WO2020259535A1 - 抗cd47/抗tigit双特异抗体及其制备方法和应用 - Google Patents

抗cd47/抗tigit双特异抗体及其制备方法和应用 Download PDF

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WO2020259535A1
WO2020259535A1 PCT/CN2020/097924 CN2020097924W WO2020259535A1 WO 2020259535 A1 WO2020259535 A1 WO 2020259535A1 CN 2020097924 W CN2020097924 W CN 2020097924W WO 2020259535 A1 WO2020259535 A1 WO 2020259535A1
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antigen binding
seq
tigit
antibody
fragment
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PCT/CN2020/097924
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French (fr)
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殷刘松
李中道
周铁林
方卓
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南京金斯瑞生物科技有限公司
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Priority to US17/618,609 priority Critical patent/US20220267475A1/en
Priority to CN202080045774.XA priority patent/CN114007646B/zh
Priority to EP20832938.3A priority patent/EP3991744A4/en
Publication of WO2020259535A1 publication Critical patent/WO2020259535A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • 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
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention belongs to the field of antibodies, and specifically relates to bispecific antibodies and preparation methods and applications thereof.
  • the bispecific antibodies comprise a first antigen binding portion that specifically binds CD47, and a second antigen binding portion that specifically binds TIGIT.
  • the mammalian immune system is a host defense system that protects it from microbial infections and prevents cancer (Chen et al., Frontiers Immunol. 9:320 (2018)).
  • the immune system is spread throughout the body and is an extremely complex network system composed of different immune cells, specific tissues and organs and exerts a synergistic effect.
  • the immune system is functioning normally, the diseased cells in the host body will be recognized and eliminated from the healthy cells, thereby ensuring the stability of the body's environment. Therefore, maintaining the integrity of the immune system is essential to maintaining our own health.
  • the immune system can be divided into two categories, namely humoral immunity and cell-mediated immunity. Antibodies and other biological macromolecules regulate humoral immunity. In contrast, the regulation of cellular immunity is achieved at the cellular level, involving the activation of macrophages, natural killer cells and antigen-specific killer T cells.
  • the first signal is antigen-mediated.
  • T cell receptors specifically recognize and bind to antigen peptides presented by MHC on the surface of antigen presenting cells (APC)
  • APC antigen presenting cells
  • the second signal is provided by the interaction between antigen-presenting cells and costimulatory molecules expressed on the surface of T cells.
  • the second signal pathway is very important for activating immune cells.
  • co-stimulatory and co-inhibitory receptors participate in the second signal pathway, which performs immune response and regulation of antigen-receptor presentation, while maintaining autoantigen immune tolerance, while balancing positive and negative signals to maximize Improve immune response to invaders (Chen and Flies, Nat. Rev. Immunol. 13:227-42 (2013); Ewing et al., Int. J. Cardiol. 168: 1965-74 (2013); Liu et al.
  • TIGIT the full name of T cell immunoglobulin and ITIM domain protein, is an inhibitory receptor shared by T cells and NK cells containing Ig and ITIM domains. TIGIT is highly expressed in T cells and natural killer (NK) cells. TIGIT, CD96, CD226 and related ligands together form an immunomodulatory signaling pathway.
  • the CD226/TIGIT/CD96 signaling pathway also contains costimulatory receptors and co-inhibitory receptors, and these receptors share some or all of the ligands, of which CD226 is a costimulatory receptor , When combined with a ligand, it transmits a stimulus signal, while TIGIT and CD96 are co-inhibitory receptors, and when combined with a related ligand, it transmits an inhibitory signal.
  • TIGIT has two ligands, CD155 and CD122, the latter two are also CD226 ligands. These two ligands are expressed in APC cells, T cells and tumor cells.
  • the ligands of CD96 include CD155 and CD111.
  • TIGIT is significantly higher than that of TIGIT and CD122 ligand, and it is also significantly higher than that of CD226 or CD96 and ligand CD155. Similar to PD-1 and CTLA-4 receptors, TIGIT is also an important inhibitory immune receptor. Inhibition of TIGIT can promote the proliferation and function of T cells; blocking TIGIT can also enhance the anti-tumor immune response mediated by NK cells. Thus inhibiting tumor growth. Therefore, monoclonal antibodies targeting the inhibitory receptor TIGIT can significantly enhance the effect of tumor immunotherapy.
  • CD47 also known as integrin-related protein, is a transmembrane protein encoded by the CD47 gene and belongs to the immunoglobulin superfamily. CD47 is widely expressed on the surface of normal cells and can interact with signal regulatory protein ⁇ (SIRP ⁇ ), thrombospondin (TSP1) and integrin (integrin), thereby mediating cell apoptosis, proliferation, and immune responses.
  • SIRP ⁇ signal regulatory protein ⁇
  • TSP1 thrombospondin
  • integrin integrin
  • CD47 is overexpressed in blood and solid tumors, which is highly correlated with poor prognosis of clinical treatment. Therefore, the use of anti-CD47 antibodies or high-affinity SIRP ⁇ variants to block the CD47-SIRP ⁇ signaling pathway has become a potential strategy to promote the phagocytosis of tumor cells by macrophages.
  • anti-CD47 antibodies have a high risk of binding to healthy cells, especially red blood cells, increasing the risk of blood toxicity.
  • more and more studies have shown that blocking CD47 alone is not enough to generate anti-tumor immunity in immunocompetent hosts.
  • the present invention provides an isolated bispecific binding protein comprising a first antigen binding portion that specifically binds CD47 and a second antigen binding portion that specifically binds TIGIT.
  • the present invention provides an isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof, the bispecific antigen binding protein or fragment thereof includes (a) a first antigen binding portion, including a heavy chain variable region (V H) and light chain variable region (V L), V H and V L, which form an antigen binding site that specifically binds to CD47; and (b) a second antigen binding portion comprising the specific binding of TIGIT Single domain antibody (sdAb); wherein the first antigen binding portion and the second antigen binding portion are fused to each other.
  • V H heavy chain variable region
  • V L light chain variable region
  • sdAb Single domain antibody
  • the V H of the first antigen-binding portion includes heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 31 and SEQ ID NO: 32 and SEQ ID NO: 33 or the sequence shown in FIG respectively comprising the amino acid sequence of the mutant up to three (3, 2 or 1);
  • the first V L antigen binding portion comprises a light chain complementarity determining regions LCDR1 , LCDR2 and LCDR3, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 respectively or the sequence shown contains at most three amino acids (3, 2 or 1) mutated sequence.
  • the V H of the first antigen-binding portion includes heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 31 and SEQ ID NO: 32 and SEQ ID NO: 33 or the sequence shown in FIG respectively include substituted up to three amino acid sequence (3, 2 or 1);
  • the first V L antigen binding portion comprises a light chain complementarity determining regions LCDR1 , LCDR2 and LCDR3, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 respectively or the sequence shown contains at most three amino acids (3, 2 or 1) substitution sequence.
  • the V H of the first antigen-binding portion includes heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as SEQ ID NO: 31 and SEQ ID NO, respectively. : 32 and SEQ ID NO: 33 shown; the first V L antigen binding portion comprises a light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 amino acid sequences, the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: 34 , SEQ ID NO:35 and SEQ ID NO:36.
  • the single domain antibody of the second antigen-binding portion includes complementarity determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
  • SEQ ID NO: 41 or the sequence shown respectively contains a sequence of at most three amino acid (3, 2, or 1) mutations.
  • the single domain antibody of the second antigen-binding portion includes complementarity determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
  • SEQ ID NO: 41 or the sequence shown in SEQ ID NO: 41 contains at most three amino acid (3, 2, or 1) substitution sequences.
  • the single domain antibody of the second antigen-binding portion includes complementarity determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.
  • the antigen binding portion is a first full-length antibody comprising two heavy chains and two light chains, the heavy chain comprises a V H, the light chain comprises a V L.
  • the first antigen binding portion and the second antigen binding portion are fused.
  • the C-terminus of the second antigen-binding portion is fused to the N-terminus of at least one heavy chain of the first antigen-binding portion or the N-terminus of at least one light chain of the first antigen-binding portion.
  • the N-terminus of the second antigen-binding portion is fused to the C-terminus of at least one heavy chain of the first antigen-binding portion or the C-terminus of at least one light chain of the first antigen-binding portion.
  • the first antigen binding portion and the second antigen binding portion are fused by a peptide bond or a peptide linker.
  • the peptide linker is selected from a mutated human IgG1 hinge region or a GS linker.
  • the amino acid sequence of the peptide linker is as shown in SEQ ID NO: 26 or SEQ ID NO: 28.
  • the heavy chain of the binding portion of the first antigen comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 4 99% identical sequence
  • the light chain of the first antigen-binding portion contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 6 99% identical sequence.
  • the heavy chain of the first antigen-binding portion includes a sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 4, and the light chain of the first antigen-binding portion includes the same sequence as SEQ ID NO: : A sequence with at least 95% identity in the amino acid sequence shown in 6.
  • the heavy chain of the first antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 4, and the light chain of the first antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 6 .
  • the second antigen-binding portion comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 38 The sequence of sex. In some embodiments, the second antigen binding portion comprises a sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:38. In some specific embodiments, the second antigen binding portion comprises an amino acid sequence as shown in SEQ ID NO:38.
  • an isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof is provided, and the heavy chain of the first antigen binding portion comprises at least 80% of the amino acid sequence shown in SEQ ID NO: 4. %, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity sequence, the light chain of the first antigen-binding portion contains at least 80% of the amino acid sequence shown in SEQ ID NO: 6 %, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity; and the second antigen-binding portion contains at least 80% of the amino acid sequence shown in SEQ ID NO: 38 , 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity sequence.
  • the isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 4,
  • the light chain of the first antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 6; and the second antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 38.
  • the first antigen binding portion comprises a human, humanized or chimeric antibody or fragment thereof.
  • the second antigen binding portion comprises a single domain antibody that specifically binds to TIGIT, the single domain antibody being of camelid origin, chimeric, humanized, or human.
  • an isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof comprising an anti-CD47 antibody and an anti-TIGIT single domain antibody, the N-terminal of the anti-TIGIT single domain antibody is fused to The C-terminus of the two heavy chains of an anti-CD47 antibody, wherein the heavy chain fusion polypeptide contains at least 80%, 85%, 90%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 12 %, 98% or 99% identity sequence, the light chain polypeptide contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 6 Consistent sequence.
  • the isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof includes an anti-CD47 antibody and an anti-TIGIT single domain antibody, and the N-terminal of the anti-TIGIT single domain antibody is fused to the anti- The C-terminus of the two heavy chains of the CD47 antibody, wherein the heavy chain fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 12, and the light chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6.
  • the heavy chain fusion polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, and the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 14. 97%, 98% or 99% identity sequence, the light chain polypeptide contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 6 % Identity sequence.
  • the isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof includes an anti-CD47 antibody and an anti-TIGIT single domain antibody, and the C-terminus of the anti-TIGIT single domain antibody is fused to the The N-terminus of the two heavy chains of the CD47 antibody, wherein the heavy chain fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 14, and the light chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6.
  • an isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof comprising an anti-CD47 antibody and an anti-TIGIT single domain antibody, the N-terminal of the anti-TIGIT single domain antibody is fused to The C-terminus of the two light chains of an anti-CD47 antibody, wherein the light chain fusion polypeptide contains at least 80%, 85%, 90%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 20 %, 98% or 99% identity sequence, the heavy chain polypeptide contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 4 Consistent sequence.
  • the isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof includes an anti-CD47 antibody and an anti-TIGIT single domain antibody, and the N-terminal of the anti-TIGIT single domain antibody is fused to the anti- The C-terminus of the two light chains of the CD47 antibody, wherein the light chain fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 20, and the heavy chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4.
  • the light chain fusion polypeptide contains at least 80%, 85%, 90%, 95%, 96%, and the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 22. 97%, 98%, or 99% identity sequence
  • the heavy chain polypeptide contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 4 % Identity sequence.
  • the isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof includes an anti-CD47 antibody and an anti-TIGIT single domain antibody, and the C-terminus of the anti-TIGIT single domain antibody is fused to the The N-terminus of the two light chains of the CD47 antibody, wherein the light chain fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 22, and the heavy chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4.
  • the present invention provides an isolated polynucleotide encoding the anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof. According to those skilled in the art, changing (e.g., substitution, deletion, etc.) the sequence encoding the protein does not change the amino acid of the protein.
  • a vector comprising the isolated polynucleotide encoding the isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof.
  • Vectors known to those skilled in the art such as plasmids, phage vectors or viral vectors.
  • the vector is a recombinant expression vector, such as a plasmid.
  • These vectors include any elements to support the functions of their conventional expression vectors, such as promoters, ribosome binding elements, terminators, enhancers, selectable markers, and origins of replication.
  • the promoter can be a conventional promoter, an inducible promoter or a repressible promoter.
  • expression vectors are known in the art to be able to deliver nucleic acids into cells and can be used to produce antibodies or antigen-binding fragments thereof in cells. According to the methods in the embodiments of the present invention, conventional cloning techniques or artificial gene synthesis can be used to produce recombinant expression vectors.
  • a host cell containing the above-mentioned isolated polynucleotide or vector is provided.
  • any host cell conventional in the art can be used for the expression of antibodies or antigen-binding fragments thereof.
  • the host cell is E. coli TG1 or BL21 (used to express scFv or Fab antibodies), CHO-DG44, CHO-3E7, CHO-K1 or HEK293.
  • the recombinant expression vector is transfected into host cells by conventional methods (such as chemical transfection, thermal transfection, or electrotransfection), and stably integrated into the host cell genome, so the recombinant nucleic acid can be effectively expressed.
  • the present invention provides a method for producing an isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragments thereof, which comprises culturing the bispecific antigen binding protein or its fragments encoding the present invention under suitable conditions. Fragmented polynucleic acid host cells, recovering antibodies or fragments thereof from the cell or cell culture medium. The expressed antibody or its fragment can be obtained from cells or extracted and purified by conventional methods in the art.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or fragment thereof and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, etc. that can be administered safely, and these substances are suitable for human and/or animal administration without excessive disadvantages Side effects are also suitable for maintaining the vitality of the drugs or active agents in them.
  • various carriers well-known in the art can be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, and polyols. , Alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water, etc.
  • the pharmaceutical composition provided by the present invention can be made into clinically acceptable dosage forms such as powder and injection.
  • the pharmaceutical composition of the present invention can be administered to the subject by any appropriate route, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or inhalation, transdermal, etc. Route administration.
  • the present invention provides a method of treating a subject suffering from or at risk of suffering from a disease associated with abnormal expression of CD47 and/or TIGIT, comprising administering to the subject an effective amount of any of the above-mentioned pharmaceutical compositions .
  • the present invention provides the use of the anti-CD47/anti-TIGIT bispecific antigen binding protein or fragments, polynucleotides, vectors, and host cells thereof in the preparation of drugs for diseases related to abnormal expression of CD47 and/or TIGIT .
  • the disease associated with CD47 and/or TIGIT is cancer.
  • the cancer is a solid tumor, such as rectal cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma , Melanoma, head and neck cancer, etc.
  • the cancer is a solid tumor, such as pancreatic cancer, non-small cell lung cancer, ovarian cancer, melanoma, breast cancer, gastric cancer, colorectal cancer, prostate cancer, and uterine cancer.
  • the above method further comprises administering to the individual additional tumor treatment, such as surgery, radiation therapy, chemotherapy, immunotherapy, hormone therapy, or a combination thereof.
  • the TIGIT single domain antibody is connected to the end of the heavy or light chain of the anti-CD47 monoclonal antibody in a specific connection mode, and the resulting anti-CD47/anti-TIGIT bispecific antigen binding protein has a significantly increased affinity for the TIGIT antigen
  • the biological activity of this bispecific antibody to TIGIT blocking is also significantly enhanced, which shows that the increase of the affinity of the bispecific antibody to TIGIT antigen can enhance the corresponding biological activity.
  • this bispecific antibody can also block the CD47 signaling pathway, so it can simultaneously block two tumor immune escape methods.
  • Antigen binding protein fragment means antibody fragments and antibody analogs, which usually include at least part of the antigen binding region or variable region (for example, one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody.
  • antigen-binding protein fragments capable of binding CD47 or a part thereof including but not limited to sdAb (single domain antibody), Fab (for example, the antibody is obtained by papain digestion), F(ab') 2 (for example, by pepsin Digested), Fv or scFv (for example, obtained by molecular biology techniques).
  • Single domain antibody refers to a single antigen binding polypeptide with three complementarity determining regions (CDRs). These single domain antibodies alone can bind to antigen without pairing with corresponding CDR-containing polypeptides. In some cases, single domain antibodies are artificially engineered from camel heavy chain antibodies and are called "V H H segments”. Cartilaginous fish also have heavy chain antibodies (IgNAR, the abbreviation of Immunoglobulin new antigen receptor), from which single domain antibodies called “V NAR segments” can also be produced.
  • IgNAR the abbreviation of Immunoglobulin new antigen receptor
  • Camelidae sdAb is the smallest known antigen-binding antibody fragment (see eg, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8: 1013-26 (2013)).
  • the basic V H H has the following structure from N-terminal to C-terminal: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 are framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.
  • the anti-TIGIT single domain antibody involved in the present invention refers to a single domain antibody that can specifically bind to TIGIT, especially a single domain antibody that binds to human TIGIT.
  • the anti-TIGIT single domain antibody of the present invention can be selected from the anti-TIGIT single domain antibody specifically described in the patent application PCT/CN2018/124979.
  • a “full-length antibody” refers to an antibody having four full-length chains, including heavy and light chains including an Fc region.
  • the anti-CD47 antibody involved in the present invention refers to an antibody that specifically binds to CD47, especially an antibody that binds to human CD47.
  • the anti-CD47 antibody of the present invention can be selected from the anti-CD47 antibodies specifically described in PCT/CN2019/072929.
  • the construction, expression, extraction and purification methods of the anti-CD47 antibody of the present invention can refer to patent application PCT/CN2019/072929.
  • “Mutation” refers to a polypeptide in which an antigen binding protein or protein fragment contains one or more (several) amino acid residues at one or more (several) positions, that is, a polypeptide that is substituted, inserted, and/or deleted. Substitution refers to replacing the amino acid occupying a certain position with a different amino acid; deletion refers to removing the amino acid occupying a certain position; and insertion refers to adding 1-5 amino acids adjacent to the amino acid occupying a certain position and afterwards.
  • amino acid sequence identity is defined as comparing sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the candidate sequence is similar to the specific peptide or polypeptide sequence.
  • Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.
  • GS linker refers to the GS combination of glycine (G) and serine (S), used to link multiple proteins together to form a fusion protein.
  • G glycine
  • S serine
  • the commonly used GS combination is (GGGGS)n, and the length of the linker sequence is changed by changing the size of n.
  • GGGGS most GS combinations use (GGGGS)3.
  • glycine and serine can also generate different linker sequences through other combinations, such as the G9-linker used in the present invention, and the GS combination is GGGGSGGGS.
  • FIG. 3 The TIGIT/CD155 blocking bioassay system is used to determine the TIGIT blocking activity of samples
  • TIGIT single domain antibody is fused to the N or C end of the heavy or light chain of the anti-CD47 monoclonal antibody through two linker sequences ((E-linker: EPKSSDKTHTSPPSP or G9-linker: GGGGSGGGS).
  • E-linker EPKSSDKTHTSPPSP or G9-linker: GGGGSGGGS.
  • Each bispecific antibody structure consists of two The same fusion polypeptide chain and two identical natural polypeptide chains are composed, and the DNA sequence expressing each polypeptide chain is inserted into the pTT5 vector between EcoRI and HindIII restriction sites.
  • Each plasmid also includes the protein secreted into the growth medium The secretion signal sequence of TIGIT.
  • the TIGIT single domain antibody was fused to the N-terminus of the IgG4-Fc portion with site mutations (S228P and L235E) as a control for in vitro biological activity determination.
  • the plasmid expressing the bispecific antibody protein is shown in Table 3. Show.
  • the CHO-3E7 host cells were transfected with the expression plasmid, they were cultured in an incubator at 37°C and 100 rpm for 6 days. The supernatant was extracted by centrifugation, and the bispecific antibody protein was purified with a Protein A column.
  • the CD47 monoclonal antibody is composed of a heavy chain H0 and a light chain L0.
  • the TIGIT single domain antibody is linked to the N or C end of the heavy or light chain of the CD47 monoclonal antibody through two linker sequences (E-linker: EPKSSDKTHTSPPSP or G9-linker: GGGGSGGGS) to generate a series of different bispecific antibodies.
  • E-linker EPKSSDKTHTSPPSP or G9-linker: GGGGSGGGS
  • GGGGSGGGS GGGGSGGGS
  • TIGIT single domain antibody with The N-terminus of the H0 heavy chain is fused to form a new polypeptide H2; (3).
  • the TIGIT single-domain antibody is fused with the C-terminus of the L0 light chain to form a new polypeptide L1; (4).
  • the TIGIT single-domain antibody is combined with the L0 light chain.
  • the N-terminus of the chain is fused to form a new polypeptide L2.
  • the G9-linker linker sequence was then used to construct the following fusion proteins: (1).
  • the TIGIT single domain antibody was fused to the C-terminus of the H0 heavy chain to produce a new polypeptide called H3; (2).
  • TIGIT single The domain antibody is fused with the N-terminus of the H0 heavy chain to form a new polypeptide H4; (3).
  • the TIGIT single-domain antibody is fused with the C-terminus of the L0 light chain to form a new polypeptide L3; (4).
  • the Fc of human IgG4 was modified by site mutation (S228P and L235E), and then the TIGIT single domain antibody was connected to the N-terminus of the Fc portion of human IgG4 to produce a new fusion protein H5, thereby constructing sdAb-TIGIT- Fc fusion protein of IgG4PE.
  • E-linker linker DNA sequence (SEQ ID NO: 25)
  • IgG4 Fc DNA sequence (SEQ ID NO: 29)
  • IgG4 Fc amino acid sequence (SEQ ID NO: 30)
  • the bispecific antibody produced when TIGIT single domain antibody is fused to the N-terminus or C-terminus of the heavy or light chain of CD47 monoclonal antibody (mAb) is incubated with CHO-K1 cells expressing TIGIT antigen
  • FACS detection compared with the TIGIT single domain antibody control fused to IgG4 Fc (sdAb-TIGIT-IgG4PE), when the TIGIT single domain antibody is fused to the N of the heavy or light chain of the CD47 monoclonal antibody (mAb)
  • the affinity of the bispecific antibody produced at the end to the TIGIT antigen was significantly higher than that of the single domain antibody control ( Figure 1).
  • the affinity of the bispecific antibody to the TIGIT antigen is lower than that of the single domain antibody control. Therefore, when the TIGIT single domain antibody is connected to the N-terminus of the CD47 monoclonal antibody, it will enhance the binding of the TIGIT single domain antibody to the TIGIT antigen, and when the TIGIT single domain antibody is connected to the C-terminus of the CD47 monoclonal antibody, it will reduce TIGIT. The affinity of the single domain antibody to the TIGIT antigen.
  • the bispecific antibody produced when the TIGIT single domain antibody is fused to the end of the heavy or light chain of a CD47 monoclonal antibody (mAb) is incubated with CHO-K1 cells expressing the CD47 antigen and detected by FACS found that, as compared with the control monoclonal antibody CD47, EC 50 values for all samples bispecific antibodies bind to CD47 antigen are higher than the EC 50 value of monoclonal antibody binding to CD47 CD47 antigen (FIG. 2).
  • TIGIT/CD155 blocking bioassay system can be used to determine the biological activity of antibodies or other biological agents that can block the interaction of TIGIT/CD155.
  • the test consists of two genetically engineered cell lines: TIGIT effector cells, namely jurkat T cells that express human TIGIT and a luciferase reporter gene driven by a natural promoter that responds to TCR activation and CD226 costimulation; CD155aAPC/CHO-K1 cells are CHO-K1 cells that express human CD155 and a cell surface protein that can activate the TCR complex in an antigen-independent manner.
  • TIGIT inhibits CD226 activation and promoter-mediated luminescence.
  • the addition of anti-TIGIT antibody can block the interaction between TIGIT and CD155 or inhibit the ability of TIGIT to prevent CD226 from forming homodimers, thereby restoring promoter-mediated luminescence.
  • the effector cell line Jurkat T cells were first spread in a 96-well plate, and then the anti-TIGIT monoclonal antibody sample and the stimulation cell line CD155aAPC/CHO-K1 cells were added. The system was incubated at 37 degrees for 6 hours. Then add Bio-Glo TM fluorescence detection reagent, and incubate at room temperature for 5-10 minutes. Finally, use a chemical fluorescence signal plate reader to read the fluorescence signal in the 96-well plate.
  • This experiment uses the form of 8-concentration triple wells, the relative fluorescence value is taken as the y-axis, and the concentration of the antibody sample is taken as the x-axis, and a four-parameter curve is drawn.
  • GraphPad Prism software was used to analyze the curve and obtain the EC 50 value of the anti-TIGIT monoclonal antibody sample.
  • the BD FACSCalibur flow cytometry line was then used to analyze the cells in the cell plate.
  • the percentage of phagocytosis is calculated by dividing the number of PKH26 and CD11b double positive cells by the number of PKH26 single positive cells.
  • the dose-effect curve uses the percentage of phagocytosis as the y-axis, the concentration of anti-CD47 antibody as the x-axis, and uses the GraphPad Prism software to analyze the EC 50 value and other curve parameters.
  • the biological activity determination results based on the TIGIT/CD155 blocker showed that the bispecific antibody TIGIT-G9-HN has higher biological activity than the TIGIT single domain antibody control (sdAb-TIGIT-IgG4PE) ( Figure 3).
  • the TIGIT single domain antibody in this bispecific antibody is fused to the N-terminus of the heavy chain of the CD47 monoclonal antibody (mAb), and the FACS affinity analysis shows that its affinity is significantly higher than that of the TIGIT single domain antibody control, and the affinity is consistent with in vitro biological activity It further confirmed that this bispecific antibody enhanced the activity of TIGIT single domain antibody.

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Abstract

抗CD47/抗TIGIT双特异抗体及其制备方法和应用，所述双特异抗体包括（a）第一抗原结合部分，包括重链可变区（V H）和轻链可变区（V L），其中V H和V L形成特异性结合CD47的抗原结合位点；以及（b）第二抗原结合部分，包含特异性结合TIGIT的单域抗体（sdAb）；其中第一抗原结合部分和第二抗原结合部分相互融合。双特异性抗体能同时阻断两种肿瘤免疫逃逸的方式，因此在肿瘤免疫治疗方面具有较好的效果。

Description

抗CD47/抗TIGIT双特异抗体及其制备方法和应用 技术领域
本发明属于抗体领域，具体涉及双特异性抗体及其制备方法和应用，所述双特异性抗体包含第一抗原结合部分特异性结合CD47，第二抗原结合部分特异性结合TIGIT。
背景技术
哺乳动物的免疫系统是一套宿主防御系统，使其免受微生物感染和防止癌症发生(Chen et al.,Frontiers Immunol.9:320(2018))。免疫系统遍布全身，是一个及其复杂的网络系统，由不同的免疫细胞，特定的组织和器官组成并发挥协同效应。当免疫系统正常发挥功能时，宿主体内病变的细胞就会被从健康的细胞中识别出来并被清除掉，从而保证机体内环境的稳定。因此，保持免疫系统的完整性，对于维持我们自身的健康至关重要。相反地，免疫系统的失控会导致自身免疫疾病，炎症和癌症等(Ribas et al.,Cancer Discovery 5:915-9(2015)；Yao and Chen,Eur.J.Immunol.43:576-9(2013))。免疫系统可以分为两类，即体液免疫和细胞介导的免疫。抗体和其他生物大分子调控体液免疫。与之相对，细胞免疫的调节是通过细胞层面实现的，涉及到巨噬细胞，自然杀伤细胞和抗原特异性杀伤T细胞的活化。
活化和抑制免疫应答主要是由两条独立的信号通路调节的(Gorentla and Zhong,J.Clin.Cell.Immunol.(2012)；Huse,J.Cell Sci.122:1269-73(2009)；Mizota et al.,J.Anesthesia 27:80-7(2013))。第一信号是抗原介导的。当T细胞受体特异性的识别并结合抗原呈递细胞(APC)表面MHC提呈的抗原肽时，产生第一信号。第二信号则由抗原呈递细胞和T细胞表面表达的共刺激分子间的相互作用提供。当第一、第二信号依次被活化后，T细胞才可以杀伤肿瘤。若缺乏第二信号，T细胞将进入无反应状态或免疫耐受，甚至引起细胞程序性死亡。
如上所述，第二信号通路对于活化免疫细胞非常重要。具体来讲，共刺激和共抑制受体参与了第二信号通路，对抗原-受体的呈递进行免疫应答和调节，在保持自身抗原免疫耐受的同时，平衡阳性和阴性信号，最大限度地提高对入侵者的免疫反应(Chen and Flies,Nat.Rev.Immunol.13:227-42(2013)；Ewing et al.,Int.J.Cardiol.168:1965-74(2013)；Liu et al.,Immunol.Invest.45:813-31(2016)；Shen et al.,Frontiers in Biosci.24:96-132(2019)；Zhang and Vignali,Immunity 44:1034-51(2016))。
TIGIT,全称为T细胞免疫球蛋白和ITIM结构域蛋白，是含Ig及ITIM结构域的T细胞和NK细胞共有的抑制性受体。TIGIT在T细胞和自然杀伤(NK)细胞中高表达。TIGIT，CD96，CD226和相关配体一起组成了一个免疫调节信号通路。与CD28/CTLA-4信号通路类 似，CD226/TIGIT/CD96信号通路也包含共刺激性受体和共抑制性受体，并且这些受体共享部分或全部配体，其中CD226属于共刺激性受体，与配体结合后传递刺激信号，而TIGIT和CD96属于共抑制性受体，与相关配体结合后传递抑制信号。TIGIT有两种配体，即CD155和CD122，后两者同时也是CD226的配体。这两个配体在APC细胞，T细胞和肿瘤细胞中均有表达。CD96的配体包括CD155和CD111。TIGIT与CD155配体的亲和力，显著高于TIGIT与CD122配体的亲和力，同时也明显高于CD226或CD96与配体CD155的亲和力。与PD-1和CTLA-4受体类似,TIGIT也是一个重要的抑制性免疫受体,抑制TIGIT可促进T细胞的增殖和功能；阻断TIGIT也可增强NK细胞介导的抗肿瘤免疫应答，从而抑制肿瘤生长。因此,靶向抑制性受体TIGIT的单克隆抗体，可以明显增强肿瘤免疫治疗效果。
CD47,也被称为整合素相关蛋白，是由CD47基因编码的跨膜蛋白，属于免疫球蛋白超家族。CD47广泛表达于正常细胞表面，可与信号调节蛋白α(SIRPα)、血小板反应素(TSP1)以及整合素(integrin)相互作用，从而介导细胞凋亡、增殖、免疫等反应。CD47是一种先天免疫检测点受体，与主要表达在巨噬细胞核树突状细胞上的SIRPα结合后，向巨噬细胞释放出“不要吃我”的信号，抑制吞噬作用，从而避免机体免疫系统攻击。癌细胞通过上调CD47的表达来防止吞噬作用，从而逃避免疫监视。CD47在血液和实体肿瘤中过度表达，这与临床治疗预后差有高度相关性。因此，利用抗CD47抗体或高亲和力SIRPα变体来阻断CD47-SIRPα信号通路已成为促进巨噬细胞吞噬肿瘤细胞的一种潜在策略。然而，鉴于CD47的广泛表达，抗CD47抗体有很高的风险结合健康细胞，尤其是红细胞，增加血液毒性的风险。同时，越来越多的研究表明，单独阻断CD47并不足以在免疫活性宿主中产生抗肿瘤免疫。此外，斯坦福大学的研究人员报道，SIRPα治疗干扰CD47/SIRPα通路不会诱导吞噬作用(Sockolosky et al.,PNAS 113:E2646-2654(2016))。因此，考虑到癌症治疗的有效性和安全性，抗CD47抗体需要进一步的优化以提高肿瘤靶向特异性。
发明内容
一方面，本发明提供了一种分离的双特异性结合蛋白，所述蛋白包括第一抗原结合部分特异性结合CD47和第二抗原结合部分特异性结合TIGIT。具体地，本发明提供一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，所述双特异性抗原结合蛋白或其片段包括(a)第一抗原结合部分，包括重链可变区(V H)和轻链可变区(V L)，其中V H和V L形成特异性结合CD47的抗原结合位点；以及(b)第二抗原结合部分，包括特异性结合TIGIT的单域抗体(sdAb)；其中第一抗原结合部分和第二抗原结合部分相互融合。
在一些实施方案中，所述第一抗原结合部分的V H包括重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示或所示序列分别包含至多三个氨基酸(3个、2个或1个)突变的序列；所述第一抗原结合部分的V L包括轻链互补决定区LCDR1、LCDR2和LCDR3，所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示或所示序列分别包含至多三个氨基酸(3个、2个或1个)突变的序列。在一些实施方案中，所述第一抗原结合部分的V H包括重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示或所示序列分别包含至多三个氨基酸(3个、2个或1个)取代的序列；所述第一抗原结合部分的V L包括轻链互补决定区LCDR1、LCDR2和LCDR3，所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示或所示序列分别包含至多三个氨基酸(3个、2个或1个)取代的序列。在一些具体实施方案中，所述第一抗原结合部分的V H包括重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示；所述第一抗原结合部分的V L包括轻链互补决定区LCDR1、LCDR2和LCDR3，所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示。
在一些实施方案中，所述第二抗原结合部分的单域抗体包括互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示或所示序列分别包含至多三个氨基酸(3个、2个或1个)突变的序列。在一些实施方案中，所述第二抗原结合部分的单域抗体包括互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示或所示序列分别包含至多三个氨基酸(3个、2个或1个)取代的序列。在一些具体实施方案中，所述第二抗原结合部分的单域抗体包括互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示。
在一些实施方案中，所述第一抗原结合部分是一种全长抗体，包括两条重链和两条轻链，所述重链包括V H，所述轻链包括V L。
在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分是融合的。在一些具体的实施方案中，所述第二抗原结合部分的C端融合至第一抗原结合部分至少一条重链的N端 或第一抗原结合部分至少一条轻链的N端。在一些实施方案中，所述第二抗原结合部分的N端融合至第一抗原结合部分至少一条重链的C端或第一抗原结合部分至少一条轻链的C端。
在一些实施方案中，所述第一抗原结合部分与第二抗原结合部分是通过肽键或肽接头融合的。在一些实施方案中，所述肽接头选自突变的人IgG1铰链区或GS接头。在一些优选实施方案中，所述肽接头的氨基酸序列如SEQ ID NO:26或SEQ ID NO:28所示。
在一些实施方案中，所述第一抗原的结合部分的重链包含与SEQ ID NO:4所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述第一抗原结合部分的轻链包含与SEQ ID NO:6所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些实施方案中，所述第一抗原结合部分的重链包含与SEQ ID NO:4所示氨基酸序列至少95％一致性的序列，所述第一抗原结合部分的轻链包含与SEQ ID NO:6所示氨基酸序列至少95％一致性的序列。在一些具体实施方案中，所述第一抗原结合部分的重链包含如SEQ ID NO:4所示氨基酸序列，所述第一抗原结合部分的轻链包含如SEQ ID NO:6所示氨基酸序列。
在一些实施方案中，所述第二抗原结合部分包含与SEQ ID NO：38所示的氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些实施方案中，所述第二抗原结合部分包含与SEQ ID NO:38所示的氨基酸序列至少95％一致性的序列。在一些具体的实施方案中，所述第二抗原结合部分包含如SEQ ID NO:38所示的氨基酸序列。
在一些实施方案中，提供了一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，所述第一抗原结合部分的重链包含与SEQ ID NO:4所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，所述第一抗原结合部分的轻链包含与SEQ ID NO:6所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列；以及所述第二抗原结合部分包含与SEQ ID NO：38所示的氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，所述第一抗原结合部分的重链包含如SEQ ID NO:4所示氨基酸序列，所述第一抗原结合部分的轻链包含如SEQ ID NO:6所示氨基酸序列；以及所述第二抗原结合部分包含如SEQ ID NO:38所示的氨基酸序列。
在一些实施方案中，所述第一抗原结合部分包含人、人源化或嵌合抗体或其片段。在一些实施方案中，所述第二抗原结合部分包含特异性结合TIGIT的单域抗体，所述单域抗体是骆驼源的、嵌合的、人源化的或人的。
在一些实施方案中，提供了一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的N端融合至抗CD47 抗体两条重链的C端，其中重链融合多肽包含与SEQ ID NO:8或SEQ ID NO:12所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，轻链多肽包含与SEQ ID NO:6所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的N端融合至抗CD47抗体两条重链的C端，其中重链融合多肽包含如SEQ ID NO:8或SEQ ID NO:12所示的氨基酸序列，轻链多肽包含如SEQ ID NO:6所示的氨基酸序列。
在一些实施方案中，提供了又一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的C端融合至抗CD47抗体两条重链的N端，其中重链融合多肽包含与SEQ ID NO:10或SEQ ID NO:14所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，轻链多肽包含与SEQ ID NO:6所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的C端融合至抗CD47抗体两条重链的N端，其中重链融合多肽包含如SEQ ID NO:10或SEQ ID NO:14所示的氨基酸序列，轻链多肽包含如SEQ ID NO:6所示的氨基酸序列。
在一些实施方案中，提供了一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的N端融合至抗CD47抗体两条轻链的C端，其中轻链融合多肽包含与SEQ ID NO:16或SEQ ID NO:20所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，重链多肽包含与SEQ ID NO:4所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的N端融合至抗CD47抗体两条轻链的C端，其中轻链融合多肽包含如SEQ ID NO:16或SEQ ID NO:20所示的氨基酸序列，重链多肽包含如SEQ ID NO:4所示的氨基酸序列。
在一些实施方案中，提供了又一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的C端融合至抗CD47抗体两条轻链的N端，其中轻链融合多肽包含与SEQ ID NO:18或SEQ ID NO:22所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列，重链多肽包含与SEQ ID NO:4所示氨基酸序列至少80％、85％、90％、95％、96％、97％、98％或99％一致性的序列。在一些具体实施方案中，所述分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其 片段，包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的C端融合至抗CD47抗体两条轻链的N端，其中轻链融合多肽包含如SEQ ID NO:18或SEQ ID NO:22所示的氨基酸序列，重链多肽包含如SEQ ID NO:4所示的氨基酸序列。
另一方面，本发明提供了一种编码上述抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段的分离的多核苷酸。根据本领域技术人员公知的，改变(如，替换、删除等)编码蛋白的序列不会改变蛋白的氨基酸。
进一步，提供了包含上述编码分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段的分离多核苷酸的载体。本领域技术人员公知的载体，如质粒、噬菌体载体或病毒载体。在一些具体实施方案中，所述载体是重组表达载体，如质粒。这些载体包括任意元件以支撑其常规表达载体的功能，如，包括启动子、核糖体结合元件、终止子、增强子、选择性标记以及复制起点。其中启动子可以是常规启动子、诱导启动子或可抑制启动子。本领域公知许多表达载体能够将核酸传递至细胞内，并且能用于在细胞内产生抗体或其抗原结合片段。根据本发明实施例中的方法，常规的克隆技术或人工基因合成均可用于产生重组表达载体。
进一步，提供了包含上述分离的多核苷酸或载体的宿主细胞。在本发明从任何本领域常规的宿主细胞均可用于抗体或其抗原结合片段的表达。在一些实施方案中，所述宿主细胞是E.coli TG1或BL21(用于表达如scFv或Fab抗体)，CHO-DG44、CHO-3E7、CHO-K1或者HEK293。按照特定实施例，通过常规方法(如化学转染、热转染或电转染等方法)将重组表达载体转染入宿主细胞，稳定整合到宿主细胞基因组，因此能有效表达重组的核酸。
另一方面，本发明提供了一种生产分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段的方法，包括在适合的条件下，培养包含编码本发明双特异性抗原结合蛋白或其片段的多核酸的宿主细胞，从细胞或细胞培养液中回收抗体或其片段。表达抗体或其片段的可以细胞中获取或者本领域常规的方法进行提取纯化。
另一方面，本发明提供了一种药物组合物，所述药物组合物包含上述分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段和药学上可接受的载体。所述“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。本发明所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组 合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
另一方面，本发明提供了一种治疗患有或有风险患有与CD47和/或TIGIT异常表达相关疾病的受试者的方法，包括给受试者施用有效量的任何上述的药物组合物。
另一方面，本发明提供了所述抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段、多核苷酸、载体、宿主细胞在制备与CD47和/或TIGIT异常表达相关疾病的药物中的应用。
在一些实施方案中，所述与CD47和/或TIGIT相关的疾病是癌症。在一些实施方案中，所述癌症是实体瘤，例如直肠癌、非小细胞肺癌、小细胞肺癌、肾细胞癌、卵巢癌、乳腺癌、胰腺癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈癌等。在优选地实施方案中，所述癌症是实体瘤，例如胰腺癌、非小细胞肺癌、卵巢癌、黑色素瘤、乳腺癌、胃癌、结直肠癌、前列腺癌和子宫癌。
在一些实施方案中，上述方法进一步包括给个体施用额外的肿瘤治疗，例如手术、放射治疗、化疗、免疫疗法、激素疗法或其组合。
本发明中，TIGIT单域抗体以特定的连接方式连接至抗CD47单克隆抗体的重链或轻链的末端，所产生的抗CD47/抗TIGIT双特异性抗原结合蛋白对TIGIT抗原的亲和力明显增加，同时，这种双特异抗体对TIGIT阻断的生物活性也明显增强，这说明双特异抗体对TIGIT抗原亲和力的增加能增强相应的生物活性。同时，这种双特异抗体也能阻断CD47信号通路，所以能同时阻断两种肿瘤免疫逃逸方式。
术语解释
“抗原结合蛋白片段”意即抗体的片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。例如，能够结合CD47或其部分的抗原结合蛋白片段，包括但不限于sdAb(单域抗体)、Fab(例如，抗体经木瓜蛋白酶消化而得到)、F(ab’) 2(例如，通过胃蛋白酶消化得到)、Fv或scFv(例如通过分子生物学技术得到)。
“单域抗体(sdAb)”是指具有三个互补决定区(CDRs)的单抗原结合多肽。这些单域抗体单独能够结合抗原而不用配对相应包含CDR的多肽。在一些情况下，单域抗体是从骆驼的重链抗体中人造工程制作出来的，称为“V HH区段”。软骨鱼也有重链抗体(IgNAR，免疫球蛋白新抗原感受器Immunoglobulin new antigen receptor的缩写)，从该类抗体也可以制作出称为“V NAR区段”的单域抗体。骆驼科sdAb是公知的一种最小的抗原结合抗体片段(参见e.g.,Hamers-Casterman et al.,Nature 363:446-8(1993)；Greenberg et al.,Nature 374:168-73(1995)；Hassanzadeh-Ghassabeh et al.,Nanomedicine(Lond),8:1013-26(2013))。基 本的V HH从N端到C端具有如下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中FR1到FR4分别是框架区1到4，和CDR1到CDR3是指互补决定区1到3。本发明中涉及的抗TIGIT单域抗体是指能特异性结合TIGIT的单域抗体，特别是结合人TIGIT的单域抗体。本发明所述的抗TIGIT单域抗体可选自专利申请PCT/CN2018/124979中具体描述的抗TIGIT单域抗体。本发明所述抗TIGIT单域抗体的构建、表达、提取及纯化方法可参考专利申请PCT/CN2018/124979。
“全长抗体”是指具有四条全长链的抗体，包括包含Fc区的重链和轻链。本发明中涉及的抗CD47抗体是指能特异性结合CD47的抗体，特别是结合人CD47的抗体。本发明所述抗CD47抗体可选自PCT/CN2019/072929中具体描述的抗CD47抗体。本发明所述抗CD47抗体的构建、表达、提取及纯化方法可参考专利申请PCT/CN2019/072929。
“突变”是指抗原结合蛋白或蛋白片段包含一个或多个(数个)位置的一个或多个(数个)氨基酸残基的变更，即取代、插入和/或缺失的多肽。取代是指用不同的氨基酸替代占据某位置的氨基酸；缺失是指除去占据某位置的氨基酸；而插入是指在占据某位置的氨基酸邻接处且在之后添加1-5个氨基酸。
“氨基酸序列一致性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
“GS接头”是指甘氨酸(G)和丝氨酸(S)的GS组合，用于将多个蛋白连接在一起形成融合蛋白。常用的GS组合是(GGGGS)n，通过改变n的大小来改变接头序列的长度，其中，大部分GS组合采用(GGGGS)3。同时，甘氨酸和丝氨酸还可以通过其他组合产生不同的接头序列，比如在本发明中使用的G9-linker，GS组合为GGGGSGGGS。
附图说明
图1流式细胞仪测定样品与表达TIGIT的CHO-K1细胞之间的亲和力
图2流式细胞仪测定样品与表达CD47的CHO-K1细胞之间的亲和力
图3 TIGIT/CD155阻断生物测定系统用于测定样品的TIGIT阻断活性
图4抗CD47抗体的细胞吞噬实验测试双特异抗体TIGIT-G9-HN的活性
具体实施例
下面结合具体实施方式，详细描述本发明。应理解，这些实施方式仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。除非另有说明，下文描述的实施例的方法和材料均为可以通过市场购买获得的常规产品。
实施例1构建和表达抗CD47/抗TIGIT双特异性抗体
我们设计了一系列抗CD47/抗TIGIT双特异性抗体，分别使用抗CD47单克隆抗体(mAb)和TIGIT单域抗体(sdAb)，两种抗体的序列见表1和表2。TIGIT单域抗体通过两种接头序列((E-linker:EPKSSDKTHTSPPSP或G9-linker:GGGGSGGGS)融合到抗CD47单克隆抗体重链或轻链的N或C端。每个双特异性抗体结构由两条相同的融合多肽链和两条相同的天然多肽链组成，表达每个多肽链的DNA序列插入EcoRI和HindIII限制位点之间的pTT5载体上。每个质粒还包括分泌到生长培养基的蛋白质的分泌信号序列。将TIGIT单域抗体融合到具有位点突变(S228P和L235E)的IgG4-Fc部分的N端，作为体外生物活性测定的对照。表达双特异性抗体蛋白的质粒如表3所示。
表1：抗CD47单克隆抗体的DNA和氨基酸序列
Figure PCTCN2020097924-appb-000001
Figure PCTCN2020097924-appb-000002
Figure PCTCN2020097924-appb-000003
表2：TIGIT单域抗体的DNA和氨基酸序列
Figure PCTCN2020097924-appb-000004
Figure PCTCN2020097924-appb-000005
CHO-3E7宿主细胞在转染了表达质粒后，在37℃和100rpm的培养箱中培养6天。采用离心法提取上清液，用Protein A柱纯化双特异性抗体蛋白。
如上所述，CD47单克隆抗体由重链H0和轻链L0组成。TIGIT单域抗体通过两种接头序列(E-linker:EPKSSDKTHTSPPSP或G9-linker:GGGGSGGGS)连接到CD47单克隆抗体重链或轻链的N或C端，从而产生一系列不同的双特异抗体。首先使用E-linker接头序列构建产生以下融合蛋白：(1).将TIGIT单域抗体与H0重链的C端融合，产生新的多肽，称为H1；(2).将TIGIT单域抗体与H0重链的N端融合，形成新的多肽H2；(3).将TIGIT单域抗体与L0轻链的C端融合，形成新的多肽L1；(4).将TIGIT单域抗体与L0轻链的N端融合，形成新的多肽L2。类似地，接着使用G9-linker接头序列构建产生以下融合蛋白：(1).将TIGIT单域抗体与H0重链的C端融合，产生新的多肽，称为H3；(2).将TIGIT单域抗体与H0重链的N端融合，形成新的多肽H4；(3).将TIGIT单域抗体与L0轻链的C端融合，形成新的多肽L3；(4).将TIGIT单域抗体与L0轻链的N端融合，形成新的多肽L4。
将这些构建的重链融合蛋白H1,H2,H3和H4，分别与未经改造的亲本轻链多肽链L0组合，或者将这些构建的轻链融合蛋白L1,L2,L3和L4，分别与未经改造的重链多肽链H0组合从而产生一系列双特异抗体。将重链融合蛋白H1与亲本轻链L0组合产生双特异抗体TIGIT-E-HC，将重链融合蛋白H2与亲本轻链L0组合产生双特异抗体TIGIT-E-HN，将重链融合蛋白H3与亲本轻链L0组合产生双特异抗体TIGIT-G9-HC，将重链融合蛋白H4与亲本轻链L0组合产生双特异抗体TIGIT-G9-HN，将轻链融合蛋白L1与亲本重链H0组合产生双特异抗体TIGIT-E-LC，将轻链融合蛋白L2与亲本重链H0组合产生双特异抗体TIGIT-E-LN，将轻链融合蛋白L3与亲本重链H0组合产生双特异抗体TIGIT-G9-LC，将轻链融合蛋白L4与亲本重链H0组合产生双特异抗体TIGIT-G9-LN。通过位点突变(S228P和L235E)将人的IgG4的Fc改造，然后将TIGIT单域抗体与人的IgG4的Fc部分的N端连接,产生了新的融合蛋白H5，从而构建了sdAb-TIGIT-IgG4PE的Fc融合蛋白。
表3：构建双特异抗体的质粒和蛋白
Figure PCTCN2020097924-appb-000006
分泌信号肽的DNA序列(SEQ ID NO:1)
Figure PCTCN2020097924-appb-000007
分泌信号肽的氨基酸序列(SEQ ID NO:2)
Figure PCTCN2020097924-appb-000008
多肽链H1的DNA序列(SEQ ID NO:7)
Figure PCTCN2020097924-appb-000009
Figure PCTCN2020097924-appb-000010
多肽链H1的氨基酸序列(SEQ ID NO:8)
Figure PCTCN2020097924-appb-000011
多肽链H2的DNA序列(SEQ ID NO:9)
Figure PCTCN2020097924-appb-000012
Figure PCTCN2020097924-appb-000013
多肽链H2的氨基酸序列(SEQ ID NO:10)
Figure PCTCN2020097924-appb-000014
多肽链H3的DNA序列(SEQ ID NO:11)
Figure PCTCN2020097924-appb-000015
Figure PCTCN2020097924-appb-000016
多肽链H3的氨基酸序列(SEQ ID NO:12)
Figure PCTCN2020097924-appb-000017
多肽链H4的DNA序列(SEQ ID NO:13)
Figure PCTCN2020097924-appb-000018
Figure PCTCN2020097924-appb-000019
多肽链H4的氨基酸序列(SEQ ID NO:14)
Figure PCTCN2020097924-appb-000020
多肽链L1的DNA序列(SEQ ID NO:15)
Figure PCTCN2020097924-appb-000021
多肽链L1的氨基酸序列(SEQ ID NO:16)
Figure PCTCN2020097924-appb-000022
Figure PCTCN2020097924-appb-000023
多肽链L2的DNA序列(SEQ ID NO:17)
Figure PCTCN2020097924-appb-000024
多肽链L2的氨基酸序列(SEQ ID NO:18)
Figure PCTCN2020097924-appb-000025
多肽链L3的DNA序列(SEQ ID NO:19)
Figure PCTCN2020097924-appb-000026
Figure PCTCN2020097924-appb-000027
多肽链L3的氨基酸序列(SEQ ID NO:20)
Figure PCTCN2020097924-appb-000028
多肽链L4的DNA序列(SEQ ID NO:21)
Figure PCTCN2020097924-appb-000029
多肽链L4的氨基酸序列(SEQ ID NO：22)
Figure PCTCN2020097924-appb-000030
多肽链H5的DNA序列(SEQ ID NO:23)
Figure PCTCN2020097924-appb-000031
Figure PCTCN2020097924-appb-000032
多肽链H5的氨基酸序列(SEQ ID NO:24)
Figure PCTCN2020097924-appb-000033
E-linker接头DNA序列(SEQ ID NO:25)
Figure PCTCN2020097924-appb-000034
E-linker接头氨基酸序列(SEQ ID NO:26)
Figure PCTCN2020097924-appb-000035
G9-linker接头DNA序列(SEQ ID NO:27)
Figure PCTCN2020097924-appb-000036
G9-linker接头氨基酸序列(SEQ ID NO:28)
Figure PCTCN2020097924-appb-000037
IgG4 Fc DNA序列(SEQ ID NO:29)
Figure PCTCN2020097924-appb-000038
IgG4 Fc氨基酸序列(SEQ ID NO:30)
Figure PCTCN2020097924-appb-000039
实施例2 FACS亲和力分析
对于构建的一系列双特异性抗体样品，使用流式细胞仪测定这些样品对抗原的亲和力。样品起始浓度为300nm，进行3倍的梯度稀释，然后分别测试不同浓度的样品与CHO-K1细胞上表达的TIGIT抗原或者CD47抗原的亲和力。接着用几何平均值生成抗体-抗原结合曲线，并利用GRAPHPAD Prism V6.02软件绘制四个参数的原始数据，并编制最佳拟合值程序对EC 50进行分析。
对于TIGIT抗原的亲和力分析，TIGIT单域抗体融合到CD47单克隆抗体(mAb)的重链或者轻链的N端或C端时产生的双特异抗体与表达TIGIT抗原的CHO-K1细胞上孵育后，通过FACS检测发现，与融合到IgG4 Fc上的TIGIT单域抗体对照(sdAb-TIGIT-IgG4PE)相比，当TIGIT单域抗体融合到CD47单克隆抗体(mAb)的重链或者轻链的N端时产生的双特异性抗体与TIGIT抗原的亲和力显著高于单域抗体对照(图1)。而当TIGIT单域抗体融合到CD47单克隆抗体(mAb)的重链或者轻链的C端时产生的双特异抗体与TIGIT抗原的亲和力则低于单域抗体对照。所以，TIGIT单域抗体连接到CD47单克隆抗体的N端时，会增强TIGIT单域抗体与TIGIT抗原的结合，而当TIGIT单域抗体连接到CD47单克隆抗体的C端时，则会降低TIGIT单域抗体与TIGIT抗原的亲和力。
对于CD47抗原的亲和力分析，TIGIT单域抗体融合到CD47单克隆抗体(mAb)的重链或者轻链的末端时产生的双特异抗体与表达CD47抗原的CHO-K1细胞上孵育后，通过FACS检测发现，与CD47单克隆抗体对照相比，所有的双特异抗体样品与CD47抗原结合的EC 50值都高于CD47单克隆抗体与CD47抗原结合的EC 50值(图2)。这种亲和力差异，暗示着TIGIT单域抗体连接到CD47单克隆抗体上，会在一定程度上干扰CD47单克隆抗体与CD47抗原的结合，从而导致构建的双特异抗体和CD47抗原的亲和力降低。
实施例3体外生物活性测定
对于CD47/TIGIT双特异性抗体的体外生物活性测定，由于没有一种能同时检测CD47和TIGIT两种阻断剂的分析系统，因此使用Promega检测试剂盒进行TIGIT阻断剂的生物测定，然后使用抗CD47抗体的细胞吞噬实验测试双特异抗体的活性。
Promega公司的TIGIT/CD155阻断生物测定系统可用于测定抗体或其他能阻断TIGIT/CD155相互作用的生物制剂的生物活性。该试验由两种基因工程细胞系组成：TIGIT效应细胞，即表达人TIGIT以及一个对TCR激活和CD226共刺激均会产生应答的天然启动子所驱动的萤光素酶报告基因的jurkat T细胞；CD155aAPC/CHO-K1细胞，即表达人CD155以及一种能以不依赖于抗原方式激活TCR复合物的细胞表面蛋白的CHO-K1细胞。当两种细胞类型共同培养时，TIGIT抑制CD226激活和启动子介导的发光。加入抗TIGIT抗体可阻断TIGIT与CD155的相互作用或抑制TIGIT阻止CD226形成同源二聚体的能力，从而恢复启动子介导的发光。
测试抗TIGIT抗体活性时，首先将效应细胞系Jurkat T细胞铺在96孔板内，随后加入抗TIGIT的单抗样品和刺激细胞系CD155aAPC/CHO-K1细胞。将该体系在37度下孵育6个小时。然后加入Bio-Glo TM荧光检测试剂，并在室温下孵育5-10分钟。最后使用化学荧光信号读板机读取96孔板中的荧光信号。该实验使用8浓度三复孔的形式，相对荧光值作为y-轴，抗体样品的浓度作为x-轴，画出四参数曲线。使用GraphPad Prism软件分析该曲线并得出抗TIGIT单抗样品的EC 50值。
对于抗CD47抗体的细胞吞噬实验，首先使用浓度梯度的方法从人体外周血中提取PBMC。再使用全单核细胞分离试剂盒(Miltenyi Biotech)从PBMC中分离出单核细胞。这些单核细胞在14天的时间内使用GM-CSF刺激成巨噬细胞。在第14天，将HL60细胞用PKH26染料染色并种在96孔培养板中，使用Accutase将MDM从培养皿上消化下来，然后把MDM加入到种有PKH26染过的HL60的培养板中，另外再加入梯度稀释的抗CD47的抗体样品，并在37度孵育1个小时，让细胞吞噬反应进行。一个小时后，将MDM从细胞培养皿上消化下来并使用荧光标记的抗CD11b的抗体去染MDM。然后使用BD FACSCalibur流式细胞系分析细胞板中的细胞。吞噬百分比由PKH26和CD11b双阳的细胞数量除以PKH26单阳的细胞数量计算而来。量效曲线图使用吞噬百分比做y轴，抗CD47抗体的浓度作为x轴，并使用GraphPad Prism软件分析得出EC 50值以及其他曲线参数。
基于TIGIT/CD155阻断剂的生物活性测定结果显示，双特异抗体TIGIT-G9-HN比TIGIT单域抗体对照(sdAb-TIGIT-IgG4PE)的生物活性要高(图3)。这个双特异抗体中的TIGIT单域抗体融合在CD47单克隆抗体(mAb)的重链N端，而FACS亲和力分析结果显示它的亲和力明显高于TIGIT单域抗体对照，亲和力和体外生物活性的一致性，进一步证实了这个双特异抗体增强了TIGIT单域抗体的活性。
抗CD47抗体的细胞吞噬实验结果显示，双特异抗体TIGIT-G9-HN的EC 50值略低于CD47对照抗体(图4)。而FACS亲和力分析结果显示它的亲和力低于CD47单克隆抗体对照，这说明亲和力的降低对于这个双特异抗体的体外生物活性影响不大。

Claims (22)

  1. 一种分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段，所述双特异性抗原结合蛋白或其片段包括(a)第一抗原结合部分，包括重链可变区(V H)和轻链可变区(V L)，其中V H和V L形成特异性结合CD47的抗原结合位点；以及(b)第二抗原结合部分，包含特异性结合TIGIT的单域抗体；其中第一抗原结合部分和第二抗原结合部分相互融合。
  2. 如权利要求1所述的分离的双特异性抗原结合蛋白或其片段，所述第一抗原结合部分的V H包括重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示或所示序列分别包含至多三个氨基酸突变的序列；所述第一抗原结合部分的V L包括轻链互补决定区LCDR1、LCDR2和LCDR3，所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示或所示序列分别包含至多三个氨基酸突变的序列。
  3. 如权利要求1所述的分离的双特异性抗原结合蛋白或其片段，所述第二抗原结合部分的单域抗体包括互补决定区CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示或所示序列分别包含至多三个氨基酸突变的序列。
  4. 如权利要求1所述的分离的双特异性抗原结合蛋白或其片段，所述第一抗原结合部分是一种全长抗体，包括两条重链和两条轻链，所述重链包括V H，所述轻链包括V L。
  5. 如权利要求1-4中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述第二抗原结合部分的C端融合至第一抗原结合部分至少一条重链的N端或第一抗原结合部分至少一条轻链的N端。
  6. 如权利要求1-4中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述第二抗原结合部分的N端融合至第一抗原结合部分至少一条重链的C端或第一抗原结合部分至少一条轻链的C端。
  7. 如权利要求1-6中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述第一抗原结合部分与第二抗原结合部分是通过肽键或肽接头融合的。
  8. 如权利要求7所述的分离的双特异性抗原结合蛋白或其片段，所述肽接头选自突变的人IgG1铰链区或GS接头，优选地，所述肽接头的氨基酸序列如SEQ ID NO:26或SEQ ID NO:28所示。
  9. 如权利要求1-8中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述第一抗原的结合部分的重链包含与SEQ ID NO:4所示氨基酸序列至少95％一致性的序列，所述第一抗原结合部分的轻链包含与SEQ ID NO:6所示氨基酸序列至少95％一致性的序列。
  10. 如权利要求1-9中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述第二抗原结合部分包含与SEQ ID NO:38所示的氨基酸序列至少95％一致性的序列。
  11. 如权利要求1-10中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述第一抗原结合部分包含人、人源化或嵌合抗体或其片段，所述第二抗原结合部分的单域抗体是骆驼源的、嵌合的、人源化的或人的。
  12. 如权利要求1-11中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述双特异性抗原结合蛋白或其片段包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的N端融合至抗CD47抗体两条重链的C端，其中重链融合多肽包含与SEQ ID NO:8或SEQ ID NO:12所示氨基酸序列至少95％一致性的序列，轻链多肽包含与SEQ ID NO:6所示氨基酸序列至少95％一致性的序列。
  13. 如权利要求1-11中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述双特异性抗原结合蛋白或其片段包括CD47抗体和TIGIT单域抗体，所述抗TIGIT单域抗体的C端融合至抗CD47抗体两条重链的N端，其中重链融合多肽包含与SEQ ID NO:10或SEQ ID NO:14所示氨基酸序列至少95％一致性的序列，轻链多肽包含与SEQ ID NO:6所示氨基酸序列至少95％一致性的序列。
  14. 如权利要求1-11中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述双特异性抗原结合蛋白或其片段包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的N端融合至抗CD47抗体两条轻链的C端，其中轻链融合多肽包含与SEQ ID NO:16或SEQ ID NO:20所示氨基酸序列至少95％一致性的序列，重链多肽包含与SEQ ID NO:4所示氨基酸序列至少95％一致性的序列。
  15. 如权利要求1-11中任一项所述的分离的双特异性抗原结合蛋白或其片段，所述双特异性抗原结合蛋白或片段包括抗CD47抗体和抗TIGIT单域抗体，所述抗TIGIT单域抗体的C端融合至抗CD47抗体两条轻链的N端，其中轻链融合多肽包含与SEQ ID NO:18或SEQ ID NO:22所示氨基酸序列至少95％一致性的序列，重链多肽包含与SEQ ID NO:4所示氨基酸序列至少95％一致性的序列。
  16. 一种编码权利要求1-15中任一项所述的双特异性抗原结合蛋白或其片段的分离的多核苷酸。
  17. 包含权利要求16所述分离多核苷酸的载体。
  18. 包含权利要求16所述分离的多核苷酸或权利要求17所述载体的宿主细胞。
  19. 一种生产分离的抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段的方法，包括在适合的条件下，培养权利要求18所述的宿主细胞，从细胞或细胞培养液中回收抗体或其片段。
  20. 一种药物组合物，所述药物组合物包含权利要求1-15中任一项所述双特异性抗原结合蛋白或其片段和药学上可接受的载体。
  21. 如权利要求1-15中任一项所述抗CD47/抗TIGIT双特异性抗原结合蛋白或其片段、权利要求16所述多核苷酸、权利要求17所述载体以及权利要求18所述宿主细胞在制备与CD47和/或TIGIT异常表达相关疾病的药物中的应用。
  22. 如权利要求21所述的应用，所述与CD47和/或TIGIT异常表达相关疾病是癌症，优选地，所述癌症为实体瘤。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022268168A1 (zh) * 2021-06-23 2022-12-29 迈威(上海)生物科技股份有限公司 靶向lag-3和pd-l1的新型双特异抗体及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7369127B2 (ja) * 2017-12-28 2023-10-25 ナンジン レジェンド バイオテック カンパニー，リミテッド Ｔｉｇｉｔに対する単一ドメイン抗体及びその変異体
TW202216778A (zh) 2020-07-15 2022-05-01 美商安進公司 Ｔｉｇｉｔ及ｃｄ１１２ｒ阻斷

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103665165A (zh) * 2013-08-28 2014-03-26 江苏匡亚生物医药科技有限公司 一种靶向人CD47-SIRPα信号通路的双特异性抗体及其制备方法和用途
WO2018224951A2 (en) * 2017-06-05 2018-12-13 Janssen Biotech, Inc. Engineered multispecific antibodies and other multimeric proteins with asymmetrical ch2-ch3 region mutations
CN109071620A (zh) * 2015-05-28 2018-12-21 昂科梅德制药有限公司 Tigit结合剂和其用途
WO2019129054A1 (zh) * 2017-12-27 2019-07-04 信达生物制药(苏州)有限公司 三链抗体、其制备方法及其用途
WO2019129221A1 (en) * 2017-12-28 2019-07-04 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against tigit
WO2019144895A1 (en) * 2018-01-24 2019-08-01 Nanjing Legend Biotech Co., Ltd. Anti-cd47 antibodies that do not cause significant red blood cell agglutination
CN110352200A (zh) * 2018-02-06 2019-10-18 天境生物 抗具有Ig和ITIM结构域的T细胞免疫受体(TIGIT)的抗体及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5758004B2 (ja) * 2010-08-24 2015-08-05 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ ジスルフィドによって安定化されたＦｖ断片を含む二重特異性抗体
WO2018014260A1 (en) * 2016-07-20 2018-01-25 Nanjing Legend Biotech Co., Ltd. Multispecific antigen binding proteins and methods of use thereof
CN111836832A (zh) * 2018-01-08 2020-10-27 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103665165A (zh) * 2013-08-28 2014-03-26 江苏匡亚生物医药科技有限公司 一种靶向人CD47-SIRPα信号通路的双特异性抗体及其制备方法和用途
CN109071620A (zh) * 2015-05-28 2018-12-21 昂科梅德制药有限公司 Tigit结合剂和其用途
WO2018224951A2 (en) * 2017-06-05 2018-12-13 Janssen Biotech, Inc. Engineered multispecific antibodies and other multimeric proteins with asymmetrical ch2-ch3 region mutations
WO2019129054A1 (zh) * 2017-12-27 2019-07-04 信达生物制药(苏州)有限公司 三链抗体、其制备方法及其用途
WO2019129221A1 (en) * 2017-12-28 2019-07-04 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against tigit
WO2019144895A1 (en) * 2018-01-24 2019-08-01 Nanjing Legend Biotech Co., Ltd. Anti-cd47 antibodies that do not cause significant red blood cell agglutination
CN110352200A (zh) * 2018-02-06 2019-10-18 天境生物 抗具有Ig和ITIM结构域的T细胞免疫受体(TIGIT)的抗体及其应用

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL., FRONTIERS IMMUNOL, vol. 9, 2018, pages 320
CHENFLIES, NAT. REV. IMMUNOL., vol. 13, 2013, pages 227 - 42
EWING ET AL., INT. J. CARDIOL., vol. 168, 2013, pages 1965 - 74
GORENTLAZHONG, J. CLIN. CELL. IMMUNOL., vol. 122, 2012, pages 1269 - 73
GREENBERG ET AL., NATURE, vol. 374, 1995, pages 168 - 73
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446 - 8
HASSANZADEH-GHASSABEH ET AL., NANOMEDICINE (LOND, vol. 8, 2013, pages 1013 - 26
HUSE, J. CELL SCI., vol. 122, 2009, pages 1269 - 73
LIU ET AL., IMMUNOL. INVEST., vol. 45, 2016, pages 813 - 31
MIZOTA ET AL., J. ANESTHESIA, vol. 27, 2013, pages 80 - 7
RIBAS ET AL., CANCER DISCOVERY, vol. 5, 2015, pages 915 - 9
See also references of EP3991744A4
SHEN ET AL., FRONTIERS IN BIOSCI., vol. 24, 2019, pages 96 - 132
SOCKOLOSKY ET AL., PNAS, vol. 113, 2016, pages E2646 - 2654
YAOCHEN, EUR. J. IMMUNOL., vol. 43, 2013, pages 576 - 9
ZHANGVIGNALI, IMMUNITY, vol. 44, 2016, pages 1034 - 51

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022268168A1 (zh) * 2021-06-23 2022-12-29 迈威(上海)生物科技股份有限公司 靶向lag-3和pd-l1的新型双特异抗体及其应用

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