WO2019109876A1 - 抗pd‐l1/抗cd47天然抗体结构样异源二聚体形式双特异抗体及其制备 - Google Patents

抗pd‐l1/抗cd47天然抗体结构样异源二聚体形式双特异抗体及其制备 Download PDF

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WO2019109876A1
WO2019109876A1 PCT/CN2018/118800 CN2018118800W WO2019109876A1 WO 2019109876 A1 WO2019109876 A1 WO 2019109876A1 CN 2018118800 W CN2018118800 W CN 2018118800W WO 2019109876 A1 WO2019109876 A1 WO 2019109876A1
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chain
replaced
antigen binding
cells
amino acid
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PCT/CN2018/118800
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French (fr)
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刘家望
杨亚平
宋楠萌
金孟燮
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北京韩美药品有限公司
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Priority to SG11202005216XA priority Critical patent/SG11202005216XA/en
Priority to EA202091364A priority patent/EA202091364A1/ru
Priority to MX2020005792A priority patent/MX2020005792A/es
Priority to EP18887151.1A priority patent/EP3722322A4/en
Priority to AU2018378529A priority patent/AU2018378529A1/en
Priority to CA3084626A priority patent/CA3084626A1/en
Priority to CN201880070773.3A priority patent/CN111615521A/zh
Priority to BR112020011295-0A priority patent/BR112020011295A2/pt
Application filed by 北京韩美药品有限公司 filed Critical 北京韩美药品有限公司
Priority to KR1020207019202A priority patent/KR20200093639A/ko
Priority to JP2020549852A priority patent/JP7231641B2/ja
Priority to US16/769,108 priority patent/US11739151B2/en
Publication of WO2019109876A1 publication Critical patent/WO2019109876A1/zh
Priority to IL275135A priority patent/IL275135A/en
Priority to ZA2020/03382A priority patent/ZA202003382B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • 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
    • C07K2317/55—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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • the present invention relates to a bispecific antibody in the form of an anti-PD-L1/anti-CD47 natural antibody structural heterodimer and preparation thereof.
  • the present invention provides a highly stable heterodimeric form of an anti-PD-L1/anti-CD47 bispecific antibody having native IgG characteristics and no heavy light chain mismatch and a process for its preparation.
  • Programmed death ligand-1 is a ligand for programmed death-1 (PD-1), which belongs to the B7 family and is induced in A variety of immune cell surfaces, including T cells, B cells, monocytes, macrophages, DC cells, and endothelial cells, epidermal cells, and the like.
  • PD-L1 binds to PD-1 and is mainly involved in the negative regulation of T cell activation, which can regulate the intensity and duration of immune response.
  • PD-L1 acts as a ligand for CD80, transmitting a negative regulatory signal to T cells and inducing T cell immune tolerance (Autoimmun Rev, 2013, 12(11): 1091-1100.
  • PD-L1 and PD-1 can mediate and maintain the autoimmune tolerance of the body tissues, prevent the excessive activation of the immune system during the inflammatory reaction, and damage the self tissue, which has a positive effect on avoiding the occurrence of autoimmune diseases. In pathological conditions, it is involved in the development of tumor immunity and various autoimmune diseases.
  • PD-L1 is highly expressed in a variety of tumor tissues
  • PD-1 is highly expressed in tumor infiltrating lymphocytes
  • overexpression of PD-L1 and PD-1 is closely related to the clinical prognosis of tumors (Anticancer Agents Med Chem) .2015;15(3):307-13.Hematol Oncol Stem Cell Ther.2014 Mar;7(1):1-17.Trends Mol Med.2015 Jan;21(1):24-33.Immunity.2013 Jul 25;39(1):61-73.J Clin Oncol.2015 Jun 10;33(17):1974-82.).
  • the PD-L1 monoclonal antibody was used to block the interaction of PD-L1/PD-1 and CD80/PD-L1, and showed good anti-tumor effects in both preclinical studies and clinical trials.
  • PD-L1 monoclonal antibody has been approved for the treatment of various tumors such as non-small cell lung cancer and urothelial carcinoma.
  • only a small percentage of cancer patients can benefit from such monoclonal antibody therapy, and most patients do not respond to such monoclonal antibodies (Expert Opin Ther Targets. 2014 Dec; 18(12): 1407-20. Oncology (Williston Park). 2014 Nov;28 Suppl 3:15-28.).
  • CD47 also known as integrin-related protein, is a 50Kd transmembrane protein belonging to the immunoglobulin superfamily. Widely expressed on a variety of cells, but significantly enhanced expression on a variety of tumor cells (Proc Natl Acad Sci U S A, 2012, 109 (17): 6662-6667).
  • the ligand for CD47 is signal-regulatory protein ⁇ (SIRP ⁇ ), which is mainly expressed in macrophages. It binds to CD47 and transmits a signal of “don't eat me” to inhibit the phagocytosis of macrophages (Curr Opin Immunol, 2009, 21(1): 47-52).
  • SIRP ⁇ signal-regulatory protein ⁇
  • the anti-tumor effect can be exerted by blocking the CD47-SIRP ⁇ signaling pathway by using an anti-CD47 antibody.
  • a variety of anti-CD47 monoclonal antibodies have been introduced into clinical research for the treatment of a variety of hematological and solid tumors. However, since CD47 is also expressed on the surface of red blood cells, these anti-CD47 treatments may cause adverse reactions such as severe anemia and thrombocytopenia, and bioavailability is low.
  • the present invention provides a novel bifunctional antibody capable of simultaneously blocking PD-L1 and CD47 with a highly stable heterodimeric form having natural IgG structural features and no heavy light chain mismatch, and a preparation method thereof.
  • the bifunctional antibody tends to selectively bind to tumor cells that simultaneously express PD-L1 and CD47, thereby exerting a highly effective and specific killing effect with low toxic side effects.
  • a first aspect of the invention relates to a heterodimeric antibody in the form of a heterodimer comprising a first Fc chain and a second Fc chain, and a first antigen binding functional region capable of specifically binding to PD-L1 and a second antigen binding domain that specifically binds to CD47;
  • first Fc chain and the second Fc chain are both immunoglobulin G Fc fragments comprising amino acid substitutions, and the first Fc chain and the second Fc chain together constitute heterodimerization capable of binding to an Fc receptor body;
  • first Fc chain and the second Fc chain are respectively linked to the first antigen binding functional region and the second antigen binding functional region by a covalent bond or a linker;
  • any one of the first Fc chain and the second Fc chain comprises amino acid substitutions at positions 366 and 399, and the other comprises amino acid substitutions at positions 351, 407 and 409, wherein the amino acid positions are according to Kabat EU Index number system number.
  • the first Fc chain and the second Fc are defined herein for the purpose of distinguishing between the two Fc strands present, and do not imply that they differ in importance or order. Meanwhile, the linkage of the first Fc chain and the second Fc chain to the first antigen-binding functional region and the second antigen-binding functional region is also arbitrary, that is, the first Fc chain can be linked to the first antigen-binding functional region, or The second antigen binds to the functional region, as does the second Fc chain.
  • the first Fc chain and the second Fc chain amino acid are replaced by
  • K409C K409P, K409S, K409F, K409V, K409Q or K409R.
  • amino acid substitutions comprise:
  • any one of the first Fc chain and the second Fc chain is replaced by T366L and D399R, and the other is replaced by L351E, Y407L and K409V;
  • any one of the first Fc chain and the second Fc chain is replaced by T366L and D399C, and the other is replaced by L351G, Y407L and K409C;
  • any one of the first Fc chain and the second Fc chain is replaced by T366L and D399C, and the other is replaced by L351Y, Y407A and K409P;
  • any one of the first Fc chain and the second Fc chain is replaced by T366P and D399N, and the other is replaced by L351V, Y407P and K409S;
  • any one of the first Fc chain and the second Fc chain is replaced by T366W and D399G, and the other is replaced by L351D, Y407P and K409S;
  • any one of the first Fc chain and the second Fc chain is replaced by T366P and D399I, and the other is replaced by L351P, Y407F and K409F;
  • any one of the first Fc chain and the second Fc chain is replaced by T366V and D399T, and the other is replaced by L351K, Y407T and K409Q;
  • amino acid substitutions comprise:
  • any one of the first Fc chain and the second Fc chain is replaced by T366L and K409V, and the other is replaced by L351E, Y407L and D399R;
  • any one of the first Fc chain and the second Fc chain is replaced by T366L and K409C, and the other is replaced by L351G, Y407L and D399C;
  • any one of the first Fc chain and the second Fc chain is replaced by T366L and K409P, and the other is replaced by L351Y, Y407A and D399C;
  • any one of the first Fc chain and the second Fc chain is replaced by T366P and K409S, and the other is replaced by L351V, Y407P and D399N;
  • any one of the first Fc chain and the second Fc chain is replaced by T366W and K409S, and the other is replaced by L351D, Y407P and D399G;
  • any one of the first Fc chain and the second Fc chain is replaced by T366P and K409F, and the other is replaced by L351P, Y407F and D399I;
  • any one of the first Fc chain and the second Fc chain is replaced by T366V and K409Q, and the other is replaced by L351K, Y407T and D399T;
  • the amino acid of any of the first Fc chain and the second Fc chain is replaced by T366L and D399R, and the other amino acid is replaced by L351E, Y407L, and K409V.
  • the first antigen binding domain and the second antigen binding domain are selected from the group consisting of a Fab fragment, a scFv fragment, a variable domain fragment Fv, and a heavy chain variable region fragment VHH of a heavy chain antibody.
  • the first antigen binding domain and the second antigen binding domain are both Fab fragments.
  • one of the first antigen binding domain and the second antigen binding domain is a Fab fragment and the other is a scFv.
  • the Fab fragment comprises a different first heavy chain variable region and a second heavy chain variable region, and a different first light chain variable region and a second light chain variable region.
  • the first Fc chain and the first antigen binding functional region covalently linked thereto and the second Fc chain and the second antigen antigen binding functional region covalently linked thereto are free from other polypeptides
  • the source dimer is less than 50% by weight based on the weight of all polypeptide chains.
  • the first antigen binding domain comprises the amino acid sequences of SEQ ID Nos: 2 and 6.
  • the second antigen binding domain comprises the amino acid sequences of SEQ ID Nos: 10 and 12.
  • the first antigen binding domain further comprises the amino acid sequences of SEQ ID Nos: 4 and 8.
  • the second antigen binding domain further comprises the amino acid sequences of SEQ ID NOs: 4 and 14.
  • the amino acid sequence of the bispecific antibody is the corresponding combination of SEQ ID Nos: 2, 4, 6, 8, 10, 12, and 14. As SEQ ID Nos: 2, 4, 6, and 8 are combined with each other, SEQ ID Nos: 10, 4, 12, and 14 are combined with each other, and then, the combined two are further combined to form a bispecific antibody of the present invention.
  • a second aspect of the invention relates to an isolated polynucleotide encoding a bispecific antibody in the form of a heterodimer as described in the first aspect.
  • the nucleotide sequence encoding the amino acid of the first antigen binding domain is selected from the group consisting of: SEQ ID NOs: 1 and 5.
  • the nucleotide sequence encoding the amino acid of the second antigen binding domain is selected from the group consisting of: SEQ ID NOs: 9 and 11.
  • nucleotide sequence encoding the amino acid of the first antigen binding domain is further selected from the group consisting of: SEQ ID NOs: 3 and 7.
  • nucleotide sequence encoding the amino acid of the second antigen binding domain is further selected from the group consisting of: SEQ ID NOs: 3 and 13.
  • the sequence of the polynucleotide is the corresponding combination of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13. As SEQ ID Nos: 1, 3, 5 and 7 are combined with each other, SEQ ID Nos: 9, 3, 11 and 13 are combined with each other.
  • a third aspect of the invention relates to a recombinant expression vector comprising the isolated polynucleotide of the second aspect.
  • the expression vector is a plasmid vector X0GC engineered based on pC DNA.
  • a fourth aspect of the invention relates to a host cell comprising the isolated polynucleotide of the second aspect, or the recombinant expression vector of the third aspect.
  • the host cell is selected from the group consisting of human embryonic kidney cell HEK293 or HEK293T, HEK293F, HEK293E engineered on HEK293 cell basis; hamster ovary cell CHO or CHO-S, CHO modified based on CHO cells. -dhfr - , CHO/DG44, ExpiCHO; Escherichia coli or Escherichia coli-based E.
  • yeast or yeast-based Pichia pastoris Saccharomyces cerevisiae , Kluyveromyces cerevisiae, Hansenula polymorpha
  • insect cells or cells modified based on insect cells High5, SF9 plant cells; mammalian breast cells, somatic cells.
  • a fifth aspect of the invention relates to a composition
  • a composition comprising the bispecific antibody in the form of a heterodimer according to the first aspect or the isolated polynucleotide of the second aspect or the recombination of the third aspect
  • An expression vector or host cell of the fourth aspect and a pharmaceutically acceptable carrier.
  • a sixth aspect of the invention relates to a method of producing a bispecific antibody in the form of a heterodimer according to the first aspect, comprising the steps of:
  • the host cell is selected from the group consisting of human embryonic kidney cell HEK293 or HEK293T, HEK293F, HEK293F engineered on HEK293 cell basis; hamster ovary cell CHO or CHO-S, CHO modified on CHO cell basis -dhfr - , CHO/DG44, ExpiCHO; Escherichia coli or Escherichia coli-based E.
  • yeast or yeast-based Pichia pastoris Saccharomyces cerevisiae , Kluyveromyces cerevisiae, Hansenula polymorpha
  • insect cells or cells modified based on insect cells High5, SF9 plant cells; mammalian breast cells, somatic cells.
  • the reducing step comprises 1) performing a reduction reaction in the presence of a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine or others Chemical derivative; 2) removal of reducing agent, for example, reduction reaction at 4 ° C for a minimum of 3 hours in the presence of 0.1 mM or higher concentration of dithiothreitol.
  • a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine or others Chemical derivative
  • removal of reducing agent for example, reduction reaction at 4 ° C for a minimum of 3 hours in the presence of 0.1 mM or higher concentration of dithiothreitol.
  • the oxidizing step is oxidizing in air, and also includes performing an oxidation reaction in the presence of an oxidizing agent selected from the group consisting of: L-dehydroascorbic acid or a chemical derivative thereof, for example, at a concentration of 0.5 mM or higher.
  • an oxidizing agent selected from the group consisting of: L-dehydroascorbic acid or a chemical derivative thereof, for example, at a concentration of 0.5 mM or higher.
  • Oxidation reaction at 4 ° C for a minimum of 5 hours in the presence of dehydroascorbic acid.
  • the method further comprises the step of isolating the purification.
  • a seventh aspect of the invention relates to the bispecific antibody of the heterodimeric form of the first aspect and/or the isolated polynucleotide of the second aspect and/or the recombinant expression vector of the third aspect and The use of the host cell of the fourth aspect and/or the composition of the fifth aspect for the preparation of a medicament for preventing and/or treating a disease in a subject.
  • the eighth aspect of the invention relates to the bispecific antibody of the heterodimeric form of the first aspect and/or the isolated polynucleotide of the second aspect and/or the recombinant expression vector of the third aspect and The host cell of the fourth aspect and/or the composition of the fifth aspect, which is used as a medicament for preventing and/or treating a disease in a subject.
  • a ninth aspect of the invention relates to a method of preventing and/or treating a disease comprising the heterodimeric antibody of the heterologous dimer form of the first aspect and/or the isolated polynucleotide of the second aspect And/or the recombinant expression vector of the third aspect and/or the host cell of the fourth aspect and/or the composition of the fifth aspect are administered to a subject in need thereof.
  • the subject is a mammal, preferably a human subject.
  • the disease is selected from the group consisting of a leukemia, a lymphoma, a myeloma, a brain tumor, a head and neck squamous cell carcinoma, a non-small cell lung cancer, a nasopharyngeal cancer, an esophageal cancer, a gastric cancer, a pancreatic cancer, Gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, bone cancer.
  • the present invention devised a novel anti-PD-L1/anti-CD47 natural antibody structure-like heterodimeric form bispecific antibody which has the characteristics of natural IgG and has no heavy light chain mismatch and is highly stable heterologous Anti-PD-L1/anti-CD47 bispecific antibody in the form of a polymer.
  • the bispecific antibody prepared by the invention can simultaneously bind two target molecules PD-L1 and CD47, and can be applied to a complex disease treatment to exert a better effect than a single therapeutic agent.
  • the bispecific antibody as a single therapeutic molecule not only facilitates the use of patients and medical workers, but also simplifies the complicated new drug development process.
  • Figure 1 shows the elution peak chromatogram of anti-PD-L1-Fc1.
  • Figure 2 shows the elution peak chromatogram of anti-CD47-Fc2.
  • Figure 3 shows the structure of an anti-PD-L1/anti-CD47 heterodimeric antibody molecule.
  • Figure 4 A half antibody molecular structure diagram showing one heavy chain and one light chain.
  • FIG. 1 Results of SEC-HPLC analysis of a half-antibody molecule of one heavy chain and one light chain.
  • Panels A and B show the results of anti-PD-L1 half antibody molecules and anti-CD47 half antibody molecules, respectively.
  • Figure 6 shows the results of SEC-HPLC analysis of anti-PD-L1/anti-CD47 heterodimeric antibody molecules.
  • Figure 7 shows the results of RPC analysis of anti-PD-L1/anti-CD47 heterodimeric antibody molecules.
  • Figure 8. shows the results of CE analysis of anti-PD-L1/anti-CD47 heterodimeric antibody molecules.
  • Panel A shows the affinity of anti-PD-L1/anti-CD47 heterodimer antibody for PD-L1
  • Panel B shows the affinity of anti-PD-L1/anti-CD47 heterodimer antibody for CD47.
  • Figure 10 shows that the combination of PD-L1 mAb and CD47 does not bind both PD-L1 and CD47, and only the anti-PD-L1/anti-CD47 heterodimeric antibody has the activity of binding both antigens simultaneously.
  • Panels A and B show the binding activity of CD47 mAb and anti-PD-L1/anti-CD47 heterodimer to HCC827 and RBC, respectively.
  • Figure 12 shows the T cell regulatory activity of anti-PD-L1/anti-CD47 heterodimeric antibodies.
  • Figure 13 Shows the anti-PD-L1/anti-CD47 heterodimeric antibody-mediated phagocytic activity of macrophages on tumor cells.
  • Covalent linkage refers to a bispecific antibody in the form of a heterodimer. Between two Fc chains, between any Fc chain and the antigen-binding functional region to which it is linked, is linked by a covalent bond into a molecule.
  • the Fc chain comprises a first antigen binding functional region and a second antigen binding functional region joined by one or more covalent linkages (eg, a disulfide bond chain); the first Fc chain and the second Fc chain are respectively a valency linkage (such as an imine bond or an amide bond) attached to an antigen binding functional region;
  • An antigen-binding domain refers to a region that can specifically interact with a target molecule such as an antigen, and its action is highly selective, and a sequence that recognizes a target molecule generally does not recognize other molecular sequences.
  • Representative antigen binding functional regions include: a variable region of an antibody, a structural allosteric domain of an antibody variable region, a binding domain of a receptor, a ligand binding domain, or an enzyme binding domain.
  • One or more disulfide linkages mean that the first Fc chain and the second Fc chain are joined by one or more disulfide linkages to form a heterodimeric fragment.
  • the formation of one or more disulfide bonds may be the first Fc chain and the second Fc chain or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are synthesized in the same cell.
  • the first Fc chain and the second Fc chain or the first Fc chain and the second Fc chain and the antigen-binding functional regions linked thereto are separately synthesized in different cells, and then formed by in vitro reductive oxidation.
  • the first Fc chain and the second Fc chain refer to a binding fragment formed by covalent linkage, the covalent linkage comprising a disulfide bond, each strand comprising at least a portion of an immunoglobulin heavy chain constant region; and the first Fc chain and The second Fc chain differs in amino acid sequence and includes at least one amino acid difference.
  • the first Fc chain and the second Fc chain in the invention there is a strong mutual repulsion between the same chains, and there is an attraction between the different chains, so when co-expressed in cells, the first Fc chain and the first The di-Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions, are more prone to form heterodimers.
  • first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are expressed in two host cells, respectively, the first Fc chain or the first Fc chain and The linked antigen binding domain does not tend to form a homodimer, and the second Fc chain or the second Fc chain and its associated antigen binding domain do not tend to form homodimers.
  • first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are expressed in two host cells, respectively, and in the presence of a reducing agent
  • the proportion of homodimers is less than 50%, ie the ratio of monomer (one Fc chain or one Fc chain and its associated antigen binding functional region) is greater than 50%.
  • the immunoglobulin is a symmetrical structure with four polypeptide chains. Two of the same heavy chains with larger relative molecular weights contain 450-550 amino acid residues, and the relative molecular mass is between 55,000 and 70,000 Da. The same light chain (L chain) with a short, relatively small molecular weight, containing about 210 amino acid residues, has a relative molecular mass of about 24,000 Da.
  • the different immunoglobulin heavy and light chains vary greatly in the sequence of about 110 amino acids near the N-terminus, called the variable region (V region), while the remaining amino acid sequences near the C-terminus are relatively stable, called constant Zone (constant region, zone C).
  • variable region in the heavy chain accounts for about 1/4 of the length of the heavy chain, and the constant region accounts for about 3/4 of the length of the heavy chain.
  • IgG ⁇
  • IgA ⁇
  • IgD ⁇
  • IgM ⁇
  • IgE ⁇
  • the constant region is both the backbone of the immunoglobulin molecule and one of the sites that activate the immune response.
  • the class of antibodies of the invention can be converted by known methods if desired.
  • an antibody of the invention that is initially IgM can be classified into an IgG antibody of the invention.
  • class switching techniques can be used to convert one IgG subclass to another, such as from IgGl to IgG2.
  • the effector functions of the antibodies of the invention can be switched by isotypes to, for example, IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies for various therapeutic uses.
  • the antibody of the invention is an IgGl antibody, such as IgGl, kappa.
  • a portion of the constant region in the present invention includes at least a region in which the first Fc chain and the second Fc chain interact, which region is a part of amino acids in the CH3 region for IgG, including at least GLN347, TYR349, THR350, LEU 351 , SER 354, ARG 355, ASP 356, GLU 357, LYS 360, SER 364, THR 366, LEU 368, LYS 370, ASN390, LYS392, THR394, PRO395, VAL 397, ASP399, SER400, PHE405, TYR407, LYS409, LYS439 .
  • the attachment of the first Fc chain and the second Fc chain to an antigen-binding functional region by a covalent bond or a linker, respectively, means that the first Fc chain and the second Fc chain are respectively linked to an antibody antigen by a covalent bond or a linker.
  • a covalent bond is an atom.
  • the atoms of the same element or different elements may be bonded by a covalent bond, and the covalent bond between the first Fc chain and the second Fc chain of the present invention includes, but is not limited to, an amino group of one molecule of amino acid and another molecule.
  • An amide bond formed by a dehydration reaction of an amino acid, or an aldehyde group of ethylene glycol or polyethylene glycol or other compound or a multimer thereof forms an amide bond or an imine bond with an amino group of one molecule of an amino acid
  • the linker is An amino acid sequence in which two polypeptide chains are joined by a covalent bond or a compound or a multimer of a compound, wherein an amino acid sequence includes, but is not limited to, a small peptide such as GGGGSGGGGSGGGGS, and the first Fc chain is passed through an amide bond.
  • the first Fc chain and the second Fc chain are more prone to form a heterodimer without tending to form a homodimer, respectively, since the same polypeptide chain is in the first Fc chain and the second Fc chain.
  • first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are expressed in two host cells, respectively, the first Fc chain or the first Fc chain and The linked antigen binding domain does not tend to form a homodimer, and the second Fc chain or the second Fc chain and its associated antigen binding domain do not tend to form homodimers.
  • the Kabat EU index numbering system means that Kabat uses a method to assign a number to each amino acid of an antibody sequence and such a method of specifying the numbering of each residue has become a standard method in the art.
  • the Kabat protocol can be extended to other antibodies not present in his study, based on conserved amino acids, the target antibody is aligned to one of the consensus sequences identified by Kabat.
  • the Fc domain refers to a fragment crystallizable (Fc), which corresponds to the CH2 and CH3 domains of Ig, and is a site where Ig interacts with an effector molecule or a cell.
  • Fc fragment crystallizable
  • IgG is an abbreviation for Immunoglobulin G (IgG) and is a major antibody component of serum. According to the difference in antigenicity of the r chain in IgG molecules, human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4.
  • a semi-antibody molecule refers to a structure in which a heavy chain of an antibody and a light chain are formed, wherein a heavy chain and a light chain may be linked by a covalent bond or may not be linked by a covalent bond, and is a monovalent antibody structure that recognizes an antigen. .
  • a Fab fragment is a molecular recognition sequence, which is a fragment of antigen binding (Fab), which corresponds to two arms of an antibody molecule and consists of a complete light and heavy chain VH and CH1 domains.
  • scFv is a molecular recognition sequence which is a structural isomer of an antibody fragment obtained by genetic engineering of a light chain variable region and a heavy chain variable region of an antibody.
  • the extracellular domain of a membrane receptor is a molecular recognition sequence, and the membrane receptor usually includes an extracellular region located outside the cell that recognizes and binds to the corresponding antigen or ligand, and anchors the receptor to the transmembrane region of the cell surface.
  • a ligand for a cell membrane receptor refers to a protein, small peptide or compound that can be recognized and bound by the extracellular region of the membrane receptor.
  • Cytokines are low-molecular-weight soluble proteins produced by various cells induced by immunogens, mitogens or other stimulators. They regulate innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, and damaged tissue repair. Features. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors and the like.
  • the protein expression tag refers to an amino acid sequence added at the N-terminus or C-terminus of the target protein, which may be a small peptide or a long amino acid.
  • the addition of the tag may facilitate the correct folding of the protein, and may facilitate the separation and purification of the protein. It is beneficial to reduce the degradation of proteins in cells.
  • Commonly used labels include, but are not limited to, HA, SUMO, His, GST, GFP, and Flag.
  • the antibody to be used in the heterodimeric form of the bispecific antibody of the present invention is not limited at all.
  • antibodies known in the art to be useful in the treatment and/or prevention of diseases are useful in the present invention.
  • a bispecific antibody in the form of a heterodimer of the invention may have one or more substitutions, deletions, additions and/or insertions.
  • certain amino acids can replace other amino acids in the structure of the protein without significant loss of ability to bind to other polypeptides (eg, antigens) or cells. Since the binding ability and protein properties determine the biological functional activity of the protein, certain amino acid sequence substitutions can be made on the protein sequence without significant loss of their biological utility or activity.
  • a polypeptide variant contains one or more conservative substitutions.
  • Constant substitution refers to the replacement of an amino acid by another amino acid having similar properties such that one skilled in the art of peptide chemistry can expect substantially no change in the secondary structure and hydrophilic nature of the polypeptide.
  • Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into account various of the foregoing features are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine ; and valine, leucine and isoleucine.
  • identity has the meaning commonly known in the art, and those skilled in the art are also familiar with the rules and criteria for determining the identity between different sequences, referring to sequence alignment and introduction of gaps (if necessary, After the maximum percent homology is obtained, the residues of the polynucleotide or polypeptide sequence variant are the same percentage of the non-variant sequence. .
  • identity in the case where the definition of identity is satisfied, it is also required that the obtained variant sequence has the biological activity possessed by the parent sequence. Methods and means for screening variant sequences using the above activities are well known to those skilled in the art. Such variant sequences can be readily obtained by those skilled in the art in light of the teachings of the present disclosure.
  • the polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least the polynucleotide or polypeptide described herein. About 98%, or at least about 99%, or at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the polynucleotide or polypeptide identity. Due to the redundancy of the genetic code, variants of these sequences encoding the same amino acid sequence will be present.
  • polynucleotide compositions are provided which are capable of hybridizing to a polynucleotide sequence provided by the invention, or a fragment thereof, or a complement thereof, under moderate to high stringency conditions.
  • Hybridization techniques are well known in the art of molecular biology.
  • suitable moderately stringent conditions for testing the hybridization of a polynucleotide of the invention to other polynucleotides include pre-washing in a solution of 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); Hybridization was carried out at 50-60 ° C, 5 x SSC, overnight; and washed twice with 2 x, 0.5 x and 0.2 x SSC containing 0.1% SDS for 20 minutes at 65 °C.
  • suitable high stringency hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60-65 ° C or 65-70 ° C.
  • the host cell of the invention may be all cells used for expression of a foreign gene, including but not limited to E. coli, yeast, insect cells, plant cells, mammalian cells.
  • Vectors of the invention include vectors that can replicate in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes.
  • a vector comprising a polynucleotide of the invention is a vector suitable for propagation or replication of a polynucleotide, or a vector suitable for expression of a polypeptide of the invention.
  • Such vectors are known in the art and are commercially available.
  • Vector includes both a shuttle vector and an expression vector.
  • plasmid constructs also include an origin of replication (such as the origin of the replicated ColE1) and a selectable marker (such as ampicillin or tetracycline resistance) for plasmid replication and selection in bacteria, respectively.
  • Expression vector refers to a vector comprising control sequences or regulatory elements required for expression of an antibody of the invention, including antibody fragments, in a bacterial or eukaryotic cell.
  • the vector of the present invention may be all vectors for expression of a foreign gene, including but not limited to a plasmid vector, wherein the plasmid vector comprises at least an origin of replication, a promoter, a gene of interest, a multiple cloning site, a selection marker gene, preferably
  • the vector of the present invention includes, but is not limited to, a plasmid vector obtained based on pC DNA engineering, such as a X0GC vector.
  • Subjects of the invention include birds, reptiles, mammals, and the like.
  • the mammal comprises a rodent, a primate, preferably, the primate comprises a human.
  • the scope of the diseases involved in the present invention includes, but is not limited to, tumors.
  • the tumors include: leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, Esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer , bone cancer.
  • a pharmaceutically acceptable carrier means a pharmaceutical carrier conventionally used in the pharmaceutical field, such as diluents, excipients and water, fillers such as starch, sucrose, lactose, microcrystalline cellulose, etc.; binders such as cellulose Derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrating agents such as sodium carboxymethyl starch, hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate and sodium hydrogencarbonate ; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate; adsorption carriers such as aged soil and soap clay; lubricants such as talc, calcium and magnesium stearate, micronized silica gel and Polyethylene glycol and the like. It is also possible to add other adjuvants such as flavoring agents, sweeteners and the like to the composition.
  • the invention relates to the following technical solutions.
  • a heterodimeric antibody in the form of a heterodimer comprising a first antigen binding domain capable of specifically binding to PD-L1 and a second antigen binding domain capable of specifically binding to CD47
  • the bispecific antibody comprises a first Fc chain and a second Fc chain linked by one or more disulfide linkages, the first Fc chain and the second Fc chain being linked to each other by a covalent bond or a linker
  • the PD-L1 antigen binding functional region and the CD47 antigen binding functional region, or the first Fc chain and the second Fc chain are respectively linked to the CD47 antigen binding functional region and the PD-L1 antigen binding functional region by a covalent bond or a linker
  • the first Fc chain and the second Fc chain comprise a substitution of 5 amino acids at the following position
  • the first Fc chain and the second Fc chain comprising the above amino acid substitutions are more likely to form heterodimers with each other without tending to form homodimers, respectively.
  • amino acid positions are numbered according to the Kabat EU index numbering system.
  • bispecific antibody in the form of a heterodimer according to the first aspect, wherein the first Fc chain and the second Fc chain amino acid are replaced by the following,
  • cysteine asparagine, isoleucine, glycine, arginine, threonine or alanine;
  • d) at position 407 is replaced by leucine, alanine, valine, phenylalanine, threonine or histidine;
  • bispecific antibody in the form of a heterodimer according to claim 1 or 2, wherein the amino acid substitution comprises:
  • bispecific antibody in the form of a heterodimer according to any one of claims 1 to 3, wherein the amino acid substitution comprises:
  • the bispecific antibody in the form of a heterodimer according to the first aspect, wherein the amino acid substitution of the first Fc chain is T366L and D399R, and the amino acid substitution of the second Fc chain is L351E, Y407L and K409V.
  • bispecific antibody in the form of a heterodimer according to any one of claims 1 to 5, wherein the Fc chain is derived from IgG.
  • bispecific antibody in the form of a heterodimer according to any one of claims 1 to 7, wherein the PD-L1 and CD47 antigen-binding functional regions are both Fab fragments.
  • heterodimeric antibody in the form of a heterodimer according to any one of claims 1 to 7, wherein the PD-L1 and CD47 antigen binding functional regions are one Fab fragment and the other is scFv.
  • heterodimeric antibody of the heterodimeric form according to any one of claims 7-9, wherein the Fab fragment comprises a different first heavy chain variable region and a second heavy chain variable region, and Different first light chain variable regions and second light chain variable regions.
  • bispecific antibody in the form of a heterodimer according to any one of claims 1 to 11, wherein the amino acid sequence of the bispecific antibody is selected from the group consisting of: SEQ ID NO. 2, 4, 6, 8, 10 , 12 and 14.
  • a recombinant expression vector comprising the isolated polynucleotide of claim 13 or 14.
  • a host cell comprising the isolated polynucleotide of any one of claims 13 or 14, or the recombinant expression vector of claim 15 or 16.
  • the host cell according to claim 17, which is selected from the group consisting of human embryonic kidney cell HEK293 or HEK293T, HEK293E, HEK293F modified based on HEK293 cells; hamster ovary cell CHO or modified based on CHO cells Obtained CHO-S, CHO-dhfr - , CHO/DG44, ExpiCHO; Escherichia coli or Escherichia coli-based E.
  • a composition comprising the heterodimeric antibody of the heterodimeric form of any one of claims 1 to 12 or the isolated polynucleotide of claim 13 or 14 or the technical solution 15 Or the recombinant expression vector of claim 16 or the host cell of claim 17 or 18, and a pharmaceutically acceptable carrier.
  • the host cell is selected from the group consisting of human embryonic kidney cell HEK293 or HEK293T, HEK293F, HEK293F modified based on HEK293 cells; hamster ovary cell CHO or CHO cell-based transformation And obtained CHO-S, CHO-dhfr - , CHO/DG44, ExpiCHO; Escherichia coli or Escherichia coli-based Escherichia coli BL21, BL21 (DE3), Rosetta, Origami; yeast or yeast-based transformation The obtained Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces cerevisiae, Hansenula polymorpha; insect cells or cells modified based on insect cells High5, SF9; plant cells; mammalian mammary cells, somatic cells.
  • the reducing step comprises 1) adding a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyl) Phosphine or other chemical derivative, 2) reduction reaction at 4 ° C for a minimum of 3 hours in the presence of 0.1 mM or higher concentration of dithiothreitol, 3) removal of the reducing agent, as by desalting.
  • a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyl) Phosphine or other chemical derivative
  • the oxidizing step comprises 1) oxidizing in air, and also including adding an oxidizing agent selected from the group consisting of: L-dehydroascorbic acid or other chemical derivative 2)
  • the oxidation reaction is carried out in the presence of 0.5 mM or higher of L-dehydroascorbic acid at 4 ° C for a minimum of 5 hours.
  • bispecific antibody in the form of a heterodimer according to any one of claims 1 to 12 and/or the isolated polynucleotide of claim 13 or 14 and/or the technical solution 15 or 16
  • bispecific antibody in the form of a heterodimer according to any one of claims 1 to 12 and/or the isolated polynucleotide of claim 13 or 14 and/or the technical solution 15 or 16
  • a method for preventing and/or treating a disease comprising the separation of the heterodimeric antibody of the heterodimeric form according to any one of claims 1 to 12 and/or the method of claim 13 or The polynucleotide and/or the recombinant expression vector of claim 15 or 16 and/or the composition of the host cell of claim 17 or 18 or the composition of claim 19 is administered to a subject in need thereof .
  • heterodimeric antibody The use of the heterodimeric antibody, the isolated polynucleotide, the recombinant expression vector, the host cell or the composition, or the method 27 of the heterodimeric form of the invention of claim 26
  • the method wherein the subject is a mammal, preferably a human subject.
  • the disease is selected from the group consisting of a leukemia, a lymphoma, a myeloma, a brain tumor, a head and neck squamous cell carcinoma, a non-small cell lung cancer, a nasopharyngeal cancer, an esophageal cancer, a gastric cancer, a pancreatic cancer, Gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, bone cancer.
  • the following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.
  • the various antibodies used in the following examples of the invention are all derived from standard antibodies of the commercial route.
  • X0GC expression vectors containing antibody heavy and light chains against human PD-L1, respectively, were constructed, wherein the antibody variable region sequence was derived from http://www.imgt.org/mAb-DB/mAbcard? AbId 472, the heavy chain constant region is human IgG1 (Fc1, wherein the N297A mutation to eliminate the ADCC/CDC effect).
  • the light chain variable region nucleotide sequence is shown in SEQ ID NO. 1, the amino acid sequence is shown in SEQ ID NO: 2; the light chain constant region nucleotide sequence is shown in SEQ ID NO. 3, and the amino acid sequence is as SEQ. ID NO: 4; the heavy chain variable region nucleotide sequence is shown in SEQ ID NO.
  • the amino acid sequence is shown in SEQ ID NO: 6; the heavy chain constant region nucleotide sequence is as SEQ ID NO. As shown, the amino acid sequence is shown as SEQ ID NO: 8.
  • the light chain variable region and the light chain constant region, the heavy chain variable region, and the heavy chain constant region were respectively amplified by a PCR method.
  • NEC Phusion Ultra-Fidelity DNA Polymerase (F-530L) was used for all PCR reactions in this application. PCR primers are routinely designed according to the principle of base complementation and the need for restriction sites.
  • the reaction system was: H 2 O 8.9 ⁇ l, 5 ⁇ Phusion ultra-fidelity DNA polymerase buffer 4 ⁇ l, 1 mM dNTP 4 ⁇ l, upstream primer 1 ⁇ l, downstream primer 1 ⁇ l, Phusion ultra-fidelity DNA polymerase 0.1 ⁇ l, template 1 ⁇ l.
  • the variable region and constant region PCR products were electrophoresed on a 1.5% agarose gel and the corresponding fragments were recovered using a DNA recovery kit (Promega, A9282, the same below).
  • the recovered variable region fragment and the constant region fragment are used as templates, and the variable region upstream primer and the constant region downstream primer are used, and then a round of PCR reaction is carried out, and then the corresponding fragment is recovered to obtain a full-length fragment of the light chain or the heavy chain.
  • the X0GC vector and the full-length fragment were digested with EcoRI (NEB, Cat. No. R3101L) and HindIII (NEB, Cat. No. R3104L).
  • the digestion reaction system was: 10 ⁇ buffer 3 2 ⁇ l, EcoRI and Hind III each 0.5 ⁇ l, and the gel was recovered.
  • the full length fragment was 3 ⁇ l, H 2 O 14.5 ⁇ l.
  • the digestion system was reacted at 37 ° C for 3 hours.
  • the digested product was ligated with T4 DNA ligase (NEB, Cat. No. M0202V).
  • the reaction system was: 10 ⁇ ligase buffer 2 ⁇ l, ligase 0.5 ⁇ l, and the full-length fragment obtained by gel recovery was 3 ⁇ l.
  • X0GC vector 3 ⁇ l, H 2 O 11.5 ⁇ l.
  • the reaction was allowed to react at room temperature for 12 hours.
  • the ligation product was transformed into E. coli competent cell DH5 ⁇ (Tiangen, CB104, the same below).
  • An X0GC expression vector against the heavy and light chains of the human PD-L1 antibody was obtained for expression of the heavy chain (Fc1) and light chain of the antibody in eukaryotic cells, respectively.
  • the present invention simultaneously constructs an X0GC expression vector containing an antibody heavy and a light chain of anti-human CD47, respectively, wherein the antibody variable region sequence is derived from WO2016109415A1.
  • the light chain variable region nucleotide sequence is shown in SEQ ID NO. 9, the amino acid sequence is shown as SEQ ID NO: 10; the light chain constant region nucleotide sequence is shown in SEQ ID NO. 3, and the amino acid sequence is SEQ. ID NO: 4; the heavy chain variable region nucleotide sequence is shown in SEQ ID NO. 11, the amino acid sequence is shown as SEQ ID NO: 12; the heavy chain constant region nucleotide sequence is as SEQ ID NO. As shown, the amino acid sequence is shown in SEQ ID NO: 14.
  • An X0GC expression vector for the heavy and light chain of anti-human CD47 antibody was obtained for expression of the heavy chain (Fc2) and light chain of the antibody in eukaryotic cells, respectively.
  • the expression vectors are the heavy and light chains containing human anti-PD-L1 antibody transfected 293F cells (FreeStyle TM 293F Cells, NO R79007, invitrogen), respectively, further comprising an anti-human CD47 antibody heavy chain and light
  • the expression vector for the strand was also transfected into 293F cells. Cells are seeded one day before transfection, the day of transfection the cells were collected by centrifugation, the cells were resuspended in fresh FreeStyle TM 293 expression medium (FreeStyle TM 293 Expression Medium, NO 12338001, Gibco) in a cell density of 200 * 105 Cells / mL.
  • the plasmid was added to the transfection volume at a final concentration of 36.67 ug/mL and gently mixed; then linear PEI (polyethyleneimine, linear, MW 25000, Cat. No. 43896, Alfa Aesar) was added at a final concentration of 55 ug/mL. Mix well. Thereafter, the cells were placed in a cell culture incubator and incubated at 37 ° C for 1 hour at 120 rpm. A 19-fold transfection volume of fresh medium was then added. Continue to incubate at 37 ° C with a 120 rpm shaker. The cell culture supernatant transfected for 5-6 days was collected by centrifugation.
  • linear PEI polyethyleneimine, linear, MW 25000, Cat. No. 43896, Alfa Aesar
  • the amount of expression was determined by ELISA.
  • the precipitate was removed by filtration through a 0.2 ⁇ m filter before applying column chromatography. This step was carried out at 4 °C.
  • Purification was carried out at 4 °C using an AKTA explorer 100 protein purification system (GE Healthcare) and an affinity chromatography column rProtein A Sepharose Fast Flow (16 mm I.D., 22 ml, GE Healthcare).
  • the column was first equilibrated with mobile phase A (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.4), and the supernatant of the above treated cells was loaded after the baseline was stabilized at a flow rate of 5 ml/min.
  • the sample was equilibrated with mobile phase A.
  • the samples were anti-PD-L1 expression products and anti-CD47 expression products, respectively.
  • PBS phosphate buffer saline
  • the obtained anti-PD-L1 and anti-CD47 purified product solutions were separately adjusted to 1 mg/ml with the PBS, and 1/200 times the final volume of 1 M DTT was added (the final concentration of DTT was 0.1 mM, 0.5 mM, 1 mM, 2 mM, respectively). 5 mM, 10 mM, 20 mM), reduction at 4 ° C (3-8 hours), the disulfide bond is opened by the reduction process, anti-PD-L1 and antibody homodimer molecules contained in the anti-CD47 product The disulfide bond in the hinge region is also opened, forming a half-antibody molecule containing a heavy chain and a light chain, and the structure is shown in FIG.
  • the reduced sample was analyzed by SEC-HPLC (TOSOH, TSKgel superSW3000) containing 1 mM DTT reducing agent in mobile phase buffer. As shown in Fig. 5, the ratio of anti-PD-L1 and anti-CD47 homodimer molecules was smaller than that. 10%, the proportion of half antibody molecules is greater than 90%.
  • the reduced anti-PD-L1 and anti-CD47 half-antibody molecules are then mixed in an equimolar ratio and subjected to a recombination reaction (0.5-24 hours) at 4 ° C.
  • anti-PD-L1 and anti-CD47 half-antibody molecules A bispecific antibody containing a heterodimer of anti-PD-L1 and anti-CD47 half-antibody molecules is formed by non-covalent interaction between CH2 and CH3, and then the protein solution is concentrated by ultrafiltration through an ultrafiltration concentrating tube.
  • the molecular weight cut-off is 10KDa
  • the oxidation reaction is carried out by air or an oxidizing agent to reform the disulfide bond of the bispecific antibody of the heterodimer.
  • the conditions of the oxidation reaction included placing the sample in air for 1 day, 3 days, and 4 days, and adding an oxidizing agent 100 mM L-dehydroascorbic acid (final protein concentration 1 mg/ml, oxidant final concentration 0.5 mM, 1 mM, 5 mM, 10 mM) at 4 ° C conditions. The oxidation was carried out, and the reaction was carried out for 24 hours.
  • the anti-PD-L1/anti-CD47 heterodimeric antibody molecule obtained by the reductive oxidation of the anti-PD-L1 and anti-CD47 expression products was concentrated by ultrafiltration in a ultrafiltration concentrating tube (nominal molecular weight cutoff 10 KDa), and the solution was replaced with 10 mM. Sodium phosphate buffer, pH 5.8. Purification was carried out at 4 °C using an AKTA explorer 100 protein purification system (GE Healthcare) and an ion chromatography column Source 15S (16 mm I.D., 17 ml, GE Healthcare). The column was first equilibrated with mobile phase A (10 mM sodium phosphate, pH 7.0).
  • the protein solution treated as described above was loaded at a flow rate of 3 ml/min and equilibrated with mobile phase A after loading. Afterwards, 20 column volumes (0% B-100% B, 170 min, flow rate 2 ml/min) were washed with a gradient of A (10 mM sodium phosphate, pH 5.8) to B (10 mM sodium phosphate, pH 5.8), and the main peaks were collected and collected.
  • the purified product was subjected to purity analysis by SEC-HPLC, and the purity was 99.3% as shown in Fig. 6.
  • the RPC-HPLC Thermo Fisher, MAbPac RP
  • Thermo Fisher, MAbPac RP was carried out, and the result was as shown in Fig. 7, the purity was 100%;
  • the ability of the anti-PD-L1/anti-CD47 heterodimer antibody to bind to a single antigen was determined by enzyme-linked immunosorbent assay (ELISA).
  • Wash PBST 5 times A heterodimeric antibody sample diluted in a PBST containing 1% BSA and a control were added, and 100 ⁇ L was added to each well, and incubated at 25 ° C for 1 hour. Wash PBST 5 times. Then, horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, Cat. No. AP309P) diluted 1:10000 in PBST containing 1% BSA was added, 100 ⁇ L per well was added, and incubation was carried out for 1 hour at 25 °C. Wash PBST 5 times. The colorimetric substrate TMB was added at 100 ⁇ L/well and developed for 10 minutes at room temperature. Color development was stopped by adding 1 M H 2 SO 4 at 100 ⁇ L/well. The absorbance at 450 nm was read on a microplate reader.
  • the anti-PD-L1/anti-CD47 heterodimer antibody has high affinity for PD-L1, and is equivalent to the antigen-binding activity of PD-L1 bivalent monoclonal antibody;
  • the anti-PD-L1/anti-CD47 heterodimer antibody has a high affinity for CD47 and is comparable to the antigen binding activity of CD47 bivalent monoclonal antibody.
  • the simultaneous binding ability of the anti-PD-L1/anti-CD47 heterodimer antibody to two different antigens was determined by enzyme-linked immunosorbent assay (ELISA).
  • a heterodimeric antibody sample diluted in a PBST containing 1% BSA and a control were added, and 100 ⁇ L was added to each well, and incubated at 25 ° C for 1 hour. Wash PBST 5 times. Then, biotin-labeled PD-1-Fc (Beijing Hanmei Pharmaceutical Co., Ltd.) diluted in 1% BSA in PBST was added, 0.5 ⁇ g/mL, 100 ⁇ L per well, and incubated at 25 ° C for 1 hour. Streptavidin-horseradish peroxidase conjugate (BD Pharmingen, Cat. No.
  • the combination of PD-L1 mAb and CD47 could not bind to PD-L1, CD47 at the same time, and only the anti-PD-L1/anti-CD47 heterodimer antibody had the activity of binding both antigens simultaneously.
  • HCC827 tumor cells express PD-L1 and CD47, and red blood cells (RBC) express only CD47.
  • RBC red blood cells
  • HCC827 and RBC were mixed together and flow cytometry (FCM) was used to detect the selectivity of heterodimer binding to the two cells in the mixed cells.
  • HCC827 cells purchased from ATCC
  • RBC cells taken from healthy population
  • HCC827 was mixed with 1 ⁇ 10 6 cells per tube
  • RBC was mixed with 10 ⁇ 10 6 cells per tube
  • resuspended in 200 ⁇ L of cold DPBS containing 2% FBS GIBCO, Cat. No. 14190-136
  • Diluted heterodimeric antibody samples as well as controls.
  • the flow tube was incubated on ice for 30 minutes. Wash twice with PBS containing 2% FBS.
  • the cells were resuspended in 200 ⁇ L of 2% FBS and 1:1000 diluted FITC-labeled anti-human IgG antibody (Beijing Zhongshan Jinqiao, Cat. No. ZF0306) in cold PBS. Incubate on ice for 30 minutes in the dark. Wash twice with PBS containing 2% FBS. The cells were resuspended in 500 ⁇ L of cold PBS, and the cell suspension was subjected to detection analysis on a flow cytometer (BD, FACS Calibur) to read the fluorescence intensity of each of the two cells in the mixed cells.
  • BD flow cytometer
  • the regulatory activity of anti-PD-L1/anti-CD47 heterodimer antibody on T cell immune response was determined by mixed lymphocyte reaction (MLR).
  • IL-4 Beijing Yiqiao Shenzhou, article number 11846-HNAE. Incubate for 3 days, change to medium, and incubate for another 3 days. The medium was then changed to complete medium (RPMI 1640 containing 10% FBS) containing 100 ng/ml GM-CSF, 100 ng/ml IL-4, and 20 ng/ml TNF- ⁇ , and incubated for 1 day. That is, DC cells are obtained.
  • the collected human DC cells and human T cells were resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in 96-well plates, and the inoculated DC cells and T cells were 1 ⁇ 10 4 /well, respectively. ⁇ 10 5 /well, mixed culture.
  • a heterodimeric antibody sample diluted with the complete medium sequence and a control were added.
  • the plate was incubated for 5 days in a 37 ° C carbon dioxide incubator. After the incubation was completed, the supernatant in the well was taken out and the cytokine IFN- ⁇ (RayBiotech, article number ELH-IFNg) was detected according to the kit manual.
  • human T cells activate the secretion of IFN- ⁇ under the stimulation of allogeneic DC cells.
  • Addition of PD-L1 antibody enhances T cell activation and promotes cytokine secretion.
  • the anti-PD-L1/anti-CD47 heterodimer antibody also showed strong T cell regulatory activity, and significantly promoted the secretion of the cytokine IFN- ⁇ .
  • Preparation of mature human macrophages Resuscitation was collected from human PBMC cells (Lonza, Cat. No. CC-2702). Human PBMC cells were resuspended in serum-free RPMI 1640 medium at a cell density of 5 ⁇ 10 6 /mL and seeded in cell culture flasks and incubated in a 37 ° C carbon dioxide incubator for 90 minutes. The culture supernatant and suspension cells were discarded, and the adherent cells were cultured in complete medium (RPMI 1640 containing 10% FBS), and 25 ng/ml M-CSF (Beijing Yishen Shenzhou, Cat. No. 11792-HNAN) was added and incubated for 7 days. .
  • complete medium RPMI 1640 containing 10% FBS
  • M-CSF Beijing Yishen Shenzhou, Cat. No. 11792-HNAN
  • Macrophages were then harvested and resuspended in complete medium (RPMI 1640 containing 10% FBS) containing 25 ng/ml M-CSF and 50 ng/ml IFN- ⁇ (Beijing Yiqiao Shenzhou, Cat. No. 11725-HNAS).
  • complete medium RPMI 1640 containing 10% FBS
  • the cell suspension was inoculated into a 48-well cell culture plate at 50,000 cells per well, and 1 angel macrophage was incubated for maturity.
  • CFSE CFSE was diluted with PBS to a working concentration of 5 ⁇ M and pre-warmed at 37 ° C, centrifuged at 1000 rpm for 5 minutes to collect Raji cells, resuspended Raji with pre-warmed CFSE working solution, and incubated at 37 ° C for 15 minutes. Wash once with complete medium, resuspend in complete medium, incubate for 30 minutes, wash twice with complete medium, and resuspend in complete medium for later use.
  • the 48-well plates were washed 3 times with complete medium.
  • the CFSE-stained Raji cells were pre-incubated with the heterodimer sample to be tested and the control for 15 minutes, then added to a 48-well culture plate, and incubated for 2 hours in a 37 ° C carbon dioxide incubator. After the incubation, the 48-well plates were washed 3 times with complete medium, and added to the germ agglutinin, alexa fluor 555 (Life technologies, Cat. No. W32464) diluted in complete medium, 10 ⁇ g/ml, and incubated for 15 minutes in the dark. The 48-well plates were washed 3 times with complete medium and fixed with 4% paraformaldehyde for 15 minutes.
  • the 48-well plates were washed 3 times with complete medium and complete medium was added. Photographing with a fluorescence microscope and counting the number of cells. The phagocytic index (%) was calculated as the number of green-labeled Raji cells phagocytized/the number of red-labeled macrophages present x 100.
  • the anti-PD-L1/anti-CD47 heterodimer antibody mediates macrophage phagocytosis of Raji tumor cells, comparable to the activity of CD47 mAb.

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Abstract

提供了一种具有天然IgG特点、并且没有重轻链错配的高度稳定的异源二聚体形式的抗PD-L1/抗CD47双特异抗体及其制备方法。其中双特异抗体的第一Fc链和第二Fc链中的任意一条在366位及399位上包含氨基酸替换，另一条在351位、407位及409位上包含氨基酸替换。

Description

抗PD‐L1/抗CD47天然抗体结构样异源二聚体形式双特异抗体及其制备 技术领域
本发明涉及抗PD-L1/抗CD47天然抗体结构样异源二聚体形式双特异抗体及其制备。具体而言，本发明提供了一种具有天然IgG特点、并且没有重轻链错配的高度稳定的异源二聚体形式的抗PD-L1/抗CD47双特异抗体及其制备方法。
背景技术
程序性死亡配体-1(programmed death ligand 1,PD-L1)是免疫检查点程序性死亡受体-1(programmed death-1，PD-1)的配体，属于B7家族，诱导性表达于多种免疫细胞表面，包括T细胞、B细胞、单核细胞、巨噬细胞、DC细胞以及内皮细胞、表皮细胞等。PD-L1与PD-1结合后，主要参与T细胞活化的负调控，可以调节免疫应答的强弱程度和持续时间。PD-L1除了作为PD-1的配体以外，还能作为CD80的配体，向T细胞传递负调控信号，诱导T细胞免疫耐受(Autoimmun Rev,2013,12(11):1091-1100.Front Immunol,2013,4:481.Nat Rev Cancer,2012,12(4):252-264.Trends Mol Med.2015 Jan；21(1):24-33.Clin Cancer Res.2012 Dec 15；18(24):6580-7.)。在正常情况下，PD-L1和PD-1可以介导和维持机体组织的自身免疫耐受，防止在炎症反应过程中免疫系统过度活化伤害自身组织，对避免自身免疫性疾病的发生具有积极作用；在病理情况下，其参与肿瘤免疫以及多种自身免疫病的发生发展过程。多项研究报道，PD-L1在多种肿瘤组织中高表达，PD-1在肿瘤浸润淋巴细胞中高表达，且PD-L1和PD-1的过表达与肿瘤不良临床预后密切相关(Anticancer Agents Med Chem.2015；15(3):307-13.Hematol Oncol Stem Cell Ther.2014 Mar；7(1):1-17.Trends Mol Med.2015 Jan；21(1):24-33.Immunity.2013 Jul 25；39(1):61-73.J Clin Oncol.2015 Jun 10；33(17):1974-82.)。利用PD-L1单克隆抗体阻断PD-L1/PD-1以及CD80/PD-L1的相互作用，在临床前实验研究和临床中都显示出了良好的抗肿瘤效果。目前PD-L1单克隆抗体已经被批准用于治疗非小细胞肺癌和尿路上皮癌等多种肿瘤。但是，只有一小部分肿瘤病人能受益于此类单抗疗法，多数病人对此类单抗没有应答(Expert Opin Ther Targets.2014 Dec；18(12):1407-20.Oncology(Williston Park).2014 Nov；28 Suppl 3:15-28.)。
CD47，又叫整合素相关蛋白，是一种大小为50Kd的跨膜蛋白，属于免疫球蛋白 超家族。广泛表达于多种细胞上，但在多种肿瘤细胞上表达明显增强(Proc Natl Acad Sci U S A，2012，109(17):6662-6667)。CD47的配体为信号调节蛋白α(signal-regulatory proteinα,SIRPα)，主要表达于巨噬细胞，与CD47结合后传递“不要吃我”的信号，抑制巨噬细胞的吞噬作用(Curr Opin Immunol,2009,21(1):47-52)。通过使用抗CD47抗体可以阻断CD47-SIRPα信号通路，从而发挥抗肿瘤作用。目前已有多种抗CD47单克隆抗体进入了临床研究阶段，用于治疗多种血液肿瘤和实体瘤。但由于红细胞等表面也表达CD47，这些抗CD47治疗可能会导致较严重的贫血和血小板减少等不良反应，并且生物利用度较低。
本领域仍然有必要研究一种同时阻断PD-L1和CD47信号通路的新型治疗药物。
发明内容
本发明提供了一种新的具有天然IgG结构特点、并且没有重轻链错配的高度稳定的异源二聚体形式的能同时阻断PD-L1和CD47的双功能抗体及其制备方法，该双功能抗体倾向于选择性结合同时高表达PD-L1和CD47的肿瘤细胞，从而发挥高效、特异的杀伤效果，同时具有较低的毒副作用。
本发明的第一方面涉及一种异源二聚体形式的双特异抗体，其包含第一Fc链和第二Fc链，以及能与PD-L1特异性结合的第一个抗原结合功能区和能与CD47特异性结合的第二个抗原结合功能区；
其中所述第一Fc链和第二Fc链均为包含氨基酸替换的免疫球蛋白G Fc片段，并且所述第一Fc链及第二Fc链共同构成可以与Fc受体结合的异源二聚体；
其中所述第一Fc链和第二Fc链通过共价键或连接体分别连接到所述第一抗原结合功能区和第二抗原结合功能区；
并且其中所述第一Fc链和第二Fc链中的任意一条在366位及399位上包含氨基酸替换，另一条在351位、407位及409位上包含氨基酸替换，其中氨基酸位置根据Kabat EU指数编号系统编号。
本文中第一Fc链及第二Fc仅出于区分存在的两条Fc链的目的而定义，并不意味着其重要性不同或次序存在差异。同时，第一Fc链和第二Fc链与第一抗原结合功能区和第二抗原结合功能区的连接也是任意的，即第一Fc链既可以与第一抗原结合功能区连接，也可以与第二抗原结合功能区连接，第二Fc链也是如此。
在一些实施方案中，第一Fc链及第二Fc链氨基酸替换如下，
a)L351G、L351Y、L351V、L351P、L351D、L351E、L351K或L351W；
b)T366L、T366P、T366W或T366V；
c)D399C、D399N、D399I、D399G、D399R、D399T或D399A；
d)Y407L、Y407A、Y407P、Y407F、Y407T或Y407H；和
e)K409C、K409P、K409S、K409F、K409V、K409Q或K409R。
在一些实施方案中，氨基酸替换包括：
a)第一Fc链和第二Fc链中的任意一条为T366L及D399R替换，另一条为L351E、Y407L及K409V替换；
b)第一Fc链和第二Fc链中的任意一条为T366L及D399C替换，另一条为L351G、Y407L及K409C替换；
c)第一Fc链和第二Fc链中的任意一条为T366L及D399C替换，另一条为L351Y、Y407A及K409P替换；
d)第一Fc链和第二Fc链中的任意一条为T366P及D399N替换，另一条为L351V、Y407P及K409S替换；
e)第一Fc链和第二Fc链中的任意一条为T366W及D399G替换，另一条为L351D、Y407P及K409S替换；
f)第一Fc链和第二Fc链中的任意一条为T366P及D399I替换，另一条为L351P、Y407F及K409F替换；
g)第一Fc链和第二Fc链中的任意一条为T366V及D399T替换，另一条为L351K、Y407T及K409Q替换；
h)第一Fc链和第二Fc链中的任意一条为T366L及D399A替换，另一条为L351W、Y407H及K409R替换。
在一些实施方案中，氨基酸替换包括：
a)第一Fc链和第二Fc链中的任意一条为T366L及K409V替换，另一条为L351E、Y407L及D399R替换；
b)第一Fc链和第二Fc链中的任意一条为T366L及K409C替换，另一条为L351G、Y407L及D399C替换；
c)第一Fc链和第二Fc链中的任意一条为T366L及K409P替换，另一条为L351Y、Y407A及D399C替换；
d)第一Fc链和第二Fc链中的任意一条为T366P及K409S替换，另一条为L351V、 Y407P及D399N替换；
e)第一Fc链和第二Fc链中的任意一条为T366W及K409S替换，另一条为L351D、Y407P及D399G替换；
f)第一Fc链和第二Fc链中的任意一条为T366P及K409F替换，另一条为L351P、Y407F及D399I替换；
g)第一Fc链和第二Fc链中的任意一条为T366V及K409Q替换，另一条为L351K、Y407T及D399T替换；
h)第一Fc链和第二Fc链中的任意一条为T366L及K409R替换，另一条为L351W、Y407H及D399A替换。
在一些实施方案中，第一Fc链和第二Fc链中的任意一条的氨基酸替换为T366L和D399R，另一条的氨基酸替换为L351E、Y407L和K409V。
在一些实施方案中，第一个抗原结合功能区和第二个抗原结合功能区选自Fab片段、scFv片段、可变结构域片段Fv和重链抗体的重链可变区片段VHH。
在一些实施方案中，第一个抗原结合功能区和第二个抗原结合功能区都是Fab片段。
在一些实施方案中，第一个抗原结合功能区和第二个抗原结合功能区中一个是Fab片段，另一个是scFv。
在一些实施方案中，Fab片段包含不同的第一重链可变区及第二重链可变区，以及不同的第一轻链可变区及第二轻链可变区。
在一些实施方案中，第一Fc链及与其共价相连的第一抗原结合功能区，和第二Fc链及与其共价相连的第二抗原结合功能区，在存在还原剂的溶液中且所述溶液中除所述第一Fc链及与其共价相连的第一抗原结合功能区和第二Fc链及与其共价相连的第二抗原抗原结合功能区以外不含其它多肽时，其形成同源二聚体的基于所有多肽链的重量比例均低于50％。
在一些实施方案中，第一抗原结合功能区包含SEQ ID NO:2和6的氨基酸序列。
在一些实施方案中，第二抗原结合功能区包含SEQ ID NO:10和12的氨基酸序列。
在一些实施方案中，第一抗原结合功能区进一步包含SEQ ID NO:4和8的氨基酸序列。
在一些实施方案中，第二抗原结合功能区进一步包含SEQ ID NO:4和14的氨基酸序列。
在一些实施方案中，双特异抗体的氨基酸序列是SEQ ID NO：2、4、6、8、10、12 和14的相应组合。如SEQ ID No:2、4、6和8相互组合，SEQ ID No:10、4、12和14相互组合，然后，组合后的二者再组合，以形成本发明的双特异性抗体。
本发明的第二方面涉及一种分离的多核苷酸，其编码如第一方面所述的异源二聚体形式的双特异抗体。
在一些实施方案中，编码第一抗原结合功能区氨基酸的核苷酸序列选自：SEQ ID NO:1和5。
在一些实施方案中，编码第二抗原结合功能区氨基酸的核苷酸序列选自：SEQ ID NO:9和11。
在一些实施方案中，编码第一抗原结合功能区氨基酸的核苷酸序列进一步选自：SEQ ID NO:3和7。
在一些实施方案中，编码第二抗原结合功能区氨基酸的核苷酸序列进一步选自：SEQ ID NO:3和13。
在一些实施方案中，多核苷酸的序列是SEQ ID NO:1、3、5、7、9、11和13的相应组合。如SEQ ID No:1、3、5和7相互组合，SEQ ID No:9、3、11和13相互组合。
本发明的第三方面涉及一种重组表达载体，其包含第二方面所述的分离的多核苷酸。
在一些实施方案中，表达载体为基于pCDNA改造得到的质粒载体X0GC。
本发明的第四方面涉及一种宿主细胞，其包含第二方面所述的分离的多核苷酸，或第三方面所述的重组表达载体。
在一些实施方案中，宿主细胞选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO；大肠杆菌或以大肠杆菌为基础改造得到的大肠杆菌BL21、BL21(DE3)、Rosetta、Origami；酵母菌或以酵母为基础改造得到的毕赤酵母、酿酒酵母、乳酸克鲁维亚酵母、多形汉逊酵母；昆虫细胞或以昆虫细胞为基础改造得到的细胞High5、SF9；植物细胞；哺乳动物乳腺细胞、体细胞。
本发明的第五方面涉及一种组合物，其包含第一方面所述的异源二聚体形式的双特异抗体或第二方面所述的分离的多核苷酸或第三方面所述的重组表达载体或第四方面所述的宿主细胞，及药学上可接受的载体。
本发明的第六方面涉及一种生产如第一方面所述的异源二聚体形式的双特异抗体的方法，其包括步骤：
1)将第二方面所述的分离的多核苷酸或第三方面所述的重组表达载体分别在宿主细 胞中进行表达；
2)将在宿主细胞中分别表达的蛋白进行还原；以及
3)将还原的蛋白混合，然后将混合物进行氧化。
在一些实施方案中，宿主细胞选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO；大肠杆菌或以大肠杆菌为基础改造得到的大肠杆菌BL21、BL21(DE3)、Rosetta、Origami；酵母菌或以酵母为基础改造得到的毕赤酵母、酿酒酵母、乳酸克鲁维亚酵母、多形汉逊酵母；昆虫细胞或以昆虫细胞为基础改造得到的细胞High5、SF9；植物细胞；哺乳动物乳腺细胞、体细胞。
在一些实施方案中，还原步骤包括1)在还原剂存在下进行还原反应，所述还原剂选自：2-巯基乙胺、二硫苏糖醇、三(2-羧乙基)膦或其他化学衍生物；2)去除还原剂，例如在0.1mM或更高浓度二硫苏糖醇存在下在4℃条件下进行还原反应最少3小时。所述还原剂及还原反应条件的限定也适用于本文涉及还原剂及还原反应使用的其它情况。
在一些实施方案中，氧化步骤为在空气中氧化，也包括在氧化剂存在下进行氧化反应，所述氧化剂选自：L-脱氢抗坏血酸或其化学衍生物，例如在0.5mM或更高浓度L-脱氢抗坏血酸存在下在4℃条件下进行氧化反应最少5小时。
在一些实施方案中，所述的方法还包括分离纯化的步骤。
本发明的第七方面涉及第一方面所述的异源二聚体形式的双特异抗体和/或第二方面所述的分离的多核苷酸和/或第三方面所述的重组表达载体和/或第四方面所述的宿主细胞和/或第五方面所述的组合物在制备用于预防和/或治疗受试者疾病的药物中的用途。
本发明的第八方面涉及第一方面所述的异源二聚体形式的双特异抗体和/或第二方面所述的分离的多核苷酸和/或第三方面所述的重组表达载体和/或第四方面所述的宿主细胞和/或第五方面所述的组合物，其用做用于预防和/或治疗受试者疾病的药物。
本发明的第九方面涉及一种预防和/或治疗疾病的方法，包括将第一方面所述的异源二聚体形式的双特异抗体和/或第二方面所述的分离的多核苷酸和/或第三方面所述的重组表达载体和/或第四方面所述的宿主细胞和/或第五方面所述的组合物施予有需求的受试者。
在一些实施方案中，受试者是哺乳动物，优选地，人类受试者。
在一些实施方案中，所述疾病选自如下肿瘤：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、 结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤、小细胞肺癌、骨癌。
本发明设计了一种全新的抗PD-L1/抗CD47天然抗体结构样异源二聚体形式双特异抗体，其具有天然IgG特点，并且没有重轻链错配，是高度稳定的异源二聚体形式的抗PD-L1/抗CD47双特异抗体。本发明制备的双特异抗体能同时结合两种靶分子PD-L1和CD47，将其应用于复杂疾病治疗时可发挥比单一治疗剂更好的效果。同时，相对于多个药物的组合治疗，该双特异抗体作为单一治疗分子不仅方便了患者和医疗工作者的使用，也简化了复杂的新药开发流程。
附图说明
图1：示出了抗PD-L1-Fc1的洗脱峰色谱图。
图2：示出了抗CD47-Fc2的洗脱峰色谱图。
图3：示出了抗PD-L1/抗CD47异源二聚体抗体分子的结构。
图4.示出了一条重链和一条轻链的半抗体分子结构图。
图5.示出了一条重链和一条轻链的半抗体分子的SEC-HPLC分析结果。其中A图和B图分别表示抗PD-L1半抗体分子和抗CD47半抗体分子的结果。
图6.示出了抗PD-L1/抗CD47异源二聚体抗体分子的SEC-HPLC分析结果。
图7.示出了抗PD-L1/抗CD47异源二聚体抗体分子的RPC分析结果。
图8.示出了抗PD-L1/抗CD47异源二聚体抗体分子的CE分析结果。
图9.A图示出了抗PD-L1/抗CD47异源二聚体抗体对PD-L1的亲和力，B图示出了抗PD-L1/抗CD47异源二聚体抗体对CD47的亲和力。
图10.示出了PD-L1单抗和CD47的组合不能同时结合PD-L1和CD47，只有抗PD-L1/抗CD47异源二聚体抗体具有同时结合两种抗原的活性。
图11.A图和B图分别示出了CD47单抗和抗PD-L1/抗CD47异源二聚体对HCC827和RBC的结合活性。
图12.示出了抗PD-L1/抗CD47异源二聚体抗体的T细胞调控活性。
图13.示出了抗PD-L1/抗CD47异源二聚体抗体介导的巨噬细胞对肿瘤细胞的吞噬活性。
具体实施方式
定义：
共价连接是指异源二聚体形式的双特异抗体中，两个Fc链之间，任一个Fc链及与其相连接的抗原结合功能区之间，是通过共价键而连接成为一个分子。其中Fc链包含通过一个或多个通过共价连接(如二硫键链)而连接的第一抗原结合功能区及第二抗原结合功能区；该第一Fc链及第二Fc链分别通过共价连接(如亚胺键或酰胺键)而连接到一个抗原结合功能区上；
抗原结合功能区是指可以与目标分子如抗原发生特异性相互作用的区域，其作用具有高度选择性，识别一种目标分子的序列通常不能识别其他分子序列。代表性的抗原结合功能区包括：抗体的可变区、抗体可变区的结构变构体、受体的结合域、配体结合域或酶结合域。
一个或多个二硫键链间连接是指第一Fc链及第二Fc链通过一个或多个二硫键链间连接，形成异源二聚体片段。在本发明中，一个或多个二硫键的形成可以是第一Fc链及第二Fc链或者第一Fc链及第二Fc链及其相连接的抗原结合功能区在同一个细胞内合成时形成，也可以是第一Fc链及第二Fc链或者第一Fc链及第二Fc链及其相连接的抗原结合功能区在不同细胞内分别合成，之后通过体外还原氧化的方法形成。
第一Fc链及第二Fc链是指通过共价连接而组成结合片段，共价连接包括二硫键，每条链至少包含免疫球蛋白重链恒定区的一部分；并且该第一Fc链及第二Fc链在氨基酸序列上是不同的，至少包括了一位氨基酸的不同。在此发明中的第一Fc链及第二Fc链，相同链之间存在强烈的相互排斥作用，而不同链之间存在吸引作用，因此当在细胞内共同表达时，第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区，更倾向于形成异源二聚体。将第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区分别在两个宿主细胞内表达时，第一Fc链或者第一Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体，第二Fc链或者第二Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体。在本发明中，当第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区分别在两个宿主细胞内表达时，并且在存在还原剂时，同源二聚体的比例低于50％，即单体(一条Fc链或者一条Fc链及其相连接的抗原结合功能区)比例大于50％。
免疫球蛋白是具有四条多肽链的对称结构，其中两条较长、相对分子量较大的相同的重链，含450～550个氨基酸残基，相对分子质量在55000～70000Da之间；两条较短、相对分子量较小的相同的轻链(L链)，含约210个氨基酸残基，相对分子质量约24000Da。 不同的免疫球蛋白重链和轻链在靠近N端的约110个氨基酸的序列变化很大，称为可变区(variable region，V区)，而靠近C端的其余氨基酸序列相对稳定，称为恒定区(constant region，C区)。重链中可变区约占重链长度的1/4，恒定区约占重链长度的3/4。对于已知五种Ig来说，IgG(γ)、IgA(α)、IgD(δ)、IgM(μ)和IgE(ε)，其中前三类Ig的H链内有三个恒定区，即CH1、CH2和CH3组成。后两类(IgM和IgE)的H链中有一个VH区和四个恒定区，即CH1至CH4。恒定区既是免疫球蛋白分子的骨架，又是激活免疫反应的部位之一。虽然本发明实施例涉及IgG，但本领域技术人员知晓，如果希望的话，可以通过已知方法转换本发明的抗体的类别。例如，最初是IgM的本发明抗体可以类别转换为本发明的IgG抗体。此外，类别转换技术可以用来将一个IgG亚类转化成另一亚类，例如从IgGl转换到IgG2。因此，本发明的抗体的效应子功能可以通过同种型切换变为例如IgG1、IgG2、IgG3、IgG4、IgD、IgA、IgE或IgM抗体，用于各种治疗用途。在一个实施例中，本发明的抗体是IgG1抗体，例如IgG1,κ。
本发明中的恒定区一部分至少包括了第一Fc链和第二Fc链相互作用的区域，该区域对于IgG来说，是位于CH3区域的一部分氨基酸，至少包括GLN347、TYR349、THR 350、LEU 351、SER 354、ARG 355、ASP 356、GLU 357、LYS 360、SER 364、THR 366、LEU 368、LYS 370、ASN390、LYS392、THR394、PRO395、VAL 397、ASP399、SER400、PHE405、TYR407、LYS409、LYS439。
第一Fc链及第二Fc链分别通过共价键或连接体连接到一个抗原结合功能区上是指第一Fc链及第二Fc链分别通过共价键或连接体连接到一个抗体的抗原结合片段，或可以识别抗原的单链抗体，或可以识别抗原的其他抗体片段变构体，或可以识别配体的受体，或可以识别受体的配体，其中所述共价键是指是化学键的一种，两个或多个原子共同使用它们的外层电子，在理想情况下达到电子饱和的状态，由此组成比较稳定的化学结构叫做共价键，或者说共价键是原子间通过共用电子对所形成的相互作用。同一种的元素的原子或不同元素的都可以通过共价键结合，对于本发明的第一Fc链及第二Fc链间的共价键，包括但不限于一分子氨基酸的氨基与另一分子氨基酸的羧基脱水反应形成的酰胺键，或者乙二醇或聚乙二醇或其他化合物或其多聚物的醛基与一分子氨基酸的氨基形成酰胺键或亚胺键，其中连接体是可以将两条多肽链通过共价键连接起来的一段氨基酸序列或者一种化合物或者一种化合物的多聚体，其中一段氨基酸序列包括但不限于一段小肽，如GGGGSGGGGSGGGGS，通过酰胺键将第一Fc链或第二Fc链，以及可以识别抗原的单链抗体，或可以识别抗原的其他抗体片段结构变构体连接起来
第一Fc链与第二Fc链更倾向于形成异源二聚体而不倾向于各自形成同源二聚体是指，由于在第一Fc链与第二Fc链中，相同的多肽链间存在互相排斥的作用，而不同的多肽链间存在吸引作用，因此当在细胞内共同表达时，第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区，更倾向于形成异源二聚体。将第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区分别在两个宿主细胞内表达时，第一Fc链或者第一Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体，第二Fc链或者第二Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体。
Kabat EU指数编号系统是指，Kabat利用一种方法将一个编号指定给抗体序列的每个氨基酸并且这种指定每个残基的编号的方法已经成为本领域的标准方法。Kabat方案可以延伸到不存在于他的研究中的其它抗体，基于保守的氨基酸，将目标抗体与Kabat鉴定的共有序列之一进行比对。
Fc结构域是指可结晶段(fragment crystallizable,Fc)，相当于Ig的CH2和CH3结构域，是Ig与效应分子或者细胞相互作用的部位。
IgG是免疫球蛋白G(Immunoglobulin G，IgG)的缩写，是血清主要的抗体成分，根据IgG分子中的r链抗原性差异，人IgG有四个亚型：IgG1、IgG2、IgG3、IgG4。
半抗体分子是指抗体的一条重链与一条轻链形成的结构，其中重链与轻链间可以通过共价键连接，也可以不通过共价键连接，是一种识别抗原的单价抗体结构。
Fab片段是一种分子识别序列，是抗原结合片段(fragment of antigen binding,Fab)，相当于抗体分子的两个臂，由一个完整的轻链和重链的VH和CH1结构域组成。scFv是一种分子识别序列，是一种由抗体的轻链可变区与重链可变区通过基因工程改造而得到的抗体片段的结构异构体。膜受体的细胞外区是一种分子识别序列，膜受体通常包括位于细胞外部的可以识别并结合相应抗原或者配体的细胞外区域，将受体锚定在细胞表面的跨膜区，以及在胞内的具有激酶活性或者可以传递信号通路的胞内区。细胞膜受体的配体是指能被膜受体胞外区识别并结合的蛋白质，小肽或化合物。细胞因子是免疫原、丝裂原或其他刺激剂诱导多种细胞产生的低分子量可溶性蛋白质，具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种功能。细胞因子可被分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。蛋白表达标签指在目标蛋白的N端或C端加入的一段氨基酸序列，可以是小肽也可以是长的氨基酸，标签的加入可以有利于蛋白质的正确折叠，可以有利于蛋白质的分离纯化， 可以有利于降低蛋白质在胞内的降解，常用的标签包括但不限于HA、SUMO、His、GST、GFP、Flag。
可应用于本发明的异源二聚体形式的双特性抗体中的抗体并无任何限制。优选地，现有技术中已知可以用于治疗和/或预防疾病的抗体均可以用于本发明。
本发明的异源二聚体形式的双特性抗体可具有一个或多个替换、缺失、添加和/或插入。例如，某些氨基酸可以替换在蛋白质结构中的其它氨基酸而没有明显损失与其它多肽(如抗原)或细胞结合的能力。由于结合能力和蛋白性质决定了蛋白的生物功能活性，可以在蛋白序列上进行某些氨基酸序列的替换而不会明显损失它们的生物效用或活性。
在许多情况中，多肽变体含有一个或多个保守替换。“保守替换”是指其中氨基酸被其它具有类似性质的氨基酸所替换，使得肽化学领域中技术人员可预期多肽的二级结构和亲水性质基本上不发生变化。
氨基酸替换通常是基于氨基酸侧链取代基的相对相似性，如它们的疏水性、亲水性、电荷、大小等。考虑了各种前述特征的示例性替换是本领域技术人员公知的并包括：精氨酸和赖氨酸；谷氨酸和天冬氨酸；丝氨酸和苏氨酸；谷氨酰胺和天冬酰胺；以及缬氨酸、亮氨酸和异亮氨酸。
本发明使用的术语“同一性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同一性的规则、标准，是指在序列比对和引入缺口(如果必要的话，以获得最大百分比同源性)后，多核苷酸或多肽序列变体的残基与非变体序列的相同的百分比。。在本发明中，在满足同一性限定的情况下，还需要所获得的变体序列具有母体序列所具有的生物活性。本领域技术人员公知如何利用上述活性筛选变体序列的方法和手段。本领域技术人员可以在本申请公开内容的教导下容易地获得这样的变体序列。在具体实施方式中，多核苷酸和多肽变体与本文所述的多核苷酸或多肽具有至少约70％、至少约75％、至少约80％、至少约90％、至少约95％、至少约98％、或至少约99％，或至少约99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的多核苷酸或多肽同一性。由于遗传密码的冗余性，将存在编码相同氨基酸序列的这些序列的变体。
本发明的另一个实施方式中，提供了能够在中度至高度严格条件下与本发明提供的多核苷酸序列或其片段或其互补序列相杂交的多核苷酸组合物。杂交技术是分子生物学领域公知的。为了说明的目的，用于测试本发明的多核苷酸与其他多核苷酸杂交的合适中等严格条件包括在5×SSC、0.5％SDS、1.0mM EDTA(pH8.0)的溶液中预洗；在50-60℃、5×SSC、过夜的条件下杂交；再于65℃下20分钟用含0.1％SDS的2×、0.5×和0.2×的SSC各洗 涤两次。本领域技术人员理解，可容易地操纵杂交的严格性，例如通过改变杂交溶液的盐含量和/或进行杂交的温度。例如，在另一个实施方式中，合适的高严格杂交条件包括上述的条件，所不同的是杂交温度升高了，例如达到60-65℃或65-70℃。
本发明的宿主细胞可以是用于外源基因表达的所有细胞，包括但不限于大肠杆菌，酵母，昆虫细胞，植物细胞，哺乳动物细胞。
本发明的载体包括可以在任何类型的细胞或生物体中进行复制的载体，包括如质粒、噬菌体、粘粒和迷你染色体。在一些实施方式中，包括本发明多核苷酸的载体是适合于多核苷酸繁殖或复制的载体，或者是适合于表达本发明多肽的载体。这样的载体是本领域已知并可以购买的。
“载体”包括穿梭载体和表达载体。通常，质粒构建体也包括分别用于细菌中质粒复制和选择的复制起点(如复制的ColE1起点)和选择标记(如氨苄青霉素或四环素抗性)。“表达载体”是指包含用于在细菌或真核细胞中表达本发明的抗体包括抗体片段所需要的控制序列或调控元件的载体。
本发明的载体可以是用于外源基因表达的所有载体，包括但不限于质粒载体，其中质粒载体至少包含复制起始位点、启动子、目的基因、多克隆位点、筛选标记基因，优选地，本发明所述载体包括但不限于基于pCDNA改造得到的质粒载体，比如X0GC载体。
本发明的受试者包括禽类、爬行动物、哺乳动物等。优选地，哺乳动物包括啮齿类动物、灵长类动物，优选地，灵长类动物包括人。
本发明所涉及的疾病的范围包括但不限于肿瘤，优选的，所述肿瘤包括：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤、小细胞肺癌、骨癌。
药学上可接受的载体是指是指药学领域常规的药物载体，例如：稀释剂、赋形剂和水等，填充剂如淀粉、蔗糖，乳糖、微晶纤维素等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；润湿剂如甘油；崩解剂如羧甲基淀粉钠，羟丙纤维素，交联羧甲基纤维素，琼脂、碳酸钙和碳酸氢钠；吸收促进剂如季铵化合物；表面活性剂如十六烷醇，十二烷基硫酸钠；吸附载体如高龄土和皂粘土；润滑剂如滑石粉、硬脂酸钙和镁、微粉硅胶和聚乙二醇等。另外还可以在组合物中加入其它辅剂如香味剂、甜味剂等。
在一些实施方案中，本发明涉及下述技术方案。
技术方案1、一种异源二聚体形式的双特异抗体，其包含能与PD-L1特异性结合的第一个抗原结合功能区和能与CD47特异性结合的第二个抗原结合功能区，其中所述双特异抗体包含通过一个或多个二硫键链间连接的第一Fc链及第二Fc链，该第一Fc链和第二Fc链通过共价键或连接体分别连接到PD-L1抗原结合功能区和CD47抗原结合功能区上，或者该第一Fc链和第二Fc链通过共价键或连接体分别连接到CD47抗原结合功能区和PD-L1抗原结合功能区上；并且第一Fc链和第二Fc链在如下位置包含5个氨基酸的替换，
1)第一Fc链上366位及399位氨基酸替换，第二Fc链上351位、407位及409位氨基酸的替换；或者
2)第一Fc链上366位及409位氨基酸替换，第二Fc链上351位、399位及407位氨基酸替换；
包含上述氨基酸替换的第一Fc链与第二Fc链更倾向于互相形成异源二聚体而不倾向于各自形成同源二聚体，
其中氨基酸位置根据Kabat EU指数编号系统编号。
技术方案2、如技术方案1所述的异源二聚体形式的双特异抗体，其中第一Fc链及第二Fc链氨基酸替换如下，
a)351位上替换为甘氨酸、酪氨酸、缬氨酸、脯氨酸、天冬氨酸、谷氨酸，赖氨酸或色氨酸；
b)366位上替换为亮氨酸、脯氨酸、色氨酸或缬氨酸；
c)399位上替换为半胱氨酸、天冬酰胺、异亮氨酸、甘氨酸、精氨酸、苏氨酸或丙氨酸；
d)407位上替换为亮氨酸、丙氨酸、脯氨酸、苯丙氨酸，苏氨酸或组氨酸；和
e)409位上替换为半胱氨酸、脯氨酸、丝氨酸、苯丙氨酸、缬氨酸、谷氨酰胺或精氨酸。
技术方案3.如技术方案1或2所述的异源二聚体形式的双特异抗体，其中氨基酸替换包括：
a)第一Fc链T366L及D399R替换，第二Fc链L351E、Y407L及K409V替换；
b)第一Fc链T366L及D399C替换，第二Fc链L351G、Y407L及K409C替换；
c)第一Fc链T366L及D399C替换，第二Fc链L351Y、Y407A及K409P替换；
d)第一Fc链T366P及D399N替换，第二Fc链L351V、Y407P及K409S替换；
e)第一Fc链T366W及D399G替换，第二Fc链L351D、Y407P及K409S替换；
f)第一Fc链T366P及D399I替换，第二Fc链L351P、Y407F及K409F替换；
g)第一Fc链T366V及D399T替换，第二Fc链L351K、Y407T及K409Q替换；
h)第一Fc链T366L及D399A替换，第二Fc链L351W、Y407H及K409R替换。
技术方案4.如技术方案1-3任一项所述的异源二聚体形式的双特异抗体，其中氨基酸替换包括：
a)第一Fc链T366L及K409V替换，第二Fc链L351E、Y407L及D399R替换；
b)第一Fc链T366L及K409C替换，第二Fc链L351G、Y407L及D399C替换；
c)第一Fc链T366L及K409P替换，第二Fc链L351Y、Y407A及D399C替换；
d)第一Fc链T366P及K409S替换，第二Fc链L351V、Y407P及D399N替换；
e)第一Fc链T366W及K409S替换，第二Fc链L351D、Y407P及D399G替换；
f)第一Fc链T366P及K409F替换，第二Fc链L351P、Y407F及D399I替换；
g)第一Fc链T366V及K409Q替换，第二Fc链L351K、Y407T及D399T替换；
h)第一Fc链T366L及K409R替换，第二Fc链L351W、Y407H及D399A替换。
技术方案5.如技术方案1所述的异源二聚体形式的双特异抗体，其中第一Fc链的氨基酸替换为T366L和D399R，第二Fc链的氨基酸替换为L351E、Y407L和K409V。
技术方案6.如技术方案1-5任一项所述的异源二聚体形式的双特异抗体，其中Fc链来源于IgG。
技术方案7.如技术方案1-6任一项所述的异源二聚体形式的双特异抗体，其中PD-L1和CD47抗原结合功能区是Fab片段或scFv片段。
技术方案8.如技术方案1-7任一项所述的异源二聚体形式的双特异抗体，其中PD-L1和CD47抗原结合功能区都是Fab片段。
技术方案9.如技术方案1-7任一项所述的异源二聚体形式的双特异抗体，其中PD-L1和CD47抗原结合功能区一个是Fab片段，另一个是scFv。
技术方案10.如技术方案7-9任一项所述的异源二聚体形式的双特异抗体，其Fab片段包含不同的第一重链可变区及第二重链可变区，以及不同的第一轻链可变区及第二轻链可变区。
技术方案11,如技术方案1-10任一项所述的异源二聚体形式的双特异抗体，其中第一Fc链及其相连接的PD-L1抗原结合功能区和第二Fc链及其相连接的CD47抗原结合功能区，或者第一Fc链及其相连接的CD47抗原结合功能区和第二Fc链及其相连接的 PD-L1抗原结合功能区，在单独存在并且同时存在还原剂时其形成同源二聚体的重量比例均低于50％。
技术方案12.如技术方案1-11任一项所述的异源二聚体形式的双特异抗体，其中双特异抗体的氨基酸序列选自：SEQ ID NO.2、4、6、8、10、12和14。
技术方案13.一种分离的多核苷酸，其编码如技术方案1-12任一项所述的异源二聚体形式的双特异抗体。
技术方案14.如技术方案13所述的分离的多核苷酸，其序列选自：SEQ ID NO：1、3、5、7、9、11和13。
技术方案15.一种重组表达载体，其包含技术方案13或14所述的分离的多核苷酸。
技术方案16.如技术方案15所述的重组表达载体，其中表达载体为基于pCDNA改造得到的质粒载体X0GC。
技术方案17.一种宿主细胞，其包含技术方案13或14所述的分离的多核苷酸，或技术方案15或16所述的重组表达载体。
技术方案18.如技术方案17所述的宿主细胞，其选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293E、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO；大肠杆菌或以大肠杆菌为基础改造得到的大肠杆菌BL21、BL21(DE3)、Rosetta、Origami；酵母菌或以酵母为基础改造得到的毕赤酵母、酿酒酵母、乳酸克鲁维亚酵母、多形汉逊酵母；昆虫细胞或以昆虫细胞为基础改造得到的细胞High5、SF9；植物细胞；哺乳动物乳腺细胞、体细胞。
技术方案19.一种组合物，其包含技术方案1-12任一项所述的异源二聚体形式的双特异抗体或技术方案13或14所述的分离的多核苷酸或技术方案15或16所述的重组表达载体或技术方案17或18所述的宿主细胞，及药学上可接受的载体。
技术方案20.一种生产如技术方案1-12任一项所述的异源二聚体形式的双特异抗体的方法，其包括步骤：
1)将技术方案13或14所述的分离的多核苷酸或技术方案15或16所述的重组表达载体分别在宿主细胞中进行表达；
2)将在宿主细胞中分别表达的蛋白进行还原；以及
3)将还原的蛋白混合，然后将混合物进行氧化。
技术方案21.如技术方案20所述的方法，其中宿主细胞选自人胚肾细胞HEK293或 以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO；大肠杆菌或以大肠杆菌为基础改造得到的大肠杆菌BL21、BL21(DE3)、Rosetta、Origami；酵母菌或以酵母为基础改造得到的毕赤酵母、酿酒酵母、乳酸克鲁维亚酵母、多形汉逊酵母；昆虫细胞或以昆虫细胞为基础改造得到的细胞High5、SF9；植物细胞；哺乳动物乳腺细胞、体细胞。
技术方案22.如技术方案20或21所述的方法，其中还原步骤包括1)加入还原剂，所述还原剂选自：2-巯基乙胺、二硫苏糖醇、三(2-羧乙基)膦或其他化学衍生物，2)在0.1mM或更高浓度二硫苏糖醇存在下在4℃条件下进行还原反应最少3小时，3)去除还原剂，如通过脱盐进行。
技术方案23.如技术方案20-22任一项所述的方法，其中氧化步骤包括1)在空气中氧化，也包括加入氧化剂，所述氧化剂选自：L-脱氢抗坏血酸或其他化学衍生物，2)在0.5mM或更高浓度L-脱氢抗坏血酸存在下在4℃条件下进行氧化反应最少5小时。
技术方案24.如技术方案20-23任一项所述的方法，其还包括分离纯化的步骤。
技术方案25.技术方案1-12任一项所述的异源二聚体形式的双特异抗体和/或技术方案13或14所述的分离的多核苷酸和/或技术方案15或16所述的重组表达载体和/或技术方案17或18所述的宿主细胞和/或技术方案19所述的组合物在制备用于预防和/或治疗受试者疾病的药物中的用途。
技术方案26.技术方案1-12任一项所述的异源二聚体形式的双特异抗体和/或技术方案13或14所述的分离的多核苷酸和/或技术方案15或16所述的重组表达载体和/或技术方案17或18所述的宿主细胞和/或技术方案19所述的组合物，其用做用于预防和/或治疗受试者疾病的药物。
技术方案27.一种预防和/或治疗疾病的方法，包括将技术方案1-12任一项所述的异源二聚体形式的双特异抗体和/或技术方案13或14所述的分离的多核苷酸和/或技术方案15或16所述的重组表达载体和/或技术方案17或18所述的宿主细胞和/或技术方案19所述的组合物施予有需求的受试者。
技术方案28.如技术方案25所述的用途，技术方案26所述的异源二聚体形式的双特异抗体、分离的多核苷酸、重组表达载体、宿主细胞或组合物，或技术方案27所述的方法，其中受试者是哺乳动物，优选地，人类受试者。
技术方案29.如技术方案25所述的用途，技术方案26所述的异源二聚体形式的双 特异抗体、分离的多核苷酸、重组表达载体、宿主细胞或组合物，或技术方案27所述的方法，其中所述疾病选自如下肿瘤：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤、小细胞肺癌、骨癌。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。本发明下述实施例中使用的各种抗体均来源于商业途径的标准抗体。
实施例1抗PD-L1/抗CD47异源二聚体抗体分子的载体构建
构建分别含抗人PD-L1的抗体重链和轻链的X0GC表达载体，其中抗体可变区序列来源于http://www.imgt.org/mAb-DB/mAbcard？AbId＝472，重链恒定区为人IgG1(Fc1,其中N297A突变以消除ADCC/CDC效应)。轻链可变区核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO:2所示；轻链恒定区核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO:4所示；重链可变区核苷酸序列如SEQ ID NO.5所示，氨基酸序列如SEQ ID NO:6所示；重链恒定区核苷酸序列如SEQ ID NO.7所示，氨基酸序列如SEQ ID NO:8所示。通过PCR的方法分别扩增轻链可变区以及轻链恒定区，重链可变区以及重链恒定区。本申请中所有PCR反应均使用NEB公司的Phusion超保真DNA聚合酶(F-530L)。PCR引物根据碱基互补原则以及酶切位点的需要进行常规设计。反应体系均为：H 2O 8.9μl，5×Phusion超保真DNA聚合酶缓冲液4μl，1mM dNTP 4μl，上游引物1μl，下游引物1μl，Phusion超保真DNA聚合酶0.1μl，模板1μl。将可变区及恒定区PCR产物，经1.5％琼脂糖凝胶电泳后用DNA回收试剂盒(Promega，A9282，下同)回收相应片段。以回收的可变区片段与恒定区片段作为模板，使用可变区上游引物及恒定区下游引物，再进行一轮PCR反应，然后再回收相应片段，得到轻链或者重链的全长片段。将X0GC载体及全长片段，用EcoRI(NEB，货号R3101L)及HindIII(NEB，货号R3104L)酶切，酶切反应体系为：10×缓冲液3 2μl，EcoRI及HindIII各0.5μl，胶回收获得的全长片段3μl，H 2O 14.5μl。酶切体系于37℃条件下反应3小时。将酶切产物用T4DNA连接酶(NEB，货号M0202V)连接(下同)，反应体系为：10×连接酶缓冲液2μl，连接酶0.5μl，胶回收获得的全长片段3μl，胶回收获得的X0GC载体3μl，H 2O 11.5μl。连接于室温反应12小时。 将连接产物转化大肠杆菌感受态细胞DH5α(天根，CB104，下同)。获得抗人PD-L1抗体重链和轻链的X0GC表达载体，分别用于在真核细胞中表达抗体的重链(Fc1)和轻链。
本发明同时构建了分别含抗人CD47的抗体重链和轻链的X0GC表达载体，其中抗体可变区序列来源于WO2016109415A1。轻链可变区核苷酸序列如SEQ ID NO.9所示，氨基酸序列如SEQ ID NO:10所示；轻链恒定区核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO:4所示；重链可变区核苷酸序列如SEQ ID NO.11所示，氨基酸序列如SEQ ID NO:12所示；重链恒定区核苷酸序列如SEQ ID NO.13所示，氨基酸序列如SEQ ID NO:14所示。获得抗人CD47抗体重链和轻链的X0GC表达载体，分别用于在真核细胞中表达抗体的重链(Fc2)和轻链。
实施例2抗PD-L1/抗CD47异源二聚体抗体分子的表达
分别将含抗人PD-L1抗体的重链和轻链的表达载体转染293F细胞(FreeStyle TM293-F Cells,货号R79007,invitrogen)，另外分别将含抗人CD47的抗体的重链和轻链的表达载体也转染293F细胞。转染前一天接种细胞，转染当天将细胞离心收集细胞，将细胞重悬于新鲜的FreeStyle TM293表达培养基(FreeStyle TM293 Expression Medium，货号12338001，Gibco)中，细胞密度为200*10 5细胞/mL。按照转染体积加入质粒，终浓度为36.67ug/mL，轻轻混匀；然后加入线性PEI(聚乙烯亚胺，线形，M.W.25000，货号43896，Alfa Aesar)，终浓度为55ug/mL，轻轻混匀。之后放入细胞培养箱，120rpm摇床37℃培养1小时。之后加入19倍转染体积的新鲜培养基。继续120rpm摇床37℃培养。离心收集转染5～6天的细胞培养上清。
通过ELISA的方法测定表达量。在应用层析柱纯化之前，以0.2μm滤膜过滤以去除沉淀物。此步骤在4℃下进行。
实施例3抗PD-L1/抗CD47异源二聚体抗体分子表达产物的纯化
采用AKTA explorer 100型蛋白纯化系统(GE Healthcare)以及亲和色谱柱rProtein A Sepharose Fast Flow(16mm I.D.，22ml，GE Healthcare)于4℃下进行纯化。首先以流动相A(20mM磷酸钠缓冲液，150mM氯化钠，pH 7.4)平衡色谱柱，在基线稳定后将经过上述处理的细胞上清液进行上样，流速为5ml/min，并在上样后以流动相A进行平衡。样品分别为抗PD-L1表达产物和抗CD47表达产物。之后，首先以流动相B1(含有0.5M精氨酸的流动相A)冲洗5个柱体积；然后以流动相B2(100mM柠檬酸，pH 3.0) 洗脱5个柱体积，收集洗脱峰即为目的蛋白峰；以上洗脱步骤流速都为5ml/min。抗PD-L1-Fc1的洗脱峰色谱图如图1所示，抗CD47-Fc2的洗脱峰如图2所示。收集标示的洗脱峰(图示灰色区域)，并通过滴加1M醋酸钠溶液将pH调至5.0。
实施例4抗PD-L1/抗CD47异源二聚体抗体分子的制备和纯化
抗PD-L1/抗CD47异源二聚体抗体分子的结构如图3所示。
将上述rProtein A Sepharose Fast Flow(16mm I.D.，22ml，GE Healthcare)方法纯化获得的产物进行体外重组以获得异源二聚体。首先将上述纯化收集的蛋白溶液通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为磷酸盐缓冲液(phosphate buffer saline，PBS)PBS(pH＝7.4)。将获得的抗PD-L1和抗CD47纯化产物溶液分别加所述PBS调整到1mg/ml,加入1/200倍终体积的1M DTT(DTT终浓度分别为0.1mM、0.5mM、1mM、2mM、5mM、10mM、20mM)，在4℃条件下进行还原(3-8小时)，通过还原的过程，二硫键被打开，抗PD-L1以及抗CD47产物中含有的抗体同源二聚体分子铰链区二硫键也打开，形成了含有一条重链和一条轻链的半抗体分子，结构如图4所示。还原的样品经流动相缓冲液中包含1mM DTT还原剂的SEC-HPLC(TOSOH，TSKgel superSW3000)分析，结果如图5所示，抗PD-L1和抗CD47的同源二聚体分子比例均小于10％，半抗体分子比例均大于90％。
然后将还原的抗PD-L1以及抗CD47半抗体分子等摩尔比例混合，在4℃条件下进行重组反应(0.5-24小时)，在重组的过程中，抗PD-L1及抗CD47半抗体分子通过CH2以及CH3间的非共价作用力形成了同时含有抗PD-L1以及抗CD47半抗体分子的异源二聚体的双特异抗体，之后将蛋白溶液通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为磷酸盐溶液(PBS，pH＝7.4)终止还原，通过空气或者氧化剂进行氧化反应，使异源二聚体的双特异抗体的二硫键重新形成。氧化反应的条件包括空气中放置样品1天、3天、4天，加入氧化剂100mM L-脱氢抗坏血酸(蛋白终浓度1mg/ml,氧化剂终浓度0.5mM、1mM、5mM、10mM)在4℃条件下进行氧化，反应进行24小时。
上述抗PD-L1以及抗CD47表达产物经还原氧化得到的抗PD-L1/抗CD47异源二聚体抗体分子通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为10mM磷酸钠缓冲液，pH 5.8。采用AKTA explorer 100型蛋白纯化系统(GE Healthcare)以及离子色谱柱Source 15S(16mm I.D.，17ml，GE Healthcare)于4℃下进行纯化。首先以流动相A(10mM磷酸钠，pH 7.0)平衡色谱柱，在基线稳定后将经过上述处理的蛋白溶 液进行上样，流速为3ml/min，并在上样后以流动相A进行平衡，之后以A(10mM磷酸钠，pH 5.8)到B(10mM磷酸钠，pH 5.8)梯度冲洗20个柱体积(0％B-100％B，170min，流速2ml/min),收集洗脱主峰，收集的蛋白溶液通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为磷酸盐溶液(PBS，pH＝7.4)，过滤除菌4℃保存。将纯化产物通过SEC-HPLC进行纯度分析，结果如图6所示，纯度为99.3％；进行RPC-HPLC(Thermo Fisher,MAbPac RP)分析，结果如图7所示，纯度为100％；进行CE分析，结果如图8所示，纯度为97.1％。
实施例5.抗PD-L1/抗CD47异源二聚体抗体的靶点结合活性
用酶联免疫吸附试验(ELISA)测定抗PD-L1/抗CD47异源二聚体抗体与单个抗原的结合能力。
ELISA具体实施过程如下：用pH＝9.6的碳酸盐缓冲溶液(0.05M)在96孔高吸附酶标板(Costar，货号42592)上包被重组人PD-L1(北京义翘神州,货号10084-H08H)或者人CD47(北京百普赛斯，货号CD7-H5227)，包被浓度为1μg/mL，每孔100μL，包被在4℃过夜进行。PBST洗涤5次。用含1％BSA的PBST按300μL/孔封闭，25℃孵育1小时。PBST洗涤5次。加入序列稀释在含1％BSA的PBST里的异源二聚体抗体样品以及对照，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。然后加入1:10000稀释在含1％BSA的PBST里的辣根过氧化物酶标记的抗人IgG抗体(Chemicon，货号AP309P)，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。加入比色底物TMB，100μL/孔，室温显色10分钟。加入1M H 2SO 4，100μL/孔，终止显色。在酶标仪上读取450nm处的吸光度。
结果如图9的A图所示，抗PD-L1/抗CD47异源二聚体抗体具有对PD-L1的高亲和力，与PD-L1双价单抗的抗原结合活性相当；如图9的B图所示，抗PD-L1/抗CD47异源二聚体抗体具有对CD47的高亲和力，与CD47双价单抗的抗原结合活性相当。
实施例6.抗PD-L1/抗CD47异源二聚体抗体的双靶点同时结合活性
用酶联免疫吸附试验(ELISA)测定抗PD-L1/抗CD47异源二聚体抗体与两个不同抗原的同时结合能力。
ELISA具体实施过程如下：用pH＝9.6的碳酸盐缓冲溶液在96孔高吸附酶标板上包被重组人CD47(北京百普赛斯，货号CD7-H5227)，包被浓度为1μg/mL，每孔100μL，包被在4℃过夜进行。PBST洗涤5次。用含1％BSA的PBST按300μL/孔封闭，25℃孵 育1小时。PBST洗涤5次。加入序列稀释在含1％BSA的PBST里的异源二聚体抗体样品以及对照，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。然后加入稀释在含1％BSA的PBST里的生物素标记的PD-1-Fc(北京韩美药品)，0.5μg/mL，每孔100μL，25℃孵育1小时。加入1:1000稀释在含1％BSA的PBST里的链霉亲和素-辣根过氧化物酶偶联物(BD Pharmingen，货号554066)，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。加入比色底物TMB，100μL/孔，室温显色10分钟。加入1M H 2SO 4，100μL/孔，终止显色。在酶标仪上读取450nm处的吸光度。
结果如图10所示，PD-L1单抗和CD47的组合不能同时结合PD-L1，CD47，只有抗PD-L1/抗CD47异源二聚体抗体具有同时结合两种抗原的活性。
实施例7.抗PD-L1/抗CD47异源二聚体抗体与肿瘤细胞/红细胞的结合
HCC827肿瘤细胞表达PD-L1和CD47，红细胞(RBC)仅表达CD47。将HCC827和RBC混合在一起，用流式细胞术(FCM)检测异源二聚体对混合细胞中两种细胞的结合是否存在选择性。
该方法具体实施过程如下：收集HCC827细胞(购自ATCC)，RBC细胞(采自健康人群)。用含2％FBS(Hyclone，货号SH30084.03)的冷PBS(GIBCO，货号14190-235)洗涤一次。将HCC827按每管1×10 6个细胞，RBC按每管10×10 6个细胞混合在一起，重悬于200μL含2％FBS的冷DPBS(GIBCO，货号14190-136)中，且加入序列稀释的异源二聚体抗体样品以及对照。流式管于冰上孵育30分钟。用含2％FBS的PBS洗涤两次。再重悬于200μL含2％FBS以及1:1000稀释的FITC标记的抗人IgG抗体(北京中杉金桥，货号ZF0306)的冷PBS中。冰上避光孵育30分钟。用含2％FBS的PBS洗涤两次。再重悬于500μL冷PBS中，该细胞悬液于流式细胞仪(BD,FACS Calibur)上进行检测分析，读取混合细胞中两种细胞各自的荧光强度。
结果如图11所示，CD47单抗与混合在一起的HCC827和RBC的结合基本没有什么差异(A图)；而抗PD-L1/抗CD47异源二聚体倾向与表达PD-L1和CD47的HCC827细胞结合，而与仅表达CD47的RBC结合较弱，显示出了结合的选择性(B图)。
实施例8.抗PD-L1/抗CD47异源二聚体抗体的T细胞调控活性
用混合淋巴细胞反应(MLR)测定抗PD-L1/抗CD47异源二聚体抗体对T细胞免疫反应的调控活性。
人树突状细胞(DC)的获取：复苏收集人PBMC细胞(Lonza，货号CC-2702)。将人PBMC细胞按细胞密度为5×10 6/mL重悬于无血清的RPMI 1640培养基(GIBCO，货号22400-089)并接种在细胞培养瓶中，在37℃二氧化碳培养箱中孵育90分钟。弃除培养上清及悬浮细胞，贴壁细胞在完全培养基(RPMI 1640含10％FBS)中培养，并加入100ng/ml GM-CSF(北京义翘神州,货号10015-HNAH)和100ng/ml IL-4(北京义翘神州,货号11846-HNAE)。孵育3天，换液，再孵育3天。然后将培养基更换为完全培养基(RPMI 1640含10％FBS)中含100ng/ml GM-CSF、100ng/ml IL-4以及20ng/ml TNF-α，孵育1天。即得DC细胞。
人T细胞的获取：复苏收集人PBMC细胞，保证此PBMC与诱导DC细胞的PBMC来自不同个体。参照Pan T细胞分离试剂盒(Miltenyi Biotech，货号5150414820)使用说明书分离人T细胞。简要的说，先用PBS洗涤PBMC一次，再将PBMC按10 7细胞每40μL分离缓冲液(PBS含2mM EDTA,0.5％BSA,pH＝7.2)重悬(以下使用量均按10 7细胞计)，并加入10μL Pan T cell Biotin Antibody Cocktail(Pan T细胞生物素抗体混合物)，在4℃孵育5分钟。再加入30μL分离缓冲液和20μL Pan T cell MicroBead Cocktail(Pan T细胞微珠混合物)，在4℃孵育10分钟。过MACS分离柱，即得T细胞。
将收集到的人DC细胞和人T细胞重悬于完全培养基(RPMI 1640含10％FBS)中，接种于96孔板，接种的DC细胞和T细胞分别为1×10 4/孔，1×10 5/孔，混合培养。并加入用完全培养基序列稀释的异源二聚体抗体样品以及对照。将培养板置于37℃二氧化碳培养箱中孵育5天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IFN-γ(RayBiotech,货号ELH-IFNg)。
如图12所示，人T细胞在同种异体DC细胞的刺激下，会活化分泌IFN-γ。加入PD-L1抗体会增强T细胞的活化，促进细胞因子的分泌。抗PD-L1/抗CD47异源二聚体抗体也显示了很强的T细胞调控活性，显著的促进细胞因子IFN-γ的分泌。
实施例9.抗PD-L1/抗CD47异源二聚体抗体介导的巨噬细胞对肿瘤细胞的吞噬活性
成熟人巨噬细胞的制备：复苏收集人PBMC细胞(Lonza，货号CC-2702)。将人PBMC细胞按细胞密度为5×10 6/mL重悬于无血清的RPMI 1640培养基并接种在细胞培养瓶中，在37℃二氧化碳培养箱中孵育90分钟。弃除培养上清及悬浮细胞，贴壁细胞在完全培养基(RPMI 1640含10％FBS)中培养，并加入25ng/ml M-CSF(北京义翘神州,货号11792-HNAN)，孵育7天。然后收取巨噬细胞，重悬于含25ng/ml M-CSF和50ng/ml IFN-γ (北京义翘神州，货号11725-HNAS)的完全培养基(RPMI 1640含10％FBS)中。将此细胞悬液接种到48孔细胞培养板中，每孔50000个细胞，孵育1天使巨噬细胞成熟，备用。
参照CFSE试剂盒(Life technology，货号C34554)使用说明书染色Raji肿瘤细胞。简要的说，用PBS稀释CFSE到工作浓度5μM并在37℃预热,1000rpm离心5分钟收集Raji细胞，用预热的CFSE工作溶液重悬Raji，在37℃孵育15分钟。用完全培养基洗涤1次，再重悬于完全培养基中，孵育30分钟，再用完全培养基洗涤2次，再重悬于完全培养基备用。
将48孔板用完全培养基洗涤3次。将CFSE染色后的Raji细胞与待测的异源二聚体样品以及对照预先孵育15分钟，然后加入48孔培养板中，在37℃二氧化碳培养箱中孵育2小时。孵育结束后，将48孔板用完全培养基洗涤3次，加入稀释在完全培养基中的wheat germ agglutinin,alexa fluor 555(Life technologies，货号W32464)，10μg/ml，避光孵育15分钟。将48孔板再用完全培养基洗涤3次，加入4％多聚甲醛固定15分钟。将48孔板再用完全培养基洗涤3次，加入完全培养基。荧光显微镜拍照，计细胞数。吞噬指数(％)的计算方法为：被吞噬的绿色标记的Raji细胞的数目/存在的红色标记的巨噬细胞数目×100。
如图13所示，抗PD-L1/抗CD47异源二聚体抗体能介导巨噬细胞吞噬Raji肿瘤细胞，与CD47单抗的活性相当。

Claims (34)

  1. 一种异源二聚体形式的双特异抗体，其包含第一Fc链和第二Fc链，以及能与PD-L1特异性结合的第一个抗原结合功能区和能与CD47特异性结合的第二个抗原结合功能区；
    其中所述第一Fc链和第二Fc链均为包含氨基酸替换的免疫球蛋白G Fc片段，并且所述第一Fc链及第二Fc链共同构成可以与Fc受体结合的异源二聚体；
    其中所述第一Fc链和第二Fc链通过共价键或连接体分别连接到所述第一抗原结合功能区和第二抗原结合功能区；
    并且其中所述第一Fc链和第二Fc链中的任意一条在366位及399位上包含氨基酸替换，另一条在351位、407位及409位上包含氨基酸替换，其中氨基酸位置根据KabatEU指数编号系统编号。
  2. 如权利要求1所述的异源二聚体形式的双特异抗体，其中第一Fc链及第二Fc链氨基酸替换如下，
    a)L351G、L351Y、L351V、L351P、L351D、L351E、L351K或L351W；
    b)T366L、T366P、T366W或T366V；
    c)D399C、D399N、D399I、D399G、D399R、D399T或D399A；
    d)Y407L、Y407A、Y407P、Y407F、Y407T或Y407H；和
    e)K409C、K409P、K409S、K409F、K409V、K409Q或K409R。
  3. 如权利要求1或2所述的异源二聚体形式的双特异抗体，其中氨基酸替换包括：
    a)第一Fc链和第二Fc链中的任意一条为T366L及D399R替换，另一条为L351E、Y407L及K409V替换；
    b)第一Fc链和第二Fc链中的任意一条为T366L及D399C替换，另一条为L351G、Y407L及K409C替换；
    c)第一Fc链和第二Fc链中的任意一条为T366L及D399C替换，另一条为L351Y、Y407A及K409P替换；
    d)第一Fc链和第二Fc链中的任意一条为T366P及D399N替换，另一条为L351V、Y407P及K409S替换；
    e)第一Fc链和第二Fc链中的任意一条为T366W及D399G替换，另一条为L351D、Y407P及K409S替换；
    f)第一Fc链和第二Fc链中的任意一条为T366P及D399I替换，另一条为L351P、Y407F及K409F替换；
    g)第一Fc链和第二Fc链中的任意一条为T366V及D399T替换，另一条为L351K、Y407T及K409Q替换；
    h)第一Fc链和第二Fc链中的任意一条为T366L及D399A替换，另一条为L351W、Y407H及K409R替换。
  4. 如权利要求1-3任一项所述的异源二聚体形式的双特异抗体，其中氨基酸替换包括：
    a)第一Fc链和第二Fc链中的任意一条为T366L及K409V替换，另一条为L351E、Y407L及D399R替换；
    b)第一Fc链和第二Fc链中的任意一条为T366L及K409C替换，另一条为L351G、Y407L及D399C替换；
    c)第一Fc链和第二Fc链中的任意一条为T366L及K409P替换，另一条为L351Y、Y407A及D399C替换；
    d)第一Fc链和第二Fc链中的任意一条为T366P及K409S替换，另一条为L351V、Y407P及D399N替换；
    e)第一Fc链和第二Fc链中的任意一条为T366W及K409S替换，另一条为L351D、Y407P及D399G替换；
    f)第一Fc链和第二Fc链中的任意一条为T366P及K409F替换，另一条为L351P、Y407F及D399I替换；
    g)第一Fc链和第二Fc链中的任意一条为T366V及K409Q替换，另一条为L351K、Y407T及D399T替换；
    h)第一Fc链和第二Fc链中的任意一条为T366L及K409R替换，另一条为L351W、Y407H及D399A替换。
  5. 如权利要求1-4任一项所述的异源二聚体形式的双特异抗体，其中第一Fc链和第二Fc链中的任意一条的氨基酸替换为T366L和D399R，另一条的氨基酸替换为L351E、Y407L和K409V。
  6. 如权利要求1-5任一项所述的异源二聚体形式的双特异抗体，其中第一个抗原结合功能区和第二个抗原结合功能区选自Fab片段、scFv片段、可变结构域片段Fv和重链抗体的重链可变区片段VHH。
  7. 如权利要求1-6任一项所述的异源二聚体形式的双特异抗体，其中第一个抗原结合功能区和第二个抗原结合功能区都是Fab片段。
  8. 如权利要求1-6任一项所述的异源二聚体形式的双特异抗体，其中第一个抗原结合功能区和第二个抗原结合功能区中一个是Fab片段，另一个是scFv。
  9. 如权利要求7任一项所述的异源二聚体形式的双特异抗体，其Fab片段包含不同的第一重链可变区及第二重链可变区，以及不同的第一轻链可变区及第二轻链可变区。
  10. 如权利要求1-9任一项所述的异源二聚体形式的双特异抗体，其中第一Fc链及与其共价相连的第一抗原结合功能区，和第二Fc链及与其共价相连的第二抗原结合功能区，在存在还原剂的溶液中且所述溶液中除所述第一Fc链及与其共价相连的第一抗原结合功能区和第二Fc链及与其共价相连的第二抗原抗原结合功能区以外不含其它多肽时，其形成同源二聚体的基于所有多肽链的重量比例均低于50％。
  11. 如权利要求1-10任一项所述的异源二聚体形式的双特异抗体，其中第一抗原结合功能区包含SEQ ID NO：2和6的氨基酸序列。
  12. 如权利要求1-10任一项所述的异源二聚体形式的双特异抗体，其中第二抗原结合功能区包含SEQ ID NO：10和12的氨基酸序列。
  13. 如权利要求11所述的异源二聚体形式的双特异抗体，其中第一抗原结合功能区进一步包含SEQ ID NO：4和8的氨基酸序列。
  14. 如权利要求12所述的异源二聚体形式的双特异抗体，其中第二抗原结合功能区进一步包含SEQ ID NO：4和14的氨基酸序列。
  15. 一种分离的多核苷酸，其编码如权利要求1-14任一项所述的异源二聚体形式的双特异抗体。
  16. 如权利要求15所述的分离的多核苷酸，其中编码第一抗原结合功能区氨基酸的核苷酸序列包含SEQ ID NO：1和5。
  17. 如权利要求15所述的分离的多核苷酸，其中编码第二抗原结合功能区氨基酸的核苷酸序列包含SEQ ID NO：9和11。
  18. 如权利要求16所述的分离的多核苷酸，其中编码第一抗原结合功能区氨基酸的核苷酸序列进一步包含SEQ ID NO：3和7。
  19. 如权利要求17所述的分离的多核苷酸，其中编码第二抗原结合功能区氨基酸的核苷酸序列进一步包含SEQ ID NO：3和13。
  20. 一种重组表达载体，其包含权利要求15-19任一项所述的分离的多核苷酸。
  21. 如权利要求20所述的重组表达载体，其中表达载体为基于pCDNA改造得到的质粒载体X0GC。
  22. 一种宿主细胞，其包含权利要求15-19任一项所述的分离的多核苷酸，或权利要求20或21所述的重组表达载体。
  23. 如权利要求22所述的宿主细胞，其选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293E、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO；大肠杆菌或以大肠杆菌为基础改造得到的大肠杆菌BL21、BL21(DE3)、Rosetta、Origami；酵母菌或以酵母为基础改造得到的毕赤酵母、酿酒酵母、乳酸克鲁维亚酵母、多形汉逊酵母；昆虫细胞或以昆虫细胞为基础改造得到的细胞High5、SF9；植物细胞；哺乳动物乳腺细胞、体细胞。
  24. 一种组合物，其包含权利要求1-14任一项所述的异源二聚体形式的双特异抗体或权利要求15-19任一项所述的分离的多核苷酸或权利要求20或21所述的重组表达载体或权利要求22或23所述的宿主细胞，及药学上可接受的载体。
  25. 一种生产如权利要求1-14任一项所述的异源二聚体形式的双特异抗体的方法，其包括步骤：
    1)将权利要求15-19任一项所述的分离的多核苷酸或权利要求20或21所述的重组表达载体分别在宿主细胞中进行表达；
    2)将在宿主细胞中分别表达的蛋白进行还原；以及
    3)将还原的蛋白混合，然后将混合物进行氧化。
  26. 如权利要求25所述的方法，其中宿主细胞选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO；大肠杆菌或以大肠杆菌为基础改造得到的大肠杆菌BL21、BL21(DE3)、Rosetta、Origami；酵母菌或以酵母为基础改造得到的毕赤酵母、酿酒酵母、乳酸克鲁维亚酵母、多形汉逊酵母；昆虫细胞或以昆虫细胞为基础改造得到的细胞High5、SF9；植物细胞；哺乳动物乳腺细胞、体细胞。
  27. 如权利要求25或26所述的方法，其中还原步骤包括1)在还原剂存在下进行还原反应，所述还原剂选自：2-巯基乙胺、二硫苏糖醇、三(2-羧乙基)膦或其他化学衍生物；2)去除还原剂。
  28. 如权利要求25-27任一项所述的方法，其中氧化步骤为在空气中氧化，也包括在氧化剂存在下进行氧化反应，所述氧化剂选自：L-脱氢抗坏血酸或其化学衍生物。
  29. 如权利要求25-28任一项所述的方法，其还包括分离纯化的步骤。
  30. 权利要求1-14任一项所述的异源二聚体形式的双特异抗体和/或权利要求15-19任一项所述的分离的多核苷酸和/或权利要求20或21所述的重组表达载体和/或权利要求22或23所述的宿主细胞和/或权利要求24所述的组合物在制备用于预防和/或治疗受试者疾病的药物中的用途。
  31. 权利要求1-14任一项所述的异源二聚体形式的双特异抗体和/或权利要求15-19任一项所述的分离的多核苷酸和/或权利要求20或21所述的重组表达载体和/或权利要求22或23所述的宿主细胞和/或权利要求24所述的组合物，其用做用于预防和/或治疗受试者疾病的药物。
  32. 一种预防和/或治疗疾病的方法，包括将权利要求1-14任一项所述的异源二聚体形式的双特异抗体和/或权利要求15-19任一项所述的分离的多核苷酸和/或权利要求20或21所述的重组表达载体和/或权利要求22或23所述的宿主细胞和/或权利要求24所述的组合物施予有需求的受试者。
  33. 如权利要求30所述的用途，权利要求31所述的异源二聚体形式的双特异抗体、分离的多核苷酸、重组表达载体、宿主细胞或组合物，或权利要求32所述的方法，其中受试者是哺乳动物，优选地，人类受试者。
  34. 如权利要求30所述的用途，权利要求31所述的异源二聚体形式的双特异抗体、分离的多核苷酸、重组表达载体、宿主细胞或组合物，或权利要求32所述的方法，其中所述疾病选自如下肿瘤：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤、小细胞肺癌、骨癌。
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WO2021124073A1 (en) * 2019-12-17 2021-06-24 Pfizer Inc. Antibodies specific for cd47, pd-l1, and uses thereof
TWI784373B (zh) * 2019-12-17 2022-11-21 美商輝瑞大藥廠 對cd47、pd-l1具特異性之抗體及其用途
US11702474B2 (en) 2019-12-17 2023-07-18 Pfizer Inc. Antibodies specific for CD47, PD-L1, and uses thereof
WO2022076987A1 (en) * 2020-10-07 2022-04-14 Celgene Corporation Bispecific antibody treatment of lymphoid malignant neoplasm conditions
WO2022148412A1 (zh) * 2021-01-08 2022-07-14 北京韩美药品有限公司 特异性结合cd47的抗体及其抗原结合片段

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