WO2023151613A1 - 一种双特异性抗原结合分子及其应用 - Google Patents

一种双特异性抗原结合分子及其应用 Download PDF

Info

Publication number
WO2023151613A1
WO2023151613A1 PCT/CN2023/075148 CN2023075148W WO2023151613A1 WO 2023151613 A1 WO2023151613 A1 WO 2023151613A1 CN 2023075148 W CN2023075148 W CN 2023075148W WO 2023151613 A1 WO2023151613 A1 WO 2023151613A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
seq
terminus
linker
variable region
Prior art date
Application number
PCT/CN2023/075148
Other languages
English (en)
French (fr)
Inventor
杨柳青
李瑞梅
钱宏亮
Original Assignee
上海齐鲁制药研究中心有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海齐鲁制药研究中心有限公司 filed Critical 上海齐鲁制药研究中心有限公司
Priority to CN202380017523.4A priority Critical patent/CN118591564A/zh
Priority to MX2024009008A priority patent/MX2024009008A/es
Priority to AU2023217790A priority patent/AU2023217790A1/en
Priority to KR1020247026956A priority patent/KR20240141178A/ko
Publication of WO2023151613A1 publication Critical patent/WO2023151613A1/zh

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/50—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
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present disclosure belongs to the field of immunology, and the present disclosure relates to a bispecific antigen-binding molecule.
  • the present disclosure also relates to related coding nucleic acids, vectors, host cells, drugs, and related applications in the treatment of cancer.
  • Monoclonal antibodies have been widely used to treat a variety of diseases including tumors, but mechanisms such as tumor immune evasion limit the long-term effectiveness of monoclonal antibody therapy.
  • One solution is to develop bispecific antibodies, which can bind two different antigens or two different epitopes, so that they can simultaneously block different signaling pathways in the development of tumors, or directly target immune cells to tumor cells , thus potentially producing stronger cytotoxicity and better avoiding immune evasion.
  • Various forms of bispecific antibodies have been developed, however there is still a need to develop new forms of bispecific antibodies.
  • SCLC Small cell lung cancer
  • the only topotecan recommended by the FDA is limited due to its hematological toxicity, and the treatment response rate is not satisfactory, only 5-24%, and the median survival time is less than 25 weeks. At present, there is no specific third-line treatment plan recommended, so a new effective treatment drug is urgently needed.
  • Delta-like protein-3 (Delta-like 3), also known as DLL3, is a protein encoded by the DLL3 gene and is one of the ligands of the Notch family. The protein is involved in the Notch-regulated signaling pathway, resulting in signals from the Notch pathway that promote unrestricted growth of tumor cells.
  • DLL3 is expressed on the surface of tumor cells in about 85% of patients with small cell lung cancer and large cell neuroendocrine carcinoma, and is also highly expressed in glioblastoma multiforme, melanoma, pancreatic cancer and rectal cancer. However, it is not expressed in healthy tissues and non-neuroendocrine tumors, so DLL3 is an ideal target for small cell lung cancer.
  • T cell engagers T cell-engager, TCE double antibody
  • TCE T cell engager
  • the first object of the present disclosure is to provide a novel form of bispecific antigen-binding molecule, the second antigen-binding part of which is wrapped between the first antigen-binding part and the Fc structure, which can reduce the exposure of the second antigen-binding part , thereby reducing the risk of non-specific cytokine release.
  • One form of the bispecific antigen binding molecule comprises:
  • a first polypeptide comprising: (i) an antigen-binding fragment (Fab) heavy chain domain specific for a first antigen, (ii) a single-chain antibody (scFv) structure capable of specifically binding a second antigen domain, and (iii) a first Fc domain;
  • Fab antigen-binding fragment
  • scFv single-chain antibody
  • Fab fragment antigen-binding
  • the antigen-binding fragment (Fab) heavy chain domain and the antigen-binding fragment (Fab) light chain domain form a first binding site for the first antigen
  • the single-chain antibody (scFv) domain forms a first binding site for the first antigen.
  • the second binding site for the second antigen, the first Fc domain and the second Fc domain associate with each other.
  • said single chain antibody (scFv) domain comprises a second heavy chain variable region and a second light chain variable region;
  • the second heavy chain variable region is connected to the second light chain variable region through a first linker or directly, wherein:
  • the C-terminus of the second heavy chain variable region is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the second light chain variable region;
  • the C-terminus of the second light chain variable region is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the second heavy chain variable region;
  • the C-terminus of the second heavy chain variable region is fused to the N-terminus of the second light chain variable region;
  • the C-terminus of the second light chain variable region is fused to the N-terminus of the second heavy chain variable region;
  • the first linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain ;
  • the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, and the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain;
  • the first CH3 domain comprises a "knob” structure
  • the second CH3 domain comprises a "hole” structure
  • the "knob” structure comprises amino acids Substitutions S354C and T366W
  • the "hole” structure comprising amino acid substitutions Y349C, T366S, L368A and Y407V;
  • the Fc domain is derived from IgG1;
  • the single-chain antibody (scFv) domain is connected to the first Fc domain through a second linker or directly, wherein:
  • the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the second linker, and the C-terminus of the second linker is fused to the N-terminus of the first Fc domain;
  • the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the first Fc domain;
  • the second linker comprises the amino acid sequence EPKSS (SEQ ID NO: 34).
  • the antigen-binding fragment (Fab) heavy chain domain comprises a first heavy chain variable region of an immunoglobulin and a CH1 domain, and the C-terminus of the first heavy chain variable region is connected to the CH1 structure The N-terminal fusion of the domain;
  • the antigen-binding fragment (Fab) light chain domain comprises the first light chain variable region of an immunoglobulin and the light chain constant region, the C-terminus of the first light chain variable region is connected to the light chain constant region N-terminal fusion of chain constant region;
  • the heavy chain domain of the antigen-binding fragment is connected to the single-chain antibody (scFv) domain through a third linker or directly, wherein:
  • the C-terminus of the heavy chain domain of the antigen-binding fragment (Fab) is fused to the N-terminus of the third linker, and the C-terminus of the third linker is fused to the N-terminus of the single-chain antibody (scFv) domain; or
  • the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the third linker, and the C-terminus of the third linker is fused to the N-terminus of the antigen-binding fragment (Fab) heavy chain domain; or
  • the C-terminus of the fragment antigen-binding (Fab) heavy chain domain is fused to the N-terminus of the single-chain antibody (scFv) domain;
  • the C-terminal of the single-chain antibody (scFv) domain is fused to the N-terminal of the antigen-binding fragment (Fab) heavy chain domain;
  • the third linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • the first polypeptide comprises the following structure: Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain or scFv domain-third linker-Fab heavy chain domain-second linker-first Fc domain;
  • the first polypeptide comprises the following structure: first heavy chain variable region-CH1-third linker-second heavy chain variable region-first linker-second light chain variable region-second linker - first CH2 - first CH3; or first heavy chain variable region - CH1 - third linker - second light chain variable region - first linker - second heavy chain variable region - second linker - first CH2-the first CH3; or the second heavy chain variable region-the first linker-the second light chain variable region-CH1-the third linker-the first heavy chain variable region-the second linker-the first CH2-the first One CH3; or the second light chain variable region-the first linker-the second heavy chain variable region-CH1-the third linker-the first heavy chain variable region-the second linker-the first CH2-the first CH3;
  • the second polypeptide includes the following structure: second CH2-second CH3;
  • the third polypeptide includes the following structure: first light chain variable region-light chain constant region.
  • the bispecific antigen binding molecule comprises one or more amino acid substitutions selected from the group consisting of: (i) L234A and L235A; (ii) H435R; preferably, the H435R substitution is the second most Substitutions on peptides.
  • the second antigen is CD3, preferably CD3 ⁇ ; preferably, the single-chain antibody (scFv) domain comprises a sequence such as HCDR1 shown in SEQ ID NO: 24, a sequence such as SEQ ID NO: HCDR2 shown in 25, sequence such as HCDR3 shown in SEQ ID NO: 26, sequence such as LCDR1 shown in SEQ ID NO: 27, sequence such as LCDR2 shown in SEQ ID NO: 28 and sequence such as SEQ ID NO: 29 LCDR3 shown;
  • the single-chain antibody (scFv) domain comprises a second heavy chain variable region with a sequence as shown in SEQ ID NO: 21 and a second light chain variable region with a sequence as shown in SEQ ID NO: 22 ;
  • the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the first Fc domain comprises the amino acid sequence shown in SEQ ID NO: 31; the second Fc domain comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the first antigen is DLL3; preferably, the antigen-binding fragment (Fab) heavy chain domain comprises a sequence of HCDR1 as shown in SEQ ID NO: 13, and a sequence as shown in SEQ ID NO: 14 The HCDR2 shown and the HCDR3 whose sequence is shown in SEQ ID NO: 15; the antigen-binding fragment (Fab) light chain domain includes LCDR1 whose sequence is shown in SEQ ID NO: 16, and whose sequence is shown in SEQ ID NO: 17 LCDR2 and LCDR3 whose sequence is shown in SEQ ID NO: 18;
  • the antigen-binding fragment (Fab) heavy chain domain comprises the first heavy chain variable region shown in SEQ ID NO: 11;
  • the antigen-binding fragment (Fab) light chain domain comprises the sequence such as The first light chain variable region shown in SEQ ID NO: 12;
  • the heavy chain domain of the antigen-binding fragment comprises a sequence such as the amino acid sequence shown in SEQ ID NO: 33; the light chain domain of the antigen-binding fragment (Fab) comprises a sequence such as SEQ ID NO: 4 Amino acid sequence shown.
  • the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2; the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3; the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4 amino acid sequence.
  • bispecific antigen-binding molecule comprises two homologous polypeptides, each polypeptide comprising: (i) a Nanobody (VHH) domain capable of specifically binding a first antigen, (ii) capable of specifically binding A single chain antibody (scFv) domain that binds a second antigen, and (iii) an Fc domain; the Fc domains of the two polypeptides associate with each other.
  • VHH Nanobody
  • scFv single chain antibody
  • Fc domains of the two polypeptides associate with each other.
  • said single chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region;
  • the heavy chain variable region is connected to the light chain variable region through a first linker or directly connected, wherein:
  • the C-terminus of the heavy chain variable region is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region;
  • the C-terminus of the light chain variable region is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the heavy chain variable region;
  • the C-terminus of the heavy chain variable region is fused to the N-terminus of the light chain variable region;
  • the C-terminus of the light chain variable region is fused to the N-terminus of the heavy chain variable region;
  • the first linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • the Fc domain comprises a CH2 domain and a CH3 domain of an immunoglobulin, and the C-terminus of the CH2 domain is fused to the N-terminus of the CH3 domain;
  • the Fc domain is derived from IgG1;
  • the Fc domain is connected to the single-chain antibody (scFv) domain through a second linker or directly, wherein:
  • the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the second linker, and the C-terminus of the second linker is fused to the N-terminus of the Fc domain;
  • the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the Fc domain;
  • the second linker comprises the amino acid sequence EPKSS (SEQ ID NO: 34).
  • said Nanobody (VHH) domain is linked to said single chain antibody (scFv) domain via a third linker or directly, wherein:
  • VHH Nanobody
  • scFv single-chain antibody
  • the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of the Nanobody (VHH) domain;
  • VHH Nanobody
  • scFv single-chain antibody
  • the N-terminus of the single-chain antibody (scFv) domain is fused to the C-terminus of the Nanobody (VHH) domain;
  • the third linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • each polypeptide comprises the following structure: VHH domain-third linker-scFv domain-second linker-Fc domain or scFv domain-third linker-VHH domain-second linker-Fc Domain;
  • each polypeptide comprises the following structure: VHH domain-third linker-heavy chain variable region-first linker-light chain variable region-second linker-CH2-CH3, or
  • the bispecific antigen binding molecule comprises the following amino acid substitutions: L234A and L235A.
  • the second antigen is CD3, preferably CD3 ⁇ ; preferably, the single-chain antibody (scFv) domain comprises a sequence such as HCDR1 shown in SEQ ID NO: 24, a sequence such as SEQ ID NO: HCDR2 shown in 25, sequence such as HCDR3 shown in SEQ ID NO: 26, sequence such as LCDR1 shown in SEQ ID NO: 27, sequence such as LCDR2 shown in SEQ ID NO: 28 and sequence such as SEQ ID NO: 29 LCDR3 shown;
  • the single-chain antibody (scFv) domain comprises a heavy chain variable region as shown in SEQ ID NO: 21 and a light chain variable region as shown in SEQ ID NO: 22;
  • the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the Fc domain comprises the amino acid sequence shown in SEQ ID NO:30.
  • the first antigen is DLL3; preferably, the Nanobody (VHH) domain comprises HCDR1 whose sequence is shown in SEQ ID NO: 6, and HCDR2 whose sequence is shown in SEQ ID NO: 7 and the HCDR3 whose sequence is shown in SEQ ID NO: 8;
  • VHH Nanobody
  • said Nanobody (VHH) domain comprises the amino acid sequence shown in SEQ ID NO: 32.
  • each polypeptide of the bispecific antigen binding molecule comprises the amino acid sequence shown in SEQ ID NO:1.
  • the second object of the present disclosure is to provide an anti-DLL3 (preferably anti-DLL3 and CD3) bispecific antigen-binding molecule, which can recruit T cells to tumor sites by targeting immune cell surface antigens (preferably CD3 antigen), It specifically kills tumor cells with high expression of DLL3, while the release level of cytokines in vitro is low, and the safety is good.
  • an anti-DLL3 preferably anti-DLL3 and CD3 bispecific antigen-binding molecule
  • the present disclosure provides a bispecific antigen-binding molecule that binds to a specific epitope, which can bind to:
  • (i) is identical to or overlaps with an epitope directed against a Nanobody comprising HCDR1 having the sequence set forth in SEQ ID NO: 6, HCDR2 having the sequence set forth in SEQ ID NO: 7, and HCDR3 having the sequence set forth in SEQ ID NO: 8 ;or
  • HCDR1 with a sequence as shown in SEQ ID NO: 13, HCDR2 with a sequence as shown in SEQ ID NO: 14, HCDR3 with a sequence as shown in SEQ ID NO: 15, and a sequence as shown in SEQ ID NO: 16
  • the LCDR1, the LCDR2 whose sequence is shown in SEQ ID NO: 17, and the LCDR3 whose sequence is shown in SEQ ID NO: 18 have the same or overlapping epitopes.
  • the epitope to which the bispecific antigen binding molecule is capable of binding :
  • the bispecific antigen binding molecule is capable of binding CD3, preferably CD3 ⁇ .
  • the present disclosure also provides a bispecific antigen binding molecule comprising:
  • the first binding moiety comprises:
  • HCDR1 whose sequence is shown in SEQ ID NO: 6, HCDR2 whose sequence is shown in SEQ ID NO: 7, and HCDR3 whose sequence is shown in SEQ ID NO: 8; or
  • the sequence is HCDR1 as shown in SEQ ID NO: 13, the sequence is HCDR2 as shown in SEQ ID NO: 14, the sequence is HCDR3 as shown in SEQ ID NO: 15, the sequence is LCDR1 as shown in SEQ ID NO: 16 , LCDR2 whose sequence is shown in SEQ ID NO: 17 and LCDR3 whose sequence is shown in SEQ ID NO: 18.
  • said first binding moiety comprises:
  • said second binding moiety is capable of binding CD3, preferably CD3 ⁇ .
  • the present disclosure also provides nucleic acid molecules encoding the aforementioned bispecific antigen-binding molecules.
  • the present disclosure also provides an expression vector comprising the aforementioned nucleic acid molecule.
  • the present disclosure also provides a host cell, which comprises the aforementioned nucleic acid molecule or expression vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or Yeast; more preferably, the mammalian cells are CHO cells, Expi293 or HEK293 cells.
  • the present disclosure also provides a method for preparing a bispecific antigen-binding molecule, the method comprising: culturing the aforementioned host cells under suitable conditions.
  • the present disclosure also provides antibody-drug conjugates, which are formed by conjugating the aforementioned bispecific antigen-binding molecules with other biologically active molecules; preferably, the other biologically active molecules are small-molecule drugs; preferably, the bispecific The specific antigen-binding molecule is linked to the other biologically active molecule via a linker.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned bispecific antigen-binding molecules, nucleic acid molecules, expression vectors, host cells and/or antibody drug conjugates.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more additional therapeutic agents.
  • the present disclosure also provides the use of the aforementioned bispecific antigen-binding molecules, nucleic acid molecules, expression vectors, host cells and/or antibody-drug conjugates in the preparation of drugs for treating, alleviating and/or preventing tumors.
  • said tumor is a DLL3 positive tumor.
  • the present disclosure also provides a method for inducing the death of a cell expressing DLL3, the method comprising allowing the cell to interact with the aforementioned bispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell, antibody-drug conjugate and/or drug
  • the composition is contacted, and the cells expressing DLL3 are tumor cells.
  • the present disclosure also provides a method for treating a disease associated with expression of DLL3 in a subject, the method comprising administering the aforementioned bispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell, antibody to a subject in need Drug conjugates and/or pharmaceutical compositions.
  • the disease is a tumor.
  • the tumor/tumor cells described in the present disclosure are selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of the above tumors.
  • the method further comprises administering to the subject an additional therapeutic agent.
  • the technical solution of the present disclosure has the following beneficial effects: while maintaining a strong tumor cell killing ability in vitro, the release level of non-specific cytokines in vitro is weak or non-existent, and the safety is good.
  • Figure 1 shows the structures of the two bispecific antibodies constructed in the examples.
  • Figure 1A is the double antibody 1
  • Figure 1B is the double antibody 2.
  • Figure 2 shows the binding of BsAb1 and BsAb2 to SHP-77 cells expressing hDLL3.
  • Figure 3 shows the binding of BsAb1 and BsAb2 to Jurkat cells naturally expressing hCD3.
  • Figure 4 shows the cytotoxicity test results (TDCC) of CD3 + T cells to SHP-77 cells mediated by bisantibody 1 and bisantibody 2.
  • Figure 5 shows the cytotoxicity test results (TDCC) of CD3 + T cells to NCI-H82 cells mediated by double antibody 1 and double antibody 2.
  • Figure 6 shows the experimental results of cytokine release with or without target cells in the presence or absence of bisantibody 1 and bisantibody 2.
  • Figure 6A- Figure 6E shows the cytokine release results of the double antibody 1 molecule, which are IFN ⁇ , TNF ⁇ , IL-10, IL-6 and IL4 respectively;
  • Figure 6F- Figure 6J shows the cytokine release results of the double antibody 2 molecule, respectively IFN ⁇ , TNF ⁇ , IL-10, IL-6 and IL4.
  • Figure 7 is a schematic diagram of mouse tumor inoculation locations in the in vivo drug efficacy experiment of BsAb 2.
  • Figure 8 shows the effect of BsAb 2 on the tumor growth weight of the SHP-77 small cell lung cancer model.
  • the term “about” is meant to include ⁇ 20%, or in some cases ⁇ 10%, or in some cases ⁇ 5%, or within ⁇ 1% in some cases, or ⁇ 0.1% in some cases.
  • antibody herein may include whole antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragments thereof (i.e., antigen-binding portions) or single chains thereof, and may also include whole antibodies or antigen-binding fragments thereof or single chains thereof.
  • a product with antigen-specific binding ability formed on the basis of chain modification such as linking other peptides, rearrangement of functional units, etc.).
  • an antibody typically refers to a Y-tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions .
  • Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and constant regions.
  • IgA Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chain constant domains are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • IgG and IgA can be further divided into different For example, IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • this constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain, CH4).
  • CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length.
  • Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.
  • variable region exhibits significant variation in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding.
  • the variable regions of each light chain/heavy chain pair form the antibody combining site such that an intact IgG antibody has two binding sites (ie it is bivalent).
  • the variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions of extreme variability known as hypervariable regions (HVR) or, more commonly, Complementarity determining region (CDR), VH and VL each have four framework regions FR, denoted by FR1, FR2, FR3, FR4 respectively.
  • the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (or light chain variable domain): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)- FR4.
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 refers to the framework region (Frame) 1-4, and wherein CDR1-CDR3 refers to the complementarity determining region 1-3, respectively.
  • VHH refers to a variable antigen binding domain of a heavy chain antibody from the family Camelidae (camelidae, dromedary, llama, alpaca, etc.).
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (for example via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • a scFv may have the VL and VH variable regions described in any order (eg relative to the N-terminus and C-terminus of the polypeptide), the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.
  • Fc is used to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxy-terminus of the heavy chain, e.g., the IgG Fc domain comprises IgG CH2 and IgG CH3 constant domains.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index.
  • association refers to a functional relationship between two or more polypeptide chains and/or two or more portions of a single polypeptide chain.
  • association means that two or more polypeptides (or parts of a single polypeptide) are associated with each other, for example, non-covalently through molecular interactions and/or through one or more binary Sulfur bridges or chemical cross-links are covalently associated, resulting in a functional antigen-binding domain.
  • Examples of possible associations in an antigen binding molecule include, but are not limited to, associations between Fc regions in an Fc domain, associations between VH and VL regions in a Fab or Fv, and associations in a Fab. Association between CH1 and CL.
  • knock-into-hole refers to a modification used to promote the association of the two polypeptide chains of Fc, which comprises a “knob” ( knob) modification and a "hole” modification in the other of the two polypeptide chains of Fc.
  • This technique is described, for example, in US 5,731,168 and US 7,695,936.
  • the method involves introducing a bump ("knob") at the interface of a first polypeptide chain and a corresponding cavity (“cavity”) in the interface of a second polypeptide chain, so that the bump can be placed in the cavity This promotes heterodimer formation and hinders homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide chain with larger side chains such as tyrosine or tryptophan.
  • a complementary cavity of the same or similar size as the bump is created in the interface of the second polypeptide chain by replacing large amino acid side chains with smaller amino acid side chains (eg alanine or threonine).
  • an amino acid residue is replaced with an amino acid residue with a larger side chain volume, determined by This creates a bulge within the CH3 domain of the first polypeptide chain, which can fit into a cavity within the CH3 domain of the second polypeptide chain, and in the CH3 domain of the second polypeptide chain of the Fc domain, an amino acid residue with Substitution of amino acid residues with smaller side chain volumes thereby creating a cavity within the CH3 domain of the second polypeptide chain in which the bulge within the CH3 domain of the first polypeptide chain can be accommodated.
  • said amino acid residues with larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • said amino acid residues with smaller side chain volumes are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
  • linker refers to any means for joining two different functional units (eg antigen-binding fragments).
  • Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers.
  • sequence of the polypeptide linker is not limited.
  • Polypeptide linkers are preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences. Linkers can be long or short depending on the particular construct.
  • linkers linking different functional units preferably comprise flexible peptide linkers, such as glycine-serine peptide linkers.
  • the linker comprises the amino acid sequence (G 4 S) n or (G 4 S) n A, wherein n is any integer selected from 1-10, preferably the amino acid sequence (G 4 S) 3 or (G 4 S) 3 A.
  • the linker joining the VH and VL domains to form a VH-VL or a VL-VH scFv domain preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker.
  • the linker comprises the amino acid sequence (G 4 S) n or (G 4 S) n A, wherein n is any integer selected from 1-10, preferably the amino acid sequence (G 4 S) 3 or (G 4S ).
  • Antibody may be used in the broadest sense herein and may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies (CDR- grafted antibody), human antibody (including recombinantly produced human antibody), recombinantly produced antibody, intrabody, multispecific antibody, bispecific antibody, monovalent antibody, multivalent antibody, anti-idiotype antibody, synthetic antibody ( Including muteins and variants thereof) and the like.
  • monoclonal antibody refers to a substantially homogeneous antibody produced by a single cell clone that only targets a specific epitope.
  • Monoclonal antibodies can be prepared using various techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology or a combination of the above technologies, etc.
  • humanized antibody refers to an antibody in which all or part of the amino acids other than the CDRs of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible so long as they do not eliminate the ability of the antibody to bind a particular antigen.
  • a “humanized” antibody retains similar antigen specificity to the original antibody.
  • chimeric antibody refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, eg, antibodies in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.
  • antibody fragment includes at least a portion of an intact antibody.
  • a "fragment" of an antibody molecule includes an "antigen-binding fragment” of an antibody, and the term “antigen-binding fragment” refers to a fragment of an immunoglobulin or antibody that specifically binds to a selected antigen or an immunogenic determining portion thereof. Or reacted polypeptide fragments, or fusion protein products further derived from such fragments, such as single-chain antibodies, extracellular binding regions in chimeric antigen receptors, etc.
  • Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to: variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear Antibodies, single-chain antibodies (scFv), bispecific antibodies or multispecific antibodies formed from antibody fragments, etc.
  • an antigen refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment.
  • an antigen can include any immunogenic fragment or determinant of a selected target, including single-epitope, multi-epitope, single-structure domains, multiple domains, complete extracellular domains (ECDs), or proteins.
  • ECDs extracellular domains
  • Peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof can constitute antigens.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens, among others.
  • Antigen can also refer to a molecule that elicits an immune response.
  • antigen or cells or preparations containing the antigen can be used to generate antibodies specific for an antigenic determinant.
  • the antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein) or protein Constructs (eg, Fc antigens).
  • the antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art.
  • the DNA encoding the antigen may be genomic or non-genomic (eg, cDNA), and may encode at least a portion of the ECD sufficient to elicit an immunogenic response.
  • Any vector may be used to transform the cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.
  • epitopes and "antigenic determinant” refer to the site on an antigen that specifically binds to an immunoglobulin or an antibody.
  • Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon denaturing solvent treatment. Epitopes generally exist in a unique spatial conformation and comprise at least 3-15 amino acids. Methods for determining the epitope bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • bispecific refers to the ability of an antigen binding molecule to specifically bind two different antigenic determinants.
  • antigen binding molecule in its broadest sense refers to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, eg fragments thereof.
  • bispecific antigen binding molecule refers to a binding molecule (eg, an antibody or a molecule comprising an antibody fragment) specific for two different antigens (or epitopes), preferably a bispecific antibody.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • variable regions in the present disclosure When making antibodies, binding molecules, bispecific binding molecules, or multispecific binding molecules, the constant regions are not particularly limited, and constant regions known to those skilled in the art or obtained by themselves can be used. Amino acid mutations (for example, mutations that increase or decrease binding to Fc ⁇ receptors or FcRn) can be introduced in the constant region portion.
  • the method for obtaining the binding molecule, antigen-binding fragment, antibody, bispecific binding molecule or multispecific binding molecule of the present disclosure is not particularly limited, and can be obtained by any method, such as Cold Spring Harbor's Antibody Experimental Technical Guide, chapters 5-8 and 15 chapters.
  • the binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules or multispecific binding molecules of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid that secretes the antibody can be purified and collected using conventional techniques.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.
  • ADC antibody drug conjugate
  • ADC antibody drug conjugate
  • ADC refers to an antibody to which a therapeutically active substance or active pharmaceutical ingredient (API) has been covalently coupled such that the therapeutically active substance or active pharmaceutical ingredient (API) can be targeted to the binding target of the antibody to demonstrate its pharmacological functions.
  • the therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing cells targeted by the ADC, preferably malignant or cancer cells.
  • Covalent attachment of therapeutically active substances, active pharmaceutical ingredients or cytotoxins can be performed in a non-site-specific manner using standard chemical linkers that couple the payload to lysine or cysteine residues, or preferably, conjugating This is done in a site-specific manner, which allows complete control over the conjugation site as well as the drug to antibody ratio of the resulting ADC.
  • amino acid substitution or “substitution” or “substitution” means the replacement of an amino acid at a specified position in a parent polypeptide sequence with another amino acid.
  • affinity refers to the overall relationship between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The strength of the sum of covalent interactions.
  • KD refers to the dissociation constant for a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultrafast centrifugation and flow cytometry, etc.
  • biological activity refers to the ability of an antibody to bind antigen and cause a measurable biological response, which can be measured in vitro or in vivo.
  • the pharmaceutical composition of the present disclosure can be prepared by mixing with appropriate inert pharmaceutically acceptable carriers, media, etc. as necessary.
  • suitable inert pharmaceutically acceptable carriers for example: physiological saline, sterilized water, excipients, stabilizers, antioxidants (such as ascorbic acid, etc.), buffers, preservatives, surfactants, chelating agents (such as EDTA, etc.) or adhesives, etc.
  • physiological saline sterilized water, excipients, stabilizers, antioxidants (such as ascorbic acid, etc.), buffers, preservatives, surfactants, chelating agents (such as EDTA, etc.) or adhesives, etc.
  • other low molecular weight polypeptides proteins such as serum albumin, gelatin and immunoglobulin, glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine and lysine can also be contained.
  • Amino acids such as acids, sugars such as polysaccharides and monosaccharides or carbohydrates, sugar alcohols such as mannitol and sorbitol.
  • physiological saline isotonic solution containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and Can be mixed with appropriate co-solvents, such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), non-ionic surfactants (polysorbate 80, polysorbate 20, poloxamer 188, HCO- 50) and so on.
  • hyaluronidase in the preparation, subcutaneous administration of a larger liquid volume is also possible.
  • binding molecules or antigen-binding fragments of the present disclosure can be used in combination with other drugs, and the active ingredients can be mixed together to form a single administration unit, or can be used independently as administration units.
  • an effective amount refers to the dose of a pharmaceutical formulation of an antibody or fragment of the present disclosure which, when administered to a patient in single or multiple doses, produces the desired effect in a treated patient.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as ethnic differences; body weight, age and health; the particular disease involved; the severity of the disease; the response of the individual patient; The specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (such as cynomolgus or rhesus or other types of rhesus monkeys), mice, pigs, horses, donkeys, cows, sheep, rats, and any species poultry.
  • the term “disease” or “condition” or “disorder” and the like refers to any change or disorder that damages or interferes with the normal function of a cell, tissue or organ.
  • the “disease” includes but is not limited to: tumor, pathogenic infection, autoimmune disease, T cell dysfunction disease, or immune tolerance defect (such as transplant rejection).
  • neoplastic refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, without inducing or inhibiting normal cell proliferation.
  • treatment refers to clinical intervention in an attempt to alter the course of a disease caused by an individual or a cell, either for prevention or for intervention in the course of clinical pathology.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing down the progression of the disease, improving or relieving the disease, remission or improving the prognosis, etc.
  • bispecific antigen-binding molecule constructed in the examples is to link the heavy chain of anti-DLL3 nanobody or anti-DLL3 full-length antibody Fab segment with the binding domain of human T cell receptor subunit CD3 ⁇ through a flexible linker form.
  • the anti-DLL3 nanobody is hDLL3-3-1-NA, and its sequence is shown in SEQ ID NO:5.
  • the anti-DLL3 full-length antibody is H2-39E2D11-NA, its heavy chain sequence is shown in SEQ ID NO:9, and its light chain sequence is shown in SEQ ID NO:10.
  • the CD3 ⁇ binding domain is derived from the full-length antibody h160C9AA, its heavy chain sequence is shown in SEQ ID NO:19, and its light chain sequence is shown in SEQ ID NO:20.
  • amino acid substitutions of L234A and L235A have been carried out in the Fc segment of the finally constructed bispecific antibody.
  • the heavy chain variable region and the light chain variable region of h160C9AA are connected via a flexible linker to form a single-chain antibody scFv, which has the structure: VH-(G 4 S) 3 -VL, and its sequence is shown in SEQ ID NO:23.
  • the scFv is then fused to the C-terminus of the heavy chain of the Fab segment of the DLL3 full-length antibody or the DLL3 nanobody through a flexible linker.
  • bispecific antibody When the scFv is fused to the C-terminus of the DLL3 Nanobody, the formed bispecific antibody is named as biantibody 1, which contains two homologous chains.
  • the sequence of the fused chain is shown in SEQ ID NO: 1, A schematic diagram is shown in Figure 1A.
  • bispecific antibody When the scFv is fused to the C-terminus of the heavy chain of the Fab segment of the full-length antibody DLL3, the resulting bispecific antibody is named biantibody 2, which contains a light chain (ie, the third polypeptide) and two heterologous heavy chains ( That is, the first polypeptide and the second polypeptide).
  • the heavy chain containing scFv is designed as a "knob" structure (named "knob” structure heavy chain of BsAb 2), including amino acid substitutions at two positions of S354C and T366W .
  • the heavy chain that does not contain scFv is designed as a "hole” structure (named the “hole” structure heavy chain of double antibody 2), including Y349C, T366S, L368A and Amino acid substitutions at four positions in Y407V. And, in order to facilitate the purification of the bispecific antibody, the heavy chain of the "hole” structure is also substituted with H435R.
  • the sequence of the heavy chain of the transformed "section” structure is shown in SEQ ID NO:2
  • the sequence of the heavy chain of the "hole” structure is shown in SEQ ID NO:3
  • the sequence of the light chain is shown in SEQ ID NO:4 (nomenclature is the light chain of Bsantibody 2)
  • the schematic diagram is shown in Figure 1B.
  • BsAb 1 and BsAb2 are summarized in Table 1 and Table 2, respectively.
  • Example 2 Construction of anti-CD3-DLL3 bispecific antibody and its transient transfection expression in eukaryotic cells
  • the gene fragments of the aforementioned bispecific antibody molecules were respectively cloned into PTT5 expression vectors to prepare transfection-grade expression plasmids.
  • Expi293F TM cells (Thermo Fisher Scientific) were cultured in serum-free medium, and the cells were seeded in shake flasks (Corning Inc.), and cultured on a shaker at 37°C in an environment of 8% CO 2 . Adjust the cell density, mix the recombinant expression vector containing the target gene fragment and the PEI transfection reagent in an appropriate ratio, and add it into the cell culture shaker flask, collect the expression supernatant after 6 days of cell culture, and remove the cell debris by high-speed centrifugation, and use Protein A column for affinity purification. Rinse the column with PBS until the A280 reading drops to baseline.
  • FACS was used to detect the binding of anti-CD3-DLL3 bispecific antibody to SHP-77 cells expressing hDLL3 and T lymphocytes (Jurkat) naturally expressing hCD3.
  • SHP-77 cells ATCC, CRL-2195
  • Jurkat cells ATCC, TIB-152
  • the medium of SHP-77 cells and Jurkat cells are RPMI1640+10% FBS, use T75 cell culture flask at 37°C 5% CO 2 incubator culture. Wash the SHP-77 cells with sterile DPBS when the cells are used, digest with 0.25% trypsin EDTA for about 5 minutes, stop with complete medium, and put them into a 50mL centrifuge tube. Jurkat cells were placed directly into 50mL centrifuge tubes without digestion.
  • FCS file from the flow cytometer, use flowjo software to analyze the average fluorescence intensity of the PE channel of each sample (hereinafter referred to as MFI), and import the average fluorescence intensity obtained from the analysis into Graphpad to analyze the half-major binding concentration of the antibody to the cell (hereinafter referred to as EC 50 ), the results are shown in Table 3, Figure 2 (SHP-77 cells) and Figure 3 (Jurkat cells).
  • the binding ability of the two double-antibody molecules to the Jurkat cell line was weaker than that of the SHP-77 cell line, and the binding ability of the double-antibody 2 molecule to the two cells was weaker than that of the double-antibody 1 molecule.
  • the CM5 chip was first activated with EDC and NHS, Mouse mAb against human Fc was then fixed and blocked with ethanolamine.
  • the anti-CD3-DLL3 bispecific antibody molecules were diluted to 0.5 ⁇ g/mL with HBS-EP+(10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% P20) buffer, and captured at a flow rate of 10 ⁇ L/min for 45s.
  • Human source/cynomolgus monkey source DLL3 was serially diluted two-fold to a serial concentration (100nM-0.78nM), bound at a flow rate of 50 ⁇ L/min for 90s, and dissociated for 450s.
  • the CM5 chip was used to directly immobilize the human-derived/cynomolgus monkey Methods of Source CD3 Molecules.
  • the bispecific antibody molecule was diluted twice to a serial concentration (100nM-0.39nM) with HBS-EP+ (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% P20) buffer, and combined at a flow rate of 50 ⁇ L/min for 90s , dissociate for 360s.
  • the experimental results show that the two double antibody molecules can bind to both human/cynomolgus monkey-derived DLL3 and human/cynomolgus monkey-derived CD3.
  • the DLL3 end of the anti-2 molecule has a stronger affinity than the CD3 end.
  • T cell-mediated cytotoxicity assay the target cell is SHP-77
  • SHP-77 cells were cultured, and the culture medium of SHP-77 cells was RPMI1640+10% FBS, and cultured in a T75 cell culture flask placed in a 5% CO 2 incubator at 37°C.
  • CD3 + T cells were sorted from fresh PBMCs with a T cell negative selection kit (StemCell, Cat. No. 17951), counted and the cell density was adjusted to 5E5/mL with RPMI1640+10% FBS. Spread the effector cells into a 96-well plate, 100 ⁇ L/well, and culture at 37°C with 5% CO 2 .
  • the initial concentration is 110nM (11X), and dilute down 5 times.
  • the lymphocytes in the plate were aspirated with a pipette tip, 100 ⁇ L of CTG detection reagent was added to each well, shaken at 300 rpm and incubated in the dark for 10 minutes, and the chemiluminescence was read on the Envision.
  • Cell killing % 1-(sample well reading value-T cell well reading value)/(maximum signal value-T cell well reading value)
  • the reading value of the T cell well is the reading value of the well only adding T cells but not adding target cells SHP-77.
  • GraphPad software calculates EC 50 and maximum lethality, and the results are shown in Table 6 and Figure 4. The maximum killing ability of the two double-antibody molecules on SHP-77 cells can reach 100%, and the half effective concentration of double-antibody 1 molecule is lower than double-antibody 2 molecule.
  • T cell-mediated cytotoxicity assay the target cell is NCI-H82
  • NCI-H82 cells (ATCC, HTB-175) were cultured.
  • the culture medium of NCI-H82 cells was RPMI1640+10% FBS, and cultured in a 5% CO 2 incubator at 37° C. using a T75 cell culture flask. Centrifuge at 1000rpm for 5 minutes and resuspend the cells in DPBS. Count the cells and adjust the cell density to 1E6/ml, add 30nM CellTrace Far Red Cell Proliferation Kit, and incubate in a 5% CO 2 incubator at 37°C for 20 minutes for staining. Add an equal volume of complete medium, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and wash once with DPBS.
  • the target cells were resuspended with complete medium, counted, adjusted the cell density to 2E5/mL and plated in a round bottom culture plate (Corning, Cat. No. 3799), 50 ⁇ L/well.
  • CD3 + T cells were sorted from fresh PBMCs with a T cell negative selection kit (StemCell, Cat. No. 17951), counted and adjusted to 2E6/mL with RPMI1640+10% FBS.
  • Antibody dilution and sample loading Dilute the antibody with complete medium, the initial concentration is 110nM (11X), and dilute down by 5 times. Add the diluted antibody to the cell culture plate, 10 ⁇ L/well, so the initial concentration is 10 nM. Incubate at 37 °C 5% CO2 for 48 hours. Add 10 ⁇ L of life-death dye PI to each well, the final concentration of PI is 4 ⁇ g/mL, mix well, and stain at room temperature for 10 minutes. The cells were filtered with 300 mesh gauze, and the ratio of APC channel (stained to target cells) and PE channel (stained to dead and alive) was detected by flow cytometry.
  • Target cell death % (Far Red+PI+cells)/[(Far Red+PI+cells)+(Far Red+PI-cells)]
  • % cell killing target cell death % - target cell spontaneous death %
  • the target cell spontaneous death % is the number of dead cells in the well with only target cells (NCI-H82) and no T cells
  • GraphPad software calculates EC 50 and maximum lethality, and the results are shown in Table 6 and Figure 5.
  • the literature reports that the expression of DLL3 in NCI-H82 cells is only 1/3 of the expression of DLL3 in SHP-77 cells, so the maximum killing ability of the two double antibody molecules on NCI-H82 cells is far weaker than that of SHP-77 cells, both are about 15%; The half effective concentration of 1 molecule of double antibody is lower than that of 2 molecules of double antibody.
  • T cell-mediated cytotoxicity test results of anti-CD3-DLL3 bispecific antibody
  • SHP-77 cells were cultured, and the culture medium of SHP-77 cells was RPMI1640+10% FBS, and cultured in a T75 cell culture flask placed in a 5% CO 2 incubator at 37°C.
  • the initial concentration is 110nM (11X), and dilute down 5 times.
  • the culture plate was centrifuged at 400g for 10 minutes, and 80 ⁇ L of the supernatant was frozen and stored at -80°C for later use.
  • the cytokine standard was diluted twice, the highest concentration was 5000pg/mL, and the lowest concentration was 20pg/mL. Vortex and mix the microbeads (beads), mix human Th1/Th2 cytokine capture beads 1:1, mix 6 kinds of cytokine beads in equal proportions, add to 96-well plate (corning 3799), 40 ⁇ L/well. Add the prepared standard or the thawed sample to the 96-well plate, 40 ⁇ L/well.
  • 6J show the release results of IFN ⁇ , TNF ⁇ , IL-10, IL-6 and IL4 cytokines of BsAb 2 molecules with or without target cells .), the level of release of various cytokines induced by the double antibody 2 molecule was weaker than that of the double antibody 1 molecule, and the cytokine release levels of the two double antibody molecules were weak or absent under the condition of only PBMC, compared with PBMC and SHP- 77 tumor cell co-incubation conditions have a suitable safety window.
  • the thermal stability of different antibodies in pH7.4PBS buffer was detected by NanoDSF (differential fluorescence scanning technology).
  • the sample concentration is about 1mg/ml, and it is detected by Prometheus NT.Plex (nano DSF).
  • Each sample was centrifuged at 10,000g for 10 minutes before detection.
  • Add 40 ⁇ l of sample to each well of the sample plate (the sample loading volume of the instrument is 10 ⁇ l, and each sample has a duplicate well).
  • the scanning temperature starts at 30°C and ends at 95°C, and the scanning rate is 0.5°C/min.
  • Table 7 Both biantibody molecules showed good thermal stability, and the aggregation temperature (Tagg) of biantibody 2 molecule was higher than that of biantibody 1 molecule.
  • Two naive cynomolgus monkeys were used in the experiment, with free access to water.
  • the dosage of the bispecific antibody is 1 mg/kg, and the intravenous infusion is completed within 30 minutes.
  • Blood collection time points are Pre-dose, 5min (during infusion), 30min (end of administration), 2hr, 4hr, 6hr, 24hr(1d), 48hr(2d), 72hr(3d), 120hr(5d), 168hr( 7d), 336hr (14d), 504hr (21d), 672hr (28d).
  • Collect the whole blood sample in a polyethylene tube without anticoagulant place it at room temperature for about 1 hour, centrifuge at 6000g at 25°C, and immediately divide it into two parts (PK sample and cytokine detection sample) and immediately place it on dry ice and transfer to -80°C refrigerator for long-term storage.
  • the concentration of DLL3/CD3 complete molecule in serum was detected by ELISA method.
  • the concentration of DLL3/CD3 intact molecules was quantified based on the color response.
  • MD company's M5 plate reader detects the absorbance value at a wavelength of 450nm, and uses softmax software to process the data.
  • the unit of the concentration in the above table is ⁇ g/mL, and BLQ is lower than the detection limit.
  • the half-life of the bispecific antibody BsAb 2 in monkeys is 99.9 ⁇ 21h, the Cmax is 25.3 ⁇ 1.2 ⁇ g/mL, and the AUC is 60.4 ⁇ 0.9day* ⁇ g/mL. Therefore, the bispecific antibody is stable in monkeys and does not There is obvious off-target binding, and the pharmacokinetic properties are good.
  • Example 7 Pharmacokinetic experiment of anti-CD3-DLL3 bispecific antibody accompanied by cytokine detection
  • the detection kit is Muti-Analyte Flow assay kit (Biolegend, product number 740391). Before use, add 250 ⁇ L of buffer solution to the lyophilized NHP Th cytokine, mix well, and let stand at room temperature for 10 minutes. Dilute the standard 1:4 with the assay buffer in the kit, and dilute the PK serum sample 1:4 with the assay buffer in the kit. Add 10mL LEGENDplex Assay Buffer to lyophilized Matrix B, dissolve at room temperature for 15 minutes and set aside.
  • Example 8 In vivo drug efficacy experiment of anti-CD3-DLL3 bispecific antibody
  • the experimental animals were all kept in an independent ventilated box with constant temperature and humidity.
  • the temperature of the breeding room was 20.0-26.0°C, the humidity was 40-70%, and the alternating time of day and night was 12h/12h.
  • PBMCs are derived from normal human peripheral blood. 48 hours after inoculation of tumor cells, PBMCs (donor number 5039) were removed from the cryopreservation solution and washed twice with PBS, and then transplanted into mice at 0.8 ⁇ 10 7 /100 ⁇ L/tail vein to establish human Derived immune cells to reconstitute the mouse model. The cell survival rates before and after inoculation were 97.7% and 95.8%, respectively.
  • mice On the 3rd day after tumor inoculation, when the average tumor volume reached 87.91 mm, 35 mice were randomly divided into 5 groups according to the tumor volume, and were divided into human IgG1 (AA) negative control group (purchased from Baiying Bio, Cat. No. B109802), 0.5 mg/kg double antibody 2 treatment group, 0.1mg/kg double antibody 2 treatment group, 0.02mg/kg double antibody 2 treatment group and 0.004mg/kg double antibody 2 treatment group, 7 rats in each group.
  • the day of grouping was defined as D0 day, and intraperitoneal injection was started on D0 day, once every three days, 6 times (see Table 10).
  • T/C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the PBS control group at a certain time point.
  • T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the PBS control group at a specific time point, respectively.
  • the experimental results such as tumor volume, mouse body weight, and tumor weight of animals in each group are expressed as mean ⁇ standard error (Mean ⁇ SEM). Independent samples T-test was used to compare whether there were significant differences between different treatment groups and the control group. Data were analyzed using SPSS. P ⁇ 0.05 means significant difference.
  • the graphing software is Graphpad Prism.
  • the test compared with the hIgG1 (AA) of the control group G1 group, the test
  • double antibody 2 compared with the hIgG1(AA) control group of G1 group, double antibody 2 (0.5 mg/kg) in G2 group, double antibody 2 (0.1 mg/kg) in G3 group, and double antibody 2 (0.02 mg/kg) in G4 group mg/kg) showed obvious growth inhibitory effects on the tumor volume and tumor weight of mice; and this tumor inhibitory effect was exerted through the killing of tumors by T cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本公开提供了双特异性抗原结合分子的形式，还提供了基于该形式构建的针对DLL3的双特异性抗原结合分子、包含所述双特异性抗原结合分子的药物组合物及其在治疗肿瘤方面的相关应用。

Description

一种双特异性抗原结合分子及其应用
本公开要求申请日为2022/02/10的中国专利申请2022101238168的优先权，本公开引用上述中国专利申请的全文。
技术领域
本公开属于免疫学领域，本公开涉及一种双特异性抗原结合分子。本公开还涉及相关的编码核酸、载体、宿主细胞、药物，以及在治疗癌症方面的相关应用。
背景技术
单克隆抗体已广泛应用于治疗包括肿瘤在内的多种疾病，但肿瘤免疫逃避等机制限制了单抗治疗的长期有效性。一个解决办法是开发双特异性抗体，这类抗体能够结合两种不同抗原或两个不同表位，从而能够同时阻断肿瘤发生发展过程中的不同信号通路，或者直接将免疫细胞靶向肿瘤细胞，因而可能产生更强的细胞毒性、更好地避免免疫逃避。目前已经开发了多种形式的双特异性抗体，然而仍然存在开发双特异性抗体的新的形式的需求。
小细胞肺癌(SCLC)约占肺癌的15％-20％，其特征在于高血管分布，肿瘤快速生长，基因组不稳定，早期转移性传播等。据估计，每年确诊的小细胞肺癌患者数超过23.4万例，每年在全球造成的死亡人数约25万人。手术、放疗或者两者联合等局部治疗的方法，几乎不可能将SCLC完全治愈。以铂类为基础的联合化疗依然是治疗的基石，虽然对一线化疗的反应率很高，但复发率也很高，对于局限期的患者中位生存期只有14-20个月，广泛期则只有9-11个月，而复发的患者，生存期更短，也几乎无治疗可选。唯一被FDA推荐的拓扑替康由于其血液学毒性受到限制，治疗反应率也不尽人意，只有5-24％，中位生存期不足25周。目前暂无具体的三线治疗方案推荐，因此急需一种新的有效的治疗药物。
δ样蛋白-3(Delta-like 3)也称为DLL3，是一种由DLL3基因编码的蛋白质，是Notch家族的配体之一。该蛋白参与影响Notch调节信号通路，导致Notch通路发出的信号促使肿瘤细胞不受限制地生长。研究发现，DLL3在大约85％的小细胞肺癌和大细胞神经内分泌癌患者的肿瘤细胞表面表达，另外还高表达于多形性胶质母细胞瘤、黑色素瘤、胰腺癌和直肠癌等。但在健康组织和非神经内分泌肿瘤中不表达，所以DLL3是一个比较理想的小细胞肺癌靶点。
靶向CD3抗原和肿瘤相关抗原的双特异性抗体能够将T细胞与肿瘤细胞在空间上拉近，利用T细胞对肿瘤细胞实现特异性的杀伤作用，这类抗体被称为T细胞衔接器(T cell-engager,TCE双抗)。在临床阶段的双特异性抗体中，有接近一半的候选物靶向CD3抗原，研究TCE双抗的安全性和有效性。虽然相关研究取得了一定进展，但仍存在许多挑战，例如如何平衡抗肿瘤活性和安全性的问题。开发高效低毒的新型T细胞双特异性抗体就显得尤为关键。
因此，需要开发双特异性抗体的新形式，并利用其进一步开发高效低毒的双特异性抗体。
发明概述
本公开的第一个目的在于提供一种具有新颖形式的双特异性抗原结合分子，其第二抗原结合部分包裹在第一抗原结合部分与Fc结构之间，可以减少第二抗原结合部分的暴露，从而减弱非特异细胞因子释放的风险。该双特异性抗原结合分子的一个形式是包含：
(A)第一多肽，其包含：(i)特异性针对第一抗原的抗原结合片段(Fab)重链结构域，(ii)能够特异性结合第二抗原的单链抗体(scFv)结构域，和(iii)第一Fc结构域；
(B)第二多肽，其包含：第二Fc结构域；
(C)第三多肽，其包含：特异性针对第一抗原的抗原结合片段(Fab)轻链结构域；
所述抗原结合片段(Fab)重链结构域与所述抗原结合片段(Fab)轻链结构域形成针对第一抗原的第一结合位点，所述单链抗体(scFv)结构域形成针对第二抗原的第二结合位点，所述第一Fc结构域和所述第二Fc结构域相互缔合。
在一个实施方案中，所述单链抗体(scFv)结构域包含第二重链可变区和第二轻链可变区；
优选地，所述第二重链可变区与所述第二轻链可变区通过第一接头连接或者直接连接，其中：
第二重链可变区的C端与第一接头的N端融合，第一接头的C端与第二轻链可变区的N端融合；或者
第二轻链可变区的C端与第一接头的N端融合，第一接头的C端与第二重链可变区的N端融合；或者
第二重链可变区的C端与第二轻链可变区的N端融合；或者
第二轻链可变区的C端与第二重链可变区的N端融合；
更优选地，所述第一接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
在一个实施方案中，所述第一Fc结构域包含免疫球蛋白的第一CH2结构域和第一CH3结构域，所述第一CH2结构域的C端与第一CH3结构域的N端融合；所述第二Fc结构域包含免疫球蛋白的第二CH2结构域和第二CH3结构域，所述第二CH2结构域的C端与第二CH3结构域的N端融合；
优选地，所述第一CH3结构域包含“节”(knob)结构，所述第二CH3结构域包含“穴”(hole)结构；更优选地，所述“节”(knob)结构包含氨基酸取代S354C和T366W，所述“穴”(hole)结构包含氨基酸取代Y349C、T366S、L368A和Y407V；
优选地，所述Fc结构域来源于IgG1；
优选地，所述单链抗体(scFv)结构域与所述第一Fc结构域通过第二接头连接或者直接连接，其中：
单链抗体(scFv)结构域的C端与第二接头的N端融合，第二接头的C端与第一Fc结构域的N端融合；或者
单链抗体(scFv)结构域的C端与第一Fc结构域的N端融合；
更优选地，所述第二接头包含氨基酸序列EPKSS(SEQ ID NO：34)。
在一个实施方案中，所述抗原结合片段(Fab)重链结构域包含免疫球蛋白的第一重链可变区和CH1结构域，所述第一重链可变区的C端与CH1结构域的N端融合；所述抗原结合片段(Fab)轻链结构域包含免疫球蛋白的第一轻链可变区和轻链恒定区，所述第一轻链可变区的C端与轻链恒定区的N端融合；
优选地，所述抗原结合片段(Fab)重链结构域与所述单链抗体(scFv)结构域通过第三接头连接或者直接连接，其中：
抗原结合片段(Fab)重链结构域的C端与第三接头的N端融合，第三接头的C端与单链抗体(scFv)结构域的N端融合；或者
单链抗体(scFv)结构域的C端与第三接头的N端融合，第三接头的C端与抗原结合片段(Fab)重链结构域的N端融合；或者
抗原结合片段(Fab)重链结构域的C端与单链抗体(scFv)结构域的N端融合；或者
单链抗体(scFv)结构域的C端与抗原结合片段(Fab)重链结构域的N端融合；
更优选地，所述第三接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
在一个实施方案中，所述第一多肽包含如下结构：Fab重链结构域-第三接头-scFv结构域-第二接头-第一Fc结构域或者scFv结构域-第三接头-Fab重链结构域-第二接头-第一Fc结构域；
优选地，所述第一多肽包含如下结构：第一重链可变区-CH1-第三接头-第二重链可变区-第一接头-第二轻链可变区-第二接头-第一CH2-第一CH3；或者第一重链可变区-CH1-第三接头-第二轻链可变区-第一接头-第二重链可变区-第二接头-第一CH2-第一CH3；或者第二重链可变区-第一接头-第二轻链可变区-CH1-第三接头-第一重链可变区-第二接头-第一CH2-第一CH3；或者第二轻链可变区-第一接头-第二重链可变区-CH1-第三接头-第一重链可变区-第二接头-第一CH2-第一CH3；所述第二多肽包含如下结构：第二CH2-第二CH3；所述第三多肽包含如下结构：第一轻链可变区-轻链恒定区。
在一个实施方案中，所述双特异性抗原结合分子包含一个或多个选自以下组的氨基酸取代：(i)L234A和L235A；(ii)H435R；优选地，所述H435R取代是第二多肽上的取代。
在一个实施方案中，所述第二抗原为CD3，优选为CD3ε；优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：24所示的HCDR1、序列如SEQ ID NO：25所示的HCDR2、序列如SEQ ID NO：26所示的HCDR3、序列如SEQ ID NO：27所示的LCDR1、序列如SEQ ID NO：28所示的LCDR2和序列如SEQ ID NO：29所示的LCDR3；
更优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：21所示的第二重链可变区和序列如SEQ ID NO：22所示的第二轻链可变区；
更优选地，所述单链抗体(scFv)结构域包含SEQ ID NO：23所示的氨基酸序列。
在一个实施方案中，所述第一Fc结构域包含SEQ ID NO：31所示的氨基酸序列；所述第二Fc结构域包含SEQ ID NO：3所示的氨基酸序列。
在一个实施方案中，所述第一抗原为DLL3；优选地，所述抗原结合片段(Fab)重链结构域包含序列如SEQ ID NO：13所示的HCDR1、序列如SEQ ID NO：14所示的HCDR2和序列如SEQ ID NO：15所示的HCDR3；所述抗原结合片段(Fab)轻链结构域包含序列如SEQ ID NO：16所示的LCDR1、序列如SEQ ID NO：17所示的LCDR2和序列如SEQ ID NO：18所示的LCDR3；
更优选地，所述抗原结合片段(Fab)重链结构域包含序列如SEQ ID NO：11所示的第一重链可变区；所述抗原结合片段(Fab)轻链结构域包含序列如SEQ ID NO：12所示的第一轻链可变区；
更优选地，所述抗原结合片段(Fab)重链结构域包含序列如SEQ ID NO：33所示的氨基酸序列；所述抗原结合片段(Fab)轻链结构域包含序列如SEQ ID NO：4所示的氨基酸序列。
在一个实施方案中，第一多肽包含SEQ ID NO：2所示的氨基酸序列；第二多肽包含SEQ ID NO：3所示的氨基酸序列；第三多肽包含SEQ ID NO：4所示的氨基酸序列。
该双特异性抗原结合分子的另一个形式是包含同源的两条多肽，每条多肽包含：(i)能够特异性结合第一抗原的纳米抗体(VHH)结构域，(ii)能够特异性结合第二抗原的单链抗体(scFv)结构域，和(iii)Fc结构域；所述两条多肽的Fc结构域相互缔合。
在一个实施方案中，所述单链抗体(scFv)结构域包含重链可变区和轻链可变区；
优选地，所述重链可变区与所述轻链可变区通过第一接头连接或者直接连接，其中：
重链可变区的C端与第一接头的N端融合，第一接头的C端与轻链可变区的N端融合；或者
轻链可变区的C端与第一接头的N端融合，第一接头的C端与重链可变区的N端融合；或者
重链可变区的C端与轻链可变区的N端融合；或者
轻链可变区的C端与重链可变区的N端融合；
更优选地，所述第一接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
在一个实施方案中，所述Fc结构域包含免疫球蛋白的CH2结构域和CH3结构域，所述CH2结构域的C端与CH3结构域的N端融合；
优选地，所述Fc结构域来源于IgG1；
优选地，所述Fc结构域与所述单链抗体(scFv)结构域通过第二接头连接或者直接连接，其中：
单链抗体(scFv)结构域的C端与第二接头的N端融合，第二接头的C端与Fc结构域的N端融合；或者
单链抗体(scFv)结构域的C端与Fc结构域的N端融合；
更优选地，所述第二接头包含氨基酸序列EPKSS(SEQ ID NO：34)。
在一个实施方案中，所述纳米抗体(VHH)结构域与所述单链抗体(scFv)结构域通过第三接头连接或者直接连接，其中：
纳米抗体(VHH)结构域的C端与第三接头的N端融合，第三接头的C端与单链抗体(scFv)结构域的N端融合；或者
单链抗体(scFv)结构域的C端与第三接头的N端融合，第三接头的C端与纳米抗体(VHH)结构域的N端融合；或者
纳米抗体(VHH)结构域的C端与单链抗体(scFv)结构域的N端融合；或者
单链抗体(scFv)结构域的N端与纳米抗体(VHH)结构域的C端融合；
更优选地，所述第三接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
在一个实施方案中，每条多肽包含如下结构：VHH结构域-第三接头-scFv结构域-第二接头-Fc结构域或者scFv结构域-第三接头-VHH结构域-第二接头-Fc结构域；
优选地，每条多肽包含如下结构：VHH结构域-第三接头-重链可变区-第一接头-轻链可变区-第二接头-CH2-CH3，或者
VHH结构域-第三接头-轻链可变区-第一接头-重链可变区-第二接头-CH2-CH3，或者
重链可变区-第一接头-轻链可变区-第三接头-VHH结构域-第二接头-CH2-CH3，或者
轻链可变区-第一接头-重链可变区-第三接头-VHH结构域-第二接头-CH2-CH3。
在一个实施方案中，所述双特异性抗原结合分子包含以下氨基酸取代：L234A和L235A。
在一个实施方案中，所述第二抗原为CD3，优选为CD3ε；优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：24所示的HCDR1、序列如SEQ ID NO：25所示的HCDR2、序列如SEQ ID NO：26所示的HCDR3、序列如SEQ ID NO：27所示的LCDR1、序列如SEQ ID NO：28所示的LCDR2和序列如SEQ ID NO：29所示的LCDR3；
更优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：21所示的重链可变区和序列如SEQ ID NO：22所示的轻链可变区；
更优选地，所述单链抗体(scFv)结构域包含SEQ ID NO：23所示的氨基酸序列。
在一个实施方案中，所述Fc结构域包含SEQ ID NO：30所示的氨基酸序列。
在一个实施方案中，所述第一抗原为DLL3；优选地，所述纳米抗体(VHH)结构域包含序列如SEQ ID NO：6所示的HCDR1、序列如SEQ ID NO：7所示的HCDR2和序列如SEQ ID NO：8所示的HCDR3；
更优选地，所述纳米抗体(VHH)结构域包含SEQ ID NO：32所示的氨基酸序列。
在一个实施方案中，所述双特异性抗原结合分子的每条多肽包含SEQ ID NO：1所示的氨基酸序列。
本公开的第二个目的在于提供一种抗DLL3(优选抗DLL3和CD3)的双特异性抗原结合分子，该分子能够通过靶向免疫细胞表面抗原(优选CD3抗原)招募T细胞到肿瘤部位，特异性地杀伤高表达DLL3的肿瘤细胞，而体外细胞因子的释放水平较低，安全性好。
基于此，本公开提供了一种结合特定表位的双特异性抗原结合分子，其能够结合的表位：
(i)与包含序列如SEQ ID NO：6所示的HCDR1、序列如SEQ ID NO：7所示的HCDR2和序列如SEQ ID NO：8所示的HCDR3的纳米抗体针对的表位相同或重叠；或者
(ii)与包含序列如SEQ ID NO：13所示的HCDR1、序列如SEQ ID NO：14所示的HCDR2、序列如SEQ ID NO：15所示的HCDR3、序列如SEQ ID NO：16所示的LCDR1、序列如SEQ ID NO：17所示的LCDR2和序列如SEQ ID NO：18所示的LCDR3的抗体针对的表位相同或重叠。
在一个实施方案中，所述双特异性抗原结合分子能够结合的表位：
(i)与包含SEQ ID NO：32所示的氨基酸序列的纳米抗体针对的表位相同或重叠；或者
(ii)与包含序列如SEQ ID NO：11所示的第一重链可变区和序列如SEQ ID NO：12所示的第一轻链可变区的抗体针对的表位相同或重叠。
在一个实施方案中，所述双特异性抗原结合分子能与CD3结合，优选能与CD3ε结合。
本公开还提供了一种双特异性抗原结合分子，其包含：
(A)能够特异性结合DLL3的第一结合部分；以及
(B)第二结合部分，所述第二结合部分特异性结合的抗原或表位与第一结合部分不同；
所述第一结合部分包含：
(i)序列如SEQ ID NO：6所示的HCDR1、序列如SEQ ID NO：7所示的HCDR2和序列如SEQ ID NO：8所示的HCDR3；或者
(ii)序列如SEQ ID NO：13所示的HCDR1、序列如SEQ ID NO：14所示的HCDR2、序列如SEQ ID NO：15所示的HCDR3、序列如SEQ ID NO：16所示的LCDR1、序列如SEQ ID NO：17所示的LCDR2和序列如SEQ ID NO：18所示的LCDR3。
在一个实施方案中，所述第一结合部分包含：
(i)SEQ ID NO：32所示的氨基酸序列；或者
(ii)序列如SEQ ID NO：11所示的第一重链可变区和序列如SEQ ID NO：12所示的第一轻链可变区。
在一个实施方案中，所述第二结合部分能与CD3结合，优选能与CD3ε结合。
本公开还提供了核酸分子，其编码前述双特异性抗原结合分子。
本公开还提供了表达载体，其包含前述核酸分子。
本公开还提供了宿主细胞，其包含前述核酸分子或者表达载体；优选地，所述宿主细胞为原核细胞或真核细胞；所述原核细胞优选大肠杆菌；所述真核细胞优选哺乳动物细胞或酵母；更优选地，所述哺乳动物细胞为CHO细胞、Expi293或HEK293细胞。
本公开还提供了制备双特异性抗原结合分子的方法，所述方法包括：在适合的条件下培养前述宿主细胞。
本公开还提供了抗体药物偶联物，其是将前述双特异性抗原结合分子与其他生物活性分子偶联形成；优选地，所述其他生物活性分子为小分子药物；优选地，所述双特异性抗原结合分子与所述其他生物活性分子通过接头连接。
本公开还提供了药物组合物，其包含前述双特异性抗原结合分子、核酸分子、表达载体、宿主细胞和/或抗体药物偶联物。
在一个实施方案中，所述药物组合物还包含药学上可接受的载体。
在一个实施方案中，所述药物组合物还包含一种或多种额外的治疗剂。
本公开还提供了前述双特异性抗原结合分子、核酸分子、表达载体、宿主细胞和/或抗体药物偶联物在制备治疗、缓解和/或预防肿瘤的药物中的用途。优选地，所述肿瘤是DLL3阳性的肿瘤。
本公开还提供一种诱导表达DLL3的细胞死亡的方法，所述方法包括使所述细胞与前述双特异性抗原结合分子、核酸分子、表达载体、宿主细胞、抗体药物偶联物和/或药物组合物接触，所述表达DLL3的细胞是肿瘤细胞。
本公开还提供一种治疗受试者中与表达DLL3相关的疾病的方法，所述方法包括向有需要的受试者施用前述双特异性抗原结合分子、核酸分子、表达载体、宿主细胞、抗体药物偶联物和/或药物组合物。优选地，所述疾病是肿瘤。
优选地，本公开所述肿瘤/肿瘤细胞选自：小细胞肺癌、胶质母细胞瘤、神经内分泌癌、黑色素瘤、胰腺癌、直肠癌以及上述肿瘤的转移癌。
在一个实施方案中，所述方法还包括向所述受试者给予额外的治疗剂。
本公开所称取代优选采用EU编号系统表示。
本公开的技术方案具有如下有益效果：保持较强体外肿瘤细胞杀伤能力的同时，体外非特异细胞因子释放水平较弱或无，安全性好。
附图说明
附图更进一步说明了本说明书所公开的新特性。参照这些附图将能更好地理解本说明书中所公开的特性和优点，但应当理解，这些附图仅用于说明本文所公开原理的具体的实施方案，而非意欲对所附权利要求的范围加以限制。
图1显示了实施例中构建的2个双特异性抗体的结构。图1A为双抗1，图1B为双抗2。
图2显示了双抗1和双抗2与表达hDLL3的SHP-77细胞的结合情况。
图3显示了双抗1和双抗2与天然表达hCD3的Jurkat细胞的结合情况。
图4显示了双抗1和双抗2介导的CD3+T细胞对SHP-77细胞的细胞毒性实验结果(TDCC)。
图5显示了双抗1和双抗2介导的CD3+T细胞对NCI-H82细胞的细胞毒性实验结果(TDCC)。
图6显示了双抗1和双抗2在有无靶细胞的条件下细胞因子释放的实验结果。图6A-图6E为双抗1分子的细胞因子释放结果，分别为IFNγ，TNFα，IL-10，IL-6和IL4；图6F-图6J为双抗2分子的细胞因子释放结果，分别为IFNγ，TNFα，IL-10，IL-6和IL4。
图7是双抗2体内药效实验中小鼠肿瘤接种位置示意图。
图8显示了双抗2对SHP-77小细胞肺癌模型肿瘤生长重量的影响。
发明详述
术语
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文，所述引用的程度就如同已特定地和个别地指示将各个别公布、专利或专利申请以引用的方式并入本文。
在下文详细描述本公开前，应理解本公开不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本公开的范围。除非另外定义，本文中使用的所有技术和科学术语与本公开所属领域中普通技术人员通常的理解具有相同的含义。
本文所公开的某些实施方案包含了数值范围，并且本公开的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本公开的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时，术语“约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
本文中的术语“抗体”可以包含完整抗体(例如全长单克隆抗体)及其任何抗原结合片段(即抗原结合部分)或其单链，还可以包含在完整抗体或其抗原结合片段或其单链的基础上进行改造(例如连接其他肽段、功能单位重排等)而形成的具有抗原特异性结合能力的产物。
在一个实施方案中，抗体典型是指包含通过共价二硫键和非共价相互作用保持在一起的两条重(H)多肽链和两条轻(L)多肽链的Y型四聚蛋白。天然IgG抗体即具有这样的结构。每条轻链由一个可变结构域(VL)和一个恒定结构域(CL)组成。每条重链包含一个可变结构域(VH)和恒定区。
本领域已知五个主要类别的抗体：IgA，IgD，IgE，IgG和IgM，对应的重链恒定结构域分别被称为α，δ，ε，γ和μ，IgG和IgA可以进一步分为不同的亚类，例如IgG可分为IgG1，IgG2，IgG3，IgG4，IgA可分为IgA1和IgA2。来自任何脊椎动物物种的抗体的轻链基于其恒定结构域的氨基酸序列可以被分配到两种明显相异的类型之一，称为κ和λ。
在IgG、IgA和IgD抗体的情形中，该恒定区包含称为CH1、CH2和CH3的三个结构域(IgM和IgE具有第四结构域CH4)。在IgG、IgA和IgD类别中，CH1和CH2结构域被柔性铰链区分离，该铰链区是可变长度的富含脯氨酸和半胱氨酸的区段。每类抗体进一步包含由配对半胱氨酸残基形成的链间和链内二硫键。
术语“可变区”或“可变结构域”显示出从一种抗体到另一种抗体的氨基酸组成的显著变化，并且主要负责抗原识别和结合。每个轻链/重链对的可变区形成抗体结合位点，使得完整的IgG抗体具有两个结合位点(即它是二价的)。重链的可变区(VH)和轻链的可变区(VL)结构域各包含具有极端变异性的三个区域，被称为高变区(HVR)，或更通常地，被称为互补决定区(CDR)，VH和VL各有4个骨架区FR，分别用FR1，FR2，FR3，FR4表示。因此，CDR和FR序列通常出现在重链可变结构域(或轻链可变结构域)的以下序列中：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
术语“纳米抗体”是指具有下述(通用)结构的氨基酸序列：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。其中，FR1-FR4分 别是指构架区(Frame)1-4，并且其中CDR1-CDR3分别是指互补决定区1-3。“VHH”涉及来自骆驼科(骆驼、单峰骆驼、美洲驼、羊驼等)重链抗体的可变抗原结合结构域。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，scFv可以以任何顺序(例如相对于多肽的N-末端和C-末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
术语“Fc”用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。虽然IgG重链的Fc区的边界可以略微变化，但是人IgG重链Fc区通常定义为自Cys226或Pro230延伸至重链的羧基端，例如，IgG Fc域包含IgG CH2和IgG CH3恒定域。除非本文中另外指定，Fc区或恒定区中氨基酸残基的编号方式依照EU编号系统，也称作EU索引。
术语“缔合”(association)是指两个或更多个多肽链和/或单一多肽链的两个或更多个部分之间的功能性关系。特别地，所述术语“缔合”意指两个或更多个多肽(或单一多肽的部分)彼此缔合，例如，通过分子相互作用非共价缔合和/或通过一个或多个二硫桥或化学交联共价缔合，从而产生功能性抗原结合结构域。抗原结合分子中可能存在的缔合的实例包括(但不限于)Fc结构域中的Fc区之间的缔合、Fab或Fv中的VH区和VL区之间的缔合、以及Fab中的CH1和CL之间的缔合。
术语“节-入-穴”(Knob-into-Hole)是指一种用于促进Fc的两条多肽链缔合的修饰，其包含在Fc的两个多肽链之一中的“节”(knob)修饰和在Fc的两个多肽链之另一中的“穴”(hole)修饰。该技术记载于例如US 5,731,168和US 7,695,936。一般地，该方法牵涉在第一多肽链的界面处引入隆起(“节”)并在第二多肽链的界面中引入相应的空腔(“穴”)，使得隆起可以置于空腔中从而促进异二聚体形成并阻碍同二聚体形成。通过将来自第一多肽链界面的小氨基酸侧链用更大的侧链(例如酪氨酸或色氨酸)替换来构建隆起。在第二多肽链的界面中创建具有与隆起相同或相似大小的互补性空腔，其通过将大氨基酸侧链用更小的氨基酸侧链(例如丙氨酸或苏氨酸)替换进行。
因而，在一个具体的实施方案中，在本公开的双特异性抗体的Fc域的第一多肽链的CH3域中，一个氨基酸残基用具有更大侧链体积的氨基酸残基替换，由此在第一多肽链的CH3域内生成隆起，其可安置于第二多肽链的CH3域内的空腔中，而且在Fc域的第二多肽链的CH3域中，一个氨基酸残基用具有更小侧链体积的氨基酸残基替换，由此在第二多肽链的CH3域内生成空腔，其中可安置第一多肽链的CH3域内的隆起。优选地，所述具有更大侧链体积的氨基酸残基选自下组：精氨酸(R)，苯丙氨酸(F)，酪氨酸(Y)，和色氨酸(W)。优选地，所述具有更小侧链体积的氨基酸残基选自下组：丙氨酸(A)，丝氨酸(S)，苏氨酸(T)，和缬氨酸(V)。
术语“接头”是指用于连接两个不同功能单元(例如抗原结合片段)的任何工具。接头的类型包括但不限于化学接头和多肽接头。多肽接头的序列不受限制。多肽接头优选是非免疫原性和柔性的，例如包含丝氨酸和甘氨酸序列的那些。取决于具体的构建体，接头可以长或短。
根据本公开，连接不同功能单元的接头优选包含柔性肽接头，例如甘氨酸-丝氨酸肽接头。在一个实施方案中，接头包含氨基酸序列(G4S)n或(G4S)nA，其中n是1-10中的任意整数选择，优选包含氨基酸序列(G4S)3或(G4S)3A。连接VH和VL结构域以形成VH-VL或VL-VH的scFv结构域的接头优选包含柔性肽接头，例如甘氨酸-丝氨酸肽接头。在一个实施方案中，接头包含氨基酸序列(G4S)n或(G4S)nA，其中n是1-10中的任意整数选择，优选包含氨基酸序列(G4S)3或(G4S)。
本文中“抗体”可在最广的意义上使用，可包括如多克隆抗体(polyclonal antibodies)、单克隆抗体、嵌合抗体、人源化抗体及灵长类化抗体、CDR移植抗体(CDR-grafted antibody)、人类抗体(包括重组产生的人类抗体)、重组产生的抗体、胞内抗体、多特异性抗体、双特异性抗体、单价抗体、多价抗体、抗个体基因型抗体、合成抗体(包括突变蛋白及其变体)等等。
术语“单克隆抗体”(或称“单抗”)指由单一细胞克隆产生的基本均质、仅针对某一特定抗原表位的抗体。单克隆抗体可以使用本领域中已知的多种技术制备，包括杂交瘤技术、重组技术、噬菌体展示技术、转基因动物、合成技术或上述技术的组合等。
需说明的是，本公开的抗体和双特异性抗原结合分子的可变区的CDR和FR的划分是根据Kabat定义确定的。而其他命名和编号系统，例如Chothia、IMGT或AHo等，也是本领域技术人员已知的。因此，以本公开的单抗序列为基础，包含任何命名系统衍生的一种或多种CDR的人源化抗体均明确地保持在本公开的范围内。
术语“人源化抗体”是指其中非人抗体(如小鼠抗体)CDR以外的所有或部分氨基酸被源自人免疫球蛋白的相应氨基酸置换的抗体。氨基酸的少量添加、缺失、插入、取代或修饰是容许的，只要它们不消除抗体结合特定抗原的能力。 “人源化”抗体保持与原抗体类似的抗原特异性。
术语“嵌合抗体”是指其中可变区源自一个物种而恒定区源自另一物种的抗体，例如其中可变区源自小鼠抗体而恒定区源自人抗体的抗体。
术语“抗体片段”包含完整抗体的至少一部分。如在此所使用，抗体分子的“片段”包括抗体的“抗原结合片段”，并且术语“抗原结合片段”是指免疫球蛋白或抗体中与所选抗原或其免疫原性决定部分特异性结合或反应的多肽片段，或由此片段进一步衍生的融合蛋白产物，例如单链抗体，嵌合抗原受体中的胞外结合区等。示例性的抗体片段或其抗原结合片段包括但不限于：可变轻链片段、可变重链片段、Fab片段、F(ab’)2片段、Fd片段、Fv片段、单结构域抗体、线性抗体、单链抗体(scFv)及由抗体片段形成的双特异性抗体或多特异性抗体等。
术语“抗原”是指被抗体或抗体结合片段识别并特异性结合的物质，广义上，抗原可以包括所选靶标的任何免疫原性片段或决定簇，包括单表位、多表位、单结构域、多结构域、完整的胞外结构域(ECD)或蛋白质。肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合均可构成抗原。非限制性示例性抗原包括肿瘤抗原或病原体抗原等。“抗原”也可以指引发免疫反应的分子。任何形式的抗原或含有该抗原的细胞或制剂都可以用于生成对抗原决定簇具有特异性的抗体。抗原可以是分离的全长蛋白质、细胞表面蛋白(例如，用在其表面上表达至少一部分抗原的细胞进行免疫的)、或可溶性蛋白质(例如，仅用该蛋白质的ECD部分进行免疫的)或蛋白质构建体(例如，Fc抗原)。该抗原可以在基因修饰的细胞中产生。前述任何抗原可以单独或与本领域已知的一种或多种免疫原性增强佐剂组合使用。编码该抗原的DNA可以是基因组的或非基因组的(例如，cDNA)，并且可以编码足以引起免疫原性应答的至少一部分ECD。可以使用任何载体来转化其中表达抗原的细胞，所述载体包括但不限于腺病毒载体、慢病毒载体、质粒以及非病毒载体如阳离子脂质。
术语“表位”、“抗原决定簇”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象存在并且包括至少3-15个氨基酸。由给定的抗体确定其结合的表位的方法是本领域熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术，例如X射线晶体分析法和二维核磁共振等。
术语“双特异性的”是指抗原结合分子能够特异性结合两个不同的抗原决定簇。术语“抗原结合分子”在其最广泛的含义上指特异性结合抗原决定簇的分子。抗原结合分子的实例是免疫球蛋白及其衍生物，例如片段。术语“双特异性抗原结合分子”指对两种不同抗原(或表位)具有特异性的结合分子(例如抗体或包含抗体片段的分子)，优选双特异性抗体。
术语“特异性结合”是指该结合对抗原具有选择性，且可以与不想要或非特异的相互作用区分开。可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术来测定抗体结合特异性抗原决定簇的能力，该其他技术例如表面等离振子共振(SPR)技术(在BIAcore仪器上分析)。
本公开中的可变区制作抗体、结合分子、双特异性结合分子或多特异性结合分子时，恒定区没有特别限定，可以使用本领域技术人员公知的恒定区或者自行获得的恒定区，还可以在恒定区部分导入氨基酸突变(例如提高或降低与Fcγ受体或FcRn的结合的突变)。
获得本公开的结合分子、抗原结合片段、抗体、双特异性结合分子或多特异性结合分子的方法没有特别限制，可通过任意方法获得，例如冷泉港的抗体实验技术指南，5-8章和15章。本公开的结合分子、抗原结合片段、抗体、双特异性结合分子或多特异性结合分子可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。
术语“抗体药物偶联物”(ADC)是指已共价偶联治疗活性物质或活性药物成分(API)的抗体，从而治疗活性物质或活性药物成分(API)可以靶向至抗体的结合靶标以表现出其药理学功能。治疗活性物质或活性药物成分可以是能够杀死ADC靶向的细胞的细胞毒素，优选恶性或癌细胞。治疗活性物质、活性药物成分或细胞毒素的共价连接可以以非位点特异性方式利用偶联有效载荷至赖氨酸或半胱氨酸残基的标准化学接头进行，或者优选地，缀合以位点特异性方式进行，其允许完全控制缀合位点以及产生的ADC的药物比抗体比例。
术语“氨基酸取代”或“取代”或“替代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。
术语“亲和力”或“结合亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非 共价相互作用总和的强度。术语“KD”是指特定的抗体-抗原相互作用的解离常数。可以使用本领域已知的各种技术来确定结合亲和力，例如表面等离子体共振、生物层干涉法、双极化干涉法、静态光散射、动态光散射、等温滴定量热法、ELISA、分析超速离心和流式细胞术等。
术语“生物学活性”指抗体结合抗原并导致可测量的生物学反应的能力，所述生物学反应可以在体外或体内进行测量。
本公开的药物组合物，可以根据需要与对其为惰性的适当的药学上可接受的载体、介质等进行混和而制剂化。例如：生理盐水、灭菌水、赋形剂、稳定剂、抗氧化剂(如抗坏血酸等)、缓冲剂、防腐剂、表面活性剂、螯合剂(如EDTA等)或粘合剂等。另外，还可以含有其它低分子量的多肽、血清白蛋白、明胶和免疫球蛋白等蛋白质、甘氨酸、谷氨酰胺、天冬酰胺、谷氨酸、天冬氨酸、蛋氨酸、精氨酸和赖氨酸等氨基酸、多糖和单糖等糖类或碳水化物、甘露糖醇和山梨糖醇等糖醇。在制为注射用水溶液时，可举出例如生理盐水、含葡萄糖和其它辅药的等渗液，例如，D-山梨糖醇、D-甘露糖、D-甘露糖醇、氯化钠，还可以与适当的助溶剂，例如醇(乙醇等)、多元醇(丙二醇、PEG等)、非离子型表面活性剂(聚山梨醇酯80、聚山梨醇酯20、泊洛沙姆188、HCO-50)等并用。另外，通过在制剂中混合透明质酸酶(hyaluronidase)，还可以进行更大液量的皮下给药。
本公开的结合分子或抗原结合片段可与其他药物联合使用，活性成分可以混合在一起形成单一的给药单元，也可分别独立成为给药单元，分别使用。
术语“有效量”指本公开的抗体或片段的药物制剂的剂量，其以单一或多次剂量施用患者后，在治疗的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如人种差异；体重、年龄和健康状况；涉及的具体疾病；疾病的严重程度；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
本文所用的术语“个体”或“受试者”是指任何动物，例如哺乳动物或有袋动物。本公开的个体包括但不限于人类、非人类灵长类动物(例如食蟹猴或恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如，所述的“疾病”包括但不限于：肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的，不诱导或抑制正常细胞增殖。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的的疾病过程中的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
具体实施方式
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
实施例1.抗CD3-DLL3双特异性抗体的设计及序列
实施例中构建的双特异性抗原结合分子(以下简称双特异性抗体)是将抗DLL3纳米抗体或抗DLL3全长抗体Fab段重链与人T细胞受体亚基CD3ε的结合结构域通过柔性接头连接形成。其中，抗DLL3纳米抗体为hDLL3-3-1-NA，序列如SEQ ID NO:5所示。抗DLL3全长抗体为H2-39E2D11-NA，其重链序列如SEQ ID NO:9所示，轻链序列如SEQ ID NO:10所示。CD3ε结合结构域来自于全长抗体h160C9AA，其重链序列如SEQ ID NO:19所示，轻链序列如SEQ ID NO:20所示。为降低抗体的ADCC活性，最终构建的双特异性抗体的Fc段均已进行了L234A和L235A的氨基酸取代。
将h160C9AA的重链可变区和轻链可变区经由柔性接头连接形成单链抗体scFv，其结构为：VH-(G4S)3-VL，序列如SEQ ID NO:23所示。所述scFv再通过柔性连接头融合到DLL3全长抗体Fab段重链或DLL3纳米抗体的C末端。
当scFv融合至DLL3纳米抗体的C末端时，则所形成的双特异性抗体被命名为双抗1，包含同源的两条链，融合后的链的序列如SEQ ID NO:1所示，示意图见图1A。
当scFv融合至DLL3全长抗体Fab段重链的C末端时，则所形成的双特异性抗体被命名为双抗2，包含一条轻链(即第三多肽)和异源的两条重链(即第一多肽和第二多肽)。异源的两条重链中，含有scFv的重链被设计为“节”(knob)结构(命名为双抗2的“节”结构重链)，包括S354C和T366W两个位点的氨基酸取代。异源的两条重链中，不含有scFv的重链被设计为“穴”(hole)结构(命名为双抗2的“穴”结构重链)，包括Y349C、T366S、L368A和 Y407V四个位点的氨基酸取代。以及，为便于双特异性抗体的纯化，“穴”结构的重链还要进行H435R的取代。改造后的“节”结构重链的序列如SEQ ID NO:2所示，“穴”结构重链的序列如SEQ ID NO:3所示，轻链序列如SEQ ID NO:4所示(命名为双抗2的轻链)，示意图见图1B。
双抗1和双抗2的结构以及相关的分子序列分别总结于表1和表2。
表1双特异性抗体的结构
表2双特异性抗体的氨基酸序列


实施例2.抗CD3-DLL3双特异性抗体的构建及其在真核细胞中的瞬时转染表达
将前述双特异性抗体分子的基因片段分别克隆到PTT5表达载体中，制备转染级别的表达质粒。
在无血清培养基中培养Expi293FTM细胞(Thermo Fisher Scientific)，将细胞接种在摇瓶(Corning Inc.)中， 并在37℃，8％CO2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Protein A柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，换液到PBS分装备用。对最终纯化的抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例3.抗CD3-DLL3双特异性抗体的亲和力检测试验
A.抗CD3-DLL3双特异性抗体与表达hDLL3和hCD3的细胞亲和力检测
使用FACS检测抗CD3-DLL3双特异性抗体与表达hDLL3的SHP-77细胞、天然表达hCD3的T淋巴细胞(Jurkat)的结合情况。
培养SHP-77细胞(ATCC，CRL-2195)和Jurkat细胞(ATCC，TIB-152)，SHP-77细胞和Jurkat细胞的培养基均为RPMI1640+10％FBS，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时使用无菌DPBS洗SHP-77细胞，0.25％胰酶EDTA消化约5分钟后用完全培养基终止，放入50mL离心管中。Jurkat细胞直接放入50mL离心管中，无需消化。
将SHP-77及Jurkat 1000rpm转速常温离心5分钟，弃上清，用100μL 1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL。将细胞铺到96孔圆底培养板(corning，货号3799)中，1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。使用1％BSA(in PBS)稀释待测抗体及阴性对照IgG1LALA(购买自百英生物，货号B109802)，起始浓度为100nM，10倍往下稀释7个浓度。用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:200稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm 4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)，结果如表3，图2(SHP-77细胞)及图3(Jurkat细胞)所示。两个双抗分子对Jurkat细胞株的结合能力均弱于SHP-77细胞株，且双抗2分子对两种细胞的结合能力均弱于双抗1分子。
表3双特异性抗体分别与hDLL3(SHP-77细胞株)和hCD3(Jurkat细胞株)的亲和力
B.抗CD3-DLL3双特异性抗体的体外重组蛋白结合亲和力和动力学
使用Biacore 8K仪器分析抗CD3-DLL3双特异性抗体分子与人源/食蟹猴源DLL3和人源/食蟹猴源CD3的亲和力和动力学性质。
为测定与人源DLL3(购自恺佧，货号DLL-HM103)/食蟹猴源DLL3(购自恺佧，货号DLL-RM103)的亲和力与动力学性质，CM5芯片先用EDC和NHS活化，然后固定抗人Fc的鼠单抗，再用乙醇胺封闭。抗CD3-DLL3双特异性抗体分子用HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％P20)缓冲液稀释至0.5μg/mL，以10μL/min的流速捕获45s。人源/食蟹猴源DLL3两倍逐级稀释至系列浓度(100nM-0.78nM)，以50μL/min的流速结合90s，解离450s。
每一轮实验结束后，使用3M MgCl2溶液冲洗以30μL/min的流速冲洗30s，将捕获的抗体连同抗原一起去除，完成芯片的再生。原始数据使用Biacore Insight Evaluation Software(3.0.12.15655)软件进行分析，以(1∶1)Langmuir模型进行拟合，得到的双特异性抗体亲和力和动力学实验数据如表4所示。
表4抗CD3-DLL3双特异性抗体与人源/食蟹猴源DLL3蛋白的结合亲和力和动力学

为测定与人源CD3(购自Acro，货号CDD-H52W1)/食蟹猴源CD3(购自Acro，货号CDD-C52W4)的亲和力与动力学性质，采用CM5芯片直接固化人源/食蟹猴源CD3分子的方法。CM5芯片先用EDC和NHS活化，人源/食蟹猴源CD3分子用PH＝5的醋酸盐溶液稀释至1μg/ml，以10μL/min的流速固化60s，再用乙醇胺封闭抗CD3-DLL3双特异性抗体分子用HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％P20)缓冲液两倍比稀释至系列浓度(100nM-0.39nM)，以50μL/min的流速结合90s，解离360s。
每一轮实验结束后，使用3M MgCl2溶液冲洗，以30μL/min的流速冲洗30s，将抗CD3-DLL3双特异性抗体分子去除，完成芯片的再生。原始数据使用Biacore Insight Evaluation Software(3.0.12.15655)软件进行分析，以(1∶1)Langmuir模型进行拟合，得到的双特异性抗体亲和力和动力学实验数据如表5所示。
表5抗CD3-DLL3双特异性抗体与人源/食蟹猴源CD3蛋白的结合亲和力和动力学
实验结果显示，两个双抗分子对人源/食蟹猴源DLL3及人源/食蟹猴源CD3均有结合，双抗1分子的CD3端亲和力强于DLL3端或与DLL3端相当；双抗2分子的DLL3端亲和力强于CD3端。
实施例4.抗CD3-DLL3双特异性抗体的体外功能实验
A.T细胞介导的细胞毒性实验(TDCC)，靶细胞为SHP-77
培养SHP-77细胞，SHP-77细胞的培养基为RPMI1640+10％FBS，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时使用无菌DPBS洗细胞，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。放入50mL离心管中，1000rpm转速离心5分钟，弃上清，使用完全培养基重悬并计数细胞，将细胞密度调整到5E4/mL。
将靶细胞铺到greiner黑色底透壁不透96孔板(货号655090)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+10％FBS将细胞密度调整到5E5/mL。将效应细胞铺到96孔板中，100μL/孔，37℃5％CO2培养。
用完全培养基稀释抗体，起始浓度为110nM(11X)，5倍往下稀释。将稀释好的抗体加入到细胞培养板中，20μL/孔，故起始终浓度为10nM。37℃5％CO2培养48小时。用枪头将板子中的淋巴细胞吸去，每孔加入100μL CTG检测试剂，300rpm摇晃避光孵育10分钟，Envision上读取化学发光。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝1-(样品孔读值-T细胞孔读值)/(最大信号值-T细胞孔读值)
其中T细胞孔读值为只加T细胞不加靶细胞SHP-77的孔的读值。
GraphPad软件计算EC50和最大杀伤，结果如表6及图4所示。两个双抗分子对SHP-77细胞最大杀伤能力均能达到100％，半数有效浓度双抗1分子低于双抗2分子。
B.T细胞介导的细胞毒性实验(TDCC)，靶细胞为NCI-H82
培养NCI-H82细胞(ATCC，HTB-175)，NCI-H82细胞的培养基为RPMI1640+10％FBS，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。1000rpm转速离心5分钟，DPBS重悬细胞。计数细胞并将细胞密度调整到1E6/ml,加入30nM CellTrace Far Red Cell Proliferation Kit，37℃5％CO2培养箱培养染色20分钟。加入等体积完全培养基，1000rpm离心5分钟，弃上清，DPBS洗涤一遍。
用完全培养基重悬靶细胞，计数，将细胞密度调整到2E5/mL并铺到圆底培养板(corning，货号3799)中，50μL/孔。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+10％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，100μL/孔，37℃5％CO2培养。
抗体稀释及加样：用完全培养基稀释抗体，起始浓度为110nM(11X),5倍往下稀释。将稀释好的抗体加入到细胞培养板中，10μL/孔，故起始终浓度为10nM。37℃5％CO2培养48小时。每孔加入10μL死活染料PI，PI终浓度为4μg/mL，混匀，常温染色10分钟。300目纱布过滤细胞，流式细胞仪检测APC通道(染靶细胞)及PE通道(染死活)的比例。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
靶细胞死亡％＝(Far Red+PI+细胞)/[(Far Red+PI+细胞)+(Far Red+PI-细胞)]
细胞杀伤％＝靶细胞死亡％-靶细胞自发死亡％
其中，靶细胞自发死亡％为只加靶细胞(NCI-H82)不加T细胞的孔死亡的细胞数
GraphPad软件计算EC50和最大杀伤，结果如表6及图5所示。文献报道NCI-H82细胞DLL3表达量只有SHP-77细胞DLL3表达量的1/3，因此两个双抗分子对NCI-H82细胞最大杀伤能力远弱于SHP-77细胞，均为15％左右；半数有效浓度双抗1分子低于双抗2分子。
表6抗CD3-DLL3双特异性抗体的T细胞介导的细胞毒性实验(TDCC)结果
C.细胞因子释放实验
培养SHP-77细胞，SHP-77细胞的培养基为RPMI1640+10％FBS，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时使用无菌DPBS洗细胞，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。放入50mL离心管中，1000rpm转速离心5分钟，弃上清，使用完全培养基重悬并计数细胞，将细胞密度调整到1E5/mL。将SHP-77细胞铺到96孔板(corning 3799)中，100μL/孔，37℃5％CO2培养。
购买新鲜PBMC，细胞计数，并用RPMI1640+10％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，100μL/孔，37℃5％CO2培养。
用完全培养基稀释抗体，起始浓度为110nM(11X)，5倍往下稀释。将稀释好的抗体加入到细胞培养板中，20μL/孔，故起始终浓度为10nM。37℃5％CO2培养48小时。
将培养板400g转速离心10分钟，取80μL上清冻存在-80℃备用。
按照CBA流式试剂盒(BD，551809)说明书稀释细胞因子标准品，两倍稀释，最高浓度为5000pg/mL,最低浓度为20pg/mL。涡旋混匀微珠(beads)，1:1混合human Th1/Th2cytokine capture beads，6种细胞因子beads等比例混合，加入到96孔板中(corning 3799),40μL/孔。将配置好的标准品或者解冻后的样品加到96孔板中，40μL/孔。将human Th1/Th2-II PE检测试剂加入到96孔板中，1100rpm转速避光摇晃5分钟，室温避光孵育3小时。Wash buffer洗板，160μL/孔，洗两遍。300目纱布过滤样本，流式检测APC通道及PE通道荧光读值。
从仪器上导出FCS文件，导入FCAP软件进行分析每个样本细胞因子释放量，结果如图6所示(图6A-图6E分别为有无靶细胞的情况下双抗1分子的IFNγ，TNFα，IL-10，IL-6和IL4细胞因子释放结果；图6F-图6J分别为有无靶细胞的情况下双抗2分子的IFNγ，TNFα，IL-10，IL-6和IL4细胞因子释放结果。)，双抗2分子诱导各类细胞因子释放的水平均弱于双抗1分子，两个双抗分子在仅有PBMC的条件下细胞因子释放水平均较弱或无，与PBMC和SHP-77肿瘤细胞共孵育条件有合适的安全窗。
实施例5.抗CD3-DLL3双特异性抗体的物理稳定性测试
利用NanoDSF(差示荧光扫描技术)检测不同抗体在PH7.4PBS缓冲液中的热稳定性。样品浓度在1mg/ml左右，利用Prometheus NT.Plex(nano DSF)进行检测。检测前，将各个样品10000g离心10分钟。样品板每个孔加入40μl样品(仪器上样量为10μl，每个样品均有一个复孔)。扫描温度从30℃开始到95℃结束，扫描速率0.5℃/min。实验结果如表7所示。两个双抗分子均表现出良好的热稳定性，双抗2分子的开始聚集温度(Tagg)高于双抗1分子。
表7抗CD3-DLL3双特异性抗体的NanoDSF检测结果
实施例6.抗CD3-DLL3双特异性抗体的药代动力学实验
实验用naive食蟹猴两只，自由饮水。双特异性抗体给药剂量为1mg/kg，30min完成静脉输注。采血时间点为Pre-dose，5min(滴注中)，30min(给药结束)，2hr，4hr，6hr，24hr(1d)，48hr(2d)，72hr(3d)，120hr(5d)，168hr(7d)，336hr(14d)，504hr(21d)，672hr(28d)。将全血样品收集在无抗凝剂的聚乙烯管中，室温放置约1小时，6000g，25℃离心，立即分装两份(PK样品及细胞因子检测样本)立即置于干冰上，转移至-80℃冰箱长期保存。
采用ELISA法检测血清中的DLL3/CD3完整分子的浓度。使用人源DLL3蛋白按1μg/ml浓度包被96孔板，每孔100μL，4℃放置过夜；每孔200μL PBST洗板3次，加入300μL封闭试剂5％奶粉，37℃孵育1h；每孔200μL PBST洗板3次，加入100μL待测样品，37℃孵育1h；每孔300μL PBST洗板6次，再加入100μL Biotin-CD3e(1:10000)，SA-HRP(1:10000)和TMB，避光放置10min后，加入100μL终止液停止显色反应。根据颜色反应定量检测DLL3/CD3完整分子的浓度。
MD公司的M5读板仪检测450nm波长的吸光度值，并使用softmax软件处理数据。
采用Phoenix Winnolin 8.2软件对双特异性抗体双抗2的猴血清浓度进行计算，得到药代参数，如下表8所示。
表8双特异性抗体双抗2的猴血清浓度和PK参数
上表中浓度的单位均为μg/mL，BLQ为低于检测限。
双特异性抗体双抗2在猴体内的半衰期为99.9±21h，Cmax为25.3±1.2μg/mL，AUC为60.4±0.9day*μg/mL，因此该双特异性抗体在猴体内性质稳定，不存在明显的脱靶结合，药代性质良好。
实施例7.抗CD3-DLL3双特异性抗体的药代动力学实验伴随细胞因子检测
检测试剂盒为Muti-Analyte Flow assay kit(Biolegend，货号740391)使用前，用250μL缓冲液加入到冻干的NHP Th细胞因子中，混匀，室温静置10分钟。用试剂盒中的assay buffer按照1:4稀释标准品，用试剂盒中的assay buffer按照1:4稀释PK血清样本。将10mL LEGENDplex Assay Buffer加入到lyophilized Matrix B中，室温溶解15分钟备用。
将25μL Matrix B及25μL标准品加入到标准品孔中，将25μL assay buffer及25μL血清样品加入到样品孔中。涡旋混匀检测微珠(beads)，然后将各种检测beads 1:1混合，并用assay buffer稀释到工作浓度，每孔加 25μL。封口膜封板，800rpm转速摇晃避光孵育2小时。250g转速离心5分钟，弃上清，200μL/孔wash buffer洗涤。加25μL检测抗体到板子中，封口膜封板，600rpm室温摇晃避光孵育1小时，加25μL SA-PE,封口膜封板，600rpm室温摇晃避光孵育0.5小时。250g转速室温离心5分钟，弃上清，200μL/孔wash buffer洗涤。1％BSA(in PBS)重悬样本，300目纱布过滤，流式检测。
导出FCS文件，用LENEGDplex 8.0软件分析样本的细胞因子释放量，结果如表9所示。
表9抗CD3-DLL3双特异性抗体的药代动力学实验伴随细胞因子检测结果
实验结果显示，双抗2伴随食蟹猴药代动力学检测各类细胞因子，整体释放水平很低，均<300pg/ml，安全性较好。
实施例8.抗CD3-DLL3双特异性抗体的体内药效实验
实验动物均饲养于恒温恒湿的独立通风盒内，饲养室温度20.0-26.0℃，湿度40-70％，昼夜明暗交替时间12h/12h。
人肺癌细胞SHP-77复苏，收集对数生长期的SHP-77细胞，去除培养液并用PBS洗两次后接种(荷瘤前、荷瘤后SHP-77细胞存活率分别为：98.8％及96.6％)，接种量：5×106/100μL/只，与基质胶1:1混合后接种NCG小鼠皮下，接种位置：小鼠右前肢(图7所示3号位)。
PBMC来源于正常人外周血，在接种肿瘤细胞48小时后，PBMC(捐赠者编号5039)去除冻存液并用PBS洗两次后以0.8×107/100μL/只尾静脉移植小鼠，建立人源免疫细胞重建小鼠模型。接种前、接种后细胞存活率分别为：97.7％及95.8％。
肿瘤接种后第3天，平均肿瘤体积达到87.91mm3时，35只小鼠根据肿瘤体积随机分成5组，分为human IgG1(AA)阴性对照组(购买自百英生物，货号B109802)，0.5mg/kg双抗2治疗组，0.1mg/kg双抗2治疗组，0.02mg/kg双抗2治疗组和0.004mg/kg双抗2治疗组，每组7只。分组当天定义为D0天，并于D0天开始腹腔注射给药，每三天一次，给药6次(见表10)。
表10实验分组及给药方案
给药后每天监测动物日常行为表现，共进行17天。整个实验过程中，用游标卡尺每周测量2次肿瘤长径和宽径，肿瘤体积(mm3)＝0.5×(肿瘤长径×肿瘤短径2)计算。相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增值率，即在某一时间点，治疗组和PBS对照组相对肿瘤体积或瘤重的百分比值。T和C分别为治疗组和PBS对照组在某一特定时间点的肿瘤体积(TV)或瘤重(TW)。各组动物的肿瘤体积、小鼠体重、肿瘤重量等实验结果以平均值±标准误差(Mean±SEM)表示。采用独立样本T检验比较不同治疗组与对照组相比有无显著性差异。数据使用SPSS进行分析。P<0.05为具有显著性差异。作图软件为Graphpad prism。
根据药效终点肿瘤体积及肿瘤重量综合数据统计分析(见表11，图8)，与对照组G1组hIgG1(AA)相比，测试 药双抗2的G2、G3、G4三个较高剂量组(0.5mg/kg、0.1mg/kg、0.02mg/kg)对肿瘤体积和瘤重的抑瘤率分别均达到90％以上，具有极显著的肿瘤生长抑制效果(***P＜0.001)；仅最低剂量G5组双抗2(0.004mg/kg)对肿瘤的生长无显著抑制作用(TGI＝17.31％，P＞0.05)。
在当前的测试系统下，与G1组hIgG1(AA)对照组相比，G2组双抗2(0.5mg/kg)、G3组双抗2(0.1mg/kg)、G4组双抗2(0.02mg/kg)对小鼠肿瘤体积及肿瘤瘤重均表现出明显的生长抑制效果；且这种肿瘤抑制作用是通过T细胞对肿瘤的杀伤来发挥作用。
表11各给药组肿瘤体积大小及抑瘤率
上文所述的本公开的实施方案仅为示例性的，任何本领域技术人员都可以认识到或者可以确定无数的特定化合物、材料和操作的等价物，而不需要进行超出常规的试验。所有这些等价物都是在本公开范围之内的，并且被权利要求所包含。

Claims (12)

  1. 一种双特异性抗原结合分子，其包含：
    (A)第一多肽，其包含：(i)特异性针对第一抗原的抗原结合片段(Fab)重链结构域，(ii)能够特异性结合第二抗原的单链抗体(scFv)结构域，和(iii)第一Fc结构域；
    (B)第二多肽，其包含：第二Fc结构域；
    (C)第三多肽，其包含：特异性针对第一抗原的抗原结合片段(Fab)轻链结构域；
    所述抗原结合片段(Fab)重链结构域与所述抗原结合片段(Fab)轻链结构域形成针对第一抗原的第一结合位点，所述单链抗体(scFv)结构域形成针对第二抗原的第二结合位点，所述第一Fc结构域和所述第二Fc结构域相互缔合。
  2. 如权利要求1所述的双特异性抗原结合分子，其具有以下特征中的一种或多种：
    (1)所述第一抗原为DLL3；
    优选地，所述抗原结合片段(Fab)重链结构域包含序列如SEQ ID NO：13所示的HCDR1、序列如SEQ ID NO：14所示的HCDR2和序列如SEQ ID NO：15所示的HCDR3；所述抗原结合片段(Fab)轻链结构域包含序列如SEQ ID NO：16所示的LCDR1、序列如SEQ ID NO：17所示的LCDR2和序列如SEQ ID NO：18所示的LCDR3；
    更优选地，所述抗原结合片段(Fab)重链结构域包含序列如SEQ ID NO：11所示的第一重链可变区；所述抗原结合片段(Fab)轻链结构域包含序列如SEQ ID NO：12所示的第一轻链可变区；
    更优选地，所述抗原结合片段(Fab)重链结构域包含序列如SEQ ID NO：33所示的氨基酸序列；所述抗原结合片段(Fab)轻链结构域包含序列如SEQ ID NO：4所示的氨基酸序列；
    (2)所述单链抗体(scFv)结构域包含第二重链可变区和第二轻链可变区；
    优选地，所述第二重链可变区与所述第二轻链可变区通过第一接头连接或者直接连接，其中：
    第二重链可变区的C端与第一接头的N端融合，第一接头的C端与第二轻链可变区的N端融合；或者
    第二轻链可变区的C端与第一接头的N端融合，第一接头的C端与第二重链可变区的N端融合；或者
    第二重链可变区的C端与第二轻链可变区的N端融合；或者
    第二轻链可变区的C端与第二重链可变区的N端融合；
    更优选地，所述第一接头包含氨基酸序列(G4S)n，n为1-10中的任意整数；
    (3)所述第二抗原为CD3，
    优选为CD3ε；
    优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：24所示的HCDR1、序列如SEQ ID NO：25所示的HCDR2、序列如SEQ ID NO：26所示的HCDR3、序列如SEQ ID NO：27所示的LCDR1、序列如SEQ ID NO：28所示的LCDR2和序列如SEQ ID NO：29所示的LCDR3；
    更优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：21所示的第二重链可变区和序列如SEQ ID NO：22所示的第二轻链可变区；
    更优选地，所述单链抗体(scFv)结构域包含SEQ ID NO：23所示的氨基酸序列；
    (4)所述第一Fc结构域包含免疫球蛋白的第一CH2结构域和第一CH3结构域，所述第一CH2结构域的C端与第一CH3结构域的N端融合；所述第二Fc结构域包含免疫球蛋白的第二CH2结构域和第二CH3结构域，所述第二CH2结构域的C端与第二CH3结构域的N端融合；
    优选地，所述第一CH3结构域包含“节”(knob)结构，所述第二CH3结构域包含“穴”(hole)结构；
    更优选地，所述“节”(knob)结构包含氨基酸取代S354C和T366W，所述“穴”(hole)结构包含氨基酸取代Y349C、T366S、L368A和Y407V；
    优选地，所述Fc结构域来源于IgG1；
    优选地，所述单链抗体(scFv)结构域与所述第一Fc结构域通过第二接头连接或者直接连接，其中：
    单链抗体(scFv)结构域的C端与第二接头的N端融合，第二接头的C端与第一Fc结构域的N端融合；或者
    单链抗体(scFv)结构域的C端与第一Fc结构域的N端融合；
    更优选地，所述第二接头包含氨基酸序列EPKSS(SEQ ID NO：34)；
    更优选地，所述第一Fc结构域包含SEQ ID NO：31所示的氨基酸序列；所述第二Fc结构域包含SEQ ID NO：3所示的氨基酸序列；
    (5)所述抗原结合片段(Fab)重链结构域包含免疫球蛋白的第一重链可变区和CH1结构域，所述第一重链可变区的C端与CH1结构域的N端融合；所述抗原结合片段(Fab)轻链结构域包含免疫球蛋白的第一轻链可变区和轻链恒定区，所述第一轻链可变区的C端与轻链恒定区的N端融合；
    优选地，所述抗原结合片段(Fab)重链结构域与所述单链抗体(scFv)结构域通过第三接头连接或者直接连接，其中：
    抗原结合片段(Fab)重链结构域的C端与第三接头的N端融合，第三接头的C端与单链抗体(scFv)结构域的N端融合；或者
    单链抗体(scFv)结构域的C端与第三接头的N端融合，第三接头的C端与抗原结合片段(Fab)重链结构域的N端融合；或者
    抗原结合片段(Fab)重链结构域的C端与单链抗体(scFv)结构域的N端融合；或者
    单链抗体(scFv)结构域的C端与抗原结合片段(Fab)重链结构域的N端融合；
    更优选地，所述第三接头包含氨基酸序列(G4S)n，n为1-10中的任意整数；
    (6)所述第一多肽包含如下结构：
    Fab重链结构域-第三接头-scFv结构域-第二接头-第一Fc结构域或者scFv结构域-第三接头-Fab重链结构域-第二接头-第一Fc结构域；
    优选地，所述第一多肽包含如下结构：
    第一重链可变区-CH1-第三接头-第二重链可变区-第一接头-第二轻链可变区-第二接头-第一CH2-第一CH3；或者
    第一重链可变区-CH1-第三接头-第二轻链可变区-第一接头-第二重链可变区-第二接头-第一CH2-第一CH3；或者
    第二重链可变区-第一接头-第二轻链可变区-CH1-第三接头-第一重链可变区-第二接头-第一CH2-第一CH3；或者
    第二轻链可变区-第一接头-第二重链可变区-CH1-第三接头-第一重链可变区-第二接头-第一CH2-第一CH3；
    所述第二多肽包含如下结构：第二CH2-第二CH3；所述第三多肽包含如下结构：第一轻链可变区-轻链恒定区；
    更优选地，所述第一多肽包含SEQ ID NO：2所示的氨基酸序列；所述第二多肽包含SEQ ID NO：3所示的氨基酸序列；所述第三多肽包含SEQ ID NO：4所示的氨基酸序列；
    (7)所述双特异性抗原结合分子包含一个或多个选自以下组的氨基酸取代：(i)L234A和L235A；(ii)H435R；优选地，所述H435R取代是第二多肽上的取代；和/或
    (8)所述双特异性抗原结合分子结合的表位：
    (i)与包含序列如SEQ ID NO：13所示的HCDR1、序列如SEQ ID NO：14所示的HCDR2、序列如SEQ ID NO：15所示的HCDR3、序列如SEQ ID NO：16所示的LCDR1、序列如SEQ ID NO：17所示的LCDR2和序列如SEQ ID NO：18所示的LCDR3的抗体针对的表位相同或重叠；或者
    (ii)与包含序列如SEQ ID NO：11所示的第一重链可变区和序列如SEQ ID NO：12所示的第一轻链可变区的抗体针对的表位相同或重叠。
  3. 一种双特异性抗原结合分子，其包含同源的两条多肽，每条多肽包含：(i)能够特异性结合第一抗原的纳米抗体(VHH)结构域，(ii)能够特异性结合第二抗原的单链抗体(scFv)结构域，和(iii)Fc结构域；所述两条多肽的Fc结构域相互缔合。
  4. 如权利要求3所述的双特异性抗原结合分子，其具有以下特征中的一种或多种：
    (1)所述第一抗原为DLL3；
    优选地，所述纳米抗体(VHH)结构域包含序列如SEQ ID NO：6所示的HCDR1、序列如SEQ ID NO：7所示的HCDR2和序列如SEQ ID NO：8所示的HCDR3；
    更优选地，所述纳米抗体(VHH)结构域包含SEQ ID NO：32所示的氨基酸序列；
    (2)所述单链抗体(scFv)结构域包含重链可变区和轻链可变区；
    优选地，所述重链可变区与所述轻链可变区通过第一接头连接或者直接连接，其中：
    重链可变区的C端与第一接头的N端融合，第一接头的C端与轻链可变区的N端融合；或者
    轻链可变区的C端与第一接头的N端融合，第一接头的C端与重链可变区的N端融合；或者
    重链可变区的C端与轻链可变区的N端融合；或者
    轻链可变区的C端与重链可变区的N端融合；
    更优选地，所述第一接头包含氨基酸序列(G4S)n，n为1-10中的任意整数；
    (3)所述第二抗原为CD3，
    优选为CD3ε；
    优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：24所示的HCDR1、序列如SEQ ID NO：25所示的HCDR2、序列如SEQ ID NO：26所示的HCDR3、序列如SEQ ID NO：27所示的LCDR1、序列如SEQ ID NO：28所示的LCDR2和序列如SEQ ID NO：29所示的LCDR3；
    更优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：21所示的重链可变区和序列如SEQ ID NO：22所示的轻链可变区；
    更优选地，所述单链抗体(scFv)结构域包含SEQ ID NO：23所示的氨基酸序列；
    (4)所述Fc结构域包含免疫球蛋白的CH2结构域和CH3结构域，所述CH2结构域的C端与CH3结构域的N端融合；
    优选地，所述Fc结构域来源于IgG1；
    优选地，所述Fc结构域与所述单链抗体(scFv)结构域通过第二接头连接或者直接连接，其中：
    单链抗体(scFv)结构域的C端与第二接头的N端融合，第二接头的C端与Fc结构域的N端融合；或者
    单链抗体(scFv)结构域的C端与Fc结构域的N端融合；
    更优选地，所述第二接头包含氨基酸序列EPKSS(SEQ ID NO：34)；
    更优选地，所述Fc结构域包含SEQ ID NO：30所示的氨基酸序列；
    (5)所述纳米抗体(VHH)结构域与所述单链抗体(scFv)结构域通过第三接头连接或者直接连接，其中：
    纳米抗体(VHH)结构域的C端与第三接头的N端融合，第三接头的C端与单链抗体(scFv)结构域的N端融合；或者
    单链抗体(scFv)结构域的C端与第三接头的N端融合，第三接头的C端与纳米抗体(VHH)结构域的N端融合；或者
    纳米抗体(VHH)结构域的C端与单链抗体(scFv)结构域的N端融合；或者
    单链抗体(scFv)结构域的N端与纳米抗体(VHH)结构域的C端融合；
    更优选地，所述第三接头包含氨基酸序列(G4S)n，n为1-10中的任意整数；
    (6)所述每条多肽包含如下结构：VHH结构域-第三接头-scFv结构域-第二接头-Fc结构域或者scFv结构域-第三接头-VHH结构域-第二接头-Fc结构域；
    优选地，所述每条多肽包含如下结构：
    VHH结构域-第三接头-重链可变区-第一接头-轻链可变区-第二接头-CH2-CH3，或者
    VHH结构域-第三接头-轻链可变区-第一接头-重链可变区-第二接头-CH2-CH3，或者
    重链可变区-第一接头-轻链可变区-第三接头-VHH结构域-第二接头-CH2-CH3，或者
    轻链可变区-第一接头-重链可变区-第三接头-VHH结构域-第二接头-CH2-CH3；
    更优选地，所述每条多肽包含SEQ ID NO：1所示的氨基酸序列；
    (7)所述双特异性抗原结合分子包含以下氨基酸取代：L234A和L235A；和/或
    (8)所述双特异性抗原结合分子结合的表位：
    (i)与包含序列如SEQ ID NO：6所示的HCDR1、序列如SEQ ID NO：7所示的HCDR2和序列如SEQ ID NO：8所示的HCDR3的纳米抗体针对的表位相同或重叠；或者
    (ii)与包含SEQ ID NO：32所示的氨基酸序列的纳米抗体针对的表位相同或重叠。
  5. 一种双特异性抗原结合分子，其包含：
    (A)能够特异性结合DLL3的第一结合部分；以及
    (B)第二结合部分，所述第二结合部分特异性结合的抗原或表位与第一结合部分不同；
    所述第一结合部分包含：
    (i)序列如SEQ ID NO：6所示的HCDR1、序列如SEQ ID NO：7所示的HCDR2和序列如SEQ ID NO：8所示的HCDR3；或者
    (ii)序列如SEQ ID NO：13所示的HCDR1、序列如SEQ ID NO：14所示的HCDR2、序列如SEQ ID NO：15所示的HCDR3、序列如SEQ ID NO：16所示的LCDR1、序列如SEQ ID NO：17所示的LCDR2和序列如SEQ ID NO：18所示的LCDR3；或者
    (iii)SEQ ID NO：32所示的氨基酸序列；或者
    (iv)序列如SEQ ID NO：11所示的第一重链可变区和序列如SEQ ID NO：12所示的第一轻链可变区。
  6. 编码前述任一权利要求所述的双特异性抗原结合分子的核酸，或者包含所述核酸的表达载体，或者包含所述核酸或所述表达载体的宿主细胞；优选地，所述宿主细胞为原核细胞(优选大肠杆菌)，或者真核细胞(优选哺乳动物细胞或酵母；进一步优选地，所述哺乳动物细胞为CHO细胞、Expi293或HEK293细胞)。
  7. 制备权利要求1-5任一项所述的双特异性抗原结合分子的方法，所述方法包括：在适合的条件下培养权利要求6所述的宿主细胞。
  8. 抗体药物偶联物，其是将权利要求1-5任一项所述的双特异性抗原结合分子与其他生物活性分子偶联形成；优选地，所述其他生物活性分子为小分子药物；优选地，所述双特异性抗原结合分子与所述其他生物活性分子通过接头连接。
  9. 药物组合物，其包含权利要求1-5任一项所述的双特异性抗原结合分子，或者权利要求6所述的核酸、表达载体或宿主细胞，或者权利要求8所述的抗体药物偶联物；
    优选地，所述药物组合物还包含药学上可接受的载体；
    优选地，所述药物组合物还包含一种或多种额外的治疗剂。
  10. 权利要求1-5任一项所述的双特异性抗原结合分子、权利要求6所述的核酸、表达载体或宿主细胞、或者权利要求8所述的抗体药物偶联物在制备治疗、缓解和/或预防肿瘤的药物中的用途；
    优选地，所述肿瘤是DLL3阳性的肿瘤；更优选地，所述肿瘤选自：小细胞肺癌、胶质母细胞瘤、神经内分泌癌、黑色素瘤、胰腺癌、直肠癌以及上述肿瘤的转移癌。
  11. 一种诱导表达DLL3的细胞死亡的方法，所述方法包括使所述细胞与权利要求1-5任一项所述的双特异性抗原结合分子、权利要求6所述的核酸、表达载体或宿主细胞、权利要求8所述的抗体药物偶联物、或者权利要求9所述的药物组合物接触，所述表达DLL3的细胞是肿瘤细胞；
    优选地，所述肿瘤细胞是选自以下肿瘤的细胞：小细胞肺癌、胶质母细胞瘤、神经内分泌癌、黑色素瘤、胰腺癌、直肠癌以及上述肿瘤的转移癌。
  12. 一种治疗受试者中与表达DLL3相关的疾病的方法，所述方法包括向有需要的受试者施用权利要求1-5任一项所述的双特异性抗原结合分子、权利要求6所述的核酸、表达载体或宿主细胞、权利要求8所述的抗体药物偶联物、或者权利要求9所述的药物组合物；
    优选地，所述疾病是肿瘤；更优选地，所述肿瘤是小细胞肺癌、胶质母细胞瘤、神经内分泌癌、黑色素瘤、胰腺癌、直肠癌以及上述肿瘤的转移癌；
    优选地，所述方法还包括向所述受试者给予额外的治疗剂。
PCT/CN2023/075148 2022-02-10 2023-02-09 一种双特异性抗原结合分子及其应用 WO2023151613A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202380017523.4A CN118591564A (zh) 2022-02-10 2023-02-09 一种双特异性抗原结合分子及其应用
MX2024009008A MX2024009008A (es) 2022-02-10 2023-02-09 Molecula de enlace de antigeno biespecifico y uso de la misma.
AU2023217790A AU2023217790A1 (en) 2022-02-10 2023-02-09 Bispecific antigen-binding molecule and use thereof
KR1020247026956A KR20240141178A (ko) 2022-02-10 2023-02-09 이중특이적 항원-결합 분자 및 이의 용도

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210123816 2022-02-10
CN202210123816.8 2022-02-10

Publications (1)

Publication Number Publication Date
WO2023151613A1 true WO2023151613A1 (zh) 2023-08-17

Family

ID=87563652

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/075148 WO2023151613A1 (zh) 2022-02-10 2023-02-09 一种双特异性抗原结合分子及其应用

Country Status (6)

Country Link
KR (1) KR20240141178A (zh)
CN (1) CN118591564A (zh)
AU (1) AU2023217790A1 (zh)
MX (1) MX2024009008A (zh)
TW (1) TW202342534A (zh)
WO (1) WO2023151613A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
CN108271376A (zh) * 2015-07-31 2018-07-10 安进研发（慕尼黑）股份有限公司 结合dll3和cd3的双特异性抗体构建体
US20200087412A1 (en) * 2018-09-14 2020-03-19 Shanghai tongji hospital Asymmetric Bispecific Antibody
CN112513092A (zh) * 2018-06-09 2021-03-16 勃林格殷格翰国际有限公司 Dll3-cd3双特异性抗体
WO2021115456A1 (en) * 2019-12-11 2021-06-17 Wuxi Biologics (Shanghai) Co., Ltd. BI-FUNCTIONAL ANTIBODY AGAINST PD-L1 AND TGFβ
WO2021155380A1 (en) * 2020-01-31 2021-08-05 Gensun Biopharma Inc. Bispecific t cell engagers
WO2022237647A1 (zh) * 2021-05-08 2022-11-17 上海齐鲁制药研究中心有限公司 针对dll3的结合分子及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US7695936B2 (en) 1995-03-01 2010-04-13 Genentech, Inc. Knobs and holes heteromeric polypeptides
CN108271376A (zh) * 2015-07-31 2018-07-10 安进研发（慕尼黑）股份有限公司 结合dll3和cd3的双特异性抗体构建体
CN112513092A (zh) * 2018-06-09 2021-03-16 勃林格殷格翰国际有限公司 Dll3-cd3双特异性抗体
US20200087412A1 (en) * 2018-09-14 2020-03-19 Shanghai tongji hospital Asymmetric Bispecific Antibody
WO2021115456A1 (en) * 2019-12-11 2021-06-17 Wuxi Biologics (Shanghai) Co., Ltd. BI-FUNCTIONAL ANTIBODY AGAINST PD-L1 AND TGFβ
WO2021155380A1 (en) * 2020-01-31 2021-08-05 Gensun Biopharma Inc. Bispecific t cell engagers
WO2022237647A1 (zh) * 2021-05-08 2022-11-17 上海齐鲁制药研究中心有限公司 针对dll3的结合分子及其应用

Also Published As

Publication number Publication date
TW202342534A (zh) 2023-11-01
KR20240141178A (ko) 2024-09-25
AU2023217790A1 (en) 2024-08-22
MX2024009008A (es) 2024-07-31
CN118591564A (zh) 2024-09-03

Similar Documents

Publication Publication Date Title
JP6906653B2 (ja) 三重特異性結合タンパク質と使用方法
ES2784131T3 (es) Polipéptidos de unión beta del receptor PDGF
BR112020015479A2 (pt) Anticorpos anticlaudina 18.2 e usos dos mesmos
US20180243435A1 (en) Anti-dll3 antibody drug conjugates and methods of use
WO2021042694A1 (zh) 抗vegf单域抗体及其应用
WO2019024911A1 (zh) B7h3抗体-药物偶联物及其医药用途
US20220356246A1 (en) Anti-ROR1 antibodies and preparation method and uses thereof
TWI821699B (zh) 抗b7h4抗體及其雙抗和應用
US11814441B2 (en) Bi-specific antigen-binding polypeptides
WO2022068914A1 (zh) 一种含抗体药物偶联物的药物组合物及其用途
TW201731879A (zh) 抗５ｔ４抗體和抗體－藥物共軛體
BR112020021280A2 (pt) anticorpos anti-dll3 e usos dos mesmos
BR112020016469A2 (pt) Anticorpos do receptor anti-folato 1 e seus usos
CN111683966A (zh) 与cd22结合的重链抗体
JP2024109911A (ja) チェックポイント分子に対する多重特異性結合性構築物およびその使用
CN116323657B (zh) 同时靶向PD-L1和TGFβ的双功能分子及其医药用途
CN115175694A (zh) 抗baff受体抗体及其用途
WO2022237647A1 (zh) 针对dll3的结合分子及其应用
WO2023151613A1 (zh) 一种双特异性抗原结合分子及其应用
WO2022068894A1 (zh) 同时靶向pd-l1和vegf的双功能分子及其医药用途
WO2023185732A1 (zh) 包含抗Claudin18.2和CD3双特异性抗体的制剂及其制备方法和用途
EP4223777A1 (en) Anti-cd3 antibody and uses thereof
WO2023025303A1 (zh) 抗cldn-18.2抗体药物偶联物及其用途
CN115594762A (zh) 一种铁蛋白重链抗体及其用途
EA043281B1 (ru) Анти-cd47 антитела и их применение

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23752393

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202380017523.4

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: MX/A/2024/009008

Country of ref document: MX

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112024015524

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20247026956

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2023217790

Country of ref document: AU

Date of ref document: 20230209

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2023752393

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023752393

Country of ref document: EP

Effective date: 20240910