WO2022243261A1 - Molécules de liaison à l'antigène cd40 agonistes ciblant cea - Google Patents

Molécules de liaison à l'antigène cd40 agonistes ciblant cea Download PDF

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WO2022243261A1
WO2022243261A1 PCT/EP2022/063226 EP2022063226W WO2022243261A1 WO 2022243261 A1 WO2022243261 A1 WO 2022243261A1 EP 2022063226 W EP2022063226 W EP 2022063226W WO 2022243261 A1 WO2022243261 A1 WO 2022243261A1
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seq
amino acid
acid sequence
antigen binding
bispecific antigen
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PCT/EP2022/063226
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Harald Duerr
Moritz RAPP
Eva Carina SUM
Christine TRUMPFHELLER
Pablo Umaña
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication of WO2022243261A1 publication Critical patent/WO2022243261A1/fr

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    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the invention relates to new bispecific antigen binding molecules, comprising at least one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA), at least two Fab fragments capable of specific binding to CD40, and Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function. Further aspects of the invention are methods of producing these molecules and methods of using the same.
  • CEA carcinoembryonic antigen
  • Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function.
  • TCR T-cell antigen receptor
  • APCs antigen-presenting cells
  • TNFR family costimulators show promise for several therapeutic applications in multiple fields including cancer, infectious disease, transplantation, and autoimmunity.
  • the TNFR family member CD40 plays a key role in triggering immune responses by inducing maturation, survival, antigen presentation, cytokine production, and expression of costimulatory molecules of APCs,
  • CD40 receptor plays a crucial role for activating dendritic cells (DCs) and enhancing their T cell (cross-) priming capabilities (R. French et al., Nat Med. 1999, 5(5), 548-53; E. Sotomayor et al., Nat Med 1999, 5(7), 780-7).
  • DCs dendritic cells
  • cross- cross- priming capabilities
  • CD40 agonistic antibodies were shown to possess a short serum half-life (R. Vonderheide et al., Oncoimmunology 2013, 2:e23033). Both observations can presumably be attributed to the broad CD40 receptor expression on a wide range of hematopoietic and non-hematopoietic cells.
  • the available pre-clinical and clinical data clearly demonstrate that there is a high clinical need for effective agonists of CD40 that are able to induce and enhance effective endogenous immune responses to cancer.
  • CD40 antibodies can only be administered in relatively low doses due to dose-limiting toxicities such as cytokine release syndrome and thrombocyte/ endothelial cell activation, resulting in an insufficient activation of the pathway on target APCs and a narrow therapeutic index.
  • FAP-CD40 selectively induces CD40 agonism in the presence of the prolyl endopeptidase fibroblast activation protein a (FAP), which is expressed in the tumor stroma on cancer-associated fibroblasts (CAFs), but not in healthy adult tissues.
  • FAP prolyl endopeptidase fibroblast activation protein a
  • CAFs cancer-associated fibroblasts
  • Tumor stroma-associated FAP expression can be observed in the vast majority of carcinomas (W. Brennen et al., Mol Cancer Ther 2012, 11(2), 257-66).
  • tumor indications such as gastric adenocarcinoma or colorectal cancer, in which a substantial fraction of tumors can be classified as “stroma-poor” (S. Zunder et al., Cellular Oncology 2019, 42(5), 717-725; N. Kemi et al., Br J Cancer 2018, 119(4), 435-439).
  • stroma-poor S. Zunder et al., Cellular Oncology 2019, 42(5), 717-725; N. Kemi et al., Br J Cancer 2018, 119(4), 435-439.
  • CEA carcinoembryonic antigen
  • the use of a CEA-targeted CD40 agonistic antigen binding molecule may be beneficial.
  • the invention relates to new bispecific antigen binding molecules capable of specific binding to CD40 and carcinoembryonic antigen (CEA) and thus combine a moiety capable of binding to CEA with a moiety capable of agonistic binding to CD40, wherein the activation of APCs through CD40 is provided by cross-linking through CEA expressed on tumor cells.
  • the bispecific antigen binding molecules are capable of specific binding to CD40 and CEA (CEA-CD40) and thus are able to bind DCs and CEA-expressing tumor- derived material simultaneously and may thereby facilitate the delivery of tumor antigen to DCs via the CD40 receptor and increase tumor-specific T cell priming.
  • the bispecific antigen binding molecules of the invention increase the delivery of tumor-derived CEA-expressing extracellular vesicles (EVs) to DCs, consequently enhancing cross-priming of T cells against antigen contained in the EVs.
  • EVs extracellular vesicles
  • the bispecific antigen binding molecules of this invention may thus be able to trigger the CD40 receptor not only effectively, but also very selectively at the CEA-expressing tumor site while overcoming the need for FcyR cross-linking and thereby reducing side effects which leads to a favorable safety profile and high potency.
  • the bispecific antigen binding molecules of the invention are thus able to activate CD40 exclusively in presence of its tumor target CEA.
  • CEA is known to be highly expressed by many tumor types including breast cancer, colorectal carcinoma or pancreatic carcinoma (S. Hammarstrom, Semin Cancer Biol 1999, 9(2), 67-81). Importantly, CEA expression in non-malignant tissues is low and mainly restricted to sites that are not well accessible for systemically administered therapeutic antibodies such as the apical surface of the lung and intestinal epithelium.
  • the present invention relates to bispecific antigen binding molecules comprising
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, and (c) one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA) connected to the C-terminus of the Fc region.
  • CEA carcinoembryonic antigen
  • the invention provides a bispecific antigen binding molecule, comprising
  • VHCD40 heavy chain variable region
  • CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1
  • CDR-H2 comprising the amino acid sequence of SEQ ID NO:2
  • CDR-H3 comprising the amino acid sequence of SEQ ID NO:3
  • VLCD40 light chain variable region
  • the bispecific antigen binding molecule comprises at least two Fab fragments capable of specific binding to CD40 each comprising (i) a heavy chain variable region (VHCD40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9 and SEQ ID NO: 10, and
  • VLCD40 light chain variable region
  • the antigen binding domains capable of specific binding to CD40 comprise
  • VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO: 11, or
  • the antigen binding domains capable of specific binding to CD40 comprise
  • the bispecific antigen binding molecule comprises at least two Fab fragments capable of specific binding to CD40 each comprising a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO: 11.
  • the Fab fragments capable of specific binding to CD40 bind to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:57.
  • Fab fragments capable of specific binding to CD40 comprise (i) a heavy chain variable region (VHCD40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, and (ii) a light chain variable region (VLCD40) comprising the amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • the bispecific antigen binding molecule as defined herein before comprises at least two Fab fragments capable of specific binding to CD40 comprising
  • VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO:22, or
  • VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO:23, or
  • the Fab frgments capable of specific binding to CD40 comprise a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO:21 or wherein the antigen binding domain capable of specific binding to CD40 comprises a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO:21.
  • a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to CD40 bind to murine CD40 and comprise (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:27, and a light chain variable region (VLCD40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:30.
  • the Fab fragments capable of specific binding to CD40 each comprise a VH comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:32.
  • bispecific antigen binding molecule as defined herein before, wherein the antigen binding domain capable of specific binding to CEA comprises
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • a bispecific antigen binding molecule comprising a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:39 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:40 or a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:47 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:48.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region comprising an amino acid sequence of SEQ ID NO:47
  • VLCEA light chain variable region
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:39 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:40.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • a bispecific antigen binding molecule as defined herein before, wherein the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:51, and a light chain variable region (VLCEA) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:55 and a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:56.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • bispecific antigen binding molecule comprising
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • one antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:39 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:40 or a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:47 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:48.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region comprising an amino acid sequence of SEQ ID NO:47
  • VLCEA light chain variable region
  • the bispecific antigen binding molecule is a humanized or a chimeric antibody.
  • the bispecific antigen binding molecule comprises an IgG Fc region, particularly an IgGl Fc region or an IgG4 Fc region.
  • the Fc region comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor and/or effector function.
  • a bispecific antigen binding molecule as defined herein before, wherein the first subunit of the Fc region comprises knobs and the second subunit of the Fc region comprises holes according to the knobs into holes method.
  • the first subunit of the Fc region comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc region comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index), or (ii) the first subunit of the Fc region comprises the amino acid substitutions K392D and K409D (numbering according to Kabat EU index) and the second subunit of the Fc region comprises the amino acid substitutions E356K and D399K (numbering according to Kabat EU index).
  • a bispecific antigen binding molecule wherein the first subunit of the Fc region comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc region comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
  • bispecific antigen binding molecule that comprises bivalent binding towards CD40 and monovalent binding towards CEA (2+1 format).
  • bispecific antigen binding molecule as described herein before, wherein the bispecific antigen binding molecule comprises
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the two Fab fragments capable of specific binding to CD40 fused to the N-termini of the Fc region, and
  • the antigen binding domain capable of specific binding to CEA connected to the C-terminus of the Fc region is a cross-fab fragment.
  • a bispecific antigen binding molecule wherein the bispecific antigen binding molecule comprises
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the two Fab fragments capable of specific binding to CD40 fused to the N-termini of the Fc region, and
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule consists of
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the first Fab fragment (aa) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the first subunit and the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the second subunit, and
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule comprises
  • each heavy chain comprising a VH and CHI domain of a Fab fragment capable of specific binding to CD40, and a Fc region subunit comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40
  • the Fab fragment capable of specific binding to CEA is a cross-Fab fragment comprising a VL-CH1 chain and a VH-Ckappa chain, and wherein the VH- Ckappa chain or the VL-CH1 chain is connected to the C-terminus of one of the two heavy chains of (i).
  • a bispecific antigen binding molecule comprising a cross-fab fragment capable specific binding to CEA, wherein the VH-Ckappa chain is fused to the C-terminus of one of the two heavy chains of (i).
  • a bispecific antigen binding molecule comprising a cross-fab fragment capable specific binding to CEA, wherein the VL-CH1 chain is fused to the C-terminus of one of the two heavy chains of (i).
  • a bispecific antigen binding molecule that comprises trivalent binding towards CD40 and monovalent binding towards CEA (3+1 format).
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule consists of
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the first Fab fragment (aa) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the first subunit and wherein the third Fab fragment (ac) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the second Fab fragment (ab), which is in turn fused at its C-terminus to the N-terminus of the second subunit of the Fc, and
  • bispecific antigen binding molecule that comprises tetravalent binding towards CD40 and monovalent binding towards CEA (4+1 format).
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule consists of
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function
  • the third Fab fragment (ac) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment (aa), which is in turn fused at its C-terminus to the N-terminus of the first subunit of the Fc region
  • the fourth Fab fragment (ad) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the second Fab fragment (ab), which is in turn fused at its C-terminus to the N-terminus of the second subunit of the Fc
  • isolated nucleic acid encoding a bispecific antigen binding molecule as described herein before.
  • the invention further provides a vector, particularly an expression vector, comprising the isolated nucleic acid of the invention and a host cell comprising the isolated nucleic acid or the expression vector of the invention.
  • the host cell is a eukaryotic cell, particularly a mammalian cell.
  • a method of producing a bispecific antigen binding molecule or an antibody as described herein before comprising culturing the host cell as described above under conditions suitable for the expression of the bispecific antigen binding molecule, and isolating the bispecific antigen binding molecule.
  • the invention also encompasses the bispecific antigen binding molecule that specifically binds to CD40 and to CEA produced by the method of the invention.
  • the invention further provides a pharmaceutical composition comprising a bispecific antigen binding molecule as described herein before or the antibody as described herein before and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an additional therapeutic agent.
  • bispecific antigen binding molecule or the antibody as described herein before or the pharmaceutical composition comprising the bispecific antigen binding molecule, for use as a medicament.
  • a bispecific antigen binding molecule as described herein before or the pharmaceutical composition of the invention, for use
  • the bispecific antigen binding molecule as described herein before or the pharmaceutical composition of the invention for use in treating or delaying the progression of cancer.
  • the invention provides the bispecific antigen binding molecule as described herein before for use in the treatment of cancer, wherein the bispecific antigen binding molecule is administered in combination with a chemotherapeutic agent, radiation and/ or other agents for use in cancer immunotherapy.
  • the bispecific antigen binding molecule as described herein is for use in the treatment of cancer, wherein the bispecific antigen binding molecule is for administration in combination with an agent blocking PD-Ll/PD-1 interaction.
  • the bispecific antigen binding molecule as described herein before or the pharmaceutical composition of the invention for use in up-regulating or prolonging cytotoxic T cell activity.
  • the invention provides a method of inhibiting the growth of tumor cells in an individual comprising administering to the individual an effective amount of the bispecific antigen binding molecule as described herein before, or the pharmaceutical composition of the invention, to inhibit the growth of the tumor cells.
  • the invention provides a method of treating or delaying cancer in an individual comprising administering to the individual an effective amount of the bispecific antigen binding molecule as described herein before, or the pharmaceutical composition of the invention.
  • the bispecific antigen binding molecule as described herein before for the manufacture of a medicament for the treatment of a disease in an individual in need thereof, in particular for the manufacture of a medicament for the treatment of cancer, as well as a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form.
  • the disease is cancer.
  • the individual is a mammal, particularly a human.
  • Figures 1A to ID, 1G and 1H show schematic representations of the bispecific antigen binding molecules, which specifically bind to CD40 and to human CEA (huCEA).
  • the molecules are of the human IgGl subclass and contain the PGLALA mutation in their Fc regions for abrogation of Fey receptor binding.
  • Figure IE to IF show the FAP-targeted human CD40 agonist FAP-CD40 and the non-targeted control molecule DP47-CD40 that were used for comparison to the CEA-CD40 molecules.
  • 1A shows a schematic representation of a bispecific CEA-CD40 antibody (0817-A5H1EL1D xFab) in the 2+1 format consisting of two human CD40 binding Fab domains combined with the huCEA binding Fab domain A5H1EL1D fused at the C-terminus of one of the heavy chains (bivalent for human CD40 and monovalent for huCEA).
  • the black point symbolizes knob- into-hole mutations.
  • IB shows a schematic representation of a bispecific CEA-CD40 antibody (FGK4.5-A5B7 xFab) in the 2+1 format consisting of two murine CD40 binding Fab domains combined with the huCEA binding Fab domain A5B7 fused at the C- terminus of one of the heavy chains (bivalent for murine CD40 and monovalent for huCEA).
  • the black point symbolizes knob-into-hole mutations.
  • 1C shows a schematic representation of a bispecific CEA-CD40 antibody (0817-T84.66 xFab) in the 2+1 format consisting of two human CD40 binding Fab domains combined with the huCEA binding Fab domain T84.66 fused at the C-terminus of one of the heavy chains (bivalent for human CD40 and monovalent for human CEA).
  • the black point symbolizes knob-into-hole mutations.
  • ID shows a schematic representation of a bispecific CEA-CD40 antibody (FGK4.5-T84.66 xFab) in the 2+1 format consisting of two murine CD40 binding Fab domains combined with the huCEA binding Fab domain T84.66 fused at the C-terminus of one of the heavy chains (bivalent for murine CD40 and monovalent for huCEA).
  • the black point symbolizes knob-into-hole mutations.
  • IE shows a schematic representation of a bispecific FAP-CD40 antibody (0817-FAP xFab) in the 2+1 format consisting of two human CD40 binding Fab domains combined with the FAP binding Fab domain 28H1 fused at the C-terminus of one of the heavy chains (bivalent for human CD40 and monovalent for FAP).
  • the black point symbolizes knob-into-hole mutations.
  • Fig. IF shows a schematic representation of a bispecific DP47-CD40 antibody (0817-DP47 xFab) in the 2+1 format consisting of two human CD40 binding Fab domains combined with the DP47 Fab domain (non-binding domain) fused at the C-terminus of one of the heavy chains.
  • Fig. 1G shows a schematic representation of a bispecific FAP-CD40 antibody in the 3+1 format consisting of three human CD40 binding Fab domains combined with a huCEA binding xFab domain fused at the C-terminus of one of the heavy chains (trivalent for human CD40 and monovalent for huCEA).
  • the black point symbolizes knob-into-hole mutations.
  • Fig. 1G shows a schematic representation of a bispecific FAP-CD40 antibody in the 3+1 format consisting of three human CD40 binding Fab domains combined with a huCEA binding xFab domain fused at the C-terminus of one of the heavy chains (trivalent for human CD40 and monovalent for huCEA).
  • the black point symbolizes knob-into-hole mutations.
  • Fig. 1G shows a schematic representation of a bispecific FAP-CD40 antibody in the 3+1 format consisting of three human CD40 binding Fab domains combined with a huCEA binding xFab domain
  • 1H shows a schematic representation of a bispecific FAP-CD40 antibody in the 4+1 format consisting of four human CD40 binding Fab domains combined with a huCEA binding xFab domain fused at the C-terminus of one of the heavy chains (tetravalent for human CD40 and monovalent for huCEA).
  • the black point symbolizes knob-into-hole mutations.
  • Figures 2A to 2P show the binding of CEA-targeted anti-human CD40 antibodies to human CEA (Fig. 2A to 2H) and to human CD40 (Fig. 21 to 2P).
  • Fig. 2A CEA-CD40 0817-A5H1EL1D
  • Fig. 2B CEA-CD40 0817-T84.66
  • Fig. 2C FAP-CD40 0817-28H1
  • Fig. 2D DP47-CD40 0817-DP47
  • the CEA-CD40 construct with the T84.66 binding domain shows stronger huCEA binding than the CEA-CD40 construct with the A5H1EL1D anti-huCEA domain (Fig. 2B vs. Fig. 2A). In contrast, no binding of the CEA-targeted anti-CD40 antibodies to MC38 cells was detected.
  • Fig. 21 CEA-CD400817-A5H1EL1D
  • Fig. 2J CEA-CD400817- T84.66
  • Fig. 2K FAP-CD40 0817-28H1
  • Fig. 21 CEA-CD400817-A5H1EL1D
  • Fig. 2J CEA-CD400817- T84.66
  • Fig. 2K FAP-CD40 0817-28H1
  • DP47-CD40 0817-DP47 show binding to splenic DCs isolated from human CD40 transgenic (huCD40tg) mice (similar expression pattern of human CD40 and mouse CD40 receptor).
  • Fig. 2M CEA-CD40 0817-A5H1EL1D
  • Fig. 2N CEA-CD400817-T84.66
  • Fig. 20 FAP-CD400817-28H1
  • Fig. 2P DP47-CD400817-DP47
  • the two CEA-targeted anti-human CD40 antibodies show similar binding to human CD40, irrespective of their anti-huCEA binding domains.
  • the binding of the non-CEA targeted control molecule DP47- CD40 and FAP-CD40 to human CD40 was comparable to that of the CEA-CD40 molecules.
  • No binding of the CEA-targeted anti-human CD40 antibodies to DCs from wild type mice was detected.
  • Shown are mean (SD) values of technical duplicates of median fluorescence intensity (MFI) values of phycoerythrin (PE)-labeled anti -human IgG Fey fragment-specific goat IgG F(ab' )2 fragment which was used as secondary detection antibody.
  • MFI was measured by flow cytometry. For the curve fit nonlinear least squares regression using a variable slope model was applied. The x-axis shows the concentration of antibody constructs.
  • Figures 3A to 3D show the in vitro activation of huCD40tg DCs by CEA-targeted anti -human CD40 and anti -mouse CD40 constructs in the presence of huCEA-coated or uncoated Dynabeads ® after 24 hours.
  • the CEA-CD40 antibodies induced an increase of DC activation marker expression (CD86, CD70, CD80).
  • the CEA-targeted anti-human CD40 molecules induced a stronger activation marker increase compared to the CEA-targeted anti-murine CD40 antibodies (Fig. 3A to 3C).
  • FIG. 4 shows the T cell priming of Ovalbumin (OVA)-pulsed huCD40tg DCs activated by the CEA-CD40 molecule 0817-A5H1EL1D.
  • DCs isolated from huCD40tg mice, treated with DEC205-OVA conjugate and stimulated with 0817-A5H1EL1D as well as huCEA-coated beads induced an increase in the number of OVA-specific T cells.
  • huCEA uncoated beads
  • no T cell proliferation was induced by DCs stimulated with the CEA-CD40 molecule.
  • Figures 5A and 5B show the enhanced delivery of huCEA + beads or huCEA + extracellular vesicles (EVs) to DCs mediated by CEA-CD40.
  • Fig. 5A shows confocal images of MuTu DC Is, a green fluorescent protein + murine DC cell line derived from CD8 + splenic DC tumors, incubated with 3 nM of the CEA-targeted anti-murine CD40 antibody FGK4.5-A5B7 and CEA-coated red fluorescent beads (left image) or non-coated red fluorescent beads (right image).
  • CEA-CD40 delivery of huCEA-coated beads to MuTu DC Is was enhanced compared to uncoated beads.
  • Fig. 5A shows confocal images of MuTu DC Is, a green fluorescent protein + murine DC cell line derived from CD8 + splenic DC tumors, incubated with 3 nM of the CEA-targeted anti-murine CD
  • 5B shows the signal of Alexa Fluor 555 (AF555)-labelled tumor-derived CEA + EVs or non-CEA-expressing EVs in splenic huCD40tg DCs that have been incubated with EVs and either 10 nM of CEA-CD40 (0817-A5H1EL1D) or FAP-CD40.
  • AF555 Alexa Fluor 555
  • the tumor-derived EVs were isolated from in vitro cultures of MC38 tumor cells expressing huCEA and OVA (MC38-huCEA-OVA) that were transfected with either Cas9 protein and scrambled control guide RNA (CEA + EVs) or with Cas9 and a guide RNA targeted against huCEA in order to disrupt CEA expression (CEA K0 EVs).
  • CEA-CD40 significantly enhanced delivery of huCEA + EVs to DCs in comparison to FAP-CD40. This effect could not be observed when EVs were lacking huCEA expression (Fig 5B).
  • Fig. 5B mean (SD) AF555 MFI values of viable CD1 lc + DCs measured by flow cytometry are shown.
  • Figures 6A to 6H show the effects of enhanced delivery of huCEA + EVs mediated by CEA-CD40 on OVA-specific OT-1 T cell priming.
  • Fig. 6A to 6D show priming of carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-1 T cells that were co-cultured with huCD40tg DCs pre-incubated with 10 nM CEA-CD40 (0817-A5H1EL1D ) or FAP- CD40 and huCEA + tumor-derived EVs (obtained from MC38-huCEA-OVA tumor cells).
  • CFSE carboxyfluorescein succinimidyl ester
  • T cell proliferation and activation were measured by quantifying OT-1 T cell numbers, CFSE dilution and PD-1 as well as CD25 expression of OT-1 T cells.
  • T cell priming was significantly enhanced compared to conditions in which FAP-CD40 and huCEA + EVs were added.
  • Shown are total number of CD8 + CD3 + OT-1 T cells (Fig. 6A), fraction of proliferated (CFSE low ) CD8 + CD3 + OT-1 T cells (Fig. 6B), MFI of CD25 (Fig. 6C) and PD-1 (Fig. 6D) on viable CD8 + CD3 + OT-1 T cells measured by flow cytometry.
  • Fig. 6A total number of CD8 + CD3 + OT-1 T cells
  • Fig. 6B fraction of proliferated CD8 + CD3 + OT-1 T cells
  • MFI of CD25 Fig. 6C
  • PD-1 Fig. 6D
  • 6F to 6H show that increased T cell priming in CEA-CD40 (0817- A5H1EL1D) + huCEA + EV conditions does not result from enhanced DC activation.
  • the MFI of DC activation markers CD86, CD70 and murine CD40 were not altered in conditions with huCEA + EVs and CEA-CD40 versus untreated or huCEA + EV + FAP- CD40-treated conditions.
  • DCs were activated by adding CEA-CD40 artificially cross-linked with an anti-human Fc F(ab’)2 fragment. Shown are DC MFI values of the activation markers CD86, CD70 and CD40 measured by flow cytometry. For Fig.
  • Fig. 7A shows a scheme of the experimental setup of the human cross-priming assay as described in Example 3.5.
  • Human DC Is were differentiated and sorted from human cord blood stem cells.
  • Human MV3-CEA melanoma cells were pulsed with the short MART-1 peptide.
  • CEA-CD40 (0817-A5H1EL1D) or FAP-CD40 and EVs derived from MV3-CEA-MART-1 tumor cells were incubated with the DC Is for 3 hours.
  • MART-1 luciferase reporter T cells expressing the MART- 1 -specific TCR DMF5 and a luciferase gene under control of the IL-2 promoter, were added to DC Is. TCR activation and concomitant IL-2 signaling were measured after 6 hours by bioluminescence assay.
  • FIG. 7B shows the increase in IL-2 signaling of MART- 1 -specific T cells cocultured with DCs treated with MV3-CEA-MART-1 -derived EVs (+ CEA+ EVs) or without EVs (- EVs) and CEA-CD40 (0817-A5H1EL1D) or FAP-CD40. Results were normalized to the corresponding untreated conditions in which no antibody was added. Shown are mean and SD values of technical triplicates. Statistical significance was calculated using unpaired twotailed t-test. Results are representative of three experiments. In Fig. 7C the expression profile of the representative DC activation markers CD70, CD80 and CD86 after 9 hours is shown.
  • Cord blood-derived DC Is were incubated with CEA+ EVs and targeted CD40 agonists as described herein before. As positive control, DCs were incubated with poly(TC). Results were confirmed using two different cord blood donors.
  • CEA carcinoembryonic antigen
  • DC dendritic cell
  • EV extracellular vesicle
  • FAP fibroblast activation protein
  • antigen binding molecule refers in its broadest sense to a molecule that specifically binds an antigenic determinant.
  • antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.
  • CEA or "moiety capable of specific binding to CEA” refers to a polypeptide molecule that specifically binds to the target cell antigen CEA.
  • the antigen binding domain is able to activate signaling through its target cell antigen.
  • the antigen binding domain is able to direct the entity to which it is attached (e.g. the CD40 agonist) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant.
  • Antigen binding domains capable of specific binding to CEA include antibodies and fragments thereof as further defined herein.
  • antigen binding domains capable of specific binding to a target cell antigen include scaffold antigen binding proteins as further defined herein, e.g. binding domains which are based on designed repeat proteins or designed repeat domains (see e.g. WO 2002/020565).
  • the term "antigen binding domain capable of specific binding to a target cell antigen” refers to the part of the molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen.
  • An antigen binding domain capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also called antibody variable regions).
  • an antigen binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the "antigen binding domain capable of specific binding to a target cell antigen” can also be a Fab fragment or a cross-Fab fragment.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • 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 and/or bind the same epitope, except for possible variant antibodies, e.g. containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • bispecific antibody denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.
  • bispecific means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants.
  • a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
  • a bispecific antigen binding molecule as described herein can also form part of a multispecific antibody.
  • valent as used within the current application denotes the presence of a specified number of binding sites specific for one distinct antigenic determinant in an antigen binding molecule that are specific for one distinct antigenic determinant.
  • bivalent tetravalent
  • hexavalent denote the presence of two binding sites, four binding sites, and six binding sites specific for a certain antigenic determinant, respectively, in an antigen binding molecule.
  • the bispecific antigen binding molecules according to the invention can be monovalent for a certain antigenic determinant, meaning that they have only one binding site for said antigenic determinant or they can be bivalent or tetravalent for a certain antigenic determinant, meaning that they have two binding sites or four binding sites, respectively, for said antigenic determinant.
  • full length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region.
  • each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region.
  • the heavy chain of an antibody may be assigned to one of five types, called a (IgA), d (IgD), e (IgE), g (IgG), or m (IgM), some of which may be further divided into subtypes, e.g. g ⁇ (IgGl), g2 (IgG2), g3 (IgG3), g4 (IgG4), al (IgAl) and a2 (IgA2).
  • the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and single domain antibodies.
  • scFv single domain antibodies.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, 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).
  • Single-domain antibodies are antibody fragments comprising ah or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain.
  • Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteins from the antibody hinge region.
  • Fab’-SH are Fab’ fragments wherein the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region. According to the present invention, the term “Fab fragment” also includes “cross-Fab fragments” or “crossover Fab fragments” as defined below.
  • cross-Fab fragment or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.
  • Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL).
  • This crossover Fab molecule is also referred to as CrossFab (VLVH>.
  • the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI).
  • This crossover Fab molecule is also referred to as CrossFab (CLCHI).
  • a “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CHl -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL-CHl -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.
  • Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain.
  • these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
  • a “crossover single chain Fab fragment” or “x-scFab” is a is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CHl and b) VL-CH1 -linker- VH-CL; wherein VH and VL form together an antigen -binding site which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids.
  • these x-scFab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
  • a “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N- terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96).
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.
  • fibronectin and designed ankyrin repeat proteins have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et ak, Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008).
  • a scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), a serum transferrin (/ra//.s-body); a designed ankyrin repeat protein (DARPin), a variable domain of antibody light chain or heavy chain (single-domain antibody, sdAb), a variable domain of antibody heavy chain (nanobody, aVH), VNAR fragments, a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), a human gamma- crystallin or ubiquitin (Affilin molecules); a kunitz type domain of human protease inhibitors, microbodies such
  • CTLA-4 Cytotoxic T Lymphocyte-associated Antigen 4
  • CTLA-4 is a CD28-family receptor expressed on mainly CD4 + T-cells. Its extracellular domain has a variable domain- like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties.
  • CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies (e.g. US7166697B1). Evibodies are around the same size as the isolated variable region of an antibody (e.g. a domain antibody). For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001).
  • Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633.
  • An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen.
  • the domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818A1. Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulfide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). A transferrin is a monomeric serum transport glycoprotein.
  • Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop.
  • engineered transferrin scaffolds include the Trans-body.
  • Designed Ankyrin Repeat Proteins are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton.
  • a single ankyrin repeat is a 33 residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat.
  • a single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain.
  • the first single domains were derived from the variable domain of the antibody heavy chain from camelids (nanobodies or VHH fragments).
  • the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.
  • Fibronectin is a scaffold which can be engineered to bind to antigen.
  • Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the .beta. -sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest.
  • Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther.
  • Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and conotoxin and knottins.
  • the microproteins have a loop which can beengineered to include upto 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see W02008098796.
  • an “antigen binding molecule that binds to the same epitope” as a reference molecule refers to an antigen binding molecule that blocks binding of the reference molecule to its antigen in a competition assay by 50% or more, and conversely, the reference molecule blocks binding of the antigen binding molecule to its antigen in a competition assay by 50% or more.
  • An “antigen binding molecule that does not bind to the same epitope” as a reference molecule refers to an antigen binding molecule that does not block binding of the reference molecule to its antigen in a competition assay by 50% or more, and conversely, the reference molecule does not block binding of the antigen binding molecule to its antigen in a competition assay by 50% or more.
  • an antigen binding domain or “antigen -binding site” refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions).
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • antigenic determinant is synonymous with “antigen” and “epitope,” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex.
  • Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the proteins useful as antigens herein can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the antigen is a human protein.
  • the term encompasses the “full-length”, unprocessed protein as well as any form of the protein that results from processing in the cell.
  • the term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants.
  • ELISA enzyme- linked immunosorbent assay
  • SPR Surface Plasmon Resonance
  • the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g. by SPR.
  • an molecule that binds to the antigen has a dissociation constant (Kd) of ⁇ 1 mM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 8 M or less, e.g. from 10 8 M to 10 13 M, e.g. from 10 9 M to 10 13 M).
  • Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (kofif and kon, respectively).
  • Kd dissociation constant
  • equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).
  • an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • a “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, in particular a target cell in a tumor such as a cancer cell or a cell of the tumor stroma.
  • the target cell antigen is a tumor-associated antigen.
  • the tumor target cell antigen is Carcinoembryonic Antigen (CEA).
  • Carcinoembroynic antigen also known as Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the amino acid sequence of human CEA is shown in UniProt accession no. P06731 (version 151, SEQ ID NO:59).
  • CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein N.
  • CEA has now been identified in several normal adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urogential tracts, and cells of colon, cervix, sweat glands, and prostate (Nap et ah, Tumour Biol., 9(2-3): 145- 53, 1988; Nap et ah, Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelial origin, as well as their metastases, contain CEA as a tumor associated antigen.
  • CEA While the presence of CEA itself does not indicate transformation to a cancerous cell, the distribution of CEA is indicative.
  • CEA is generally expressed on the apical surface of the cell (Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)), making it inaccessible to antibody in the blood stream.
  • CEA tends to be expressed over the entire surface of cancerous cells (Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)). This change of expression pattern makes CEA accessible to antibody binding in cancerous cells.
  • CEA expression increases in cancerous cells.
  • CEA expression promotes increased intercellular adhesions, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003).
  • CRC colorectal carcinoma
  • NSCLC non-small cell lung cancer
  • HER3 non-small cell lung cancer
  • CEA is readily cleaved from the cell surface and shed into the blood stream from tumors, either directly or via the lymphatics. Because of this property, the level of serum CEA has been used as a clinical marker for diagnosis of cancers and screening for recurrence of cancers, particularly colorectal cancer (Goldenberg D M., The International Journal of Biological Markers, 7:183-188, 1992; Chau L, et al., J Clin Oncol., 22:1420- 1429, 2004; Flamini et al., Clin Cancer Res; 12(23):6985-6988, 2006).
  • anti-CEA antigen binding molecule or “antigen binding molecule capable of specific binding to CEA” refers to an antigen binding molecule that is capable of binding to CEA with sufficient affinity such that the antigen binding molecule is useful as a diagnostic and/or therapeutic agent in targeting CEA.
  • the antigen binding molecule includes but is not limited to, antibodies, Fab molecules, crossover Fab molecules, single chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, and VH and scaffold antigen binding protein.
  • an antigen binding molecule that is capable of specific binding to CEA has a dissociation constant (K d ) of ⁇ 1 mM, ⁇ 500 nM, ⁇ 200 nM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 8 M or less, e.g.
  • an anti-CEA antigen binding molecule binds to CEA from different species. In certain aspects, the anti-CEA antigen binding molecule binds to human and cynomolgus CEA. In further certain aspects, an anti-CEA antigen binding molecule binds to the A2 domain of hu CEA which consists of the amino acid sequence of SEQ ID NO:60. In another certain aspects, an anti-CEA antigen binding molecule binds to the Al domain of hu CEA which consists of the amino acid sequence of SEQ ID NO:61.
  • FAP Fibroblast activation protein
  • Prolyl endopeptidase FAP or Seprase refers to any native FAP from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the term encompasses “full-length,” unprocessed FAP as well as any form of FAP that results from processing in the cell.
  • the term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants.
  • the antigen binding molecule of the invention is capable of specific binding to human, mouse and/or cynomolgus FAP.
  • the amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version 149, SEQ ID NO:62), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2.
  • the extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760.
  • the amino acid sequence of a His-tagged human FAP ECD is shown in SEQ ID NOs 63.
  • the amino acid sequence of mouse FAP is shown in UniProt accession no.
  • mouse FAP (version 126, SEQ ID NO:64), or NCBI RefSeq NP 032012.1.
  • the extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761.
  • SEQ ID NO:65 shows the amino acid of a His-tagged mouse FAP ECD.
  • SEQ ID NO:66 shows the amino acid of a His-tagged cynomolgus FAP ECD.
  • an anti-FAP binding molecule of the invention binds to the extracellular domain of FAP.
  • variable region or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen.
  • variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).
  • CDRs complementarity determining regions
  • antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3).
  • Exemplary CDRs herein include:
  • the CDRs are determined according to Rabat et al., supra.
  • One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra , McCallum, supra , or any other scientifically accepted nomenclature system.
  • “Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs).
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR- H2(CDR-L2)-FR3 - CDR-H3 (CDR-L3 )-FR4.
  • 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.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m respectively..
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non -human antibody refers to an antibody that has undergone humanization.
  • Other forms of "humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
  • CHI domain denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 118 to EU position 215 (EU numbering system according to Rabat).
  • a CHI domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 67).
  • a segment having the amino acid sequence of EPKSC (SEQ ID NO: 163) is following to link the CHI domain to the hinge region, but in some cases where the antigen binding molecule comprises a CHI domain with a free C-terminal end (for example in a cross-fab fragment) a variant of the segment consisting of for example the amino acid sequence EPKSCD (SEQ ID NO:69) or EPKSCS (SEQ ID NO:70) may be included.
  • hinge region denotes the part of an antibody heavy chain polypeptide that joins in a wild-type antibody heavy chain the CHI domain and the CH2 domain, e. g. from about position 216 to about position 230 according to the EU number system of Kabat, or from about position 226 to about position 230 according to the EU number system of Kabat.
  • the hinge regions of other IgG subclasses can be determined by aligning with the hinge-region cysteine residues of the IgGl subclass sequence.
  • the hinge region is normally a dimeric molecule consisting of two polypeptides with identical amino acid sequence.
  • the hinge region generally comprises up to 25 amino acid residues and is flexible allowing the associated target binding sites to move independently.
  • the hinge region can be subdivided into three domains: the upper, the middle, and the lower hinge domain (see e.g. Roux, et al., J. Immunol. 161 (1998) 4083).
  • the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 71), wherein X is either S or P.
  • the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 72), wherein X is either S or P.
  • the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 73), wherein X is either S or P.
  • Fc domain or “Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain.
  • the “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. (EU numbering system according to Kabat).
  • a CH2 domain has the amino acid sequence of APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 74).
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc- region. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206.
  • a carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain.
  • the “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 according to EU numbering system according to Kabat of an IgG).
  • the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 75).
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • wild-type Fc domain denotes an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include a native human IgGl Fc-region (non- A and A allotypes), native human IgG2 Fc- region, native human IgG3 Fc-region, and native human IgG4 Fc-region as well as naturally occurring variants thereof.
  • Wild-type Fc-regions are denoted in SEQ ID NO:76 (IgGl, Caucasian allotype), SEQ ID NO:77 (IgGl, afroamerican allotype), SEQ ID NO:78 (IgG2), SEQ ID NO:79 (IgG3) and SEQ ID NO:80 (IgG4).
  • the term “variant (human) Fc domain” denotes an amino acid sequence which differs from that of a “wild-type” (human) Fc domain amino acid sequence by virtue of at least one “amino acid mutation”.
  • the variant Fc-region has at least one amino acid mutation compared to a native Fc- region, e.g.
  • the (variant) Fc-region has at least about 95 % homology with a wild-type Fc-region.
  • the “knob-into-hole” technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain
  • the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain.
  • the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C
  • the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C.
  • a "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity).
  • one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function.
  • Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et ak, Science 247:1306-10 (1990)).
  • effector function refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
  • Fc receptor binding dependent effector functions can be mediated by the interaction of the Fc-region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells.
  • Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) (see e.g. Van de Winkel, J.G. and Anderson, C.L., J. Leukoc. Biol. 49 (1991) 511-524).
  • ADCC antibody dependent cell mediated cytotoxicity
  • FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcyR. Fc receptor binding is described e.g. in Ravetch, J. V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492, Capel, P.J., et ak, Immunomethods 4 (1994) 25-34; de Haas, M., et ak, J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J.E., et ak, Ann. Hematok 76 (1998) 231-248.
  • FcyR Fc-region of IgG antibodies
  • FcyRI binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils.
  • Modification in the Fc-region IgG at least at one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to EU index of Kabat) reduce binding to FcyRI.
  • FcyRIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process.
  • FcyRIIB seems to play a role in inhibitory processes and is found on B cells, macrophages and on mast cells and eosinophils. On B-cells it seems to function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class.
  • FcyRIIB acts to inhibit phagocytosis as mediated through FcyRIIA.
  • the B-form may help to suppress activation of these cells through IgE binding to its separate receptor.
  • Reduced binding for FcyRIIA is found e.g. for antibodies comprising an IgG Fc-region with mutations at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to EU index of Kabat).
  • FcyRIII (CD 16) binds IgG with medium to low affinity and exists as two types.
  • FcyRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC.
  • FcyRIIIB is highly expressed on neutrophils. Reduced binding to FcyRIIIA is found e.g.
  • antibodies comprising an IgG Fc-region with mutation at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbering according to EU index of Kabat).
  • ADCC antibody-dependent cellular cytotoxicity
  • Fc receptor binding refers to lysis of target cells by an antibody as reported herein in the presence of effector cells.
  • the capacity of the antibody to induce the initial steps mediating ADCC is investigated by measuring their binding to Fey receptors expressing cells, such as cells, recombinantly expressing FcyRI and/or FcyRIIA or NK cells (expressing essentially FcyRIIIA). In particular, binding to FcyR on NK cells is measured.
  • an “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
  • Activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
  • a particular activating Fc receptor is human FcyRIIIa (see UniProt accession no. P08637, version 141).
  • CD40 refers to any native CD40 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses “full-length,” unprocessed CD40 as well as any form of CD40 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of CD40, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human CD40 is shown in SEQ ID NO:57 (Uniprot P25942, version 200) and the amino acid sequence of an exemplary mouse CD40 is shown in SEQ ID NO:58 (Uniprot P27512, version 160).
  • the CD40 antigen is a 50 kDa cell surface glycoprotein which belongs to the Tumor Necrosis Factor Receptor (TNF-R) family. (Stamenkovic et al. (1989), EMBO J. 8: 1403-10). CD40 is expressed in many normal and tumor cell types, including B lymphocytes, dendritic cells, monocytes, macrophages, thymus epithelium, endothelial cells, fibroblasts, and smooth muscle cells. CD40 is expressed in all B-lymphomas and in 70% of all solid tumors and is up-regulated in antigen presenting cells (APCs) by maturation signals, such as IFN-gamma and GM- CSF.
  • APCs antigen presenting cells
  • CD40 activation also induces differentiation of monocytes into functional dendritic cells (DCs) and enhances cytolytic activity of NK cells through APC-CD40 induced cytokines.
  • DCs functional dendritic cells
  • CD40 plays an essential role in the initiation and enhancement of immune responses by inducing maturation of APCs, secretion of helper cytokines, upregulation of costimulatory molecules, and enhancement of effector functions.
  • CD40 agonist as used herein includes any moiety that agonizes the
  • CD40/CD40L interaction refers preferably to human CD40, thus the CD40 agonist is preferably an agonist of human CD40.
  • the moiety will be an agonistic CD40 antibody or antibody fragment.
  • anti-CD40 antibody refers to an antibody that is capable of binding CD40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD40.
  • the extent of binding of an anti-CD40 antibody to an unrelated, non-CD40 protein is less than about 10% of the binding of the antibody to CD40 as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS).
  • RIA radioimmunoassay
  • FACS flow cytometry
  • an antibody that binds to CD40 has a dissociation constant (KD) of ⁇ ImM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 6 M or less, e.g. from 10 68 M to 10 13 M, e.g., from 10 8 M to 10 10 M).
  • KD dissociation constant
  • peptide linker refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids.
  • Peptide linkers are known in the art or are described herein.
  • Suitable, non -immunogenic linker peptides are, for example, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 2 and 4, in particular 2, i.e.
  • GGGGS GGGGGGS
  • SEQ ID NO: 81 GGGGSGGGGS
  • SEQ ID NO: 82 SGGGGSGGGG
  • SEQ ID NO:83 GGGGS GGGGS GGGG
  • GGGGS GGGGS GGGG SEQ ID NO:84
  • GSPGSSSSGS SEQ ID NO:85
  • G4S 3
  • SEQ ID NO:86 SEQ ID NO:86
  • G4S SEQ ID NO:87
  • GSGSGS SEQ ID NO:88
  • GSGSGNGS SEQ ID NO:89
  • GGSGSGSG SEQ ID NO: 90
  • GGSGSG SEQ ID NO:91
  • GGSG SEQ ID NO: 92
  • GGSGNGSG SEQ ID NO:93
  • GGNGSGSGSG SEQ ID NO:94
  • GGNGSG SEQ ID NO:95
  • Peptide linkers of particular interest are (G4S) (SEQ ID NO: 81), (G 4 S) 2 or GGGGSGGGGS (SEQ ID NO:82), (G4S) 3 (SEQ ID NO:86) and (G4S (SEQ ID NO:87).
  • amino acid denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
  • alanine three letter code: ala, one letter code: A
  • arginine arg, R
  • fused or “connected” is meant that the components (e.g. a heavy chain of an antibody and a Fab fragment) are linked by peptide bonds, either directly or via one or more peptide linkers.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference 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. SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • Amino acid sequence variants of the TNF ligand trimer-containing antigen binding molecules may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecules, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • Sites of interest for substitutional mutagenesis include the HVRs and Framework (FRs).
  • amino acid side chain classes (1) to (6) Conservative substitutions are provided in Table B under the heading “Preferred Substitutions” and further described below in reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. TABLE A Amino acids may be grouped according to common side-chain properties:
  • amino acid sequence variants includes substantial variants wherein there are amino acid substitutions in one or more hypervariable region residues of a parent antigen binding molecule (e.g . a humanized or human antibody).
  • a parent antigen binding molecule e.g . a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule and/or will have substantially retained certain biological properties of the parent antigen binding molecule.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antigen binding molecules displayed on phage and screened for a particular biological activity (e.g. binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antigen binding molecule to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science , 244:1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antigen binding molecule complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include bispecific antigen binding molecules of the invention with an N-terminal methionyl residue. Other insertional variants of the molecule include the fusion to the N- or C-terminus to a polypeptide which increases the serum half-life of the bispecific antigen binding molecules.
  • the bispecific antigen binding molecules provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecules may be conveniently obtained by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the TNF ligand trimer-containing antigen binding molecule comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. ⁇ B ⁇ OH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in TNF family ligand trimer-containing antigen binding molecule may be made in order to create variants with certain improved properties.
  • variants of bispecific antigen binding molecules or antibodies of the invention are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • Such fucosylation variants may have improved ADCC function, see e.g. US Patent Publication Nos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • variants of the bispecific antigen binding molecules or antibodies of the invention are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc.
  • Such variants may have reduced fucosylation and/or improved ADCC function., see for example WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546
  • variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
  • Such antibody variants may have improved CDC function and are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • cysteine engineered variants of the bispecific antigen binding molecules of the invention e.g., “thioMAbs,” in which one or more residues of the molecule are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the molecule.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antigen binding molecules may be generated as described, e.g., in U.S. Patent No. 7,521,541.
  • nucleic acid or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides.
  • Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule.
  • the sequence of bases is typically represented from 5’ to 3’.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • DNA deoxyribonucleic acid
  • cDNA complementary DNA
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid molecule may be linear or circular.
  • nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms.
  • the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038/nm.4356 or EP 2 101 823 Bl).
  • 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.
  • isolated nucleic acid encoding a bispecific antigen binding molecule or antibody refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the bispecific antigen binding molecule or antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).
  • expression cassette refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
  • vector or "expression vector” is synonymous with "expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell.
  • 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.
  • the expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery.
  • the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention.
  • Host cells include cultured cells, e.g.
  • mammalian cultured cells such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • an “effective amount” of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
  • a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
  • mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
  • domesticated animals e.g. cows, sheep, cats, dogs, and horses
  • primates e.g. humans and non-human primates such as monkeys
  • rabbits e.g. mice and rats
  • rodents e.g. mice and rats
  • pharmaceutical composition or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the molecules of the invention are used to delay development of a disease or to slow the progression of a disease.
  • cancer refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the
  • an “advanced” cancer is one which has spread outside the site or organ of origin, either by local invasion or metastasis. Accordingly, the term “advanced” cancer includes both locally advanced and metastatic disease.
  • a “recurrent” cancer is one which has regrown, either at the initial site or at a distant site, after a response to initial therapy, such as surgery.
  • a “locally recurrent” cancer is cancer that returns after treatment in the same place as a previously treated cancer.
  • An “operable” or “resectable” cancer is cancer which is confined to the primary organ and suitable for surgery (resection).
  • a “non-resectable” or “unresectable” cancer is not able to be removed (resected) by surgery.
  • chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer.
  • the chemotherapeutic agent is an antimetabolite.
  • the antimetabolite is selected from the group consisting of Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine, Cytarabine, Fluorouracil, Floxuridine, and Gemcitabine.
  • the antimetabolite is capecitabine or gemcitabine.
  • the antimetabolite is fluorouracil.
  • the chemotherapeutic agent is an agent that affects microtubule formation.
  • the agent that affects microtubule formation is selected from the group consisting of: paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere, etoposide, and teniposide.
  • the chemotherapeutic agent is an alkylating agent such as cyclophosphamide.
  • the chemotherapeutic agent is a cytotoxic antibiotic such as a topoisomerase II inhibitor.
  • the topoisomerase II inhibitor is doxorubicin.
  • the invention relates to new bispecific antigen binding molecules, comprising (a) at least two Fab fragments capable of specific binding to capable of specific binding to CD40, (b) Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function and (c) one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA) connected to the C-terminus of the Fc region.
  • CEA carcinoembryonic antigen
  • bispecific antigen binding molecules possess particularly advantageous properties such as producibility, stability, binding affinity, biological activity, targeting efficiency, reduced internalization, superior pharmacokinetic (PK) properties, and compared to classical CD40 agonistic antibodies, reduced toxicity, an extended dosage range that can be given to a patient and thereby a possibly enhanced efficacy.
  • PK pharmacokinetic
  • the invention provides bispecific antigen binding molecules that are characterized by targeted agonistic binding to CD40.
  • the bispecific antigen binding molecule is a CD40 agonist that is targeted against CEA.
  • the bispecific antigen binding molecules of the invention comprise a Fc region composed of a first and a second subunit capable of stable association which comprises mutations that reduce effector function. The use of the Fc region comprising mutations that reduce or abolish effector function will prevent unspecific agonism by crosslinking via Fc receptors and will prevent ADCC of CD40 + cells.
  • the bispecific antigen binding molecules as described herein possess advantages over conventional antibodies capable of specific binding to CD40 in that they selectively induce immune response at the target cells, which are typically cancer cells or tumor stroma.
  • the bispecific antigen binding molecules are thus characterized by CEA-targeted agonistic binding to CD40.
  • the bispecific antigen binding molecules are able to activate dendritic cells (DCs) and to foster tumor-specific T cell priming without causing systemic side effects.
  • DCs dendritic cells
  • the bispecific antigen binding molecules described herein increase the co-localization of CEA + expressing tumor material and DCs.
  • EVs tumor-derived extracellular vesicles
  • a bispecific antigen binding molecule comprising
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, and (c) one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA) connected to the C-terminus of the Fc region.
  • CEA carcinoembryonic antigen
  • the antigen binding domains capable of specific binding to CD40 are Fab fragments.
  • a bispecific antigen binding molecule comprising
  • VHCD40 heavy chain variable region
  • CDR-H1 comprising the amino acid sequence of SEQ ID NO:l
  • CDR-H2 comprising the amino acid sequence of SEQ ID NO:2
  • CDR-H3 comprising the amino acid sequence of SEQ ID NO:3
  • VLCD40 light chain variable region
  • Fab fragments capable of specific binding to CD40 each comprise
  • VHCD40 heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10, and
  • VLCD40 light chain variable region
  • a bispecific antigen binding molecule wherein the Fab fragments capable of specific binding to CD40 each comprise (a) a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO: 11, or (b) a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 12, or (c) a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO: 13, or (d) a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO: 14, or (e) a VH comprising the amino acid sequence of SEQ ID NO:8 and a VL comprising the amino acid sequence of SEQ ID NO: 11, or (f) a VH comprising the amino acid sequence of SEQ ID NO:8 and a VL comprising the amino acid sequence of the amino
  • Fab fragments capable of specific binding to CD40 each comprise a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO: 11.
  • a bispecific antigen binding molecule wherein wherein the Fab fragments capable of specific binding to CD40 each comprise (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO:21, or (b) a VH comprising the amino acid sequence of SEQ ID NO: 16 and a VL comprising the amino acid sequence of SEQ ID NO:21, or (c) a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO:21, or (d) a VH comprising the amino acid sequence of SEQ ID NO:18 and a VL comprising the amino acid sequence of SEQ ID NO:21, or (e) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO:22, or (f) a VH comprising the amino acid sequence of SEQ ID NO: 16 and
  • a bispecific antigen binding molecule wherein the Fab fragments capable of specific binding to CD40 each comprise a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO:21 or wherein the antigen binding domain capable of specific binding to CD40 comprises a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO:21.
  • Fab fragments capable of specific binding to CD40 each comprise a heavy chain variable region (VHCD40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:27, and a light chain variable region (VLCD40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:30.
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • Fab fragments capable of specific binding to CD40 each comprise a VH comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:32.
  • a bispecific antigen binding molecule wherein the antigen binding domain capable of specific binding to CD40 comprises
  • VHCD40 heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, and
  • VLCD40 light chain variable region
  • a bispecific antigen binding molecule as described hereinbefore, wherein the antigen binding domain capable of specific binding to CEA binds to the A1 domain of carcinoembryonic antigen (CEACAM5).
  • CEACAM5 carcinoembryonic antigen
  • the antigen binding domain capable of specific binding to CEA binds to the domain comprising the amino acid sequence of SEQ ID NO:61.
  • a bispecific antigen binding molecule as described hereinbefore, wherein the antigen binding domain capable of specific binding to CEA comprises
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • a bispecific antigen binding molecule as described hereinbefore, wherein the antigen binding domain capable of specific binding to CEA binds to the A2 domain of carcinoembryonic antigen (CEACAM5).
  • CEACAM5 carcinoembryonic antigen
  • the antigen binding domain capable of specific binding to CEA binds to the domain comprising the amino acid sequence of SEQ ID NO:60.
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:35, and a light chain variable region (VLCEA) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:38.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:42, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable region (VLCEA) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:46.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:39 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:40 or a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:47 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:48.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region comprising an amino acid sequence of SEQ ID NO:47
  • VLCEA light chain variable region
  • CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:39 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:40.
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:47 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:48.
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:51, and a light chain variable region (VLCEA) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:54.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:55 and a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:56.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • a bispecific antigen binding molecule wherein (i) the two Fab fragments capable of specific binding to CD40 each comprise a heavy chain variable region (VHCD40) comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region (VLCD40) comprising the amino acid sequence of SEQ ID NO: 11, and
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:39 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:40.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • a bispecific antigen binding molecule wherein
  • the two Fab fragments capable of specific binding to CD40 each comprise a heavy chain variable region (VHCD40) comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region (VLCD40) comprising the amino acid sequence of SEQ ID NO: 11, and
  • the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising an amino acid sequence of SEQ ID NO:47 and a light chain variable region (VLCEA) comprising an amino acid sequence of SEQ ID NO:48.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • the antigen binding domain capable of specific binding to CEA is a cross-fab fragment connected to the C-terminus of the Fc region.
  • Bispecific antigen binding molecules bivalent for binding to CD40 and monovalent for binding to CEA (2+1 format)
  • a bispecific antigen binding molecule comprising
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule comprises bivalent binding to CD40 and monovalent binding to CEA.
  • bispecific antigen binding molecule as described herein before, wherein the bispecific antigen binding molecule comprises
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the two Fab fragments capable of specific binding to CD40 fused to the N-termini of the Fc region, and (c) one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA) fused to one of the C-termini of the Fc region.
  • CEA carcinoembryonic antigen
  • the antigen binding domain capable of specific binding to CEA connected to the C-terminus of the Fc region is a cross-fab fragment.
  • a bispecific antigen binding molecule wherein the bispecific antigen binding molecule comprises
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the two Fab fragments capable of specific binding to CD40 fused to the N-termini of the Fc region, and
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule consists of
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the first Fab fragment (aa) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the first subunit and the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the second subunit, and
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule comprises
  • each heavy chain comprising a VH and CHI domain of a Fab fragment capable of specific binding to CD40, and a Fc region subunit comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40
  • the Fab fragment capable of specific binding to CEA is a cross-Fab fragment comprising a VL-CH1 chain and a VH-Ckappa chain, and wherein the VH- Ckappa chain or the VL-CH1 chain is connected to the C-terminus of one of the two heavy chains of (i).
  • a bispecific antigen binding molecule comprising a cross-fab fragment capable specific binding to CEA, wherein the VH-Ckappa chain is fused to the C-terminus of one of the two heavy chains of (i).
  • a bispecific antigen binding molecule comprising a cross-fab fragment capable specific binding to CEA, wherein the VL-CH1 chain is fused to the C-terminus of one of the two heavy chains of (i).
  • a bispecific antigen binding molecule comprising
  • each heavy chain comprising a VH and CHI domain of a Fab fragment capable of specific binding to CD40 and a Fc region subunit
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and (c) a cross-fab fragment capable of specific binding to FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a), optionally via a peptide linker.
  • VH-CL (VH-Ckappa) chain is connected to the C-terminus of the Fc knob heavy chain.
  • a bispecific antigen binding molecule comprising
  • each heavy chain comprising a VH and CHI domain of a Fab fragment capable of specific binding to CD40 and a Fc region subunit
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and (c) a cross-fab fragment capable of specific binding to FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VL-CH1 chain is connected to the C-terminus of one of the two heavy chains of (a), optionally via a peptide linker.
  • the VL-CH1 chain is connected to the C-terminus of the Fc knob heavy chain.
  • the peptide linker is selected from GGGGS (SEQ ID NO:81)
  • GGGGSGGGGS S GGGGS GGGG (SEQ ID NO:83), GGGGSGGGGSGGGG (SEQ ID NO:84), GSPGSSSSGS (SEQ ID NO:85), (G4S (SEQ ID NO:86), (G4S (SEQ ID NO:87), GSGSGSGS (SEQ ID NO:88), GSGSGNGS (SEQ ID NO: 89), GGSGSGSG (SEQ ID NO: 90), GGSGSG (SEQ ID NO:91), GGSG (SEQ ID NO: 92), GGSGNGSG (SEQ ID NO:93), GGNGSGSG (SEQ ID NO:94) and GGNGSG (SEQ ID NO:95).
  • Peptide linkers of particular interest are (G4S) (SEQ ID NO:81), (G4S)2 or GGGGSGGGGS (SEQ ID NO:82), (G4S) 3 (SEQ ID NO:86) and (G4S (SEQ ID NO:87).
  • the invention provides a bispecific antigen binding molecule comprising
  • the invention provides a bispecific antigen binding molecule comprising
  • a bispecific antigen binding molecule comprising
  • a heavy chain comprising the VH and CHI domain of a Fab fragment capable of specific binding to CD40 and a Fc region subunit
  • a heavy chain comprising a VH and CHI domain of a Fab fragment capable of specific binding to CD40, a VL and CHI domain of a Fab fragment capable of specific binding to CEA and a Fc region subunit
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and (d) a light chain comprising a VH and CL domain of a Fab fragment capable of specific binding to CEA.
  • Bispecific antigen binding molecules trivalent for binding to CD40 and monovalent for binding to the target cell antigen (3+1 format)
  • a bispecific antigen binding molecule that comprises trivalent binding towards CD40 and monovalent binding towards CEA (3+1 format).
  • bispecific antigen binding molecule wherein the bispecific antigen binding molecule consists of
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, wherein the first Fab fragment (aa) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the first subunit and wherein the third Fab fragment (ac) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the second Fab fragment (ab), which is in turn fused at its C-terminus to the N-terminus of the second subunit of the Fc, and
  • a bispecific antigen binding molecule comprising
  • a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to CD40 fused at its N-terminus to the VH-CH1 chain of a second Fab fragment capable of specific binding to CD40, optionally via a peptide linker, and a Fc region subunit,
  • a heavy chain comprising a VH-CH1 domain of a Fab fragment capable of specific binding to CD40, a Fc region subunit, and a VH-CL chain of a cross-Fab fragment capable of specific binding to CEA fused to the C-terminus of the Fc region subunit, optionally via a peptide linker, (c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and
  • a bispecific antigen binding molecule comprising (a) a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to CD40 fused at its N-terminus to the VH-CH1 chain of a second Fab fragment capable of specific binding to CD40, optionally via a peptide linker, a Fc region subunit, and a VH-CL chain of a Fab fragment capable of specific binding to CEA fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and (d) a light chain comprising a VL and CHI domain of a Fab fragment capable of specific binding to CEA.
  • a bispecific antigen binding molecule comprising
  • a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to CD40 fused at its N-terminus to the VH-CH1 chain of a second Fab fragment capable of specific binding to CD40, optionally via a peptide linker, and a Fc region subunit,
  • a heavy chain comprising a VH-CH1 domain of a Fab fragment capable of specific binding to CD40, a Fc region subunit, and a VL-CH1 chain of a Fab fragment capable of specific binding to CEA fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40
  • a light chain comprising a VH and CL domain of a Fab fragment capable of specific binding to CEA.
  • a bispecific antigen binding molecule comprising
  • a heavy chain comprising a VH-CHl chain of a first Fab fragment capable of specific binding to CD40 fused at its N-terminus to the VH-CHl chain of a second Fab fragment capable of specific binding to CD40, optionally via a peptide linker, a Fc region subunit, and a VL-CHl chain of a Fab fragment capable of specific binding to CEA fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and (d) a light chain comprising a VH and CL domain of a Fab fragment capable of specific binding to CEA.
  • Bispecific antigen binding molecules tetravalent for binding to CD40 and monovalent for binding to the target cell antigen (4+1 format)
  • a bispecific antigen binding molecule that comprises tetravalent binding towards CD40 and monovalent binding towards CEA (4+1 format).
  • a bispecific antigen binding molecule wherein the bispecific antigen binding molecule consists of
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function
  • the third Fab fragment (ac) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CHl chain of the first Fab fragment (aa), which is in turn fused at its C-terminus to the N-terminus of the first subunit of the Fc region
  • the fourth Fab fragment (ad) is fused at the C-terminus of the VH-CHl chain to the N-terminus of the VH-CHl chain of the second Fab fragment (ab), which is in turn fused at its C-terminus to the N-terminus of the second subunit of the Fc
  • a cross-Fab fragment capable of specific binding to CEA fused to the C-terminus of the first subunit of the Fc region.
  • a bispecific antigen binding molecule wherein the four antigen binding domains capable of specific binding to CD40 are Fab fragments and each two thereof are fused to each other at the heavy chain, optionally via a peptide linker.
  • the peptide linker comprises the amino acid sequence of SEQ ID NO: 82. More particularly, the antigen binding molecule comprises two heavy chains comprising each a VHCHl -peptide linker-VHCHl fragment. In a particular aspect, the peptide linker has the amino acid sequence of SEQ ID NO: 82.
  • the bispecific antigen binding molecule comprises (a) four light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40,
  • each of the heavy chain comprises a VH and CHI domain of a Fab fragment capable of specific binding to CD40 fused to a VH and CHI domain of a second Fab fragment capable of specific binding to CD40, and a Fc region subunit, and (c) a cross-fab fragment capable of specific binding to CEA, wherein the VH domain is connected via a peptide linker to the C-terminus of one of the heavy chains.
  • the bispecific antigen binding molecule comprises
  • each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40
  • each of the heavy chain comprises a VH and CHI domain of a Fab fragment capable of specific binding to CD40 fused to a VH and CHI domain of a second Fab fragment capable of specific binding to CD40, and a Fc region subunit, and (c) a cross-fab fragment capable of specific binding to CEA, wherein the VL domain is connected via a peptide linker to the C-terminus of one of the heavy chains.
  • the peptide linker comprises an amino acid sequence selected from SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:86 and SEQ ID NO:87. More particularly, the peptide linker comprises the SEQ ID NO: 82. Fc domain modifications reducing Fc receptor binding and/or effector function
  • the bispecific antigen binding molecules of the invention further comprise a Fc domain composed of a first and a second subunit capable of stable association.
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the Fc domain confers favorable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Accordingly, in particular embodiments the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG Fc domain, in particular an IgGl Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgGl Fc domain.
  • the Fc domain exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGl Fc domain (or the bispecific antigen binding molecule of the invention comprising a native IgGl Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGl Fc domain (or the bispecific antigen binding molecule of the invention comprising a native IgGl Fc domain).
  • the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function.
  • the Fc receptor is an Fey receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • the Fc receptor is an inhibitory Fc receptor.
  • the Fc receptor is an inhibitory human Fey receptor, more specifically human FcyRIIB.
  • the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion.
  • the effector function is ADCC.
  • the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGl Fc domain.
  • Substantially similar binding to FcRn is achieved when the Fc domain (or the the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGl Fc domain (or the the bispecific antigen binding molecule of the invention comprising a native IgGl Fc domain) to FcRn.
  • the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
  • the Fc domain of the bispecific antigen binding molecule of the invention comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.
  • the bispecific antigen binding molecule of the invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to bispecific antibodies of the invention comprising a non-engineered Fc domain.
  • the Fc receptor is an Fey receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an inhibitory Fc receptor.
  • the Fc receptor is an inhibitory human Fey receptor, more specifically human FcyRIIB In some aspects the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • binding to each of these receptors is reduced.
  • binding affinity to a complement component, specifically binding affinity to Clq is also reduced.
  • binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e.
  • the Fc domain or the bispecific antigen binding molecule of the invention comprising said Fc domain
  • the Fc domain, or the bispecific antigen binding molecule of the invention comprising said Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity.
  • the Fc domain of the bispecific antigen binding molecule of the invention is engineered to have reduced effector function, as compared to a non-engineered Fc domain.
  • the reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dendritic cell maturation, or reduced T cell priming.
  • CDC complement dependent cytotoxicity
  • ADCC reduced antibody-dependent cell-mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • reduced immune complex-mediated antigen uptake by antigen-presenting cells reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dend
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • Certain antibody variants with improved or diminished binding to FcRs are described (e.g. U.S. Patent No. 6,737,056; WO 2004/056312, and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604).
  • the Fc domain comprises an amino acid substitution at a position of E233, L234, L235, N297, P331 and P329.
  • the Fc domain comprises the amino acid substitutions L234A and L235A (“LALA”).
  • the Fc domain is an IgGl Fc domain, particularly a human IgGl Fc domain.
  • the Fc domain comprises an amino acid substitution at position P329.
  • the amino acid substitution is P329A or P329G, particularly P329G.
  • the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D or P331S.
  • the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”).
  • P329G LALA amino acid mutations L234A, L235A and P329G
  • the “P329GLALA” combination of amino acid substitutions almost completely abolishes Fey receptor binding of a human IgGl Fc domain, as described in PCT Patent Application No. WO 2012/130831 Al. Said document also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
  • Such antibody is an IgGl with mutations L234A and L235A or with mutations L234A, L235A and P329G (numbering according to EU index of Kabat et al , Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • the Fc domain is an IgG4 Fc domain.
  • the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P.
  • the Fc domain is an IgG4 Fc domain comprising amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).
  • Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US 2005/0014934.
  • Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826). See also Duncan, A.R. and Winter, G, Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
  • Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression.
  • a suitable such binding assay is described herein.
  • binding affinity of Fc domains or cell activating bispecific antigen binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor. Effector function of an Fc domain, or bispecific antigen binding molecules of the invention comprising an Fc domain, can be measured by methods known in the art.
  • a suitable assay for measuring ADCC is described herein.
  • ACTITM non-radioactive cytotoxicity assay for flow cytometry CellTechnology, Inc. Mountain View, CA
  • CytoTox 96 ® non-radioactive cytotoxicity assay Promega, Madison, WI
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo , e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
  • the invention relates to the bispecific antigen binding molecule (a) at least one antigen binding domain capable of specific binding to CD40, (b) at least one antigen binding domain capable of specific binding to FAP, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor, in particular towards Fey receptor.
  • the invention in another aspect, relates to the bispecific antigen binding molecule comprising (a) at least one antigen binding domain capable of specific binding to CD40, (b) at least one antigen binding domain capable of specific binding to a target cell antigen, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitution that reduces effector function.
  • the Fc domain is of human IgGl subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).
  • the bispecific antigen binding molecules of the invention comprise different antigen binding sites, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain may be comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antigen binding molecules of the invention in recombinant production, it will thus be advantageous to introduce in the Fc domain of the bispecific antigen binding molecules of the invention a modification promoting the association of the desired polypeptides.
  • the invention relates to the bispecific antigen binding molecule comprising (a) at least two Fab fragments capable of specific binding to CD40, (b) one antigen binding domain capable of specific binding to CEA, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain.
  • the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.
  • the invention relates to the bispecific antigen binding molecule comprising (a) at least two Fab fragments capable of specific binding to CD40, (b) one antigen binding domain capable of specific binding to CEA, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method.
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
  • the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V).
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C)
  • the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C).
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
  • a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • the C-terminus of the heavy chain of the bispecific antibody as reported herein can be a complete C-terminus ending with the amino acid residues PGK.
  • the C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed.
  • the C-terminus of the heavy chain is a shortened C-terminus ending PG.
  • a bispecific antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein comprises the C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index).
  • a bispecific antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index).
  • the invention relates to a bispecific antigen binding molecule comprising (a) at least two Fab fragments capable of specific binding to CD40, (b) a cross- Fab fragment capable of specific binding to CEA, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein in one of the Fab fragments either the variable domains VH and VL or the constant domains CHI and CL are exchanged.
  • the bispecific antibodies are prepared according to the Crossmab technology.
  • Multispecific antibodies with a domain replacement/exchange in one binding arm are described in detail in W02009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. They clearly reduce the byproducts caused by the mismatch of a light chain against a first antigen with the wrong heavy chain against the second antigen (compared to approaches without such domain exchange).
  • the bispecific antigen binding molecule comprising (a) a first Fab fragment capable of specific binding to CD40, (b) a second Fab fragment capable of specific binding to FAP, and (c) a Fc domain composed of a first and a second subunit capable of stable association, can contain different charged amino acid substitutions (so-called “charged residues”). These modifications are introduced in the crossed or non-crossed CHI and CL domains.
  • the invention relates to a bispecific antigen binding molecule, wherein in one of CL domains the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and/or wherein the amino acid at position 124 (EU numbering) has been substituted by lysine (K) and wherein in one of the CHI domains the amino acids at position 147 (EU numbering) and/or at position 213 (EU numbering) have been substituted by glutamic acid (E).
  • the application further provides isolated nucleic acid encoding a bispecific antigen binding molecule as described herein or a fragment thereof or isolated nucleic acid encoding an antibody as described herein.
  • the isolated polynucleotides encoding bispecific antigen binding molecules of the invention may be expressed as a single polynucleotide that encodes the entire antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional antigen binding molecule.
  • the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide from the heavy chain portion of the immunoglobulin.
  • the heavy chain polypeptides When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoglobulin.
  • the isolated polynucleotide encodes a polypeptide comprised in the bispecific molecule as described herein.
  • an isolated polynucleotide encoding a bispecific antigen binding molecule, (a) at least two Fab fragments capable of specific binding to CD40,
  • a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, and (c) one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA) connected to the C-terminus of the Fc region.
  • CEA carcinoembryonic antigen
  • RNA for example, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • RNA of the present invention may be single stranded or double stranded.
  • Bispecific antigen binding molecules as described herein may be obtained, for example, by recombinant production.
  • For recombinant production one or more polynucleotide encoding the bispecific antigen binding molecule or polypeptide fragments thereof are provided.
  • the one or more polynucleotide encoding the bispecific antigen binding molecule are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • Such polynucleotide may be readily isolated and sequenced using conventional procedures.
  • a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided.
  • the expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment.
  • the expression vector includes an expression cassette into which the polynucleotide encoding the bispecific antigen binding molecule or polypeptide fragments thereof (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements.
  • a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids.
  • a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region.
  • Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions, e.g.
  • a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage.
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the bispecific antigen binding molecule of the invention or polypeptide fragments thereof, or variants or derivatives thereof.
  • Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g.
  • a polypeptide is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
  • transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus).
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.
  • tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins).
  • inducible promoters e.g. promoters inducible tetracyclins
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • the expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).
  • LTRs retroviral long terminal repeats
  • AAV adeno-associated viral inverted terminal repeats
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the bispecific antigen binding molecule of the invention or polypeptide fragments thereof.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide.
  • the native signal peptide e.g.
  • an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse b-glucuronidase.
  • DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the fusion protein may be included within or at the ends of the polynucleotide encoding a bispecific antigen binding molecule of the invention or polypeptide fragments thereof.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising one or more vectors of the invention.
  • the polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively.
  • a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a bispecific antigen binding molecule of the invention of the invention.
  • the term "host cell” refers to any kind of cellular system which can be engineered to generate the fusion proteins of the invention or fragments thereof.
  • Host cells suitable for replicating and for supporting expression of antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the antigen binding molecule for clinical applications.
  • Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like.
  • polypeptides may be produced in bacteria in particular when glycosylation is not needed.
  • the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et ah, Nat Biotech 24, 210-215 (2006).
  • Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates).
  • invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et ah, J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3 A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et ah, Annals N.Y.
  • COS-7 monkey kidney CV1 line transformed by SV40
  • MRC 5 cells MRC 5 cells
  • FS4 cells Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfir- CHO cells (Urlaub et ak, Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
  • CHO Chinese hamster ovary
  • dhfir- CHO cells Urlaub et ak, Proc Natl Acad Sci USA 77, 4216 (1980)
  • myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
  • Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the art to express foreign genes in these systems.
  • Cells expressing a polypeptide comprising either the heavy or the light chain of an immunoglobulin may be engineered so as to also express the other of the immunoglobulin chains such that the expressed product is an immunoglobulin that has both a heavy and a light chain.
  • a method of producing a bispecific antigen binding molecule of the invention or polypeptide fragments thereof comprises culturing a host cell comprising polynucleotides encoding the bispecific antigen binding molecule of the invention or polypeptide fragments thereof, as provided herein, under conditions suitable for expression of the bi specific antigen binding molecule of the invention or polypeptide fragments thereof, and recovering the bispecific antigen binding molecule of the invention or polypeptide fragments thereof from the host cell (or host cell culture medium).
  • Bispecific molecules of the invention prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art.
  • affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the bispecific antigen binding molecule binds.
  • a matrix with protein A or protein G may be used.
  • Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an antigen binding molecule essentially as described in the examples.
  • the purity of the bispecific antigen binding molecule or fragments thereof can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.
  • the bispecific antigen binding molecules expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing and non-reducing SDS-PAGE.
  • antigen binding molecules provided herein may be characterized for their binding properties and/or biological activity by various assays known in the art. In particular, they are characterized by the assays described in more detail in the examples.
  • Binding of the bispecific antigen binding molecule provided herein to the corresponding target expressing cells may be evaluated for example by using a murine fibroblast cell line expressing human Fibroblast Activation Protein (FAP) and flow cytometry (FACS) analysis. Binding of the bispecific antigen binding molecules provided herein to CD40 may be determined by using murine splenic dendritic cells (DCs) isolated from human CD40 transgenic (huCD40tg) mice as described in Example 2.2.
  • DCs murine splenic dendritic cells isolated from human CD40 transgenic mice as described in Example 2.2.
  • Bispecific antigen binding molecules of the invention are tested for biological activity.
  • Biological activity may include efficacy and specificity of the bispecific antigen binding molecules.
  • Efficacy and specificity are demonstrated by assays showing agonistic signaling through the CD40 receptor upon binding of the target antigen.
  • in vitro T cell priming assays are conducted using dendritic cells (DCs) that have been incubated with the bispecific antigen binding molecules.
  • DCs dendritic cells
  • the invention provides pharmaceutical compositions comprising any of the bispecific antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods.
  • a pharmaceutical composition comprises any of the bispecific antigen binding molecules provided herein and at least one pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises any of the bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
  • compositions of the present invention comprise a therapeutically effective amount of one or more bispecific antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one bispecific antigen binding molecule according to the invention and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed.
  • compositions are lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable excipient includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers and combinations thereof, as would be known to one of ordinary skill in the art.
  • compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intra-lesional, intravenous, intra-arterial, intramuscular, intrathecal or intraperitoneal injection.
  • the bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Sterile injectable solutions are prepared by incorporating the antigen binding molecules of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum- drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monos
  • Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • compositions comprising the bispecific antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the bispecific antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form.
  • Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g. those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
  • composition herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • bispecific antigen binding molecules may be used in therapeutic methods.
  • bispecific antigen binding molecules of the invention can be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • bispecific antigen binding molecules provided herein for use as a medicament are provided.
  • bispecific antigen binding molecule as described herein before or the pharmaceutical composition of the invention, for use
  • bispecific antigen binding molecules for use in treating a disease, in particular for use in the treatment of cancer, are provided.
  • the bispecific antigen binding molecules described herein are for use in treating or delaying growth of CEA-expressing tumors
  • the bispecific antigen binding molecules described herein are for use in treating gastric adenocarcinoma or colorectal cancer.
  • bispecific antigen binding molecules of the invention for use in a method of treatment are provided.
  • the invention provides a bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof.
  • the invention provides a bispecific antigen binding molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the bispecific antigen binding molecule.
  • the disease to be treated is cancer.
  • the subject, patient, or “individual” in need of treatment is typically a mammal, more specifically a human.
  • a method for i) in activating dendritic cells (DCs), (ii) stimulating tumor-specific T cell response, (iii) causing apoptosis of tumor cells, (iv) treating of cancer, (v) delaying progression of cancer, (vi) prolonging the survival of a patient suffering from cancer, or (vii) treating of infections wherein the method comprises administering a therapeutically effective amount of the bispecific antigen binding molecule of the invention to an individual in need thereof.
  • the invention provides for the use of the bispecific antigen binding molecule of the invention in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof.
  • the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament.
  • the disease to be treated is a proliferative disorder, particularly cancer.
  • cancers include, but are not limited to, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
  • cancer examples include carcinoma, lymphoma (e.g., Hodgkin’s and non-Hodgkin’s lymphoma), blastoma, sarcoma, and leukemia.
  • Other cell proliferation disorders that can be treated using the bispecific antigen binding molecule or antibody of the invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system.
  • the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.
  • the cancer to be treated is gastric adenocarcinoma or colorectal cancer.
  • an amount of the bispecific antigen binding molecule or antibody of the invention that provides a physiological change is considered an "effective amount” or a "therapeutically effective amount”.
  • the appropriate dosage of a bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the specific molecule, the severity and course of the disease, whether the bispecific antigen binding molecule of the invention is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the bispecific antigen binding molecule, and the discretion of the attending physician.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • the bispecific antigen binding molecule of the invention is suitably administered to the patient at one time or over a series of treatments.
  • about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg - 10 mg/kg) of the bispecific antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • a dose may also comprise from about 1 pg/kg body weight, about 5 pg/kg body weight, about 10 pg/kg body weight, about 50 pg/kg body weight, about 100 pg/kg body weight, about 200 pg/kg body weight, about 350 pg/kg body weight, about 500 pg/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein.
  • a range of about 0.1 mg/kg body weight to about 20 mg/kg body weight, about 5 pg/kg body weight to about 1 mg/kg body weight etc. can be administered, based on the numbers described above.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the fusion protein).
  • the bispecific antigen binding molecule will be administered every three weeks.
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the bispecific antigen binding molecule of the invention will generally be used in an amount effective to achieve the intended purpose.
  • the bispecific antigen binding molecule of the invention, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the ICso as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the bispecific antigen binding molecule of the invention which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.1 to 1 mg/kg/day.
  • Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
  • the effective local concentration of the bispecific antigen binding molecule or antibody of the invention may not be related to plasma concentration.
  • One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • a therapeutically effective dose of the bispecific antigen binding molecule of the invention described herein will generally provide therapeutic benefit without causing substantial toxicity.
  • Toxicity and therapeutic efficacy of a fusion protein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LDso (the dose lethal to 50% of a population) and the EDso (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50.
  • Bispecific antigen binding molecules that exhibit large therapeutic indices are preferred. In one aspect, the the bispecific antigen binding molecule or antibody of the invention exhibits a high therapeutic index.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans.
  • the dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et ah, 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).
  • the attending physician for patients treated with fusion proteins of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
  • the bispecific antigen binding molecule of the invention may be administered in combination with one or more other agents in therapy.
  • the bispecific antigen binding molecule or antibody of the invention of the invention may be co-administered with at least one additional therapeutic agent.
  • therapeutic agent encompasses any agent that can be administered for treating a symptom or disease in an individual in need of such treatment.
  • additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • an additional therapeutic agent is another anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti angiogenic agent.
  • an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic or cytostatic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers.
  • bispecific antigen binding molecules of the invention or pharmaceutical compositions comprising them for use in the treatment of cancer wherein the bispecific antigen binding molecule is administered in combination with a chemotherapeutic agent, radiation and/ or other agents for use in cancer immunotherapy.
  • Such other agents are suitably present in combination in amounts that are effective for the purpose intended.
  • the effective amount of such other agents depends on the amount of fusion protein used, the type of disorder or treatment, and other factors discussed above.
  • the bispecific antigen binding molecule or antibody of the invention are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the bispecific antigen binding molecule or antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • bispecific antigen binding molecule as described herein before for use in the treatment of cancer, wherein the bispecific antigen binding molecule is administered in combination with another immunomodulator.
  • immunomodulator refers to any substance including a monoclonal antibody that effects the immune system.
  • the molecules of the inventions can be considered immunomodulators.
  • Immunomodulator s can be used as anti -neoplastic agents for the treatment of cancer.
  • immunomodulators include, but are not limited to anti-CTLA4 antibodies (e.g. ipilimumab), anti -PD 1 antibodies (e.g. nivolumab or pembrolizumab), PD-L1 antibodies (e.g. atezolizumab, avelumab or durvalumab), OX-40 antibodies, 4- IBB antibodies and GITR antibodies.
  • the bispecific antigen binding molecule as described herein before for use in the treatment of cancer wherein the bispecific antigen binding molecule is administered in combination with an agent blocking PD-Ll/PD-1 interaction.
  • the agent blocking PD- Ll/PD-1 interaction is an anti-PD-Ll antibody or an anti-PDl antibody. More particularly, the agent blocking PD-Ll/PD-1 interaction is an anti-PD-Ll antibody, in particular an anti- PD-Ll antibody selected from the group consisting of atezolizumab, durvalumab, pembrolizumab and nivolumab.
  • the agent blocking PD-Ll/PD-1 interaction is atezolizumab (MPDL3280A, RG7446).
  • the agent blocking PD-Ll/PD-1 interaction is an anti-PDl antibody, in particular an anti-PDl antibody selected from pembrolizumab or nivolumab.
  • Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above.
  • bispecific antigen binding molecules as described herein before are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the bispecific antigen binding molecule can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a bispecific antigen binding molecule of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a bispecific antigen binding molecule of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • Ringer's solution such as phosphate
  • a bispecific antigen binding molecule comprising (a) at least two Fab fragments capable of specific binding to CD40, (b) a Fc region composed of a first and a second subunit capable of stable association comprising one or more amino acid substitution that reduces the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, and (c) one antigen binding domain capable of specific binding to carcinoembryonic antigen (CEA) connected to the C-terminus of the Fc region.
  • CEA carcinoembryonic antigen
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • bispecific antigen binding molecule of any one of paras 1 to 3, wherein the Fab fragments capable of specific binding to CD40 each comprise
  • VH comprising the amino acid sequence of SEQ ID NO:8 and a VL comprising the amino acid sequence of SEQ ID NO: 11, or
  • VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO: 11, or
  • bispecific antigen binding molecule of paras 1 or 2 wherein Fab fragments capable of specific binding to CD40 each comprise
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO:22, or
  • VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO:23, or
  • Fab fragments capable of specific binding to CD40 each comprise a heavy chain variable region (VHCD40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:27, and a light chain variable region (VLCD40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:30.
  • VHCD40 heavy chain variable region
  • VLCD40 light chain variable region
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • VHCEA heavy chain variable region comprising an amino acid sequence of SEQ ID NO:47
  • VLCEA light chain variable region
  • the bispecific antigen binding molecule of any one of paras 1 to 10, wherein the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:51, and a light chain variable region (VLCEA) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:54.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • bispecific antigen binding molecule of any one of paras 1 to 10 or 13, wherein the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:55 and a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:56.
  • VHCEA heavy chain variable region
  • VLCEA light chain variable region
  • bispecific antigen binding molecule of any one of paras 1 to 16, wherein the bispecific antigen binding molecule comprises bivalent binding to CD40 and monovalent binding to CEA.
  • An expression vector comprising the isolated nucleic acid of para 18.
  • a host cell comprising isolated nucleic acid of para 18 or the expression vector of para 19.
  • a method of producing a bispecific antigen binding molecule comprising culturing the host cell of para 20 under conditions suitable for the expression of the bispecific antigen binding molecule, and isolating the bispecific antigen binding molecule.
  • a pharmaceutical composition comprising the bispecific antigen binding molecule of any one of paras 1 to 17 and a pharmaceutically acceptable carrier.
  • a method of treating an individual having cancer comprising administering to the individual an effective amount of the bispecific antigen binding molecule of any one of paras 1 to 17, or the pharmaceutical composition of para 22.
  • DNA sequences were determined by double strand sequencing.
  • Desired gene segments were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. In cases where no exact gene sequence was available, oligonucleotide primers were designed based on sequences from closest homologues and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed with a 5’ -end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells.
  • HEK293-EBNA cells that grow in suspension were co-transfected with the respective expression vectors using polyethylenimine as a transfection reagent.
  • Bispecific antibodies were generated by transient transfection of HEK293 EBNA cells.
  • Cells were centrifuged and medium replaced by pre-warmed CD CHO medium.
  • Expression vectors were mixed in CD CHO medium, PEI was added, the solution vortexed and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to shake flask and incubated for 3 hours at 37°C in an incubator with a 5% CO2 atmosphere.
  • Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (MabSelectSure-SepharoseTM, GE Healthcare, Sweden) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric bispecific antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0.
  • Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at -20°C or -80°C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.
  • MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator
  • the NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer’s instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.
  • Size exclusion chromatography for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4/K2HPO4, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2 x PBS on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.
  • This section describes the characterization of the multispecific antibodies with VH/VL or CH/CL exchange (CrossMabs) with emphasis on their correct assembly.
  • the expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact CrossMabs and deglycosylated/FabALACTICA or alternatively deglycosylated/GingisKHAN digested CrossMabs.
  • ESI-MS electrospray ionization mass spectrometry
  • the CrossMabs were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37°C for up to 17 h at a protein concentration of 1 mg/ml.
  • the FabALACTICA or GingisKHAN (Genovis AB; Sweden) digestions were performed in the buffers supplied by the vendor with 100 pg deglycosylated CrossMabs.
  • Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a Tri Versa NanoMate source (Advion).
  • Bispecific antibodies targeting CD40 and CEA were prepared in 2+1 format consisting of two CD40 binding moieties combined with one CEA binding moiety at the C -terminus of an Fc ( Figures 1A to ID).
  • the bispecific CD40-CEA antibodies included humanized CD40 clone P1AE0817 ( Figures 1A and 1C), however for animal studies corresponding molecules with murine CD40 clone FGK4.5 ( Figures IB and ID) were prepared.
  • Anti-CEA antibody A5B7 is for example disclosed by M. J. Banfield et al, Proteins 1997, 29(2), 161-171 and its structure can be found as PDB ID:lCLO in the Protein structural database PDB (www.rcsb.org, H.M.
  • All genes are transiently expressed under control of a chimeric MPSV promoter consisting of the MPSV core promoter combined with the CMV promoter enhancer fragment.
  • the expression cassette also contains a synthetic polyA signal at the 3’ end of the cDNAs.
  • the expression vector also contains the oriP region for episomal replication in EBNA (Epstein Barr Virus Nuclear Antigen) containing host cells.
  • the bispecific antigen binding molecules targeting CEA and CD40 were expressed by transient transfection of HEK cells grown in suspension with expression vectors encoding the 4 different peptide chains.
  • Transfection into HEK293-F cells was performed according to the cell supplier's instructions using Maxiprep (Qiagen) preparations of the antibody vectors, F17 medium (Invitrogen, USA), Peipro (Poly science Europe GmbH) and an initial cell density of 1-2 million viable cells/ml in serum free FreeStyle 293 expression medium (Invitrogen).
  • Cell culture supernatants were harvested after 7 days of cultivation in shake flasks or stirred fermenters by centrifugation at 14000 g for 30 minutes and filtered through a 0.22 pm filter.
  • the bispecific antibodies were purified from cell culture supernatants by Protein A affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on a Mab Select SuRe resin equilibrated with PBS buffer (10 mM NaiHPCri, 1 mM KH2PO4,
  • bispecific antibodies were purified by Protein A affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden), followed by cation exchange chromatography (POROS® 50HS IEX), and hydrophobic interaction chromatography (PHENYL SEPHAROSETM).
  • PHENYL SEPHAROSETM Protein A affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden)
  • POROS® 50HS IEX cation exchange chromatography
  • PHENYL SEPHAROSETM hydrophobic interaction chromatography
  • MC38 and MC38-huCEA cells were obtained from City of Hope (Mizobata S et al., Cancer Immunol Immunother 2000, 49(6), 285-295). MC38 and MC38-huCEA cells were cultured in lx Dulbecco's Modified Eagle's Medium (DMEM) (gibco, Cat. No. 42430-025) supplemented with 10 % (v/v) Fetal Bovine Serum (FBS) (life technologies, Cat. No. 16140, Lot No. 1797306A) and 1% (v/v) Penicillin Streptomycin (gibco, Cat. No. 15070- 063).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • Penicillin Streptomycin gibco, Cat. No. 15070- 063
  • huCEA-expressing cells 500 pg/mL Geneticin (gibco, Cat. No. 10131035) was added in the medium of MC38-huCEA cells. MC38 and MC38-huCEA cells were detached by using enzyme-free Cell Dissociation Buffer (gibco, Cat. No.
  • 0.3xl0 5 MC38-huCEA or MC38 cells were added in 200 m ⁇ of lx DMEM + 10 % FBS to each well of a round-bottom 96-well plate (greiner bio-one, cellstar, Cat. No. 650185). Plates were centrifuged 5 minutes at 1500 rpm and supernatants were flicked off. Cells were washed once with 200 pL of 4 °C cold FACS buffer (eBioscience, Cat. No. GO- 4222-26).
  • the C-terminal xFab domain is directed against FAP, while in the DP47-CD40 molecule the non-binding DP47 domain was used.
  • Cells were incubated with the different molecules for 120 minutes at 4 °C. Afterwards the cells were washed three times with 200 pL 4°C cold FACS buffer.
  • Cells were further stained with 50 pL/well of 4 °C cold secondary antibody solution (1:50 dilution of secondary antibody) containing R-Phycoerythrin (PE) conjugated AffmiPure F(ab')2 Fragment Goat Anti-Human IgG, Fey Fragment Specific (Jackson ImmunoResearch, Cat. No. 109-116-098) and incubated for 60 minutes at 4 °C in the dark. Cells were washed with 200 m ⁇ FACS buffer and finally resuspended in 85 pL/well FACS- buffer containing 0.2 pg/mL 4',6-diamidino-2-phenylindole (DAPI) (Roche, Cat. No. 10236276001) and acquired the same day using a 5-laser LSR-Fortessa (BD Bioscience with DIVA software). Data analysis was performed using the FlowJo version 10 software (FlowJo LLC).
  • the bispecific CEA-CD40 molecules bind to huCEA-expressing MC38 cells, but not to CEA-negative MC38 cells.
  • the FAP- targeted CD40 agonist FAP-CD40 and the non-targeted control antibody DP47-CD40 did not show binding to MC38-huCEA.
  • a stronger huCEA binding was observed for the human CEA-CD40 molecule containing the T84.66 huCEA binding domain in comparison to the human CEA-CD40 molecule with the A5H1EL1D anti-huCEA binder.
  • the binding of the human CEA-CD40 molecules to human CD40 was tested using murine splenic dendritic cells (DCs) isolated from human CD40 transgenic (huCD40tg) mice (mice with similar human and murine CD40 receptor expression pattern; C57BL/6 background; generated by Taconic). As negative control, binding to DCs from C57BL/6 wild type mice was assessed.
  • DCs murine splenic dendritic cells isolated from human CD40 transgenic mice
  • the spleen from a huCD40tg or wild type mouse was put into one well of a 6-well plate containing 2.5 mL Hank's Balanced Salt Solution (HBSS) with Calcium 2+ (gibco, Cat. No. 14025-05), 0.1 mg/ml collagenase D (end concentration 0.1 mg/mL) (Sigma-Aldrich, Cat. No. 11088866001) 0.05 mg/mL DNase solution (Sigma-Aldrich, D5025-150KU).
  • HBSS Hank's Balanced Salt Solution
  • 0.1 mg/ml collagenase D end concentration 0.1 mg/mL
  • DNase solution Sigma-Aldrich, D5025-150KU
  • the spleen was ballooned and subsequently, with the help of scissors, tom into small pieces.
  • 50 pL of 0.5 M ethyl enediaminetetraacetic acid (EDTA) (Applichem, Cat. No. A4892.1000) were added, followed by a second incubation step at 37°C for five minutes.
  • the solution containing splenocytes and small pieces of splenic tissue was filtered through a 40 pm filter (Corning, Cat. No. 352340) into a 50 mL polypropylene centrifuge tube. Splenic tissue pieces were smashed through the filter with the end of a 3 mL syringe plug.
  • the 50 mL tube was centrifuged at 1500 rpm for 5 minutes at room temperature, the supernatant was discarded and 1 mL of lx cell lysis buffer (diluted 1 : 10 with distilled water) (BD, Cat. No. 555899) was added to the splenocytes in order to lyse the red blood cells.
  • 20 mL of R10 medium consisting of Roswell Park Memorial Institute medium (RPMI) 1640 (gibco, Cat. No. 31870-025) supplied with 10 % (v/v) FBS, 1% (v/v) Penicillin Streptomycin (gibco, Cat. No.
  • mice were used according to the manufacturer’s instruction to isolate DCs.
  • 0.25x105 huCD40tg or wild type DCs were added in 200 pi of R10 to each well of a round-bottom 96-well plate (greiner bio-one, cellstar, Cat. No. 650185). Plates were centrifuged 5 minutes at, 1500 rpm and supernatants were flicked off.
  • Cells were further stained with 50 pL/well of 4 °C cold secondary antibody solution (1:50 dilution of secondary antibody) containing R-Phycoerythrin (PE) conjugated AffmiPure F(ab')2 Fragment Goat Anti-Human IgG, Fey Fragment Specific (Jackson ImmunoResearch, Cat. No. 109-116-098) and incubated for 60 minutes at 4 °C in the dark. Cells were washed with 200 pi FACS buffer and finally resuspended in 85 pL/well FACS- buffer containing 0.2 pg/mL DAPI (Roche, Cat. No.
  • FIG. 21 to 2P show that the CEA-CD40 molecules 0817-A5H1EL1D and 0817- T84.66, as well as the human FAP-CD40 and DP47-CD40 antibody bind to huCD40tg DCs. As all the tested antibodies are bivalent for human CD40 and contain the same anti human CD40 domain (0817 domain), the binding behavior of all molecules was very similar. In contrast, no binding was observed to DCs of wild type mice.
  • CD40 Ligation of CD40 induces DC maturation as well as activation and promotes survival.
  • CD40 signaling costimulatory molecule expression on the surface of DCs is increased (S. Quezada et al., Annu Revlmmunol. 2004, 22, 307-328; S. Danese et al., Gut. 2004, 53, 1035-1043; G. Bishop et al., Adv Exp Med Biol. 2007, 597, 131-151).
  • DCs isolated from spleens of huCD40tg mice, which express both the human and murine CD40 receptor were incubated with the huCEA-dependent agonistic anti-CD40 antibodies and either huCEA-coated beads or non- coated beads.
  • DC activation was measured by FACS.
  • DCs were isolated from the spleen of a huCD40tg mouse as described in Example 2.2. 0.25xl0 5 huCD40tg DCs were added in 100 m ⁇ of R10 to each well of a 96-well flat- bottom plate (TPP, Cat. No. 92696). Streptavidin Dynabeads ® (ThermoFisher Scientific, Cat. No.: 11205D) were coated with biotinylated huCEA (biotinylated A2-B2 or A3-B3 CEA domains produced in-house) (binding capacity of 6.5xl0 4 beads: 0.01 pg of protein) according to the manufacturer’s instructions.
  • biotinylated huCEA biotinylated A2-B2 or A3-B3 CEA domains produced in-house
  • beads were coated with the A3-B3 CEA domain, which is recognized by T84.66.
  • beads were coated with the biotinylated A2-B2 domain.
  • CEA-coated or non-coated beads were added to the huCD40tg DCs in a beadxell ratio of 2: 1 in 50 m ⁇ of R10 medium. Usage of beads coated with huCEA instead of huCEA-expressing cells provides a more stable and reproducible system, since fluctuating quality of the cells and secretion of cellular products that might influence DC activation status represent factors that potentially distort results.
  • CEA-targeted anti-human CD40 and anti-murine CD40 antibodies were added in 50 m ⁇ of R10 medium to the DCs (in duplicates). After 24 hours, DCs were analyzed by flow cytometry. For flow cytometry staining DCs were transferred into a 96-well round-bottom plate, washed once with PBS and incubated with 50 m ⁇ of 3 pg/mL of Fc receptor blocking Mouse IgG Isotype Control (ThermoFisher Scientific, Cat. No.l0400C) in FACS buffer. After 10 minutes of incubation at 4°C, cells were washed with PBS and 50 m ⁇ of a mixture of fluorescently labelled antibodies in lx Brilliant Stain Buffer (BD, Cat. No. 563794) was added to the cells. Table 3 shows the fluorescently labelled antibodies used.
  • Table 3 Fluorescently labelled antibodies used to measure murine huCD40tg DC activation
  • Figures 3A to 3D show the CEA-dependent upregulation of DC cell activation markers CD86 (Figure 3A), CD70 (Figure 3B), CD80 ( Figure 3C) and murine CD40 (moCD40) ( Figure 3D) by the bispecific CEA-CD40 molecules.
  • the CEA-CD40 molecules recognizing human CD40 induced a stronger upregulation of the activation markers CD86, CD70 and CD80 compared to the CEA-CD40 molecules recognizing murine CD40.
  • upregulation of murine CD40 could only be measured for the CEA- CD40 molecules binding to human CD40, as the CEA-CD40 molecules against murine CD40 compete with the fluorescently labelled flow cytometry antibody used for detection of murine CD40 on DCs.
  • T cells of these mice all possess a transgenic TCR recognizing the OVA-derived SIINFEKL peptide in the context of H2-Kb; C57BL/6-Tg(TcraTcrb)l lOOMjb/Crl, Charles River) and added to the pulsed DCs. On day five of the experiment, T cell proliferation was assessed by quantifying CD8 + T cell numbers.
  • DCs were isolated from the spleens of huCD40tg mice as described in Example 2.2. Subsequently 0.25xl0 5 DCs were pulsed with OVA.
  • the Ova Antigen Delivery Reagent (Miltenyi, Cat. No. 130-094-663) in combination with a biotinylated anti-mouse DEC205 antibody (Miltenyi, clone NLDC-145, Cat. No. 130-101-854) was used according to the manufacturer’s protocol for this purpose.
  • DCs were incubated with a biotinylated antibody that binds to the DEC205 receptor, which is highly expressed on CD8 + cross- presenting DCs (M.
  • the spleen of an OT-1 mouse was smashed through a 40 pm filter with the end of a 3 mL syringe plug into a 50 mL tube.
  • the filter was washed with R10 and the splenocytes were centrifuged at 1500 rpm for 5 minutes at room temperature.
  • 1 mL of lx cell lysis buffer (diluted 1:10 with distilled water) was added to the cells and after four minutes of incubation at room temperature, 20 mL of R10 were added.
  • the tube was centrifuged at 1500 rpm for 5 minutes at room temperature and the supernatant was discarded.
  • CD8 + cells were isolated in a negative selection process using the mouse CD8a+ T Cell Isolation Kit (Miltenyi, Cat. No. 130-104-075) and LS columns in combination with a QuadroMACSTM Separator according to the manufacturer’s instructions.
  • CD8 + T cells were resuspended in R10 and 0.5xl0 5 cells were added in 100 pi R10 to the OVA-pulsed DCs. On day 5 of the assay, T cell numbers were quantified by flow cytometry.
  • CEA-CD40 antibody 0817-A5H1EL1D (P1AE5314) induced a CEA-dependent proliferation of OT-1 T cells.
  • DCs activated with CEA- CD40 in a CEA-specific manner possess an increased potential to cross-prime CD8 + T cells.
  • CEA-CD40 can bind concomitantly to DCs and huCEA + expressing tumor material, we wanted to test, whether CEA-CD40 could enhance antigen presentation and -Ill- subsequent T cell priming not only by increasing DC activation but also by promoting delivery of tumor antigen to DCs.
  • MuTu DCs a murine green fluorescent protein-expressing DC cell line derived from CD8 + splenic cDCs (SA Fuertes Marraco et al., Front Immunol 2012, 3, 331) with fluorescent huCEA-coated beads and the CEA- targeted anti-murine CD40 antibody FGK4.5-A5B7 (P1AE4998).
  • MuTu DCs and beads were assessed after 2 hours by confocal microscopy.
  • MuTu DCs were grown in IMDMIO consisting of Iscove's Modified Dulbecco's Medium (IMDM, gibco, Cat. No. 31980022) supplemented with 10% (v/v) FBS, 1% (v/v) Penicillin Streptomycin, 10 mM hydroxy ethyl piperazineethanesulfonic acid (HEPES, gibco, Cat. No. 15630106) and 50 mM beta-Mercaptoethanol. MuTu DCs were detached with PBS containing 5 mM EDTA and 2.0xl0 5 MuTu DCs were seeded in 400 m ⁇ IMDMIO on a glass slide (Thermo, Cat. No.
  • the glass slide with the fixed cells was mounted on a coverslip (Thermo, J1800AMNZ) with Fluoromount-G (Thermo, 00-4958-02) and cells were imaged the next day by confocal microscopy using a Zeiss inverted LSM 800 microscope (Plan-Apochromat 63x/1.4 Oil DIC M27 objective) and the Zen software (Zeiss). Images were analyzed with Imaris (Bitplane; Oxford Instruments).
  • Figure 5A shows that CEA-CD40 can induce a strong co-localization of MuTu DCs with huCEA-coated beads but not with non-coated beads.
  • CEA-targeted anti-CD40 antigen binding molecules can enhance not only the delivery of huCEA + beads but also of huCEA + tumor-derived extracellular vesicles (EVs) to DCs.
  • EVs are known to be an extraordinary good source of tumor antigen for induction of tumor-specific T cell priming.
  • injection of EVs isolated from in vitro autologous tumor cell cultures prior to tumor challenge protected mice from tumor establishment.
  • the prophylactic anti -tumor effects of EVs were superior to those of irradiated tumor cells or tumor lysates.
  • administration of EV-loaded DCs induced better anti -tumor immunity in comparison to administration of EVs alone (O Markov et ah, Front Pharmacol 2019,
  • EVs from in vitro cultured MC38-huCEA tumor cells expressing OVA as model antigen MC38-huCEA-OVA.
  • CRISPR/Cas9 clustered regularly interspaced short palindromic repeats/Cas9-induced knockout (KO) of huCEA
  • MC38-huCEA KO-OVA huCEA KO EVs
  • AF555 Alexa Fluor 555
  • MC38-huCEA cells were engineered to express OVA using the pcDNA3.1 MYC- OVA[49-41 l]-IRES-Puro HTS#1641 plasmid (ProQinase).
  • MC38-huCEA-OVA cells were cultured in DMEM + 10% FBS (v/v) + 1% (v/v) Penicillin Streptomycin. 300 pg/ml Geneticin and 1.5 m/ml Puromycin were added to the medium to select for huCEA and OVA transgene expression.
  • a knockout of huCEA was performed in MC38-huCEA-OVA cells by CRISPR/Cas9 technology using the following target DNA sequence: 5’- AACATCATCCAGAATGACAC-3’ (single guide RNA by Invitrogen,
  • CRISPR620861 SGM (MC38-huCEAKO-OVA cells).
  • scr scrambled guide RNA
  • MC38-huCEA-OVA or MC38-huCEAKO-OVA cells were seeded per T150 flask (TPP, Cat. No. 9015) in 20 ml of 0.1 pm filtered DMEM + 10 % EV-depleted FBS (gibco, Cat. No. A2720803) + 1 % Penicillin-Streptomycin and cultured for four days.
  • Supernatants were harvested and pre-cleared by sequential centrifugation at 500 g for 5 min at 4°C, 2000 g for 5 min at 4°C and 4600 g for 20 min at 4°C.
  • Pre-cleared supernatants were subsequently ultracentrifuged using a Thermo Sorvall Ultracentrifuge with a SureSpin 630 rotor (Thermo, 79368). Ultracentrifugation was performed at 134000 g for 70 min at 4°C to pellet EVs. EV pellets were resuspended in 0.1 pm filtered PBS and stored at -80°C.
  • Nanoparticle tracking analysis was employed using a NanoSight NS300 instrument (Malvern Panalytical). For the NanoSight analysis five subsequent measurements over 60 seconds were performed per sample and samples were diluted between 1 : 100 and 1 : 1000 in order to obtain concentrations of 40-80 particles per frame.
  • EVs were incubated with 20 ng/ml of Alexa Fluor 555 NHS Ester dye (Invitrogen, Cat. No. A20009) in a 0.1 M sodium bicarbonate solution for one hour at room temperature with continuous rotation to covalently stain EV lipid membrane proteins. Unbound dye was removed from the EVs by size exclusion chromatography using qEV columns (iZON, Cat. No. SP2) according to the manufacturer’s instructions.
  • lxlO 9 AF555 labelled EVs isolated from MC38-huCEA-OVA or MC38-huCEAKO-OVA cells were added to 0.25xl0 5 splenic huCD40tg DCs (isolated as described in Example 2.2) together with 10 nM FAP-CD40 or CEA-CD40 antibody (0817-A5H1EL1D, P1AE5314). After a three to four hour incubation, DCs were washed and resuspended in flow cytometry staining buffer containing 0.2 pg/mL DAPI. The signal of the labelled EVs was immediately quantified in the population of viable DCs employing flow cytometry.
  • Figure 5B shows that addition of CEA-CD40 significantly increased the labelled huCEA + EV (isolated from MC38-huCEA-OVA cells) signal in DCs after 3 hours in comparison to FAP-CD40 conditions.
  • EVs produced by MC38- huCEAKO-OVA cells were used, no enhanced CEA-CD40-mediated EV delivery could be detected.
  • OT-1 T cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) prior to addition to the DCs.
  • CFSE carboxyfluorescein succinimidyl ester
  • OT-1 T cells were resuspended at a concentration of 2xl0 7 cells/mL in pre-warmed PBS. 10 mM CFSE solution (CellTraceTM CFSE Cell Proliferation Kit, ThermoFisher, Cat. No.
  • C34554 was 5000-fold diluted in pre-warmed PBS and added to the cells resuspended in PBS in a 1 : 1 ratio (CFSE end concentration 1 mM). After a short vortex, cells were incubated for five minutes at room temperature. The labelling reaction was stopped by adding 40 mL of pre-warmed R10 medium to the cells. After two washing steps with PBS, labelled OT-1 T cells were resuspended in R10 and added to the DCs. DC activation was assessed 24 hours after addition of EVs in combination with CEA- CD40 or FAP-CD40.
  • CEA-CD40 XL As a positive control for DC activation, DCs were incubated with 10 nM CEA-CD40 artificially crosslinked by a goat anti-human IgGFcy fragment specific F(ab’)2 fragment (Jackson ImmunoResearch, Cat. No. 109-006-008) (CEA-CD40 XL).
  • DC activation was measured by flow cytometry (staining as described in Example 3.1, staining panel as described in Table 3).
  • OT-1 T cell priming was assessed after 72 hours of co-incubation of T cells and DCs by flow cytometry (staining as described in Example 3.2, staining panel as described in Table 4).
  • CFSE dilution as a measure of T cell proliferation
  • activation markers of live CD 19- CD3 + CD8 + cells were analyzed.
  • T cell numbers were quantified using counting beads as described in Example 3.2.
  • Figures 6A to 6D show that CEA-CD40 in combination with huCEA + EVs greatly enhanced both proliferation and activation of OT-1 T cells compared to conditions with FAP-CD40 and huCEA + EVs.
  • T cell numbers were eight-fold increased in average ( Figure 6A), consistent with a significantly higher fraction of proliferated, CFSE low T cells ( Figure 6B).
  • the expression of CD25 and PD-1 on OT-1 T cells was substantially elevated in CEA-CD40 versus FAP-CD40 conditions ( Figures 6C and 6D).
  • Increased CEA-CD40-mediated T cell priming was dependent on the presence of huCEA on the EVs.
  • huCEA + EVs cannot induce CEA-CD40 cross-linking and concomitant DC activation as their low huCEA surface density and their small size are insufficient to trigger CEA-CD40 clustering.
  • CEA-CD40-mediated enhanced delivery of tumor material such as tumor-derived EVs to DCs
  • CEA-CD40 may therefore possess a novel dual mode of action to promote tumor-specific T cell priming by (i) activating DCs in a CEA-specific manner ( Figure 3) and (ii) increasing delivery of tumor material and tumor antigen to DCs ( Figures 5 and 6).
  • CEA-CD40 fostered human DC-mediated cross-priming of T cells against human tumor EV-derived antigen
  • CD34 + cord blood stem cells (STEMCELL, 70008) were expanded for 7 days in StemSpan SFEM medium (STEMCELL, 09650) supplemented with 20 ng/mL huIL-3 (Peprotech, 200-03), 100 ng/mL huSCF (Peprotech, 300-07), 100 ng/mL huFlt3L (Peprotech, 300-19) and 50 ng/mL huTPO (Peprotech, 300-18).
  • MS-5 stromal cells obtained from DSMZ (ACC 441) were treated for 3 hours with 10 pg/ml proliferation-inhibiting Mitomycin (Sigma, M4287).
  • MS-5 cells were added per well of a 96-well flat bottom plate in 100 m ⁇ MS-5 medium (MEM-alpha (gibco, 15070-063) supplemented with 10 % FBS (gibco, 16140), 1 % Penicillin-Streptomycin (gibco, 11548876) and 2 mM Sodium Pyruvate (gibco, 11360-039).
  • MEM-alpha gibco, 15070-063
  • FBS gibco, 16140
  • Penicillin-Streptomycin gibco, 11548876
  • 2 mM Sodium Pyruvate gibco, 11360-039
  • differentiated cord blood-derived DC Is CD45 + CD14 Clec9A + CD141 + cells
  • a FACSAriaTM III BD Bioscience with DIVA software
  • Sorted DCls were rested overnight.
  • 0.4xl0 5 DCls were incubated with lxlO 8 - 1X10 9 MV3-CEA-MART-1 EVs in combination with 10 nM FAP- CD40 or CEA-CD40 for three hours.
  • DCls were washed and 0.8xl0 5 MART-1 Luciferase reporter T cells were added.
  • TCR signaling was measured by Bioluminescence assay using the Bio-Glo-NLTM Luciferase Assay System (Promega, J3082) according to the manufacturer’s instructions.
  • MART-1 reporter cells were generated by Promega: Jurkat cells were engineered to stably express CD8 and the MART- 1 -specific TCR DMF5, while endogenous CD4 and the endogenous TCR were knocked out.
  • a Luciferase gene under control of the IL-2 promoter was introduced.
  • MV3 cells were received from DJ Ruiter (Amsterdam, Netherlands) and engineered at R1CZ to stably express CEA.
  • HLA-A*02:01 + MV3-CEA cells We pulsed the CEA + human melanoma cell line HLA-A*02:01 + MV3-CEA cells with 100 nM of HLA-A*02:01-restricted short MART-1 peptide (ELAGIGILTV) 2 hours before EV isolation. Peptide pulsing was necessary as MV3 cells do not express any of the T cell-defined melanoma antigens commonly used in immuno-oncology studies (Schadendorf et ah, Br. J. Cancer 1996, 74, 194-199). Supernatants were harvested and EVs were isolated from MV3-CEA-MART-1 tumor cells according to the protocol published by Squadrito et al. (Nat. Methods.
  • MART-1 reporter cells are Jurkat T cells engineered to express CD8 and the MART- 1 -specific TCR DMF5, while the endogenous TCR and CD4 were knocked out.
  • MART-1 reporter cells express Luciferase under control of the IL-2 promoter. Therefore, MART- 1 -specific TCR activation and consecutive IL-2 signaling were measured by bioluminescence assay (see experimental scheme in Figure 7A).

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Abstract

L'invention concerne de nouvelles molécules bispécifiques de liaison à l'antigène, comprenant (a) au moins deux fragments Fab capables de se lier spécifiquement à un CD40, (b) une région Fc composée d'une première et d'un second sous-motif capable d'une association stable comprenant une ou plusieurs substitutions d'acides aminés qui réduisent l'affinité de liaison de la molécule de liaison à l'antigène à un récepteur Fc et/ou à une fonction effectrice et (c) un domaine de liaison à l'antigène capable de se lier spécifiquement à l'antigène carcino-embryonnaire (CEA) relié à l'extrémité C-terminale de la région Fc.
PCT/EP2022/063226 2021-05-19 2022-05-17 Molécules de liaison à l'antigène cd40 agonistes ciblant cea WO2022243261A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024213533A1 (fr) * 2023-04-13 2024-10-17 Alligator Bioscience Ab Polythérapies

Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1993001161A1 (fr) 1991-07-11 1993-01-21 Pfizer Limited Procede de preparation d'intermediaires de sertraline
WO1993016185A2 (fr) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Proteine de liaison biosynthetique pour marqueur de cancer
WO1994029351A2 (fr) 1993-06-16 1994-12-22 Celltech Limited Anticorps
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
WO1997030087A1 (fr) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation d'anticorps glycosyles
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1998058964A1 (fr) 1997-06-24 1998-12-30 Genentech, Inc. Procedes et compositions concernant des glycoproteines galactosylees
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
WO1999022764A1 (fr) 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
WO2002020565A2 (fr) 2000-09-08 2002-03-14 Universität Zürich Groupes de proteines a domaines de repetition comprenant des modules de repetition
US6420548B1 (en) 1999-10-04 2002-07-16 Medicago Inc. Method for regulating transcription of foreign genes
WO2003011878A2 (fr) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2004056312A2 (fr) 2002-12-16 2004-07-08 Genentech, Inc. Variants d'immunoglobuline et utilisations
US6818418B1 (en) 1998-12-10 2004-11-16 Compound Therapeutics, Inc. Protein scaffolds for antibody mimics and other binding proteins
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005056764A2 (fr) 2003-12-05 2005-06-23 Compound Therapeutics, Inc. Inhibiteurs des recepteurs du facteur de croissance endothelial vasculaire de type 2
EP1641818A1 (fr) 2003-07-04 2006-04-05 Affibody AB Polypeptides presentant une affinite de liaison pour le recepteur 2 du facteur de croissance epidermique humain (her2)
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
US7166697B1 (en) 1998-03-06 2007-01-23 Diatech Pty. Ltd. V-like domain binding molecules
US7250297B1 (en) 1997-09-26 2007-07-31 Pieris Ag Anticalins
US20070224633A1 (en) 2003-08-25 2007-09-27 Pieris Ag Muteins of Tear Lipocalin
US7371826B2 (en) 1999-01-15 2008-05-13 Genentech, Inc. Polypeptide variants with altered effector function
US20080139791A1 (en) 1998-12-10 2008-06-12 Adnexus Therapeutics, Inc. Pharmaceutically acceptable Fn3 Polypeptides for human treatments
WO2008098796A1 (fr) 2007-02-16 2008-08-21 Nascacell Technologies Ag Polypeptide comprenant un fragment de protéine knottine
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
WO2009080252A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009089004A1 (fr) 2008-01-07 2009-07-16 Amgen Inc. Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique
WO2012130831A1 (fr) 2011-03-29 2012-10-04 Roche Glycart Ag Variants de fc d'anticorps
WO2016075278A1 (fr) 2014-11-14 2016-05-19 F. Hoffmann-La Roche Ag Molécules de liaison à l'antigène comprenant un ligand trimèrique de la famille du tnf
EP2101823B1 (fr) 2007-01-09 2016-11-23 CureVac AG Anticorps code par un arn
WO2018127473A1 (fr) * 2017-01-03 2018-07-12 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à l'antigène comprenant un clone 20h4.9 anti-4-1bb
WO2018185045A1 (fr) * 2017-04-04 2018-10-11 F. Hoffmann-La Roche Ag Nouvelles molécules bispécifiques de liaison à l'antigène capables de se lier spécifiquement à cd40 et à fap
WO2020070035A1 (fr) * 2018-10-01 2020-04-09 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à l'antigène ayant une liaison trivalent à cd40
WO2020260329A1 (fr) 2019-06-26 2020-12-30 F. Hoffmann-La Roche Ag Fusion d'un anticorps se liant à cea et 4-1bbl

Patent Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
US6417429B1 (en) 1989-10-27 2002-07-09 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1993001161A1 (fr) 1991-07-11 1993-01-21 Pfizer Limited Procede de preparation d'intermediaires de sertraline
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993016185A2 (fr) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Proteine de liaison biosynthetique pour marqueur de cancer
WO1994029351A2 (fr) 1993-06-16 1994-12-22 Celltech Limited Anticorps
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5821333A (en) 1995-03-01 1998-10-13 Genetech, Inc. Method for making heteromultimeric polypeptides
US7695936B2 (en) 1995-03-01 2010-04-13 Genentech, Inc. Knobs and holes heteromeric polypeptides
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
WO1997030087A1 (fr) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation d'anticorps glycosyles
WO1998058964A1 (fr) 1997-06-24 1998-12-30 Genentech, Inc. Procedes et compositions concernant des glycoproteines galactosylees
US7250297B1 (en) 1997-09-26 2007-07-31 Pieris Ag Anticalins
WO1999022764A1 (fr) 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
US7166697B1 (en) 1998-03-06 2007-01-23 Diatech Pty. Ltd. V-like domain binding molecules
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US20080139791A1 (en) 1998-12-10 2008-06-12 Adnexus Therapeutics, Inc. Pharmaceutically acceptable Fn3 Polypeptides for human treatments
US6818418B1 (en) 1998-12-10 2004-11-16 Compound Therapeutics, Inc. Protein scaffolds for antibody mimics and other binding proteins
US7332581B2 (en) 1999-01-15 2008-02-19 Genentech, Inc. Polypeptide variants with altered effector function
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US7371826B2 (en) 1999-01-15 2008-05-13 Genentech, Inc. Polypeptide variants with altered effector function
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
US6420548B1 (en) 1999-10-04 2002-07-16 Medicago Inc. Method for regulating transcription of foreign genes
WO2002020565A2 (fr) 2000-09-08 2002-03-14 Universität Zürich Groupes de proteines a domaines de repetition comprenant des modules de repetition
US20040132028A1 (en) 2000-09-08 2004-07-08 Stumpp Michael Tobias Collection of repeat proteins comprising repeat modules
WO2003011878A2 (fr) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
WO2004056312A2 (fr) 2002-12-16 2004-07-08 Genentech, Inc. Variants d'immunoglobuline et utilisations
EP1641818A1 (fr) 2003-07-04 2006-04-05 Affibody AB Polypeptides presentant une affinite de liaison pour le recepteur 2 du facteur de croissance epidermique humain (her2)
US20070224633A1 (en) 2003-08-25 2007-09-27 Pieris Ag Muteins of Tear Lipocalin
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005056764A2 (fr) 2003-12-05 2005-06-23 Compound Therapeutics, Inc. Inhibiteurs des recepteurs du facteur de croissance endothelial vasculaire de type 2
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
EP2101823B1 (fr) 2007-01-09 2016-11-23 CureVac AG Anticorps code par un arn
WO2008098796A1 (fr) 2007-02-16 2008-08-21 Nascacell Technologies Ag Polypeptide comprenant un fragment de protéine knottine
WO2009080252A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009089004A1 (fr) 2008-01-07 2009-07-16 Amgen Inc. Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique
WO2012130831A1 (fr) 2011-03-29 2012-10-04 Roche Glycart Ag Variants de fc d'anticorps
WO2016075278A1 (fr) 2014-11-14 2016-05-19 F. Hoffmann-La Roche Ag Molécules de liaison à l'antigène comprenant un ligand trimèrique de la famille du tnf
WO2018127473A1 (fr) * 2017-01-03 2018-07-12 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à l'antigène comprenant un clone 20h4.9 anti-4-1bb
WO2018185045A1 (fr) * 2017-04-04 2018-10-11 F. Hoffmann-La Roche Ag Nouvelles molécules bispécifiques de liaison à l'antigène capables de se lier spécifiquement à cd40 et à fap
WO2020070035A1 (fr) * 2018-10-01 2020-04-09 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à l'antigène ayant une liaison trivalent à cd40
WO2020260329A1 (fr) 2019-06-26 2020-12-30 F. Hoffmann-La Roche Ag Fusion d'un anticorps se liant à cea et 4-1bbl

Non-Patent Citations (87)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1990, MACK PRINTING COMPANY
ARMOUR, K.L. ET AL., EUR. J. IMMUNOL., vol. 29, 1999, pages 2613 - 2624
BARR T A ET AL: "Co-stimulatory agonists as immunological adjuvants", VACCINE, ELSEVIER, AMSTERDAM, NL, vol. 24, no. 17, 24 April 2006 (2006-04-24), pages 3399 - 3407, XP025151500, ISSN: 0264-410X, [retrieved on 20060424], DOI: 10.1016/J.VACCINE.2006.02.022 *
BERINSTEIN N. L., J CLIN ONCOL., vol. 20, 2002, pages 2197 - 2207
BIOCHIM BIOPHYS ACTA, vol. 1482, 2000, pages 337 - 350
BOWIE, J. U. ET AL., SCIENCE, vol. 247, 1990, pages 1306 - 10
BRUGGEMANN ET AL., J EXP MED, vol. 166, 1987, pages 1351 - 1361
BURTON, MOL. IMMUNOL., vol. 22
CAPEL, P.J. ET AL., IMMUNOMETHODS, vol. 113, 1994, pages 269 - 315
CARTER, J IMMUNOL METH, vol. 248, 2001, pages 7 - 15
CARTER, J IMMUNOL METHODS, vol. 248, 2001, pages 7 - 15
CHAU I. ET AL., J CLIN ONCOL., vol. 22, 2004, pages 1420 - 1429
CLYNES ET AL., PROC NATL ACAD SCI USA, vol. 95, 1998, pages 652 - 656
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DANESE ET AL., GUT, vol. 53, 2004, pages 1035 - 1043
DE HAAS, M. ET AL., J. LAB. CLIN. MED., vol. 126, 1995, pages 330 - 341
DUNCAN, A.R.WINTER, G., NATURE, vol. 322, 1988, pages 738 - 740
E. SOTOMAYOR ET AL., NAT MED, vol. 5, no. 7, 1999, pages 780 - 7
ELGUETA R. ET AL., IMMUNOL REV., vol. 229, no. 1, 2009, pages 152 - 72
EXPERT OPIN. BIOL. THER., vol. 5, 2005, pages 783 - 797
EXPERT OPINION ON INVESTIGATIONAL DRUGS, vol. 16, no. 6, June 2007 (2007-06-01), pages 909 - 917
FINGL ET AL., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 1975, pages 1
FLAMINI ET AL., CLIN CANCER RES, vol. 12, no. 23, 2006, pages 6985 - 6988
G. BISHOP ET AL., ADV EXP MED BIOL., vol. 597, 2007, pages 131 - 151
GEBAUERSKERRA: "Engineered protein scaffolds as next-generation antibody therapeutics", CURR OPIN CHEM BIO, vol. 13, 2009, pages 245 - 255
GERNGROSS, NAT BIOTECH, vol. 22, 2004, pages 1409 - 1414
GESSNER, J.E. ET AL., ANN. HEMATOL., vol. 76, 1998, pages 231 - 248
GOLDENBERG D M., THE INTERNATIONAL JOURNAL OF BIOLOGICAL MARKERS, vol. 7, 1992, pages 183 - 188
GOLDFREEDMAN, J EXP MED., vol. 121, 1965, pages 439 - 462
GRAHAM ET AL., J GEN VIROL, vol. 36, 1977, pages 59
GUYER, R.L. ET AL., J. IMMUNOL., vol. 117, 1976, pages 587 - 593
H.M. BERMAN ET AL., THE PROTEIN DATA BANK, NUCLEIC ACIDS RESEARCH, vol. 28, 2000, pages 235 - 242
HAMMARSTROM S., SEMIN CANCER BIOL., vol. 9, no. 2, 1999, pages 67 - 81
HEELEY, ENDOCR RES, vol. 28, 2002, pages 217 - 229
HELLSTROM ET AL., PROC NATL ACAD SCI USA, vol. 82, 1985, pages 1499 - 1502
HELLSTROM ET AL., PROC NATL ACAD SCI USA, vol. 83, 1986, pages 7059 - 7063
HOLLINGER ET AL., PROC NATL ACAD SCI USA, vol. 90, 1993, pages 6444 - 6448
HOUSTON, J.S., METHODS IN ENZYMOL., vol. 203, 1991, pages 46 - 96
HUDSON ET AL., NAT MED, vol. 9, 2003, pages 129 - 134
J WOLFERS ET AL., NAT MED, vol. 7, no. 3, 2001, pages 297 - 303
J. BIOL. CHEM, vol. 274, 1999, pages 24066 - 24073
J. MOL. BIOL., vol. 332, 2003, pages 489 - 503
J. MOL. BIOL., vol. 369, 2007, pages 1015 - 1028
KABAT, E.A. ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION
KIM, J.K. ET AL., J. IMMUNOL., vol. 24, 1994, pages 2429 - 2434
LI ET AL., NAT BIOTECH, vol. 24, 2006, pages 210 - 215
LILJEBLAD ET AL., GLYCO J, vol. 17, 2000, pages 323 - 329
M. J. BANFIELD ET AL., PROTEINS, vol. 29, no. 2, 1997, pages 161 - 171
M. LAHOUD ET AL., INT IMMUNOL., vol. 12, no. 5, 2000, pages 731 - 735
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MARSHALL J., SEMIN ONCOL., vol. 30, 2003, pages 30 - 6
MATHER ET AL., ANNALS N.Y. ACAD SCI, vol. 383, 1982, pages 44 - 68
MATHER, BIOL REPROD, vol. 23, 1980, pages 243 - 251
MIZOBATA S ET AL., CANCER IMMUNOL IMMUNOTHER, vol. 49, no. 6, 2000, pages 285 - 295
N. KEMI ET AL., BR J CANCER, vol. 119, no. 4, 2018, pages 435 - 439
NAP ET AL., CANCER RES., vol. 52, no. 8, 1992, pages 2329 - 23339
NAP ET AL., TUMOUR BIOL., vol. 9, no. 2-3, 1988, pages 145 - 53
NATURE BIOTECHNOLOGY, vol. 23, no. 12, pages 1556 - 1561
O MARKOV ET AL., FRONT PHARMACOL, vol. 10, 2019, pages 1152
PNAS, vol. 100, no. 4, 2003, pages 1700 - 1705
PROTEIN ENG. DES. SEL., vol. 17, 2004, pages 455 - 462
PROTEIN ENG. DES. SEL., vol. 18, 2005, pages 435 - 444
R. FRENCH ET AL., NAT MED., vol. 5, no. 5, 1999, pages 548 - 53
R. VONDERHEIDE ET AL., ONCOIMMUNOLOGY, vol. 2, 2013, pages e23033
RAVETCH, J.V.KINET, J.P., ANNU. REV. IMMUNOL., vol. 9
RIDGWAY ET AL., PROT ENG, vol. 9, 1996, pages 617 - 621
ROUX ET AL., J. IMMUNOL., vol. 161, 1998, pages 4083
S. HAMMARSTROM, SEMIN CANCER BIOL, vol. 9, no. 2, 1999, pages 67 - 81
S. QUEZADA ET AL., ANNU REVLMMUNOL., vol. 22, 2004, pages 307 - 328
S. ZUNDER ET AL., CELLULAR ONCOLOGY, vol. 42, no. 5, 2019, pages 717 - 725
SA FUERTES MARRACO ET AL., FRONT IMMUNOL, vol. 3, 2012, pages 331
SAMBROOK ET AL.: "Molecular cloning: A laboratory manual", 1989, GREENE PUBLISHING ASSOCIATES AND WILEY
SCHADENDORF ET AL., BR. J. CANCER, vol. 74, 1996, pages 194 - 199
SCHAEFER, W. ET AL., PNAS, vol. 108, 2011, pages 11187 - 1191
SHIELDS, R.L. ET AL., J. BIOL. CHEM., vol. 276, 2001, pages 6591 - 6604
SQUADRITO ET AL., NAT. METHODS, vol. 15, no. 3, 2018, pages 183 - 186
STADLER, NATURE MEDICINE, 12 June 2017 (2017-06-12)
STAMENKOVIC ET AL., EMBO J., vol. 8, 1989, pages 1403 - 10
STUBENRAUCH ET AL., DRUG METABOLISM AND DISPOSITION, vol. 38, 2010, pages 84 - 91
STUMPP ET AL.: "Darpins: A new generation of protein therapeutics", DRUG DISCOVERY TODAY, vol. 13, 2008, pages 695 - 701, XP023440383, DOI: 10.1016/j.drudis.2008.04.013
SUM ET AL., CLIN. CANCER RES., 2021
URLAUB ET AL., PROC NATL ACAD SCI USA, vol. 77, 1980, pages 4216
VAN DE WINKEL, J.G.ANDERSON, C.L, J. LEUKOC. BIOL., vol. 49, 1991, pages 511 - 524
W. BRENNEN ET AL., MOL CANCER THER, vol. 11, no. 2, 2012, pages 257 - 66
WATTS T.H., ANNU. REV. IMMUNOL., vol. 23, 2005, pages 23 - 68
WRIGHT ET AL., TIBTECH, vol. 15, 1997, pages 26 - 32
YAZAKIWU: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 255 - 268

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* Cited by examiner, † Cited by third party
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