WO2022253248A1 - 抗cd3抗体变异体、融合蛋白及应用 - Google Patents

抗cd3抗体变异体、融合蛋白及应用 Download PDF

Info

Publication number
WO2022253248A1
WO2022253248A1 PCT/CN2022/096501 CN2022096501W WO2022253248A1 WO 2022253248 A1 WO2022253248 A1 WO 2022253248A1 CN 2022096501 W CN2022096501 W CN 2022096501W WO 2022253248 A1 WO2022253248 A1 WO 2022253248A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
fragment
antibody
taa
variable region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/096501
Other languages
English (en)
French (fr)
Inventor
屈向东
潘琴
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qure Biotechnology (Shanghai) Co Ltd
Original Assignee
Qure Biotechnology (Shanghai) Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qure Biotechnology (Shanghai) Co Ltd filed Critical Qure Biotechnology (Shanghai) Co Ltd
Priority to EP22815292.2A priority Critical patent/EP4349870A4/en
Priority to US18/566,817 priority patent/US20240294640A1/en
Priority to JP2023574487A priority patent/JP7734994B2/ja
Publication of WO2022253248A1 publication Critical patent/WO2022253248A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the technical field of biomedicine, in particular to anti-CD3 antibody variants, fusion proteins and applications.
  • T lymphocytes are lymphatic stem cells derived from bone marrow. After differentiation and maturation in the thymus, they are distributed to immune organs and tissues throughout the body through lymphatic and blood circulation to exert immune functions. T cells are highly efficient killer cells that can rapidly destroy virus-infected cells and cancer cells. The killing effect of T cells requires the formation of immune synapses, and this process is highly dependent on the complex formed by TCR recognition of MHC molecules on the surface of antigen-presenting cells and the presented antigen peptides. Activated immune synapses will release cytotoxins and cytokines to play a killing role.
  • bispecific or multispecific antibody-directed T cells In the process of immune synapse formation, limited by the distance between T cells and target cells, bispecific or multispecific antibody-directed T cells must simulate the formation of immune synapses, and one end bridges T cells by targeting CD3.
  • Cell TCR receptor the other end bridges the target cell by targeting the target cell surface antigen.
  • the CD3 molecule is expressed on the surface of all mature T cells and binds non-covalently with TCR to form a complete TCR-CD3 complex, which jointly participates in the immune response to antigen stimulation. It is currently the most widely used and most successful bispecific antibody. Triggering molecules on the surface of immune effector cells.
  • Bispecific antibodies targeting CD3 can bind CD3 on the surface of T cells and antigens on the surface of tumor cells, thereby shortening the distance between cytotoxic T cells (Tc or CTL) and tumor cells, and directly activating T cells. Induce T cells to directly kill cancer cells without relying on the dual activation signals of traditional T cells.
  • OKT3 is an antibody with milestone influence.
  • SP34 is currently one of the few published CD3 antibodies that can have species reactivity with monkeys.
  • the scientific community has made new breakthroughs in the field of double antibodies, thus developing different types of new CD3 double antibodies (CD3-BsAb).
  • CD3-BsAb new CD3 double antibodies
  • Amgen's Blinatumomab (CD19xCD3 in BiTE format) was approved by the FDA in 2014 and is currently the most successful CD3-BsAb double antibody. This medicine is used to treat patients with relapsed or refractory acute lymphoblastic leukemia (ALL). Compared with standard chemotherapy, more than 40% of patients treated with blinatumomab can achieve complete or partial remission (CR/PR), and the median overall survival can be improved by several months. In addition to Blinatumomab, many other CD3-BsAbs are currently undergoing clinical trials, such as CD19, CD20, CD38, BCMA, CD33, and CD123, among others.
  • CRS cytokine storm
  • Interleukin-15 is a 14–15kDa cytokine that is important for the function of NK cells, NKT cells and memory CD8+ T cells.
  • IL-15 combines with its receptor IL-15R ⁇ to produce a complex IL-15 superagonist (IL-15SA) with extremely high biological potency, which is then transduced and transported to target cells.
  • IL-15SA strongly activates cells expressing IL-15R in response, especially NK cells/T cells, thereby promoting antitumor and antiviral functions.
  • IL-15 has a wide range of immunoregulatory effects, and can participate in the regulation of the survival, proliferation and function of T cells, especially NK cells and memory CD8+ T cells.
  • IL-15 is very similar to IL-2 in structure, and both belong to the helical cytokine family.
  • the heterotrimeric receptor for IL-15 shares IL-2R/IL-15R ⁇ (CD122) and common ⁇ c chain (CD132) with the IL-2 receptor, due to these common receptor components and shared JAK 1/JAK 3/ STAT 5 signaling pathway, IL-15 has the same functions as IL-2, including stimulating T cell proliferation, generating cytotoxic T lymphocytes, stimulating B cells to synthesize immunoglobulins, and NK cell production and continued survival.
  • IL-2 and IL-15 have distinct and often competing roles in many adaptive immune responses. There are four main points: 1. IL-2 can regulate the activation of Tregs cells, but IL-15 cannot. 2.
  • IL-2 can suppress T cell responses by activating the induced cell death of CD8+ effector T cells.
  • IL-15 plays an essential role in the differentiation of NK, effector CD8+ and memory phenotype CD8+ T cells. 4. Preclinical studies have shown that the toxicity of IL-15 is different from that of IL-2. Compared with IL-2, IL-15 has almost no vascular capillary leakage. These factors make IL-15 a more potential cytokine in tumor immune strategies.
  • IL-15 super agonist N-803 which is a fusion of IL-15 protein N72D mutation combined with IL-15Ra and IgG1Fc co-expression Protein
  • ImmunityBio submitted to the FDA a marketing application for the IL-15 superagonist N-803, which is used in combination with Bacillus Calmette-Guerin (BCG) to treat non-muscle invasive bladder cancer (NMIBC) that does not respond to BCG.
  • BCG Bacillus Calmette-Guerin
  • NMIBC non-muscle invasive bladder cancer
  • Nivolumab PD-1 antibody, Opdivo
  • N-803 PD-1 antibody, Opdivo
  • SHR-1501 IL-15 fusion protein
  • SHR-1316 PD-L1 monoclonal antibody drug
  • the first object of the present invention is to provide a variant of an anti-CD3 antibody, the anti-CD3 antibody comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising: VHCDR1, VHCDR2, VHCDR3;
  • the light chain variable region includes: VLCDR1, VLCDR2, VLCDR3;
  • the amino acid sequence of the anti-CD3 antibody includes VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 shown in SEQ ID NO: 29-34;
  • the mutation site of the antibody variant includes any 1 or more of the following sites: the tenth site of the sequence shown in SEQ ID NO: 30, the second and seventh positions in the sequence shown in SEQ ID NO: 31, SEQ ID NO: 31
  • the mutation refers to amino acid substitution.
  • the mutation site of the variant includes any one or more than two of the following sites: A in the tenth position in the sequence shown in SEQ ID NO: 30 is replaced by E, in the sequence shown in SEQ ID NO: 31
  • the G in the second position is replaced by S
  • the S in the seventh position is replaced by G
  • the N in the third position in the sequence shown in SEQ ID NO: 33 is replaced by W
  • the K in the fourth position is replaced by L
  • SEQ ID NO: 33 In the sequence shown in NO:34, the Y at the fourth position is replaced by N or R
  • the S at the fifth position is replaced by K
  • the N at the sixth position is replaced by G
  • the L at the seventh position is replaced by G.
  • amino acid sequence of VHCDR2 is shown in SEQ ID NO:35; or, the amino acid sequence of VHCDR3 is shown in SEQ ID NO:36; or, the amino acid sequence of VLCDR2 is shown in SEQ ID NO:37 or 38; or, The amino acid sequence of VLCDR3 is shown in SEQ ID NO: 39, 40 or 41.
  • amino acid sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are as shown in any one of the following sequences:
  • the heavy chain variable region and/or the light chain variable region of the antibody variant is selected from the heavy chain variable region and/or the light chain variable region of the sequence shown in SEQ ID NO: 1, 2 or 3 , or have at least 90% sequence identity therewith.
  • the mutation site includes any one or two or more of the following combinations, Mutation sites are numbered according to EU, the heavy chain variable region is represented by VH, and the light chain variable region is represented by VL:
  • the anti-CD3 antibody variant further comprises: a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4 or variants thereof; and a light chain selected from human ⁇ , ⁇ chains or variants thereof chain constant region; the heavy chain constant region comprises: an Fc fragment or a variant thereof.
  • the antibody variant is a scFv comprising a heavy chain variable region, a light chain variable region, and a linking fragment connecting the heavy chain variable region and the light chain variable region, the linking fragment
  • the amino acid sequence of is preferably several GGGGS repeats, more preferably 3 GGGGS repeats.
  • Another object of the present invention is to provide the application of the anti-CD3 antibody variant in the preparation of medicaments for inhibiting or treating cancer.
  • the cancer is selected from or occurs in the following cancers: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, bone marrow, lymphoma, leukemia, thyroid, endometrium, uterus, bladder , neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel Cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • cancers colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, bone marrow, lymphoma, leukemia, thyroid, endometrium, uterus, bladder , neuroendocrine, head and neck, liver, nasoph
  • Another object of the present invention is to provide a nucleic acid molecule encoding the anti-CD3 antibody variant.
  • Another object of the present invention is to provide an expression vector comprising the above-mentioned nucleic acid molecule.
  • Another object of the present invention is to provide an anti-tumor-associated antigen (TAA)/anti-CD3 protein molecule, which is in the form of a heterodimer, including a first monomer and a second monomer;
  • TAA anti-tumor-associated antigen
  • the first monomer includes: (a) Fd fragment; (b) light chain fragment and first Fc chain;
  • the second monomer includes: (a) Fd fragment; (b) light chain fragment, anti-CD3 antibody fragment and a second Fc chain;
  • the light chain fragment includes a VL domain and a CL domain;
  • the Fd fragment includes a VH domain and a CH1 domain; in the first monomer or in the second monomer,
  • the light chain fragment is paired with the Fd fragment to form an anti-TAA Fab domain; wherein, the light chain fragment of the first monomer is fused with the first Fc chain; the N-terminal of the anti-CD3 antibody fragment is fused with The light chain fragment of the second monomer is fused, and the C-terminus is fused with the second Fc chain;
  • the first monomer includes: (a) Fd fragment; (b) light chain fragment, cytokine functional region, first Fc chain;
  • the second monomer includes: (a) Fd fragment; (b) light chain fragment, anti-CD3 antibody fragment, and the second Fc chain;
  • the cytokine functional region includes IL-15 and IL-15Ra; wherein, the N-terminal of the cytokine functional region is connected to the light chain of the first monomer chain fragment fusion, the C-terminal is fused with the first Fc chain; the N-terminal of the anti-CD3 antibody fragment is fused with the light chain fragment of the second monomer, and the C-terminal is fused with the second Fc chain;
  • the first monomer includes: an anti-TAA antibody fragment, a cytokine functional region, and a first Fc chain
  • the second monomer includes: an anti-TAA antibody fragment, an anti-CD3 antibody fragment, and a second Fc chain; wherein , the N-terminal of the cytokine functional region is fused with the anti-TAA antibody fragment of the first monomer, and the C-terminal is fused with the first Fc chain; the N-terminal of the anti-CD3 antibody fragment is fused with the second monomer
  • the anti-TAA antibody fragment of the body is fused, and the C-terminus is fused with the second Fc chain;
  • the first Fc chain is interchangeable with the second Fc chain.
  • the anti-TAA antibody fragment is in the form of scFv, comprising: a heavy chain variable region (VH), a light chain variable region (VL), and a variable region connecting the heavy chain variable region and the light chain variable region from the N-terminal to the C-terminal of the peptide chain, the amino acid fusion sequence of the anti-TAA antibody fragment is "VH ⁇ VL", or "VL ⁇ VH”; " ⁇ " indicates the connecting fragment; or, the The anti-CD3 antibody fragment is in the form of scFv, comprising: a heavy chain variable region (VH), a light chain variable region (VL), and a connecting fragment connecting the heavy chain variable region and the light chain variable region; From the N-terminal to the C-terminal of the peptide chain, the fusion sequence of the amino acids of the anti-CD3 antibody fragment is "VH ⁇ VL", or "VL-VH”; " ⁇ " indicates the linking fragment; or, from the N-terminal of the peptide
  • the amino acid sequence of the anti-CD3 antibody fragment is selected from CD3-binding specific antibodies, antibody fragments, single domain antibodies or humanized forms thereof; preferably, the amino acid sequence of the anti-CD3 antibody fragment is selected from SP34, OKT3, UCTH1 or derivatives thereof.
  • the anti-CD3 antibody fragment includes a heavy chain variable region and a light chain variable region;
  • the heavy chain variable region of the anti-CD3 antibody fragment includes: VHCDR1, VHCDR2, VHCDR3; the light chain variable region of the anti-CD3 antibody fragment
  • the chain variable region includes: VLCDR1, VLCDR2, VLCDR3; the amino acid sequence of said VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 is as shown in any set of sequences in the anti-CD3 antibody variant according to claim 5.
  • IL-15Ra and IL-15 form an IL-15/IL-15Ra complex;
  • IL-15 includes: IL-15 and its mutations, truncations and various derivatives that can bind IL-15Ra;
  • IL-15Ra Including: IL-15Ra and its mutations, truncations and various derivatives that can bind IL-15;
  • the IL-15/IL-15Ra complex includes, but is not limited to, any combination of mutations shown below
  • the counting method is calculated as the first position according to the first amino acid of the amino acid sequence of IL-15 or IL-15Ra; preferably, the parental sequence of the IL-15 is as shown in SEQ ID NO: 44; the IL The parental sequence of -15Ra is shown in SEQ ID NO:45:
  • the IL-15 includes but is not limited to any of the mutations shown in any of the following combinations, and the counting method starts from the first amino acid of the amino acid sequence of IL-15 as the first position; preferably, the IL-15
  • the maternal sequence of 15 is shown in SEQ ID NO:44:
  • the TAA is selected from CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB or Glypican-3.
  • the first Fc chain and the second Fc chain are polymerized to form an Fc segment;
  • the Fc segment is selected from Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc or variants thereof, preferably IgG1 Fc, or Human IgG4 Fc or a variant thereof;
  • the protein molecule is in the form of an Fc heterodimer; preferably, the Fc heterodimer comprises, but is not limited to, a combination of the following mutations, which are counted according to EU :
  • the protein molecule comprises an Fc segment, and the Fc segment selects to eliminate the immune effector function, including but not limited to a combination of the following mutations, the following mutations are counted according to EU:
  • the anti-tumor-associated antigen (TAA)/anti-CD3 protein molecule is obtained by fusion of amino acid fragments shown in any one of the following sequences:
  • Another object of the present invention is to provide a nucleic acid molecule encoding the anti-tumor-associated antigen (TAA)/anti-CD3 protein molecule.
  • Another object of the present invention is to provide the application of the anti-tumor-associated antigen (TAA)/anti-CD3 protein molecule in the preparation of medicines for inhibiting or treating cancer
  • the cancer includes cancers selected from the following or occurring in The following sites: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, bone marrow cancer, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, neuroendocrine, head and neck, liver, nasopharynx , testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, Sarcoma, mesothelioma, and myelodysplastic syndrome.
  • IL-15 includes: IL-15 and its mutation, truncation and various derivatives capable of binding IL-15Ra;
  • IL-15Ra includes: IL-15Ra and its mutation, truncation and various derivatives thereof capable of binding IL-15;
  • the IL-15/IL-15Ra complex includes, but is not limited to, the mutations shown in any of the following combinations, and the counting method is counted as the first position from the first amino acid of the amino acid sequence of IL-15 or IL-15Ra;
  • the parent sequence of the IL-15 is shown in SEQ ID NO: 44;
  • the parent sequence of the IL-15Ra is shown in SEQ ID NO: 45:
  • the anti-tumor-associated antigen (TAA) and fusion protein containing IL-15/IL-15Ra, its IL-15 includes but not limited to the mutation mode shown in any of the following combinations, and the counting mode is based on IL-15
  • the first amino acid of the amino acid sequence is counted as the first position; preferably, the parental sequence of IL-15 is shown in SEQ ID NO:44:
  • the anti-tumor-associated antigen (TAA) and fusion protein containing IL-15/IL-15Ra comprises an Fc segment, and the Fc segment is selected to eliminate immune effector functions, including but not limited to a combination of the following mutations, the following mutations For counting according to EU::
  • the anti-tumor-associated antigen (TAA) and the fusion protein containing IL-15/IL-15Ra, the amino acid sequence of any one of its polypeptide chains is shown in SEQ ID NO: 28, or has at least 90% of its sequence identity.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the anti-tumor-associated antigen (TAA) and fusion protein containing IL-15/IL-15Ra.
  • TAA anti-tumor-associated antigen
  • Another object of the present invention is to provide the application of the anti-tumor-associated antigen (TAA) and the fusion protein containing IL-15/IL-15Ra in the preparation of drugs for inhibiting or treating cancer
  • the cancer includes selected from The following cancers may occur in the following sites: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, bone marrow, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, neuroendocrine, head and neck liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma tumors, gliomas, sarcomas, mesotheliomas, and myelodysplastic syndromes.
  • the present invention has the following beneficial effects:
  • the present invention mutates existing anti-CD3 antibodies to obtain new anti-CD3 antibody variants.
  • the present invention also provides an antibody molecule with a novel structure, which has better biological activity.
  • Figure 1 is a graph showing the binding of CD3 antibody SP34 and its mutants to Jurkat cells detected by FACS.
  • Fig. 2 is a design diagram of the molecular form of the anti-TAA (tumor-associated antigen) and anti-CD3 bispecific antibody.
  • Figure 3 is the molecular design diagram of QP372337241461.
  • Figure 4 is the molecular design diagram of QP374437481461.
  • Figure 5 and Figure 6 are the results of FACS detection of CLDN18.2/CD3 double antibody and other molecules binding to CLDN18.2.
  • Figure 7 and Figure 8 are the results of FACS detection of CLDN18.2/CD3 double antibody and other molecules binding to CD3.
  • Figure 9 and Figure 10 are graphs showing the results of PBMC killing human gastric cancer cell NUGC4-CLDN18.2 cells mediated by CLDN18.2/CD3 antibody and other molecules.
  • Figure 11 is a graph showing the results of PBMC killing human lung cancer cells HCC827-CLDN18.2 cells mediated by molecules such as CLDN18.2/CD3 antibody.
  • Figure 12 is a graph showing the tumor growth inhibition curve of CLDN18.2/CD3 biantibody molecules in CD3EGD HuGEMM mice subcutaneously transplanted colorectal cancer cells MC38-hCLDN18.2 in vivo animal model.
  • Fig. 13 is a graph showing the change in body weight of the mice in each group after the subcutaneous transplantation of colorectal cancer cells MC38-hCLDN18.2 in vivo in CD3EGD HuGEMM mice.
  • Figure 14 and Figure 15 are graphs showing the inhibition of tumor growth by CLDN18.2/CD3 double antibody molecules in the PBMC humanized NOG mouse X-CLDN18.2/MIA PaCa-2 subcutaneous xenograft tumor model.
  • Fig. 16 to Fig. 19 are design diagrams of the structural form of a type of anti-TAA/CD3 and IL15/IL15Ra-containing multifunctional fusion protein.
  • Fig. 20 to Fig. 27 are design diagrams of structural forms of another type of anti-TAA/CD3 and IL15/IL15Ra-containing multifunctional fusion protein.
  • Fig. 28 is a structural diagram of TAA/IL15 bifunctional fusion protein activated by tumor-targeted T cells/NK cells.
  • Figure 29 is a graph showing the results of FACS detection of CLDN18.2/CD3/IL15 multifunctional fusion protein binding to CLDN18.2.
  • Figure 30 is a graph showing the results of FACS detection of CLDN18.2/CD3/IL15 and CLDN18.2/IL15 fusion protein binding to CLDN18.2.
  • Figure 31 is a graph showing the results of FACS detection of CLDN18.2/CD3/IL15 multifunctional fusion protein binding to CD3.
  • Figure 32 is a graph showing the results of FACS detection of CLDN18.2/CD3/IL15 and CLDN18.2/IL15 fusion protein binding to CD3.
  • Figure 33, Figure 34, and Figure 35 are graphs showing the results of the Mo7e cell proliferation assay to detect the activity of the multifunctional fusion protein IL15.
  • Figure 36 and Figure 37 are graphs showing the results of CLDN18.2/CD3/IL15-mediated PBMC killing of human gastric cancer cell NUGC4-CLDN18.2 cells.
  • Figure 38 is a graph showing the results of CLDN18.2/CD3/IL15-mediated PBMC killing of human lung cancer cells HCC827-CLDN18.2 cells.
  • an anti-tumor-associated antigen (TAA)/anti-CD3 bispecific antibody refers to an antibody targeting both TAA and CD3.
  • fused refers to linking components directly by peptide bonds or via linker fragments, or by intermolecular interactions. Within a single peptide chain, fusion refers to direct linking by peptide bonds or linking via linking fragments.
  • Multifunctional fusion protein refers to a protein comprising two or more antigen-binding domains capable of binding two or more different epitopes (e.g., two, three or more different epitopes ), the multifunctional fusion protein can also contain cytokines (such as IL-15, IL-15Ra) and the like.
  • Fusion position refers to the position of the functional region or structural domain in the peptide chain, indicating the connection sequence of the functional fragments on the peptide chain.
  • polypeptide refers to amino acid chains of any length, including proteins and fragments thereof.
  • the present invention discloses a polypeptide as a sequence of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal orientation.
  • amino acid residue sequences are designated by three-letter or one-letter codes as follows: alanine (Ala,A), arginine (Arg,R), asparagine ((Asn,N) , Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic acid (Glu, E), Glycine (Gly, G), Histidine (His, H), isoleucine (I1e, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe , F), proline (Pro,P), serine (Ser,S), threonine (Thr,T), tryptophan (Trp,W), tyrosine (Tyr,Y) and valine (Val,V).
  • single-chain refers to a molecule comprising amino acids linearly linked by peptide bonds.
  • variant refers to a polypeptide or polynucleotide that differs from the included amino acids or nucleotides but retains essential properties. Usually the differences between variants or between variants and the parental antibody are limited and the amino acid sequences are very similar overall.
  • the antibody or antibody fragment before mutation is referred to as parent antibody, and the antibody or antibody fragment after mutation is referred to as variant. The variant still has antigen binding activity.
  • antibody refers to an immunoglobulin molecule (immunoglobulin, Ig) comprising at least one antigen-binding site and capable of specifically binding an antigen.
  • antigen is a substance that can induce an immune response in the body and specifically binds to an antibody.
  • the binding of antibody and antigen is mediated by the interaction formed between them, including hydrogen bond, van der Waals force, ionic bond and hydrophobic bond.
  • the region on the surface of an antigen that binds to an antibody is called an "antigenic determinant” or "epitope".
  • each antigen has multiple determinants.
  • antibody referred to in the present invention is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, antibodies comprising at least two different antigen-binding domains Multispecific antibodies (eg, bispecific antibodies).
  • Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies of other origin.
  • the antibodies of the present invention can be derived from any animal, including but not limited to immunoglobulin molecules of humans, non-human primates, mice, rats, cows, horses, chickens, camels, and alpacas. Antibodies may contain additional alterations, such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations.
  • Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of antibodies, and immunoglobulin molecules comprising any other modification to the antigen recognition site, so long as these antibodies exhibit the desired biological activity.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain at least one antigen-binding domain.
  • the basic structure of an antibody is a Y-shaped monomer composed of two identical heavy chains (heavy chain, H) and two identical light chains (light chain, L) connected by disulfide bonds. Each chain is composed of 2 to 5 structural domains (also known as functional regions) of about 110 amino acids with similar sequences but different functions.
  • the amino acid sequence of the light chain and the heavy chain near the N-terminus of the antibody molecule changes greatly, and the formed domain is called the variable region (variable region, V region); the relatively constant region near the C-terminal amino acid sequence is called the constant region (constant region) , area C).
  • VH and VL The V regions of the heavy chain and light chain are called VH and VL, respectively, and each of VH and VL has three regions whose amino acid composition and sequence are highly variable, called hypervariable region (HVR); the formation of this region is related to the expression of antigen
  • the complementary spatial conformation is also called complementarity determining region (CDR).
  • the three CDRs of VH are represented by VHCDR1, VHCDR2, and VHCDR3, and the three CDRs of VL are represented by VLCDR1, VLCDR2, and VLCDR3, respectively.
  • a total of 6 CDRs of VH and VL together form the antigen-binding site.
  • the diversity of amino acids in the CDR region is the molecular basis for the specific binding of antibodies to a large number of different antigens.
  • VH and VL each have four framework regions (or framework regions), which are represented by FR1, FR2, FR3, and FR4, respectively.
  • Each VH and VL consists of three CDRs and four FRs, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • human immunoglobulins can be divided into 5 classes: IgM, IgG, IgA, IgD, IgE. It can be further divided into different subclasses (isotypes), for example, human IgG can be divided into IgG1, IgG2, IgG3, IgG4; IgA can be divided into IgA1 and IgA2. IgM, IgD, and IgE have not yet been found to have subclasses.
  • light chains can be classified into kappa chains and lambda chains.
  • Antibodies of the invention may be of any class (eg, IgM, IgG, IgA, IgD, IgE) or subclass (eg, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2).
  • class eg, IgM, IgG, IgA, IgD, IgE
  • subclass eg, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2
  • the hinge region (hinge region) is located between CH1 and CH2 and is rich in proline, so it is easy to stretch and bend, and can change the distance between the two arms of the Y shape, which is conducive to the simultaneous binding of the two arms to the antigenic epitope.
  • Fab fragment is a fragment of antigen binding (Fab), which means an antibody fragment composed of VL, VH, CL and CH1 domains, which binds to a single antigenic epitope (monovalent).
  • Fab antigen binding
  • Fab fragment of antigen binding
  • Fd fragment means an antibody fragment consisting of VH and CH1 domains.
  • Fc fragment crystallizable, which has no antigen-binding activity and is the interaction between an antibody and an effector molecule or a cell surface Fc receptor (FcR).
  • FcR cell surface Fc receptor
  • the site of action. Fc fragments bind to cells with corresponding Fc receptors on their surface and produce different biological effects.
  • ADCC effect antibody-dependent cell-mediated cytotoxicity
  • the Fab segment of the antibody binds to the antigenic epitope of the virus-infected cell or tumor cell, and its Fc segment interacts with the killer cell (NK FcR binding on the surface of cells, macrophages, etc.) mediates killer cells to directly kill target cells.
  • NK FcR binding on the surface of cells, macrophages, etc.
  • the Fc fragment includes the constant region polypeptides of the antibody except the heavy chain constant region CH1, that is, the two constant region domains CH2 and CH3 at the carboxy-terminal of the heavy chain constant region of human immunoglobulin IgA, IgD, and IgG, and human immunoglobulin IgE and The three constant region domains CH2, CH3 and CH4 at the carboxy-terminal end of the IgM heavy chain constant region.
  • the Fc fragment is usually selected from Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc or variants thereof, preferably from IgG1 Fc, or Human IgG4 Fc or variants thereof
  • a preferred mutation design is a "Knob-in-hole" style mutation design.
  • Tumor-associated antigen refers to the antigen molecules present on tumor cells or normal cells, including: embryonic protein, glycoprotein antigen, squamous cell antigen, etc., which are often used in the diagnosis of clinical tumors. Tumor-associated antigens are not unique to tumor cells, and can be synthesized in a small amount by normal cells, but are highly expressed when tumor cells proliferate, so they are called "associated antigens”.
  • Tumor-associated antigens can be, for example, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB or Glypican-3.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors useful in recombinant DNA techniques are usually in the form of plasmids
  • IL-15 may be mutants or fragments thereof.
  • the present invention uses the existing CD3 antibody SP34 as a parent antibody, and mutates its variable region to obtain CD3 antibody variants.
  • the present invention also provides several protein molecules targeting different targets and having different configurations.
  • IL-15 ⁇ IL15Ra can adopt the following sequence:
  • IL15Ra ⁇ IL-15 can adopt the following sequence:
  • Example 1 Obtaining anti-CD3 antibody variants, molecular cloning, transient expression, and protein purification
  • amino acid sequence is as follows:
  • the underlined part is the heavy chain variable region of the antibody, the wavy part is the light chain variable region, and the italicized part between the heavy chain variable region and the light chain variable region is the connecting sequence.
  • the amino acid sequence of the variable region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the parts in bold and thick lines at the bottom show HCDR1, HCDR3, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.
  • the comparison of the CDR sequences of the parent antibody and its mutants is shown in the table below:
  • Protein expression adjust the cell density of 293E cells to 1 ⁇ 10 6 /ml.
  • the amount of plasmid to be transfected is 100 ⁇ g/100ml of cells, and the mass ratio of PEI to plasmid is 2:1.
  • the plasmid and PEI were mixed and allowed to stand for 15 minutes.
  • the plasmid and PEI mixture was slowly added to the 293E cells, and cultured in a shaker with 8% CO 2 , 120 rpm, and 37°C.
  • the cell supernatant was collected and purified by centrifuging at 4700rpm for 20min in a horizontal centrifuge.
  • Protein affinity chromatography the supernatant of the cell culture medium was centrifuged at high speed, and the protein A chromatography column of GE was used for affinity chromatography. Chromatography uses an equilibration buffer of 1 ⁇ PBS (pH7.4). After the cell supernatant is loaded and combined, it is washed with PBS until the ultraviolet rays return to the baseline, and then the target protein is eluted with an elution buffer of 0.1M glycine (pH3.0).
  • QP3679 is the mother humanized anti-CD3 antibody SP34, and the yield is 68.64mg/L. Higher, the protein may be more stable, and the purity of the protein detected by SEC-HPLC is very good.
  • Example 2 FACS detection of anti-CD3 antibody variants binding to Jurkat cells naturally expressing CD3
  • FACS detection of anti-CD3 antibody binding activity Human lymphocytic leukemia cells (Jurkat cells) express natural CD3, and the binding of anti-CD3 antibody and its mutants to Jurkat cells was detected by FACS (flow cytometry, fluorescence activated cell sorter). Seed 1E5/well Jurkat cells in a U-shaped 96-well plate, wash once with ice PBS, centrifuge at 1200 rpm for 3 min; after washing, add 200 ⁇ L/well of 3% FBS/PBS blocking solution, and incubate on ice for 1 h.
  • FACS flow cytometry, fluorescence activated cell sorter
  • Example 3 Design of a bispecific antibody format containing anti-TAA (tumor-associated antigen) and anti-CD3
  • the therapeutic effect of the CD3 double antibody is mainly by combining the tumor-associated antigen (TAA) expressed on tumor cells and the CD3 target on T cells, cross-linking these two cells to form an immune synapse, resulting in the activation of T cells, thereby killing tumor cells.
  • TAA tumor-associated antigen
  • Multivalent TAA antibodies can improve target selectivity, especially if the target is not specific enough.
  • the main direction to improve the safety of CD3 double antibody is to improve the selectivity of TAA, and at the same time, selecting the appropriate CD3 antibody affinity can maintain the efficacy of the drug while reducing toxic side effects, thereby improving the selectivity of tumors and reducing the targeting of healthy cells.
  • the molecular form design of the anti-TAA (tumor-associated antigen) and anti-CD3 bispecific antibody provided by the present invention is shown in FIG. 2 .
  • the anti-TAA and anti-CD3 bispecific antibody molecule shown in FIG. 2 includes: a first monomer (the left part in FIG. 2 ) and a second monomer (the right part in FIG. 2 ).
  • the first monomer includes two chains, chain 1: it has an Fd fragment (including VH and CH1); chain 2: it has a light chain fragment (including VL and CL), and the first Fc chain.
  • the second monomer also includes 2 chains, chain 1: it has Fd fragment (including VH and CH1); chain 2: it has light chain fragment (including VL and CL), anti-CD3 antibody fragment (Fig. CD3 Antibody in the form of scFv), and a second Fc chain.
  • the light chain fragment and the Fd fragment are respectively paired to form an anti-TAA Fab domain (to realize the function of the TAA Antibody in FIG. 2 ).
  • the light chain fragment of the first monomer is fused with the first Fc chain; at the same time, the N-terminal of the anti-CD3 antibody fragment is fused with the light chain fragment of the second monomer, and the C-terminal of the anti-CD3 antibody fragment is fused with the second Fc chain chain fusion.
  • the first Fc chain and the second Fc chain polymerize to form an Fc domain.
  • the Fc domain may be selected from Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc or variants thereof.
  • TAA selects CLDN18.2 and designs anti-CLDN18.2/CD3 bispecific antibody.
  • the sequence of CLDN18.2 antibody can refer to the sequence of antibody QP14611463 in patent document CN202010344676.8, and the anti-CD3 antibody uses SP34 Antibody light and heavy chain sequences of sequence and its variants QP3685, QP3689, QP3690.
  • the anti-CD3 antibody sequence of QP374537493746 is selected from SP34
  • the anti-CD3 antibody sequence of QP090837493746, QP090937493746, and QP091037493746 is selected from QP3685, QP3689, and QP3690. Protein expression and sequence numbers are shown in the table below.
  • the molecular design of QP372337241461 is shown in Figure 3.
  • the molecular design of QP374437481461 is shown in Figure 4.
  • Primer design Use the online software DNAWorks (v3.2.4) ( http://helixweb.nih.gov/dnaworks/ ) to design multiple primers to synthesize gene fragments containing the required recombination.
  • Fragment splicing According to the instruction manual of Primer STAR GXL DNA polymerase from TaKaRa Company, use the multiple primers designed above to amplify by PCR to obtain the gene fragments required for recombination.
  • the first step of PCR 50 ⁇ L of PCR reaction system including 10 ⁇ L PrimeSTAR GXL Buffer (5 ⁇ ); 4 ⁇ L dNTP Mixture (2.5 mmol L-1); 1 ⁇ L each of the primers shown above; 1 ⁇ L PrimeSTAR GXL DNA Polymerase.
  • the PCR reaction conditions were 98°C for 2min, 98°C for 20s, 55°C for 15s, 68°C for 30s, 30 cycles; 68°C for 5min.
  • the second step of PCR use the PCR product of the first step as a template, and carry out PCR amplification with the first and last primers, and the conditions are the same as the first step. PCR construction to amplify the target fragment.
  • Expression vector pQD use some special restriction enzymes, such as BsmBI, to design and construct expression vector pQD (with signal peptide), BsmBI digestion vector, gel recovery spare.
  • Recombination construction of expression vectors Recombination of target gene fragments and expression vector pQD (with signal peptide fragments) recovered by BsmBI enzyme digestion were added to DH5 ⁇ competent cells at a ratio of 3:1, ice bathed at 0°C for 30 minutes, heat shocked at 42°C for 90s, added 5 Double the volume of LB medium, incubate at 37°C for 45min, spread on LB-Amp plate, incubate overnight at 37°C, pick single clones and send them for sequencing to obtain the target clones.
  • the cell density to 1 ⁇ 10 6 /ml by 293E cells.
  • the amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1.
  • the plasmid and PEI were mixed and allowed to stand for 15 minutes.
  • the plasmid and PEI mixture was slowly added to the 293E cells, and cultured in a shaker with 8% CO2, 120 rpm, and 37°C.
  • the cell supernatant was collected and purified by centrifuging at 4700rpm for 20min in a horizontal centrifuge.
  • Protein affinity chromatography the supernatant of the cell culture medium was centrifuged at high speed, and the protein A chromatography column of GE was used for affinity chromatography. Chromatography uses an equilibration buffer of 1 ⁇ PBS (pH7.4). After the cell supernatant is loaded and combined, it is washed with PBS until the ultraviolet rays return to the baseline, and then the target protein is eluted with an elution buffer of 0.1M glycine (pH3.0).
  • Human lymphocytic leukemia cells (Jurkat cells) express natural CD3, and the binding of anti-CD3 antibody and its mutants to Jurkat cells was detected by FACS. Seed 1E5/well Jurkat cells in a U-shaped 96-well plate, wash once with ice PBS, centrifuge at 1200 rpm for 3 min; after washing, add 200 ⁇ L/well of 3% FBS/PBS blocking solution, and incubate on ice for 1 h.
  • Example 6 CLDN18.2/CD3 bispecific antibody-mediated killing of human gastric cancer cells by T cells
  • the target cell was selected as the human gastric cancer cell line NUGC4-CLDN18.2 stably expressing CLDN18.2.
  • Effector cells were human PBMCs. According to the E:T ratio of 10:1, add different concentrations of antibodies, culture at 37°C, 5% CO 2 for 48h, and use CytoTox Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) detects LDH in the cell culture supernatant and quantifies the cytotoxicity %.
  • the maximum lysis rate (100%) of the target cells is that the target cells are treated with 1% Triton X-100 so that the cells are lysed to release all LDH.
  • %Cytotoxicity [(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]*100.
  • Example 7 CLDN18.2/CD3 bispecific antibody-mediated killing of human lung cancer cells by T cells
  • the target cell was selected as the human lung cancer cell line HCC827-CLDN18.2 stably expressing CLDN18.2.
  • Effector cells were human PBMCs. According to the E:T ratio of 10:1, add different concentrations of antibodies, culture at 37°C, 5% CO2 for 48h, and use CytoTox Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) detects LDH in the cell culture supernatant and quantifies the cytotoxicity %.
  • the maximum lysis rate (100%) of the target cells is that the target cells are treated with 1% Triton X-100 so that the cells are lysed to release all LDH.
  • %Cytotoxicity [(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]*100.
  • CD3 humanized mouse CD3EDG HuGEMM
  • Colorectal cancer cells MC38-hCLDN18.2 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration for inoculation.
  • Each experimental mouse was subcutaneously inoculated with 5 ⁇ 10 6 MC38-hCLDN18.2 cells on the right back, and the tumor growth was observed regularly.
  • the mice were randomly divided into groups according to the tumor size and the weight of the mice.
  • the dosing regimen is as follows:
  • the tumor inhibition rate is shown in Table 7, and the tumor inhibition curve is shown in Figure 12.
  • Figure 13 shows the changes in body weight of mice in each group after administration. The results showed that the body weight of the mice in the administration group did not change substantially and grew well, which proved that the drug safety was good.
  • CLDN18.2/CD3 bi-antibody inhibits tumor growth in the in vivo animal model of colorectal cancer cells MC38-hCLDN18.2 subcutaneously transplanted into CD3EGD HuGEMM mice
  • mice A total of 36 mice (24plus50%) were needed for the experiment. Animals were provided by qualified suppliers. Tumor inoculation: 5*10 6 X-CLDN18.2/MIA PaCa-2 and 2*10 6 PBMC cells were inoculated on the right side of the neck of each mouse. At the same time as the vaccination, the experimental animals were marked with ear tags as the only confirmation mark for subsequent experiments. Waiting for tumor growth, when the average tumor volume reached about 60-100mm3 , randomized group dosing began. The experimental grouping and dosing regimen are shown in Table 8.
  • Table 8 Dosing regimens for evaluating the inhibitory effect of anti-CLDN18.2/CD3 biantibody molecules on tumor growth
  • the drug was administered 5 times, and the tumor was measured twice a week.
  • the tumor inhibition rate is shown in Table 9, and the tumor inhibition curve is shown in Figure 14.
  • the tumor inhibition rate (TGI) of 4mpk reached 92.3%
  • the tumor inhibition rate (TGI 69.3%) of QP374537493746 high-dose group (8mpk).
  • Figure 15 shows the changes in body weight of mice in each group after administration. The results showed that the body weight of the mice in the administration group did not change substantially and grew well, which proved that the drug safety was good.
  • CD3 bis-antibodies have shown great potential in hematological tumors, CD3 bis-antibodies still face some challenges in the treatment of solid tumors.
  • CD3 dual-antibody therapy is associated with T cell infiltration and immunosuppression in solid tumor tissues. When there are few infiltrating T cells, it is easy to develop resistance to CD3 dual-antibody therapy.
  • IL15 is a soluble cytokine that activates T cells and NK cells and mediates the proliferation and survival of these cells.
  • the present invention designs the TAA/CD3/IL15 multifunctional fusion protein in the hope that the cytokine IL15 component in it can stimulate the proliferation of immune cells including T cells and change the immune microenvironment of the tumor.
  • Example 10 Design of anti-TAA (tumor-associated antigen), anti-CD3 and multifunctional fusion protein containing IL15/IL15Ra
  • the invention provides multiple anti-TAA (tumor-associated antigen), anti-CD3 and multifunctional fusion proteins containing IL15/IL15Ra.
  • the first type of multifunctional fusion protein can refer to Figure 16 to Figure 19, the fusion protein molecule includes the first monomer (the left part in the figure) and the second monomer (the right part in the figure).
  • the first monomer includes 2 chains, chain 1: it has Fd fragment (including VH and CH1); chain 2: it has light chain fragment (including VL and CL), cytokine domain (including IL15 and IL15Ra) , the first Fc chain.
  • the second monomer includes two chains, chain 1: it has Fd fragments (including VH and CH1); chain 2: it has light chain fragments (including VL and CL), anti-CD3 antibody fragments (CD3 Antibody in the figure) , which is in scFv format), and a second Fc chain.
  • the light chain fragment and the Fd fragment are respectively paired to form an anti-TAA Fab domain (to realize the function of the Anti-TAA Antibody in the figure).
  • the N-terminal of the cytokine functional region is fused with the light chain fragment of the first monomer, and the C-terminal is fused with the first Fc chain; the N-terminal of the anti-CD3 antibody fragment is fused with the light chain fragment of the second monomer, and the C-terminal is fused with the second Fc chain fragment.
  • Fc chain fusion The first Fc chain and the second Fc chain polymerize to form an Fc domain.
  • an anti-CD3 antibody fragment in scFv format may comprise: a heavy chain variable region (VH), a light chain variable region (VL), and a linker connecting the heavy chain variable region and the light chain variable region fragment.
  • VH heavy chain variable region
  • VL light chain variable region
  • linker connecting the heavy chain variable region and the light chain variable region fragment.
  • the amino acid fusion sequence of the anti-CD3 antibody fragment can be "VH(CD3) ⁇ VL(CD3)", or "VL(CD3) ⁇ VH(CD3)"; " ⁇ ” Represents a connected fragment.
  • the fusion sequence of amino acid fragments in the cytokine functional region may be "IL-15-IL-15Ra", or "IL-15Ra-IL-15".
  • the four polypeptide chains of the fusion protein shown in Figure 16 can be described as follows: (1) the first polypeptide chain: VH-CH1; (2) the second polypeptide chain: VL-CL- IL15Ra-IL15 -the first Fc chain ; (3) The third polypeptide chain: VL-CL- VH(CD3)-VL(CD3) -the second Fc chain; (4) The fourth polypeptide chain: VH-CH1. "-" indicates linking by peptide bond or connecting fragment.
  • the four polypeptide chains of the fusion protein shown in Figure 17 are described as follows: (1) the first polypeptide chain: VH-CH1; (2) the second polypeptide chain: VL-CL- IL15-IL15Ra -the first Fc chain; (3) The third polypeptide chain: VL-CL- VH(CD3)-VL(CD3) -the second Fc chain; (4) The fourth polypeptide chain: VH-CH1.
  • the four polypeptide chains of the fusion protein shown in Figure 18 are described as follows: (1) the first polypeptide chain: VH-CH1; (2) the second polypeptide chain: VL-CL- IL15Ra-IL15 -the first Fc chain; (3) The third polypeptide chain: VL-CL- VL(CD3)VH(CD3) -the second Fc chain; (4) The fourth polypeptide chain: VH-CH1.
  • the four polypeptide chains of the fusion protein shown in Figure 19 are described as follows: (1) the first polypeptide chain: VH-CH1; (2) the second polypeptide chain: VL-CL- IL15-IL15Ra -the first Fc chain; (3) The third polypeptide chain: VL-CL- VL(CD3)VH(CD3) -the second Fc chain; (4) The fourth polypeptide chain: VH-CH1.
  • the second type of multifunctional fusion protein can refer to Figure 20 to Figure 27, the protein molecule includes: the first monomer (the left part in the figure) and the second monomer (the right part in the figure).
  • the first monomer comprises: anti-TAA antibody fragment (in the form of scFv, including VH and VL domains), cytokine functional region (including IL15 and IL15Ra), the first Fc chain;
  • the second monomer comprises: anti-TAA antibody fragment ( In the form of scFv, including VH and VL domains), an anti-CD3 antibody fragment (CD3 Antibody in the figure, which is in the form of scFv), and the second Fc chain.
  • the N-terminal of the cytokine functional region is fused with the anti-TAA antibody fragment of the first monomer, and the C-terminal is fused with the first Fc chain; the N-terminal of the anti-CD3 antibody fragment is fused with the anti-TAA antibody fragment of the second monomer, and the C-terminal is fused with the anti-TAA antibody fragment of the second monomer.
  • Second Fc chain fusion The first Fc chain and the second Fc chain polymerize to form an Fc domain.
  • an anti-CD3 antibody fragment in the form of scFv can include: a heavy chain variable region (VH), a light chain variable region (VL), and a linking heavy chain variable region and light chain Linking segments of variable regions.
  • VH heavy chain variable region
  • VL light chain variable region
  • VL linking heavy chain variable region and light chain Linking segments of variable regions.
  • the amino acid fusion sequence of the anti-CD3 antibody fragment can be "VH(CD3) ⁇ VL(CD3)", or "VL(CD3) ⁇ VH(CD3)”; " ⁇ ” Represents a connected fragment.
  • the fusion sequence of amino acid fragments in the cytokine functional region may be "IL-15-IL-15Ra", or "IL-15Ra-IL-15".
  • the anti-TAA antibody fragment in the form of scFv includes: a heavy chain variable region (VH), a light chain variable region (VL), and a connecting fragment connecting the heavy chain variable region and the light chain variable region; From the N-terminal to the C-terminal of the peptide chain, the amino acid fusion sequence of the anti-TAA antibody fragment can be "VH(TAA) ⁇ VL(TAA)", or "VL(TAA) ⁇ VH(TAA)"; " ⁇ " means Connect fragments.
  • the domains of the antibody molecules shown in Figure 20 to Figure 27 use the same amino acid sequence, the difference lies in the fusion sequence or configuration.
  • the two polypeptide chains of the fusion protein are described as follows: (1) the first polypeptide chain: VL(TAA)-VH(TAA)-IL15-IL15Ra-the first Fc chain; (2) the second Two polypeptide chains: VL(TAA)-VH(TAA)-VH(CD3)-VL(CD3)-second Fc chain.
  • the present invention also provides a TAA/IL15 bifunctional fusion protein activated by tumor-targeted T cells/NK cells, whose structural form can be referred to in Figure 28.
  • the difference is that Anti-CD3 antibody fragments were replaced with cytokine domains (including IL15 and IL15Ra).
  • the protein molecule has a symmetrical structural form.
  • Example 11 Molecular cloning, transient expression, and protein purification of CLDN18.2/CD3/IL15 multifunctional fusion protein
  • CLDN18.2/CD3/IL15 or CLDN18.2/IL15 multifunctional fusion protein wherein the CLDN18.2 antibody can refer to the sequence of antibody QP14611463 in the patent document CN202010344676.8, and the anti-CD3 antibody uses SP34 light,
  • the heavy chain sequence, protein expression and sequence number are shown in the table below.
  • IL15/IL15Ra-FC from Hengrui Pharmaceutical Company was constructed as a control, and the IL15/IL15Ra-FC protein number was QP33123313.
  • Clones were designed as shown in Table 10, and full-length expression vectors were constructed.
  • protein expression and protein purification please refer to Example 3.
  • Example 12 FACS detection of CLDN18.2/CD3/IL15 multifunctional fusion protein and CLDN18.2 binding activity
  • Example 13 FACS detection of CLDN18.2/CD3/IL15 multifunctional fusion protein and CD3 binding activity
  • Lymphocytic leukemia cells (Jurkat cells) express natural CD3, and the binding of anti-CD3 antibody and its mutants to Jurkat cells is detected by FACS. Seed 1E5/well Jurkat cells in a U-shaped 96-well plate, wash once with ice PBS, centrifuge at 1200 rpm for 3 min; after washing, add 200 ⁇ L/well of 3% FBS/PBS blocking solution, and incubate on ice for 1 h.
  • Example 14 Mo7e cell proliferation assay to evaluate the activity of multifunctional fusion protein IL15
  • Mo7e human giant cell leukemia cell line
  • IL-15R ⁇ cytokine growth-dependent cells.
  • Previous studies have shown that resting NK and naive T cells express the intermediate affinity IL-15R ⁇ phenotype, and the results of cytokine IL15/IL15Ra on the proliferation of Mo7e (IL-15R ⁇ ) cells are consistent with the proliferation of unstimulated PBMC (Mol Cancer Ther; 11(6) June 2012).
  • the Mo7e cell proliferation assay is used to evaluate the activity of the multifunctional fusion protein IL15. The method and results are as follows:
  • Mo7e cells human giant cell leukemia cell line
  • CCK-8 Cell Resource Center
  • recombinant Human GM-CSF was purchased from perprotech, Cat. No. 300-03
  • human IgG was purchased from Sigma, Cat. No. I4506; other antibodies were prepared in-house.
  • Mo7e cells were cultured in RPMI1640 containing 10% FBS, 2mM L-glutamine and 8ng/ml GM-CSF in a 5% CO 2 incubator at 37°C; collected Mo7e cells, centrifuged at 800rpm for 5 minutes and discarded Wash the cells twice with RPMI1640 medium without GM-CSF; resuspend the cells with RPMI1640 medium without GM-CSF and count them, and inoculate 2 ⁇ 10 cells in 80 ⁇ l per well in a 96-well plate , incubate in a 5% CO 2 incubator at 37°C for 1 hour; dilute each medicinal medium to be tested by a 4-fold gradient, mix 20 ⁇ l of each well with the cell suspension, and incubate in a 5% CO 2 incubator at 37°C for 3 1 day; add 10 ⁇ l of CCK-8 reagent to each well of the 96-well plate to be tested, and incubate in a 5% CO 2 incubator at 37°C for 3
  • Example 15 T cells killing human gastric cancer cells mediated by CLDN18.2/CD3/IL15 multifunctional fusion protein
  • the target cell was selected as the human gastric cancer cell line NUGC4-CLDN18.2 stably expressing CLDN18.2.
  • Effector cells were human PBMCs. According to the E:T ratio of 10:1, add different concentrations of antibodies, culture at 37°C, 5% CO 2 for 48h, and use CytoTox Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) detects LDH in the cell culture supernatant and quantifies the cytotoxicity %.
  • the maximum lysis rate (100%) of the target cells is that the target cells are treated with 1% Triton X-100 so that the cells are lysed to release all LDH.
  • %Cytotoxicity [(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]*100.
  • QP374508993746, QP090109003746, QP09023688, QP36670903, QP09040905, QP09060905, QP09040907 and QP09060907 can all mediate PBMC killing of human gastric cancer cell NUGC4-CLDN18.
  • QP374508993746 and QP090109003746 mediated the activity of PBMC to kill human lung cancer cell NUGC4-CLDN18.2 cells significantly better than the control molecule AMG910.
  • the activity of PBMCs mediated by other molecules to kill human lung cancer cell NUGC4-CLDN18.2 cells was comparable to that of the control molecule AMG910.
  • Example 16 T cell killing of human lung cancer cells mediated by CLDN18.2/CD3/IL15 multifunctional fusion protein
  • the target cell was selected as the human lung cancer cell line HCC827-CLDN18.2 stably expressing CLDN18.2.
  • Effector cells were human PBMCs. According to the E:T ratio of 10:1, add different concentrations of antibodies, culture at 37°C, 5% CO 2 for 48h, and use CytoTox Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) detects LDH in the cell culture supernatant and quantifies the cytotoxicity %.
  • the maximum lysis rate (100%) of the target cells is that the target cells are treated with 1% Triton X-100 so that the cells are lysed to release all LDH.
  • QP090109003746-mediated PBMC killing activity of human lung cancer cells HCC827-CLDN18.2 cells was significantly better than that of the control molecule AMG910.
  • the activity of PBMCs mediated by other molecules to kill human lung cancer cells HCC827-CLDN18.2 cells was comparable to that of the control molecule AMG910.
  • the anti-CD3 antibody fragments were selected from the antibody light and heavy chain sequences of SP34 variants QP3685, QP3689, and QP3690, respectively, and the cloned, expressed, and purified proteins were designed as shown in Table 10 in Example 11. The results showed that the molecules constructed using the antibody light and heavy chain sequences of QP3685, QP3689, and QP3690 also had CLDN18.2 binding activity and IL15 activity, anti-CD3 molecules also had CD3 binding activity, and also had the ability to mediate T cell killing of human gastric cancer cells or lung cancer cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供了抗CD3抗体变异体、融合蛋白及应用。通过对抗CD3抗体进行突变获得其变异体。提供的融合蛋白包括：(1)抗肿瘤相关抗原(TAA)/抗CD3双特异抗体；(2)抗肿瘤相关抗原(TAA)、抗CD3及含IL15/IL15Ra的多功能融合蛋白；(3)抗肿瘤相关抗原(TAA)及含IL15/IL15Ra的融合蛋白。还提供了编码抗体分子的核酸分子及载体，以及抗体分子的制药用途。

Description

抗CD3抗体变异体、融合蛋白及应用 技术领域
本发明涉及生物医药技术领域，具体涉及抗CD3抗体变异体、融合蛋白及应用。
背景技术
T淋巴细胞(T lymphocyte)简称T细胞，是由来源于骨髓的淋巴干细胞，在胸腺中分化、发育成熟后，通过淋巴和血液循环而分布到全身的免疫器官和组织中发挥免疫功能。T细胞是一种高效的杀伤细胞，能迅速消灭病毒感染细胞和癌细胞。T细胞杀伤作用需要免疫突触的形成，而这一过程高度依赖于TCR识别抗原递呈细胞表面MHC分子与所递呈的抗原肽形成的复合物。激活的免疫突触将释放细胞毒素和细胞因子起到杀伤作用。在免疫突触形成过程中，受T细胞与靶细胞之间距离的限制，双特异性或多特异性抗体定向T细胞就要模拟免疫突触的形成，一端通过靶向CD3来实现桥连T细胞TCR受体，另一端桥连靶细胞通过靶向靶细胞表面抗原来实现。CD3分子表达于所有成熟T细胞表面，并与TCR呈非共价结合，形成完整的TCR-CD3复合物，共同参与对抗原刺激的免疫应答，是目前在双特异性抗体中应用最多且最成功的免疫效应细胞表面的触发分子。靶向CD3的双特异性抗体则能够分别结合T细胞表面CD3和肿瘤细胞表面抗原，从而拉近细胞毒性T细胞(cytotoxic T cell,Tc或CTL)与肿瘤细胞的距离，并直接激活T细胞，诱导T细胞直接杀伤癌细胞，而不再依赖于传统的T细胞的双重激活信号。
在已公开的CD3抗体中，OKT3是具有里程碑式影响力的抗体，作为第一个上市的治疗性抗体，其开创了单克隆抗体治疗疾病的先河。SP34是目前为数不多公开的可以和猴子具有种属反应的CD3抗体。近年来，科学界在双抗领域有了新的突破，从而发展出不同种类的新型CD3双抗(CD3-BsAb)。目前，已有100多种不同结构的CD3-BsAb被成功开发出来。众多的形式决定了抗体的诸多特性，例如半衰期、免疫原性、疗效反应类型、穿透实体瘤能力等等。安进的Blinatumomab(BiTE格式的CD19xCD3)于2014年获得FDA批准上市，是目前最成功的CD3-BsAb双抗。该药用于治疗复发或难治的急性淋巴细胞白血病(ALL)患者。与标准化疗相比，40％以上接受Blinatumomab治疗的患者能达到完全或部分缓解(CR/PR)，中位总生存期能提高多几个月。除Blinatumomab外，目前还有许多其他CD3-BsAb正在进行临床试验，例如CD19、CD20、CD38、BCMA、CD33和CD123等等。此外，在一个1/2期临床试验中，患有急性骨髓性白血病(AML)的患者使用MacroGenics公司的Flotetuzumab(CD123xCD3-BsAb)进行治疗，其总有效率(ORR)为30％。另一项Abbvie/Genmab公司的Epcoritamab(CD20xCD3-BsAb)的1/2期临床实验中，对弥漫性淋巴大B细胞瘤(DLBCL)患者显示出44％的完全缓解(CR)和11％的部分缓解(PR)，而滤泡性淋巴瘤患者则有100％的缓解(PR)。其他多个CD20xCD3双抗也获得了类似的 优异结果，例如，在NSG小鼠中，再生元公司的REGN1979(CD20xCD3-BsAb)比罗氏的Rituximab(CD20单抗)能更好地控制肿瘤生长，进一步证明了CD3双抗类药物的疗效。罗氏的CD20xCD3T细胞结合双特异性抗体glofitamab(前称CD20-TCB)治疗复发或难治性(R/R)弥漫性大B细胞淋巴瘤(DLBCL)患者的最新数据，该扩展研究纳入了过度预治疗且高度难治的DLBCL患者，58.3％的患者对其初始治疗无效，约三分之一(33.1％)的患者之前接受过CAR-T细胞治疗。根据独立审查委员会(IRC)评估，中位随访12.6个月，39.4％的患者(61/155)达到CR(主要疗效终点)，一半患者(51.6％；80/155)达到总体缓解(部分缓解[PR]+完全缓解[CR]的患者比例；次要疗效终点)，安全性方面，细胞因子释放综合征(CRS)是最常见的不良事件，发生在63.0％的患者中。CRS事件是可预测的，通常级别较低(主要是1级[47.4％]或2级[11.7％])，在初始剂量时发生，只有一名患者因CRS停止glofitamab治疗。3级及以上CRS的发生率较低(3.9％)，无5级事件。glofitamab具有一种“2:1”的新颖结构模式，含有2个靶向CD20的Fab区，一个靶向结合CD3的Fab区。
安进公司的Blinatumomab在临床试验中显示细胞因子风暴(CRS)为其最重要的毒副反应之一。CD19表达的肿瘤细胞，与大量健康的B细胞和T细胞处于同一环境时，会使CD3-BsAb介导的T细胞活化突发性地进行，从而过度释放炎性细胞因子，如IFN-γ、IL-6和TNF-α等，最终导致从发热到多重器官衰竭等症状。然而，CRS不是Blinatumomab独有的毒副作用。事实上，在大部分CD3-BsAb和CAR-T治疗中，CRS都经常出现。在人源化小鼠模型中表明，CD3-BsAb诱导CRS的主要介质物是被激活T细胞所产生的TNF-α，从而导致monocytes细胞大量分泌炎性细胞因子。研究发现，阻断上游TNF-α和其下游的IL-1β或IL-6都能够有效地缓解CRS。另外，在小鼠和猴子模型中进行的几项临床前研究表明，通过降低CD3的亲和力(即使用“较弱”的CD3)，也可降低细胞因子风暴的程度。
白细胞介素-15(interleukin-15，IL-15)是一种长14–15kDa的细胞因子，对于NK细胞、NKT细胞和memory CD8+T细胞功能非常重要。IL-15通过与其受体IL-15Rα结合产生具有极高生物效价的复合物IL-15超激动剂(IL-15SA)后一起被转导转运至靶细胞。IL-15SA强烈激活响应表达IL-15R的细胞，特别是NK细胞/T细胞，从而促进抗肿瘤和抗病毒功能。IL-15具有广泛的免疫调节作用，能够参与调节T细胞，特别是NK细胞、记忆性CD8+T细胞的存活、增殖与功能。IL-15与IL-2结构十分相似，同属于螺旋型细胞因子家族。IL-15的异三聚体受体与IL-2受体共用IL-2R/IL-15Rβ(CD122)和共同γc链(CD132)，由于这些共同的受体成分和共用JAK 1/JAK 3/STAT 5信号通路，IL-15与IL-2具有相同的功能，包括刺激T细胞增殖、产生细胞毒性T淋巴细胞、刺激B细胞合成免疫球蛋白以及NK细胞的产生和持续存活。在许多适应性免疫应答中，IL-2和IL-15具有独特且常为竞争性的作用。主要有以下四点：1、IL-2可以调节Tregs细胞的激活，而IL-15则不能调节。2、IL-2可以通过激活诱导的CD8+效应T细胞的细胞死亡从而抑制T细胞反应。3、IL-15对NK、效应细胞CD8+和记忆表型CD8+T细胞的分化起着必不可少的作用。4、临床前研究标明，IL-15与IL-2的毒性不同，与IL-2相比，IL-15几乎观察不到血 管毛细血管渗漏。这些因素使得IL-15成为肿瘤免疫策略中更具潜力的细胞因子。目前有多个IL-15靶点产品仍处于临床试验阶段，其中最值得关注的是IL-15超级激动剂N-803，为IL-15蛋白N72D突变后结合IL-15Ra及IgG1Fc共同表达的融合蛋白，2022年5月23日，ImmunityBio向FDA递交IL-15超级激动剂N-803的上市申请，用于与卡介苗(BCG)联合治疗卡介苗不响应的非肌肉浸润性膀胱癌(NMIBC)。2021年公布的临床数据，N-803+BCG联合治疗效果显著，CR率达到71％(59/83)，平均CR维持时间为24.1个月。一项1期临床实验显示，Nivolumab(PD-1抗体，Opdivo)联合N-803治疗转移性非小细胞肺癌的能够明显延长患者长期生存。基因泰克与Xencor公司合作开发IL-15细胞因子
Figure PCTCN2022096501-appb-000001
目前正在进行
Figure PCTCN2022096501-appb-000002
联用Tecentriq临床研究。恒瑞医药的SHR-1501(IL-15融合蛋白)于2019年5月14日获批开展SHR-1501临床试验。此外，SHR-1316(PD-L1单抗药物)将联合SHR-1501用于治疗失败的晚期恶性肿瘤患者。
目前用于治疗的CD3双特异性抗体的开发需求仍未得到满足，怎么解决在实体瘤中所面临的挑战，提高CD3-BsAb双抗的安全性，都需要继续进行开发研究。
发明的公开
本发明的第一目的是提供一种抗CD3抗体变异体，所述抗CD3抗体包括：重链可变区和轻链可变区；所述重链可变区包括：VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包括：VLCDR1、VLCDR2、VLCDR3；所述抗CD3抗体的氨基酸序列包括SEQ ID NO：29-34所示的VHCDR1、VHCDR2、VHCDR3、VLCDR1、VLCDR2和VLCDR3；所述抗CD3抗体变异体的突变位点包括以下任意1个或2个以上位点：SEQ ID NO：30所示序列的第十位点，SEQ ID NO：31所示序列的第二、七位点，SEQ ID NO：33所示序列的第三、四位点，SEQ ID NO：34所示序列的第四、五、六、七位点。
优选地，所述的突变是指氨基酸被取代。
优选地，变异体的突变位点包括以下任意1个或2个以上位点：SEQ ID NO：30所示序列中第十位的A被取代为E，SEQ ID NO：31所示序列中第二位的G被取代为S、第七位的S被取代为G，SEQ ID NO：33所示序列中第三位的N被取代为W、第四位的K被取代为L，SEQ ID NO：34所示序列中第四位的Y被取代为N或R、第五位的S被取代为K、第六位的N被取代为G、第七位的L被取代为G。
优选地，VHCDR2的氨基酸序列如SEQ ID NO:35所示；或者，VHCDR3的氨基酸序列如SEQ ID NO:36所示；或者，VLCDR2的氨基酸序列如SEQ ID NO:37或38所示；或者，VLCDR3的氨基酸序列如SEQ ID NO:39、40或41所示。
优选地，所述VHCDR1、VHCDR2、VHCDR3、VLCDR1、VLCDR2和VLCDR3的氨基酸序列如以下任意一组序列所示：
(1)SEQ ID NO：29、35、31、32、37、39；
(2)SEQ ID NO：29、35、36、32、37、40；
(3)SEQ ID NO：29、35、36、32、38、41。
优选地，所述抗体变异体的重链可变区和/或轻链可变区选自SEQ ID NO：1、2或3所示序列的重链可变区和/或轻链可变区，或与其具有至少90％序列同一性。
优选地，所述重链可变区和所述轻链可变区之间具有用于形成二硫键的突变，所述突变的位点包括以下组合形式中的任意一种或两种以上，突变位点根据EU编号，所述重链可变区以VH表示，所述轻链可变区以VL表示：
Figure PCTCN2022096501-appb-000003
优选地，所述抗CD3抗体变异体还包含：选自人源IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区；以及选自人源κ、λ链或其变体的轻链恒定区；所述重链恒定区包含：Fc片段或其变体。
优选地，所述抗体变异体为scFv，其包含重链可变区、轻链可变区、以及连接所述重链可变区和所述轻链可变区的连接片段，所述连接片段的氨基酸序列优选为若干个GGGGS重复，更优选为3个GGGGS重复。
本发明的另一目的是提供所述的抗CD3抗体变异体在制备用于抑制或治疗癌症的药物中的应用。
优选地，所述癌症选自以下癌症或发生于以下部位：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤、以及骨髓增生异常综合症。
本发明的另一目的是提供一种核酸分子，其编码所述的抗CD3抗体变异体。
本发明的另一目的是提供一种表达载体，其包含上述的核酸分子。
本发明的另一目的是提供一种抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其为异二聚体形式，包括第一单体和第二单体；
所述第一单体包括：(a)Fd片段；(b)轻链片段及第一Fc链；所述第二单体包括：(a)Fd片段；(b)轻链片段、抗CD3抗体片段以及第二Fc链；所述轻链片段包括VL结构域和CL结构域；所述Fd片段包括VH结构域和CH1结构域；在所述第一单体或所述第二单体中，所述轻链片段与所述Fd片段配对形成抗 TAA的Fab结构域；其中，所述第一单体的轻链片段与所述第一Fc链融合；所述抗CD3抗体片段的N端与所述第二单体的轻链片段融合，C端与所述第二Fc链融合；
或者，所述第一单体包括：(a)Fd片段；(b)轻链片段、细胞因子功能区、第一Fc链；所述第二单体包括：(a)Fd片段；(b)轻链片段、抗CD3抗体片段、以及第二Fc链；所述细胞因子功能区包括IL-15及IL-15Ra；其中，所述细胞因子功能区的N端与所述第一单体的轻链片段融合，C端与所述第一Fc链融合；所述抗CD3抗体片段的N端与所述第二单体的轻链片段融合，C端与所述第二Fc链融合；
或者，所述第一单体包括：抗TAA抗体片段、细胞因子功能区、第一Fc链；所述第二单体包括：抗TAA抗体片段、抗CD3抗体片段、以及第二Fc链；其中，所述细胞因子功能区的N端与所述第一单体的抗TAA抗体片段融合，C端与所述第一Fc链融合；所述抗CD3抗体片段的N端与所述第二单体的抗TAA抗体片段融合，C端与所述第二Fc链融合；
所述第一Fc链与所述第二Fc链能够互换。
优选地，所述抗TAA抗体片段为scFv形式，包括：重链可变区(VH)、轻链可变区(VL)、以及连接所述重链可变区和所述轻链可变区的连接片段；自肽链的N端到C端，所述抗TAA抗体片段的氨基酸融合顺序为“VH～VL”，或者，“VL～VH”；“～”表示连接片段；或者，所述抗CD3抗体片段为scFv形式，包括：重链可变区(VH)、轻链可变区(VL)、以及连接所述重链可变区和所述轻链可变区的连接片段；自肽链的N端到C端，所述抗CD3抗体片段的氨基酸的融合顺序为“VH～VL”，或者，“VL～VH”；“～”表示连接片段；或者，自肽链的N端到C端，所述细胞因子功能区的氨基酸片段的融合顺序为“IL-15～IL-15Ra”，或者，“IL-15Ra～IL-15”；“～”表示连接片段；优选地，所述IL-15～IL-15Ra的序列如SEQ ID NO:42所示，所述IL-15Ra～IL-15的序列如SEQ ID NO:43所示。
优选地，所述抗CD3抗体片段的氨基酸序列选自结合CD3的特异性抗体、抗体片段、单域抗体或其人源化形式；优选的，所述抗CD3抗体片段的氨基酸序列选自SP34、OKT3、UCTH1或其衍生物。
优选地，所述抗CD3抗体片段包括重链可变区和轻链可变区；所述抗CD3抗体片段的重链可变区包括：VHCDR1、VHCDR2、VHCDR3；所述抗CD3抗体片段的轻链可变区包括：VLCDR1、VLCDR2、VLCDR3；所述VHCDR1、VHCDR2、VHCDR3、VLCDR1、VLCDR2和VLCDR3的氨基酸序列如权利要求5所述的抗CD3抗体变异体中的任意一组序列所示。
优选地，IL-15Ra和IL-15形成IL-15/IL-15Ra复合物；IL-15包括：IL-15及其能结合IL-15Ra的突变，截短及各种衍生物；IL-15Ra包括：IL-15Ra及其能结合IL-15的突变，截短及各种衍生物；优选地，所述IL-15/IL-15Ra复合物包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15或IL-15Ra的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示；所述IL-15Ra的母本序列如SEQ ID NO:45所示：
组合 IL15 IL15Ra
1 wt D96
2 wt D96/P97
3 wt D96/P97/A98
4 E87C D96/C97
5 E87C D96/P97/C98
6 E87C D96/C97/A98
7 V49C S40C
8 L52C S40C
9 E89C K34C
10 Q48C G38C
11 E53C L42C
12 C42S A37C
13 L45C G38C
14 L45C A37C
优选地，所述IL-15包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示：
组合 IL15突变
1 N1D
2 N4D
3 D8N
4 D30N
5 D61N
6 E64Q
7 N65D
8 Q108E
9 N1D/D61N
10 N1D/E64Q
11 N4D/D61N
12 N4D/E64Q
13 D8N/D61N
14 D8N/E64Q
15 D61N/E64Q
16 E64Q/Q108E
17 N1D/N4D/D8N
18 D61N/E64Q/N65D
19 N1D/D61N/E64Q/Q108E
20 N4D/D61N/E64Q/Q108E
优选地，所述TAA选自CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1、CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB或Glypican-3。
优选地，所述第一Fc链和所述第二Fc链聚合形成Fc段；所述Fc段选自Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc或其变异体，优选自IgG1 Fc、或Human IgG4 Fc或其变异体；所述 蛋白分子为Fc异源二聚体形式；优选地，所述Fc异源二聚体包含但不限于以下突变的组合，以下突变为根据EU计数：
Figure PCTCN2022096501-appb-000004
优选地，所述蛋白分子包含Fc段，Fc段选择消除免疫效应功能，包含但不限于以下突变的组合，以下突变为根据EU计数：
Figure PCTCN2022096501-appb-000005
优选地，所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子由以下任意一组序列所示的氨基酸片段融合得到：
(1)SEQ ID NO:05、SEQ ID NO:06、SEQ ID NO:07；
(2)SEQ ID NO:08、SEQ ID NO:06、SEQ ID NO:07；
(3)SEQ ID NO:09、SEQ ID NO:06、SEQ ID NO:07；
(4)SEQ ID NO:10、SEQ ID NO:06、SEQ ID NO:07；
(5)SEQ ID NO:05、SEQ ID NO:17、SEQ ID NO:07；
(6)SEQ ID NO:01、SEQ ID NO:18、SEQ ID NO:07；
(7)SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:07；
(8)SEQ ID NO:19、SEQ ID NO:18、SEQ ID NO:07；
(9)SEQ ID NO:20、SEQ ID NO:21；
(10)SEQ ID NO:22、SEQ ID NO:21；
(11)SEQ ID NO:20、SEQ ID NO:23；
(12)SEQ ID NO:22、SEQ ID NO:23；
(13)SEQ ID NO:24、SEQ ID NO:25；
(14)SEQ ID NO:26、SEQ ID NO:25；
(15)SEQ ID NO:24、SEQ ID NO:27；
(16)SEQ ID NO:26、SEQ ID NO:27。
本发明的另一目的是提供一种核酸分子，其编码所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子。
本发明的另一目的是提供所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子在制备用于抑制或治疗癌症的药物中的应用，所述癌症包括选自以下的癌症或发生于以下部位：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤、以及骨髓增生异常综合症。
本发明的另一目的是提供一种抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其包括两条多肽链，任意一条多肽链包括：抗TAA抗体片段、细胞因子功能区、Fc片段；所述抗TAA抗体片段为scFv形式；所述细胞因子功能区包括IL-15及IL-15Ra；所述细胞因子功能区的N端与所述抗TAA抗体片段融合，C端与所述Fc片段融合。
优选地，所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其IL-15Ra和IL-15形成IL-15/IL-15Ra复合物；IL-15包括：IL-15及其能结合IL-15Ra的突变，截短及各种衍生物；IL-15Ra包括：IL-15Ra及其能结合IL-15的突变，截短及各种衍生物；优选地，所述IL-15/IL-15Ra复合物包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15或IL-15Ra的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示；所述IL-15Ra的母本序列如SEQ ID NO:45所示：
组合 IL15 IL15Ra
1 wt D96
2 wt D96/P97
3 wt D96/P97/A98
4 E87C D96/C97
5 E87C D96/P97/C98
6 E87C D96/C97/A98
7 V49C S40C
8 L52C S40C
9 E89C K34C
10 Q48C G38C
11 E53C L42C
12 C42S A37C
13 L45C G38C
14 L45C A37C
优选地，所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其IL-15包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示：
组合 IL15突变
1 N1D
2 N4D
3 D8N
4 D30N
5 D61N
6 E64Q
7 N65D
8 Q108E
9 N1D/D61N
10 N1D/E64Q
11 N4D/D61N
12 N4D/E64Q
13 D8N/D61N
14 D8N/E64Q
15 D61N/E64Q
16 E64Q/Q108E
17 N1D/N4D/D8N
18 D61N/E64Q/N65D
19 N1D/D61N/E64Q/Q108E
20 N4D/D61N/E64Q/Q108E
优选地，所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其包含Fc段，Fc段选择消除免疫效应功能，包含但不限于以下突变的组合，以下突变为根据EU计数：：
Figure PCTCN2022096501-appb-000006
优选地，所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其任意一条多肽链的氨基酸序列如SEQ ID NO:28所示，或与其具有至少90％序列同一性。
本发明的另一目的是提供一种核酸分子，其编码所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白。
本发明的另一目的是提供所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白在制备用于抑制或治疗癌症的药物中的应用，所述癌症包括选自以下的癌症或发生于以下部位：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤、以及骨髓增生异常综合症。
相对于现有技术，本发明具有以下有益效果：
(1)本发明对现有抗CD3抗体进行突变，获得了新的抗CD3抗体变异体。
(2)本发明还提供了具有新颖结构的抗体分子，其具有较好的生物学活性。
附图的简要说明
图1为FACS检测CD3抗体SP34及其突变体与Jurkat细胞结合图。
图2为抗TAA(肿瘤相关抗原)及抗CD3的双特异抗体分子形式设计图。
图3为QP372337241461的分子设计图。
图4为QP374437481461的分子设计图。
图5、图6为FACS检测CLDN18.2/CD3双抗等分子结合CLDN18.2结果图。
图7、图8为FACS检测CLDN18.2/CD3双抗等分子结合CD3结果图。
图9、图10为CLDN18.2/CD3抗体等分子介导的PBMC杀伤人胃癌细胞NUGC4-CLDN18.2细胞结果图。
图11为CLDN18.2/CD3抗体等分子介导的PBMC杀伤人肺癌细胞HCC827-CLDN18.2细胞结果图。
图12为CLDN18.2/CD3双抗分子在CD3EGD HuGEMM小鼠皮下移植结直肠癌细胞MC38-hCLDN18.2体内动物模型中抑制肿瘤生长曲线图。
图13为在CD3EGD HuGEMM小鼠皮下移植结直肠癌细胞MC38-hCLDN18.2体内动物模型给药后各组小鼠体重变化曲线图。
图14、图15为CLDN18.2/CD3双抗分子在PBMC人源化NOG小鼠X-CLDN18.2/MIA PaCa-2皮下移植瘤模型中抑制肿瘤生长曲线图。
图16至图19为一类型抗TAA/CD3及含IL15/IL15Ra的多功能融合蛋白的结构形式设计图。
图20至图27为另一类型的抗TAA/CD3及含IL15/IL15Ra的多功能融合蛋白的结构形式设计图。
图28为肿瘤靶向T细胞/NK细胞激活的TAA/IL15双功能融合蛋白的结构形式图。
图29为FACS检测CLDN18.2/CD3/IL15多功能融合蛋白结合CLDN18.2结果图。
图30为FACS检测CLDN18.2/CD3/IL15及CLDN18.2/IL15融合蛋白结合CLDN18.2结果图。
图31为FACS检测CLDN18.2/CD3/IL15多功能融合蛋白结合CD3结果图。
图32为FACS检测CLDN18.2/CD3/IL15及CLDN18.2/IL15融合蛋白结合CD3结果图。
图33、图34、图35为Mo7e细胞增殖实验检测多功能融合蛋白IL15活性结果图。
图36、图37为CLDN18.2/CD3/IL15介导的PBMC杀伤人胃癌细胞NUGC4-CLDN18.2细胞结果图。
图38为CLDN18.2/CD3/IL15介导的PBMC杀伤人肺癌细胞HCC827-CLDN18.2细胞结果图。
实现本发明的最佳方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。
本实验中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的 条件。未注明具体来源的试剂，为市场购买的常规试剂。
本文中，“/”是指“和”。例如抗肿瘤相关抗原(TAA)/抗CD3双特异抗体是指抗体同时靶向TAA和CD3。
术语“融合”指由肽键直接连接或借助连接片段连接组分，或通过分子间相互作用进行融合。在单条肽链中，融合是指由肽键直接连接或借助连接片段连接。“多功能融合蛋白”是指包含两个或更多个抗原结合结构域的蛋白，能够结合两个或更多个不同的表位(例如，两个、三个或更多个不同的表位)，多功能融合蛋白还可以包含细胞因子(例如IL-15、IL-15Ra)等。“融合位置”是指功能区或者结构域在肽链中的位置，指示该肽链上各功能片段的连接顺序。
术语“多肽”是指任何长度的氨基酸链，包括蛋白质及其片段。本发明将多肽公开为氨基酸残基序列。那些序列按氨基末端到羧基末端的方向从左到右书写。根据标准命名法，氨基酸残基序列以三字母或单字母代码命名，如下所示：丙氨酸(Ala,A)、精氨酸(Arg,R)、天冬酞胺((Asn,N)、天冬氨酸(Asp，D)、半肌氨酸(Cys,C)、谷氨酞胺(Gln,Q)、谷氨酸(Glu,E)、甘氨酸(Gly,G)、组氨酸(His,H)、异亮氨酸(I1e，I)、亮氨酸(Leu,L)、赖氨酸(Lys,K)、甲硫氨酸(Met,M)、苯丙氨酸(Phe，F)、脯氨酸(Pro,P)、丝氨酸(Ser,S)、苏氨酸(Thr,T)、色氨酸(Trp,W)、酪氨酸(Tyr,Y)和缬氨酸(Val,V)。
术语“单链”指包含由肽键线性连接氨基酸的分子。
术语“变异体”或“突变体”是指与包含的氨基酸或核苷酸有所不同但保持基本特性的多肽或多核昔酸。通常变异体之间或者变异体与母本抗体之间的差异是有限的，氨基酸序列总体上非常相似。本说明书中，将变异前的抗体或抗体片段称为母本抗体，变异后的抗体或抗体片段称为变异体。变异体仍具有抗原结合活性。
术语“抗体”(antibody，Ab)指包含至少一个抗原结合位点并能特异性结合抗原的免疫球蛋白分子(immunoglobulin，Ig)。
术语“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。
本发明所提及的术语“抗体”以其最广泛的含义理解，并包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同的抗原结合结构域的多特异性抗体(例如，双特异性抗体)。抗体还包括鼠源抗体、人源化抗体、嵌合抗体、人抗体以及其它来源的抗体。本发明的抗体可以来源于任何动物，包括但不限于人、非人灵长类动物、小鼠、大鼠、牛、马、鸡、骆驼、羊驼的免疫球蛋白分子等。抗体可以含有另外的改变，如非天然氨基酸，Fc效应子功能突变和糖基化位点突变。抗体还包括翻译后修饰的抗体、包含抗体的抗原决定簇的融合蛋白，以及包含对抗原识别位点的任何其它修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。换句话说，抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合结构域的分子。
抗体的基本结构是由两条完全相同的重链(heavy chain，H)和两条完全相同的轻链(light chain，L)通过二硫键连接的呈Y形的单体。每条链分别由2～5个约含110个氨基酸，序列相似但功能不同的结构域(又称功能区)组成。抗体分子中轻链和重链靠近N端的氨基酸序列变化较大，形成的结构域称为可变区(variable region，V区)；靠近C端的氨基酸序列相对恒定的区域称为恒定区(constant region，C区)。
重链和轻链的V区分别称为VH和VL，VH和VL各有3个区域的氨基酸组成和排列顺序高度可变，称为高变区(hypervariable region，HVR)；该区域形成与抗原表位互补的空间构象，又被称为互补决定区(complementarity determining region，CDR)。VH的3个CDR分别用VHCDR1、VHCDR2、VHCDR3表示，VL的3个CDR分别用VLCDR1、VLCDR2、VLCDR3表示。VH和VL共6个CDR共同组成抗原结合部位(antigen-binding site)。CDR区氨基酸的多样性是抗体与数量庞大的不同抗原特异性结合的分子基础。V区中CDR之外的氨基酸组成和排列顺序相对变化不大，称为骨架区(framework region，FR)。VH和VL各有4个骨架区(或称框架区)，分别用FR1、FR2、FR3、FR4表示。每个VH和VL有三个CDR和四个FR组成，从氨基端到羧基端排列顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
根据抗体重链恒定区的氨基酸序列，可以将人免疫球蛋白分为5类：IgM、IgG、IgA、IgD、IgE。其还可以进一步分成不同的亚类(同种型)，如人IgG可以分为IgG1、IgG2、IgG3、IgG4；IgA可分为IgA1和IgA2。IgM、IgD、IgE尚未发现有亚类。根据轻链氨基酸序列，可将轻链分类为κ链和λ链。本发明的抗体可以是任何种类(如IgM、IgG、IgA、IgD、IgE)或亚类(如IgG1、IgG2、IgG3、IgG4、IgA1、IgA2)。
重链和轻链的恒定区分别称为CH和CL。IgG、IgA、IgD的重链恒定区有CH1、CH2、CH3三个结构域，IgM、IgE的重链恒定区有CH1、CH2、CH3、CH4四个结构域。
铰链区(hinge region)位于CH1和CH2之间，含有丰富的脯氨酸，因此易伸展弯曲，能改变Y形两个臂之间的距离，有利于两臂同时结合抗原表位。
术语“Fab片段”为抗原结合片段(fragment of antigen binding,Fab)，意指由VL、VH、CL和CH1结构域组成的抗体片段，与单个抗原表位结合(单价)。本领域技术人员可知，在一定条件下，抗体分子链的某些部分易被蛋白水解酶水解为各种片段。木瓜蛋白酶从铰链区的近N端，将抗体分子水解为2个完全相同的抗原结合片段(Fab)和一个可结晶片段(Fc)。
术语“Fd片段”意指由VH和CH1结构域组成的抗体片段。
术语“Fc”、“Fc段”、“Fc片段”“Fc结构域”，是指可结晶片段(fragment crystallizable)，无抗原结合活性，是抗体与效应分子或细胞表面Fc受体(FcR)相互作用的部位。Fc片段与表面具有相应Fc受体的细胞结合，产生不同的生物学作用。在ADCC效应中(抗体依赖的细胞介导的细胞毒性作用，antibody-dependent cell-mediated cytotoxicity)，抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的FcR结合，介导杀伤细胞直接杀伤靶细胞。Fc片段 包含抗体除重链恒定区CH1之外的恒定区多肽，即人免疫球蛋白IgA、IgD、IgG重链恒定区羧基端的两个恒定区结构域CH2及CH3，以及人免疫球蛋白IgE和IgM重链恒定区羧基端的三个恒定区结构域CH2，CH3及CH4。Fc片段常选自Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc或其变异体，优选自IgG1 Fc、或Human IgG4 Fc或其变异体
Fc可由两条链组成，本文中将Fc片段的两条链描述为第一Fc链和第二Fc链，第一Fc链和第二Fc链可以分别进行突变，本发明并不做特别限定。Fc片段也可指Fc结构域中的单条多肽链。抗体的Fc段可选择消除免疫效应功能，包含但不限于以下突变的组合(根据EU计数)。
Figure PCTCN2022096501-appb-000007
经突变设计的Fc变体之间可以形成空间填充效应、静电转向、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体。优选的突变设计为“Knob-in-hole"形式的突变设计。
scFv(single chain antibody fragment)，或称单链抗体，由抗体重链可变区和轻链可变区通过连接片段(linker)连接而成。“连接片段”还可将IL-15与IL-15Ra连接、CD3抗体的VH与VL连接，并保证蛋白质的正确折叠和肽稳定性。“连接片段”优选为(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多。 如果连接片段序列太短，可能影响两蛋白高级结构的折叠，从而相互干扰；如果连接肽序列太长，又涉及免疫原性的问题，因为连接肽序列本身就是新的抗原。
肿瘤相关抗原(tumor-associated antigen，TAA)，是指在肿瘤细胞或正常细胞上存在的抗原分子，包括：胚胎性蛋白、糖蛋白抗原、鳞状细胞抗原等，常用于临床肿瘤的诊断。肿瘤相关抗原并非肿瘤细胞所特有，正常细胞可微量合成，而在肿瘤细胞增殖时高度表达，因此称为“相关抗原”。肿瘤相关抗原例如可以是CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1、CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB或Glypican-3。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，其中附加的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。此外，某些载体能够指导与它们可操作地连接的基因的表达。通常，在重组DNA技术中有用的表达载体通常呈质粒的形式
术语“IL-15”、“IL-15Ra”可以是其突变体或片段。
术语“第一”、“第二”仅用于描述目的，并不指示或暗示其重要性程度。
本发明以现有CD3抗体SP34为母本抗体，对其可变区进行突变，获得了CD3抗体变异体。本发明另外还提供了数种针对不同靶点及具有不同构型的蛋白分子。
“IL-15～IL15Ra”可采用以下序列：
SEQ ID NO:42
Figure PCTCN2022096501-appb-000008
“IL15Ra～IL-15”可采用以下序列：
SEQ ID NO:43
Figure PCTCN2022096501-appb-000009
一、抗CD3抗体变异体获得及一种抗TAA(肿瘤相关抗原)/CD3双特异抗体的形式设计
实施例1：抗CD3抗体变异体获得，分子克隆，瞬转表达，及蛋白纯化
1.分子克隆：以人源化抗CD3抗体SP34(Wileman et al.,1990；US 8236308)为母本抗体，对其CDR进行突变，筛选稳定性高的抗体，获得3条变异体。将CD3母本抗体SP34及其变异体的轻重链可变区通过连接序列构建成单链抗体VH-VL并在其C端融合人IgG1的FC段，通过分子克隆构建装入真核表达载体pQD。克隆编号及蛋白序列编号如下表所示：
表1：CD3抗体SP34及其突变体抗体编号
Figure PCTCN2022096501-appb-000010
氨基酸序列如下所示：
SEQ ID NO:01 QD3685
Figure PCTCN2022096501-appb-000011
SEQ ID NO:02 QD3689
Figure PCTCN2022096501-appb-000012
SEQ ID NO:03 QD3690
Figure PCTCN2022096501-appb-000013
Figure PCTCN2022096501-appb-000014
SEQ ID NO:04 QD3679(hSP34VH-VL-FC)
Figure PCTCN2022096501-appb-000015
注：下划线部分的氨基酸为抗体的重链可变区，波浪线部分为轻链可变区，重链可变区和轻链可变区之间的斜体部分为连接序列。可变区氨基酸顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，加粗并且底部粗线的部分分别示出HCDR1、HCDR3、HCDR3、LCDR1、LCDR2、LCDR3。母本抗体及其突变体CDR序列比较如下表所示：
表2：母本抗体及其突变体CDR序列比较
Figure PCTCN2022096501-appb-000016
2.蛋白表达：293E细胞调整细胞密度为1×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养。转染第五到六天，水平离 心机4700rpm离心20min收集细胞上清纯化。
3.蛋白纯化：
蛋白亲和层析法：细胞培养液经过高速离心后取上清，利用GE的Protein A层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后利用洗脱缓冲液0.1M甘氨酸(pH3.0)洗脱目的蛋白，利用Tris调节pH至中性保存；蛋白体积排阻层析：将离子交换所得产物超滤浓缩后进行体积排阻层析，如利用GE的Superdex200凝胶进行分离，以去除可能的聚体及其它成分，获得高纯度的目的产物。所得蛋白纯度分析可以通过SDS-PAGE及SEC-HPLC检测进行分析。蛋白浓度通过紫外分光光度法测定。
细胞表达蛋白产量及纯度如下表所示，QP3679即母本人源化抗CD3抗体SP34，产量为68.64mg/L，抗CD3抗体突变体均表现出比母本抗体QP3679的表达产量，显示突变体产量更高，可能蛋白更稳定，同时SEC-HPLC检测蛋白纯度均很好。
表3：SP34抗体及其变异体瞬转产量及蛋白纯度
Figure PCTCN2022096501-appb-000017
实施例2：FACS检测抗CD3抗体变异体结合天然表达CD3的Jurkat细胞
FACS检测抗CD3抗体结合活性：人淋巴细胞白血病细胞(Jurkat细胞)表达天然的CD3，通过FACS(流式细胞术，fluorescence activated cell sorter)检测抗CD3抗体及其突变体对Jurkat细胞的结合。种1E5/孔Jurkat细胞于U型96孔板，冰PBS洗一遍，1200rpm，离心3min；清洗结束后，按200μL/孔加入3％FBS/PBS封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育不同浓度样品，冰上孵育2h，冰PBS洗3遍；孵育PE-anti human FC抗体，按照1:200稀释比例，50μl/孔，充分混匀后，冰上1h，冰PBS洗3遍。PBS 200μl/孔重悬细胞，FACS仪器读取平均荧光值，使用graphpad prism软件分析结果。
结果如图1所示，抗CD3抗体SP34及其突变体QP3685、QP3689、QP3690均保持了jurkat结合活性。
实施例3一种含抗TAA(肿瘤相关抗原)及抗CD3的双特异抗体形式设计
CD3双抗发挥治疗作用，主要是通过同时结合肿瘤细胞上表达的肿瘤相关抗原(TAA)和T细胞上的CD3靶点，将这两种细胞交联形成免疫突触，导致T细胞被活化，从而杀死肿瘤细胞。多价TAA抗体可以提高对靶点的选择性，尤其是在靶点不够特异的情况下。提高CD3双抗安全性主要方向是提高TAA 的选择性，同时选择适当的CD3抗体亲和力可以保持药效同时降低毒副作用，从而提高肿瘤的选择性和减少靶向健康细胞。
本发明提供的抗TAA(肿瘤相关抗原)及抗CD3的双特异抗体分子形式设计如图2所示。
图2所示的抗TAA及抗CD3的双特异抗体分子包括：第一单体(图2中左侧部分)和第二单体(图2中右侧部分)。第一单体包括2条链，分别为链1：其具有Fd片段(包括VH和CH1)；链2：其具有轻链片段(包括VL和CL)、第一Fc链。第二单体也包括2条链，分别为链1：其具有Fd片段(包括VH和CH1)；链2：其具有轻链片段(包括VL和CL)、抗CD3抗体片段(图2中的CD3 Antibody，其为scFv形式)、以及第二Fc链。在第一单体或第二单体中，轻链片段与Fd片段分别配对形成抗TAA的Fab结构域(用以实现图2中的TAA Antibody的功能)。与常规抗体不同，第一单体的轻链片段与第一Fc链融合；同时抗CD3抗体片段的N端与第二单体的轻链片段融合，抗CD3抗体片段的C端与第二Fc链融合。第一Fc链和所述第二Fc链聚合形成Fc结构域。Fc结构域可选自Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc或其变异体。
双特异抗体的分子克隆，瞬转表达，及蛋白纯化：
1.分子克隆：
根据图2所示，TAA选择CLDN18.2，设计抗CLDN18.2/CD3双特异性抗体，其中CLDN18.2抗体序列可参阅专利文献CN202010344676.8中的抗体QP14611463的序列，抗CD3抗体分别使用SP34序列及其变异体QP3685、QP3689、QP3690的抗体轻、重链序列。QP374537493746的抗CD3抗体序列选择SP34，QP090837493746、QP090937493746、QP091037493746的抗CD3抗体序列选自QP3685、QP3689、QP3690。蛋白表达及序列号如下表所示。
表4：双特异抗体的克隆设计
Figure PCTCN2022096501-appb-000018
同时为了比较，构建了另外2种形式CLDN18.2/CD3双抗分子QP372337241461及QP374437481461， 及根据专利US20200055932A1，构建安进公司的CLDN18.2/CD3双抗分子AMG910，将AMG910蛋白编号为QP3693。蛋白表达及序列号如下表所示。
表5：双特异抗体的分子克隆
Figure PCTCN2022096501-appb-000019
QP372337241461的分子设计如图3所示。QP374437481461的分子设计如图4所示。
2.克隆构建方法：
如表4、表5所示设计克隆，构建全长表达载体。引物设计：利用在线软件DNAWorks(v3.2.4)( http://helixweb.nih.gov/dnaworks/)设计多条引物合成含重组所需基因片段。片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，PCR扩增得到重组所需基因片段。第一步PCR：PCR反应体系50μL包括10μL PrimerSTAR GXL Buffer(5×)；4μL dNTP Mixture(2.5mmol·L-1)；引物如上所示各1μL；1μL PrimeSTAR GXL DNA Polymerase。PCR反应条件为98℃2min，98℃20s，55℃15s，68℃30s，30个循环；68℃5min。第二步PCR：以第一步PCR产物为模板，首尾两条引物进行PCR扩增，条件同第一步。PCR搭建扩增目的片段。表达载体pQD构建及酶切：利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pQD(带信号肽)，BsmBI酶切载体，切胶回收备用。重组构建表达载体：重组目的基因片段与BsmBI酶切回收表达载体pQD(带信号肽片段)按3:1比例分别加入DH5α感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB medium，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
3.蛋白表达：
通过293E细胞调整细胞密度为1×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO2，120rpm，37℃的摇床中培养。转染第五到六天，水平离心机4700rpm离心20min收集细胞上清纯化。
4.蛋白纯化：
蛋白亲和层析法：细胞培养液经过高速离心后取上清，利用GE的Protein A层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后利用洗脱缓冲液0.1M甘氨酸(pH3.0)洗脱目的蛋白，利用Tris调节pH至中性保存；蛋白体积排阻层析：将离 子交换所得产物超滤浓缩后进行体积排阻层析，如利用GE的Superdex200凝胶进行分离，以去除可能的聚体及其它成分，获得高纯度的目的产物。所得蛋白纯度分析可以通过SDS-PAGE及SEC-HPLC检测进行分析。蛋白浓度通过紫外分光光度法测定。整个纯化过程严格控内毒素，纯化完成的蛋白内毒素含量小于1EU/mg。
结果显示，CLDN18.2/CD3双特异抗体QP374537493746、QP090837493746、QP090937493746及QP091037493746细胞表达产量高，HPLC-SEC纯度大于95％，具有可生产性。
实施例4：FACS检测双特异抗体与CLDN18.2结合活性
实验步骤：
取稳定表达CLDN18.2的细胞株CHOS-CLDN18.2，种1E5/孔细胞CHOS-CLDN18.2于U型96孔板，冰PBS洗一遍，1200rpm，离心3min；清洗结束后，按200μL/孔加入3％FBS/PBS封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育不同浓度样品，冰上孵育2h冰PBS洗3遍；孵育PE-anti human FC抗体，按照1:200稀释比例，50μl/孔，充分混匀后，冰上1h，冰PBS洗3遍。PBS 200μl/孔重悬细胞，FACS仪器读取平均荧光值，使用graphpad prism软件分析结果。
如图5、图6所示，结果显示：CLDN18.2/CD3双特异抗体QP374537493746、QP090837493746、QP090937493746及QP091037493746均结合CLDN18.2。如图5所示，本发明图2形式双抗QP374537493746、QP090837493746、QP090937493746及QP091037493746结合TAA靶点CLDN18.2亲和力与抗CLDN18.2抗体IgG形式QP14611463相当，明显强于对照抗体AMG910(1:1形式)QP3693，符合设计预期。
实施例5：FACS检测双特异抗体与CD3结合活性
人淋巴细胞白血病细胞(Jurkat细胞)表达天然的CD3，通过FACS检测抗CD3抗体及其突变体对Jurkat细胞的结合。种1E5/孔Jurkat细胞于U型96孔板，冰PBS洗一遍，1200rpm，离心3min；清洗结束后，按200μL/孔加入3％FBS/PBS封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育不同浓度样品，冰上孵育2h，冰PBS洗3遍；孵育PE-anti human FC抗体，按照1:200稀释比例，50μl/孔，充分混匀后，冰上1h，冰PBS洗3遍。PBS 200μl/孔重悬细胞，FACS仪器读取平均荧光值，使用graphpad prism软件分析结果。
结果如图7、图8所示，结果显示CLDN18.2/CD3双特异抗体QP374537493746、QP090837493746、QP090937493746及QP091037493746均结合CD3。如图7所示，本发明图2形式双抗QP374537493746、QP090837493746、QP090937493746及QP091037493746结合CD3亲和力分成2类，其中QP374537493746、QP090837493746结合CD3亲和力较弱，QP090937493746及QP091037493746亲和力较强，但是这2类分子结合CD3亲和力都明显弱于对照抗体AMG910(1:1形式)QP3693，这与我们期望降低CD3抗体亲和力以提高CD3双抗安全性的预期符合。
实施例6：CLDN18.2/CD3双特异抗体介导的T细胞杀伤人胃癌细胞
实验方法：选择靶细胞为稳定表达CLDN18.2的人胃癌细胞系NUGC4-CLDN18.2。效应细胞为人PBMC。按照E:T比例10：1，再加入不同浓度抗体，37℃，5％CO 2培养48h，用CytoTox
Figure PCTCN2022096501-appb-000020
Non-Radioactive Cytotoxicity Assay(Promega，G1780-1000assays)检测细胞培养上清中的LDH，定量细胞毒％。靶细胞的最大裂解率(100％)为1％Triton X-100处理靶细胞使细胞裂解释放全部LDH。设置靶细胞自发、效应细胞自发、靶细胞+效应细胞自发等对照孔。按照公式％Cytotoxicity＝[(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]×100分析数据。
结果如图9和图10所示，QP374537493746、QP090837493746、QP090937493746及QP091037493746均能介导的PBMC杀伤人胃癌细胞NUGC4-CLDN18.2细胞。如图9所示，本发明图2形式双抗QP374537493746、QP090837493746、QP090937493746及QP091037493746介导的PBMC杀伤人胃癌细胞NUGC4-CLDN18.2细胞活性均强于对照分子QP3693(AMG910)。虽然本发明所选的CD3抗体结合CD3的亲和力都弱于AMG910分子的CD3抗体(参阅图7)，但是并不影响对靶细胞的杀伤能力。
实施例7：CLDN18.2/CD3双特异抗体介导的T细胞杀伤人肺癌细胞
实验方法：选择靶细胞为稳定表达CLDN18.2的人肺癌细胞系HCC827-CLDN18.2。效应细胞为人PBMC。按照E:T比例10：1，再加入不同浓度抗体，37℃，5％CO2培养48h，用CytoTox
Figure PCTCN2022096501-appb-000021
Non-Radioactive Cytotoxicity Assay(Promega，G1780-1000 assays)检测细胞培养上清中的LDH，定量细胞毒％。靶细胞的最大裂解率(100％)为1％Triton X-100处理靶细胞使细胞裂解释放全部LDH。设置靶细胞自发、效应细胞自发、靶细胞+效应细胞自发等对照孔。按照公式％Cytotoxicity＝[(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]×100分析数据。
结果如图11所示，CD3双抗分子QP374537493746、QP090837493746、QP090937493746及QP091037493746均能介导的PBMC杀伤人肺癌细胞HCC827-CLDN18.2细胞，本发明图2形式双抗QP374537493746、QP090837493746、QP090937493746及QP091037493746介导的PBMC杀伤人肺癌细胞HCC827-CLDN18.2细胞活性均强于对照分子QP3693(AMG910)。在HCC827-CLDN18.2细胞上重现了NUGC4-CLDN18.2的结果(参见图9)。
实施例8：CD3人源化小鼠模型动物药效
在CD3人源化小鼠(CD3EDG HuGEMM)模型中评价抗CLDN18.2/CD3双抗分子对肿瘤生长的抑制作用。收集指数生长期的结直肠癌细胞MC38-hCLDN18.2细胞，PBS重悬至适合浓度接种。每只实验小鼠于右侧背部皮下接种5×10 6MC38-hCLDN18.2细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积约100mm 3时根据肿瘤大小和小鼠体重随机分组给药。给药方案如下：
表6：评价抗CLDN18.2/CD3双抗分子对肿瘤生长的抑制作用的给药方案
组别 给药组 小鼠数 给药剂量 给药方 给药次
    量 药物 (mg/kg) 式 数
1 Vehicle 6 PBS -- ip BIW x 6
2 AMG910 6 AMG910 0.7 ip BIW x 6
3 QP374537493746 6 QP374537493746 0.6 ip BIW x 6
4 QP372337241461 6 QP372337241461 1.2 ip BIW x 6
5 QP374437481461 6 QP374437481461 1.2 ip BIW x 6
按照上表所示方案，给药6次，每周量瘤2次，肿瘤抑制率见表7，肿瘤抑制曲线见图12，结果显示，给药后第21天，QP374537493746在低剂量组0.6mpk的肿瘤抑制率(TGI)达到了89.29％，优于或相当于对照抗体AMG910在0.7mpk组(AMG910 0.7mpk组的摩尔浓度等同于QP374537493746 1.2mpk)的肿瘤抑制率(TGI＝86.5％)，即QP374537493746使用一半剂量即可达到与对照抗体AMG910相同的肿瘤抑制效果。图13为给药后各组小鼠体重变化，结果显示，给药组小鼠体重基本无变化，生长良好，证明药物安全性良好。
表7：CLDN18.2/CD3双抗分子在CD3EGD HuGEMM小鼠皮下移植结直肠癌细胞MC38-hCLDN18.2体内动物模型中抑制肿瘤生长
Figure PCTCN2022096501-appb-000022
*Note:％T/C＝(delta T/delta C)×100,％TGI＝100-％T/C.
实施例9：人源化NOG小鼠模型动物药效
实验目的：评价受试物在PBMC人源化NOG小鼠X-CLDN18.2/MIA PaCa-2皮下移植瘤模型中的体内药效。细胞培养：X-CLDN18.2/MIA PaCa-2细胞体外培养，培养条件为添加10％胎牛血清的培养基，37℃，5％CO 2。一周常规传代2次。当细胞维持在指数增长期时，细胞存活率大于95％时，收取细胞，计数，活细胞百分比，接种。动物:NOG小鼠，雌性，6-8周龄，体重18-20克。实验共需36只(24plus50％)。动物由具有资质的供应商提供。肿瘤接种：在每只小鼠的右侧颈背部混合接种5*10 6个X-CLDN18.2/MIA PaCa-2和2*10 6个PBMC细胞。接种同时将实验动物进行耳标号标记，作为后续实验的唯一确认标志。等待肿瘤生长，肿瘤平均体积达到约60-100mm 3时开始进行随机分组给药。实验分组和给药方案见表8。
表8：评价抗CLDN18.2/CD3双抗分子对肿瘤生长的抑制作用的给药方案
组别 给药组 小鼠数 给药剂量 给药方 给药次
    量 药物 (mg/kg) 式 数
1 Vehicle 6 PBS -- iv BIW x 5
2 AMG910 6 AMG910 5 iv BIW x 5
3 QP374537493746 6 QP374537493746 8 iv BIW x 5
4 QP372337241461 6 QP372337241461 4 iv BIW x 5
按照上表所示方案，给药5次，每周量瘤2次，肿瘤抑制率见表9，肿瘤抑制曲线见图14，结果显示，给药后第15天结果显示，QP374537493746在低剂量组4mpk的肿瘤抑制率(TGI)达到了92.3％，在高剂量组8mpk的肿瘤抑制率(TGI)达到了＝99.8％，都要明显优于对照抗体AMG910在5mpk组(AMG9105mpk组的摩尔浓度等同于QP374537493746高剂量组8mpk)的肿瘤抑制率(TGI＝69.3％)。图15为给药后各组小鼠体重变化，结果显示，给药组小鼠体重基本无变化，生长良好，证明药物安全性良好。
表9：CLDN18.2/CD3双抗分子在PBMC人源化NOG小鼠X-CLDN18.2/MIA PaCa-2皮下移植瘤模型中抑制肿瘤生长
Figure PCTCN2022096501-appb-000023
*Note:％T/C＝(delta T/delta C)×100,％TGI＝100-％T/C.
二：抗TAA(肿瘤相关抗原)、抗CD3抗体及含细胞因子IL15/IL15Ra的多功能融合蛋白形式设计
虽然CD3双抗在血液肿瘤方面显示出巨大的潜力，然而，CD3双抗在实体瘤的治疗上依然面临着一些挑战。CD3双抗治疗与实体瘤组织T细胞浸润和免疫抑制相关，浸润T细胞少的情况下，容易对CD3双抗治疗产生抵抗。IL15是一种可溶性细胞因子，能激活T细胞和NK细胞，并介导这些细胞的增殖和存活。本发明设计了TAA/CD3/IL15多功能融合蛋白，以期其中的细胞因子IL15组分可以刺激包括T细胞的免疫细胞增殖，改变肿瘤的免疫微环境。
实施例10：抗TAA(肿瘤相关抗原)、抗CD3及含IL15/IL15Ra的多功能融合蛋白形式设计
本发明提供多种抗TAA(肿瘤相关抗原)、抗CD3及含IL15/IL15Ra的多功能融合蛋白。
1.第一种类型的多功能融合蛋白可参阅图16至图19，融合蛋白分子包括第一单体(图中左侧部分)和第二单体(图中右侧部分)。第一单体包括2条链，分别为链1：其具有Fd片段(包括VH和CH1)；链2：其具有轻链片段(包括VL和CL)、细胞因子功能区(包括IL15和IL15Ra)、第一Fc链。第二单体包括2条链，分别为链1：其具有Fd片段(包括VH和CH1)；链2：其具有轻链片段(包括VL和CL)、抗CD3抗体片段(图中的CD3 Antibody，其为scFv形式)、以及第二Fc链。在第一单体或第二单 体中，轻链片段与Fd片段分别配对形成抗TAA的Fab结构域(用以实现图中的Anti-TAA Antibody的功能)。细胞因子功能区的N端与第一单体的轻链片段融合，C端与第一Fc链融合；抗CD3抗体片段的N端与第二单体的轻链片段融合，C端与第二Fc链融合。第一Fc链和第二Fc链聚合形成Fc结构域。
图16至图19中，scFv形式的抗CD3抗体片段可包括：重链可变区(VH)、轻链可变区(VL)、以及连接重链可变区和轻链可变区的连接片段。自肽链的N端到C端，抗CD3抗体片段的氨基酸的融合顺序可以是“VH(CD3)～VL(CD3)”，或者，“VL(CD3)～VH(CD3)”；“～”表示连接片段。细胞因子功能区的氨基酸片段的融合顺序可以是“IL-15～IL-15Ra”，或者，“IL-15Ra～IL-15”。
图16所示的融合蛋白的4条多肽链可描述如下：(1)第一条多肽链：VH-CH1；(2)第二条多肽链：VL-CL- IL15Ra-IL15-第一Fc链；(3)第三条多肽链：VL-CL- VH(CD3)-VL(CD3)-第二Fc链；(4)第四条多肽链：VH-CH1。“-”表示以肽键或连接片段进行连接。
图17所示的融合蛋白的4条多肽链描述如下：(1)第一条多肽链：VH-CH1；(2)第二条多肽链：VL-CL- IL15-IL15Ra-第一Fc链；(3)第三条多肽链：VL-CL- VH(CD3)-VL(CD3)-第二Fc链；(4)第四条多肽链：VH-CH1。
图18所示的融合蛋白的4条多肽链描述如下：(1)第一条多肽链：VH-CH1；(2)第二条多肽链：VL-CL- IL15Ra-IL15-第一Fc链；(3)第三条多肽链：VL-CL- VL(CD3)VH(CD3)-第二Fc链；(4)第四条多肽链：VH-CH1。
图19所示的融合蛋白的4条多肽链描述如下：(1)第一条多肽链：VH-CH1；(2)第二条多肽链：VL-CL- IL15-IL15Ra-第一Fc链；(3)第三条多肽链：VL-CL- VL(CD3)VH(CD3)-第二Fc链；(4)第四条多肽链：VH-CH1。
2.第二种类型的多功能融合蛋白可参阅图20至图27，蛋白分子包括：第一单体(图中左侧部分)和第二单体(图中右侧部分)。第一单体包括：抗TAA抗体片段(为scFv形式，包括VH和VL结构域)、细胞因子功能区(包括IL15和IL15Ra)、第一Fc链；第二单体包括：抗TAA抗体片段(为scFv形式，包括VH和VL结构域)、抗CD3抗体片段(图中的CD3 Antibody，其为scFv形式)、以及第二Fc链。细胞因子功能区的N端与第一单体的抗TAA抗体片段融合，C端与第一Fc链融合；抗CD3抗体片段的N端与第二单体的抗TAA抗体片段融合，C端与第二Fc链融合。第一Fc链和第二Fc链聚合形成Fc结构域。
与第一种类型的多功能融合蛋白类似，scFv形式的抗CD3抗体片段可包括：重链可变区(VH)、轻链可变区(VL)、以及连接重链可变区和轻链可变区的连接片段。自肽链的N端到C端，抗CD3抗体片段的氨基酸的融合顺序可以是“VH(CD3)～VL(CD3)”，或者，“VL(CD3)～VH(CD3)”；“～”表示连接片段。细胞因子功能区的氨基酸片段的融合顺序可以是“IL-15～IL-15Ra”，或者，“IL-15Ra～IL-15”。另外，scFv形式的抗TAA抗体片段包括：重链可变区(VH)、轻链可变区(VL)、以及连接所述重链可 变区和所述轻链可变区的连接片段；自肽链的N端到C端，抗TAA抗体片段的氨基酸融合顺序可以是“VH(TAA)～VL(TAA)”，或者，“VL(TAA)～VH(TAA)”；“～”表示连接片段。
图20至图27所示的抗体分子各结构域采用相同的氨基酸序列，区别在于融合顺序或者构型有所区别。以图20为例子，对融合蛋白的2条多肽链描述如下：(1)第一条多肽链：VL(TAA)-VH(TAA)-IL15-IL15Ra-第一Fc链；(2)第二条多肽链：VL(TAA)-VH(TAA)-VH(CD3)-VL(CD3)-第二Fc链。
3.本发明还提供一种肿瘤靶向T细胞/NK细胞激活的TAA/IL15双功能融合蛋白，其结构形式可参阅图28，与第二种类型的多功能融合蛋白相比，区别在于将抗CD3抗体片段替换为细胞因子功能区(包括IL15和IL15Ra)。图28中，蛋白分子具有对称结构形式。
实施例11：CLDN18.2/CD3/IL15多功能融合蛋白的分子克隆，瞬转表达，及蛋白纯化
1.分子克隆：
根据实施例10所示，设计CLDN18.2/CD3/IL15或CLDN18.2/IL15多功能融合蛋白，其中CLDN18.2抗体可参阅专利文献CN202010344676.8中抗体QP14611463序列，抗CD3抗体使用SP34轻、重链序列，蛋白表达及序列号如下表所示。
表10：多功能融合蛋白的克隆设计
Figure PCTCN2022096501-appb-000024
Figure PCTCN2022096501-appb-000025
同时构建恒瑞医药公司的IL15/IL15Ra-FC作为对照，将IL15/IL15Ra-FC蛋白编号为QP33123313。
如表10所示设计克隆，构建全长表达载体。克隆构建、蛋白表达和蛋白纯化方法可参阅实施例3。
实施例12：FACS检测CLDN18.2/CD3/IL15多功能融合蛋白与CLDN18.2结合活性
实验步骤：取稳定表达CLDN18.2的细胞株CHOS-CLDN18.2，种1E5/孔细胞CHOS-CLDN18.2于U型96孔板，冰PBS洗一遍，1200rpm，离心3min；清洗结束后，按200μL/孔加入3％FBS/PBS封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育不同浓度样品，冰上孵育2h,冰PBS洗3遍；孵育PE-anti human FC抗体，按照1:200稀释比例，50ul/孔，充分混匀后，冰上1h，冰PBS洗3遍。PBS 200ul/孔重悬细胞，FACS仪器读取平均荧光值，使用graphpad prism软件分析结果。结果如图29，图30所示，结果显示CLDN18.2/CD3/IL15及CLDN18.2/IL15多功能融合蛋白均结合TAA靶点CLDN18.2，且亲和力强于AMG910类似物(1:1形式)QP3693，符合预期。
实施例13：FACS检测CLDN18.2/CD3/IL15多功能融合蛋白与CD3结合活性
淋巴细胞白血病细胞(Jurkat细胞)表达天然的CD3，通过FACS检测抗CD3抗体及其突变体对Jurkat细胞的结合。种1E5/孔Jurkat细胞于U型96孔板，冰PBS洗一遍，1200rpm，离心3min；清洗结束后，按200μL/孔加入3％FBS/PBS封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育不同浓度样品，冰上孵育2h，冰PBS洗3遍；孵育PE-anti human FC抗体，按照1:200稀释比例，50μl/孔，充分混匀后，冰上1h，冰PBS洗3遍。PBS 200ul/孔重悬细胞，FACS仪器读取平均荧光值，使用graphpad prism软件分析结果。结果如图31、图32所示，结果显示CLDN18.2/CD3/IL15多功能融合蛋白均结合CD3，且亲和力弱于对照分子AMG910(QP3693)，这与发明人期望降低CD3抗体亲和力提高CD3双抗安全性的预期符合。
实施例14：Mo7e细胞增殖实验评估多功能融合蛋白IL15活性
Mo7e(人巨细胞白血病细胞株)细胞表达IL-15Rβγ，是细胞因子生长依赖性细胞。已有研究表明，静息NK和幼稚T细胞表达中等亲和力IL-15Rβγ表型，细胞因子IL15/IL15Ra对Mo7e(IL-15Rβγ)细胞增殖实验结果与对未刺激的PBMC的增殖实验一致(Mol Cancer Ther；11(6)June 2012)。本发明用Mo7e细胞增殖实验评估多功能融合蛋白IL15活性，方法及结果如下：
实验试剂：Mo7e细胞(人巨细胞白血病细胞株)，购于中国医学科学院基础医学研究所细胞资源中心,细胞增殖及毒性检测试剂盒(CCK-8)，购自美仑生物，货号MA0218；重组人GM-CSF，购自perprotech，货号300-03；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验方法：Mo7e细胞使用含有10％FBS，2mM L-谷氨酰胺和8ng/ml GM-CSF的RPMI1640培养基 于37℃5％CO 2培养箱中培养；收集Mo7e细胞，800rpm离心5分钟倒掉上清，细胞用不含有GM-CSF的RPMI1640培养基洗涤2遍；用无GM-CSF的RPMI1640培养基重悬细胞并计数，将细胞按2×10 4个，80μl每孔接种于96孔板中，37℃5％CO 2培养箱中培养1小时；将各个待测药用培养基按4倍梯度稀释后，20μl每孔与细胞悬液混合均匀，37℃5％CO 2培养箱中培养3天；将CCK-8试剂按10μl每孔加入待测的96孔板中，37℃5％CO 2培养箱中孵育4小时；将96孔板取出，于酶标仪中检测450nm波长的吸光值。
实验结果如图33、图34、图35所示，结果显示，不同设计形式的CLDN18.2/CD3/IL15及CLDN18.2/IL15多功能融合蛋白均有IL15活性，根据分子设计不同，活性有强有弱。不同的构建形式产生了不同的IL15活性的分子，这提供了选择最好治疗窗口的机会。
实施例15：CLDN18.2/CD3/IL15多功能融合蛋白介导的T细胞杀伤人胃癌细胞
实验方法：选择靶细胞为稳定表达CLDN18.2的人胃癌细胞系NUGC4-CLDN18.2。效应细胞为人PBMC。按照E:T比例10：1，再加入不同浓度抗体，37℃，5％CO 2培养48h，用CytoTox
Figure PCTCN2022096501-appb-000026
Non-Radioactive Cytotoxicity Assay(Promega，G1780-1000assays)检测细胞培养上清中的LDH，定量细胞毒％。靶细胞的最大裂解率(100％)为1％Triton X-100处理靶细胞使细胞裂解释放全部LDH。设置靶细胞自发、效应细胞自发、靶细胞+效应细胞自发等对照孔。按照公式％Cytotoxicity＝[(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]×100分析数据。结果如图36、图37所示，QP374508993746、QP090109003746、QP09023688、QP36670903、QP09040905、QP09060905、QP09040907及QP09060907均能介导PBMC杀伤人胃癌细胞NUGC4-CLDN18.2细胞。其中，QP374508993746、QP090109003746介导PBMC杀伤人肺癌细胞NUGC4-CLDN18.2细胞活性明显优于对照分子AMG910。其他分子介导的PBMC杀伤人肺癌细胞NUGC4-CLDN18.2细胞活性与对照分子AMG910相当。
实施例16：CLDN18.2/CD3/IL15多功能融合蛋白介导的T细胞杀伤人肺癌细胞
实验方法：选择靶细胞为稳定表达CLDN18.2的人肺癌细胞系HCC827-CLDN18.2。效应细胞为人PBMC。按照E:T比例10：1，再加入不同浓度抗体，37℃，5％CO 2培养48h，用CytoTox
Figure PCTCN2022096501-appb-000027
Non-Radioactive Cytotoxicity Assay(Promega，G1780-1000assays)检测细胞培养上清中的LDH，定量细胞毒％。靶细胞的最大裂解率(100％)为1％Triton X-100处理靶细胞使细胞裂解释放全部LDH。设置靶细胞自发、效应细胞自发、靶细胞+效应细胞自发等对照孔。按照公式％Cytotoxicity＝[(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]×100分析数据。结果如图38所示，QP090109003746、QP36670903、QP09040905、QP09060905及QP09040907均能介导PBMC杀伤人肺癌细胞HCC827-CLDN18.2细胞活性。其中，QP090109003746介导的PBMC杀伤人肺癌细胞HCC827-CLDN18.2细胞活性明显优于对照分子AMG910。其他分子介导的PBMC杀伤人肺癌细胞 HCC827-CLDN18.2细胞活性与对照分子AMG910相当。
另外，在其他一些实施例中，抗CD3抗体片段分别选择SP34变异体QP3685、QP3689、QP3690的抗体轻、重链序列，如实施例11中的表10所示设计克隆并表达、纯化蛋白。结果表明，采用QP3685、QP3689、QP3690的抗体轻、重链序列构建的分子，也具有CLDN18.2结合活性和IL15活性、抗CD3分子还具有CD3结合活性，并且也具有介导T细胞杀伤人胃癌细胞或肺癌细胞的效果。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。

Claims (32)

  1. 一种抗CD3抗体变异体，所述抗CD3抗体包括：重链可变区和轻链可变区；所述重链可变区包括：VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包括：VLCDR1、VLCDR2、VLCDR3；所述抗CD3抗体的氨基酸序列包括SEQ ID NO：29-34所示的VHCDR1、VHCDR2、VHCDR3、VLCDR1、VLCDR2和VLCDR3；其特征在于：
    所述抗CD3抗体变异体的突变位点包括以下任意1个或2个以上位点：
    SEQ ID NO：30所示序列的第十位点，
    SEQ ID NO：31所示序列的第二、七位点，
    SEQ ID NO：33所示序列的第三、四位点，
    SEQ ID NO：34所示序列的第四、五、六、七位点。
  2. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，所述的突变是指氨基酸被取代。
  3. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，变异体的突变位点包括以下任意1个或2个以上位点：
    SEQ ID NO：30所示序列中第十位的A被取代为E，
    SEQ ID NO：31所示序列中第二位的G被取代为S、第七位的S被取代为G，
    SEQ ID NO：33所示序列中第三位的N被取代为W、第四位的K被取代为L，
    SEQ ID NO：34所示序列中第四位的Y被取代为N或R、第五位的S被取代为K、第六位的N被取代为G、第七位的L被取代为G。
  4. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，
    VHCDR2的氨基酸序列如SEQ ID NO:35所示；或者，
    VHCDR3的氨基酸序列如SEQ ID NO:36所示；或者，
    VLCDR2的氨基酸序列如SEQ ID NO:37或38所示；或者，
    VLCDR3的氨基酸序列如SEQ ID NO:39、40或41所示。
  5. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，所述VHCDR1、VHCDR2、VHCDR3、VLCDR1、VLCDR2和VLCDR3的氨基酸序列如以下任意一组序列所示：
    (1)SEQ ID NO：29、35、31、32、37、39；
    (2)SEQ ID NO：29、35、36、32、37、40；
    (3)SEQ ID NO：29、35、36、32、38、41。
  6. 根据权利要求5所述的抗CD3抗体变异体，其特征在于，所述抗体变异体的重链可变区 和/或轻链可变区选自SEQ ID NO：1、2或3所示序列的重链可变区和/或轻链可变区，
    或与其具有至少90％序列同一性。
  7. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，所述重链可变区和所述轻链可变区之间具有用于形成二硫键的突变，所述突变的位点包括以下组合形式中的任意一种或两种以上，突变位点根据EU编号，所述重链可变区以VH表示，所述轻链可变区以VL表示：
    Figure PCTCN2022096501-appb-100001
  8. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，所述抗CD3抗体变异体还包含：选自人源IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区；以及选自人源κ、λ链或其变体的轻链恒定区；所述重链恒定区包含：Fc片段或其变体。
  9. 根据权利要求1所述的抗CD3抗体变异体，其特征在于，所述抗体变异体为scFv，其包含重链可变区、轻链可变区、以及连接所述重链可变区和所述轻链可变区的连接片段，所述连接片段的氨基酸序列优选为若干个GGGGS重复，更优选为3个GGGGS重复。
  10. 权利要求1至9中任意一项所述的抗CD3抗体变异体在制备用于抑制或治疗癌症的药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述癌症选自以下癌症或发生于以下部位：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤、以及骨髓增生异常综合症。
  12. 一种核酸分子，其编码权利要求1至9中任意一项所述的抗CD3抗体变异体。
  13. 一种表达载体，其包含权利要求12所述的核酸分子。
  14. 一种抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，其为异二聚体形式，包括第一单体和第二单体；
    所述第一单体包括：(a)Fd片段；(b)轻链片段及第一Fc链；
    所述第二单体包括：(a)Fd片段；(b)轻链片段、抗CD3抗体片段以及第二Fc链；
    所述轻链片段包括VL结构域和CL结构域；所述Fd片段包括VH结构域和CH1结构域；在所述第一单体或所述第二单体中，所述轻链片段与所述Fd片段配对形成抗TAA的Fab结构域；其中，所述第一单体的轻链片段与所述第一Fc链融合；所述抗CD3抗体片段的N端与所述第二单体的轻链片段融合，C端与所述第二Fc链融合；
    或者，
    所述第一单体包括：(a)Fd片段；(b)轻链片段、细胞因子功能区、第一Fc链；
    所述第二单体包括：(a)Fd片段；(b)轻链片段、抗CD3抗体片段、以及第二Fc链；
    所述细胞因子功能区包括IL-15及IL-15Ra；其中，所述细胞因子功能区的N端与所述第一单体的轻链片段融合，C端与所述第一Fc链融合；所述抗CD3抗体片段的N端与所述第二单体的轻链片段融合，C端与所述第二Fc链融合；
    或者，
    所述第一单体包括：抗TAA抗体片段、细胞因子功能区、第一Fc链；
    所述第二单体包括：抗TAA抗体片段、抗CD3抗体片段、以及第二Fc链；其中，
    所述细胞因子功能区的N端与所述第一单体的抗TAA抗体片段融合，C端与所述第一Fc链融合；所述抗CD3抗体片段的N端与所述第二单体的抗TAA抗体片段融合，C端与所述第二Fc链融合；
    所述第一Fc链与所述第二Fc链能够互换。
  15. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，
    所述抗TAA抗体片段为scFv形式，包括：重链可变区(VH)、轻链可变区(VL)、以及连接所述重链可变区和所述轻链可变区的连接片段；自肽链的N端到C端，所述抗TAA抗体片段的氨基酸融合顺序为“VH～VL”，或者，“VL～VH”；“～”表示连接片段；或者，
    所述抗CD3抗体片段为scFv形式，包括：重链可变区(VH)、轻链可变区(VL)、以及连接所述重链可变区和所述轻链可变区的连接片段；自肽链的N端到C端，所述抗CD3抗体片段的氨基酸的融合顺序为“VH～VL”，或者，“VL～VH”；“～”表示连接片段；或者，
    自肽链的N端到C端，所述细胞因子功能区的氨基酸片段的融合顺序为“IL-15～IL-15Ra”，或者，“IL-15Ra～IL-15”；“～”表示连接片段；优选地，所述IL-15～IL-15Ra的序列如SEQ ID NO:42所示，所述IL-15Ra～IL-15的序列如SEQ ID NO:43所示。
  16. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，所述 抗CD3抗体片段的氨基酸序列选自结合CD3的特异性抗体、抗体片段、单域抗体或其人源化形式；优选的，所述抗CD3抗体片段的氨基酸序列选自SP34、OKT3、UCTH1或其衍生物。
  17. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，所述抗CD3抗体片段包括重链可变区和轻链可变区；所述抗CD3抗体片段的重链可变区包括：VHCDR1、VHCDR2、VHCDR3；所述抗CD3抗体片段的轻链可变区包括：VLCDR1、VLCDR2、VLCDR3；所述VHCDR1、VHCDR2、VHCDR3、VLCDR1、VLCDR2和VLCDR3的氨基酸序列如权利要求5所述的抗CD3抗体变异体中的任意一组序列所示。
  18. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，IL-15Ra和IL-15形成IL-15/IL-15Ra复合物；IL-15包括：IL-15及其能结合IL-15Ra的突变，截短及各种衍生物；IL-15Ra包括：IL-15Ra及其能结合IL-15的突变，截短及各种衍生物；优选地，所述IL-15/IL-15Ra复合物包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15或IL-15Ra的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示；所述IL-15Ra的母本序列如SEQ ID NO:45所示：
    组合 IL15 IL15Ra 1 wt D96 2 wt D96/P97 3 wt D96/P97/A98 4 E87C D96/C97 5 E87C D96/P97/C98 6 E87C D96/C97/A98 7 V49C S40C 8 L52C S40C 9 E89C K34C 10 Q48C G38C 11 E53C L42C 12 C42S A37C 13 L45C G38C 14 L45C A37C
    。
  19. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，所述IL-15包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示：
    组合 IL15突变 1 N1D 2 N4D 3 D8N 4 D30N 5 D61N 6 E64Q 7 N65D 8 Q108E 9 N1D/D61N 10 N1D/E64Q 11 N4D/D61N 12 N4D/E64Q 13 D8N/D61N 14 D8N/E64Q 15 D61N/E64Q 16 E64Q/Q108E 17 N1D/N4D/D8N 18 D61N/E64Q/N65D 19 N1D/D61N/E64Q/Q108E 20 N4D/D61N/E64Q/Q108E
    。
  20. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，所述TAA选自CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、CD239、CD276、PD-1、CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB或Glypican-3。
  21. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，所述第一Fc链和所述第二Fc链聚合形成Fc段；所述Fc段选自Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc或其变异体，优选自IgG1 Fc、或Human IgG4 Fc或其变异体；所述蛋白分子为Fc异源二聚体形式；优选地，所述Fc异源二聚体包含但不限于以下突变的组合，以下突变为根据EU计数：
    Figure PCTCN2022096501-appb-100002
  22. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，所述蛋白分子包含Fc段，Fc段选择消除免疫效应功能，包含但不限于以下突变的组合，以下突变为根据EU计数：
    Figure PCTCN2022096501-appb-100003
  23. 根据权利要求14所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子，其特征在于，由以下任意一组序列所示的氨基酸片段融合得到：
    (1)SEQ ID NO:05、SEQ ID NO:06、SEQ ID NO:07；
    (2)SEQ ID NO:08、SEQ ID NO:06、SEQ ID NO:07；
    (3)SEQ ID NO:09、SEQ ID NO:06、SEQ ID NO:07；
    (4)SEQ ID NO:10、SEQ ID NO:06、SEQ ID NO:07；
    (5)SEQ ID NO:05、SEQ ID NO:17、SEQ ID NO:07；
    (6)SEQ ID NO:01、SEQ ID NO:18、SEQ ID NO:07；
    (7)SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:07；
    (8)SEQ ID NO:19、SEQ ID NO:18、SEQ ID NO:07；
    (9)SEQ ID NO:20、SEQ ID NO:21；
    (10)SEQ ID NO:22、SEQ ID NO:21；
    (11)SEQ ID NO:20、SEQ ID NO:23；
    (12)SEQ ID NO:22、SEQ ID NO:23；
    (13)SEQ ID NO:24、SEQ ID NO:25；
    (14)SEQ ID NO:26、SEQ ID NO:25；
    (15)SEQ ID NO:24、SEQ ID NO:27；
    (16)SEQ ID NO:26、SEQ ID NO:27。
  24. 一种核酸分子，其编码权利要求14至23中任意一项所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子。
  25. 权利要求14至23中任意一项所述的抗肿瘤相关抗原(TAA)/抗CD3的蛋白分子在制备用于抑制或治疗癌症的药物中的应用，所述癌症包括选自以下的癌症或发生于以下部位：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤、以及骨髓增生异常综合症。
  26. 一种抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其特征在于，包括两条多肽链，任意一条多肽链包括：抗TAA抗体片段、细胞因子功能区、Fc片段；所述抗TAA抗体片段为scFv形式；所述细胞因子功能区包括IL-15及IL-15Ra；所述细胞因子功能区的N端与所述抗TAA抗体片段融合，C端与所述Fc片段融合。
  27. 根据权利要求26所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其特征在于，IL-15Ra和IL-15形成IL-15/IL-15Ra复合物；IL-15包括：IL-15及其能结合IL-15Ra的突变，截短及各种衍生物；IL-15Ra包括：IL-15Ra及其能结合IL-15的突变，截短及各种衍生物；优选地，所述IL-15/IL-15Ra复合物包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15或IL-15Ra的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示；所述IL-15Ra的母本序列如SEQ ID NO:45所示：
    组合 IL15 IL15Ra 1 wt D96 2 wt D96/P97 3 wt D96/P97/A98 4 E87C D96/C97 5 E87C D96/P97/C98 6 E87C D96/C97/A98 7 V49C S40C 8 L52C S40C 9 E89C K34C 10 Q48C G38C 11 E53C L42C 12 C42S A37C 13 L45C G38C 14 L45C A37C
    。
  28. 根据权利要求26所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其特征在于，所述IL-15包括但不限于以下任意一组合所示的突变方式，计数方式根据IL-15的氨基酸序列的第一个氨基酸开始算为第1位；优选地，所述IL-15的母本序列如SEQ ID NO:44所示：
    组合 IL15突变 1 N1D 2 N4D 3 D8N 4 D30N 5 D61N 6 E64Q 7 N65D 8 Q108E 9 N1D/D61N 10 N1D/E64Q 11 N4D/D61N 12 N4D/E64Q 13 D8N/D61N 14 D8N/E64Q 15 D61N/E64Q 16 E64Q/Q108E 17 N1D/N4D/D8N 18 D61N/E64Q/N65D 19 N1D/D61N/E64Q/Q108E 20 N4D/D61N/E64Q/Q108E
    。
  29. 根据权利要求26所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其特征在于，所述融合蛋白包含Fc段，Fc段选择消除免疫效应功能，包含但不限于以下突变的组合，以下突变为根据EU计数：：
    Figure PCTCN2022096501-appb-100004
  30. 根据权利要求26所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白，其特征在于，所述融合蛋白的任意一条多肽链的氨基酸序列如SEQ ID NO:28所示，或与其具有至少90％序列同一性。
  31. 一种核酸分子，其编码权利要求26至30中任意一项所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白。
  32. 权利要求26至30中任意一项所述的抗肿瘤相关抗原(TAA)及含IL-15/IL-15Ra的融合蛋白在制备用于抑制或治疗癌症的药物中的应用，所述癌症包括选自以下的癌症或发生于以下部位：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤、以及骨髓增生异常综合症。
PCT/CN2022/096501 2021-06-02 2022-05-31 抗cd3抗体变异体、融合蛋白及应用 Ceased WO2022253248A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP22815292.2A EP4349870A4 (en) 2021-06-02 2022-05-31 Anti-CD3 Antibody Variant, Fusion Protein and Application
US18/566,817 US20240294640A1 (en) 2021-06-02 2022-05-31 Anti-cd3 antibody variant, fusion protein, and application
JP2023574487A JP7734994B2 (ja) 2021-06-02 2022-05-31 抗ｃｄ３抗体変異体、融合タンパク質および適用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110628817 2021-06-02
CN202110628817.3 2021-06-02

Publications (1)

Publication Number Publication Date
WO2022253248A1 true WO2022253248A1 (zh) 2022-12-08

Family

ID=84240946

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/096501 Ceased WO2022253248A1 (zh) 2021-06-02 2022-05-31 抗cd3抗体变异体、融合蛋白及应用

Country Status (5)

Country Link
US (1) US20240294640A1 (zh)
EP (1) EP4349870A4 (zh)
JP (1) JP7734994B2 (zh)
CN (1) CN115433282A (zh)
WO (1) WO2022253248A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4640712A4 (en) * 2022-12-31 2026-04-08 Hefei Tg Immunopharma Co Ltd Anti-PDL1 antibodies and their use

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN121851177A (zh) * 2023-12-11 2026-04-14 合肥天港免疫药物有限公司 双特异性抗体及其应用
WO2026132786A1 (en) * 2024-12-20 2026-06-25 Centessa Pharmaceuticals (Uk) Limited Anti-cd3 binding agents and uses thereof

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090252683A1 (en) * 2005-10-11 2009-10-08 Micromet Ag Composition Comprising Cross-Species-Sepecific Antibodies and uses Thereof
CN107207610A (zh) * 2014-11-26 2017-09-26 森科股份有限公司 结合cd3和肿瘤抗原的异二聚体抗体
CN109496217A (zh) * 2016-05-27 2019-03-19 阿尔托生物科学有限公司 具有cd3结合域的基于多聚体il-15的分子的构造和表征
CN110023336A (zh) * 2016-09-23 2019-07-16 生物技术公司 用于治疗表达密蛋白之癌症疾病的与密蛋白6或密蛋白18.2和cd3结合的双特异性三价抗体
US20200055932A1 (en) 2018-08-03 2020-02-20 Amgen Research (Munich) Gmbh Antibody Constructs for CLDN18.2 and CD3
WO2020088608A1 (zh) * 2018-11-01 2020-05-07 安源医药科技（上海）有限公司 同源二聚体型双特异性抗体及其制备方法和用途
WO2021063330A1 (zh) * 2019-09-30 2021-04-08 和铂医药(苏州)有限公司 靶向cd3的抗体、双特异性抗体及其用途
CN113135996A (zh) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 一种双特异抗体及其应用
CN113185611A (zh) * 2019-12-05 2021-07-30 启愈生物技术(上海)有限公司 含有肿瘤相关抗原taa抗体的三功能融合蛋白及其应用
CN113307879A (zh) * 2020-02-27 2021-08-27 启愈生物技术(上海)有限公司 一种taa/ctla-4/il15三功能融合蛋白及其应用
CN113321738A (zh) * 2020-02-27 2021-08-31 启愈生物技术(上海)有限公司 肿瘤靶向、抗cd3和t细胞激活三功能融合蛋白及其应用
CN114106195A (zh) * 2020-08-27 2022-03-01 盛禾(中国)生物制药有限公司 一种多功能融合蛋白及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HRP20251020T1 (hr) 2016-05-20 2025-10-24 Harpoon Therapeutics, Inc. Proteini s jednolančanim varijabilnim fragmentom koji vežu cd3
EP3704155A2 (en) * 2017-11-01 2020-09-09 F. Hoffmann-La Roche AG Combination therapy with targeted ox40 agonists
KR20200142525A (ko) 2018-04-05 2020-12-22 노파르티스 아게 암에 대한 삼중특이성 결합 분자 및 이의 용도
CN120842421A (zh) * 2019-07-30 2025-10-28 魁尔斯弗生物治疗股份有限公司 双特异性抗LRRC15和CD3ε抗体

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090252683A1 (en) * 2005-10-11 2009-10-08 Micromet Ag Composition Comprising Cross-Species-Sepecific Antibodies and uses Thereof
US8236308B2 (en) 2005-10-11 2012-08-07 Micromet Ag Composition comprising cross-species-specific antibodies and uses thereof
CN107207610A (zh) * 2014-11-26 2017-09-26 森科股份有限公司 结合cd3和肿瘤抗原的异二聚体抗体
CN109496217A (zh) * 2016-05-27 2019-03-19 阿尔托生物科学有限公司 具有cd3结合域的基于多聚体il-15的分子的构造和表征
CN110023336A (zh) * 2016-09-23 2019-07-16 生物技术公司 用于治疗表达密蛋白之癌症疾病的与密蛋白6或密蛋白18.2和cd3结合的双特异性三价抗体
US20200055932A1 (en) 2018-08-03 2020-02-20 Amgen Research (Munich) Gmbh Antibody Constructs for CLDN18.2 and CD3
WO2020088608A1 (zh) * 2018-11-01 2020-05-07 安源医药科技（上海）有限公司 同源二聚体型双特异性抗体及其制备方法和用途
WO2021063330A1 (zh) * 2019-09-30 2021-04-08 和铂医药(苏州)有限公司 靶向cd3的抗体、双特异性抗体及其用途
CN113185611A (zh) * 2019-12-05 2021-07-30 启愈生物技术(上海)有限公司 含有肿瘤相关抗原taa抗体的三功能融合蛋白及其应用
CN113135996A (zh) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 一种双特异抗体及其应用
CN113307879A (zh) * 2020-02-27 2021-08-27 启愈生物技术(上海)有限公司 一种taa/ctla-4/il15三功能融合蛋白及其应用
CN113321738A (zh) * 2020-02-27 2021-08-31 启愈生物技术(上海)有限公司 肿瘤靶向、抗cd3和t细胞激活三功能融合蛋白及其应用
CN114106195A (zh) * 2020-08-27 2022-03-01 盛禾(中国)生物制药有限公司 一种多功能融合蛋白及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUO SIQI, SMELTZ RONALD B., NANAJIAN ANTHONY, HELLER RICHARD: "IL-15/IL-15Rα Heterodimeric Complex as Cancer Immunotherapy in Murine Breast Cancer Models", FRONTIERS IN IMMUNOLOGY, vol. 11, 8 February 2021 (2021-02-08), Lausanne, CH , pages 1 - 12, XP093010824, ISSN: 1664-3224, DOI: 10.3389/fimmu.2020.614667 *
See also references of EP4349870A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4640712A4 (en) * 2022-12-31 2026-04-08 Hefei Tg Immunopharma Co Ltd Anti-PDL1 antibodies and their use

Also Published As

Publication number Publication date
CN115433282A (zh) 2022-12-06
EP4349870A1 (en) 2024-04-10
US20240294640A1 (en) 2024-09-05
JP7734994B2 (ja) 2025-09-08
EP4349870A4 (en) 2025-12-31
JP2024521910A (ja) 2024-06-04

Similar Documents

Publication Publication Date Title
CN111699197B (zh) 结合cd137的单结构域抗体
CN114127111B (zh) 与nkp30结合的抗体分子及其用途
CN110382539B (zh) 用于cd16a定向的nk细胞结合的串联双抗体
RU2714733C2 (ru) Технологии мультимеризации
WO2022105832A1 (zh) 抗pd-l1纳米抗体及三功能融合蛋白
CN109476747A (zh) 结合免疫调节性蛋白和肿瘤抗原的双特异性结合蛋白
EP4378960A1 (en) Antigen targeting, anti-cd16a, and immune effector cell activating trifunctional fusion protein, and application thereof
US11987609B2 (en) Proteinaceous heterodimer and use thereof
TW201900674A (zh) 含有ＴＧＦ－β受體的融合蛋白及其醫藥用途
JP7734994B2 (ja) 抗ｃｄ３抗体変異体、融合タンパク質および適用
CN117321087A (zh) 包含与细胞因子融合用于nk细胞衔接的nkp46结合位点、癌抗原结合位点的多特异性蛋白质
JP2023539645A (ja) Ｎｋｐ３０に結合する抗体分子およびその使用
US20240383985A1 (en) Antigen targeting, anti-cd16a, and immune effector cell activating trifunctional fusion protein, and application thereof
Mohammadi et al. Immunotherapy For Researchers
WO2022171080A1 (zh) 抗cd112r抗体及其用途
WO2020199860A1 (zh) 针对程序性死亡配体的结合物及其应用
US11795230B2 (en) Anti-CD27 antibodies and use thereof
TWI894360B (zh) 抗tigit抗體及雙抗體和它們的應用
WO2026085716A1 (zh) 新型il-18替代物及其用途
WO2025167357A1 (zh) Cd80/taa融合蛋白相关的肿瘤靶向、t细胞激活分子形式及其应用
JP2025530110A (ja) 二重特異性ポリペプチド及びその使用
CN121159699A (zh) 抗Trop2/CD3的双特异性抗体及其应用
WO2023246574A1 (zh) 靶向gpc3的抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22815292

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023574487

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 18566817

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2022815292

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022815292

Country of ref document: EP

Effective date: 20240102