WO2021110110A1 - FcγRIIB亲和力增强的抗体Fc区 - Google Patents

FcγRIIB亲和力增强的抗体Fc区 Download PDF

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WO2021110110A1
WO2021110110A1 PCT/CN2020/133685 CN2020133685W WO2021110110A1 WO 2021110110 A1 WO2021110110 A1 WO 2021110110A1 CN 2020133685 W CN2020133685 W CN 2020133685W WO 2021110110 A1 WO2021110110 A1 WO 2021110110A1
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mutant
antibody
region
polypeptide fragment
mutation
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PCT/CN2020/133685
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English (en)
French (fr)
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李福彬
张燕
张咪
毕艳侠
田世豪
张慧慧
柳淑君
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上海交通大学医学院
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Priority to MX2022006670A priority Critical patent/MX2022006670A/es
Priority to CA3160574A priority patent/CA3160574A1/en
Priority to EP20897306.5A priority patent/EP4071167A4/en
Priority to US17/756,836 priority patent/US20230220042A1/en
Priority to AU2020396825A priority patent/AU2020396825A1/en
Priority to KR1020227022711A priority patent/KR20220121822A/ko
Priority to CN202080084609.5A priority patent/CN115052888A/zh
Priority to JP2022533391A priority patent/JP2023507922A/ja
Priority to BR112022010757A priority patent/BR112022010757A2/pt
Publication of WO2021110110A1 publication Critical patent/WO2021110110A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular to a series of variants of the Fc region of human IgG2 antibodies. These variants have enhanced Fc ⁇ RIIB affinity, and agonistic antibodies containing these Fc region variants are expected to have better agonistic activity.
  • biomacromolecule drugs provides new methods and possibilities for the treatment of many diseases, especially molecular targeted therapy based on antibodies and heavy chain constant regions (including Fc regions), including antibodies and heavy chain constant region fusion proteins,
  • molecular targeted therapy based on antibodies and heavy chain constant regions (including Fc regions), including antibodies and heavy chain constant region fusion proteins
  • biological macromolecules can be divided into three main categories: effector molecules that clear targets (molecules and cells), blocking molecules that block the signal pathways involved in the target, and agonistic molecules that activate the downstream signal pathways of the target.
  • effector molecules that clear targets (molecules and cells)
  • blocking molecules that block the signal pathways involved in the target
  • agonistic molecules that activate the downstream signal pathways of the target.
  • agonistic antibodies that can combine with target molecules that transmit immune activation signals on the surface of immune cells and activate important immune activation signal pathways controlled by them, thereby enhancing anti-tumor immunity. The response indirectly kills tumor cells.
  • agonistic tumor immunotherapy antibodies have proven their great potential in animal models and have become a widely accepted and promising tumor immunotherapy concept, the development of such antibodies has not been successful so far, and they are in the field of tumor immunotherapy. A major challenge now.
  • agonistic antibody activation is also a favorable means to intervene and regulate key signal pathways of other biological processes, and has broad application prospects in the field of disease prevention and treatment. For example, activation of immunosuppressive signaling pathways may help reduce inflammation and autoimmune symptoms.
  • Antibodies mainly interact with Fc ⁇ R through their constant regions, and different antibodies have different binding abilities to Fc ⁇ R. This difference also affects the function of the antibody in the body. Antibody Fc modification is an important means and trend for optimizing therapeutic antibodies. Changing the ability of the antibody Fc region to interact with key proteins such as Fc ⁇ R is a very effective method for optimizing antibody activity.
  • the present invention is based on IgG, through mutation modification of the Fc region, obtained amino acid mutations and combinations that can enhance the affinity of Fc ⁇ RIIB; Fc containing these amino acid mutations can be used to optimize the activity of antibodies, especially to improve the activity of agonistic antibodies. Agonistic activity.
  • mutant Fc polypeptide fragment has the following characteristics:
  • the mutant Fc polypeptide fragment has a mutation relative to the corresponding wild-type Fc fragment before the mutation
  • the mutant Fc region has an increased affinity for Fc ⁇ RIIB; and, the wild-type Fc is a wild-type IgG2 Fc.
  • the mutant Fc polypeptide fragment has a mutation at a site selected from the group consisting of L328, H268, S267, P271, G327, or a combination thereof relative to the corresponding wild-type Fc fragment before mutation;
  • the numbering of all amino acids is based on the IgG Eu numbering.
  • the affinity can be embodied in the degree of enrichment of the screening or the binding analysis signal.
  • the wild-type Fc fragment is the 233-332nd amino acid sequence of IgG2 based on IgG Eu numbering, and the amino acid sequence of the wild-type Fc fragment is as SEQ ID NO: 20 shown.
  • mutant Fc polypeptide fragment has an enhanced Fc ⁇ RIIB affinity compared to the wild-type Fc fragment.
  • the affinity of the mutant Fc polypeptide fragment for Fc ⁇ RIIB is more than 1 times higher than that of the wild-type Fc fragment for Fc ⁇ RIIB; preferably, it is increased by 2, 3, 4, 5, 10, 20, 50 or 100 times. the above.
  • the affinity of the mutant Fc polypeptide fragment to activated Fc ⁇ R is lower than that of Fc ⁇ RIIB.
  • the mutant Fc polypeptide fragment has an increased affinity for Fc ⁇ RIIB, and a reduced affinity for the activated Fc ⁇ R (I/A ratio is higher than that of the wild-type Fc fragment).
  • the affinity ratio (RIIB/RIIAR) of the mutant Fc polypeptide fragment to Fc ⁇ RIIB and Fc ⁇ RIIA 131R respectively is higher than that of wild-type IgG2.
  • the activated FcyR includes: FcyRI, FcyRIIA 131H , FcyRIIA 131R , FcyRIIIA 158F and FcyRIIIA 158V .
  • the mutation L328 includes L328W, L328E, L328Y, L328F, L328M, L328A, L328G, L328N, L328P, L328R, L328V; preferably L328W or L328E.
  • the mutation H268 includes H268D, H268S, H268E, H268K, H268N; preferably H268D, H268S or H268E.
  • the mutation S267 includes S267E, S267V, S267D, S267M, S267Q, S267A, S267G, S267R, S267N, S267W; preferably S267E, S267V, S267D, S267M; more preferably S267E.
  • the mutation P271 includes P271C, P271G, P271V, P271A, P271W, P271Y, P271Q, P271T, P271I, P271L, P271S; preferably P271C, P271G, P271V, P271A, P271W, P271Y, P271Q, P271T ; More preferably P271C, P271G.
  • the mutation G327 includes G327A, G327L, G327S; preferably G327A.
  • the mutant Fc polypeptide fragment has a mutation at a site selected from the following group relative to the wild-type Fc fragment: L328W, L328E, H268D, H268S, H268E, S267E, A330S, P233G, P271G, P271C, P271V, P271A, P271W, P271Y, G327A, or a combination thereof; wherein, the numbering of all amino acids is based on IgG Eu numbering.
  • the mutant Fc polypeptide fragment has a mutation at least one of the two positions L328 and H268 relative to the wild-type Fc fragment, and the specific mutations in the mutant Fc polypeptide fragment are selected From one of the following groups: L328W, L328E, H268D, H268S, H268E, H268D/S298L/L328W, S267V/S298L/L328W, V234M/S267E/S298L/L328W, A235W/V266L/S298L/L328W, V234Q/A235G/P238L/ S239V/H268D/G327A/L328E/A330S/I332T, S239V/V266L/S298L/L328W, V266L/L328W, V266L/S267D/H268D/E269D/P271Q, S267E/H268S
  • the mutant Fc polypeptide fragment has a mutation at the S267 site relative to the wild-type Fc fragment, and the specific mutation in the mutant Fc polypeptide fragment is selected from one of the following groups: S267E/A330S , S267E/S298G, P233G/S267E, V234M/S267E/S298G/I332L, P233G/S267E/S298G, V266L/S267E/E269K/P271G, P238Q/S267E, S267A/P271C, S267A/P271G, S267E/DP271C, S267E/D270H, , S267E/P271V, S267E/P271W, S267E/P271Y, S267E/S298R, S267M/P271C, S267M/P271G, S267V/S
  • the mutant Fc polypeptide fragment has a mutation at the S267 site relative to the wild-type Fc fragment, and the specific mutation in the mutant Fc polypeptide fragment is selected from one of the following groups: S267E/A330S , S267E/S298G, P233G/S267E, V234M/S267E/S298G/I332L, P233G/S267E/S298G, V266L/S267E/E269K/P271G, S267E/P271C; among them, all amino acid numbers are based on IgG Eu numbering.
  • the mutant Fc polypeptide fragment has a mutation at position P271 relative to the wild-type Fc fragment, and the specific mutation in the mutant Fc polypeptide fragment is selected from one of the following groups: V266L/S267E /E269K/P271G, V266A/P271C, V266A/P271G, S267A/P271C, S267A/P271G, S267E/P271C, S267E/P271V, S267E/P271W, S267E/P271Y, S267M/P271C, S267M/P271G, VP271G/P271C, 266G/P271C /S298R, P271G/G236V, P271G/P329S, P271G/P331C, P271G/P331T, P271G/S298D, P271G
  • the mutant Fc polypeptide fragment has a mutation at position G327 relative to the wild-type Fc fragment, and the specific mutation in the mutant Fc polypeptide fragment is selected from one of the following groups: G327A/A330R , G327A/A330V, G327A/I332A, G327A/I332C, G327A/I332E.
  • the specific mutation of the mutant Fc polypeptide fragment relative to the wild-type Fc fragment is selected from one of the following groups: H268D/S298L/L328W, S267V/S298L/L328W, V234M/S267E/S298L/ L328W, A235W/V266L/S298L/L328W, V234Q/A235G/P238L/S239V/H268D/G327A/L328E/A330S/I332T; among them, the numbering of all amino acids is based on IgG Eu numbering.
  • mutant immunoglobulin Fc region comprising the mutant Fc polypeptide fragment as described in the first aspect of the present invention.
  • the immunoglobulin is human IgG2.
  • the mutant immunoglobulin Fc region has an enhanced Fc ⁇ RIIB affinity.
  • the affinity of the mutant immunoglobulin Fc region to Fc ⁇ RIIB is more than 1 times higher than that of the wild-type IgG2 Fc region to Fc ⁇ RIIB; preferably, the affinity is increased by 2, 3, 4, 5, 10, 20 , 50 or 100 times or more.
  • the affinity can be embodied in the degree of enrichment of the screening or the binding analysis signal.
  • the affinity of the mutant immunoglobulin Fc region to activated Fc ⁇ R is lower than that of Fc ⁇ RIIB.
  • the affinity of the mutant immunoglobulin Fc region to Fc ⁇ RIIB is increased, and the affinity to activated Fc ⁇ R is reduced (I/A ratio is higher than that of wild-type IgG2 Fc Area).
  • the affinity ratio of the mutant immunoglobulin Fc region to Fc ⁇ RIIB and Fc ⁇ RIIA 131R is higher than that of wild-type IgG2.
  • an antibody comprising the mutant Fc polypeptide fragment according to the first aspect of the present invention or the mutant immunoglobulin Fc region according to the second aspect of the present invention .
  • the antibody is an antibody based on a human IgG2 backbone.
  • the antibody is an agonistic antibody.
  • the antibody specifically targets the tumor necrosis factor receptor superfamily.
  • the antibody can specifically bind to a target selected from the group consisting of CD40, DR5, OX40, CD137, CD27, CD30, GITR, HVEM, TACI, DR4, FAS, or a combination thereof.
  • the antibody can specifically bind to OX40.
  • the antigen targeted by the antibody is an immune receptor molecule, or a combination thereof.
  • the antigen targeted by the antibody is an immunosuppressive receptor molecule
  • the immunosuppressive receptor molecule is selected from PD-1, CTLA-4, VISTA, TIM-3, BTLA , LAG-3, or a combination thereof.
  • the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
  • the antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
  • a fusion protein comprising the mutant Fc polypeptide fragment according to the first aspect of the present invention, and the mutant immunoglobulin Fc according to the second aspect of the present invention. Region, or an antibody as described in the third aspect of the invention.
  • the fusion protein also includes other protein sequences or fragments thereof with receptor agonistic function.
  • the other proteins with receptor agonistic function are cytokines in the TNF gene family.
  • the other proteins with receptor agonistic function include immunoreceptor ligand molecules.
  • the other proteins with receptor agonistic function include ligand molecules of immune receptors, which are selected from CD80, CD86, ICOSL, OX40L, CD137L, CD40L, Any one of CD30L, CD27L, CD244, CD150, CD48, CD84, CD319, Ly118, or CD229, or a combination thereof.
  • the other proteins with receptor agonistic function include ligand molecules of immune receptors, and the ligand molecules of immune receptors are selected from the following group: PD-L1, PD-L2, B7- H3, B7-H4, CD47, VISTA, HVEM, GAL9, or a combination thereof.
  • the fusion protein may also include a tag sequence that assists in expression and/or purification; preferably, the tag sequence includes a 6His tag, a HA tag and/or a FLAG tag.
  • an isolated polynucleotide which encodes the mutant Fc polypeptide fragment according to the first aspect of the present invention, and the mutant Fc polypeptide fragment according to the second aspect of the present invention.
  • the vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.
  • the vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a viral vector such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a host cell contains the vector according to the sixth aspect of the present invention, or its genome integrates the polynucleotide according to the fifth aspect of the present invention;
  • the host cell expresses the mutant Fc polypeptide fragment according to the first aspect of the present invention, the mutant immunoglobulin Fc region according to the second aspect of the present invention, or the antibody according to the third aspect of the present invention, Or the recombinant protein as described in the fourth aspect of the present invention.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, insect cells, avian cells, and mammalian cells.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition may also include other drugs for treating tumors, such as cytotoxic drugs.
  • the pharmaceutical composition may also include other active substances with receptor agonistic function.
  • the pharmaceutical composition is in the form of injection.
  • mutant Fc polypeptide fragment according to the first aspect of the present invention, the mutant immunoglobulin Fc region according to the second aspect of the present invention, and the third aspect of the present invention.
  • the antibody according to the aspect, or the method of recombinant protein according to the fourth aspect of the present invention comprises the steps:
  • step (ii) Purifying and/or separating the culture obtained in step (i) to obtain the mutant Fc polypeptide fragment, mutant immunoglobulin Fc region, antibody, or recombinant protein.
  • the purification can be purified and separated by a protein A affinity column to obtain the target antibody.
  • the purification can be purified and separated by a protein G affinity column to obtain the target antibody.
  • the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
  • mutant Fc polypeptide fragment as described in the first aspect of the present invention, a mutant immunoglobulin Fc region as described in the second aspect of the present invention, as described in the third aspect of the present invention
  • Antibody, or recombinant protein as described in the fourth aspect of the present invention polynucleotide as described in the fifth aspect of the present invention, vector as described in the sixth aspect of the present invention and/or as described in the seventh aspect of the present invention
  • the host cell is used to prepare a pharmaceutical composition for tumor immunotherapy, reducing inflammation and/or reducing autoimmune symptoms.
  • a method for treating diseases including the steps of: administering the mutant Fc polypeptide fragment as described in the first aspect of the present invention to a subject in need thereof, as described in the second aspect of the present invention.
  • the subject includes mammals, preferably humans.
  • the disease is selected from the group consisting of tumors, inflammatory diseases, autoimmune diseases, or a combination thereof.
  • the cancer includes, but is not limited to, breast cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, skin cancer, melanoma, colon cancer, stomach cancer, liver cancer, esophageal cancer, kidney cancer, and throat cancer. Cancer, thyroid cancer, pancreatic cancer, testicular cancer, brain cancer, bone cancer and blood cancer (such as leukemia, chronic lymphocytic leukemia), etc.
  • the cancer includes but is not limited to basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective cancer Tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial tumor, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), lymphoma (including Hodge Gold lymphoma and non-Hodgkin’s lymphoma); melanoma; neuroblastoma; oral cancer (such as lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer Respiratory system cancer; Sarcoma; Skin cancer; Gastric cancer; Testicular cancer; Thyroid cancer; Uterine cancer; Urinary system cancer; and other
  • the method can be used to stimulate an immune response to treat tumors by inhibiting or delaying tumor growth or reducing tumor size.
  • a method for screening amino acid mutations that enhance protein-molecule binding ability includes the following steps:
  • the amino acid mutation whose ratio after sorting is significantly higher than the ratio before sorting is an amino acid mutation that can enhance the binding ability between the protein and the interacting molecule.
  • the screening method can efficiently screen various amino acid mutations, has very good convenience and effectiveness, and can be used to screen amino acid mutations that enhance various protein-protein interactions or protein-other molecule interactions.
  • the modified Fc region or Fc fragment provided by the present invention has enhanced Fc ⁇ RIIB affinity, and some of them have enhanced Fc ⁇ RIIB affinity while also having reduced affinity for activated Fc ⁇ Rs.
  • These proteins can be used to design, engineer or optimize the agonistic activity of antibodies or fusion proteins. In particular, it can be used to design the agonistic activity of antibodies or fusion proteins based on human IgG2 backbone. As a result, protein molecules with significantly enhanced agonistic activity can be provided for the treatment of tumors, inflammatory diseases, autoimmune diseases, or a combination thereof, and have significant application prospects.
  • Figure 1 shows the construction map of mammalian surface display antibody plasmids.
  • Figure 2 shows a schematic diagram of the mutation screening process. Specifically, it includes constructing a mutation library first, then transferring it into cells through retroviruses, expressing the Fc mutation library on the cell surface, sorting cells with high affinity for Fc ⁇ RIIB, extracting DNA, high-throughput sequencing (NSG), and analyzing various mutations In the enrichment situation before and after sorting, a mutation type with a relatively high enrichment fold compared with the wild type was selected.
  • Figure 3 shows the Eu number corresponding to the amino acid encoded by CH2 on human IgG2 Fc.
  • Figure 4 shows that the flow cytometer sorting can gradually enrich cells with high Fc ⁇ RIIB binding ability.
  • the figure shows the cell flow cytometric analysis results of wild-type human IgG2 cells and human IgG2 library LibraryMix cells after one and two rounds of Fc ⁇ RIIB sorting, respectively.
  • Figure 5 shows that the ability of the cells screened by the library to bind to Fc ⁇ RIIB is significantly improved.
  • the figure shows the results of cell flow cytometry analysis of wild-type human IgG2 cells and two rounds of Fc ⁇ RIIB sorting.
  • Figure 6 shows a schematic diagram of two types of combinatorial mutation libraries.
  • Type one single-point combination mutations among the four regions, namely P233-V240, V266-P271, S298-T299 and G327-I332.
  • Figure 7 shows the flow cytometric analysis results of wild-type IgG2 and the library IgG2_C01 and library IgG2_C02 after flow cytometry sorting. It shows that in the library IgG2_C01 and library IgG2_C02, the proportion of cells that bind to Fc ⁇ RIIB has increased.
  • Figure 8 shows the ability of library Cmix and library C2 cells to bind to Fc ⁇ RIIB before (“_befor") and after sorting ("_1 st “ and “_2 nd ”) by flow cytometry.
  • the results show that the library is in the library after sorting.
  • the ratio of cells bound to Fc ⁇ RIIB gradually increased (2 nd >1 st >befor).
  • Figure 9 shows the binding ability of different IgG2 antibody Fc mutants to Fc ⁇ RI (expressed as ELISA signal: absorbance at 650nm (A650)).
  • Figure 10 shows the binding ability of different IgG2 antibody Fc mutants to Fc ⁇ RIIA 131H (expressed as ELISA signal: absorbance at 650nm (A650)).
  • Figure 11 shows the binding ability of different IgG2 antibody Fc mutants to Fc ⁇ RIIA 131R (expressed as ELISA signal: absorbance at 650nm (A650)).
  • Figure 12 shows the binding ability of different IgG2 antibody Fc mutants to Fc ⁇ RIIB (expressed as ELISA signal: absorbance at 650nm (A650)).
  • Figure 13 shows the binding ability of different IgG2 antibody Fc mutants to Fc ⁇ RIIIA 158F (expressed as an ELISA signal: absorbance at 650 nm (A650)).
  • Figure 14 shows the binding ability of different IgG2 antibody Fc mutants to FcyRIIIA 158V (expressed as an ELISA signal: absorbance at 650 nm (A650)).
  • Figure 15 shows the ELISA analysis of IgG1 V11 (hIgG1V11) corresponding to the IgG2 mutant on the binding capacity of Fc ⁇ RIIA 131H and Fc ⁇ RIIB (expressed as ELISA signal: absorbance at 650nm (A650)).
  • Figure 16 shows the results of the immunoactivation activity analysis of different antibody Fc mutant anti-human OX40 antibodies, expressed as a histogram of the average fluorescence intensity of CD4 positive cells CFSE (the lower the CFSE signal, the higher the activity).
  • Figure 17 shows the results of the immunoactivation activity analysis of different antibody Fc mutant anti-human OX40 antibodies, expressed as a histogram of the fluorescence intensity of CD4-positive cells CFSE (the lower the CFSE signal, the higher the activity).
  • the enzyme reaction and purification techniques are carried out according to the manufacturer's instructions, and usually can be carried out according to methods known in the art or according to the methods described herein.
  • the nomenclature, experimental methods and techniques related to analytical chemistry, synthetic organic chemistry, and medicine and medicinal chemistry described herein are known and commonly used in the art.
  • the chemical synthesis method, chemical analysis method, pharmaceutical preparation method, compounding method and drug delivery method, and the treatment of patients all adopt standard techniques.
  • Antibody shall include, but is not limited to, glycoprotein immunoglobulin, which specifically binds to antigen and includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, Or its antigen binding portion.
  • Each H chain contains a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • Antibody "heavy chain constant region”, also known as CDs contains three domains of CH1, CH2 and CH3 and a hinge region located between the CH1 domain and the CH2 domain.
  • Each light chain includes a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into hyperdenatured regions, called complementarity determining regions (CDR), with more conservative regions called framework regions (FR) interspersed between them.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • Fc region (crystallizable fragment region) or “Fc domain” or “Fc” refers to the C-terminal region of an antibody heavy chain, which mediates the binding of immunoglobulins to host tissues or factors, including those located in the immune system Binding to Fc receptors on various cells (e.g., effector cells), or to the first component of the classical complement system (C1q). Therefore, the Fc region is a polypeptide that constitutes a part of the constant region of an antibody heavy chain excluding the first constant region immunoglobulin domain (CH1 domain).
  • the Fc region is composed of two identical protein fragments from the second (CH2 domain) and third (CH3 domain) constant domains of the two heavy chains of the antibody; IgM
  • the Fc region of IgE and IgE contains three heavy chain constant domains (CH2-CH3-CH4 domains) in each polypeptide chain.
  • the Fc region contains immunoglobulin domains CH2 and CH3 and the hinge region between CH1 and CH2.
  • the Fc region of an immunoglobulin heavy chain is defined in the present invention as a sequence stretch from the amino acid residue at position P231 of the heavy chain to the carboxy terminus, where the numbering is based on EU numbering, As in Kabat.
  • the CH2 domain of the Fc region of human IgG extends from about amino acid 231 to about amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain of the Fc region, that is, it extends from about amino acid 341 to about amino acid 447 of IgG.
  • Fc polypeptide fragment refers to a fragment contained in the Fc region.
  • the Fc polypeptide fragment referred to in the present invention refers to a fragment extending from amino acid 233 to amino acid 332 in the Fc region, or other fragments in the Fc region containing the fragment, for example Fc CH2 domain or Fc region.
  • Fc region or “Fc polypeptide fragment” may be a natural Fc or an engineered Fc.
  • Fc region or “Fc polypeptide fragment” can also refer to this region in an isolated state, or this region in a protein polypeptide containing Fc.
  • Such polypeptides are also called “Fc fusion proteins” (for example, antibodies or immunological proteins). Adhesin).
  • Fc receptor or “FcR” is a receptor that binds to the Fc region of immunoglobulins.
  • FcRs that bind IgG antibodies include receptors of the Fc ⁇ R family, including allelic variants and alternatively spliced forms of these receptors.
  • the human Fc ⁇ receptor family includes several members: Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32a), Fc ⁇ RIIB (CD32b), Fc ⁇ RIIIA (CD16a), Fc ⁇ RIIIB (CD16b). Among them, Fc ⁇ RIIB is the only inhibitory Fc ⁇ receptor, and the others are activated Fc ⁇ receptors.
  • Fc ⁇ RIII activated Fc ⁇ receptor
  • Fc ⁇ RIIIA activated Fc ⁇ RIIIA
  • Fc ⁇ RIIB activated Fc ⁇ RIIB
  • Fc ⁇ RIIIA low-affinity receptors
  • Fc ⁇ receptors Gene polymorphisms also exist in these different Fc ⁇ receptors and affect their binding affinity.
  • the most common gene polymorphisms are the R131/H131 of Fc ⁇ RIIA and the V158/F158 of Fc ⁇ RIIIA. Some of these polymorphisms have been found to be associated with a variety of diseases, and the effect of some specific therapeutic antibodies also depends on whether the patient has a specific Fc ⁇ receptor gene polymorphism.
  • sequence of the present invention should be understood to include the sequence that is substantially the same as the sequence of the present invention.
  • substantially the same sequence means that after an optimal alignment, such as: using GAP or BESTFIT programs, using the default gap
  • the value determines that two peptide sequences have at least 70, 75 or 80% sequence identity, preferably at least 90 or 95% sequence identity, and more preferably at least 97, 98 or 99% sequence identity.
  • the difference in residue positions that are not identical are conservative amino acid substitutions.
  • a “conservative amino acid substitution” refers to the substitution of an amino acid residue with another amino acid residue having a similar chemical property (for example, charge or hydrophilicity) of the side chain R group.
  • conservative amino acid substitutions will not substantially change the functional properties of the protein. If the difference between two or more amino acid sequences is a conservative substitution, the sequence identity percentage or similarity can be adjusted upward to correct for the conservative nature of the substitution. See, for example: Pearson, Methods Mol. Biol. 243: 307-31 (1994).
  • amino acid groups with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: Serine and Threonine; 3) Amide-containing side chains: Asparagine and Glutamine; 4) Aromatic side chains: Phenylalanine, Tyrosine, and Tryptophan; 5) Basic side chains: Lys Acid, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.
  • the preferred conservative amino acid base group is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid -Aspartic acid, and asparagine-glutamine.
  • the Fc region, antibody, Fc fragment or fusion protein according to the present invention may further include other possible modifications not disclosed in the present invention, as long as the above is satisfied The stated sequence requirements are substantially the same.
  • amino acid numbers of the antibodies and fragments or domains of the present invention are based on the IgG Eu numbering. See Table 1 for the corresponding mutation types and names of some of the mutants involved in the present invention.
  • Antibodies usually specifically bind to their associated antigens with high affinity (similarly, the protein of the present invention can also specifically bind to its epitope recognition molecules), which is expressed as a dissociation constant of 10 -5 -10 -11 M or less ( KD). Any KD greater than about 10 -4 M -1 is generally considered to indicate non-specific binding.
  • an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and substantially the same antigen with high affinity, which means that the KD is 10 -7 M or less, preferably 10 -8 M or less, even more preferably 5 ⁇ 10 -9 M or less, most preferably 10 -8 -10 -10 M or less, but will not bind to irrelevant antigens with high affinity.
  • the antigen shows a high degree of sequence identity with a given antigen, for example, if it shows at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% with the sequence of the given antigen Sequence identity, the antigen is "substantially the same" as the given antigen.
  • the immunoglobulin may be from any commonly known isotype.
  • IgG isotypes can be divided into subclasses in some species: IgG1, IgG2, IgG3 and IgG4 in humans, IgG1, IgG2a, IgG2b and IgG3 in mice.
  • Isotype refers to the antibody class (eg, IgM or IgG1) encoded by heavy chain constant region genes.
  • Antibody includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single chain antibodies.
  • natural IgG refers to those IgGs that can occur naturally in nature, and the sequences of these IgGs are not artificially modified. It should be noted that due to genetic polymorphism, even natural IgG can have different mutants. Regardless of whether it is mutated or not, as long as it is natural IgG that is not artificially modified, it is called natural IgG.
  • an "agonistic antibody” is an antibody that binds to and activates a receptor.
  • An example of the function of an agonistic antibody is to bind to a receptor in the tumor necrosis factor receptor (TNFR) superfamily and induce apoptosis of cells expressing the TNF receptor. Tests for determining the apoptosis-inducing effect are described in WO98/51793 and WO99/37684, both of which are specifically incorporated herein by reference.
  • the anti-CD40 agonistic antibody can indirectly kill tumor cells by binding to target molecules that transmit immune activation signals on the surface of immune cells and activate important immune activation signal pathways controlled by them, thereby enhancing the anti-tumor immune response.
  • Antigen molecules include receptor molecules and other molecules with signal or physiological functions.
  • the above-mentioned specific physiological activities include, for example: proliferation activity, survival activity, differentiation activity, transcription activity, membrane transport activity, affinity, proteolytic activity, phosphorylation/dephosphorylation activity, redox activity, transfer activity, nucleic acid degradation activity, Dehydration activity, cell death inducing activity, apoptosis inducing activity, etc., but not limited to these.
  • Parent refers to the object of transformation in the process of protein transformation.
  • the parent may be a naturally-occurring polypeptide, or a variant or modified version of a naturally-occurring polypeptide.
  • the parent Fc region of the present invention is the Fc region of natural IgG.
  • Variant refers to the protein obtained after modification of the parent in the process of protein modification. Specifically, it can be a protein derived by mutation, deletion and/or addition of parent amino acids and retains some or all functions inherent in the parent.
  • the sequence of the variant herein preferably has at least about 80% of the parent’s sequence. Homology, most preferably at least about 90% homology, more preferably at least about 95% homology.
  • Fc variant refers to an Fc sequence that differs from the parent Fc sequence due to at least one amino acid modification.
  • the Fc variant may comprise only the Fc region, or may be present in the context of antibodies, Fc fusions, isolated Fc, Fc regions, or other polypeptides substantially encoded by Fc.
  • An Fc variant may refer to the Fc polypeptide itself, a composition comprising the Fc variant polypeptide, or the amino acid sequence encoding it.
  • tumor necrosis factor receptor superfamily or “TNF receptor superfamily” herein refers to receptor polypeptides that can bind to cytokines of the TNF family. Generally speaking, these receptors are type I transmembrane receptors with one or more cysteine-rich repetitive sequences in their extracellular region.
  • cytokines in the TNF gene family include: tumor necrosis factor- ⁇ (TNF- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ or lymphotoxin), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 Ligand, 4-1BB ligand, Apo-1 ligand (also called Fas ligand or CD95 ligand), Apo-2 ligand (also called TRAIL), Apo-3 ligand (also called TWEAK) , Osteoprotegerin (OPG), APRIL, RANK ligand (also called TRANCE), and TALL-1 (also called BlyS, BAFF or THANK).
  • TNF- ⁇ tumor necrosis factor- ⁇
  • TNF- ⁇ or lymphotoxin tumor necrosis factor- ⁇
  • CD30 ligand CD27 ligand
  • CD40 ligand OX-40 Ligand
  • 4-1BB ligand 4-1BB ligand
  • Apo-1 ligand also called Fas ligand or CD
  • TNF receptor superfamily examples include: tumor necrosis factor receptor type 1 (TNFR1), tumor necrosis factor receptor type 2 (TNFR2), p75 nerve growth factor receptor (NGFR), B cell surface antigen CD40, T cell antigen OX-40, Apo-1 receptor (also called Fas or CD95), Apo-3 receptor (also called DR3, sw1-1, TRAMP and LARD), called “transmembrane activator and CAML -Receptor of "interactor” or "TACI”, BCMA protein, DR4, DR5 (or, also known as Apo-2; TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER), DR6 , DcR1 (also called TRID, LIT or TRAIL-R3), DcR2 (also called TRAIL-R4 or TRUNDD), OPG, DcR3 (also called TR6 or M68), CAR1, HVEM (also called ATAR or TR2) , GITR,
  • the “affinity ratio to inhibitory Fc ⁇ receptor and activated Fc ⁇ receptor” or “I/A ratio” in the present invention is equal to the affinity value of the constant region of the antibody heavy chain and the inhibitory Fc ⁇ receptor (for example: human Fc ⁇ RIIB) Except for the highest affinity value between the constant region of the antibody heavy chain and the activated Fc ⁇ receptor of the same species (for example, including human Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIIA, and Fc ⁇ RIIIB).
  • the “affinity” refers to the size of the binding ability between two molecules, which can usually be measured by KD. In the present invention, it can also be measured by the enrichment factor (as shown in Table 7) or the binding analysis signal (as Figure 12).
  • KD refers to the equilibrium dissociation constant of the interaction of two molecules (e.g., a specific antibody and an antigen or a ligand and a receptor).
  • Enrichment factor refers to the percentage of cells expressing antibodies containing specific Fc mutations after sorting/pre-sorting after sorting using the flow cytometric sorting method or magnetic bead sorting method provided by the present invention (Analyzed by next-generation sequencing, as shown in Table 7).
  • the "binding analysis signal” adopts the absorbance value in the ELISA analysis provided by the present invention (as shown in Figure 12).
  • Human antibodies refer to antibodies whose variable regions have framework regions and CDR regions derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutations in vitro or by somatic mutations in vivo).
  • the term "human antibody” is not intended to include antibodies in which CDR sequences from the germline of other mammalian species (such as mice) are grafted onto human framework sequences.
  • the terms "human” antibody and "fully human” antibody are used synonymously.
  • the "antibody” of the present invention includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies.
  • a “humanized” antibody refers to an antibody in which some, most or all of the amino acids other than the CDR domain of a non-human antibody are replaced by corresponding amino acids from human immunoglobulins.
  • some, most, or all of the amino acids outside the CDR domain are replaced by amino acids from human immunoglobulin, and some, most, or all of the amino acids in one or more CDR regions No change. Small additions, deletions, insertions, substitutions or modifications to amino acids are allowed, as long as they do not eliminate the ability of the antibody to bind to a specific antigen.
  • “Humanized” antibodies retain antigen specificity similar to the original antibody.
  • a “chimeric antibody” refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.
  • the human IgG2 anti-mouse CD40 antibody light chain and heavy chain full-length sequences were constructed into the retroviral vector MIGRI (Addgene, Pear, etc., Blood.) by a multi-fragment one-step rapid cloning kit (Shanghai Yisheng Biotechnology Co., Ltd., China). 1998 Nov 15.92(10):3780-92.), the primers used are shown in Table 2, and the construction map is shown in Figure 1.
  • the light chain DNA and heavy chain DNA of the complete antibody are inserted after single digestion with restriction enzyme BglII. Connected by a self-cutting Linker P2A, the end of the heavy chain DNA contains a section of transmembrane domain DNA (the DNA sequence of each fragment is shown in Table 3).
  • the plasmid expresses the antibody human IgG2 and also expresses the GFP protein, which is displayed on the surface.
  • the amino acid sequences of antibodies and their related Fc regions are shown in Table 4. Sequencing confirmed that the construction was
  • the point mutation library is to introduce mutations through degenerate primers, and design multiple degenerate primers.
  • the degenerate primer used for P233 mutation is CCACCGTGCCCAGCACCANNSGTGGCAGGACCGTCAGTC (SEQ ID NO: 21).
  • the mutant library fragments and the required ligation vector were obtained by PCR, and the one-step rapid cloning kit (Shanghai Yisheng Biotechnology Co., Ltd., China) was ligated and transformed into DH5 ⁇ to obtain the mutant library. Pick 20 colonies from each library and sequence them to confirm whether mutations have been introduced at the target location.
  • the method of combining mutation libraries is similar.
  • the primers are designed based on the enriched mutation types that have been screened from the point mutation library.
  • the intermediate degenerate primers are changed to a codon encoding a specific amino acid or a degenerate codon encoding several amino acids.
  • the sequence of the primers for example: L328W (TGCAAGGTCTCCAACAAAGGCTGGCCAGCCCCCATCGAGAAAAC, SEQ ID NO: 22), Combine2_1 (ACGTGCGTGGTGGTGGACKNGWBGVASRWSSACCHGGAGGTCCAGTTCAACTGG, SEQ ID NO: 23).
  • R stands for A/G
  • Y stands for C/T
  • M stands for A/C
  • K stands for G/T
  • S stands for G/C
  • W stands for A /T
  • H stands for A/T/C
  • B stands for G/T/C
  • V stands for G/A/C
  • D stands for G/A/T
  • N stands for A/T/C/G.
  • the staining system is scaled up in equal proportions, and the cells are finally resuspended to about 1-5 ⁇ 10 7 cells/ml.
  • the proportion of cells with strong APC fluorescence signal is controlled to be less than 1% of IgG-positive cells.
  • MACS buffer (1 ⁇ PBS containing 0.5% BSA, 2mM EDTA), incubate on ice for 15 minutes, then MACS buffer Wash the cells twice, resuspend in 100 ⁇ l MACS buffer, add 25 ⁇ l anti-Biotin beads, incubate on ice for 15 minutes, wash the cells once with MACS buffer, resuspend to 500 ⁇ l, and add to the LS column placed in a magnetic field , Collect the effluent, wash and collect with 3ml MACS buffer, and finally remove the magnetic field from the LS column. 5ml MACS buffer will quickly elute the cells retained on the sorting column and collect the positive cells, which are the cells with high I/A ratio. .
  • Enzyme-linked immunosorbent assay to analyze the binding characteristics of mutant antibodies and Fc ⁇ R
  • the antibody concentration was diluted with the primary cell culture medium containing 0.2 ⁇ g/ml anti-CD3 to 2, 0.2, 0.02 ⁇ g/ml, respectively. Transfer 100 ⁇ L of the prepared antibody mixture to the wells of humanized FC ⁇ R/OX40 spleen cells that already contain CFSE markers.
  • CD3only group with CFSE-labeled cells and anti-mouse CD3, but no other antibodies
  • CD3+CD28 group with CFSE-labeled cells and anti-mouse CD3, and anti-mouse CD28 at a concentration of 2 ⁇ g/ml Antibody (Clone 37.51 (RUO), BD Pharmaceutical)
  • final concentrations of anti-mouse CD3 and CD28 are 0.1 ⁇ g/ml and 1 ⁇ g/ml, respectively.
  • Flow cytometry to detect the proliferation of T cells transfer the cultured cells to a 96-well U bottom plate, wash twice with PBS, centrifuge at 500g for 5 minutes, shake off the supernatant, and resuspend the cells in PE anti-mouse CD4 (Clone:GK1.5, 1:500, BD) and APC anti-mouse CD8a (Clone:53-6.7, 1:500, BioLegend) in 50 ⁇ l FACS buffer (PBS containing 0.5% FBS, 2mM EDTA) Incubate on ice for 15 minutes in the dark, then wash the cells twice with PBS buffer, and resuspend in 200 ⁇ l FACS buffer containing DAPI (0.5 ⁇ g/ml, Invitrogen) and CountBright Absolute Counting Beads (Life Technologies, 2 ⁇ l/sample) , And analyzed by flow cytometry.
  • PE anti-mouse CD4 Clone:GK1.5, 1:500, BD
  • the present invention mainly provides some antibody Fc mutant sequences that enhance the interaction between human IgG2 and Fc ⁇ RIIB. At the same time, the present invention also provides methods for screening and obtaining these mutants. As shown in Figure 2, according to the above method, Fc mutants with specific affinity can be screened.
  • Example 1 Construction of human antibody IgG2_Fc point mutation library.
  • the present invention selected some sites on human IgG2_Fc_CH2, including P233-S239, V266-P271, S298-T299 and G327-I332 (Eu numbering, see Figure 3 and Table 4), and randomized the amino acids at these sites. Mutations, each site including wild type has a total of 20 amino acid types (A, R, D, C, Q, E, H, I, G, N, L, K, M, F, P, S, T , W, Y, V). Prepare plasmid libraries (Library1, Library2, Library3, Library4) corresponding to the four regions of human IgG2_Fc, respectively, and a mixed library (LibraryMix) of these four libraries.
  • Example 2 Flow cytometry analysis of cell enrichment before and after sorting.
  • the present invention uses flow cytometry sorting technology to sort cells expressing human IgG2 ( detected by anti-human IgG F(ab') 2 ) and binding to human Fc ⁇ RIIB. As shown in Figure 4, flow cytometric sorting can gradually enrich cells with high Fc ⁇ RIIB binding ability.
  • the human IgG2 library LibraryMix was sorted by the flow cytometer to analyze the cells obtained in different rounds. The proportion of cells bound to Fc ⁇ RIIB that can be detected by the flow cytometer increased after each round of sorting. It was 0.90% in the first round and 22.2% in the second round, which was 2.65 times and 65.29 times higher than the 0.34% of wild-type human IgG2, respectively.
  • the proportion of cells that bind to Fc ⁇ RIIB in Library1, Library2, Library3, Library4 and LibraryMix can be detected on the flow cytometer: 1.85% and 16.3%, respectively , 0.57%, 2.19% and 22.2%, compared with 0.34% of wild-type human IgG2, the ratio increased to 5.44 times, 47.94 times, 1.69 times, 6.44 times and 65.29 times.
  • Example 3 Next-generation sequencing analysis of IgG2Fc mutation types with improved Fc ⁇ RIIB binding ability after flow cytometry sorting.
  • the present invention selects mutations whose sequence ratio before and after sorting is increased by more than twice that of wild-type human IgG2 (enrichment factor/wild-type enrichment factor>2), that is, human IgG2 is beneficial to the improvement of its ability to bind to Fc ⁇ RIIB (relative to See Table 7 for the mutation types of wild type whose enrichment factor is increased by at least 1 fold).
  • the proportion of L328W before sorting is 0.39%
  • the proportion after sorting is 12.84%
  • the enrichment factor is 33.20, which is 22.21 times of the wild-type enrichment factor (1.49).
  • the present invention also screened and obtained some double mutation types.
  • the proportion of these amino acid mutations before and after flow cytometry was increased to more than twice that of wild-type human IgG2.
  • double mutation combinations V266A/P271C or G, S267A/P271C or G, S267E/D270H, S267E/P271C or V or W or Y, S267E/S298R, S267M/P271C or G, P271A/S298R, P271G/G236V, P271G/G329S, P271G/P331C or T, P271G/S298D or E or G or K or L or N or R, P271G/T299A or M or S or W, G327A/A330R or V, G327A/I332A or C or E, The increase ratio of L328E/I332T, etc.) is higher than
  • the Fc region, antibody, Fc fragment or fusion protein of the present invention may further include other possible combinations or modifications in addition to the amino acid mutations provided by the present invention.
  • Example 4 Next-generation sequencing analysis of favorable mutant types after magnetic bead sorting.
  • the present invention uses magnetic bead sorting technology to continue sorting the Fc ⁇ RIIB high-affinity cells obtained by the above-mentioned flow sorting, using Biotin-labeled Fc ⁇ RIIB and unlabeled activated Fc ⁇ R to mix and incubate the cells, and then use anti-Biotin-magnetic beads to sort For labeled cells, the enrichment ratio before and after the next-generation sequencing analysis can be screened for mutations with a higher I/A ratio, as shown in Table 9.
  • the method of selecting mutations is: selecting the mutations whose ratio has increased to more than twice the wild-type human IgG2 before and after the sorting, and at the same time, the part of the unbound magnetic beads (that is, the effluent when passing through the LS column) is sorted.
  • NA is not detected in the corresponding mutation type in this row of the sample in this column.
  • #1-#4 This data comes from two experiments (#3, #4) of Library2(#1), Library4(#2), LibraryMix.
  • G236D, S239D (reported according to Seung Y. Chu et al. (Molecular Immunology 45 (2008) 3926-3933)) and S298A mutation (reported according to patent US20090042291A1) can increase the affinity of human IgG1 and Fc ⁇ RIIB.
  • the second-generation sequencing analysis results of the present invention showed that the proportion of IgG2 variants containing G236D or S298A mutations did not increase after sorting, but decreased to a certain extent (Table 10); human IgG2 variants containing S239D mutations The proportion before sorting was 0.026%, but it was not detected in the sequencing results after sorting.
  • NA means that the mutant type in this row is not detected in this column of experiments, and the percentage and multiple cannot be calculated.
  • Example 5 Amino acid mutations in human IgG2 Fc polypeptide fragments that can enhance the affinity of Fc ⁇ RIIB.
  • Table 11 summarizes the amino acid mutations that can enhance the affinity of FcyRIIB screened according to the point mutation library screening method of the present invention.
  • S267G and P271C sites in the present invention are new mutation types on human IgG2 that can improve the binding ability of Fc ⁇ RIIB.
  • H268S, L328A, and G327A are all new mutation types on human IgG2 that can improve the binding ability of Fc ⁇ RIIB and have weaker affinity with other activated Fc ⁇ Rs than Fc ⁇ RIIB.
  • the Fc mutation type that can increase the affinity of Fc ⁇ RIIB (the mutation whose enrichment factor is more than 1 times higher than that of the wild type) was screened out, and two types of combinatorial mutation libraries were designed: type one, Single-point combined mutations between the four regions, namely P233-V240, V266-P271, S298-T299 and G327-I332, each of the four regions has 0-1 mutations, a total of 0-4 site combined mutations; Type two, multiple amino acid combination mutations in one region, namely P233-V240, V266-P271, S298-T299 or G327-I332 four regions, the combination of amino acid mutations within each region (0-7, 0-, respectively) 6, 0-2 and 0-6 combined mutations), as shown in Figure 6.
  • the size of the plasmid library is up to 4.67 ⁇ 10 5 .
  • Example 7 Cell enrichment of combinatorial mutation library before and after flow cytometry sorting
  • IgG2_C01 is a combinatorial mutation (type one) library with a maximum of 1 mutation in each of the four regions of P233-V240, V266-P271, S298-T299 and G327-I332;
  • IgG2_C02 is P233-V240, V266-P271, S298-T299 and The consecutive combinatorial mutation (type 2) libraries in the four regions of G327-I332 were merged into a cell library made by 3T3 cells.
  • Example 8 Next-generation sequencing analysis after flow cytometry sorting and IgG2Fc combination mutation type with improved Fc ⁇ RIIB binding ability.
  • the combinatorial mutation library constructed in the present invention was subjected to flow sorting to obtain cells with higher binding capacity to Fc ⁇ RIIB, and then the second-generation sequencing was used to analyze the different types of Fc ⁇ RIIB before and after the sorting.
  • the enrichment factor of the mutation type the results are shown in Table 12.
  • the selected mutations are enriched at a higher multiple (after the last round of sorting/before sorting), or are significantly enriched in at least one round of sorting (after this round of sorting/before the round of sorting), Or it will only be tested after the final round of sorting.
  • the enrichment factor of V266L/S298L/L328W is 21,920.33 times of wild type (after the final round of sorting/before sorting); the enrichment factor of point mutation L328W is 8.78 times of wild type (in the last round of sorting).
  • the enrichment multiples of H268D/S298L/L328W were 43.98 (after the second round of sorting/before the second round of sorting) and 5.95 (after the third round of sorting/the third round of sorting) Before); the enrichment multiple of V234M/S267E/S298L/L328W is 5.13 (after the third round of sorting/before the third round of sorting); V234Q/A235G/P238L/S239V/G327A/L328E/A330S/I332T is only at the end It was tested after one round of sorting.
  • Example 9 Enzyme-linked immunosorbent assay (ELISA) analysis of the binding characteristics of mutant antibodies and Fc ⁇ R
  • Example 11 The effect of known mutations on the Fc ⁇ R binding ability of IgG1 cannot be directly predicted based on its influence on the Fc ⁇ R binding ability of IgG2
  • G236D, S239D (reported according to Seung Y. Chu et al. (Molecular Immunology 45 (2008) 3926-3933)) and S298A mutation (reported according to patent US20090042291A1) can increase the affinity of human IgG1 and Fc ⁇ RIIB.
  • the second-generation sequencing analysis results of the present invention showed that the proportion of IgG2 variants containing G236D or S298A mutations did not increase after sorting, but decreased to a certain extent (Table 10); human IgG2 variants containing S239D mutations The proportion before sorting was 0.026%, but it was not detected in the sequencing results after sorting.
  • V11 mutation site G237D/P238D/H268D/P271G/A330R
  • F. Mimoto et al., Protein Engineering, Design & Selection vol. 26no F. Mimoto et al., Protein Engineering, Design & Selection vol. 26no
  • IgG2_M2 H268D/P271G
  • IgG2_M3 H268D/P271G/A330R
  • IgG2_M4 G236D/P238D/H268D/P271G
  • IgG2_M5 G236D/P238D/H268D/P271G/A330R

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Abstract

提供了一种改造的Fc区，所述Fc区相比于亲本Fc区具有至少一个氨基酸突变，这些改造的Fc区对FcγRIIB的亲和力较亲本Fc区对FcγRIIB的亲和力提高。还提供了包含上述改造的Fc区的抗体，特别是激动型抗体。改造的Fc区对于优化抗体的激动活性具有重要用途。

Description

FcγRIIB亲和力增强的抗体Fc区 技术领域
本发明涉及生物制药领域，特别是涉及一系列人IgG2抗体的Fc区的变体，这些变体具有增强的FcγRIIB亲和力，包含这些Fc区变体的激动型抗体可望具有更好的激动活性。
背景技术
生物大分子药物的发展为多种疾病的治疗提供了新的方法和可能，尤其是基于抗体和重链恒定区(包含Fc区)的分子靶向治疗，包括抗体和重链恒定区融合蛋白，三十多年来在生物制药领域已经取得了巨大成功并持续成为该领域的重点。
从作用方式上，生物大分子主要可以分为三类：清除靶标(分子和细胞)的效应分子、阻断靶标参与的信号通路的阻断型分子、和激活靶标下游信号通路的激动型分子。肿瘤免疫治疗近年取得了重要突破。这得益于通过阻断免疫抑制节点，提高免疫细胞活性杀灭肿瘤的抗体的使用。但是目前仍有大量癌症患者对已有治疗手段没有应答。因此，一方面需要对目前已有的肿瘤免疫治疗手段进行优化；另一方面亟需研发新的肿瘤免疫治疗药物。需要特别指出的是有一类被称为“激动型抗体”的肿瘤免疫治疗手段，能够通过结合免疫细胞表面传递免疫激活信号的靶标分子并激活其控制的重要免疫激活信号通路，进而增强抗肿瘤免疫应答间接杀死肿瘤细胞。然而，虽然激动型肿瘤免疫治疗抗体已经在动物模型中证明了其巨大潜力，并且已经成为一个被广泛接受并看好的肿瘤免疫治疗理念，但是这类抗体的研发至今尚未成功，是肿瘤免疫治疗领域当前的一个主要挑战。此外，激动型抗体激活也是干预和调控其它生物学过程的关键信号通路的有利手段，在疾病防控和治疗领域有着广泛的应用前景。例如激活免疫抑制信号通路，可能有利于减轻炎症和自身免疫症状。
抗体主要通过其恒定区与FcγR相互作用，并且不同的抗体与FcγR的结合能力也有所不同，这种差异也影响着抗体在体内的功能。抗体Fc改造是优化治疗抗体的重要手段和趋势，其中改变抗体Fc区与关键蛋白如FcγR的相互作用的能力是一种优化抗体的活性非常有效的方法。
发明内容
针对上述问题，本发明基于IgG，通过对Fc区进行突变改造，获得了可以增强FcγRIIB亲和力的氨基酸突变及组合；包含这些氨基酸突变的Fc可以用于优化抗体的活性，特别是提高激动型抗体的激动活性。
在本发明的第一方面，提供了一种突变型Fc多肽片段，所述的突变型Fc多肽片段具有以下特征：
(i)所述的突变型Fc多肽片段相对于突变前相应的野生型Fc片段具有突变；并且
(ii)与野生型Fc区相比，所述突变型Fc区与FcγRIIB的亲和力提高；并且，所述的野生型Fc为野生型IgG2的Fc。
在另一优选例中，所述的突变型Fc多肽片段相对于突变前相应的野生型Fc片段具有选自下组的位点的突变：L328、H268、S267、P271、G327，或其组合；
其中，所有氨基酸的编号以IgG Eu编号为依据。
在另一优选例中，所述的亲和力可以体现为筛选的富集程度或者结合分析信号。
在另一优选例中，所述野生型Fc片段是以IgG Eu编号为依据的IgG2的氨基酸序列中的第233位至第332位，并且所述野生型Fc片段的氨基酸序列如SEQ ID NO:20所示。
在另一优选例中，与野生型Fc片段相比，所述突变型Fc多肽片段具有增强的FcγRIIB亲和力。
在另一优选例中，突变型Fc多肽片段对FcγRIIB的亲和力较野生型Fc片段对FcγRIIB的亲和力提升1倍以上；优选地，提升2、3、4、5、10、20、50或100倍以上。
在另一优选例中，所述突变型Fc多肽片段对激活型FcγR的亲和力较FcγRIIB的亲和力低。
在另一优选例中，与野生型Fc片段相比，所述突变型Fc多肽片段与FcγRIIB的亲和力提高，并且与激活型FcγR的亲和力降低(I/A比值高于野生型Fc片段)。
在另一优选例中，所述的突变型Fc多肽片段分别与FcγRIIB和FcγRIIA 131R的亲和力的比值(RIIB/RIIAR)高于野生型IgG2。
在另一优选例中，所述的激活型FcγR包括：FcγRI、FcγRIIA 131H、FcγRIIA 131R、FcγRIIIA 158F和FcγRIIIA 158V。
在另一优选例中，突变L328包括L328W、L328E、L328Y、L328F、L328M、L328A、L328G、L328N、L328P、L328R、L328V；优选地为L328W或L328E。
在另一优选例中，突变H268包括H268D、H268S、H268E、H268K、H268N；优选地为H268D、H268S或H268E。
在另一优选例中，突变S267包括S267E、S267V、S267D、S267M、S267Q、S267A、S267G、S267R、S267N、S267W；优选地为S267E、S267V、S267D、S267M；更优选地为S267E。
在另一优选例中，突变P271包括P271C、P271G、P271V、P271A、P271W、P271Y、P271Q、P271T、P271I、P271L、P271S；优选地为P271C、P271G、P271V、P271A、P271W、P271Y、P271Q、P271T；更优选地为P271C、P271G。
在另一优选例中，突变G327包括G327A、G327L、G327S；优选地为G327A。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段具有选自下组的位点的突变：L328W、L328E、H268D、H268S、H268E、S267E、A330S、P233G、P271G、P271C、P271V、P271A、P271W、P271Y、G327A，或其组合；其中，所有氨基酸的编号以IgG Eu编号为依据。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段具有L328和H268两个位点中至少一个位点的突变，并且所述突变型Fc多肽片段中具体的突变选自下组之一：L328W、L328E、H268D、H268S、H268E、H268D/S298L/L328W、S267V/S298L/L328W、V234M/S267E/S298L/L328W、A235W/V266L/S298L/L328W、V234Q/A235G/P238L/S239V/H268D/G327A/L328E/A330S/I332T、S239V/V266L/S298L/L328W、V266L/L328W、V266L/S267D/H268D/E269D/P271Q、S267E/H268S/E269D、P233F/V266L/S298L/L328W、V266L/S298L/L328W、V234Q/A235G/P238L/S239V/G327A/L328E/A330S/I332T、V266L/S267E/H268S/E269V、V266L/S267E/H268S/E269V/P271C、V266L/S267E/H268S/E269A/P271C、S267V/S298G/L328W、V266L/S267M/H268E/P271Q、V266L/S267E/H268S/E269G/P271T、V234Q/V266L/S267D/H268D/E269D/P271Q、V234Q/A235G/P238L/S239V/S267E/H268S/E269D、V266L/S267E/H268S/P271V、V234Q/A235G/P238L/S239V/S267E/S298L/G327A/L328E/A330S/I332T、V234Q/V266L/S267E/H268S/E269V、L328E/I332T；其中，所有氨基酸的编号以IgG Eu编号为依据。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段具有S267位点的突变，并且所述突变型Fc多肽片段中具体的突变选自下组之一：S267E/A330S、S267E/S298G、P233G/S267E、V234M/S267E/S298G/I332L、P233G/S267E/S298G、V266L/S267E/E269K/P271G、P238Q/S267E、S267A/P271C、S267A/P271G、S267E/D270H、S267E/P271C、S267E/P271V、S267E/P271W、S267E/P271Y、S267E/S298R、S267M/P271C、S267M/P271G、S267V/S298L、S267E/P329R；其中，所有氨基酸的编号以IgG Eu编号为依据。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段具有S267位点的突变，并且所述突变型Fc多肽片段中具体的突变选自下组之一：S267E/A330S、S267E/S298G、P233G/S267E、V234M/S267E/S298G/I332L、P233G/S267E/S298G、V266L/S267E/E269K/P271G、S267E/P271C；其中，所有氨基酸的编号以IgG Eu编号为依据。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段具有P271位点的突变，并且所述突变型Fc多肽片段中具体的突变选自下组之一：V266L/S267E/E269K/P271G、V266A/P271C、V266A/P271G、S267A/P271C、S267A/P271G、S267E/P271C、S267E/P271V、S267E/P271W、S267E/P271Y、S267M/P271C、S267M/P271G、V266G/P271C、P271A/S298R、P271G/G236V、P271G/P329S、P271G/P331C、P271G/P331T、P271G/S298D、P271G/S298E、P271G/S298G、P271G/S298K、P271G/S298L、P271G/S298N、P271G/S298R、P271G/T299A、P271G/T299M、P271G/T299S、P271G/T299W、P271G/I332L；其中，所有氨基酸的编号以IgG Eu编号为依据。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段具有G327位点的突变，并且所述突变型Fc多肽片段中具体的突变选自下组之一：G327A/A330R、G327A/A330V、G327A/I332A、G327A/I332C、G327A/I332E。
在另一优选例中，所述的突变型Fc多肽片段相对于野生型Fc片段的具体的突变选自下组之一：H268D/S298L/L328W、S267V/S298L/L328W、V234M/S267E/S298L/L328W、A235W/V266L/S298L/L328W、V234Q/A235G/P238L/S239V/H268D/G327A/L328E/A330S/I332T；其中，所有氨基酸的编号以IgG Eu编号为依据。
在本发明的第二方面，提供了一种突变型免疫球蛋白Fc区，所述突变型免疫球蛋白Fc区包含如本发明第一方面所述的突变型Fc多肽片段。
在另一优选例中，所述的免疫球蛋白是人IgG2。
在另一优选例中，与野生型IgG2 Fc区相比，所述突变型免疫球蛋白Fc区具有增强的FcγRIIB亲和力。
在另一优选例中，所述突变型免疫球蛋白Fc区对FcγRIIB的亲和力较野生型IgG2 Fc区对FcγRIIB的亲和力提升1倍以上；优选地，提升2、3、4、5、10、20、50或100倍以上。
在另一优选例中，所述的亲和力可以体现为筛选的富集程度或者结合分析信号。
在另一优选例中，所述突变型免疫球蛋白Fc区对激活型FcγR的亲和力较FcγRIIB的亲和力低。
在另一优选例中，与野生型IgG2 Fc区相比，所述突变型免疫球蛋白Fc区与FcγRIIB的亲和力提高，并且与激活型FcγR的亲和力降低(I/A比值高于野生型IgG2 Fc区)。
在另一优选例中，所述的突变型免疫球蛋白Fc区分别与FcγRIIB和FcγRIIA 131R的亲和力的比值(RIIB/RIIAR)高于野生型IgG2。
在本发明的第三方面，提供了一种抗体，所述抗体中包含如本发明第一方面所述的突变型Fc多肽片段或如本发明第二方面所述的突变型免疫球蛋白Fc区。
在另一优选例中，所述的抗体是基于人IgG2骨架的抗体。
在另一优选例中，所述抗体是激动型抗体。
在另一优选例中，所述抗体特异性地靶向肿瘤坏死因子受体超家族。
在另一优选例中，所述抗体能够特异性地结合选自下组的靶点：CD40、DR5、OX40、CD137、CD27、CD30、GITR、HVEM、TACI、DR4、FAS，或其组合。
在另一优选例中，所述抗体能够特异性地结合OX40。
在另一优选例中，所述抗体所靶向的抗原为免疫受体分子，或其组合。
在另一优选例中，所述抗体所靶向的抗原为免疫抑制性受体分子，并且所述免疫抑制性受体分子选自于PD-1、CTLA-4、VISTA、TIM-3、BTLA、LAG-3，或其组合。
在另一优选例中，所述抗体是人抗体、人源化抗体，或嵌合抗体。
在另一优选例中，所述抗体是单克隆抗体或多克隆抗体，优选为单克隆抗体。
在本发明的第四方面，提供了一种融合蛋白，所述融合蛋白包含如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区，或如本发明第三方面所述的抗体。
在另一优选例中，所述融合蛋白中还包括其它具有受体激动功能的蛋白序列或其片段。
在另一优选例中，所述其它具有受体激动功能的蛋白为TNF基因家族中细胞因子。
在另一优选例中，所述其它具有受体激动功能的蛋白包括免疫受体的配体分子。
在另一优选例中，所述其它具有受体激动功能的蛋白包括免疫受体的配体分子，所述免疫受体的配体分子选自于CD80、CD86、ICOSL、OX40L、CD137L、CD40L、CD30L、CD27L、CD244、CD150、CD48、CD84、CD319、Ly118或者CD229中的任意一种，或其组合。
在另一优选例中，所述其它具有受体激动功能的蛋白包括免疫受体的配体分子，所述免疫受体的配体分子选自下组：PD-L1、PD-L2、B7-H3、B7-H4、CD47、VISTA、HVEM、GAL9，或其组合。
在另一优选例中，所述融合蛋白还可包括协助表达和/或纯化的标签序列；优选地，所述的标签序列包括6His标签、HA标签和/或FLAG标签。
在本发明的第五方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区、如本发明第三方面所述的抗体，或如本发明第四方面所述的重组蛋白。
在本发明的第六方面，提供了一种载体，所述载体含有如本发明第五方面所述的分离的多核苷酸。
在另一优选例中，所述的载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其它基因转移系统、或其组合。
在另一优选例中，所述载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
在本发明的第七方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第 六方面所述的载体，或其基因组中整合有如本发明第五方面所述的多核苷酸；
或者，所述宿主细胞表达如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区、如本发明第三方面所述的抗体，或如本发明第四方面所述的重组蛋白。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、昆虫细胞、鸟类细胞、哺乳动物细胞。
在本发明的第八方面，提供了一种药物组合物，所述的药物组合物包括：
(a)如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区、如本发明第三方面所述的抗体，或如本发明第四方面所述的重组蛋白；和
(b)药学上可接受的载体。
在另一优选例中，所述药物组合物中还可包括治疗肿瘤的其它药物，如细胞毒性药物。
在另一优选例中，所述药物组合物中还可包括其它具有受体激动功能的活性物质。
在另一优选例中，所述的药物组合物为注射剂型。
在本发明的第九方面，提供了一种产生如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区、如本发明第三方面所述的抗体，或如本发明第四方面所述的重组蛋白的方法，包括步骤：
(i)在合适的条件下，培养如本发明第七方面所述的宿主细胞，从而获得含所述突变型Fc多肽片段、所述突变型免疫球蛋白Fc区、所述抗体，或所述重组蛋白的培养物；和
(ii)对步骤(i)中得到的培养物进行纯化和/或分离，获得所述的所述突变型Fc多肽片段、突变型免疫球蛋白Fc区、抗体，或重组蛋白。
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。
在另一优选例中，所述纯化可以通过蛋白G亲和柱纯化分离获得目标抗体。
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。
在本发明的第十方面，提供了如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区、如本发明第三方面所述的抗体，或如本发明第四方面所述的重组蛋白、如本发明第五方面所述的多核苷酸、如本发明第六方面所述的载体和/或如本发明第七方面所述的宿主细胞的用途，用于制备一用于肿瘤免疫治疗、减轻炎症和/或减轻自身免疫症状的药物组合物。
在本发明的第十一方面，提供了一种治疗疾病的方法，包括步骤：给有所需要的对象施用如本发明第一方面所述的突变型Fc多肽片段、如本发明第二方面所述的突变型免疫球蛋白Fc区、如本发明第三方面所述的抗体，或如本发明第四方面所述的重组蛋白、如本发明第五方面所述的多核苷酸、如本发明第六方面所述的载体、如本发明第七方面所述的宿主细胞，和/或如本发明第八方面所述的药物组合物。
在另一优选例中，所述的对象包括哺乳动物，优选地是人。
在另一优选例中，所述的疾病选自下组：肿瘤、炎性疾病、自身免疫性疾病，或其组合。
在另一优选例中，所述的癌症包括但不限于乳腺癌、前列腺癌、肺癌、卵巢癌、宫颈癌、皮肤癌、黑素瘤、结肠癌、胃癌、肝癌、食道癌、肾癌、咽喉癌、甲状腺癌、胰腺癌、睾丸癌、脑癌、骨癌和血癌(如白血病、慢性淋巴细胞性白血病)等。
在另一优选例中，所述的癌症包括但不限于基底细胞癌、胆道癌、膀胱癌、骨癌、脑和中枢神经系统(CNS)癌、宫颈癌、绒毛膜癌、结肠直肠癌、结缔组织癌、消化系统癌、子宫内膜癌、食道癌、眼癌、头颈癌、胃癌、上皮内肿瘤、肾癌、喉癌、肝癌、肺癌(小细胞、大细胞)、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤)；黑素瘤；神经母细胞瘤；口腔癌(例如唇、舌头、口和咽)；卵巢癌；胰腺癌；视网膜母细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统癌；肉瘤；皮肤癌；胃癌；睾丸癌；甲状腺癌；子宫癌；泌尿系统癌；以及其它癌和肉瘤。
在另一优选例中，所述方法可用于刺激免疫反应以通过抑制或延缓肿瘤的生长或减少肿瘤的大小来治疗肿瘤。
在本发明的另一方面，还提供了一种筛选增强蛋白-分子结合能力的氨基 酸突变的方法，包括以下步骤：
1)提供亲本蛋白序列，通过PCR在亲本蛋白序列中引入氨基酸突变；
2)将亲本蛋白以及突变后的蛋白构建入表达载体，形成突变文库；
3)将表达载体转染进入哺乳动物细胞，并将亲本蛋白以及突变后的蛋白表达于细胞表面；
4)将带标记的相互作用分子和上述哺乳动物细胞孵育，通过流式细胞分选或者磁珠分选出被标记的哺乳动物细胞；
5)提取被标记的哺乳动物细胞的DNA，进行测序，分析比较分选前后序列比例的变化情况；
其中分选后的比例显著高于分选前的比例的氨基酸突变为能够增强所述蛋白和相互作用分子之间结合能力的氨基酸突变。
该筛选方法可高效地筛选各种氨基酸突变，具有非常好的便捷性和有效性，可用于筛选增强各种蛋白-蛋白相互作用或者蛋白-其它分子相互作用的氨基酸突变。
本发明的有益效果是：本发明提供的改造的Fc区或Fc片段具有增强的FcγRIIB亲和力，部分在具有增强的FcγRIIB亲和力的同时还具有降低的激活型FcγRs亲和力。这些蛋白可以用于设计、改造或优化抗体或者融合蛋白的激动活性。特别是可以用于设计基于人IgG2骨架的抗体或者融合蛋白的激动活性。由此，可提供激动活性显著增强的蛋白分子，用于肿瘤、炎性疾病、自身免疫性疾病或其组合的治疗，具备显著的应用前景。
附图说明
图1显示了哺乳动物表面展示抗体质粒构建图谱。
图2显示了突变筛选流程示意图。具体包括先构建突变文库，然后通过逆转录病毒转入细胞，Fc突变文库表达于细胞表面，细胞分选与FcγRIIB高亲和力的细胞，提取DNA，高通量测序(NSG),然后分析各种突变在分选前后的富集情况，选出与野生型相比富集倍数相对高的突变类型。
图3显示了人IgG2 Fc上CH2编码氨基酸对应的Eu编号。
图4显示了流式细胞分选仪分选可以逐步富集与FcγRIIB结合能力高的细胞。图中分别为野生型人IgG2细胞和人IgG2文库LibraryMix细胞经过一轮 和两轮FcγRIIB分选以后的细胞流式分析结果。
图5显示了经过文库筛选的细胞与FcγRIIB的结合能力明显提高。图中分别为野生型人IgG2细胞和经过两轮FcγRIIB分选以后的细胞流式分析结果。
图6显示了两种类型组合突变文库示意图。类型一，四个区域之间单点组合突变，即P233-V240、V266-P271、S298-T299以及G327-I332四个区域每个区域有0-1个突变，共0-4个位点的组合突变；类型二，一个区域内的多个氨基酸组合突变，即P233-V240、V266-P271、S298-T299或G327-I332四个区域，每个区域内部氨基酸的组合突变(分别为0-7、0-6、0-2和0-6个组合突变)。
图7显示了野生型IgG2和经过流式细胞分选后的文库IgG2_C01、文库IgG2_C02的流式分析结果，显示在文库IgG2_C01和文库IgG2_C02中，与FcγRIIB结合的细胞比例有所提高。
图8显示了文库Cmix和文库C2细胞在流式细胞分选前(“_befor”)和分选后(“_1 st”和“_2 nd”)与FcγRIIB的结合能力，结果显示分选后文库中细胞与FcγRIIB结合的比例逐步提高(2 nd>1 st>befor)。
图9显示了不同IgG2抗体Fc突变体与FcγRI的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图10显示了不同IgG2抗体Fc突变体与FcγRIIA 131H的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图11显示了不同IgG2抗体Fc突变体与FcγRIIA 131R的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图12显示了不同IgG2抗体Fc突变体与FcγRIIB的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图13显示了不同IgG2抗体Fc突变体与FcγRIIIA 158F的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图14显示了不同IgG2抗体Fc突变体与FcγRIIIA 158V的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图15显示了ELISA分析IgG1 V11(hIgG1V11)上对应到IgG2上突变体与FcγRIIA 131H以及与FcγRIIB的结合能力(表达为ELISA信号：在650nm的吸光值(A650))。
图16显示了不同抗体Fc突变体抗人OX40抗体免疫激活活性分析结果，表达为CD4阳性细胞CFSE的平均荧光强度柱状图(CFSE信号越低，活性越高)。
图17显示了不同抗体Fc突变体抗人OX40抗体免疫激活活性分析结果，表达为CD4阳性细胞CFSE的荧光强度直方图(CFSE信号越低，活性越高)。
具体实施方式
除非另有说明，否则本文中所使用的科学与技术术语应具有那些本领域普通技术人员通常理解的含义。此外，除非本文中另有要求，否则单数术语应包括复数，且复数术语应包括单数。通常，本文中所描述的细胞及组织培养、分子生物学、免疫学及蛋白质与核酸化学所涉及的命名法与其技术均是本领域已知且常用的。
本发明的方法与技术通常依据本领域已知的传统方法进行，且说明于本说明书所摘录和讨论的多种一般性及较专业性参考书中，除非另有说明。参见例如：Sambrook等人的Molecular Cloning：A Laboratory Manual，第2版(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1989))及Ausubel等人的Current Protocols in Molecular Biology(Greene Publishing Associates(1992))，及Harlow与Lane的Antibodies：A Laboratory Manual(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1990))，其内容已以引用的方式并入本文中。酶反应与纯化技术是根据制造商的说明书进行，通常可依本领域已知的方法或依本文说明的方法进行。与本文中描述的分析化学、合成有机化学、及医学与医药化学相关的命名法，及实验方法与技术是本领域已知且常用的。化学合成法、化学分析法、医药制法、调配法与药物递送法，及患者的治疗法均采用标准技术。
除非另有说明，否则下列术语具有如下定义。
“抗体”(Ab)应包括，但不仅限于，糖蛋白免疫球蛋白，其特异性结合抗原并且至少包括通过二硫键相互连接的两个重(H)链和两个轻(L)链，或其抗原结合部分。每个H链包含重链可变区(本文缩写为VH)和重链恒定区。抗体“重链恒定区”，又称CDs，包含CH1、CH2和CH3三个结构域以及位于CH1结构域与CH2结构域之间的铰链区。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区由一个结构域CL组成。VH和VL区可以进一步细分为高变性的区域，称为互补决定区(CDR)，它们之间散在着较为保守的称作框架区(FR)的区域。每个VH和VL由三个CDR和四个FR组成，从氨基端向羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包 含与抗原相互作用的结合结构域。
“Fc区”(可结晶片段区域)或“Fc结构域”或“Fc”是指抗体重链的C-末端区域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体的结合，或者与经典补体系统的第一组分(C1q)的结合。因此，Fc区是构成抗体重链恒定区中除第一恒定区免疫球蛋白结构域(CH1结构域)之外的部分的多肽。在IgG，IgA和IgD抗体同种型中，Fc区由来自抗体两条重链的第二(CH2结构域)和第三(CH3结构域)恒定结构域的两个相同的蛋白片段构成；IgM和IgE的Fc区在每个多肽链中包含三个重链恒定结构域(CH2-CH3-CH4结构域)。对于IgG，Fc区包含免疫球蛋白结构域CH2和CH3以及CH1和CH2之间的铰链区。虽然免疫球蛋白重链的Fc区的边界可以变化，但是人IgG重链Fc区在本发明中被定义为从重链位置P231的氨基酸残基到羧基端的序列段，其中该编号是根据EU编号，如在Kabat中一样。人IgG Fc区的CH2结构域从大约氨基酸231延伸至大约氨基酸340，而CH3结构域位于Fc区的CH2结构域的C末端侧，即，它从IgG的大约氨基酸341延伸至大约氨基酸447。
“Fc多肽片段”指包含在Fc区内的片段，本发明所指的Fc多肽片段是指Fc区中从氨基酸233延伸至氨基酸332的片段，或者包含该片段的其它Fc区内的片段，例如Fc CH2结构域或者Fc区。
如本文所使用的，“Fc区”或者“Fc多肽片段”可以是天然Fc或改造后的Fc。此外，“Fc区”或者“Fc多肽片段”还可以指处于分离状态的这个区域，或者处于包含Fc的蛋白多肽中的这个区域，这样的多肽又称“Fc融合蛋白”(例如，抗体或免疫粘附素)。
“Fc受体”或“FcR”是结合免疫球蛋白Fc区的受体。结合IgG抗体的FcR包括FcγR家族的受体，包括这些受体的等位基因变体和可变剪接形式。人Fcγ受体家族包括几个成员：FcγRI(CD64)、FcγRIIA(CD32a)、FcγRIIB(CD32b)、FcγRIIIA(CD16a)、FcγRIIIB(CD16b)。其中，FcγRIIB是唯一的抑制性Fcγ受体，其它均为激活型Fcγ受体。大多数天然效应器细胞类型共表达一种或多种激活型FcγR和抑制型FcγRIIB，而自然杀伤(NK)细胞选择性地表达一种激活型Fcγ受体(在小鼠中是FcγRIII，在人中是FcγRIIIA)，但在小鼠和人类中不表达抑制型FcγRIIB。这些Fcγ受体的分子结构不同，也因此对各IgG抗体亚类具有不同的亲和力。在这些Fcγ受体中FcγRI是高亲和力受体，而 FcγRIIA、FcγRIIB和FcγRIIIA是低亲和力受体。基因多态性也存在于这些不同的Fcγ受体中并影响它们的结合亲和力。最常见的基因多态性是FcγRIIA的R131/H131和FcγRIIIA的V158/F158等多态形式。这些多态形式中有的被发现与多种疾病有相关性，一些特定治疗抗体的效果也依赖于病人是否带有特定的Fcγ受体基因多态形式。
本发明所述“序列”应当理解为包括与本发明序列实质相同的序列，所述“实质相同的序列”一词指当经过最适比对后，如：采用GAP或BESTFIT程序，使用默认缺口值测定两种肽序列之间具有至少70、75或80％序列同一性，优选为至少90或95％序列同一性，更优选为至少97、98或99％序列同一性。优选的，不相同的残基位置的差异最好为保守性氨基酸取代。“保守性氨基酸取代”指其中氨基酸残基被另一种具有化学性质(例如：电荷或亲水性)类似的侧链R基的氨基酸残基取代。通常，保守性氨基酸取代实质上不会改变蛋白质的功能性质。若其中两种或多种氨基酸序列的差异在于保守性取代时，可使序列同一性百分比或相似度上调，以校正取代作用的保守性质。参见例如：Pearson，Methods Mol.Biol.243：307-31(1994)。具有相似化学性质的侧链的氨基酸基团实例包括：1)脂肪族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸、与异亮氨酸；2)脂肪族-羟基侧链：丝氨酸与苏氨酸；3)含酰胺侧链：天冬酰胺与谷氨酰胺；4)芳香族侧链：苯丙氨酸、酪氨酸、与色氨酸；5)碱性侧链：赖氨酸、精氨酸、与组氨酸；6)酸性侧链：天冬氨酸与谷氨酸；及7)含硫侧链：半胱氨酸与甲硫氨酸。优选保守性氨基酸取基组为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸、谷氨酸-天冬氨酸、及天冬酰胺-谷氨酰胺。正如上面所指出的，根据本发明的Fc区、抗体、Fc片段或者融合蛋白除了具备本发明所提供的氨基酸突变之外，也可以进一步包含本发明所未揭示的其它可能的修饰，只要满足上面所述的实质相同的序列要求。
本发明抗体及其片段或者结构域的氨基酸的序号是以IgG Eu编号为依据的。本发明中涉及的部分突变体对应的突变类型及其命名见表1。
抗体通常以高亲和力特异性结合其关联抗原(同样地，本发明的蛋白也可以特异性结合其表位识别分子)，这表现为10 -5-10 -11M或更小的解离常数(KD)。任何大于大约10 -4M -1的KD通常被认为指示非特异性结合。如本文所使用的，与抗原“特异性结合”的抗体是指以高亲和力与抗原和基本上相同的抗原结合的抗体，这意味着KD为10 -7M或更小，优选地10 -8M或更小，甚至更优选 地5×10 -9M或更小，最优选地10 -8-10 -10M或更小，但是不会以高亲和力与无关抗原结合。如果抗原与给定抗原显示高度的序列同一性，例如，如果它与给定抗原的序列显示至少80％，至少90％，优选至少95％，更优选至少97％，或甚至更优选至少99％的序列同一性，则该抗原与给定抗原是“基本上相同的”。
免疫球蛋白可能来自任何通常已知的同种型。IgG同种型在某些物种中可以被分细为亚类：人类中的IgG1，IgG2，IgG3和IgG4，小鼠中的IgG1,IgG2a,IgG2b和IgG3。“同种型”是指由重链恒定区基因编码的抗体类别(例如IgM或IgG1)。“抗体”包括例如天然存在的和非天然存在的抗体；单克隆和多克隆抗体；嵌合和人源化抗体；人或非人抗体；全合成抗体；和单链抗体。
本发明所述的“天然IgG”是指那些可以在自然界中天然存在的IgG，这些IgG的序列不是经过人工修饰的。需要说明的是，由于基因多态性的原因，即便是天然IgG也可以存在不同的突变体，不论是否突变，只要是自然状态下非经人工修饰的IgG都被称之为天然IgG。
“激动型抗体”是与受体结合并激活受体的抗体，激动型抗体的功能实例是与肿瘤坏死因子受体(TNFR)超家族中受体结合并诱导表达TNF受体的细胞凋亡。测定细胞凋亡诱导作用的试验描述在WO98/51793和WO99/37684中，该两篇文献特别在此引入作为参考。本发明具体的实施例中抗CD40的激动型抗体能够通过结合免疫细胞表面传递免疫激活信号的靶标分子并激活其控制的重要免疫激活信号通路，进而增强抗肿瘤免疫应答间接杀死肿瘤细胞。一些已经进入临床研究阶段的激动型抗体的实例可以参考专利PCT/CN2017/087620。
“激动活性”是指通过使抗体与抗原分子结合，激发该抗原分子产生信号，诱导某些特异性生理活性变化的活性。抗原分子包括受体分子和其它具有信号或生理功能的分子。上述特异性生理活性包括，例如：增殖活性、生存活性、分化活性、转录活性、膜转运活性、亲和力、蛋白分解活性、磷酸化/脱磷酸化活性、氧化还原活性、转移活性、核酸分解活性、脱水活性、细胞死亡诱导活性及凋亡诱导活性等，但不限于这些。
“亲本”指在蛋白质改造过程中，改造的对象。所述亲本可以是天然发生的多肽，或者是天然发生的多肽的变体或改造型式。在一些实施例中，本发明所述亲本Fc区为天然IgG的Fc区。
“变体”指在蛋白质改造过程中，亲本经过修饰后所获得的蛋白。具体的 可以是通过对亲本氨基酸的突变、缺失和/或添加而衍生出来的，且保留亲本中固有的一些或所有功能的蛋白质，本文变体的序列优选与亲本的序列拥有至少约80％的同源性，最优选至少约90％的同源性，更优选至少约95％的同源性。相应的是，本文所用“Fc变体”指由于至少一个氨基酸修饰而不同于亲本Fc序列的Fc序列。Fc变体可以仅包含Fc区，或者可以在抗体、Fc融合物、分离的Fc、Fc区、或基本上由Fc编码的其它多肽的背景中存在。Fc变体可以指Fc多肽本身、包含Fc变体多肽的组合物、或者编码它的氨基酸序列。
本文的″肿瘤坏死因子受体超家族″或″TNF受体超家族″，是指可与TNF家族的细胞因子结合的受体多肽。一般而言，这些受体是在其胞外区具有一个或多个半胱氨酸丰富的重复序列的I型跨膜受体。TNF基因家族中细胞因子的实例包括：肿瘤坏死因子-α(TNF-α)、肿瘤坏死因子-β(TNF-β或淋巴毒素)、CD30配体、CD27配体、CD40配体、OX-40配体、4-1BB配体、Apo-1配体(也称作Fas配体或CD95配体)、Apo-2配体(也称作TRAIL)、Apo-3配体(也称作TWEAK)、osteoprotegerin(OPG)，APRIL、RANK配体(也称作TRANCE)，和TALL-1(也称作BlyS、BAFF或THANK)。TNF受体超家族中受体的实例包括：1型肿瘤坏死因子受体(TNFR1)、2型肿瘤坏死因子受体(TNFR2)、p75神经生长因子受体(NGFR)、B细胞表面抗原CD40、T细胞抗原OX-40、Apo-1受体(也称作Fas或CD95)、Apo-3受体(也称作DR3、sw1-1、TRAMP和LARD)、称作″跨膜激活剂和CAML-相互作用子(interactor)″或″TACI″的受体、BCMA蛋白、DR4、DR5(或者，也称作Apo-2；TRAIL-R2，TR6，Tango-63，hAPO8，TRICK2或KILLER)、DR6、DcR1(也称作TRID、LIT或TRAIL-R3)、DcR2(也称作TRAIL-R4或TRUNDD)、OPG、DcR3(也称作TR6或M68)、CAR1、HVEM(也称作ATAR或TR2)、GITR、ZTNFR-5、NTR-1、TNFL1、CD30、淋巴毒素β受体(LTBr)、4-1BB受体和TR9(EP988,371 A1)。
本发明所述的“对抑制型Fcγ受体以及激活型Fcγ受体的亲和力比值”或者“I/A比值”等于抗体重链恒定区与抑制型Fcγ受体(例如：人FcγRIIB)的亲和力值除于抗体重链恒定区与同种属的激活型Fcγ受体(例如：包括人FcγRI、FcγRIIA、FcγRIIIA、FcγRIIIB)的最高亲和力值。
所述的“亲和力”是指两个分子之间的结合能力的大小，通常地可以用KD来衡量，在本发明中也可以用富集倍数(如表7)或者结合分析信号来衡量(如图12)。“KD”指两个分子(例如：特定抗体和抗原或者配体和受体)相互作用的 平衡解离常数。“富集倍数”指采用本发明所提供的流式细胞分选方法或者磁珠分选方法进行分选后，表达包含特定Fc突变的抗体的细胞在分选后的百分比/分选前的百分比(由二代测序分析，如表7所示)。“结合分析信号”采用本发明所提供的ELISA分析中的吸光值(如图12)。
“人”抗体是指这样的抗体，其可变区具有来自人种系免疫球蛋白序列的框架区和CDR区。而且，如果抗体含有恒定区，则恒定区也来自人种系免疫球蛋白序列。本发明的人抗体可以包括不是由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或定点突变或者通过体内体细胞突变引入的突变)。然而，如本文所使用的，术语“人抗体”，不意图包括来自其它哺乳动物物种(如小鼠)的种系的CDR序列被嫁接到人框架序列上的抗体。术语“人”抗体和“完全人”抗体被同义使用。
本发明“抗体”包括例如天然存在的和非天然存在的抗体；单克隆和多克隆抗体；嵌合和人源化抗体；人或非人抗体；全合成抗体。
“人源化”抗体是指其中非人抗体CDR结构域以外的一些、大部分或全部的氨基酸被来自人免疫球蛋白的相应氨基酸所替换的抗体。在人源化形式抗体的一个实施方案中，CDR结构域以外的一些、大部分或全部氨基酸被来自人免疫球蛋白的氨基酸代替，而一个或多个CDR区域内的一些、大部分或全部氨基酸都不变。允许对氨基酸进行小的添加、删除、插入、取代或修饰，只要它们不会消除抗体结合特定抗原的能力即可。“人源化”抗体保留与原始抗体相似的抗原特异性。
“嵌合抗体”是指可变区来自一个物种而恒定区来自另一个物种的抗体，例如可变区来自小鼠抗体而恒定区来自人抗体的抗体。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
表1 本发明中涉及的部分突变体对应的突变类型及其命名
Figure PCTCN2020133685-appb-000001
Figure PCTCN2020133685-appb-000002
材料与方法
哺乳动物表面展示载体构建
将人IgG2抗鼠CD40抗体轻链和重链全长序列通过多片段一步法快速克隆试剂盒(上海翊圣生物科技有限公司，中国)构建至逆转录病毒载体MIGRI(Addgene、Pear等，Blood.1998Nov 15.92(10):3780-92.)上，所用引物见表2，构建图谱如图1所示，限制性内切酶BglII单酶切后插入完整抗体的轻链DNA和重链DNA,中间以一段自剪切Linker P2A相连，其中重链DNA的末端包含一段跨膜结构域DNA(各片段的DNA序列如表3)，该质粒在表达抗体人IgG2的同时也会表达GFP蛋白，表面展示抗体及其相关Fc区的氨基酸序列如表4。测序确认构建成功。
表2.哺乳动物表面展示抗体质粒构建所用引物
Figure PCTCN2020133685-appb-000003
Figure PCTCN2020133685-appb-000004
表3.表面展示抗体各片段的DNA序列
Figure PCTCN2020133685-appb-000005
Figure PCTCN2020133685-appb-000006
表4.表面展示抗体及其相关Fc区的氨基酸序列
Figure PCTCN2020133685-appb-000007
Fc突变文库构建
点突变文库为通过简并引物引入突变，设计多条简并引物。例如，P233位点突变所用简并引物为CCACCGTGCCCAGCACCANNSGTGGCAGGACCGTCAGTC(SEQ ID NO:21)。分别将突变文库片段和所需连接载体通过PCR获得，一步法快速克隆试剂盒(上 海翊圣生物科技有限公司，中国)连接转化进入DH5α，获得突变文库。每个文库挑取20个菌落测序确认是否在目的位置引入突变。
组合突变文库的方法类似，引物为根据点突变文库已经筛选获得的具有富集的突变类型所设计，中间简并引物处改为编码特定氨基酸的密码子或者编码几个氨基酸的简并密码子组成的引物序列，例如：L328W(TGCAAGGTCTCCAACAAAGGCTGGCCAGCCCCCATCGAGAAAAC，SEQ ID NO:22)、Combine2_1(ACGTGCGTGGTGGTGGACKNGWBGVASRWSSACCHGGAGGTCCAGTTCAACTGG，SEQ ID NO:23)。
上述引物序列中除正常碱基外的符合为简并碱基：R代表A/G、Y代表C/T、M代表A/C、K代表G/T、S代表G/C、W代表A/T、H代表A/T/C、B代表G/T/C、V代表G/A/C、D代表G/A/T、N代表A/T/C/G。
稳转细胞系构建
将上述文库质粒和辅助质粒Phoenix-Eco(摩尔比2:1)与转染试剂PEI(质量比1:5)混合加入到提前铺板的Phoenix细胞(密度80％)中，6小时换成新鲜培养基，48小时后收集含有逆转录病毒的上清。用50％上述逆转录病毒上清加50％新鲜培养基感染提前铺板密度80％的3T3细胞，24小时后换液，48小时后即可获得表达抗体的稳转细胞系。
流式细胞仪分析表达抗体的稳转细胞系
胰酶消化收集3T3细胞，制备单细胞悬浮液，将约1-5×10 6个细胞重悬在含有PE-F(ab’)2Fragment Goat Anti-Human IgG(1:500,Jackson ImmunoResearch Laboratories)或者Biotin-FcγRIIB(1μg/ml,义翘神州)和Streptavidin-APC(1:500，BD Biosciences)的50μl FACS缓冲液(含有0.5％FBS和2mM EDTA的1xPBS)中，冰上孵育15分钟，然后FACS缓冲液洗涤细胞两次，重悬于200μl FACS缓冲液中，并通过流式细胞仪分析。
流式细胞分选与FcγRIIB高亲和力的细胞
按照上述流式细胞仪分析染色方法等比例放大染色体系，最终细胞重悬至约1-5×10 7个细胞/ml，上机，选取IgG表达(PE荧光信号强)，同时FcγRIIB高亲和力(APC荧光信号强)的细胞，比例控制在小于1％的IgG阳性细胞量。
磁珠分选与FcγRIIB亲和力高但与激活型FcγR的亲和力弱的细胞(I/A比值高)
胰酶消化收集3T3细胞，制备单细胞悬浮液，将约1×10 7个细胞重悬在含有Biotin-FcγRIIB(1μg/ml,义翘神州)和激活型FcγR(包括FcγRI、FcγRIIA 131H、FcγRIIA 131R、FcγRIIIA 158V、FcγRIIIA 158F，均1μg/ml，上海近岸科技有限公司)的50μl MACS缓冲液(含有0.5％BSA，2mM EDTA的1×PBS)中，在冰上孵育15分钟，然后MACS缓冲液洗涤细胞两次，重悬于100μl MACS缓冲液中，加入25μl抗Biotin微珠，在冰上孵育15分钟，MACS缓冲液洗涤细胞一次，重悬至500μl，加入到以放入磁场的LS柱中，收集流出液，3ml MACS缓冲液洗涤并收集，最后LS柱移出磁场，5ml MACS缓冲液快速将分选柱上滞留的细胞洗脱下来，收集阳性细胞，这些细胞即I/A比值高的细胞。
高通量测序分析
取目的细胞群约0.1-5×10 6个细胞，裂解，提取基因组DNA，PCR，自建库进行高通量测序(PE150或PE250)，每个样品获得约6-30万条序列，提取突变区域的核苷酸序列，将其批量翻译成对应的氨基酸序列(http://www.cbs.dtu.dk/services/VirtualRibosome/，Rasmus Wernersson.Nucl.Acids Res.2006 34:W385-W388)，计算每个样品中各突变序列所占比例：突变序列所占比例＝突变序列条数/总序列条数。
抗体表达纯化
将二代测序中的有利Fc突变体或新的组合突变体构建到抗人OX40抗体的重链中，和对应的抗体轻链一起瞬转进入HEK293S细胞表达蛋白，Protein G纯化获得不同的Fc突变抗体，SDS-PAGE检测抗体结构均完整的。抗人OX40抗体DNA序列和蛋白序列见表5和表6。
表5.野生型IgG2抗人OX40抗体DNA序列
Figure PCTCN2020133685-appb-000008
Figure PCTCN2020133685-appb-000009
表6.野生型IgG2抗人OX40抗体蛋白序列
Figure PCTCN2020133685-appb-000010
Figure PCTCN2020133685-appb-000011
酶联免疫吸附实验(ELISA)分析突变抗体与FcγR的结合特性
取2μg/mL，100μL的抗体或其突变体加入酶联板中包被过夜，弃上清，用含1％BSA的PBS封闭2小时，PBST(含0.05％吐温20的PBS)洗涤，然后加入适当浓度的Biotin标签的FcγR胞外结构域蛋白(北京义翘神州生物技术有限公司)室温孵育1小时后弃掉上清，PBST洗涤。加入Streptavidin-HRP(BD生物科技)室温孵育1小时检测Biotin蛋白，去除上清，加入显色液显色20-40分钟后测定650nm的吸光值(A650)。
不同抗体Fc突变抗人OX40抗体体外免疫激活活性分析(促进T细胞增殖)
分离人源化FCγR/OX40小鼠脾脏，制备裂解红细胞的单细胞悬液，进行CFSE标记，用含有5μM CFSE的PBS重悬细胞浓度为5×10 6/mL，37℃放置15分钟，含有5％FBS的PBS 600g，5min，洗2次，然后用原代细胞培养基(RPMI+10％FBS+1％Pen/Strep+1％HEPES+1％Sodium Pyruvate+0.1％2ME(final 50μM)+1％L-glutamine+1％Non-essential Aa)重悬细胞浓度为3×10 6/ml，每孔加入100μl细胞液，即每孔含有3×10 5个细胞。用含有0.2μg/ml抗CD3的原代细胞培养基稀释抗体浓度分别为2、0.2、0.02μg/ml。转移上述100μL配置好的抗体混合物到已经含有CFSE标记的人源化FCγR/OX40脾脏细胞孔中。其中有两组对照分别为CD3only组加CFSE标记的细胞和抗鼠CD3，但不加其它抗体；CD3+CD28组加CFSE标记的细胞和抗鼠CD3，并加入浓度为2μg/ml的抗鼠CD28抗体(Clone37.51(RUO)，BD制药)(抗鼠CD3和CD28终浓度分别为0.1μg/ml和1μg/ml)。在37℃，5％CO 2恒温细胞培养箱中孵育3天。培养结束后，收集细胞用流式检测CD4 +和CD8 +细胞数量和扩增情况。流式细胞术检测T细胞的增殖：将上述培养后的细胞转移至96孔U底板中，用PBS洗2遍，500g离心5分钟，甩去上清，将细胞重悬在含有PE anti-mouse CD4(Clone:GK1.5，1:500，BD)和APC anti-mouse CD8a(Clone:53-6.7，1:500，BioLegend)的50μl FACS缓冲液(含有0.5％FBS，2mM EDTA的PBS)中，冰上避光孵育15分 钟，然后PBS缓冲液洗涤细胞两次，重悬于含有DAPI(0.5μg/ml，Invitrogen)和CountBright Absolute Counting Beads(Life Technologies，2μl/sample)的200μl FACS缓冲液中，并通过流式细胞仪分析。
本发明主要提供了一些增强人IgG2与FcγRIIB相互作用的抗体Fc突变体序列。同时本发明也提供了筛选获得这些突变体的方法，如图2所示，根据上述方法可以筛选特定亲和性质的Fc突变体。
不仅如此，现有技术的人员根据本发明提供的上述筛选方法，可以筛选出增强各种蛋白-蛋白相互作用或者蛋白-其它分子相互作用的氨基酸突变；一般的筛选方法包括：
1)提供亲本蛋白序列，通过PCR在亲本蛋白序列中引入氨基酸突变。
2)将亲本蛋白以及突变后的蛋白构建入表达载体，形成突变文库。
3)将表达载体转染进入哺乳动物细胞，并将亲本蛋白以及突变后的蛋白表达于细胞表面。
4)将带标记的相互作用蛋白和上述哺乳动物细胞孵育，通过流式细胞分选或者磁珠分选出被标记的哺乳动物细胞。
5)提取被标记的哺乳动物细胞的DNA，进行测序，分析比较分选前后序列比例的变化情况；其中分选后的比例显著高于分选前的比例的氨基酸突变为能够增强所述蛋白和相互作用蛋白之间结合能力的氨基酸突变。
实施例1：人抗体IgG2_Fc点突变文库构建。
本发明选取了人IgG2_Fc_CH2上一些位点，包括P233-S239、V266-P271、S298-T299以及G327-I332(Eu编号，见图3和表4)，将这些位点上的氨基酸进行单点随机突变，每个位点上包括野生型共计有20种氨基酸类型(A、R、D、C、Q、E、H、I、G、N、L、K、M、F、P、S、T、W、Y、V)。制备分别对应上述人IgG2_Fc四个区域的单点随机突变的质粒文库(Library1、Library2、Library3、Library4)，以及这四个文库的混合文库(LibraryMix)。
实施例2：流式细胞分析分选前后的细胞富集情况。
上述质粒文库构建成功后，制备逆转录病毒感染3T3细胞，获得表达这些文库的稳转细胞系。本发明利用流式细胞仪分选技术分选表达人IgG2(由抗人 IgG F(ab’) 2检测)并结合人FcγRIIB的细胞。如图4所示，流式细胞分选可以逐步富集与FcγRIIB结合能力高的细胞。人IgG2文库LibraryMix经流式细胞分选仪分选不同轮次所得到的细胞分别进行分析，流式细胞仪可以检测到的与FcγRIIB结合的细胞比例在每一轮分选以后均有提高，第一轮为0.90％、第二轮为22.2％，相比较于野生型人IgG2的0.34％，分别提高到了2.65倍和65.29倍。
如图5所示，经过两轮分选以后，流式细胞仪上可以检测出Library1、Library2、Library3、Library4和LibraryMix中与FcγRIIB的结合的细胞比例均有所提高：分别达到1.85％、16.3％、0.57％、2.19％和22.2％，相比较于野生型人IgG2的0.34％比例提高到了5.44倍、47.94倍、1.69倍、6.44倍和65.29倍。
实施例3：流式细胞分选后二代测序分析与FcγRIIB结合能力提高的IgG2Fc突变类型。
本发明选取分选前后序列占比增加为野生型人IgG2两倍以上的突变(富集倍数/野生型富集倍数>2)，也就是人IgG2上对其与FcγRIIB结合能力提高有利(相对于野生型其富集倍数至少提高1倍)的突变类型，见表7。比如，L328W分选前的占比为0.39％,分选后的占比为12.84％，富集倍数为33.20，是野生型的富集倍数(1.49)的22.21倍。
表7.人IgG2上对其与FcγRIIB结合能力提高有利的突变体
Figure PCTCN2020133685-appb-000012
Figure PCTCN2020133685-appb-000013
Figure PCTCN2020133685-appb-000014
#1-3：该数据来自于Library2(#1)、Library4(#2)、LibraryMix(#3)的实验。
除了点突变之外，本发明同时也筛选获得了一些双突变类型，这些氨基酸突变在流式细胞分选前后比例增加至野生型人IgG2两倍以上。其中，双突变组合(V266A/P271C或G、S267A/P271C或G、S267E/D270H、S267E/P271C或V或W或Y、S267E/S298R、S267M/P271C或G、P271A/S298R、P271G/G236V、P271G/G329S、P271G/P331C或T、P271G/S298D或E或G或K或L或N或R、P271G/T299A或M或S或W、G327A/A330R或V、G327A/I332A或C或E、L328E/I332T等)的提高比例要高于其中任何一个点突变的提高比例，说明该突变组合中两个氨基酸对FcγRIIB的结合能力的提高均有贡献。而另外也出现突变组合如S267V/S298L，其组合突变提高的倍数约等于其中提高倍数较高的S298L突变，说明该突变组合的提高只是这一个位点突变的贡献，另一个点突变基本没有影响，见表8。由此，本发明的Fc区、抗体、Fc片段或者融合蛋白除了具备本发明所提供的氨基酸突变之外，也可以进一步包含其它可能的组合或修饰。
表8.人IgG2上对其与FcγRIIB结合能力提高有利的双突变类型
Figure PCTCN2020133685-appb-000015
Figure PCTCN2020133685-appb-000016
a.分选后突变体的富集倍数/野生型的富集倍数的比值
b.原点突变分选后突变体的富集倍数/野生型的富集倍数的比值
c.组合突变提高倍数/其中一个点突变提高倍数的比值
实施例4：磁珠分选后二代测序分析有利突变体类型。
本发明利用磁珠分选技术对上述流式分选获得的FcγRIIB高亲和力细胞继续分选，使用Biotin标记的FcγRIIB和没有标记的激活型FcγR混合与细胞孵育，然后利用抗Biotin-磁珠分选被标记的细胞，二代测序分析前后的富集比例，可以筛选到I/A比值较高的突变，见表9。挑选突变的方法为：选取分选前后比例增加至野生型人IgG2两倍以上，同时在分选未结合磁珠部分(即过LS柱时流出液)中比例 减少的突变。
表9.人IgG2上对其与FcγRIIB结合能力提高同时与激活型FcγRs的结合能力弱于FcγRIIB的突变类型
Figure PCTCN2020133685-appb-000017
Figure PCTCN2020133685-appb-000018
NA在该列样品中该行对应突变类型未检测出。
#1-#4：该数据来自于Library2(#1)、Library4(#2)、LibraryMix的两次实验(#3、#4)。
G236D、S239D(根据Seung Y.Chu等人(Molecular Immunology 45(2008)3926–3933)报道)和S298A突变(根据专利US20090042291A1报道)可以提高人IgG1与FcγRIIB的亲和力。但是，在本发明的二代测序分析结果显示，包含G236D或S298A突变的IgG2变体在分选后的占比没有增加，反而有一定降低(表10)；包含S239D突变的人IgG2变体在分选前的占比为0.026％，而在分选后测序结果中未测出。这些结果说明G236D、S239D和S298A突变不提高人IgG2的FcγRIIB结合能力，见表10。
表10.不能根据已知突变对IgG1的FcγR结合能力的影响直接预测其对IgG2的FcγR结合能力的影响
Figure PCTCN2020133685-appb-000019
NA是指该行突变体类型在该列实验中未检测出，百分比和倍数无法计算。
实施例5：人IgG2 Fc多肽片段中可增强FcγRIIB亲和力的氨基酸突变。
表11总结了根据本发明点突变文库筛选方法所筛选出来的可增强FcγRIIB亲和力的氨基酸突变。与先前专利比较发现：本发明中S267G、P271C这些位点均为人IgG2上新的可以提高FcγRIIB结合能力的突变类型。H268S、L328A、G327A这些位点均为人IgG2上新的可以提高FcγRIIB结合能力同时与其它激活型FcγRs的亲和力弱于FcγRIIB的突变类型。
表11.人IgG2中可增强FcγRIIB亲和力的氨基酸突变
Figure PCTCN2020133685-appb-000020
实施例6：人抗体IgG2_Fc组合突变文库的构建
根据前述点突变文库和高通量测序结果筛选出的能够提高FcγRIIB亲和力的Fc突变类型(富集倍数相比较野生型提高1倍以上的突变)，设计两种类型的组合突变文库：类型一，四个区域之间单点组合突变，即P233-V240、V266-P271、S298-T299以及G327-I332四个区域每个区域有0-1个突变，共0-4个位点的组合突变；类型二，一个区域内的多个氨基酸组合突变，即P233-V240、V266-P271、S298-T299或G327-I332四个区域，每个区域内部氨基酸的组合突变(分别为0-7、0-6、0-2和0-6个组合突变)，如图6所示。质粒文库的大小最大至4.67×10 5。
实施例7：组合突变文库流式细胞分选前后的细胞富集情况
根据点突变筛选高通量测序结果，构建类型一和类型二组合突变文库(实施例6)，获得表达组合突变的细胞文库，然后流式细胞分选FcγRIIB高亲和力的细胞，连续分选两至三轮。如图7所示，经过两轮分选以后，流式细胞仪上可以检测出文库IgG2_C01、文库IgG2_C02与FcγRIIB结合的细胞比例均有所提高：分别达到9.53％和26.9％，相比较于野生型人IgG2的0.23％比例提高到了41.43倍和116.96倍。其中IgG2_C01为P233-V240、V266-P271、S298-T299以及G327-I332四个区域各有最多1个突变的组合突变(类型一)文库；IgG2_C02为P233-V240、V266-P271、S298-T299和G327-I332四个区域内连续的组合突变(类型二)文库合并共同转入3T3细胞制成的细胞文库。
如图8所示，经过两轮分选以后，流式细胞仪上可以检测出文库Cmix和文库C2细胞与FcγRIIB的结合能力明显提高，每一轮分选之后其与FcγRIIB结合的细胞比例均显著增多：Cmix在第一轮分选和第二轮分选之后与FcγRIIB 结合的细胞比例分别达到1.86％和28.9％，相比较于分选前0.00756％，分别提高到了246.03倍和3822.75倍；Cmix分别达到6.46％和28.9％，相比较于分选前0.00899％，分别提高到了718.58倍和3214.68倍。文库Cmix为四个区域各有最多1个突变的组合突变(类型一)文库；文库C2为V266-P271区域的组合突变(类型二)文库。
实施例8：流式细胞分选后二代测序分析与FcγRI IB结合能力提高的IgG2Fc组合突变类型。
为了筛选与FcγRIIB结合能力提高的IgG2 Fc组合突变类型，将本发明中构建的组合突变文库进行流式分选，获得与FcγRIIB的结合能力较高的细胞，然后使用二代测序分析分选前后各突变类型的富集倍数，结果见表12。所选取的突变均有较高倍数的富集(最后一轮分选后/分选前)，或者在至少一轮分选中有明显富集(该轮分选后/该轮分选前)，或者仅在最后一轮分选后被测出。例如，V266L/S298L/L328W的富集倍数为野生型的21920.33倍(在最后一轮分选后/分选前)；点突变L328W的富集倍数为野生型的8.78倍(在最后一轮分选后/分选前)；H268D/S298L/L328W的富集倍数分别为43.98(第二轮分选后/第二轮分选前)和5.95(第三轮分选后/第三轮分选前)；V234M/S267E/S298L/L328W的富集倍数为5.13(第三轮分选后/第三轮分选前)；V234Q/A235G/P238L/S239V/G327A/L328E/A330S/I332T仅在最后一轮分选后被测出。
表12.组合突变文库中筛选出人IgG2上对其与FcγRIIB结合能力提高有利的突变体
Figure PCTCN2020133685-appb-000021
Figure PCTCN2020133685-appb-000022
#1-4：该数据来自于文库IgG2_C01(#1)、IgG2_C02(#2)、Cmix(#3)、C2(#4)的实验。NA在该列样品中该行对应突变类型未被测出。
实施例9：酶联免疫吸附实验(ELISA)分析突变抗体与FcγR的结合特性
在二代测序筛选到的有利Fc单点突变和突变组合的基础上，构建新的Fc突变组合，然后表达含有这些新的Fc突变组合的抗人OX40抗体，并使用酶联 免疫吸附实验(ELISA)分析突变抗体与FcγR的结合特性。如图9-14所示，不同的Fc突变体与FcγRI、FcγRIIA 131H、FcγRIIA 131R、FcγRIIB、FcγRIIIA 158F和FcγRIIIA 158V的结合能力。设定IgG1的与各个FcγR的结合能力为1，计算各突变体与这些FcγR的相对结合能力(该变体与该FcγR结合的ELISA吸光值除以IgG1与该FcγR结合的ELISA吸光值)，以及各突变体相对于野生型IgG2的FcγRIIB结合能力的提高倍数(该变体与FcγRIIB结合的ELISA吸光值除以野生型IgG2与FcγRIIB结合的ELISA吸光值)。如表13所示，各突变体相对于野生型IgG2的FcγRIIB结合能力的提高倍数为4.23至25.55倍。此外，计算各突变体的FcγRIIB和FcγRIIA 131R结合能力比值(RIIB/RIIAR)，结果(表13)显示这些突变体的RIIB/RIIAR比值均高于野生型IgG2(野生型IgG2：0.34；突变体：0.46-3.67)。
表13.不同IgG2抗体Fc突变体与FcγR的结合情况。
Figure PCTCN2020133685-appb-000023
Figure PCTCN2020133685-appb-000024
注：表格中RI、RIIAH、RIIAR、RIIB、RIIIAF、RIIIAV分别代表FcγRI、FcγRIIA 131H、FcγRIIA 131R、FcγRIIB、FcγRIIIA 158F、FcγRIIIA 158V。
实施例10：有利突变支持更强的免疫激活活性
分析上述包含有利突变的抗人OX40抗体(实施例9)在FCγR/OX40人源化小鼠脾脏细胞中的免疫激活活性，图16、17显示了抗体浓度为1μg/ml时，CD4 +细胞的CFSE荧光强度和对应直方图，数值越低活性越强，所选抗体的活性均优于野生型IgG2。将两次实验的不同抗体浓度的活性数值一起分析，设定只加入αCD3抗体的活性为0，加入αCD3抗体和αCD28抗体阳性对照活性为1，分别计算各突变抗体的相对活性，并计算平均值。表14显示，所选取的30个突变抗体的活性均优于野生型IgG2，活性提高为野生型的9.17至16.51倍。
表14 包含不同Fc突变的抗人OX40抗体的免疫激活活性分析
Figure PCTCN2020133685-appb-000025
Figure PCTCN2020133685-appb-000026
NA此行突变抗体未进行该列实验。
实施例11：不能根据已知突变对IgG1的FcγR结合能力的影响直接预测其对IgG2的FcγR结合能力的影响
G236D、S239D(根据Seung Y.Chu等人(Molecular Immunology 45(2008)3926–3933)报道)和S298A突变(根据专利US20090042291A1报道)可以提高人IgG1与FcγRIIB的亲和力。但是，在本发明的二代测序分析结果显示，包含G236D或S298A突变的IgG2变体在分选后的占比没有增加，反而有一定降低(表10)；包含S239D突变的人IgG2变体在分选前的占比为0.026％，而在分选后测序结果中未测出。这些结果说明G236D、S239D和S298A突变不提高人IgG2的FcγRIIB结合能力，见表10。
此外，我们还通过在IgG2中引入能够显著提高IgG1的FcγRIIB结合能力的V11突变位点(G237D/P238D/H268D/P271G/A330R)(F.Mimoto等人，Protein Engineering,Design & Selection vol.26no.10pp.589–598,2013)，获得了IgG2_M2(H268D/P271G)、IgG2_M3(H268D/P271G/A330R)、IgG2_M4(G236D/P238D/H268D/P271G)和IgG2_M5(G236D/P238D/H268D/P271G/A330R)，其中M5在IgG2中的突变对应IgG1 V11的突变。图15ELISA结果显示，这些突变体并没有使得IgG2获得和IgG1 V11一样的FcγRIIB结合能力。
因此，不能根据已知突变对IgG1的FcγR结合能力的影响直接预测其对IgG2的FcγR结合能力的影响。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种突变型Fc多肽片段，其特征在于，所述的突变型Fc多肽片段具有以下特征：
    (i)所述的突变型Fc多肽片段相对于突变前相应的野生型Fc片段具有突变；并且
    (ii)与野生型Fc区相比，所述突变型Fc区与FcγRIIB的亲和力提高；并且，所述的野生型Fc为野生型IgG2的Fc。
  2. 一种突变型免疫球蛋白Fc区，其特征在于，所述突变型免疫球蛋白Fc区包含如权利要求1所述的突变型Fc多肽片段。
  3. 一种抗体，其特征在于，所述抗体中包含如权利要求1所述的突变型Fc多肽片段或如权利要求2所述的突变型免疫球蛋白Fc区。
  4. 一种融合蛋白，其特征在于，所述融合蛋白包含如权利要求1所述的突变型Fc多肽片段、如权利要求2所述的突变型免疫球蛋白Fc区，或如权利要求3所述的抗体。
  5. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1所述的突变型Fc多肽片段、如权利要求2所述的突变型免疫球蛋白Fc区、如权利要求3所述的抗体，或如权利要求4所述的重组蛋白。
  6. 一种载体，其特征在于，所述载体含有如权利要求5所述的分离的多核苷酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求6所述的载体，或其基因组中整合有如权利要求5所述的多核苷酸；
    或者，所述宿主细胞表达如权利要求1所述的突变型Fc多肽片段、如权利要求2所述的突变型免疫球蛋白Fc区、如权利要求3所述的抗体，或如权利要求4所述的重组蛋白。
  8. 一种药物组合物，其特征在于，所述的药物组合物包括：
    (a)如权利要求1所述的突变型Fc多肽片段、如权利要求2所述的突变型免疫球蛋白Fc区、如权利要求3所述的抗体，或如权利要求4所述的重组蛋白；和
    (b)药学上可接受的载体。
  9. 一种产生如权利要求1所述的突变型Fc多肽片段、如权利要求2所述的突变型免疫球蛋白Fc区、如权利要求3所述的抗体，或如权利要求4所述的重组 蛋白的方法，其特征在于，包括步骤：
    (i)在合适的条件下，培养如权利要求7所述的宿主细胞，从而获得含所述突变型Fc多肽片段、所述突变型免疫球蛋白Fc区、所述抗体，或所述重组蛋白的培养物；和
    (ii)对步骤(i)中得到的培养物进行纯化和/或分离，获得所述的所述突变型Fc多肽片段、突变型免疫球蛋白Fc区、抗体，或重组蛋白。
  10. 如权利要求1所述的突变型Fc多肽片段、如权利要求2所述的突变型免疫球蛋白Fc区、如权利要求3所述的抗体，或如权利要求4所述的重组蛋白、如权利要求5所述的多核苷酸、如权利要求6所述的载体和/或如权利要求7所述的宿主细胞的用途，其特征在于，用于制备一用于肿瘤免疫治疗、减轻炎症和/或减轻自身免疫症状的药物组合物。
PCT/CN2020/133685 2019-12-03 2020-12-03 FcγRIIB亲和力增强的抗体Fc区 WO2021110110A1 (zh)

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MX2022006670A MX2022006670A (es) 2019-12-03 2020-12-03 Region fc de anticuerpo teniendo mayor afinidad de union al fcyriib.
CA3160574A CA3160574A1 (en) 2019-12-03 2020-12-03 Antibody fc region having enhanced binding affinty to fcyriib
EP20897306.5A EP4071167A4 (en) 2019-12-03 2020-12-03 FC REGION OF ANTIBODIES WITH ENHANCED AFFINITY TO FC?RIIB
US17/756,836 US20230220042A1 (en) 2019-12-03 2020-12-03 Antibody fc region having enhanced binding affinity to fcyriib
AU2020396825A AU2020396825A1 (en) 2019-12-03 2020-12-03 Antibody Fc region having enhanced FCγRIIB affinity
KR1020227022711A KR20220121822A (ko) 2019-12-03 2020-12-03 FcγRIIB 친화력이 강화된 항체 Fc 영역
CN202080084609.5A CN115052888A (zh) 2019-12-03 2020-12-03 FcγRIIB亲和力增强的抗体Fc区
JP2022533391A JP2023507922A (ja) 2019-12-03 2020-12-03 ＦｃγＲＩＩＢ親和性が増強された抗体Ｆｃ領域
BR112022010757A BR112022010757A2 (pt) 2019-12-03 2020-12-03 Região de anticorpo fc com afinidade de ligação aumentada para fc-gama-riib.

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