
  • mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cattle and the like.
  • the individual is a human.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration.
  • antibodies of the invention may be used as therapeutic agents.
  • agents will generally be employed to diagnose, prognose, monitor, treat, alleviate, and/or prevent a disease or pathology associated with aberrant EpCAM expression, activity and/or signaling in a subject.
  • a therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant EpCAM expression, activity and/or signaling, e.g., a cancer or other neoplastic disorder, using standard methods.
  • An antibody preparation preferably one having high specificity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target.
  • Administration of the antibody may abrogate or inhibit or interfere with the expression, activity and/or signaling function of the target (e.g., EpCAM).
  • Administration of the antibody may abrogate or inhibit or interfere with the binding of the target (e.g., EpCAM) with an endogenous ligand to which it naturally binds.
  • the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with EpCAM expression, activity and/or signaling.
  • Hematological cancers include, e.g., leukemia, lymphoma and myeloma.
  • leukemia include, by way of non-limiting example, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome.
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MPDS Myeloproliferative disorder/neoplasm
  • myelodysplasia syndrome myelodysplasia syndrome.
  • lymphoma include, by way of non-limiting example, Hodgkin’s lymphoma, both indolent and aggressive non-Hodgkin’s lymphoma, Burkitt’s lymphoma, and follicular lymphoma (small cell and large cell).
  • Certain forms of myeloma include, by way of non-limiting example, multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma.
  • Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.
  • Symptoms associated with cancers and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, often localized to the inflamed area, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue and abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation.
  • a therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular inflammatory-related disorder. Alleviation of one or more symptoms of the inflammatory-related disorder indicates that the antibody confers a clinical benefit.
  • antibodies directed against EpCAM may be used in methods known within the art relating to the localization and/or quantitation of EpCAM (e.g., for use in measuring levels of EpCAM within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies specific to EpCAM, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen-binding domain are utilized as pharmacologically active compounds (referred to hereinafter as“Therapeutics”).
  • an antibody specifically recognizing EpCAM can be used to isolate a EpCAM polypeptide, by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation.
  • Antibodies directed against the EpCAM protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • an antibody according to the invention can be used as an agent for detecting the presence of EpCAM (or a protein fragment thereof) in a sample (such as biological sample).
  • the antibody contains a detectable label.
  • Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., scFv) is used.
  • the term“labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently- labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
  • the term“biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term“biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T.
  • in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the low-affinity bispecific molecules or the anti-EpCAM antibodies (or antigen- binding fragments thereof) described herein may be prepared by any of the known protein expression and purification methods in the art.
  • the present application provides isolated nucleic acids encoding one or more of the polypeptide chains of any one of the low-affinity bispecific molecules or the anti-EpCAM antibodies (or antigen-binding fragments thereof) described herein.
  • the isolated nucleic acid comprises a first nucleic acid sequence of SEQ ID NO: 60, and a second nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 66 or SEQ ID NO: 67.
  • the isolated nucleic acids may be DNA or RNA.
  • the isolated nucleic acid comprises a first sequence of SEQ ID NO: 62, and a second sequence of SEQ ID NO: 63. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 68 or SEQ ID NO: 69.
  • the isolated nucleic acids may be DNA or RNA.
  • the isolated nucleic acid comprises a first sequence of SEQ ID NO: 64, and a second sequence of SEQ ID NO: 65. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71.
  • the isolated nucleic acids may be DNA or RNA.
  • the isolated nucleic acid comprises a nucleic acid sequence of SEQ ID NOS: 72, 73, or 75.
  • the isolated nucleic acids may be DNA or RNA.
  • the isolated nucleic acid described herein is operably linked to a promoter.
  • the promoter is a late promoter.
  • the promoter is a VV promoter.
  • the promoter is a VV late promoter.
  • the promoter is F17R.
  • the isolated nucleic acid is inserted into a vector, such as an expression vector, a viral vector, or a cloning vector.
  • a vector such as an expression vector, a viral vector, or a cloning vector.
  • the vector may be introduced into a host cell to allow expression of the nucleic acids within the host cell.
  • the expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector.
  • Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter. EF1a promoter, CMV promoter, SV40 promoter, and vaccinia virus promoter (such as F17R). Enhancer sequences may be selected to enhance the transcription of the nucleic acids. Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell.
  • the isolated nucleic acids further comprise a nucleic acid sequence encoding a signal peptide.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 53.
  • the nucleic acid sequence encoding the signal peptide comprises the nucleic acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54.
  • the oncolytic virus encoding the low-affinity bispecific engager molecules (and/or immune checkpoint modulators, cytokines, such as those described herein) is a vaccinia virus (VV).
  • VV vaccinia virus
  • Vaccinia virus is appealing for cancer gene therapy due to several characteristics. It has natural tropism towards cancer cells and the selectivity can be significantly enhanced by deleting some of the viral genes.
  • the VV is a WR strain.
  • the VV encoding the low- affinity bispecific engager molecule (and/or immune checkpoint modulators, cytokines, such as those described herein) comprises double deletion of TK and VGF genes (vvDD strain).
  • TK and VGF genes are needed for virus to replicate in normal but not in cancer cells.
  • the TK or VGF deletion may be engineered in the TK or VGF region conferring activity, respectively.
  • this can by generated by recombination of a pSEM-1 shuttle plasmid containing the bispecific engager molecule (and/or immune checkpoint modulators, cytokines, such as those described herein) into the TK gene of the VSC20 strain (VGF deleted strain) of Western Reserve vaccinia virus (WR VV).
  • VSC20 train can be constructed by inserting lacZ gene under the control of the p11 promoter into two copies of the viral VGF genes, thus inactivating VGF.
  • the shuttle vector pSEM-1 can be constructed to have the bispecific engager molecule (and/or immune checkpoint modulators, cytokines, such as those described herein) expressed under the transcriptional control of a promoter, such as the F17R late promoter which allows for sufficient viral replication before T-cell activation.
  • a promoter such as the F17R late promoter which allows for sufficient viral replication before T-cell activation.
  • the VV can further express a marker, such as DsRed2, YFP, GFP, or YFP-GFP, to allow for virus selection.
  • the infectivity monitoring marker can be expressed under the transcriptional control of the same promoter that drives the expression of the low-affinity engager molecule (and/or immune checkpoint modulators, cytokines, such as those described herein), or under the transcriptional control of a different promoter, such as Pse/I promoter, or P7.5 promoter.
  • the virus selection marker can be flanked by loxP sites in the same orientation.
  • the shuttle vectors pSEM-1 containing the bispecific engager molecule (and/or immune checkpoint modulators, cytokines, such as those described herein) can be transfected into human 143 TK- cells. Cells can then be infected with virus vSC20 at a multiplicity of infection (MOI) of, e.g., 0.1.
  • MOI multiplicity of infection
  • the selection marker e.g., YFP-GFP cassette can be removed from the recombinant viruses.
  • viruses can be passaged on a U2OS cell line expressing a cytoplasmic form of Cre recombinase (U2OS- Cre).
  • one of the selectable marker-negative (e.g., YFP-GFP- negative) clones can be selected for amplification and purification.
  • any suitable method can be used for generating inactivating mutations in a gene of interest, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to a person of skill in the art.
  • Mutation can involve modification of a nucleotide sequence, a single gene, or blocks of genes.
  • a mutation may involve a single nucleotide (such as a point mutation, which involves the removal, addition or substitution of a single nucleotide base within a DNA sequence) or it may involve the insertion or deletion of large numbers of nucleotides. Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication, or induced following exposure to chemical or physical mutagens.
  • a mutation can also be site-directed through the use of particular targeting methods that are well known to persons of skill in the art.
  • the obtained virus of the present invention can be replicated by conventional methods for viral replication, e.g. infecting host cells such as 293 cells with the virus.
  • oncolytic viral nucleic acid molecules may contain, for example, thioester bonds and/or nucleotide analogues. The modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
  • the nucleic acid molecules may be transcribed by an appropriate oncolytic vector comprising a chimeric gene that allows for the transcription of the nucleic acid molecule in the cell.
  • polynucleotides can be used for “gene targeting” or“gene therapeutic” approaches.
  • the nucleic acid molecules may also be labeled.
  • nucleic acids are well known in the art, e.g., Southern and Northern blotting, PCR or primer extension. This embodiment may be useful for screening methods for verifying successful introduction of the nucleic acid molecules described above, for example during gene therapy approaches.
  • an isolated host cell comprising the isolated nucleic acid encoding the anti-EpCAM antibody (or antigen-binding fragment thereof) described herein.
  • the host cells comprising the isolated nucleic acid described herein may be useful in expression or cloning of the low-affinity bispecific molecule or isolated nucleic acids.
  • Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
  • the expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. BioTechnology 9: 545-551 (1991).
  • eukaryotic cells in culture are also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.
  • Higher eukaryotic cells in particular, those derived from multicellular organisms can be used for expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, without limitation, invertebrate cells and insect cells, and vertebrate cells.
  • an oncolytic virus comprising the isolated nucleic acid encoding the anti-EpCAM antibody (or antigen-binding fragment thereof) described herein.
  • the vector e.g., viral vector encoding bispecific molecule or vector comprising the isolated nucleic acid described herein
  • the vector can be introduced to the host cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art.
  • the host cells comprise a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cells comprise a single vector comprising isolated nucleic acids encoding a first polypeptide and a second polypeptide.
  • the present application provides methods of expressing the low-affinity bispecific molecule or any one of the anti-EpCAM antibodies (or antigen- binding fragments thereof) described herein, comprising culturing the isolated host cell containing the vector (or transfected with the OV) and recovering the low-affinity bispecific molecule or anti-EpCAM antibody (or antigen-binding fragment thereof) from the cell culture.
  • the isolated host cells are cultured under conditions that allow expression of the low- affinity bispecific molecule or anti-EpCAM antibody (or antigen-binding fragment thereof) described herein.
  • Suitable conditions for expression of polynucleotides may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants.
  • suitable medium suitable density of host cells in the culture medium
  • suitable temperatures and humidity suitable temperatures and humidity, and absence of microorganism contaminants.
  • the polypeptides expressed in the host cell can form a dimer and thus produce a low-affinity bispecific molecule or anti-EpCAM antibody (or antigen- binding fragment thereof) described herein.
  • the polypeptide expressed in the host cell can form a polypeptide complex which is a homodimer.
  • the host cells express a first polynucleotide and a second polynucleotide
  • the first polynucleotide and the second polynucleotide can form a polypeptide complex which is a heterodimer.
  • the polypeptide complex (such as the low-affinity bispecific molecule, the anti-EpCAM antibody or antigen-binding fragment thereof) may be formed inside the host cell.
  • the dimer may be formed inside the host cell with the aid of relevant enzymes and/or cofactors.
  • the polypeptide complex may be secreted out of the cell.
  • a first polypeptide and a second polypeptide may be secreted out of the host cell and form a dimer (such as the low-affinity bispecific molecule, anti-EpCAM antibody or antigen-binding fragment thereof) outside of the host cell.
  • a first polypeptide and a second polypeptide may be separately expressed and allowed for dimerization to form the low-affinity bispecific molecule or the anti-EpCAM antibody (or antigen-binding fragment thereof) under suitable conditions.
  • the first polypeptide and the second polypeptide may be combined in a suitable buffer and allow the first protein monomer and the second protein monomer to dimerize through appropriate interactions such as hydrophobic interactions.
  • the first polypeptide and the second polypeptide may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote the dimerization of the first polypeptide and the second polypeptide.
  • the first polypeptide and the second polypeptide may be combined in a suitable vehicle and allow them to react with each other in the presence of a suitable reagent and/or catalyst.
  • the expressed polypeptide(s) and/or the polypeptide complex can be collected using any suitable methods.
  • the polypeptide(s) and/or the polypeptide complex can be expressed intracellularly, in the periplasmic space or be secreted outside of the cell into the medium. If the polypeptide and/or the polypeptide complex is expressed intracellularly, the host cells containing the polypeptide and/or the polypeptide complex may be lysed and polypeptide and/or the polypeptide complex may be isolated from the lysate by removing the unwanted debris by centrifugation or ultrafiltration. If the polypeptide and/or the polypeptide complex is secreted into periplasmic space of E.
  • the cell paste may be thawed in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)).
  • agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)).
  • the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, for example, an Amincon or Millipore Pellicon ultrafiltration unit.
  • Protease inhibitor and/or antibiotics may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminated microorganisms.
  • the expressed polypeptide(s) and/or the polypeptide complex can be further purified by a suitable method, such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see, for review, Bonner, P. L., Protein purification, published by Taylor & Francis. 2007; Janson, J. C., et al, Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998).
  • a suitable method such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography,
  • the polypeptides and/or polypeptide dimer complexes can be purified by affinity chromatography.
  • protein A chromatography or protein A/G (fusion protein of protein A and protein G) chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising a component derived from antibody CH2 domain and/or CH3 domain (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983); Zettlit, K. A., Antibody Engineering, Part V, 531-535, 2010).
  • protein G chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising IgG ⁇ 3 heavy chain (Guss et al., EMBO J. 5:1567 1575 (1986)).
  • protein L chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising K light chain (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. at al, J. Biol. Chem., 267, 2234-2239 (1992)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bakerbond ABX resin J. T. Baker, Phillipsburg, N.J. is useful for purification.
  • compositions e.g. pharmaceutical composition
  • a kit e.g., OV encoding the low-affinity bispecific molecule, anti-EpCAM antibodies or antigen-binding fragments thereof.
  • one or more viruses and/or the reagents to generate or manipulate the virus may be comprised in a kit.
  • the kit components are provided in suitable container means.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. Various combinations of components may also be comprised in a vial.
  • the kits also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful.
  • the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
  • kits may also be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • viruses for use in therapy are provided in a kit, and in some cases the viruses are essentially the sole component of the kit.
  • the kit may comprise reagents and materials to modify the desired virus.
  • the reagents and materials include expression constructs, primers for amplifying desired sequences, restriction enzymes, one or more DNAs for inclusion in the virus, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include vectors and/or DNA that encodes an engager molecule as described herein and/or regulatory elements therefor.
  • kits suitable for extracting one or more samples from an individual, or delivering the pharmaceutical composition (e.g., OV encoding the low-affinity bispecific molecule, anti-EpCAM antibodies or antigen-binding fragments thereof) to the individual.
  • the apparatus may be a syringe, scalpel, and so forth.
  • the kit in addition to the virus embodiments, also includes a second cancer therapy, such as chemotherapy, hormone therapy, and/or immunotherapy, for example.
  • a second cancer therapy such as chemotherapy, hormone therapy, and/or immunotherapy, for example.
  • the kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual.
  • kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.
  • compositions such as pharmaceutical compositions
  • suitable packaging for compositions such as OV encoding the low-affinity bispecific molecule, or anti-EpCAM antibody or antigen-binding fragments thereof
  • vials such as sealed vials
  • vessels such as ampules, bottles, jars
  • flexible packaging e.g., sealed Mylar or plastic bags
  • Embodiment 1 An oncolytic virus comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -9 M.
  • Embodiment 2 The oncolytic virus of embodiment 1, wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is similar to or about 2-10 times of the K D of the binding between the first antigen-binding domain and the tumor antigen.
  • Embodiment 3 The oncolytic virus of embodiment 1 or 2, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 10 -5 M to about 10 -8 M.
  • Embodiment 4 The oncolytic virus of embodiment 3, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M.
  • Embodiment 5 The oncolytic virus of embodiment 4, wherein the K D of the binding between the first antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -8 M to about 1 ⁇ 10 -8 M.
  • Embodiment 6 The oncolytic virus of embodiment 5, wherein the K D of the binding between the second antigen-binding domain and the cell surface molecule is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -7 M.
  • Embodiment 7 The oncolytic virus of any one of embodiments 1-6, wherein the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3).
  • the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3).
  • Embodiment 8 The oncolytic virus of embodiment 7, wherein the tumor antigen is EpCAM.
  • Embodiment 9 The oncolytic virus of embodiment 7, wherein the tumor antigen is FAP.
  • Embodiment 10 The oncolytic virus of embodiment 7, wherein the tumor antigen is EGFR.
  • Embodiment 11 The oncolytic virus of any one of embodiments 1-10, wherein the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell.
  • the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell.
  • Embodiment 12 The oncolytic virus of embodiment 11, wherein the effector cells is a T lymphocyte.
  • Embodiment 13 The oncolytic virus of embodiment 12, wherein the T lymphocyte is a cytotoxic T lymphocyte.
  • Embodiment 14 The oncolytic virus of any one of embodiments 1-13, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D.
  • Embodiment 15 The oncolytic virus of embodiment 14, wherein the cell surface molecule is CD3.
  • Embodiment 16 The oncolytic virus of any one of embodiments 1-15, wherein the first antigen-binding domain is a single chain variable fragment (scFv).
  • scFv single chain variable fragment
  • Embodiment 17 The oncolytic virus of any one of embodiments 1-16, wherein the second antigen-binding domain is a scFv.
  • Embodiment 18 The oncolytic virus of any one of embodiments 1-17, wherein the first antigen-binding domain and the second antigen-binding domain are connected by a linker.
  • Embodiment 19 The oncolytic virus of embodiment 18, wherein the first antigen- binding domain is N-terminal to the second antigen-binding domain.
  • Embodiment 20 The oncolytic virus of embodiment 18, wherein the first antigen- binding domain is C-terminal to the second antigen-binding domain.
  • Embodiment 21 The oncolytic virus of any one of embodiments 1-20, wherein the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus, Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, and Newcastle disease virus (NDV).
  • VV vaccinia virus
  • SVV Seneca Valley virus
  • HSV Herpes simplex virus 1
  • HSV2 Herpes simplex virus 2
  • myxoma virus reovirus
  • poliovirus poliovirus
  • MV measles virus
  • lentivirus retrovirus
  • morbillivirus influenza virus
  • Sinbis virus Sinbis virus
  • Embodiment 22 The oncolytic virus of embodiment 21, wherein the oncolytic virus is a vaccinia virus (VV).
  • VV vaccinia virus
  • Embodiment 23 The oncolytic virus of embodiment 22, wherein the vaccinia virus is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR).
  • MVA Ankara
  • WR Western Reserve
  • Embodiment 24 The oncolytic virus of embodiment 23, wherein the vaccinia virus is a WR strain.
  • Embodiment 25 The oncolytic virus of any one of embodiments 22-24, wherein the vaccinia virus comprises double deletion of thymidine kinase (TK) gene and vaccinia virus growth factor (VGF) gene.
  • TK thymidine kinase
  • VVF vaccinia virus growth factor
  • Embodiment 26 The oncolytic virus of any one of embodiments 1-25, wherein the oncolytic virus further comprises a second nucleic acid encoding an immune checkpoint modulator.
  • Embodiment 27 The oncolytic virus of embodiment 26, wherein the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.
  • Embodiment 28 The oncolytic virus of embodiment 26, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.
  • Embodiment 29 The oncolytic virus of embodiment 28, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4.
  • the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4.
  • Embodiment 30 The oncolytic virus of embodiment 29, wherein the immune checkpoint inhibitor is an inhibitor of PD-1.
  • Embodiment 31 The oncolytic virus of any one of embodiments 26-30, wherein the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule.
  • Embodiment 32 The oncolytic virus of any one of embodiments 26-29, wherein the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule.
  • Embodiment 33 The oncolytic virus of embodiment 32, wherein the immune checkpoint molecule is PD-L1, PD-L2, HHLA-2, CD47, or CXCR4.
  • Embodiment 34 The oncolytic virus of embodiment 33, wherein the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin, a TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin, or an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin.
  • the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin, a TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin, or an extracellular domain of SIRP ⁇ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin.
  • Embodiment 35 The oncolytic virus of embodiment 34, wherein the Fc fragment is an IgG4 Fc.
  • Embodiment 36 The oncolytic virus of any one of embodiments 26-35, wherein the oncolytic virus further comprises a third nucleic acid encoding a cytokine.
  • Embodiment 37 The oncolytic virus of embodiment 36, wherein the cytokine is GM-CSF.
  • Embodiment 38 The oncolytic virus of any one of embodiments 1-37, wherein the nucleic acid encoding the bispecific molecule is operably linked to a promoter.
  • Embodiment 39 The oncolytic virus of any one of embodiments 1-38, wherein the second nucleic acid encoding the immune checkpoint modulator and/or the third nucleic acid encoding the cytokine is operably linked to a promoter.
  • Embodiment 40 The oncolytic virus of embodiment 38 or 39, wherein the promoter is a late promoter.
  • Embodiment 41 The oncolytic virus of any one of embodiments 38-40, wherein the promoter is a vaccinia virus promoter.
  • Embodiment 42 The oncolytic virus of embodiment 41, wherein the promoter is F17R.
  • Embodiment 43 A pharmaceutical composition comprising the oncolytic virus of any one of embodiments 1-42, and a pharmaceutical acceptable carrier.
  • Embodiment 44 A method of treating a cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 43.
  • Embodiment 45 The method of embodiment 44, wherein the effective amount is about 10 5 to about 10 13 pfu.
  • Embodiment 46 The method of embodiment 45, wherein the effective amount is about 10 9 pfu.
  • Embodiment 47 The method of any one of embodiments 44-46, wherein the pharmaceutical composition is administered systemically.
  • Embodiment 48 The method of embodiment 47, wherein the pharmaceutical composition is administered intravenously.
  • Embodiment 49 The method of any one of embodiments 44-46, wherein the pharmaceutical composition is administered locally.
  • Embodiment 50 The method of embodiment 49, wherein the pharmaceutical composition is administered intratumorally.
  • Embodiment 51 The method of any one of embodiments 44-50, wherein the cancer is a solid tumor.
  • Embodiment 52 The method of embodiment 51, wherein the cancer is selected from the group consisting of colorectal cancer, lung cancer, liver cancer, skin cancer, brain cancer, and breast cancer.
  • Embodiment 53 The method of embodiment 52, wherein the skin cancer is melanoma.
  • Embodiment 54 The method of embodiment 52, wherein the brain cancer is glioblastoma.
  • Embodiment 55 The method of any one of embodiments 44-54, further comprising administering to the individual an additional cancer therapy.
  • Embodiment 56 The method of embodiment 55, wherein the additional cancer therapy is surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof.
  • Embodiment 57 The method of any one of embodiments 44-56, wherein the individual is a human.
  • Embodiment 58 A construct comprising an antigen-binding domain comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • Embodiment 59 The construct of embodiment 58, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20.
  • VH heavy chain variable region
  • VL light chain variable region
  • Embodiment 60 A construct comprising an antigen-binding domain comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.
  • VH heavy chain variable region
  • VL light chain variable region
  • Embodiment 61 The construct of embodiment 60, wherein the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 21, and/or a VL comprising the amino acid sequence of SEQ ID NO: 22.
  • Embodiment 62 A construct comprising an antigen-binding domain comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
  • VH heavy chain variable region
  • VL light chain variable region
  • Embodiment 63 The construct of embodiment 62, wherein the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 23, and/or a VL comprising the amino acid sequence of SEQ ID NO: 24.
  • Embodiment 64 The construct of any one of embodiments 58-63, wherein the construct is a full length antibody.
  • Embodiment 65 The construct of embodiment 64, wherein the full length antibody is a multispecific antibody.
  • Embodiment 66 The construct of any one of embodiments 58-65, wherein the construct is a bispecific molecule, wherein the bispecific molecule further comprises a second antigen-binding fragment specifically recognizing a cell surface molecule of an effector cell.
  • Embodiment 67 The construct of embodiment 66, wherein the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell.
  • Embodiment 68 The construct of embodiment 67, wherein the effector cell is a T lymphocyte.
  • Embodiment 69 The construct of embodiment 68, wherein the T lymphocyte is a cytotoxic T lymphocyte.
  • Embodiment 70 The construct of any one of embodiments 66-69, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D.
  • Embodiment 71 The construct of embodiment 70, wherein the cell surface molecule is CD3.
  • Embodiment 72 The construct of any one of embodiments 66-71, wherein the antigen-binding domain is a scFv.
  • Embodiment 73 The construct of any one of embodiments 66-72, wherein the second antigen-binding domain is a scFv.
  • Embodiment 74 The construct of any one of embodiments 66-73, wherein the antigen-binding domain and the second antigen-binding domain are connected by a linker.
  • Embodiment 75 The construct of any one of embodiments 66-74, wherein the antigen-binding domain is N-terminal to the second antigen-binding domain.
  • Embodiment 76 The construct of any one of embodiments 66-74, wherein the antigen-binding domain is C-terminal to the second antigen-binding domain.
  • Embodiment 77 An isolated nucleic acid encoding the construct of any one of embodiments 58-76.
  • Embodiment 78 The isolated nucleic acid of embodiment 77, comprising a first nucleic acid sequence of SEQ ID NO: 60, and a second nucleic acid sequence of SEQ ID NO: 61.
  • Embodiment 79 The isolated nucleic acid of embodiment 77, comprising a first nucleic acid sequence of SEQ ID NO: 62, and a second nucleic acid sequence of SEQ ID NO: 63.
  • Embodiment 80 The isolated nucleic acid of embodiment 77, comprising a first nucleic acid sequence of SEQ ID NO: 64, and a second nucleic acid sequence of SEQ ID NO: 65.
  • Embodiment 81 The isolated nucleic acid of embodiment 77, comprising the nucleic acid sequence of any one of SEQ ID NOs: 66-71.
  • Embodiment 82 The isolated nucleic acid of any one of embodiments 77-81, wherein the isolated nucleic acid is operably linked to a promoter.
  • Embodiment 83 The isolated nucleic acid of embodiment 82, wherein the promoter is a late promoter.
  • Embodiment 84 The isolated nucleic acid of embodiment 82 or 83, wherein the promoter is a vaccinia virus promoter.
  • Embodiment 85 The isolated nucleic acid of embodiment 84, wherein the vaccinia virus promoter is F17R.
  • Embodiment 86 An isolated host cell comprising the isolated nucleic acid of any one of embodiments 77-85.
  • Embodiment 87 An oncolytic virus comprising the isolated nucleic acid of any one of embodiments 77-85.
  • Embodiment 88 A pharmaceutical composition comprising the construct according to any one of embodiments 58-76, the isolated host cell of embodiment 86, or the oncolytic virus of embodiment 87, and a pharmaceutically acceptable carrier.
  • Embodiment 89 A method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 88.
  • Embodiment 90 The method of embodiment 89, wherein the pharmaceutical composition is administered to the individual intravenously or intratumorally.
  • Embodiment 91 The method of embodiment 89 or 90, wherein the individual is a human.
  • Embodiment 92 A pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, a second OV comprising a second nucleic acid encoding an immune checkpoint modulator, and a pharmaceutical acceptable carrier.
  • Embodiment 93 The pharmaceutical composition of embodiment 92, wherein the pharmaceutical composition comprises a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, a second OV comprising a second nucleic acid encoding an immune checkpoint modulator of any one of embodiments 27-35 and 38-42, and a pharmaceutical acceptable carrier.
  • Embodiment 94 A pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, a second OV comprising a second nucleic acid encoding a cytokine, and a pharmaceutical acceptable carrier.
  • Embodiment 95 The pharmaceutical composition of embodiment 94, wherein the pharmaceutical composition comprises a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, a second OV comprising a second nucleic acid encoding a cytokine of any one of embodiments 36, 37, and 39-42, and a pharmaceutical acceptable carrier.
  • Embodiment 96 A pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, a second OV comprising a second nucleic acid encoding an immune checkpoint modulator, a third OV comprising a third nucleic acid encoding a cytokine, and a pharmaceutical acceptable carrier.
  • Embodiment 97 The pharmaceutical composition of embodiment 96, wherein the pharmaceutical composition comprises a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, a second OV comprising a second nucleic acid encoding an immune checkpoint modulator of any one of embodiments 27-35 and 38-42, a third OV comprising a third nucleic acid encoding a cytokine of any one of embodiments 36, 37, and 39-42, and a pharmaceutical acceptable carrier.
  • Embodiment 98 In one embodiment, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of any one of embodiments 92-97.
  • Embodiment 99 A method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator, and a second pharmaceutical acceptable carrier.
  • Embodiment 100 The method of embodiment 99, wherein the method comprises administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, and a first pharmaceutical acceptable carrier, and a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator of any one of embodiments 27-35 and 38-42, and a second pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, and a first pharmaceutical acceptable carrier
  • a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator of any one of embodiments 27-35 and 38-42, and a second pharmaceutical acceptable carrier.
  • Embodiment 101 A method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine, and a second pharmaceutical acceptable carrier.
  • Embodiment 102 The method of embodiment 101, wherein the method comprises administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, and a first pharmaceutical acceptable carrier, and a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine of any one of embodiments 36, 37, and 39-42, and a second pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, and a first pharmaceutical acceptable carrier
  • a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine of any one of embodiments 36, 37, and 39-42, and a second pharmaceutical acceptable carrier.
  • Embodiment 103 A method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, and a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator, and a second pharmaceutical acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV comprising a third nucleic acid encoding a cytokine, and a third pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen
  • Embodiment 104 The method of embodiment 103, wherein the method comprises administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, and a first pharmaceutical acceptable carrier, a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding an immune checkpoint modulator of any one of embodiments 27-35 and 38-42, and a second pharmaceutical acceptable carrier, and a third pharmaceutical composition comprising a third OV comprising a third nucleic acid encoding a cytokine of any one of embodiments 36, 37, and 39-42, and a third pharmaceutical acceptable carrier.
  • a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding a bispecific molecule of any one of embodiments 1-25 and 38-42, and a first pharmaceutical acceptable carrier
  • a second pharmaceutical composition comprising a second OV comprising a
  • VV oncolytic vaccinia virus
  • TE T-cell engager
  • a low-affinity T-cell engager e.g.
  • the K D of the binding between the tumor-antigen-binding domain and the tumor antigen is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -8 M
  • the K D of the binding between the effector cell surface molecule- binding domain and the effector cell surface molecule is about 5 ⁇ 10 -7 M to about 1 ⁇ 10 -7 M) as a novel oncolytic vaccinia virus therapy, thereby opening a new avenue for development of effective oncolytic virus therapy.
  • This new strategy is applicable to improve the efficacy of various forms of oncolytic virus described herein.
  • some embodiments of the present disclosure provide a therapeutic composition that is useful for the treatment of solid tumors.
  • the following examples evaluated the efficacy of new oncolytic VV in human tumor xenograft models.
  • the T-cell engager armed-VV (TEA-VV) are useful for the treatment of, e.g., colorectal cancer, lung cancer, liver cancer, skin cancer, brain cancer, or breast cancer.
  • TEA-VV exerts its anti- tumor activity through one or two mechanisms: i) bispecific scFv directs T cells to recognize and kill tumor cells that are not infected with vaccinia virus (bystander killing), resulting in enhanced tumor lysis, and ii) CD3-scFv activates T-cells within the tumor, and the cytokines they release upon activation create a pro-inflammatory microenvironment, inhibiting tumor growth.
  • CD3-scFvs are capable of redirecting the vast number of existing T-cell clones in patients to tumor cells.
  • oncolytic virus induces local production of T- cell engagers that might allow higher concentrations at the target while reducing systemic side effects.
  • arming oncolytic VV with bispecific scFvs provides an alternative approach to engage T cells for cancer therapy and produce the desired increase in antitumor activity of current vaccinia virus therapy by inducing bystander killing (FIG. 1).
  • Example 1 Construction of hEpCAM-TEA-VV and expression of hEpCAM-TE
  • Vaccinia viruses (Western Reserve strain) encoding secretory EpCAM-scFv-human CD3-scFv (hEpCAM-TEA-VV, hereinafter also referred to as hEpCAM-CD3-VV) or GFP (GFP-VV) were generated by recombination of a version of pSEM-1 plasmid containing T- cell engagers (TE) or GFP into the TK gene of the VSC20 strain of WR vaccinia virus (WR VV).
  • TE T- cell engagers
  • WR VV WR vaccinia virus
  • the shuttle vector pSEM-1 was constructed to contain the EpCAM-scFv-human CD3-scFv (hereinafter also referred to as hEpCAM-CD3 or hEpCAM-TE), or GFP (FIG. 2).
  • the inserted hEpCAM-TE or GFP was expressed under the transcriptional control of the F17R late promoter to allow for sufficient viral replication before T-cell activation.
  • the VVs also expressed YFP-GFP to allow virus selection.
  • YFP-GFP was expressed under the transcriptional control of P7.5 promoter, and loxP sites in the same orientation flanking YFP- GFP, the selectable marker (FIG. 2).
  • the shuttle vectors pSEM-1 were firstly transfected into human 143 TK- cells. Cells were then infected with virus VSC20 at a multiplicity of infection (MOI) of 0.1. After five rounds of plaque selection and amplification to confirm the expression of hEpCAM-TE or GFP, one of the clones was selected for amplification and purification. To remove the YFP-GFP cassette from the recombinant viruses, viruses were passaged on a U2OS cell line expressing a cytoplasmic form of Cre recombinase (U2OS-Cre). After five rounds of plaque selection and amplification to confirm the expression of hEpCAM-TE or GFP, one of the YFP-GFP-negative clones was selected for amplification and purification.
  • MOI multiplicity of infection
  • hEpCAM-TEA-VV-infected tumor cells were analyzed for their ability to express secretory bispecific EpCAM-scFv-human CD3-scFv (hEpCAM-TE). Briefly, the lung cancer cell line A549 was transduced with hEpCAM-TEA-VV or GFP-VV (data not shown) at MOI 5 and at 24 hours after incubation, and the cell culture medium was collected. The collected media from hEpCAM-TEA-VV-infected tumor cells were analyzed on a SDS-PAGE gel, which showed secretory hEpCAM-TE at the molecular weight of 56KD (FIG. 3).“EpCAM- TE” lane shows the positive EpCAM-bispecific scFv control produced in 293-T cells.
  • Example 2 hEpCAM-TE encoded by hEpCAM-TEA-VV binds to EpCAM expressed on multiple cancer cell lines
  • hEpCAM-TE was used in FACS analysis of various tumor cell lines expressing EpCAM, including U87 (glioblastoma), A549 (lung carcinoma), HT-29 (colorectal adenocarcinoma), SK-BR-3 (breast carcinoma), and Huh7 (hepatoma). Briefly, the hEpCAM-TE were incubated with the respective cells and washed with PBS according to standard FACS protocol as described below. Following hEpCAM-TE staining, a PE (phycoerythrin)-labeled secondary antibody was used to detect hEpCAM-TE bound on the respective cells. As can be seen from FIG.
  • hEpCAM-TE encoded by hEpCAM-TEA-VV binds to cell surface EpCAM expressed on multiple cancer cell lines.
  • FACS assay [0531] For immunophenotyping, cells were stained with hEpCAM-TE or with fluorescein- conjugated monoclonal antibodies (Becton Dickinson, San Jose, CA) directed against CD3-, CD4-, CD8-, CD69- or FITC-labeled EpCAM, hFAP and mFAP proteins. Isotype controls were immunoglobulin G1–fluorescein isothiocyanate (IgG1-FITC; BD) and IgG1– phycoerythrin (IgG1-PE; BD).
  • hEpCAM-TE The binding affinity of hEpCAM-TE encoded by hEpCAM-TEA-VV was also tested in Western blot assay according to regular protocol.
  • hEpCAM-TE was used as the primary antibody to detect EpCAM in lysates of various tumor cell lines expressing EpCAM, including U87, A549, HT-29, SK-BR-3, and Huh7. Antibodies against actin were used as a loading control.
  • hEpCAM-TE can bind to EpCAM expressed on multiple cancer cell lines.
  • Example 4 hEpCAM-TE expression does not impair the ability of VVs to induce tumor cell lysis
  • Huh7, SK-BR-3, and HT-29 cells were infected with hEpCAM- TEA-VV at increasing MOIs (0, 0.01, 0.1, 1, or 5).
  • Huh7, SK-BR-3, and HT-29 cells infected with GFP-VV at the same MOIs were used as controls.
  • Huh7, SK-BR-3, and HT-29 viability was determined by MTS assay (see protocol below).
  • tumor cells were first plated in a 96-well tissue culture plate at 1' ⁇ 10 4 cells per well (100 ⁇ l) and incubated overnight at 37°C. The tumor cells were then infected with VVs at indicated MOIs in 2.5% FBS medium for 2 hours followed by culturing in complete medium. All samples were measured in triplicate. Unstimulated human PBMCs were incubated at 37°C for 2 hours to remove adherent cells. In some experiments, unstimulated nonadherent human PBMCs (see Example 5 below for PBMC culturing) or CD4/8 microbead-selected T cells were added to the culture at effector: target ratio of 5:1.
  • Example 5 hEpCAM-TEA-VV activates PBMC
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC populations blood samples from healthy donors were obtained in accordance to protocols approved by the Institutional Review Board of Baylor College of Medicine. Peripheral blood was processed over Ficoll gradients, and the resulting PBMCs were cultured in Roswell Park Memorial Institute 1640 (Thermo Scientific HyClone, Waltham, MA; Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated FCS and 2 mmol/L GLUTAMAX.
  • hEpCAM-TEA-VV can activate human PBMCs
  • fresh human PBMC were incubated with or without SK-BR-3 tumor cells infected with GFP-VV (control) or hEpCAM-TEA-VV at a tumor cell:PBMC ratio of 1:5.
  • the incubated PBMCs were then tested for CD69 expression, a marker illustrative of PBMC activation.
  • PBMC samples incubated with or without VV-infected tumors were stained for CD4 and CD69, or for CD8 and CD69, according to the FACS protocol described in Example 2.
  • Example 6 hEpCAM-TEA-VV induced potent tumor lysis in the presence of human PBMC
  • EpCAM-positive colorectal adenocarcinoma cancer cell line HT-29 or breast cancer cell line SK-BR-3 was transduced with hEpCAM-TEA-VV, or HN3-TEA-VV (HN3- scFv-CD3-scFv) at MOI of 0.1 and 1.
  • HN3 is an antibody clone against hepatocellular carcinoma (HCC) antigen Glypican-3 (GPC3).
  • Mock transduced tumor cells were used as a control. The respective transduced cells were co-cultured with fresh PBMC for 48 hours.
  • HT-29-GFP or SK-BR-3-GFP cell viability was measured by GFP signal.
  • Example 7 hEpCAM-TEA-VV induced potent bystander tumor lysis in the presence of human PBMC
  • EpCAM-positive colorectal adenocarcinoma cancer cell line HT-29 or breast cancer cell line SK-BR-3 was transduced with hEpCAM-TEA-VV, or HN3- TEA-VV at MOI of 1.
  • GFP-VV or mock transduced tumor cells were used as controls.
  • the cell culture medium was collected after 48 hours of culture.
  • non-infected HT-29-GFP cells were co-cultured with fresh PBMC in the presence of the cell culture media collected from the mock or VV infected HT-29.
  • Non-infected SK-BR-3-GFP cells were co-cultured with fresh PBMC in the presence of the cell culture media collected from the mock or VV infected SK-BR-3. After 48 hours of co-culture in conditioned media, HT-29-GFP or SK-BR- 3-GFP cell viability was measured by GFP signal. The results demonstrated that there was significant GFP signal decrease only in the presence of cell culture medium conditioned by hEpCAM-TEA-VV-infected HT-29 (or SK-BR-3), as measured by FACS (FIG. 9A) or immunofluorescence (FIG.9B), indicating the bystander killing of uninfected tumor cells.
  • Example 8 hEpCAM-TEA-VV inhibited HT-29 tumor growth in vivo
  • HT-29 tumor xenograft mice model was employed.
  • 4 ⁇ 10 6 HT-29 cells were inoculated subcutaneously into the right flank of NSG mice, followed by i.p. injection of 1 ⁇ 10 8 pfu of hEpCAM-TEA-VV, GFP-VV or no VV (PBS) on day 5, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 7. Mice that received PBS only or PBMC only served as controls.
  • mice that received hEpCAM-TEA-VV with PBMC showed a significant decrease in tumor growth, compared to mice received hEpCAM-TEA-VV without PBMC, GFP-VV with PBMC, PBMC only or PBS only (hEpCAM-TEA-VV/PBMC vs. GFP-VV/PBMC p ⁇ 0.0001) (FIG. 10A).
  • mice treated with hEpCAM-TEA-VV with PBMC showed a significant decrease in tumor growth, compared to mice that received hEpCAM-TEA-VV without PBMC, GFP-VV with PBMC, PBMC only, or PBS only (FIG.10B).
  • hEpCAM-TEA-VV/PBMC treatment also largely prolonged mice survival compared to groups treated by hEpCAM-TEA-VV without PBMC, GFP-VV with PBMC, PBMC only, or PBS only (FIG. 10C).
  • Example 9 hEpCAM-TEA-VV inhibited SK-BR-3 tumor growth in vivo
  • SK-BR-3 tumor xenograft model was employed.
  • 4 ⁇ 10 6 SK-BR-3 cells were inoculated subcutaneously into the right flank of NSG mice, followed by i.p. injection of 1 ⁇ 10 8 pfu of hEpCAM-TEA-VV, HN3-TEA-VV or no VV (PBS) into the right flank tumor on day 8, and i.v. implantation of 2 ⁇ 10 7 unactivated human PBMC cells on day 10. Mice that received PBS only or PBMC only served as controls.
  • HN3-TEA-VVs/PBMC and hEpCAM-TEA-VV moderately inhibited tumor growth compared to that in PBS and PBMC control mice.
  • mice that received hEpCAM-TEA-VV with PBMC showed a significant decrease in tumor growth, compared to mice received hEpCAM-TEA- VV without PBMC, HN3-TEA-VV with PBMC, PBMC or PBS only (hEpCAM-TEA- VV/PBMC vs. HN3-TEA-VV/PBMC p ⁇ 0.0001) (FIG.11).
  • Example 10 Construction of hFAP-TEA-VV and expression of hFAP-TE
  • Vaccinia viruses (Western Reserve strain) encoding secretory FAP-scFv-human CD3-scFv (hFAP-TEA-VV, hereinafter also referred to as hFAP-CD3-VV), GPC3-scFv- human CD3-scFv (hGPC3-TEA-VV, hereinafter also referred to as hGPC3-CD3-VV), or GFP (GFP-VV) were generated by recombination of a version of pSEM-1 plasmid containing T-cell engagers (TE) or GFP into the TK gene of the VSC20 strain of WR vaccinia virus (WR VV).
  • TE T-cell engagers
  • WR VV WR vaccinia virus
  • the shuttle vector pSEM-1 was constructed to contain the FAP-scFv- human CD3-scFv (hFAP-TE, or hFAP-CD3), GPC3-scFv-human CD3-scFv (hGPC3-TE, or hGPC3-CD3), or GFP (FIG. 12A).
  • the inserted T-cell engagers (or GFP) were expressed under the transcriptional control of the F17R late promoter to allow for sufficient viral replication before T-cell activation.
  • the VVs also expressed YFP-GFP to allow for virus selection.
  • YFP-GFP was expressed under the transcriptional control of P7.5 promoter, and loxP sites in the same orientation flanking YFP-GFP, the selectable marker (FIG. 12A).
  • the shuttle vectors pSEM-1 were transfected into human 143 TK- cells. Cells were then infected with virus VSC20 at a MOI of 0.1. After five rounds of plaque selection and amplification to confirm the expression of TEs (or GFP), one of the clones was selected for amplification and purification.
  • viruses were passaged on a U2OS cell line expressing a cytoplasmic form of Cre recombinase (U2OS-Cre). After five rounds of plaque selection and amplification to confirm the expression of TEs (or GFP), one of the YFP-GFP-negative clones was selected for amplification and purification. [0544] hFAP-TEA-VV infected tumor cells were analyzed for their ability to express secretory bispecific hFAP-TE.
  • SK-BR-3 or HT-29 cells were transduced with hFAP- TEA-VV, hGPC3-TEA-VV or GFP-VV at MOI of 5; and at 24 hours after incubation, the cell culture medium was collected.
  • the collected media from hFAP-TEA-VV-infected HT-29 tumor cells were purified using His column and analyzed on a SDS-PAGE gel, which showed secretory hFAP-TE at the molecular weight of 56kD (FIG. 12B).
  • Example 11 hFAP-TE expression does not impair VVs’ ability to replicate
  • hFAP-TE expression affects the ability of hFAP-TEA-VV to replicate in tumor cells
  • CV-1, SK-BR-3 and HT-29 cells were infected with hFAP-TEA-VV, hGPC3- TEA-VV, or parental vaccinia virus (VSC20).
  • Infection with hFAP-TEA-VV, hGPC3-TEA- VV, and VSC20 yielded similar amounts of virus at various time points across all three cell lines (FIG.13), suggesting that hFAP-TE expression does not interfere with VV replication.
  • Example 12 hFAP-TE expression does not impair the ability of VVs to induce tumor cell lysis
  • FAP-positive glioblastoma cell line U87-GFP was transduced with hFAP-TEA-VV or hGPC3-TEA-VV. Mock transduced tumor cells were used as a control. The respective transduced cells were co- cultured with fresh PBMC (isolated as described in Example 5) for 72 hours. After 72 hours of co-culture with PBMC, U87-GFP cell viability was measured by GFP signal. These VV infected U87-GFP cells were also stained for hCD45, a T cell activation marker, in the FACS analysis.
  • Annexin-V The vaccinia virus infected U87 cells were also measured for apoptotic markers Annexin-V and Propidium iodide (PI).
  • Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells.
  • PS phosphatidylserine
  • PI is a DNA dye that can be used as plasma membrane permeability indicator to differentiate necrotic, apoptotic and normal cells.
  • the vaccinia virus infected U87 cells were also measured for cell viability via MTS assay as described in Example 4. Again the result demonstrated significant decrease in cell viability in the hFAP-TEA-VV-infected U87 cells and only a moderate decrease in viability in hGPC3-TEA-VV infected cells in the presence of PBMC (FIG. 15B lower panel), indicating enhanced tumor lysis induced by hFAP-TEA-VV in the presence of human PBMC.
  • Example 14 hFAP-TEA-VV activated human T cells in vitro
  • the incubated PBMCs were then tested for CD69 expression, an marker illustrative of PBMC activation.
  • PBMC samples incubated with or without VV-infected tumors were stained for CD4 and CD69, or for CD8 and CD69, according to the FACS protocol described in Example 2.
  • T-cell activation As an alternative to measure T-cell activation, cell culture media was collected 24 hours post-infection by the vaccinia viruses, to determine the presence of pro-inflammatory cytokines by ELISA. Human T cells were activated by hFAP-TEs as evidenced by the production of pro-inflammatory cytokines such as IFN- ⁇ and IL-2 in the co-culture supernatant of hFAP-TEA-VV infected U87 and PBMC, compared to that of hGPC3-TEA- VV infected U87 and PBMC co-culture (p ⁇ 0.05). T cells produced little to no IFN- ⁇ or IL-2 in response to hGPC3-TEA-VV infected U87 (FIG. 16B). These results indicate that hFAP- TE expressed by infected tumor cells activate T cells.
  • Example 15 hFAP-TEA-VV induced bystander killing of tumor cells that are not infected by virus
  • FAP-positive glioblastoma cell line U87 was transduced with hFAP-TEA-VV at MOI of 1.
  • hGPC3-TEA-VV transduced or non-transduced tumor cells were used as controls.
  • the cell culture medium was collected after 48 hours of culture.
  • non-infected FAP+ U87-GFP cells were co-cultured with fresh PBMC in the presence of the respective cell culture media collected from uninfected U87, hGPC3-TEA-VV infected U87, or hFAP- TEA-VV infected U87 (FIG. 17A).
  • tumor cells used for conditioning media were infected at a range of increasing MOI from 0 to 10, respectively.
  • the conditioned media was then used for PBMC/U87 co- culture at the PBMC: U87 ratio of 5:1.
  • MTS assay showed that after 48 hours of co-culture, the effect of hFAP-TEA-VV killing of bystander cells is correlated to increasing MOI employed to infect the medium-conditioning tumor cells (FIG. 17E).
  • Example 16 hFAP-TEA-VV inhibited SK-BR-3 growth in vivo
  • SK-BR-3 tumor xenograft model was employed.
  • 4 ⁇ 10 6 SK-BR-3 cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV, hGPC3-TEA-VV or no VV (PBS) into the right flank tumor on day 8, and i.v. implantation of 2 ⁇ 10 7 unactivated human PBMC cells on day 10 (FIG. 18A).
  • PBS pfu of hFAP-TEA-VV
  • hGPC3-TEA-VV or no VV PBS
  • mice received hFAP-TEA-VV with PBMC showed a significant decrease in tumor growth, compared to mice that received FAP-TEA-VV without PBMC, hGPC3-TEA-VV with PBMC, PBMC only or PBS only (hFAP-TEA-VV/PBMC vs. hGPC3-TEA-VV/PBMC p ⁇ 0.0001) (FIG. 18B).
  • mice treated with hFAP-TEA-VV/PBMC showed a significant decrease in tumor growth, compared to mice that received hFAP-TEA-VV without PBMC, hGPC3-TEA-VV with PBMC, or PBMC/PBS only (FIG.18C).
  • hFAP-TEA-VV could inhibit SK-BR-3 tumor growth in vivo, especially in the presence of PBMC.
  • Example 17 hFAP-TEA-VV inhibited HT-29 growth in vivo
  • HT-29 tumor xenograft model was also employed.
  • 4 ⁇ 10 6 HT-29 cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV, GFP-VV or no VV (PBS) into the right flank tumor on day 5, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 7.
  • mice that received hFAP-TEA-VV with PBMC showed a significant decrease in tumor growth, compared to mice that received hFAP-TEA-VV without PBMC, GFP-VV with PBMC, PBMC only or PBS only (hFAP-TEA-VV/PBMC vs. GFP-VV/PBMC p ⁇ 0.0001) (FIG. 19A).
  • hFAP-TEA-VV/PBMC treatment also significantly prolonged the mice survival compared to groups treated by hFAP-TEA-VV without PBMC, GFP-VV with PBMC, PBMC only or PBS only (FIG. 19B), thus illustrating that hFAP-TEA-VV inhibited HT-29 tumor growth in vivo, with a greater effect in the presence of PBMC.
  • Example 18 Stromal destruction in SK-BR-3 tumor environment correlates to hFAP- TEA-VV spread
  • SK-BR-3 tumor model was employed.
  • 4 ⁇ 10 6 SK-BR-3 cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV or GPC3-TEA-VV or no VV (PBS) into the right flank tumor on day 8, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 10.
  • FACS analysis was performed with FITC-conjugated anti-mCD45 and APC- conjugated anti-mFAP, illustrating that hFAP-TEA-VV in the presence of PBMC caused a significant decrease in mFAP positive cells and slight increase in mCD45 T cells, compared to that treated with PBMC control, hGPC3-TEA-VV/PBMC and hFAP-TEA-VV/no PBMC (FIG. 20A), thus indicating the ability of hFAP-TEA-VV in tumor stromal destruction.
  • the biopsied samples were also subjected to lysis and qRT-PCR analysis, which again illustrated a significant decrease in overall mFAP mRNA levels in tumor environment of hFAP-TEA- VV/PBMC treated mice as compared to that treated with PBMC/PBS control, hGPC3-TEA- VV/PBMC and hFAP-TEA-VV/no PBMC (FIG. 20B), thus indicating effective tumor stromal destruction by hFAP-TEA-VV in the presence of T cells.
  • mice treated with hFAP-TEA-VV/PBMC showed nearly absent mouse ⁇ -SMA signal as compared to those infected with PBMC control, hGPC3-TEA-VV/PBMC and hFAP-TEA-VV/no PBMC (FIG. 20C), indicating effective tumor stromal destruction by hFAP-TEA-VV in the presence of T cells.
  • Example 19 hFAP-TEA-VV causes stromal destruction in HT-29 tumor environment
  • the HT-29 tumor model was employed.
  • 4 ⁇ 10 6 HT-29 cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV or GFP-VV or no VV (PBS) into the right flank tumor on day 5, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 7.
  • FACS analysis was performed with APC-conjugated anti-mFAP, illustrating that hFAP-TEA-VV in the presence of PBMC caused a significant decrease in mFAP positive cells compared to that treated with PBS control, PBMC control, GFP-VV/PBMC and hFAP- TEA-VV/no PBMC (FIG. 21A), thus indicating the ability of hFAP-TEA-VV in tumor stromal destruction in the presence of PBMC.
  • the biopsied sample cells from mice treated with hFAP-TEA-VV/PBMC showed nearly absent ⁇ -SMA signal as compared to those infected with PBMC control, PBS control, GFP-VV/PBMC and hFAP-TEA-VV/no PBMC (FIG. 21B), indicating effective tumor stromal destruction by hFAP-TEA-VV in the presence of T cells.
  • Example 20 hFAP-TEA-VV activated T cells in vivo in SK-BR-3 tumor environment.
  • SK- BR-3 tumor model was employed.
  • 4 ⁇ 10 6 SK-BR-3 cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV, hGPC3-TEA-VV or no VV (PBS) into the right flank tumor on day 8, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 10.
  • FACS analysis was performed with FITC-conjugated anti-hCD3, with the FACS plot illustrating that hFAP-TEA-VV in the presence of human PBMC caused a significant increase in CD3 positive human PBMC as compared to mice treated with PBMC control, hGPC3-TEA- VV/PBMC or hFAP-TEA-VV/no PBMC (FIG. 22A), thus indicating the infiltration of human PBMCs in SK-BR-3 tumor environment.
  • the biopsied tumors described earlier were also fixed and subjected to immunofluorescence for hCD3.
  • Immunofluorescent staining illustrated a significantly higher level of hCD3 in biopsied samples from mice treated with hFAP-TEA-VV/PBMC as compared to that treated with hGPC3-TEA-VV/PBMC (FIG. 22B), indicating the facilitated infiltration of human PBMCs in SK-BR-3 tumor environment by hFAP-TEA-VV.
  • DAPI was used to mark nuclei (FIG. 22B). Therefore, as illustrated by FIGS. 22A and 22B, T cell infiltration into tumor tissue was facilitated by hFAP-TEA-VV.
  • mice treated with hFAP-TEA- VV/PBMC as compared to those treated with PBS/PBMC control, hGPC3-TEA-VV/PBMC or hFAP-TEA-VV/no PBMC, indicating in vivo activation of human PBMCs in SK-BR-3 tumor environment by hFAP-TEA-VV.
  • Example 21 hFAP-TEA-VV activated T cells in vivo in HT-29 tumor environment.
  • the HT-29 tumor model was also employed.
  • 4 ⁇ 10 6 HT-29 cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of 1 ⁇ 10 8 pfu of hFAP-TEA-VV or GFP-VV or no VV (PBS) into the right flank tumor on day 5, and i.v. implantation of 2x10 7 unactivated human PBMC cells on day 7.
  • biopsies of right flanks of the mice treated with PBMC control, GFP-VV/PBMC, or hFAP-TEA-VV/PBMC were also subjected to FACS analysis for pro-inflammatory cytokines such as hINF ⁇ , the expression of which reflects activation of human PBMCs.
  • FACS analysis illustrated a significant increase of hIFN ⁇ positive cells in samples from mice treated with hFAP-TEA- VV/PBMC as compared to those treated with PBMC control or GFP-VV/PBMC, indicating in vivo activation of human PBMCs in HT-29 tumor environment by hFAP-TEA-VV (FIG. 23B).
  • Example 22 hFAP-TEA-VV distribution in NSG mice after intratumor injection
  • Example 23 Toxicity of hFAP-TEA-VV in HT-29 tumor model
  • hFAP-TEA-VV toxicity after intratumor injection the body weight of HT-29 tumor-bearing mice was monitored over the course of 24 days and at day 21 bone marrow cellularity and quadriceps mass was assessed. Mice were sacrificed, femur and tibia were dissected. Bone marrow cells were flushed out to count the viable cell number. Mouse quadriceps were removed and weighed. hFAP-TEA-VV intratumor injection (with or without the presence of PBMC) did not cause significant body weight loss compared with intratumor injection of GFP-VV/PBMC, PBMC only, or PBS control group (FIG. 25A).
  • Vaccinia viruses (Western Reserve strain) encoding secretory FAP-scFv-mouse CD3-scFv (mFAP-TEA-VV, hereinafter also referred to as mFAP-CD3-VV), EphA2-scFv- human CD3-scFv (EphA2-TEA-VV, hereinafter also referred to as EphA2-CD3-VV), or GFP (GFP-VV) were generated by recombination of a version of pSEM-1 plasmid containing T- cell engagers (TE) or GFP into the TK gene of the VSC20 strain of WR vaccinia virus (WR VV).
  • TE T- cell engagers
  • WR VV WR vaccinia virus

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Abstract

La présente invention concerne un virus oncolytique codant pour une molécule bispécifique reconnaissant spécifiquement (i) un antigène tumoral (tel que EphCAM) et (ii) une molécule de surface cellulaire d'une cellule effectrice, telle que le CD3 des cellules T. La molécule bispécifique présente une faible affinité de liaison à la fois pour l'antigène tumoral et pour la molécule de surface cellulaire. L'invention concerne également des compositions pharmaceutiques du virus oncolytique de l'invention destinées au traitement du cancer, et les utilisations médicales correspondantes. L'invention concerne également de nouveaux anticorps anti-EpCAM, et les acides nucléiques codant pour ces derniers.
PCT/US2017/050803 2016-09-09 2017-09-08 Virus oncolytique équipé de molécules d'engagement bispécifiques WO2018049248A1 (fr)

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