WO2022237897A1 - Anticorps à domaine unique anti-pd-l1 et sa protéine dérivée - Google Patents

Anticorps à domaine unique anti-pd-l1 et sa protéine dérivée Download PDF

Info

Publication number
WO2022237897A1
WO2022237897A1 PCT/CN2022/092753 CN2022092753W WO2022237897A1 WO 2022237897 A1 WO2022237897 A1 WO 2022237897A1 CN 2022092753 W CN2022092753 W CN 2022092753W WO 2022237897 A1 WO2022237897 A1 WO 2022237897A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
human
binding molecule
binding
single domain
Prior art date
Application number
PCT/CN2022/092753
Other languages
English (en)
Chinese (zh)
Inventor
曾大地
刘大涛
王荣娟
焦莎莎
张畅
张姣
王双
Original Assignee
迈威(上海)生物科技股份有限公司
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 迈威(上海)生物科技股份有限公司 filed Critical 迈威(上海)生物科技股份有限公司
Publication of WO2022237897A1 publication Critical patent/WO2022237897A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present invention belongs to the field of antibody engineering, and in particular relates to a therapeutic single-domain antibody for diagnosing or treating tumors, in particular to a single-domain antibody targeting human programmed death ligand 1 (PD-L1), its derivative protein and Its use for preparing medicines, especially its use in treating and/or preventing or diagnosing PD-L1-related diseases such as tumors.
  • PD-L1 programmed death ligand 1
  • PD-1 and its ligand PD-L1 are important targets of tumor immunity.
  • PD-1 and PD-L1 are a pair of immunosuppressive molecules, which are important components of the immune system to prevent autoimmune hyperactivity. The activation of their pathways can inhibit tumor immune response and induce tumor-specific T cell apoptosis, which is closely related to tumor development. close relationship.
  • PD-L1 programmed death ligand-1, which can bind to the receptor PD-1 on the surface of T cells to exert an immunosuppressive effect.
  • PD-L1 is an inhibitory immune checkpoint molecule expressed on the surface of various malignant tumor cells such as melanoma, non-small cell lung cancer, renal cell carcinoma, and head and neck squamous cell carcinoma. After PD-L1 binds to the immunosuppressive receptor PD-1 on the surface of T cells, it can induce apoptosis, incapacity, and exhaustion of T cells, thereby inhibiting the activation, proliferation, and anti-tumor functions of tumor antigen-specific T cells, and achieving tumor immunity. immune escape.
  • PD-1/PD-L1 blocking antibody can relieve the immunosuppressive effect of PD-L1, enhance the recognition and killing of tumor cells by immune cells T cells in the body, so as to achieve the effect of killing tumors.
  • a number of antibody drugs targeting PD-1/PD-L1 have been marketed worldwide, and are clinically effective against melanoma, lung cancer, kidney cancer, Hodgkin's lymphoma, head and neck squamous cell carcinoma, and urothelial carcinoma, etc.
  • a variety of tumors have shown good therapeutic effects.
  • the effective rate of clinical use is low. For most cancer types, if they are used alone indiscriminately, the average effective rate is only 10%. %-20%. Therefore, it is necessary to develop more effective antibody drug molecules to meet clinical needs.
  • PD-L1 antibodies include: (1) Further development of antibody molecules with higher affinity and better activity; (2) Bispecific antibodies or analogs based on this target; (3) Preliminary research It shows that the response rate of PD-L1-positive tumor patients to PD-L1 inhibitors is much higher than that of PD-L1-negative tumor patients, so effective biomarkers are needed to predict PD-L1-positive tumor patients or screen patients in advance to reduce treatment costs and possible serious adverse reactions; (4) In order to improve the response rate, combination therapy has become the development trend of tumor immunotherapy, such as combination with other tumor immunotherapy drugs, combined use with targeted drugs, chemotherapy or radiotherapy. (5) Anti-PD-L1 nanobody.
  • single domain antibodies also have some unique characteristics, such as small molecular weight, strong stability, good solubility, easy expression, strong targeting, Humanization is simple, etc., especially suitable for the development of bi/multi-specific therapeutic antibodies and the development of Car-T/M/NK and other therapies.
  • Single domain antibody (single domain antibody, sdAb) is a special type of antibody that contains only one antibody heavy chain. Similar to traditional diabodies, it can selectively bind to specific antigens. Single-domain antibodies were first discovered in camelids and later in cartilaginous fishes such as nurse sharks. Single Domain Antibody The variable region (VHH) of a single heavy chain antibody is a single functional domain that can completely bind antigen, only 12-15kDa. VHH has a simple structure, and has the advantages of high specificity, high affinity, low immunogenicity, good permeability when binding to an antigen, and the ability to access relatively hidden targets that cannot be accessed by conventional antibodies during tumor treatment.
  • single-domain antibodies have only one chain, there is no mismatch problem when diabody fusion occurs.
  • the use of single-domain antibodies as the antigen-binding sequence of bispecific antibodies has great advantages and has gradually become a research hotspot (Serge Muyldermans (2013), Annu.Rev.Biochem.82:775-797).
  • Single variable domain antibody is currently the smallest antibody molecule, also known as nanobody. It was first discovered in camel blood by Belgian scientist Hamers, R. It is a class of engineered antibody products that has attracted much attention.
  • single variable domain antibodies In addition to the antigen reactivity of monoclonal antibodies, single variable domain antibodies also have some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, strong targeting, and simple humanization. It is especially suitable for the development of bi/multi-specific therapeutic antibodies and the development of Car-T/M/NK and other therapies. At present, the development of single variable domain antibodies and/or bi/multispecific antibodies based on single variable domain antibodies has become a research and development hotspot.
  • the anti-PD-L1 antibody is mainly a complete whole antibody, and the anti-PD-L1 nanobody of the nanobody is currently only one Envafolimab (KN035) approved as an orphan drug.
  • Envafolimab (KN035) is a recombinant humanized PD-L1 single domain antibody Fc fusion protein developed by Corning Jireh, and it is also the world's first PD-L1 single domain antibody (single domain antibody) to enter the clinic.
  • KN035 has significant differentiated advantages in terms of safety, convenience, and compliance, and can effectively improve the quality of life of patients.
  • the present disclosure provides a single-domain antibody targeting human programmed death ligand 1 (PD-L1) and its derivative protein.
  • PD-L1 recombinant protein as an immunogen
  • isolate PBMC to construct a single-domain antibody heavy chain variable region phage surface display antibody library
  • human PD-L1 recombinant protein to perform solid-phase screening to obtain phage that can bind to human PD-L1 recombinant protein
  • Human-camelin chimeric antibody chB6 was prepared and humanized to obtain hzB6. Affinity maturation was carried out on the basis of hzB6, and 27 variants of humanized single domain antibodies were obtained.
  • the invention provides a binding molecule.
  • the binding molecule includes one or more human PD-L1 binding domains, and the human PD-L1 binding domains include:
  • CDR1 EYIGDRYCAG (SEQ ID NO:33) or GFTFDRYCAG (SEQ ID NO:34);
  • CDR2 MIDRHGIVRYKDSVEG (SEQ ID NO: 35);
  • CDR3 ERDPTNVTPCRPEYPDMDY (SEQ ID NO: 36), or a polypeptide obtained by replacing one or two amino acids on the basis of ERDPTNVTPCRPEYPDMDY.
  • the CDR3 has the amino acid sequence described in any one of SEQ ID NO: 36-56.
  • the one or more human PD-L1 binding domains include any amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4, 6-32.
  • the binding molecule also includes one or more non-PD-L1 binding functional components, and the human PD-L1 binding domain is connected to the non-PD-L1 binding functional components through covalent linkage or non-PD-L1 binding functional components. covalently linked.
  • the non-PD-L1 binding functional components include carrier protein, active peptide, tag peptide and chaperone protein;
  • the covalent connection includes linker connection, peptide bond connection and bifunctional group connection , said non-covalent linking includes affinity coupling.
  • the carrier protein includes BSA, OVA, HSA, KLH, and Fc, etc.
  • the active peptide includes linear peptide and cyclic peptide, and has enzyme activity, cytotoxic activity, antigen binding activity, etc.
  • the tag peptides include purification tag peptides and tracer tag peptides
  • the chaperone proteins include HSP60, HSP70 and HSP90.
  • the binding molecules are single domain antibodies, heavy chain antibodies, camel antibodies, chimeric antibodies, humanized antibodies and multispecific antibodies (including bispecific antibodies, trispecific antibodies , tetravalent antibodies, etc.), monovalent antibodies and multivalent antibodies (including bivalent antibodies, trivalent antibodies, tetravalent antibodies, etc.).
  • the multispecific antibody also has one or more specificities for other epitopes or fragments of PD-L1, and/or one or more specificities for other antigens.
  • the other antigens are selected from at least one of CTLA-4, LAG3, TIGIT, TIM3, CD47, 4-1BB, CD73, ROR1, HER2, HER3, EGFR and OX40.
  • the invention provides a binding molecule.
  • the binding molecule is a homomultimer or a heteromultimer, and includes at least one aforementioned binding molecule.
  • the multimer includes dimer, trimer and tetramer.
  • At least two monomers of the multimer respectively comprise the aforementioned human PD-L1 binding domains, and the human PD-L1 binding domains in the at least two monomers interact to make The multimer can specifically bind human PD-L1.
  • the present invention provides an anti-human PD-L1 single domain antibody. According to an embodiment of the present invention, in the single domain antibody
  • CDR1 has the amino acid sequence shown in EYIGDRYCAG (SEQ ID NO:33) or GFTFDRYCAG (SEQ ID NO:34);
  • CDR2 has the amino acid sequence shown in MIDRHGIVRYKDSVEG (SEQ ID NO: 35),
  • CDR3 has X 1 X 2 X 3 X 4 TX 5 X 6 X 7 PCRX 8 X 9 YPDMDY,
  • X1 is selected from E, D,
  • X2 is selected from R, E,
  • X 3 is selected from D, S,
  • X is selected from P, R, S,
  • X is selected from N, S, A, Q, D, T,
  • X 6 is selected from V, P,
  • X is selected from T, I, L, A , Y, Q,
  • X 8 is selected from P, A,
  • X9 is selected from E, T, S.
  • the single domain antibody is a camel single domain antibody, a chimeric single domain antibody or a humanized single domain antibody.
  • the anti-human PD-L1 single domain antibody comprises any amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4 and 6-32.
  • the present invention provides a variable region of an anti-human PD-L1 single domain antibody.
  • the variable region of the anti-human PD-L1 single domain antibody includes any amino acid sequence selected from SEQ ID NO: 1, 4 and 6-32.
  • the present invention provides a humanized anti-human PD-L1 heavy chain antibody.
  • the heavy chain antibody includes the amino acid sequence shown in SEQ ID NO: 1 or a mutant having at least 90% identity therewith and having human PD-L1 binding ability.
  • the mutation of the mutant is a combination of the following sites: A23, G44, L45, D72, N73, K75, Q81, E26, Y27, I28, G29, N103, T105, A74, L78, D100, P101, E98, R99, E110, P109 and V104.
  • the mutation of the mutant is selected from the group consisting of amino acid substitutions at the following mutation sites: A23I, G44E, L45R, D72N, N73H, K75G, Q81E, E26G, Y27F, I28T, G29F .
  • the mutation of the mutant is selected from:
  • the present invention provides an immunoconjugate.
  • the immunoconjugate includes:
  • the coupling moiety includes a detectable label, a cytotoxic molecule or a bioactive molecule.
  • the detectable labels include chemiluminescent labels, fluorescent labels, enzyme labels, radioactive labels, quantum dots and nanoparticles.
  • the cytotoxic molecules include diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin and the like.
  • bioactive molecules include cytokines, enzymes, chemotherapeutic agents, liposomes and virus particles.
  • the present invention provides a composition.
  • the composition comprises the aforementioned binding molecule, the aforementioned anti-human PD-L1 single domain antibody, the aforementioned variable region of the aforementioned anti-human PD-L1 single domain antibody, the aforementioned humanized anti-human PD- L1 heavy chain antibody or the aforementioned immunoconjugate.
  • the composition further includes biotherapeutic agents, chemotherapeutic agents, natural active ingredients and the like.
  • the composition further includes a pharmaceutically acceptable carrier and is used as a pharmaceutical composition.
  • the pharmaceutical composition is an aqueous solution, an injection, or a powder injection.
  • the present invention provides a polynucleotide.
  • the polynucleotide encodes the aforementioned binding molecule, the aforementioned anti-human PD-L1 single domain antibody, the aforementioned variable region of the aforementioned anti-human PD-L1 single domain antibody, or the aforementioned humanized anti-human PD - L1 heavy chain antibody.
  • the present invention provides a nucleic acid construct.
  • the nucleic acid construct comprises the aforementioned polynucleotide.
  • the present invention provides a host cell.
  • the host cell comprises the aforementioned polynucleotide or the aforementioned nucleic acid construct.
  • the present invention provides a method for preparing the aforementioned binding molecule, the aforementioned anti-human PD-L1 single domain antibody, the aforementioned variable region of the aforementioned anti-human PD-L1 single domain antibody, or the aforementioned humanized anti-human PD -A method for an L1 heavy chain antibody, comprising the steps of:
  • the host cell is Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, animal cells, plant cells or insect cells.
  • the present invention provides a method for preparing the aforementioned binding molecule, the aforementioned anti-human PD-L1 single domain antibody, the aforementioned variable region of the aforementioned anti-human PD-L1 single domain antibody, the aforementioned humanized anti-human PD - L1 heavy chain antibody or the aforementioned immunoconjugate. According to the embodiments of the present invention, it is prepared by recombinant expression method, chemical synthesis method or combined method of recombinant expression and chemical synthesis.
  • the chemical synthesis method is a solid phase synthesis method, a liquid phase synthesis method or a solid phase-liquid phase synthesis method.
  • the chemical synthesis method adopts an N-terminal-to-C-terminal synthesis method, a C-terminal-to-N-terminal synthesis method, or a segmented synthesis method.
  • the present invention provides the aforementioned binding molecule, the aforementioned anti-human PD-L1 single domain antibody, the aforementioned variable region of the aforementioned anti-human PD-L1 single domain antibody, or the aforementioned humanized anti-human PD-L1 heavy Use of the chain antibody, the aforementioned immunoconjugate, or the aforementioned composition in the preparation of a therapeutic PD-1/PD-L1 signaling pathway blocker.
  • the present invention provides the aforementioned binding molecule, the aforementioned anti-human PD-L1 single domain antibody, the aforementioned variable region of the aforementioned anti-human PD-L1 single domain antibody, or the aforementioned humanized anti-human PD-L1 Use of the heavy chain antibody, the aforementioned immunoconjugate, or the aforementioned composition in the preparation of a product for diagnosing, preventing, treating, or evaluating a disease related to the overexpression of the PD-1/PD-L1 signaling pathway.
  • the diseases related to the overexpression of PD-1/PD-L1 signaling pathway include dysproliferative diseases or immune-related diseases.
  • the abnormal proliferative disease is selected from tumor, cyst and hyperplasia;
  • the immune-related disease is selected from inflammation, immunodeficiency, immune tolerance and allergy.
  • the tumor is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, esophageal cancer, liver cancer, gastric cancer, kidney cancer, bladder cancer, head and neck tumors, Hodgkin's lymphoma , cervical cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, endometrial cancer, glioma, neuroendocrine tumors, malignant mesothelioma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and All microsatellite instability-high (MSI-H) solid tumors.
  • MSI-H microsatellite instability-high
  • immunoglobulin sequence is used as a generic term including full-sized antibodies, individual chains thereof, and all of them Parts, domains or fragments (including but not limited to antigen binding domains or fragments such as VHH domains or VH/VL domains, respectively).
  • sequence as used herein (for example in terms like “immunoglobulin sequence", “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence)
  • sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence)
  • sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence)
  • sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence)
  • Immunoglobulin single variable domains can be used as a method for preparing immunoglobulins containing one or more additional immunoglobulins that can serve as binding units (i.e., for the same or different epitopes of the same target and/or for one or more different targets).
  • a "binding unit”, “binding domain” or “building block” (the terms are used interchangeably) of a polypeptide of a globulin single variable domain.
  • immunoglobulin single variable domain which is used interchangeably with “single variable domain” (“SVD”), defines a domain in which the antigen binding site exists on a single immunoglobulin domain And molecules formed from a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from “regular” immunoglobulins or their fragments, in which two immunoglobulin variable domains, especially two variable domains, interact to form antigens binding site.
  • the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) interact to form the antigen binding site.
  • the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, ie a total of 6 CDRs will be involved in the formation of the antigen binding site.
  • the binding site of an immunoglobulin single variable domain is formed by a single VH or VL domain.
  • the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.
  • immunoglobulin single variable domain and “single variable domain” therefore do not include conventional immunoglobulins or fragments thereof which require the interaction of at least two variable domains to form an antigen binding site. However, these terms include fragments of conventional immunoglobulins in which the antigen binding site is formed by a single variable domain.
  • a single variable domain will be an amino acid sequence consisting essentially of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively).
  • Such single variable domains and fragments are most preferred such that they comprise an immunoglobulin fold or are capable of forming an immunoglobulin fold under suitable conditions.
  • a single variable domain may, for example, comprise a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or VHH sequence) or a suitable fragment thereof; Fragment; as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain such that the single antigen-binding unit does not need to interact with another variable domain to form a functional Antigen binding units, e.g. for variable domains present e.g. in conventional antibodies and scFv fragments that require interaction with another variable domain, e.g. through VH/VL interactions, to form a functional antigen binding domain is the case).
  • a light chain variable domain sequence e.g., a VL sequence
  • a heavy chain variable domain sequence e.g., a VH sequence or VHH sequence
  • Fragment
  • the immunoglobulin single variable domain is a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); more specifically,
  • the immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a conventional four chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody.
  • a single variable domain or an immunoglobulin single variable domain may be a (single) domain antibody (or suitable for use as a (single) domain amino acid of an antibody), a "dAb” or a dAb (or an amino acid suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to VHH); other single variable domains, or any of them Any suitable fragment of a species.
  • single variable domains may be derived from certain species of sharks (e.g. so-called "IgNAR domains", see e.g. WO 05 /18629).
  • immunoglobulin single variable domains may be (as defined herein) or suitable fragments thereof.
  • suitable fragments thereof are a registered trademark of Ablynx NV.
  • Nanobodies reference is made to the further description below, as well as to the prior art cited herein, such as, for example, in WO 08/020079( described on page 16).
  • VHHs and Nanobodies For a further description of VHHs and Nanobodies, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302), and to the following patent application mentioned as general background art: VrijeUniversiteit Brussel WO 94/04678, WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/ 44301, EP 1134231 and WO 02/48193; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituutvoor Biotechnologie (VIB); Algonomics N.V.
  • Nanobodies may be characterized in particular by the presence of one or more "signature residues" in one or more framework sequences.
  • a further description of Nanobodies, including humanization and/or camelization of Nanobodies, and other modifications, parts or fragments, derivatives or "Nanobody fusions" can be found, for example, in WO 08/101985 and WO 08/142164. ", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase the half-life of Nanobodies and their preparation.
  • the term “immunoglobulin single variable domain” or “single variable domain” includes polypeptides derived from non-human sources, preferably camelids, preferably camelid heavy chain antibodies. They may be humanized as previously described. Furthermore, the term includes polypeptides derived from non-camelid sources such as mice or humans that have been "camelized”, for example in Davies and Riechmann 1994 (FEBS 339:285-290), 1995 (Biotechnol. 13:475- 479), 1996 (Prot. Eng. 9:531-537) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231:25-38).
  • immunoglobulin single variable domain includes immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. It also includes fully human, humanized or chimeric immunoglobulin sequences. For example, it includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized immunoglobulin single variable domains, such as the camelized dAb described by Ward et al., 1989 ( See eg WO 94/04678 and Davies and Riechmann 1994, 1995 and 1996) and camelized VH.
  • immunoglobulin single variable domains may be derived in any suitable manner from any suitable source and, for example, may be naturally occurring VHH sequences (i.e. from a suitable camelid species) or synthetic or semi- Synthetic amino acid sequences, including but not limited to partially or fully “humanized” VHHs, "camelized” immunoglobulin sequences (and especially camelized VHs), and Nanobodies obtained by techniques and/or VHH: such as affinity maturation (e.g.
  • immunoglobulin sequence such as a VHH sequence
  • CDR grafting CDR grafting
  • veneering combining fragments derived from different immunoglobulin sequences, using PCR assembly of overlapping primers, and similar techniques known to the skilled artisan for engineering immunoglobulin sequences; or any suitable combination of any of the foregoing.
  • the amino acid sequence and structure of an immunoglobulin single variable domain can be considered - however not limited to - to consist of four framework regions, or "FRs”, which are referred to in the art and herein as “framework regions” respectively.
  • FRs framework regions
  • 1" or “FR1”; “Framework Region 2" or “FR2”; “Framework Region 3” or “FR3”; and “Framework Region 4" or “FR4"; CDR which are referred to in the art as “complementarity determining region 1" or “CDR1”; “complementarity determining region 2" or “CDR2”; and “complementarity determining region 3" or “CDR3", respectively.
  • the total number of amino acid residues in an immunoglobulin single variable domain may be in the range of 110-120, preferably 112-115, and most preferably 113.
  • the immunoglobulin single FR1 of the variable domain contains amino acid residues at positions 1-30
  • CDR1 of the immunoglobulin single variable domain contains amino acid residues at positions 31-35
  • FR2 of the immunoglobulin single variable domain contains Amino acids at positions 36-49
  • the CDR2 of the immunoglobulin single variable domain contains amino acid residues at positions 50-65
  • the FR3 of the immunoglobulin single variable domain contains amino acid residues at positions 66-94
  • the immune The CDR3 of a globulin single variable domain comprises amino acid residues at positions 95-102
  • the FR4 of an immunoglobulin single variable domain comprises amino acid residues at positions 103-113.
  • FR4 comprises the C-terminal amino acid sequence VTVSS, ie each of positions 109, 110, 111, 112 and 113.
  • the invention also includes ISVDs that terminate at position 109, 110, 111 or 112.
  • FR4 terminates with the C-terminal amino acid sequence VTVS (position 109-112)
  • FR4 terminates with the C-terminal amino acid sequence VTV (position 109-111)
  • FR4 terminates with the C-terminal amino acid sequence VT (position 109-110).
  • FR4 terminates with C-terminal amino acid V (position 109).
  • the C-terminal extension may be present at the C-terminus of the last (most C-terminal) FR4 of the ISVD, e.g. V109, T110, V111, S112 or S113, wherein the cysteine moiety of the present invention is preferably present at Or at the C-terminus of the C-terminal extension.
  • FR4 comprises a C-terminal amino acid sequence VTVSS and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTVSSC).
  • FR4 comprises the C-terminal amino acid sequence VTVS and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTVSC).
  • FR4 comprises the C-terminal amino acid sequence VTV and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTVC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VT and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTC). In one embodiment, FR4 comprises a C-terminal amino acid V and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VC).
  • the invention relates to a dimer as described herein, wherein said ISVD is selected from the group consisting of single domain antibodies, domain antibodies, suitable for use as single domain antibodies Amino acid sequences, amino acid sequences suitable for use as domain antibodies, dAbs, amino acid sequences suitable for use as dAbs, Nanobodies, VHH, humanized VHH, and camelized VH.
  • the ISVD comprises between 100 and 140 amino acids, such as between 110-130 amino acids.
  • phage display library refers to a "library" of phage on the surface of which exogenous peptides or proteins are expressed.
  • Foreign peptides or polypeptides are displayed on the outer surface of the phage capsid.
  • the foreign peptide can be displayed as a recombinant fusion protein incorporated as part of the phage coat protein; a recombinant fusion protein that is not normally a phage coat protein but can become incorporated into the outer surface of the capsid; or become covalently or non-covalently linked A protein or peptide based on said protein. This is achieved by inserting exogenous nucleic acid sequences into nucleic acids that can be packaged into phage particles.
  • Said exogenous nucleic acid sequence may for example be inserted into the coding sequence of the phage coat protein gene. If the foreign sequence is cloned in frame, then the protein it encodes will be expressed as part of the coat protein.
  • a library of nucleic acid sequences such as a library of antibody repertoires made from gene segments encoding the entire B-cell repertoire of one or more individuals, can thus be inserted into phage to generate a "phage library”.
  • a "peptide display library” is produced when those peptides and proteins representative of those encoded by the nucleic acid library are displayed by phage. Although a variety of phages are used in such library construction, filamentous phages are commonly used (Dunn (1996) Curr. Opin. Biotechnol. 7:547-553). See, eg, the description of phage display libraries below.
  • chimeric antibody refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species.
  • variable regions of both the light and heavy chains correspond to variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity and capacity, Instead, the constant regions are homologous to sequences in the antibody derived from another species, usually a human, to avoid eliciting an immune response in that species.
  • chimeric antibody refers to a portion of the heavy and/or light chain that is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to that derived from another antibody class or subclass.
  • the corresponding sequences in antibodies belonging to one species or belonging to another antibody class or subclass are identical or homologous, and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison SL et al., Proc. . Natl. Acad. Sci. USA, 81:6851-6855, 1984).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).
  • a primary antibody e.g., a murine antibody
  • the light chain constant region is from a second antibody (eg, a human antibody).
  • an antibody of the invention may be monospecific, bispecific or multispecific. Multispecific antibodies can be specific for different epitopes of a target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. See, eg, Tutt et al., 1991, Journal of Immunology 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. An antibody of the invention may be linked to or co-expressed with another functional molecule (eg, another peptide or protein).
  • another functional molecule eg, another peptide or protein
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to Bispecific or multispecific antibodies are produced with a second binding specificity.
  • the invention includes bispecific antibodies in which one arm of the immunoglobulin is specific for the N-terminal region of hemolysin A or a fragment thereof and the other arm of the immunoglobulin is specific for the C-terminal region of hemolysin A Either the second therapeutic agent is target specific, or binds to a therapeutic agent moiety.
  • An exemplary bispecific antibody format that may be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains are due to at least one Amino acids differ from each other, and wherein at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking said amino acid difference.
  • the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering).
  • the second CH3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU).
  • Other modifications that can be found within the second CH3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; according to EU are D356E, L358M, N384S, K392N, V397M and V422I); In the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); EU is Q355R, N384S, K392N, V397M, R409K, E419Q and V422I).
  • bispecific antibody formats are encompassed within the scope of the invention.
  • Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, e.g. scFv or bifunctional antibody based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetramers Hybridoma (Quadroma), Knob-into-Hole Structure, Common Light Chain (e.g.
  • Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into molecules with defined The composition, valence and geometry of multimeric complexes. (See eg, Kazane et al., J. Am. Chem. Soc. [Epub: 4 Dec. 2012).
  • single-domain antibody is obtained by genetic engineering methods. There are mainly three types. The first type is the heavy chain variable region obtained from camelid HCAb. It is a single folding unit that retains complete antigen-binding activity and is the smallest natural antibody fragments. The second category is the heavy chain variable region obtained from cartilaginous fish IgNARs such as sharks, denoted by VNAR. The third type is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibody, which retains the antigen binding activity, but the affinity and solubility are greatly reduced.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain and the CH3 domain, which mediate the binding of the immunoglobulin to host tissues or factors , including binding to Fc receptors located on various cells of the immune system (eg, effector cells) or binding to the first component of the classical complement system (eg, C1q), including native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region is the segment from its amino acid residue at position Cys226 or Pro230 to the carboxy-terminus, although its boundaries may vary.
  • the C-terminal lysine of the Fc region may or may not be present.
  • Fc may also refer to this region in isolation, or in the case of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein", also referred to as an "Fc fusion protein” (eg, an antibody or an immunoadhesin).
  • the native sequence Fc region of the antibody of the present invention includes human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • the Fc region contains the CH2 and CH3 constant domains of each of the antibody's two heavy chains; the IgM and IgEFc regions contain the three heavy chain constant domains in each polypeptide chain domain (CH domains 2-4).
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.
  • immunological binding refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific.
  • the strength or affinity of the immune binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, wherein the smaller the KD value, the higher the affinity.
  • KD equilibrium dissociation constant
  • the immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • association rate constant Ka or Kon
  • dissociation rate constant Kd or Koff
  • concentration a ratio of Kd/Ka is equal to the dissociation constant KD, see Davies DR et al., 1990, Annual Rev Biochem., 59:439-473.
  • KD, Ka and Kd values can be measured by any effective method.
  • the dissociation constant is measured using bioluminescence interferometry.
  • the dissociation constant can be measured using surface plasmon resonance techniques (eg Biacore) or KinExa.
  • antibody drug conjugate consists of an antibody, a linker and a drug, and the linker is a combination of cleavable linkers or a non-cleavable linker.
  • Antibodies are globular proteins that contain a series of amino acid sites for drug-linker conjugation. Due to their tertiary and quaternary structures, only solvent-accessible amino acids are available for coupling. In fact, high-yield couplings usually occur at the ⁇ -amino group of a lysine residue or the sulfhydryl group of a cysteine residue.
  • DAR drug/antibody ratio
  • the antibody constituting the antibody-drug conjugate in the present invention preferably maintains its original antigen-binding ability in the wild state.
  • the antibodies of the invention are capable of, preferably specifically, binding to an antigen.
  • targets can be specifically expressed on the surface of one or more cancer cells, with little or no expression on the surface of one or more non-cancer cells.
  • tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancer cells. Identification of such tumor-associated factors could greatly improve the specific targeting properties of antibody-based therapies for cancer.
  • conjugate refers to the association of two or more molecules. Linking can also be genetic (ie recombinant fusion). In specific contexts, the term includes reference to linking a ligand (eg, an antibody moiety) to an effector molecule. The linking can be achieved using a variety of art-recognized techniques, such as chemical or recombinant means. “Chemical means” refers to the reaction between the antibody moiety and the effector molecule such that a covalent bond is formed between the two molecules to form one molecule.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioisotopes (eg, I131, I125, Y90, Re186), chemotherapeutic agents, and toxins (such as enzymatic toxins of bacterial, fungal, plant or animal origin), or fragments thereof.
  • cytokine generally refers to a protein that is released by a population of cells, acts on another cell as an intercellular medium, or has an autocrine effect on the cell that produced the protein.
  • cytokines include lymphokines, monokines; interleukins (“IL”), such as IL-2, IL-6, IL-17A-F; tumor necrosis factors, such as TNF-alpha or TNF-beta; and Other polypeptide factors, such as leukemia inhibitory factor ("LIF").
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid 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 with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell and thereby replicate along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
  • expression vectors useful in recombinant DNA techniques are usually in the form of plasmids.
  • viral vectors eg, replication defective retroviruses, adenoviruses and adeno-associated viruses
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • a nucleic acid molecule can be single- or double-stranded, and can be cDNA.
  • polypeptide refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain.
  • One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds.
  • a “protein” may comprise one or more polypeptides.
  • host cell is a cell in which a vector can be propagated and its DNA can be expressed, which can be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that not all progeny will be identical to the parental cells as mutations may occur during replication and such progeny are included.
  • immune-related disease refers to an immune-related disease in a mammal that is caused, mediated, or otherwise contributed to by components of the mammalian immune system, and also includes a disease in which stimulating or interfering with the immune response has an ameliorating effect on the development of the disease.
  • the term includes immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumors, and the like.
  • cancer refers to or describe the physiological condition of a mammal in which a population of cells is characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, benign or malignant tumors.
  • cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma , cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, brain cancer, hepatic carcinoma and various types of head and neck cancer, neurofibromatosis type I or type II.
  • Other examples of such cancers include those that are therapy resistant, refractory or metastatic.
  • the current anti-PD-L1 antibody is mainly a complete whole antibody
  • the anti-PD-L1 nanobody of the nanobody is currently only one Envafolimab (KN035) that has been approved as an orphan drug.
  • the invention develops anti-PD-L1 nanobody with good biological activity and druggability, which provides more options for clinical development of anti-PD-L1 monoclonal antibody and multispecific antibody drug with better curative effect.
  • the present invention is based on the defects of low affinity and poor solubility caused by humanization and camelization of murine antibodies in the prior art, and uses phage surface display technology to construct an immune camel VHH antibody library, through library screening, human A humanized anti-PD-L1 single domain antibody that specifically binds to human PD-L1 protein with high affinity was obtained, with an affinity (KD) of 2.26nM, comparable to that of the control antibody KN035.
  • KD affinity
  • the basic characteristics of high affinity and specificity provide a theoretical basis and broad application prospects for the inhibitory effect of hzB6 on the PD-1/PD-L1 signaling pathway.
  • single-domain antibodies usually have low biological activity and unsatisfactory metabolic kinetics due to their monovalency and small molecular weight. So far, their clinical application is still relatively rare.
  • the humanized anti-PD-L1 single domain antibody hzB6 and its derivatives prepared by the present invention have good biological activity.
  • CHO-PD-L1 recombinant human PD-L1
  • the binding effect of human PD-1, IC50 is 7.99nM
  • the blocking of PD-1/PD-L1 signaling pathway by hzB6 was detected by using Jurkat-PD1-NFAT cells and CHO-PD-L1-CD3L cell reporter gene method, the results
  • the active EC50 is 1.99nM; hzB6 has good stability in vivo and can effectively inhibit tumor growth in hPD-1/hPD-L1/hLAG-3 transgenic mouse MC38-hPD-L1 tumor model.
  • Figure 1 FACS analysis of the binding activity of chB6 to cell surface antigens.
  • Figure 2 ELISA analysis of the binding specificity of chB6 to recombinant PD-L1.
  • Figure 3 Analysis of the binding specificity of chB6 to PD-L1 on the cell surface.
  • Figure 4 Analysis results of the inhibitory effect of hzB6 on the binding of human PD-L1 to its receptor PD-1.
  • Figure 5 Reporter gene system assessing the in vitro blocking activity of hzB6m24.
  • Fig. 6 Drug-time curve of hzB6m24 single administration in Balb/C mice.
  • Figure 7 Experimental results of antitumor efficacy of hzB6m24 on human hPD-1/hPD-L1/hLAG-3 transgenic mice subcutaneously allografted MC38-hPDL1 mouse colon cancer tumor model.
  • Example 1 Preparation of camelid single domain antibody by phage display
  • Camels were immunized with human PD-L1 recombinant protein antigen, peripheral blood mononuclear cells (PBMC) were isolated and total RNA was extracted for reverse transcription, and the variable domain of heavy chain of single domain antibody was amplified using the reverse transcription product as a template.
  • the heavy-chain of heavychain antibody, VHH was connected to a phage display vector, and electrotransfected into Escherichia coli TG1 competent cells to construct a camel immune library. Then, the constructed camel immune library was screened by solid-phase screening using PD-L1 recombinant protein to obtain specific phage-displayed single-domain antibodies.
  • VHH-B6 camel-origin single-domain antibody B6
  • SEQ ID NO.1 Amino acid sequence of camel-derived single domain antibody VHH-B6 variable region
  • Example 2 Preparation of anti-human PD-L1 chimeric single domain antibody fusion protein and control antibody
  • Design specific primers use the positive cloned phagemid as a template, and obtain the variable region gene of the camel-derived antibody VHH-B6 by PCR, and clone the variable region gene into the human Fc (IgG1, hFc) coding gene by enzyme digestion and ligation. on eukaryotic expression vectors. After obtaining the expression plasmid with the correct sequence, it was transfected into 293F cells for transient expression, purified by Protein A, and finally the human-camelid chimeric single domain antibody fusion protein (VHH-B6-human-Fc chimeric antibody, chB6 for short) was obtained.
  • the amino acid sequence of human Fc (IgG1, hFc) is shown in SEQ ID NO.2.
  • variable region gene of KN035 was fully synthesized, and the amino acid sequence of the KN035 variable region is shown in SEQ ID Shown in NO.3. And use the same strategy as above to construct the KN035-hFc fusion protein in the same form as chB6, referred to as KN035.
  • SEQ ID NO.2 Amino acid sequence of human IgG1 Fc region
  • Example 3 Analysis of binding activity of anti-human PD-L1 chimeric single domain antibody
  • chB6 was diluted to 4 ⁇ g/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s.
  • Human PD-L1 recombinant protein is used as the mobile phase to interact with the antibody captured on the surface of the chip.
  • the concentration of PD-L1 recombinant protein was 60nM.
  • the binding time of each antigen is 300s, and the final dissociation time is 300s. The results are shown in Table 1. Under the above experimental conditions, chB6 has a higher affinity for PD-L1 recombinant protein, which is similar to that of the control antibody KN035.
  • Cells (CHO-PD-L1-CD3L cells) were collected by centrifugation, and the cells were divided into 5 ⁇ 105cells/sample/100 ⁇ L, and serially diluted single-domain antibodies were added. gradient. Incubate on ice for 2 h, wash cells twice with ice-cold PBS (containing 0.05% Tween); add FITC-labeled anti-human Fc secondary antibody (Cat.: F9512, Sigma), incubate on ice for 1 h, ) Wash the cells twice, resuspend in 200 ⁇ L of flow cytometry buffer, and measure the mean fluorescence intensity (MFI) of the cells with a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER). The test results are shown in Figure 1. The binding activity of chB6 and KN035 to PD-L1 expressed on the cell surface is comparable, and their half effective binding concentration (EC50) values are 0.87nM and 1.27nM, respectively.
  • Example 4 Specificity analysis of anti-human PD-L1 chimeric single domain antibody
  • Dilute human recombinant proteins (PD-L1, PD-1, B7H3, B7H4, CTLA4, CD28, ICOS, etc.) to 1 ⁇ g/mL with PBS, coat 100 ⁇ L/well on an enzyme-linked plate, and coat overnight at 4°C; 5% BSA Blocking solution 37°C constant temperature incubator for 60min, PBST wash plate 3 times; add chB6 diluted to 1 ⁇ g/mL, react at 37°C for 60min, PBST wash plate 4 times; add 1:5000 diluted HRP-Anti-human IgG for 45min , wash the plate 4 times with PBST; finally add TMB substrate for color development, react in a constant temperature incubator at 37°C for 15 minutes, stop the reaction with 2M HCl, take 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at a wavelength of 450nm.
  • chB6 specifically binds to PD-L1 and does
  • chB6 was diluted to 4 ⁇ g/mL with PBS buffer, and flowed over the surface of the AHC probe (PALL, Cat: 18-0015) for 120 s.
  • Monkey PD-L1 recombinant protein and mouse PD-L1 recombinant protein are used as the mobile phase to interact with the antibody captured on the surface of the chip.
  • the concentration of PD-L1 recombinant protein was 60nM.
  • the binding time of each antigen is 300s, and the final dissociation time is 300s.
  • the results are shown in Table 2. Both chB6 and KN035 bind to monkey PD-L1 recombinant protein with similar affinity; they do not bind to mouse PD-L1 recombinant protein.
  • the cells were collected by centrifugation, resuspended in the medium, divided into 3 ⁇ 10 5 cells/sample/100 ⁇ L, and 20 ⁇ g/ml single domain antibody was added. Incubate on ice for 2 hours, wash cells twice with ice-cold PBS (containing 0.05% Tween); add FITC-labeled anti-human Fc secondary antibody (sigma, F9512), incubate on ice for 1 hour, wash cells with ice-cold PBS (containing 0.05% Tween) 2 times, resuspended in 200 ⁇ L flow buffer, and detected by flow cytometry.
  • the test results are shown in Figure 3.
  • the reactivity of chB6 to each tumor cell is exactly the same as that of the control antibody KN035, and both specifically bind to cell lines expressing human PD-L1, but not to non-expressing cell lines.
  • VHH-B6-human-Fc humanized antibody (VHH-B6-human-Fc humanized antibody, abbreviated as hzB6)
  • SEQ ID NO.4 The amino acid sequence of the variable region of the designed humanized single domain antibody fusion protein (VHH-B6-human-Fc humanized antibody, abbreviated as hzB6) is shown in SEQ ID NO.4, and the full-length amino acid sequence is shown in SEQ ID NO.5.
  • the nucleotide sequence of hzB6 variable region was synthesized, and the recombinant expression vector of hzB6 was constructed. After eukaryotic expression was carried out by using the constructed full-length hzB6 expression vector, the affinity was determined by BLI method, and the correlation analysis was carried out.
  • the antibody strain hzB6 was further mutated to obtain a large number of mutant antibodies.
  • the mutant sequence CDR region amino acid sequence mutation site and mutation scheme are shown in Table 3 below, and the hzB6 mutant variable region amino acid sequence is shown in Table 4 (SEQ ID NO .6 ⁇ SEQ ID NO.32), the binding and dissociation constants of hzB6 mutants and human PD-L1 are shown in Table 5.
  • Example 6 ELISA detection of the inhibitory effect of hzB6 and its mutants on the binding of human PD-L1/PD-1
  • Human PD-1-hFc (PD-1 serial number: NP_005009.2, 21aa-167aa) was diluted to 0.5 ⁇ g/mL with PBS, coated overnight at 4°C, and blocked with 5% BSA in a 37°C incubator for 60 minutes.
  • Serially dilute hzB6 and control antibody KN035, and the isotype control NC-hIgG1 (initial working concentration is 50nM, 1.5-fold dilution of 10 concentration gradients) add PD-L1-mFc (PD-L1 sequence No.: NP_054862.1, 19aa-238aa) were co-incubated and reacted in a constant temperature incubator at 37°C for 60min.
  • Example 7 Evaluation of cell blocking activity by PD-1 and PD-L1 reporter gene method
  • the blocking effect of hzB6 on PD-1 and PD-L1 pathways was detected using Jurkat-PD1-NFAT cells and CHO-PD-L1-CD3L cell reporter gene method (Reporter Gene Assay, RGA).
  • the details are as follows: the CHO-PD-L1-CD3L cells in the logarithmic growth phase were adjusted to a cell density of 5 ⁇ 10 5 cells/ml, and plated at 100 ⁇ l/well for overnight culture.
  • Antibody samples were pre-diluted step by step to 20 ⁇ g/ml with medium products, and then serially diluted by 2 times, a total of 10 points, and the diluted samples were added to overnight cultured cells, 50 ⁇ l/well.
  • the blocking activity of hzB6 and most of its mutants is basically equivalent to that of KN035, in which the EC50 of hzB6m24 is 1.99nM, while the EC50 of the control KN035 is 3.31nM.
  • the blocking activity of other mutants of hzB6 was determined by the same method, and the results showed that most of the mutants maintained the same activity level as hzB6 (EC50, Table 7); the results of hzB6m24 binding blocking activity on PD-1 and PD-L1 are shown in the figure 5.
  • Healthy female 5-week-old nude mice in a group of 3, injected the antibody into the mice through the tail vein, and the dosage was a single dose of 15mg/kg, 2h, 4h, 8h, 24h after administration, respectively , 48h, 96h, 144h, and 196h tail vein blood collection, blood samples were collected and centrifuged to separate serum, stored at -20°C until use, and its pharmacokinetic properties were investigated.
  • Example 9 Detection of antitumor efficacy of hzB6m24 on human PD-1/PD-L1/LAG-3 transgenic mice subcutaneously implanted with MC38-hPDL1 mouse colon cancer tumor model
  • Murine colon cancer tumor cells MC38-hPDL1 highly expressing human PD-L1 were inoculated into female hPD-1/hPD-L1/hLAG-3 transgenic mice (PD-1/PD-L1/LAG-3 humanized from C57 Mice, purchased from Biocytogen, Cat.130572) subcutaneously on the right anterior flank, were divided into 3 groups when the tumor grew to about 100mm 3 , and 6 mice in each group were given hzB6m24 and isotype control respectively NC-hIgG1, wherein the dosages of hzB6m24 were 6 mg/kg and 3 mg/kg respectively, and the dosage of the isotype control NC-hIgG1 was 6 mg/kg, twice a week, for a total of three times.

Landscapes

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

Abstract

L'invention concerne un anticorps à domaine unique qui cible le ligand de mort programmée humaine 1 (PD-L1) et une protéine dérivée de celui-ci. Une protéine PD-L1 humaine recombinante est utilisée en tant qu'immunogène, et des PBMC sont séparées pour construire une bibliothèque d'anticorps de présentation sur surface de phage de région variable de chaîne lourde d'anticorps à domaine unique ; la protéine PD-L1 humaine recombinante est soumise à un criblage en phase solide, de façon à obtenir un affichage de phage de VHH-B6 d'anticorps à domaine unique de camélidé, qui peut se lier à la protéine PD-L1 humaine recombinante ; l'anticorps chimérique chB6 de camélidé humain est préparé et humanisé, et hzB6 est obtenu ; la maturation d'affinité est réalisée sur la base de hzB6, et des variants de 27 anticorps à domaine unique humanisés sont obtenus et criblés pour obtenir un variant d'anticorps à domaine unique humanisé ayant une affinité élevée pour PD-L1 humain, une forte activité de blocage pour la liaison de PD-L1 humain avec le récepteur PD-1, une demi-vie in vivo longue, et de bons effets de traitement de tumeur dans un modèle animal.
PCT/CN2022/092753 2021-05-14 2022-05-13 Anticorps à domaine unique anti-pd-l1 et sa protéine dérivée WO2022237897A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110529312 2021-05-14
CN202110529312.1 2021-05-14

Publications (1)

Publication Number Publication Date
WO2022237897A1 true WO2022237897A1 (fr) 2022-11-17

Family

ID=83947716

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/092753 WO2022237897A1 (fr) 2021-05-14 2022-05-13 Anticorps à domaine unique anti-pd-l1 et sa protéine dérivée

Country Status (2)

Country Link
CN (1) CN115340605A (fr)
WO (1) WO2022237897A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107686520A (zh) * 2016-08-04 2018-02-13 信达生物制药(苏州)有限公司 抗pd‑l1纳米抗体及其应用
CN111116747A (zh) * 2015-07-31 2020-05-08 苏州康宁杰瑞生物科技有限公司 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白
US20200148772A1 (en) * 2016-03-18 2020-05-14 Nanomab Technology Limited Anti-pd-l1 nanobody, coding sequence and use thereof
CN111699200A (zh) * 2018-01-15 2020-09-22 南京传奇生物科技有限公司 针对pd-1的单域抗体和其变体
EP3786184A1 (fr) * 2018-02-14 2021-03-03 Shanghai Novamab Biopharmaceuticals Co., Ltd. Anticorps camel à domaine unique pd-l1 de type bloquant et son application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111116747A (zh) * 2015-07-31 2020-05-08 苏州康宁杰瑞生物科技有限公司 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白
US20200148772A1 (en) * 2016-03-18 2020-05-14 Nanomab Technology Limited Anti-pd-l1 nanobody, coding sequence and use thereof
CN107686520A (zh) * 2016-08-04 2018-02-13 信达生物制药(苏州)有限公司 抗pd‑l1纳米抗体及其应用
CN111699200A (zh) * 2018-01-15 2020-09-22 南京传奇生物科技有限公司 针对pd-1的单域抗体和其变体
US20200347135A1 (en) * 2018-01-15 2020-11-05 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against pd-1
EP3786184A1 (fr) * 2018-02-14 2021-03-03 Shanghai Novamab Biopharmaceuticals Co., Ltd. Anticorps camel à domaine unique pd-l1 de type bloquant et son application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YU, S. ET AL.: "Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review)", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 47, no. 2, 28 February 2021 (2021-02-28), pages 444 - 454, XP055944687, DOI: 10.3892/ijmm.2020.4817 *

Also Published As

Publication number Publication date
CN115340605A (zh) 2022-11-15

Similar Documents

Publication Publication Date Title
US11566071B2 (en) Nucleic acid molecules encoding anti-GPRC5D antibodies
US20210388081A1 (en) Antibodies targeting fc receptor-like 5 and methods of use
JP2020018298A (ja) Ｃｌｄｎ１８．２及びｃｄ３に対する抗体コンストラクト
TW201726713A (zh) 最優化抗ｃｄ３雙特異性抗體及其用途
JP7352973B2 (ja) 二重特異性抗体及びその使用
WO2018117237A1 (fr) Anticorps anti-cd3 et molécule contenant ledit anticorps
WO2021213435A1 (fr) Anticorps à domaine variable unique ciblant le ligand 1 humain de mort programmée (pd-l1) et dérivé de celui-ci
US20200216539A1 (en) Bispecific anti pd1-anti tim3 antibodies
US20240010721A1 (en) Ror1-targeting antibody or antigen-binding fragment thereof, preparation method therefor, and application thereof
CA3128104A1 (fr) Anticorps anti-pd-1, fragment de liaison a l'antigene de celui-ci et utilisation pharmaceutique associee
US11021538B2 (en) Bispecific coupled antibody, preparation method and application thereof
TW202122423A (zh) 抗ｃｄ７３抗體及組合物
CN105820250A (zh) 一种抗basigin人源化抗体及其应用
KR20220137723A (ko) 항-cd3 및 항-cd123 이중 특이성 항체 및 이의 용도
WO2021197401A1 (fr) Polypeptide de liaison à l'antigène se liant à cd47, et son utilisation
WO2022237897A1 (fr) Anticorps à domaine unique anti-pd-l1 et sa protéine dérivée
CN115109156B (zh) 一种靶向bcma的纳米抗体及其应用
AU2022273136A9 (en) Binding molecule against dll3 and use thereof
JP2024512252A (ja) Ｃｄ４７に対する単一ドメイン抗体及びその用途
JP2023524102A (ja) Ａｂｃｂ５に特異的な抗体およびその使用
WO2022253302A1 (fr) Nanoanticorps ciblant l'albumine sérique humaine (hsa) et utilisation d'un nanoanticorps
WO2022268168A1 (fr) Nouveau type d'anticorps bispécifique ciblant lag-3 et pd-l1 et son utilisation
WO2022002006A1 (fr) Protéine de liaison dans une structure fab-hcab
WO2023040940A1 (fr) Utilisation d'une protéine de liaison pvrig/tigit en combinaison avec un inhibiteur de point de contrôle immunitaire dans le traitement de cancers
CA3206838A1 (fr) Molecule de liaison pd-1 et son application

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: 22806859

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22806859

Country of ref document: EP

Kind code of ref document: A1