WO2023169524A1 - Mutant d'interleukine 2 et complexe le contenant - Google Patents

Mutant d'interleukine 2 et complexe le contenant Download PDF

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WO2023169524A1
WO2023169524A1 PCT/CN2023/080577 CN2023080577W WO2023169524A1 WO 2023169524 A1 WO2023169524 A1 WO 2023169524A1 CN 2023080577 W CN2023080577 W CN 2023080577W WO 2023169524 A1 WO2023169524 A1 WO 2023169524A1
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antigen
interleukin
antibody
amino acid
mutant
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PCT/CN2023/080577
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English (en)
Chinese (zh)
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张喆
耿梦圆
芦迪
霍永庭
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广东菲鹏制药股份有限公司
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Priority claimed from CN202210423871.9A external-priority patent/CN116769013A/zh
Application filed by 广东菲鹏制药股份有限公司 filed Critical 广东菲鹏制药股份有限公司
Publication of WO2023169524A1 publication Critical patent/WO2023169524A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

Definitions

  • the present invention relates to the technical field of molecular biology, specifically to interleukin 2 mutants and complexes containing them.
  • Cytokines play an important role in human immune regulation. They also participate in the immune regulation of tumors and are closely related to the occurrence and development of tumors. In immunotherapy, cytokines can directly act on immune effector cells in the tumor microenvironment to enhance the tumor suppressive effect. Through clinical studies and animal experiments, many cytokines have been proven to have significant anti-tumor activity, and several cytokines have been approved by the FDA.
  • Interleukin 2 is a cytokine of about 15.5-16kDa. It was first discovered by Morgan et al. in 1976 and has the effect of enhancing immunity. IL2 has many immunostimulatory and immunoregulatory functions. It can stimulate the proliferation and enhance the function of T lymphocytes, natural killer cells (NK) and B cells.
  • IL2 is one of the four ⁇ -helix bundle family of cytokines. IL2 needs to bind to its receptor to exert biological activity.
  • the IL2 receptor consists of three receptor subunits: IL2 receptor ⁇ (IL2R ⁇ , also known as CD25, 55kDa), IL2 receptor ⁇ (IL2R ⁇ , also known as CD122) and IL2 receptor gamma (IL2R ⁇ , also known as CD132).
  • IL2 receptor ⁇ is a single-channel type I membrane protein that contains two Sushi domains, which are necessary domains for IL2 to bind to it and exert biological functions.
  • IL2 has not been widely used due to toxic side effects caused by high-dose administration.
  • IL2 and its receptor subtypes due to in-depth research on IL2 and its receptor subtypes, it has been found that the selective immune stimulating ability of modified IL2 has been improved, which makes IL2 susceptible to The body was re-investigated and used to construct fusion proteins.
  • the types of fusion proteins related to IL2 and IL2 receptors need to be enriched and their stability needs to be improved.
  • one object of the present invention is to provide an interleukin 2 mutant, a conjugate containing the mutant, a complex containing the mutant, and a method for preparing the mutant, conjugate or complex. Use in compositions to stimulate the body's immune system and preparation of drugs for the treatment of cancer and autoimmune diseases.
  • a first aspect of the present invention provides an interleukin-2 mutant.
  • the amino acid at position 75 in the amino acid sequence of the interleukin 2 mutant is mutated to cysteine.
  • amino acid sequence of the wild-type interleukin 2 is shown in SEQ ID NO: 1.
  • amino acid sequence of wild-type interleukin 2 (SEQ ID NO: 1) is as follows:
  • amino acid sequence of the interleukin 2 mutant (SEQ ID NO: 25) is as follows:
  • a second aspect of the invention provides a conjugate.
  • the conjugate includes the interleukin 2 mutant of the first aspect.
  • the conjugate further comprises a first antigen binding moiety.
  • the first antigen-binding module is connected to one end of the interleukin-2 mutant directly or indirectly through a linker.
  • the first antigen-binding module is a variable region of a first antibody or an antigen-binding fragment thereof.
  • the first antibody or antigen-binding fragment thereof targets a first antigen on the surface of tumor cells or immune cells.
  • the immune cells include T cells, NK cells, B cells, monocytes, and macrophages.
  • the conjugate provided by the invention contains an interleukin 2 mutant and an antibody or an antigen-binding fragment thereof, and binds to immune cells through the antibody or an antigen-binding fragment thereof, thereby stimulating immune cells, such as stimulating T cell proliferation, to achieve the effect of enhancing immunity. , can be used to treat some autoimmune diseases.
  • the first antigen is selected from any one or more of TIGIT, CD47, PDL1, PD-1, GUCY2C or BCMA.
  • a third aspect of the invention provides a composite.
  • the complex includes the interleukin 2 mutant of the first aspect or the conjugate of the second aspect.
  • the complex further includes an interleukin 2 receptor alpha mutant, the interleukin 2 receptor alpha mutant is connected to the interleukin 2 mutant through a disulfide bond.
  • an interleukin 2 mutant is obtained by mutating serine at position 75 of wild-type interleukin 2 to cysteine, and the mutated cysteine at this position is similar to that found in leukocyte interleukin 2.
  • a disulfide bond is formed between the cysteines extending from the N-terminus of the ⁇ mutant of the receptor 2.
  • the N-terminus of the amino acid sequence of the interleukin 2 receptor alpha mutant is extended by two or three amino acids compared to the amino acid sequence of the wild-type interleukin 2 receptor alpha.
  • the disulfide bond is formed between the extended 2nd amino acid of the interleukin 2 receptor alpha mutant and the 75th amino acid of the interleukin 2 mutant; according to In an embodiment of the present invention, the second amino acid is the second amino acid extending from the N-terminus of wild-type interleukin 2 receptor ⁇ in an extending direction.
  • the amino acid sequence of the wild-type interleukin 2 receptor ⁇ is as shown in SEQ ID NO: 2 or SEQ ID NO: 3.
  • amino acid sequence of SEQ ID NO:2 is as follows:
  • amino acid sequence of SEQ ID NO:3 is as follows:
  • SEQ ID NO:2 is the amino acid sequence of the mature fully truncated form of the wild-type interleukin 2 receptor ⁇ extracellular domain
  • SEQ ID NO:3 is the mature fully truncated form of the wild-type interleukin 2 receptor ⁇ extracellular domain. form of the amino acid sequence.
  • amino acid sequence of an exemplary wild-type interleukin 2 receptor alpha mutant is shown in SEQ ID NO: 26.
  • amino acid sequence of SEQ ID NO:26 is as follows:
  • the complex further comprises a second antigen binding moiety.
  • the second antigen-binding module is connected to one end of the interleukin 2 receptor alpha mutant directly or indirectly through a linker.
  • the second antigen-binding module when the second antigen-binding module is directly connected to the N-terminus of the interleukin 2 receptor alpha mutant, the N terminus of the amino acid sequence of the interleukin 2 receptor alpha mutant Two amino acids are extended at the end, among which, the extended amino acid at position 2
  • the acid is cysteine, and the extended first amino acid is any one of non-polar fatty acid amino acids, aromatic amino acids, amino acids with uncharged R base, positively charged amino acids with R base, or negatively charged amino acids with R base. .
  • the amino acid of the interleukin 2 receptor alpha mutant extends by three amino acids.
  • the extended second amino acid is cysteine
  • the extended first and third amino acids are non-polar fatty acid amino acids, aromatic amino acids
  • the R group is uncharged. Any of amino acids, amino acids with a positive charge on the R group, or amino acids with a negative charge on the R group.
  • the linker can be a Linker, and the Linker is a flexible Linker.
  • the length and specific amino acid sequence of the flexible Linker are not limited. All flexible Linkers commonly used in the art can be used in the present invention.
  • amino acid sequence of the flexible Linker is shown in SEQ ID NO: 29.
  • amino acid sequence of SEQ ID NO:29 is as follows:
  • the non-polar fatty acid amino acid may be glycine, alanine, valine, leucine, isoleucine, methionine, etc., but the non-polar fatty acid mentioned in the present invention
  • the types of fatty acid and amino acids are not limited to these.
  • the aromatic amino acid may be phenylalanine, tryptophan, tyrosine, etc., but the types of aromatic amino acids mentioned in the present invention are not limited thereto.
  • the uncharged amino acid of the R group may be serine, threonine, cysteine, proline, aspartic acid, glutamine, etc., but the R mentioned in the present invention
  • the type of amino acid having an uncharged base is not limited to these.
  • the positively charged amino acid with the R base may be lysine, arginine, histidine, etc., but the types of positively charged amino acids with the R base mentioned in the present invention are not limited thereto.
  • the negatively charged amino acid with R base may be aspartic acid, glutamic acid, etc., but the types of negatively charged amino acids with R base mentioned in the present invention are not limited thereto.
  • the second antigen-binding moiety is a variable region of the first antibody or an antigen-binding fragment thereof.
  • the first antibody or antigen-binding fragment thereof targets a first antigen on the surface of tumor cells or immune cells.
  • the immune cells include T cells, NK cells, B cells, monocytes, and macrophages.
  • the first antigen is selected from any one or more of TIGIT, CD47, PDL1, PD-1, GUCY2C or BCMA.
  • the first antigen-binding module is selected from the light chain variable region of the first antibody or its antigen-binding fragment
  • the second antigen-binding module is selected from the group consisting of the first antibody or its antigen.
  • the heavy chain variable region of the binding fragment; or the first antigen-binding module is selected from the heavy chain variable region of the first antibody or its antigen-binding fragment
  • the second antigen-binding module is selected from the first antibody or the light chain variable region of an antigen-binding fragment thereof.
  • the first antigen-binding module is the light chain variable region (VL) of the TIGIT antibody
  • the second antigen-binding module is the heavy chain variable region (VH) of the TIGIT antibody.
  • the first antigen-binding module is the heavy chain variable region (VH) of the TIGIT antibody
  • the second antigen-binding module is the light chain variable region (VL) of the TIGIT antibody.
  • TIGIT antibodies include, but are not limited to: TIGIT antibodies, antibody tiragolumab, antibody Vibostolimab, antibody ociperlimab, etc. in the Chinese patent with application number 202211161371.9.
  • the first antigen-binding module is the light chain variable region (VL) of the CD47 antibody
  • the second antigen-binding module is the heavy chain variable region (VH) of the CD47 antibody.
  • the first antigen-binding module is the heavy chain variable region (VH) of the CD47 antibody
  • the second antigen-binding module is the light chain variable region (VL) of the CD47 antibody.
  • CD47 antibodies include, but are not limited to: CD47 antibodies in the CD47 antibodies in the Chinese patent with announcement number CN113004406B, antibodies magrolimab, CC9002, IBI188, lemzoparlimab, AK117, etc.
  • the first antigen-binding module is the light chain variable region (VL) of the PDL1 antibody
  • the second antigen-binding module is the heavy chain variable region (VH) of the PDL1 antibody.
  • the first antigen-binding module is the heavy chain variable region (VH) of the PDL1 antibody
  • the second antigen-binding module is the light chain variable region (VL) of the PDL1 antibody.
  • Exemplary PDL1 antibodies include, but are not limited to: PDL1 antibodies, antibody cemiplimab, antibody balstilima, antibody islelizumab, antibody pucotenlimab, etc. in the Chinese patent with application number 202111580114.4.
  • the first antigen-binding module is the light chain variable region (VL) of the PD-1 antibody
  • the second antigen-binding module is the heavy chain variable region (VL) of the PD-1 antibody ( VH).
  • the first antigen-binding module is the heavy chain variable region (VH) of the PD-1 antibody
  • the second antigen-binding module is the light chain variable region of the PD-1 antibody.
  • Area(VL) is the heavy chain variable region of the PD-1 antibody
  • Exemplary PD1 antibodies include, but are not limited to: PD1 antibodies, antibody pembrolizumab, antibody Nivolumab, antibody Atezolizumab, antibody avelumab, antibody Durvalumab, etc. in the international patent with announcement number WO2021121373A1.
  • the first antigen-binding module is the light chain variable region (VL) of the GUCY2C antibody
  • the second antigen-binding module is the heavy chain variable region (VH) of the GUCY2C antibody.
  • the first antigen-binding module is the heavy chain variable region (VH) of the GUCY2C antibody
  • the second antigen-binding module is the light chain variable region (VL) of the GUCY2C antibody.
  • Exemplary GUCY2C antibodies include, but are not limited to: the GUCY2C antibody in Chinese patent application number 202310004900.2, the GUCY2C antibody in patent WO2019224716A8, the antibody Indusatumab, etc.
  • the first antigen-binding module is the light chain variable region (VL) of the BCMA antibody
  • the second antigen-binding module is the heavy chain variable region (VH) of the BCMA antibody.
  • the first antigen-binding module is the heavy chain variable region (VH) of the BCMA antibody
  • the second antigen-binding module is the light chain variable region (VL) of the BCMA antibody.
  • BCMA antibodies include, but are not limited to: BCMA antibodies in Chinese patent application number 202011484592.0, antibody Belantamab, antibody MEDI2228, etc.
  • the first antigen-binding module is connected to the N-terminus of the interleukin-2 mutant directly or indirectly through a linker.
  • the second antigen-binding module is connected to the N-terminus of the interleukin 2 receptor alpha mutant directly or indirectly through a linker.
  • the complex further includes an Fc region connected to the other end of the interleukin 2 mutant or the interleukin 2 receptor alpha mutant through a hinge region.
  • the complex further includes an Fc region connected to the C-terminus of the interleukin 2 mutant or the interleukin 2 receptor alpha mutant through a hinge region.
  • the complex is a bis-antibody molecule, and the bis-antibody molecule further includes a second antibody or an antigen-binding fragment thereof, and the second antibody or an antigen-binding fragment thereof targets the surface of immune cells or tumor cells. the second antigen.
  • the second antibody and the first antibody of the diabody molecule specifically bind to different antigens.
  • the second antigen on the surface of immune cells or tumor cells includes any one or more of PDL1, CD47, PD-1, TIGIT, BCMA or GUCY2C.
  • the exemplary PDL1 antibody, CD47 antibody, PD-1 antibody, TIGIT antibody, BCMA antibody or GUCY2C antibody targeting the second antigen may be combined with the above-mentioned exemplary PDL1 antibody, CD47 antibody targeting the first antigen.
  • Antibodies, PD-1 antibodies, TIGIT antibodies, BCMA antibodies, or GUCY2C antibodies are the same.
  • the immune cells include T cells, NK cells, B cells, monocytes, and macrophages.
  • the bisantibody molecule includes a first peptide chain, a second peptide chain, a third peptide chain and a fourth peptide chain.
  • the first peptide chain is: a first antigen module-interleukin 2 mutant; the second peptide chain is: a second antigen module-interleukin 2 receptor alpha mutant- Hinge region-Fc region; the third peptide chain is: the heavy chain variable region of the second antibody-CH1-hinge region-Fc region; the fourth peptide chain is: the light chain variable region of the second antibody- C.L.
  • the first peptide chain is: first antigen module-interleukin 2 receptor alpha mutant; and the second peptide chain is: second antigen module-interleukin 2 mutant- Hinge region-Fc region; the third peptide chain is: the heavy chain variable region of the second antibody-CH1-hinge region-Fc region; the fourth peptide chain is: the light chain variable region of the second antibody- C.L.
  • amino acid sequence of the hinge region is shown in SEQ ID NO: 23.
  • the Fc region is an immunoglobulin Fc region, and the immunoglobulin Fc region is a natural amino acid sequence or a mutant sequence thereof.
  • the immunoglobulin Fc region is of human or animal origin.
  • amino acid sequence of the Fc region is shown in SEQ ID NO: 24.
  • the CL is the constant region of an immunoglobulin lambda light chain or kappa light chain.
  • the second antigen on the surface of immune cells or tumor cells is PDL1.
  • the amino acid sequence of the heavy chain variable region (VH) of the second antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the heavy chain variable region (VL) of the second antibody is As shown in SEQ ID NO:28.
  • a fourth aspect of the invention provides a nucleic acid molecule.
  • the nucleic acid molecule includes a nucleotide sequence encoding the interleukin 2 mutant of the first aspect, the conjugate of the second aspect, or the complex of the third aspect. .
  • a fifth aspect of the present invention provides a carrier.
  • the vector includes the nucleic acid molecule of the fourth aspect.
  • a sixth aspect of the present invention provides a host cell.
  • the host cell includes the nucleic acid molecule described in the fourth aspect and/or the vector described in the fifth aspect.
  • a seventh aspect of the invention provides a composition.
  • the composition includes the interleukin 2 mutant described in the first aspect, the conjugate described in the second aspect, or the complex described in the third aspect.
  • An eighth aspect of the present invention provides the use of the interleukin-2 mutant described in the first aspect, the conjugate described in the second aspect, and the complex described in the third aspect in the preparation of a composition for stimulating the body's immune system. use.
  • a ninth aspect of the present invention provides the interleukin 2 mutants described in the first aspect, the conjugates described in the second aspect, and the complexes described in the third aspect in the preparation of drugs for treating cancer and autoimmune diseases. uses in.
  • the cancer includes at least one selected from: melanoma, renal cell carcinoma, lymphoma, sarcoma, breast cancer, lung cancer, bladder cancer, colon cancer, gastric cancer, non-small cell lung cancer , head and neck cancer, skin cancer, squamous cell carcinoma, etc., but the types of cancer in the present invention are not limited to these.
  • the autoimmune disease includes at least one selected from the following: rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, multiple sclerosis, lupus erythematosus , lupus nephritis, ankylosing spondylitis, type I diabetes, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, celiac disease, scleroderma, temporal arteritis, atopic dermatitis, alopecia areata, graft versus host disease , autoimmune hepatitis, primary sclerosing cholangitis or inflammatory myopathy, etc., but the types of autoimmune diseases in the present invention are not limited to these.
  • a tenth aspect of the present invention provides a method for diagnosing, treating or preventing symptoms of cancer or autoimmune diseases in a subject, comprising administering to the subject a therapeutically effective amount of the composition described in the seventh aspect.
  • An eleventh aspect of the present invention provides a method for producing a polypeptide, comprising culturing the host cell described in the sixth aspect under conditions for expressing nucleic acid molecules.
  • the interleukin 2 mutant and the complex containing it provided by the invention improve the affinity between IL2 and IL2R ⁇ , solve the problem of free light chain, and thereby improve the purity and stability of the fusion protein.
  • Figure 1 shows an exemplary structural diagram of the disulfide bond modified IL2/IL2R ⁇ complex of the present invention.
  • Figure A shows that the heavy chain variable region (VH) of an antibody targeting tumor cells or immune cell surface antigens (such as a TIGIT antibody) is connected to the IL2R ⁇ mutant through a Linker, and then connected to the hIgG1 antibody through the Hinge (i.e., hinge region) Fc connection, the light chain variable region (VL) of the above-mentioned antibody targeting the same antigen (TIGIT antibody) is connected to the IL2 mutant through a Linker.
  • VH heavy chain variable region
  • TIGIT antibody immune cell surface antigens
  • Figure B shows that the heavy chain variable region (VH) of an antibody targeting tumor cells or immune cell surface antigens (such as a TIGIT antibody) is connected to the IL2 mutant through a Linker, and then connected to the Fc of the hIgG1 antibody through the Hinge (i.e. hinge region) To connect, the light chain variable region (VL) of the above-mentioned antibody targeting the same antigen (TIGIT antibody) is connected to the IL2R ⁇ mutant through a Linker.
  • VH heavy chain variable region
  • TIGIT antibody immune cell surface antigens
  • VH of an antibody targeting an immune cell or tumor cell surface antigen (such as a PDL1 antibody) is directly connected to the constant region (hIgG1) of an hIgG1 antibody, and an antibody targeting the same antigen (PDL1 antibody) VL is directly linked to the kappa constant region (CL) of the human immunoglobulin light chain.
  • Figure 2 shows the gel electrophoresis detection results of the purified disulfide bond-modified and unmodified IL2/IL2R ⁇ complexes prepared in Example 2 of the present invention; wherein, complexes 1 and 2 are disulfide bond-modified IL2/IL2R ⁇ complexes.
  • Complex 3 is the currently reported IL2/IL2R ⁇ complex, and complex 4 is the IL2/IL2R ⁇ complex without disulfide bond modification.
  • Figure 3 shows the SEC detection results of the IL2/IL2R ⁇ complex (complex 1) modified by the disulfide bond of the present invention.
  • Figure 4 shows the detection results of the affinity of the targeting region (the antibody targeting tumor cell surface antigen in the dual antibody molecule) using the disulfide bond modified and unmodified IL2/IL2R ⁇ complexes of the present invention; wherein, R1262 is a disulfide bond Transform IL2/IL2R ⁇ complex 1, R1115 is IL2/IL2R ⁇ complex 4, and hIgG1 is the subtype control antibody.
  • Figure 5 shows the detection results of the affinity of the targeting region (antibodies targeting immune cell surface antigens) using the disulfide bond modified and unmodified IL2/IL2R ⁇ complexes of the present invention; wherein, R1262 is the disulfide bond modified IL2/IL2R ⁇ Complex 1, R1115 is IL2/IL2R ⁇ complex 4, and hIgG1 is a subtype control antibody.
  • Figure 6 shows the blocking detection results of the binding ability of the ligand and the targeting region (antibodies targeting immune cell surface antigens) using the disulfide bond modified and unmodified IL2/IL2R ⁇ complexes of the present invention; wherein, R1262 is two Sulfur bond modified IL2/IL2R ⁇ complex 1, R1115 is IL2/IL2R ⁇ complex 4, and hIgG1 is a subtype control antibody.
  • Figure 7 shows the gel electrophoresis detection results of the purified disulfide bond-modified IL2/IL2R ⁇ complexes 5, 6, 7, 8 and complex 1 prepared in Example 5 of the present invention.
  • the disulfide bond-unmodified IL2/IL2R ⁇ Complex 4 served as a control.
  • Figure 8 shows the gel electrophoresis detection results of the purified disulfide bond-modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, 13 and complex 1 prepared in Example 5 of the present invention.
  • the disulfide bond-unmodified IL2 /IL2R ⁇ complex 4 served as a control.
  • first and second are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
  • the term "interleukin 2" or “IL2” refers to any natural IL2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., small animals). mice and rats).
  • the IL2 is human IL2
  • the amino acid sequence of an exemplary human wild-type IL2 is shown in SEQ ID NO: 1.
  • IL2 mutant is intended to include any modification of various forms of the IL2 molecule, including full-length IL2, truncated IL2, and IL2 linked to another molecule, such as by fusion or chemical coupling. form.
  • Full-length when referring to IL2 refers to the mature, native-length IL2 molecule.
  • Various forms of IL2 mutants are characterized by at least one amino acid mutation that affects the interaction of IL2 with IL2R ⁇ . Such mutations may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position.
  • IL2 mutants or variants are obtained by amino acid substitutions. Different names can be used here to represent the same mutation. For example, a mutation from Serine 75 to Cysteine may be designated 75C, S75C, or Ser75Cys.
  • IL2R ⁇ refers to interleukin 2 receptor ⁇ , also known as “ ⁇ receptor subunit”
  • IL2R ⁇ refers to interleukin 2 receptor ⁇ , also known as “ ⁇ receptor subunit”
  • IL2R ⁇ refers to interleukin 2 receptor ⁇ , also known as “ ⁇ receptor subunit”
  • IL2R ⁇ refers to interleukin 2 receptor ⁇ , also known as “ ⁇ receptor subunit”
  • IL2R ⁇ refers to interleukin 2 receptor ⁇ , also known as “ ⁇ receptor subunit”
  • IL2R ⁇ refers to the complex formed by interleukin 2 receptor ⁇ and receptor ⁇ , also known as " ⁇ and ⁇ receptor subunits”. base complex”.
  • IL2R ⁇ is human IL2R ⁇ , and the amino acid sequence of an exemplary wild-type IL2R ⁇ is shown in SEQ ID NO: 2 or 3.
  • IL2R ⁇ mutant is intended to include any form of modification of the IL2R ⁇ molecule in various forms, including full-length IL2R ⁇ , truncated IL2R ⁇ , and IL2R ⁇ linked to another molecule, such as by fusion or chemical coupling. form.
  • Full-length when referring to IL2R ⁇ refers to the mature, native-length IL2R ⁇ molecule.
  • Various forms of IL2R ⁇ mutants are characterized by at least one amino acid mutation that affects the interaction of IL2 with IL2R ⁇ . Such mutations may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position.
  • IL2R ⁇ mutants or variants are obtained by amino acid extension or substitution.
  • antibody is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., , Fab', F'(ab)2, Fv, single chain antibody, double antibody body, Fab) and the aforementioned recombinant peptide, as long as it exhibits the desired biological activity.
  • An "immunoglobulin” or “tetrameric antibody” is a tetrameric glycoprotein composed of two heavy chains and two light chains, each containing a variable region and a constant region.
  • Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single chain antibodies (scFv), single chain antibodies (scFv), Chain antibody fragments, chimeric antibodies, diabodies, tribodies, tetrabodies, microbodies, linear antibodies; chelated recombinant antibodies, tribody or bibody, intracellular antibodies, nanobodies, small Modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies or variants or derivatives thereof, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specificity for binding to the polypeptide Sexual antigens, such as one, two
  • heavy chain variable region refers to a region of an antibody molecule that includes at least one complementarity determining region (CDR) of the antibody heavy chain variable domain.
  • CDR complementarity determining region
  • the heavy chain variable region may contain one, two or three CDRs of the antibody heavy chain.
  • light chain variable region refers to a region of an antibody molecule that includes at least one complementarity determining region (CDR) of the antibody light chain variable domain.
  • the light chain variable region may contain one, two or three CDRs of the antibody light chain, which may be a kappa or lambda light chain, depending on the antibody.
  • Antibodies as described herein, also known as immunoglobulins, are tetrameric glycoproteins. In naturally occurring immunoglobulins, each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy” chain (approximately 50-70 kDa). Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the first constant domain of the heavy chain. Variable domain alignment of chains. Specific amino acid residues form the interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).
  • Immunoglobulin variable domains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, or CDRs. From N-terminus to C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain conforms to the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD.) (1987 and 1991); or Chothia and Lesk, Journal of Molecular Biology, 196:901-917, 1987; Chothia et al., Nature, 342:878-883, 1989).
  • the hypervariable region of an antibody refers to the CDR amino acid residues of the antibody that are responsible for antigen binding.
  • the hypervariable region contains amino acid residues from the CDRs, e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and the heavy chain variable domain 31-35(H1), 50-65(H2), and 95-102(H3) (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed.
  • residues from hypervariable loops e.g., residues 26-32 (L1), 50 in the light chain variable region -52(L2) and 91-96(L3) and 26-32(H1), 53-55(H2) and 96-101(H3) in the heavy chain variable domain (as Chothia et al., Molecular Biology Journal of Science and Technology 196:901-917 (1987).
  • Monoclonal antibodies refer to antibodies obtained from a substantially homogenous population of antibodies, where all antibodies in the mixture have a single amino acid sequence derived from a single clone. Monoclonal antibodies are usually highly specific and target a single antigenic site or epitope. In contrast, polyclonal antibody preparations typically include a mixture of antibodies with different amino acid sequences directed against the same or different determinants (epitopes). In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized in homogeneous cultures and are not contaminated by other immunoglobulins with different specificities and characteristics.
  • the single-letter abbreviations for non-polar fatty acid amino acids are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine ( M); the single-letter abbreviations of aromatic amino acids are phenylalanine (F), tryptophan (W), and tyrosine (Y); the single-letter abbreviations of amino acids with uncharged R groups are serine ( S), threonine (T), cysteine (C), proline (P), aspartic acid (N), glutamine (Q); single-letter abbreviation for the positively charged amino acid with R base They are lysine (K), arginine (R), and histidine (H); the single-letter abbreviations of the negatively charged amino acids with R base are aspartic acid (D) and glutamic acid (E).
  • the term "hinge” refers to the region between the CH1 and CH2 regions of the immunoglobulin heavy chain. This region includes H inter-chain disulfide bonds, is rich in proline, and does not form an alpha helix. It is easy to stretch and twist to a certain extent, which is conducive to the complementary binding between the antigen-binding site of the antibody and the antigenic epitope.
  • the term "Fc region” refers to a protein comprising heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin quality, excluding heavy and light chain variable regions, heavy chain constant region 1 (CH1) and light chain constant region (CL).
  • the Fc fragment is meant to include not only the natural amino acid sequence but also its mutant sequence.
  • the immunoglobulin Fc region can be derived from humans or animals, such as cattle, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs.
  • the term "constant region” refers to the carboxyl terminus (C-terminal) of the polypeptide chain, 3/4 or 4/5 of the H chain and 1/2 of the L chain, the number, type, and number of amino acids in this segment.
  • the arrangement order, configuration and sugar content are relatively stable, so this section is called the constant region, that is, the C region.
  • the C regions of H chain and L chain are represented by CH and CL respectively.
  • Different types of immunoglobulins have different CH lengths. IgG, IgA, and IgD have three CHs, including CH1, CH2, and CH3; IgM and IgE have four CHs, including CH1, CH2, CH3, and CH4.
  • Each heavy chain consists of a variable domain (VH) and first, second, third and fourth (optionally) constant domains (CH1, CH2, CH3, CH4 respectively).
  • VH variable domain
  • CH1, CH2, CH3, CH4 constant domains
  • natural intact antibodies are in a "Y" shape, with the stem of the "Y” structure consisting of the second and third constant regions of two heavy chains bound by disulfide bonds.
  • Each arm of the "Y" structure includes the VH and CH1 of the heavy chain (VH-CH1) and the light chain (VL-CL).
  • Mammalian light chains can be divided into lambda chains or kappa chains, and each light chain consists of a variable region (VL) and a constant region (CL).
  • nucleic acid is used interchangeably herein with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form, encompassing Nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to the reference nucleic acid, and behave in a manner similar to the reference nucleic acid Metabolized by glycosides.
  • Nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and present in a host cell or such one or more nucleic acid molecules at more positions. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated.
  • vector refers to a vehicle into which a genetic element (such as the aforementioned nucleic acid molecule) can be operatively inserted and expressed, for example, to produce a protein, RNA, or protein encoded by the genetic element. DNA, or a copy of said genetic element. Vectors can be used to transform, transduce or transfect host cells so that the genetic elements they carry can be expressed in the host cells.
  • a genetic element such as the aforementioned nucleic acid molecule
  • vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage Or M13 bacteriophage, as well as animal viruses, etc.
  • Vectors can contain a variety of expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain an origin of replication site.
  • the vector may also include components to facilitate its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats.
  • the vector may be an expression vector or a cloning vector.
  • the vector (eg, expression vector) provided by the invention contains a nucleic acid sequence encoding a fusion protein of the invention, at least one promoter operably linked to the nucleic acid sequence (eg, SV40, CMV, EF-1 ⁇ ), and at least one selectable marker.
  • the term "host cell” refers to a cell into which exogenous polynucleotides and/or vectors can be or have been introduced.
  • the host cell contains the vector, and the vector can be introduced into mammalian cells to construct host cells, and then use these host cells to express the antibodies or antigen-binding fragments provided by the invention. By culturing the host cells, the corresponding antibodies or fusion proteins can be obtained.
  • Usable mammalian cells may be CHO cells or the like.
  • composition is in a form that is effective to permit the biological activity of the active ingredient and does not contain additional ingredients that would have unacceptable toxicity to the subject to whom the composition is to be administered.
  • the above composition further includes a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to ingredients in a pharmaceutical formulation that are distinct from the active ingredients and are not toxic to the subject, including but not limited to buffers, excipients, stabilizers, and preservatives , or any physiologically compatible solvent, etc.
  • treatment means to temporarily or permanently, partially or completely eliminate, reduce, inhibit or ameliorate the clinical symptoms, manifestations or progression of an event, disease or condition.
  • compositions of the present invention when administered to a subject or exposed to a sample from a subject, aid in the diagnosis of cancer, neoplasia, or condition.
  • the terms “effective amount,” “effective dose,” or “effective dose” are defined as an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • the terms “subject” and “patient” are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment.
  • the term “subject” or “patient” refers to a mammalian subject or patient. Unless otherwise indicated, the terms “patient” or “subject” are used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, avian, goats, and sheep. In certain embodiments, the subject is human. In some embodiments, the subject is a person suspected of having cancer, an autoimmune disease or condition, and/or an infection.
  • a disulfide bond modification between IL2 and IL2R ⁇ (mutation of the 75th amino acid in the amino acid sequence of wild-type interleukin 2 (SEQ ID NO: 1) to cysteine to obtain an interleukin 2 mutant.
  • the amino acid sequence of an exemplary interleukin 2 mutant is shown in SEQ ID NO: 25); and the N-terminus of the amino acid sequence of interleukin 2 receptor ⁇ (SEQ ID NO: 2 or SEQ ID NO: 3) is extended Two or three amino acids, of which the extended second amino acid is cysteine (the second amino acid refers to the second extended direction starting from the N-terminus of wild-type interleukin 2 receptor ⁇ amino acids) to obtain an interleukin 2 receptor alpha mutant.
  • the amino acid sequence of an exemplary interleukin 2 receptor alpha mutant is shown in SEQ ID NO: 26), so that non-covalently linked IL2 and IL2R ⁇ are formed. Covalent disulfide bonding.
  • amino acid sequence of SEQ ID NO:1 is as follows:
  • amino acid sequence of SEQ ID NO:2 is as follows:
  • amino acid sequence of SEQ ID NO:3 is as follows:
  • amino acid sequence of SEQ ID NO:25 is as follows:
  • amino acid sequence of SEQ ID NO:26 is as follows:
  • FIG. 1A An exemplary structural diagram of the complex is shown in Figure 1A: the complex structure of an antibody (such as a TIGIT antibody) targeting tumor cells or immune cell surface antigens.
  • the chain variable region (VH) is connected to the IL2R ⁇ mutant through Linker (the amino acid sequence of linker is shown in SEQ ID NO: 29), and then through Hinge (i.e., hinge region).
  • the amino acid sequence of Hinge is shown in SEQ ID NO: 23.
  • VH of antibodies targeting immune cells or tumor cell surface antigens (such as PDL1 antibodies) is directly connected to the constant region (hIgG1) of hIgG1 antibodies, and the VL of antibodies targeting the same antigen (PDL1 antibodies) is directly connected to human immunoglobulin light Chain kappa (CL) linkage to prepare targeted disulfide bond-modified IL2/IL2R ⁇ complex.
  • FIG. 1B shows that the heavy chain variable region (VH) of an antibody targeting tumor cells or immune cell surface antigens (such as a TIGIT antibody) is connected to the IL2 mutant through a Linker, and then through Hinge (i.e., hinge region, amino group of Hinge).
  • VH heavy chain variable region
  • the acid sequence is shown in SEQ ID NO: 23) is connected to the Fc of the hIgG1 antibody (the amino acid sequence of the Fc region is shown in SEQ ID NO: 24), and the light chain variable region of the above-mentioned antibody targeting the same antigen (TIGIT antibody) (VL) is connected to the IL2R ⁇ mutant through a Linker; the VH of an antibody targeting immune cells or tumor cell surface antigens (such as a PDL1 antibody) is directly connected to the constant region (CH1-Hinge-Fc) of the hIgG1 antibody, and the VH of an antibody targeting the same antigen is The VL of the antibody (PDL1 antibody) is directly connected to the kappa constant region (CL) of the human immunoglobulin light chain to prepare a targeted disulfide bond-modified IL2/IL2R ⁇ complex.
  • amino acid sequence of SEQ ID NO:29 is as follows:
  • amino acid sequence of SEQ ID NO:23 is as follows:
  • amino acid sequence of SEQ ID NO:24 is as follows:
  • the inventors designed two disulfide bond-modified IL2/IL2R ⁇ complexes, namely disulfide bond-modified IL2/IL2R ⁇ complex 1 and disulfide bond-modified IL2/IL2R ⁇ complex 2; and IL2/IL2R ⁇ complex 3 is the currently reported complex, and complex 4 is the IL2/IL2R ⁇ complex without disulfide bond modification;
  • the specific composition and amino acid sequence of complexes 1, 2, 3, and 4 are as follows:
  • the sequence structure of disulfide bond modified IL2/IL2R ⁇ complex 1 is: TIGIT VH-IL2R ⁇ mutant-Fc, TIGIT VL-IL2 mutant, PDL1VH-CH1-Hinge-Fc and PDL1VL-CL; among them, TIGIT VH-IL2R ⁇ mutant
  • TIGIT-Fc is shown in SEQ ID NO: 4
  • the amino acid sequence of TIGIT VL-IL2 mutant is shown in SEQ ID NO: 5
  • the amino acid sequence of PDL1VH-CH1-Hinge-Fc is shown in SEQ ID NO: 6
  • the amino acid sequence of PDL1VL-CL is shown in SEQ ID NO: 7.
  • the amino acid sequence of VH of PDL1 is shown in SEQ ID NO: 27, and the amino acid sequence of VL of PDL1 is shown in SEQ ID NO: 28.
  • amino acid sequence of SEQ ID NO:4 is as follows:
  • amino acid sequence of SEQ ID NO:5 is as follows:
  • amino acid sequence of SEQ ID NO:6 is as follows:
  • amino acid sequence of SEQ ID NO:7 is as follows:
  • amino acid sequence of SEQ ID NO:27 is as follows:
  • amino acid sequence of SEQ ID NO:28 is as follows:
  • the sequence structure of disulfide bond modified IL2/IL2R ⁇ complex 2 is: TIGIT VH-IL2 mutant-Fc, TIGIT VL-IL2R ⁇ mutant, PDL1VH-CH1-Hinge-Fc and PDL1VL-CL; among them, TIGIT VH-IL2 mutant
  • the amino acid sequence of TIGIT-Fc is shown in SEQ ID NO: 8
  • the amino acid sequence of TIGIT VL-IL2R ⁇ mutant is shown in SEQ ID NO: 9
  • the amino acid sequence of PDL1VH-CH1-Hinge-Fc is shown in SEQ ID NO: 6
  • the amino acid sequence of PDL1VL-CL is shown in SEQ ID NO: 7.
  • amino acid sequence of SEQ ID NO:8 is as follows:
  • amino acid sequence of SEQ ID NO:9 is as follows:
  • the sequence structure of IL2/IL2R ⁇ complex 3 is: TIGIT VH-IL2R ⁇ (L42C)-Fc, TIGIT VL-IL2(F42C), PDL1VH-CH1-Hinge-Fc and PDL1VL-CL; among them, TIGIT VH-IL2R ⁇ (L42C)
  • the amino acid sequence of -Fc is shown in SEQ ID NO: 10
  • the amino acid sequence of TIGIT VL-IL2 (F42C) is shown in SEQ ID NO: 11
  • the amino acid sequence of PDL1VH-CH1-Hinge-Fc is shown in SEQ ID NO: 6 shows that the amino acid sequence of PDL1VL-CL is shown in SEQ ID NO: 7;
  • IL2R ⁇ (L42C) represents: mutating leucine at position 42 in the amino acid sequence of IL2R ⁇ to cysteine,
  • IL2 (F42C) represents: mutating IL2 The pheny
  • amino acid sequence of SEQ ID NO: 10 is as follows:
  • amino acid sequence of SEQ ID NO: 11 is as follows:
  • the sequence structure of IL2/IL2R ⁇ complex 4 without disulfide bond modification is: TIGIT VH-wild type IL2R ⁇ -Fc, TIGIT VL-wild type IL2, PDL1VH-CH1-Hinge-Fc and PDL1VL-CL; among them, TIGIT VH-wild type
  • the amino acid sequence of IL2R ⁇ -Fc is shown in SEQ ID NO: 12
  • the amino acid sequence of TIGIT VL-wild-type IL2 is shown in SEQ ID NO: 13
  • the amino acid sequence of PDL1VH-CH1-Hinge-Fc is shown in SEQ ID NO: 6
  • the amino acid sequence of PDL1VL-CL is shown in SEQ ID NO: 7.
  • amino acid sequence of SEQ ID NO: 12 is as follows:
  • amino acid sequence of SEQ ID NO: 13 is as follows:
  • Aiming at the disulfide bond-modified IL2/IL2R ⁇ complex 1 (SEQ ID NO: 4-7), disulfide bond-modified IL2/IL2R ⁇ complex 2 (SEQ ID NO: 6-9), and IL2/IL2R ⁇ complex in Example 1 3 (SEQ ID NO: 6, 7, 10, 11) and disulfide bond unmodified IL2/IL2R ⁇ complex 4 (SEQ ID NO: 6, 7, 12, 13), respectively design plasmid vectors expressing these amino acid sequences.
  • the plasmid containing the target gene forms a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293. While the plasmid is in the cell, the foreign gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130 rpm, and the cells were counted before transfection.
  • the 2E6 cells were inoculated into a 1L shake flask, and the culture system was approximately 300 mL.
  • the transiently transfected cell expression solution was centrifuged at 9000 rpm/20 min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter membrane to purify the disulfide bond-modified IL2/IL2R ⁇ complex 1 sample.
  • ProA affinity chromatography is used for purification. The process is as follows: Use AKTA york 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, and make the target Proteins are adsorbed on the chromatography column while other impurities penetrate and separate.
  • equilibration buffer 10mM PBS
  • Neutralization buffer (1M) is pre-added to the collection tube. Tris, pH8.0), the added volume of neutralizing buffer is determined based on the estimated content of the elution sample, generally 10% of the elution volume is added. Finally, the disulfide bond modified IL2/IL2R ⁇ complex 1 was obtained.
  • the disulfide bond modified IL2/IL2R ⁇ complexes 1 and 2, IL2/IL2R ⁇ complex 3, and the disulfide bond unmodified IL2/IL2R ⁇ complex 4 prepared in Example 2 were subjected to SDS-PAGE electrophoresis detection.
  • IL2/IL2R ⁇ complex 4 is an unmodified molecule with a band between the molecular weight of 25KD and 35KD, indicating the presence of free light chains; disulfide bond modified IL2/IL2R ⁇ complexes 1 and 2 There is no band between the molecular weight of 25KD and 35KD, which is the same as IL2/IL2R ⁇ complex 3, indicating that the free light chain has been successfully removed and the disulfide bond has been successfully transformed.
  • test results are shown in Figure 3.
  • the results illustrate that free light chains are successfully removed, and the purity of the disulfide bond-modified IL2/IL2R ⁇ complex 1 prepared by the present invention is high.
  • Example 4 Disulfide bond modified IL2/IL2R ⁇ complex targeting region affinity detection
  • the FCM experimental method is used to detect the binding activity of the antibody targeting the tumor cell surface antigen (anti-PDL1 antibody) in the double-antibody molecule to CHO-PDL1 cells.
  • test results are shown in Figure 4.
  • the results show that compared with the bisantibody (R1115) of disulfide bond-modified IL2/IL2R ⁇ complex 1 (R1262) and non-disulfide bond-modified IL2/IL2R ⁇ complex 4, the affinity of the two is equivalent. This shows that disulfide bond modification will not affect the affinity of the targeting region.
  • the FCM experimental method is used to detect the binding activity of the antibody (anti-TIGIT antibody) targeting immune cell surface antigens in the double-antibody molecule to CHO-TIGIT cells.
  • Antibody dilution dilute the test antibody (R1262) and positive control antibody (R1115) with 3% BSA to an initial concentration of 800nM, and dilute the subtype control antibody (hIgG1 antibody) with 3% BSA to an initial concentration of 20 ⁇ g/ mL, volume 300 ⁇ L, 3-fold gradient dilution (100 ⁇ L+200 ⁇ L), a total of 10 concentrations;
  • the test results are shown in Figure 5.
  • the results show that the disulfide bond-modified IL2/IL2R ⁇ complex 1 (R1262) has the same affinity as the disulfide bond-unmodified IL2/IL2R ⁇ complex 4 (R1115), indicating that the disulfide bond modification will not Affects the affinity of the targeting region.
  • the FCM experimental method is used to detect the antibody (anti-TIGIT antibody) targeting the surface antigen of tumor cells or immune cells in the double-antibody molecule to block the interaction between the ligand and CHO-TIGIT cells. Binding activity. Proceed as follows:
  • Antibody dilution dilute the test antibody (R1262) and positive control antibody (R1115) with 3% BSA to an initial concentration of 800nM, and dilute the subtype control antibody (hIgG1 antibody) with 3% BSA to an initial concentration of 20 ⁇ g/ mL, volume 300 ⁇ L. 3 times gradient dilution (100 ⁇ L +200 ⁇ L) 10 concentrations in total;
  • test results are shown in Figure 6.
  • the results show that compared with the disulfide bond-modified IL2/IL2R ⁇ complex 1 (R1262) and the disulfide-unmodified IL2/IL2R ⁇ complex 4 (R1115), the two have better ligand and target The blocking effect of zone binding force is equivalent.
  • Example 5 Effect of the types of amino acids at positions 1 and 3 of the N-terminal extension of the amino acid sequence of the interleukin 2 receptor ⁇ mutant on the formation of disulfide bonds introduced after modification of the IL2/IL2R ⁇ complex.
  • the second antigen-binding module When the second antigen-binding module is indirectly connected to the N-terminus of the interleukin-2 receptor alpha mutant through a linker, the N-terminus of the amino acid sequence of the interleukin-2 receptor alpha mutant is extended by three amino acids; in order to further clarify leukocytes
  • the following experiments were conducted to determine the impact of the types of amino acids at positions 1 and 3 of the N-terminal extension of the interleukin 2 receptor ⁇ mutant on the formation of disulfide bonds introduced after modification of the IL2/IL2R ⁇ complex:
  • the amino acid at position 3 selected for extension is an aromatic amino acid (take phenylalanine as an example), an amino acid with an uncharged R base (take serine as an example), an amino acid with a positively charged R base (take lysine as an example) or Any of the negatively charged amino acids in the R base (taking aspartic acid as an example), construct and prepare disulfide bond modified IL2/IL2R ⁇ complexes 5, 6, 7, and 8 (abbreviated as R1493, R1494, R1495, R1496), and the third amino acid in the N-terminal extension of the amino acid sequence of the interleukin 2 receptor alpha mutant in IL2/IL2R ⁇ complex 1 (IL2/IL2R ⁇ complex 1) is glycine (non-polar fatty acid amino acids)) together as the experimental group, and IL2/IL2R ⁇ complex 4 without disulfide bond modification was used as the control group.
  • IL2/IL2R ⁇ complex 1 is glycine (non-polar fatty acid amino acids)
  • the preparation process of disulfide bond-modified IL2/IL2R ⁇ complexes 5, 6, 7, and 8 is the same as the preparation process of disulfide bond-modified IL2/IL2R ⁇ complex 1 in Example 2.
  • the sequence structures of disulfide bond modified IL2/IL2R ⁇ complexes 5, 6, 7, and 8 are: TIGIT VH-IL2R ⁇ mutant-Fc, TIGIT VL-IL2 mutant (SEQ ID NO: 5), PDL1VH-hIgG1 (SEQ ID NO: 6) and PDL1VL-CL (SEQ ID NO: 7); among them, the amino acid sequence of the TIGIT VH-IL2R ⁇ mutant-Fc of the disulfide bond modified IL2/IL2R ⁇ complex 5, 6, 7, 8 is as follows: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 (wherein, the three amino acids extending from the N-terminus of the amino acid sequence of the interleukin 2 receptor ⁇ mutant are in bold Shown, the underlined one is the extended 3rd amino acid);
  • amino acid sequence of SEQ ID NO: 14 is as follows:
  • amino acid sequence of SEQ ID NO: 15 is as follows:
  • amino acid sequence of SEQ ID NO: 16 is as follows:
  • amino acid sequence of SEQ ID NO: 17 is as follows:
  • the obtained disulfide bond-modified IL2/IL2R ⁇ complexes 5, 6, 7, 8 and complex 1 were subjected to SDS-PAGE electrophoresis, and the disulfide bond-unmodified IL2/IL2R ⁇ complex 4 was used as a control group to explore interleukins.
  • the type of the third amino acid in the N-terminal extension of the amino acid sequence of the 2 receptor ⁇ mutant affects the formation of the disulfide bond introduced after the modification of the IL2/IL2R ⁇ complex.
  • the experimental results are shown in Figure 7.
  • the first and third amino acids selected for extension are non-polar fatty acid amino acids (taking glycine as an example), aromatic amino acids (taking phenylalanine as an example), and amino acids with uncharged R groups (taking serine as an example).
  • the preparation process of disulfide bond-modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, and 13 is the same as the preparation process of disulfide bond-modified IL2/IL2R ⁇ complex 1 in Example 2.
  • sequence structures of disulfide bond modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, and 13 are: TIGIT VH-IL2R ⁇ mutant-Fc, TIGIT VL-IL2 mutant (SEQ ID NO: 5), PDL1VH-hIgG1 (SEQ ID NO: 6) and PDL1VL-CL (SEQ ID NO: 7); among them, the amino acids of TIGIT VH-IL2R ⁇ mutant-Fc of disulfide bond modified IL2/IL2R ⁇ complex 9, 10, 11, 12, 13
  • the sequence is shown in sequence as SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 (among them, the amino acid sequence of the interleukin 2 receptor alpha mutant is The three amino acids of the N-terminal extension are shown in bold, and the underlined ones are the 1st and 3rd amino acids of the extension);
  • amino acid sequence of SEQ ID NO: 18 is as follows:
  • amino acid sequence of SEQ ID NO: 19 is as follows:
  • amino acid sequence of SEQ ID NO:20 is as follows:
  • amino acid sequence of SEQ ID NO:21 is as follows:
  • amino acid sequence of SEQ ID NO:22 is as follows:
  • the obtained disulfide bond-modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, 13 and complex 1 were subjected to SDS-PAGE electrophoresis.
  • the disulfide bond-unmodified IL2/IL2R ⁇ complex 4 was used as a control group to explore leukocytes.
  • the type of combination of amino acids at positions 1 and 3 of the N-terminal extension of the interleukin 2 receptor alpha mutant affects the formation of disulfide bonds introduced after modification of the IL2/IL2R ⁇ complex.

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Abstract

La présente invention relève du domaine technique de la biologie moléculaire, et concerne en particulier un mutant d'interleukine 2 et un complexe le contenant. La présente invention concerne un mutant d'interleukine 2, un conjugué contenant le mutant, un complexe contenant le mutant, et une utilisation du mutant, du conjugué ou du complexe dans la préparation d'une composition pour stimuler un système immunitaire d'organisme et dans la préparation d'un médicament pour le traitement de cancers et de maladies auto-immunes. Dans la présente invention, une connexion de liaison disulfure covalente est formée entre un mutant IL2 et un mutant IL2Rα, ce qui permet d'améliorer l'affinité entre l'IL2 et l'IL2Rα, de résoudre le problème de chaînes légères libres existantes dans la protéine de fusion associée, et d'améliorer en outre la pureté et la stabilité de l'IL2 et de la protéine de fusion associée à IL2Rα.
PCT/CN2023/080577 2022-03-10 2023-03-09 Mutant d'interleukine 2 et complexe le contenant WO2023169524A1 (fr)

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CN202210234287 2022-03-10
CN202210423871.9 2022-04-21
CN202210423871.9A CN116769013A (zh) 2022-03-10 2022-04-21 白细胞介素2突变体以及含有其的复合物

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050142106A1 (en) * 2003-07-18 2005-06-30 Wittrup K. D. Mutant interleukin-2 (IL-2) polypeptides
CN107266553A (zh) * 2016-04-08 2017-10-20 南京康志制药有限公司 高效人白细胞介素ⅱ突变体融合蛋白及其应用
WO2021185361A1 (fr) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et son utilisation
CN113667004A (zh) * 2020-05-14 2021-11-19 上海盖浦生物科技有限公司 一种白介素2突变体

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050142106A1 (en) * 2003-07-18 2005-06-30 Wittrup K. D. Mutant interleukin-2 (IL-2) polypeptides
CN107266553A (zh) * 2016-04-08 2017-10-20 南京康志制药有限公司 高效人白细胞介素ⅱ突变体融合蛋白及其应用
WO2021185361A1 (fr) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et son utilisation
CN113667004A (zh) * 2020-05-14 2021-11-19 上海盖浦生物科技有限公司 一种白介素2突变体

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