WO2021110110A1 - FcγRIIB亲和力增强的抗体Fc区 - Google Patents
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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
Description
Claims (10)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
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JP2022533391A JP2023507922A (ja) | 2019-12-03 | 2020-12-03 | FcγRIIB親和性が増強された抗体Fc領域 |
US17/756,836 US20230220042A1 (en) | 2019-12-03 | 2020-12-03 | Antibody fc region having enhanced binding affinity to fcyriib |
MX2022006670A MX2022006670A (es) | 2019-12-03 | 2020-12-03 | Region fc de anticuerpo teniendo mayor afinidad de union al fcyriib. |
CN202080084609.5A CN115052888A (zh) | 2019-12-03 | 2020-12-03 | FcγRIIB亲和力增强的抗体Fc区 |
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CA3160574A CA3160574A1 (en) | 2019-12-03 | 2020-12-03 | Antibody fc region having enhanced binding affinty to fcyriib |
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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US12030945B2 (en) | 2022-10-25 | 2024-07-09 | Seismic Therapeutic, Inc. | Variant IgG Fc polypeptides and uses thereof |
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