WO2021213435A1 - 一种靶向人程序性死亡配体1(pd-l1)的单可变域抗体及其衍生物 - Google Patents

一种靶向人程序性死亡配体1(pd-l1)的单可变域抗体及其衍生物 Download PDF

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WO2021213435A1
WO2021213435A1 PCT/CN2021/088674 CN2021088674W WO2021213435A1 WO 2021213435 A1 WO2021213435 A1 WO 2021213435A1 CN 2021088674 W CN2021088674 W CN 2021088674W WO 2021213435 A1 WO2021213435 A1 WO 2021213435A1
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variable domain
single variable
domain antibody
antibody
amino acid
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French (fr)
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王双
曾大地
焦莎莎
张畅
王荣娟
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Mabwell Shanghai Bioscience Co Ltd
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Priority to US17/996,802 priority patent/US20230279115A1/en
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    • 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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • 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
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of antibody drugs. Specifically, the present invention relates to a single variable domain antibody targeting human programmed death ligand 1 (PD-L1), its derivative protein and its use in the preparation of drugs, especially in Use in the treatment and/or prevention, or diagnosis of 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 an important part of the immune system to prevent autoimmune overload. The activation of their pathways has the effect of inhibiting tumor immune response and inducing tumor-specific T cell apoptosis, and is associated with tumor development Close relationship.
  • PD-1 (CD279) is a type I transmembrane protein, a member of the immunoglobulin superfamily, and is mainly expressed on activated CD4+T cells, CD8+T cells and B cells and other immune cells.
  • Its ligand PD-L1 (also known as B7-H1, CD274) belongs to the B7 family and is highly expressed in tumor infiltrating immune cells (TIC) and a variety of malignant tumor cells, such as malignant melanoma, non-small cell lung cancer, and head and neck squamous cells. Cancer etc.
  • TIC tumor infiltrating immune cells
  • malignant tumor cells such as malignant melanoma, non-small cell lung cancer, and head and neck squamous cells. Cancer etc.
  • the use of monoclonal antibodies to block PD-1 and PD-L1 pathways to treat tumors has shown good clinical efficacy and safety.
  • a variety of antibody drugs have been approved for the market, and the indications include melanoma, non-small cell
  • malignant tumors such as lung cancer and advanced renal cell carcinoma, and many ongoing clinical trials are trying to develop more new indications.
  • the PD-1/PD-L1 monoclonal antibody has shown good therapeutic effects in the clinical treatment of various malignant tumors, there are problems such as large dosage and low overall response rate.
  • the main reasons include the low expression of PD-L1 and the exhaustion of T cells in the tumor microenvironment. Therefore, it is still necessary to further develop PD-1/PD-L1 targets and develop therapeutic drugs with better clinical effects. In terms of drug development for this target, the following approaches may increase the benefits of clinical patients.
  • Single variable domain antibody is the smallest antibody molecule at present, and its molecular weight is 1/10 of that of ordinary antibody. It was originally discovered in camel blood by the Belgian scientist Hamers, R. It is a class of engineered antibody products that has attracted much attention. 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 dual/multispecific 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 hotspot.
  • Caplacizumab developed by the company, was approved by the FDA in February 2019 for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP). Acquired thrombotic thrombocytopenic purpura is characterized by excessive blood clotting in small blood vessels.
  • Caplacizumab is the first drug approved for this disease and the first to target von willebrand factor (vWF) The drug, vWF is a key protein in the blood coagulation cascade.
  • vWF von willebrand factor
  • Caplacizumab is also the first single variable domain antibody approved by the FDA.
  • Caplacizumab is a landmark event in the field of single variable domain antibodies that has officially embarked on the treatment of human diseases.
  • Corning Jerry first entered the field of single variable domain antibodies.
  • Its PD-L1 single variable domain antibody (KN035, subcutaneous administration) was approved by CFDA and FDA to enter clinical trials in 2016, and in 2017 Obtained clinical approval from the Pharmaceutical Regulatory Agency (PMDA) of Japan at the end of June.
  • KN035 is the world's first PD-L1 single variable domain antibody.
  • the PD-1/PD-L1 monoclonal antibody has shown good therapeutic effects in the clinical treatment of various malignant tumors, but there are problems such as large dosage and low overall response rate.
  • the main reasons include the low expression of PD-L1 and the exhaustion of T cells in the tumor microenvironment. Therefore, it is necessary to discover new anti-PD-L1 antibody drugs.
  • antibody molecules with better therapeutic effects may be obtained through the following approaches: (1) Further develop antibody molecules with higher affinity and better activity; (2) Bispecific antibodies or analogues based on the target (3) Develop more effective diagnostic antibodies to predict PD-L1 positive tumor patients by detecting PD-L1 expression or to screen patients in advance to reduce treatment costs and possible serious adverse reactions. With its unique properties, camel-derived single variable domain antibodies are expected to be used to solve the above problems.
  • the present disclosure provides a single variable domain antibody and its derivatives targeting human programmed death ligand 1 (PD-L1).
  • PBMCs were extracted from human PD-L1 immunized camels to construct a phage surface display VHH antibody library, and the anti-human PD-L1 specific single variable domain antibody 2-2F2 was obtained through screening and identification.
  • chimeric antibody chF2 and humanized modification were prepared The antibody hzF2 mutant.
  • the affinity of the hzF2 mutant can reach or even exceed the initial single variable domain antibody 2-2F2, can block the combination of PD-1 and PD-L1 in vitro, and can inhibit tumor growth in an in vivo test in tumor-bearing mice. in particular:
  • the present invention provides an anti-PD-L1 single variable domain antibody, characterized in that the CDR1 to CDR3 in the variable region of the single variable domain antibody are shown in SEQ ID NOs: 43-45, respectively.
  • the anti-PD-L1 single variable domain antibody of the present invention is characterized in that the single variable domain antibody does not have a constant region or has 1-3 heavy chain constant regions.
  • anti-PD-L1 single variable domain antibody of the present invention is characterized in that the amino acid sequence of the variable region of the single variable domain antibody is shown in SEQ ID NO:1.
  • the present invention provides an anti-PD-L1 single variable domain antibody, characterized in that the single variable domain antibody is a human-camel chimeric single variable domain antibody, including the single variable domain antibody described in the first aspect of the present invention.
  • Variable domain antibody variable region and human heavy chain constant region are included in the second aspect of the present invention.
  • the anti-PD-L1 single variable domain antibody of the present invention is characterized in that the chimeric single variable domain antibody has the amino acid sequence shown in SEQ ID NO: 3.
  • the present invention provides an anti-PD-L1 single variable domain antibody, which is characterized in that the single variable domain antibody is a humanized single variable domain antibody, and its variable region is as described in the first aspect of the present invention.
  • the variable region of the single variable domain antibody has been humanized and obtained.
  • the anti-PD-L1 single variable domain antibody of the present invention is characterized in that the variable region of the single variable domain antibody has the amino acid sequence shown in SEQ ID NO:7.
  • the anti-PD-L1 single variable domain antibody of the present invention is characterized in that the single variable domain antibody has the amino acid sequence shown in SEQ ID NO: 9.
  • the present invention provides an anti-PD-L1 single variable domain antibody, characterized in that the single variable domain antibody is a mutant anti-PD-L1 humanized single variable domain antibody, which is in the present invention
  • the anti-PD-L1 single variable domain antibody described in the third aspect is produced by mutating 1, 2, 3 or 4 amino acid residues in the variable region CDRs; the mutant anti-PD-L1
  • the humanized single variable domain antibody at least partially retains the specific binding ability to PD-L1.
  • the anti-PD-L1 single variable domain antibody of the present invention is characterized in that its variable region is selected from the group consisting of SEQ ID NO: 11-26.
  • the present invention provides a composition comprising one or more anti-PD-L1, the one or more anti-PD-L1 is selected from any one of the first aspect to the fourth aspect of the present invention Group of anti-PD-L1 single variable domain antibodies.
  • composition of the present invention is characterized in that it also contains a pharmaceutically acceptable carrier and is used as a pharmaceutical composition.
  • the pharmaceutical composition is a liquid, injection, or powder injection.
  • the present invention provides the application of antibodies or fragments thereof in the preparation of drugs for the treatment of dysproliferative diseases, characterized in that: the antibodies are selected from the anti-PD-L1 monomers described in any one of the first to fourth aspects of the present invention. Group of variable domain antibodies.
  • the application of the present invention is characterized in that the abnormal proliferative diseases include tumors, preferably melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, kidney cancer, colon cancer and the like.
  • the present invention provides the application of antibodies or fragments thereof in the preparation of multispecific antibodies or antibody-targeted drugs, characterized in that the antibodies are selected from the anti-PD described in any one of the first to fourth aspects of the present invention.
  • -L1 single variable domain antibody group -L1 single variable domain antibody group.
  • the present invention provides a polynucleotide encoding the anti-PD-L1 single variable domain antibody of any one of the first to fourth aspects of the present invention.
  • the present invention provides a vector comprising the polynucleotide according to the eighth aspect of the present invention.
  • the present invention provides a host cell comprising the polynucleotide according to the seventh aspect of the present invention or the vector according to the eighth aspect of the present invention.
  • the present invention provides a method for preparing an anti-PD-L1 single variable domain antibody, which comprises the following steps:
  • the present invention provides a method for preventing or treating aberrant proliferative diseases, characterized by administering to a subject in need an effective amount of the anti-PD-L1 single variable domain antibody of any one of the foregoing, The composition of any one, the multispecific antibody or antibody-targeted drug in the application of the aforementioned sixth aspect.
  • the method of the present invention is characterized in that the abnormal proliferative diseases include tumors, especially tumors related to the PD-1/PD-L1 signaling pathway.
  • the method of the present invention is characterized in that: the tumor includes melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, kidney cancer, colon cancer and the like.
  • the present invention provides a method for diagnosing or evaluating the occurrence and development of a subject's abnormal proliferative disease, which is characterized in that: a detectable sample from the subject is combined with any one of the aforementioned anti-PD-L1 drugs.
  • a detectable sample from the subject is combined with any one of the aforementioned anti-PD-L1 drugs.
  • the method of the present invention is characterized in that the abnormal proliferative diseases include tumors, especially tumors related to the PD-1/PD-L1 signaling pathway.
  • the method of the present invention is characterized in that: the tumor includes melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, kidney cancer, colon cancer and the like.
  • the present invention provides a method for predicting or evaluating the therapeutic effect of a PD-1/PD-L1 antagonist on subjects suffering from abnormal proliferative diseases, which is characterized in that: a substance selected from the following group is used to detect The expression of the subject's PD-L1: the anti-PD-L1 single variable domain antibody of any one of the foregoing, the composition of any one of the foregoing, the multispecific antibody or antibody-targeted drug in the application of the sixth aspect of the foregoing .
  • the method of the present invention is characterized in that the abnormal proliferative diseases include tumors, especially tumors related to the PD-1/PD-L1 signaling pathway.
  • the method of the present invention is characterized in that: the tumor includes melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, kidney cancer, colon cancer and the like.
  • immunoglobulin sequence is used as a general term, including full-size antibodies, their individual chains, 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 such as “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence) It should generally be understood to include both the related amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive explanation.
  • the immunoglobulin single variable domain can be used as a preparation for the preparation of one or more additional immunizations that can serve as binding units (ie, the same or different epitopes against the same target and/or against one or more different targets).
  • binding unit ie, the same or different epitopes against the same target and/or against one or more different targets.
  • binding unit binding domain
  • building unit of a polypeptide of a globulin single variable domain (these terms are used interchangeably).
  • immunoglobulin single variable domain which is used interchangeably with “single variable domain” (“SVD”), defines where the antigen binding site is present on a single immunoglobulin domain And a molecule formed by a single immunoglobulin domain. This makes immunoglobulin single variable domains different from “regular” immunoglobulins or their fragments, in which two immunoglobulin domain variable domains, especially two variable domains, interact to form an antigen Binding site.
  • the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) interact to form an antigen binding site.
  • the complementarity determining regions (CDRs) of both VH and VL will benefit the antigen binding site, that is, 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. Therefore, 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 their fragments that require at least two variable domains to interact 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 so that they contain an immunoglobulin fold or are capable of forming an immunoglobulin fold under suitable conditions. Therefore, 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 a VHH sequence) or a suitable fragment thereof.
  • a single antigen binding unit ie, a functional antigen binding unit consisting essentially of a single variable domain, so that the single antigen binding unit does not need to interact with another variable domain to form a functional
  • An antigen-binding unit for example, for a variable domain that is present in, for example, conventional antibodies and scFv fragments, and needs to interact with another variable domain, such as through VH/VL interaction, to form a functional antigen-binding domain This is the case).
  • the immunoglobulin single variable domain is a light chain variable domain sequence (for example, a VL sequence) or a heavy chain variable domain sequence (for example, 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 immunoglobulin single variable domain can be a (single) domain antibody (or suitable for use as a (single) domain Amino acids of antibodies), "dAbs” or dAbs (or amino acids suitable for use as dAbs) or Nanobodies (as defined herein and including but not limited to VHH); other single variable domains, or any of them Any suitable fragment of the species.
  • (single) domain antibodies For a general description of (single) domain antibodies, reference is also made to the prior art cited herein and to EP0368684.
  • dAb for example, refer to Ward et al., 1989 (Nature341:544-546), refer to Holt et al., 2003 (Trends Biotechnol. 21:484-490); and refer to, for example, WO 04/068820, WO 06/ 030220, WO06/003388, WO 06/059108, WO 07/049017, WO 07/085815 and other published patent applications of Domantis Ltd.
  • variable domains can be derived from certain species of sharks (for example, the so-called "IgNAR domains", see for example WO 05 /18629).
  • the immunoglobulin single variable domain can be (As defined herein) or suitable fragments thereof.
  • Is a registered trademark of Ablynx NV For a general description of Nanobodies, refer to the further description below, as well as to the prior art cited herein, such as, for example, in WO 08/020079 ( Page 16).
  • VHH and Nanobodies For further description of VHH and Nanobodies, refer to the review article of Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302), and the following patent application mentioned as general background technology: VrijeUniversiteit Brussels 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; Vlaams Instituutvoor Biotechnologie (VIB) WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527; Algonomics NV and Eberlinks WO 03/050531 of a company limited by shares; WO 01/90190 of the National Research Council of Canada; WO 03/
  • Nanobodies (especially VHH sequences and partially humanized Nanobodies) may be characterized in particular by the presence of one or more "signature residues" in one or more framework sequences.
  • Nanobodies can be found, for example, in WO 08/101985 and WO 08/142164, including the humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions" ", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to 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 camelid, preferably camelid heavy chain antibodies. As previously described, they can be humanized. In addition, 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 from different sources, 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 camelidized immunoglobulin single variable domains, such as the camelidized dAb described by Ward et al., 1989 ( See, for example, WO 94/04678 and Davies and Riechmann 1994, 1995 and 1996) and camelized VH.
  • immunoglobulin single variable domains can be derived from any suitable source in any suitable manner, and for example can be a naturally occurring VHH sequence (ie, from a suitable camelid species) or synthetic or semi- Synthetic amino acid sequences, including but not limited to partially or fully "humanized” VHH, "camelized” immunoglobulin sequences (and especially camelized VH), and Nanobodies and/or obtained by the following techniques VHH: such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences such as VHH sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, using PCR assembly of overlapping primers, and similar techniques known to the skilled person for engineering immunoglobulin sequences; or any suitable combination of any of the foregoing.
  • VHH such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences such as VHH sequences), CDR grafting, veneering, combining fragments derived from different immunoglobul
  • the amino acid sequence and structure of an immunoglobulin single variable domain can be considered-but not limited to-consisting of four framework regions or "FRs", which are referred to in the art and herein as the "framework regions, respectively" 1" or “FR1”; “framework region 2" or “FR2”; “framework region 3” or “FR3”; and “framework region 4" or “FR4"; the framework region is divided by three complementary determining regions or " CDRs are separated, 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.
  • CDRs according to Kabat can be mentioned, and CDRs are most preferably defined based on the Abm definition (which is based on OxfordMolecular's AbM antibody modeling software), because it is considered that this is defined in Kabat and Chothia The best compromise between. Refer again to the website http://www.bioinf.org.uk/abs/).
  • FR4 comprises the C-terminal amino acid sequence VTVSS, each of positions 109, 110, 111, 112, and 113.
  • the present invention also includes ISVDs that terminate at positions 109, 110, 111, or 112.
  • FR4 is terminated by the C-terminal amino acid sequence VTVS (109-112)
  • FR4 is terminated by the C-terminal amino acid sequence VTV (109-111)
  • FR4 is terminated by the C-terminal amino acid sequence VT (109-110).
  • the C-terminal extension may be present in the last (located at the C-terminal) ISVD at the last amino acid residue of FR4 such as the C-terminal of V109, T110, V111, S112 or S113, wherein the cysteine portion of the present invention is preferably present in Or located at the C-terminus of the C-terminus extension.
  • FR4 comprises the C-terminal amino acid sequence VTVSS and the C-terminal extension is cysteine (for example, the polypeptide of the invention is terminated with VTVSSC).
  • FR4 comprises the C-terminal amino acid sequence VTVS and the C-terminal extension is cysteine (for example, the polypeptide of the invention is terminated with VTVSC).
  • FR4 comprises the C-terminal amino acid sequence VTV and the C-terminal extension is cysteine (for example, the polypeptide of the invention is terminated with VTVC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VT and the C-terminal extension is cysteine (for example, the polypeptide of the invention is terminated by VTC). In one embodiment, FR4 comprises the C-terminal amino acid V and the C-terminal extension is cysteine (e.g., the polypeptide of the invention is terminated with VC).
  • the present invention relates to a dimer as described herein, wherein ISVD is a light chain variable domain sequence (VL), is a heavy chain variable domain sequence (VH), derived from a conventional four Chain antibodies or derived from heavy chain antibodies.
  • VL light chain variable domain sequence
  • VH heavy chain variable domain sequence
  • the present invention relates to a dimer as described herein, wherein the 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 contains between 100 and 140 amino acids, such as between 110 and 130 amino acids.
  • the antibody of the present invention is an anti-PD-L1 humanized single variable domain antibody with high affinity.
  • the humanized anti-PD-L1 single variable domain antibody hzF2 specifically binds to human PD-L1 protein with high affinity, and its affinity (KD) is 1.1 nM, which is equivalent to the control antibody KN035.
  • KD affinity
  • the basic characteristics of high affinity and good specificity provide a theoretical basis for the inhibitory effect of hzF2 on the PD-1/PD-L1 signaling pathway; compared with traditional monoclonal antibodies, single variable domain antibodies have more flexible application forms and are more suitable Development of dual/multispecific therapeutic antibodies.
  • the antibody of the present invention has good biological activity.
  • hzF2 can effectively bind to human PD-L1 recombinantly expressed on the cell surface, and the EC50 of the combination with CHO cells expressing human PD-L1 (CHO-PD-L1) is 1.01nM; it can effectively block recombinant human PD-L1 and its The binding effect of receptor PD-1, IC50 is 4.3nM; by using Jurkat-PD1-NFAT cells and CHO-PD-L1-CD3L cell reporter gene method to detect hzF2 blocking PD-1/PD-L1 signaling pathway, Results The active EC50 is 5.45nM; hzF2 has good stability in vivo, and can effectively inhibit tumor growth in the humanized immune system xenograft subcutaneous melanoma A375 model.
  • the present invention provides multiple mutants on the basis of hzF2. Some mutants show better effects than the initial antibody hzF2 in terms of specificity, affinity and other performance parameters. These different single variable domain antibody mutants provide PD-L1-based tumor detection, targeted therapy, drug delivery, etc. With more choices, the application potential of hzF2 has been further enriched and expanded.
  • Figure 1 ELISA to detect the inhibitory effect of single variable domain antibodies on the binding of human PD-L1 to its receptor PD-1.
  • Figure 2 FACS analysis of the binding activity of chF2 to cell surface antigens.
  • Figure 3 ELISA analysis of the binding specificity of chF2 and recombinant PD-L1.
  • Figure 4 chF2 binding specificity analysis of PD-L1 on the cell surface.
  • Figure 5 The results of the analysis of the inhibitory effect of hzF2 on the binding of human PD-L1 to its receptor PD-1.
  • Figure 6 The reporter gene system evaluates the in vitro blocking activity of the anti-PD-L1VHH antibody Fc fusion protein.
  • Figure 7 Single-dose-time curve of hzF2 in Balb/C mice.
  • Figure 8 Experimental results of anti-tumor efficacy of hzF2 on human PD-L1 transgenic mice subcutaneously transplanted MC38-hPDL1 murine colon cancer tumor model (tumor volume).
  • Figure 9 Experimental results of anti-tumor efficacy of hzF2 on human PD-L1 transgenic mice subcutaneously transplanted MC38-hPDL1 murine colon cancer tumor model (tumor weight).
  • VHH heavychain antibody
  • PBMCs are separated with a separation kit (Tianjin Haoyang, Cat:TBD2011CM), and the total RNA of PBMC is extracted to obtain cDNA, which is used as a template for subsequent amplification of VHH fragments.
  • a separation kit Teianjin Haoyang, Cat:TBD2011CM
  • primers were designed and synthesized to construct the VHH antibody library, and the antibody variable region gene sequences were amplified by PCR. Subsequently, endonuclease is used to digest the vector and the amplified antibody fragment.
  • T4 ligase connection method is used to construct the ligation product, and the ligation product is transferred to the TG1 strain by electrotransformation technology.
  • a 1.8 ⁇ 10 8 camel anti-human PD-L1VHH antibody immune library was constructed for the screening of specific anti-human PD-L1 single variable domain antibodies. In order to detect the correct rate of the library, 50 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached 100%.
  • the constructed camel immune library was screened by solid-phase screening to obtain specific phage-displayed single variable domain antibodies.
  • the original library is presented.
  • the camel immune library was transferred to 2YT medium containing ampicillin and tetracycline, cultured to the logarithmic growth phase, M13 helper phage was added, and then kanamycin was added, and it was presented overnight at a lower temperature. The next day, the culture supernatant was collected, and the phage was concentrated by PEG precipitation to obtain a high-titer antibody library display product for subsequent screening.
  • the obtained 5 single-domain VHH antibodies were induced and expressed in E. coli TG1 under the conditions of 1mM IPTG, 30°C, 150rpm overnight culture.
  • the bacterial samples induced to express were processed by ultrasonic disintegration and filtration, then affinity purification was performed using a nickel column, and ultrafiltration treatment was performed to obtain single domain VHH antibodies. Subsequently, the inhibitory effect of the single domain on the binding of human PD-L1 to its receptor PD-1 was detected by ELISA.
  • the fusion protein of the extracellular domain of human PD-1 and human Fc (PD-1-hFc, PD-1 sequence number: NP_005009.2, 21aa-167aa), at a concentration of 0.5 ⁇ g/mL, was coated overnight at 4°C , Sealed with 5% BSA in a constant temperature incubator at 37°C for 60 min.
  • the single domain VHH antibody concentration of 50, 10, 2nM
  • 1 ⁇ g/mL PD-L1-mFc were incubated together, and reacted in a constant temperature incubator at 37°C for 60 minutes.
  • VHH-F2 This molecule was selected as the initial molecule for subsequent development, abbreviated as VHH-F2.
  • the amino acid sequence of the variable region of the single variable domain antibody is shown in SEQ ID NO. 1, and the nucleotide sequence of the variable region is shown in SEQ ID NO. 2.
  • Design specific primers use the positive cloned phagemid as a template, obtain the variable region gene of camel-derived antibody VHH-F2 by PCR, and clone the variable region gene into a human Fc (IgG1, hFc) encoding gene through restriction enzyme digestion and ligation.
  • Eukaryotic expression vector After obtaining the expression plasmid with the correct sequence, it was transfected into 293F cells for transient expression, purified by Protein A, and finally obtained the human-camel chimeric single variable domain antibody fusion protein (VHH-F2-human-Fc chimeric antibody, chF2 for short). See SEQ ID NO. 3 for the full-length amino acid sequence of the chF2 antibody molecule, and SEQ ID NO. 4 for the nucleotide sequence.
  • variable region gene of KN035 was fully synthesized, and the amino acid sequence of the variable region of KN035 is shown in SEQ ID NO. 5. See SEQ ID NO. 6 for the nucleotide sequence. And use the same strategy as above to construct the Fc fusion protein of KN035 with the same form of chF2, referred to as KN035.
  • Example 4 Analysis of binding activity of anti-human PD-L1 chimeric single variable domain antibody
  • Collect the cells (CHO-PD-L1-CD3L cells) by centrifugation, divide the cells into 5 ⁇ 10 5 cells/sample/100 ⁇ L, and add serially diluted single variable domain antibodies.
  • the final concentration of the added antibody is: the highest concentration 66nM, 3 10 fold serial dilutions.
  • Example 5 Specificity analysis of anti-human PD-L1 chimeric single variable domain antibody
  • Dilute human recombinant protein (PD-L1, PD-1, B7H3, B7H4, CTLA4, CD28, ICOS, etc.) with PBS to 1 ⁇ g/mL, 100 ⁇ L/well to coat the enzyme-linked plate, and coat overnight at 4°C; 5% BSA Blocking solution in a constant temperature incubator at 37°C for 60min, wash the plate with PBST 3 times; add chF2 diluted to 1 ⁇ g/mL, react at 37°C for 60min, wash the plate 4 times with PBST; add 1:5000 diluted HRP-Anti-human IgG to react 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, use 630nm as the reference wavelength, read and record the absorbance of the well plate at 450nm wavelength A450nm-630nm.
  • the results show ( Figure 3) that chF2 specifically binds to
  • the anti-human antibody Fc segment capture antibody (AHC) biological probe was used to capture the antibody Fc segment to determine whether chF2 can bind to recombinant monkey and mouse PD-L1.
  • AHC anti-human antibody Fc segment capture antibody
  • chF2 was diluted to 4 ⁇ g/mL with PBS buffer, and passed through the surface of the AHC probe (PALL, Cat:18-0015) for 120s.
  • Monkey PD-L1 recombinant protein and mouse PD-L1 recombinant protein are used as mobile phases to interact with the antibody captured on the surface of the chip.
  • the concentration of PD-L1 recombinant protein is 60nM.
  • the cells were collected by centrifugation, divided into 3 ⁇ 10 5 cells/sample/100 ⁇ L, and 20 ⁇ g/ml single variable 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, resuspend in 200 ⁇ L flow buffer, flow cytometry detection.
  • the test results show (Figure 4) that the reactivity of chF2 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-F2 human heavy chain variable region framework with the highest homology between the variable region and the variable region of camel antibody VHH-F2 was selected, and the variable region of VHH-F2 was humanized by CDR grafting and retaining some of the supporting structural amino acids.
  • the design of a humanized single variable domain antibody fusion protein (VHH-F2-human-Fc humanized antibody, abbreviated as hzF2) variable region amino acid sequence is shown in SEQ ID NO. 7, and the full-length amino acid sequence is shown in SEQ ID NO. 9 shown.
  • the synthesized hzF2 variable region nucleotide sequence is shown in SEQ ID NO.
  • the antibody strain hzF2 was further mutated to obtain a large number of mutant antibodies.
  • the amino acid sequence of the CDR region of the mutant sequence is shown in Table 3 below, and the amino acid sequence of the variable region of the mutant is shown in Table 4 (SEQ ID NO. 11 ⁇ SEQ ID NO. 26). See SEQ ID NO. 27 to SEQ ID NO. 42 for the nucleotide sequence.
  • Table 5 shows the changes in binding and dissociation constants of some mutants.
  • Example 7 ELISA to detect the inhibitory effect of hzF2 on the binding of human PD-L1 to its receptor PD-1
  • Human PD-1-hFc (PD-1 serial number: NP_005009.2, 21aa-167aa) was diluted with PBS to 0.5 ⁇ g/mL, coated overnight at 4°C, and sealed with 5% BSA in a constant temperature incubator at 37°C for 60 minutes.
  • Stepwise dilution of hzF2 and control antibody KN035, and isotype control NC-hIgG1 starting working concentration is 50nM, 1.5 times dilution 10 concentration gradients
  • adding PD-L1-mFc (PD-L1 sequence) with a working concentration of 0.5 ⁇ g/mL No.: NP_054862.1, 19aa-238aa) co-incubate and react in a constant temperature incubator at 37°C for 60 minutes.
  • CHO-PD-L1-CD3L cells in the logarithmic growth phase are adjusted to a cell density of 5 ⁇ 10 5 cells/ml, and plated and cultured overnight at 100 ⁇ l/well.
  • the antibody sample is pre-diluted with culture medium to 20 ⁇ g/ml step by step, and then a 2-fold gradient dilution is carried out for a total of 10 points.
  • the diluted sample is added to the cells cultured overnight, 50 ⁇ l/well.
  • mice Healthy female 5-week-old nude mice, a group of 3, were injected with the antibody into the mice via the tail vein at a single dose of 15mg/kg, respectively 2h, 4h, 8h, 24h after administration , 48h, 96h, 144h, 196h tail vein blood collection, blood samples are collected and centrifuged to separate the serum, and stored at -20°C. Observe its pharmacokinetic properties.
  • Example 10 Detection of anti-tumor efficacy of hzF2 on human PD-L1 transgenic mice subcutaneously transplanted MC38-hPDL1 murine colon cancer tumor model
  • Murine colon cancer tumor cells MC38-hPDL1, which highly express human PDL1 were inoculated into female B6-hPDL1 mice (human PD-L1 transgenic mice derived from C57) subcutaneously on the right anterior flank, and grouped when the tumors grew to about 100mm3 Give hzF2, KN035 or isotype control IgG at a dose of 10 mg/kg, twice a week, 6 times.
  • the tumor volume and body weight were measured at the same time of each administration, and the relationship between the body weight and tumor volume change of the tumor-bearing mice and the administration time was recorded.
  • the tumor-bearing mice were euthanized and the tumors were stripped and weighed and photographed. Calculate the relative tumor volume ratio (T/C) and tumor growth inhibition rate (1-T/C) between the treatment group and the control group and perform statistical analysis.
  • the results showed that the tested drug hzF2 effectively inhibited tumor growth (Figure 8, Figure 9).
  • amino acid and nucleotide sequences related to this application are as follows:
  • SEQ ID NO. 1 Camel-derived single variable domain antibody VHH-F2 variable region amino acid sequence
  • Heavy chain CDR1 amino acid (SEQ ID NO.43): RSDDDGASCMG
  • Heavy chain CDR2 amino acid (SEQ ID NO.44): IIFNAGERTDYGDSVKG
  • Heavy chain CDR3 amino acid (SEQ ID NO.45): VWCGSWVARS
  • SEQ ID NO. 2 Camel-derived single variable domain antibody VHH-F2 variable region nucleotide sequence
  • SEQ ID NO. 3 Chimeric single variable domain antibody chF2 full-length amino acid sequence
  • SEQ ID NO. 4 Chimeric single variable domain antibody chF2 full-length nucleotide sequence
  • Heavy chain CDR2 amino acid KLLTTSGSTYLADSVKG
  • SEQ ID NO.7 Humanized single variable domain antibody hzF2 variable region amino acid sequence
  • SEQ ID NO. 8 Humanized single variable domain antibody hzF2 variable region nucleotide sequence
  • SEQ ID NO.9 Humanized single variable domain antibody hzF2 full-length amino acid sequence
  • SEQ ID NO.10 Humanized single variable domain antibody hzF2 full-length nucleotide sequence
  • SEQ ID NO.11 Humanized single variable domain antibody hzF2-m1 variable region amino acid sequence
  • SEQ ID NO.12 Humanized single variable domain antibody hzF2-m2 variable region amino acid sequence
  • SEQ ID NO.13 Humanized single variable domain antibody hzF2-m3 variable region amino acid sequence
  • SEQ ID NO.14 Humanized single variable domain antibody hzF2-m4 variable region amino acid sequence
  • SEQ ID NO.15 Humanized single variable domain antibody hzF2-m5 variable region amino acid sequence
  • SEQ ID NO.16 Humanized single variable domain antibody hzF2-m6 variable region amino acid sequence
  • SEQ ID NO.17 Humanized single variable domain antibody hzF2-m7 variable region amino acid sequence
  • SEQ ID NO.18 Humanized single variable domain antibody hzF2-m8 variable region amino acid sequence
  • SEQ ID NO.19 Humanized single variable domain antibody hzF2-m9 variable region amino acid sequence
  • SEQ ID NO.20 Humanized single variable domain antibody hzF2-m10 variable region amino acid sequence
  • SEQ ID NO.21 Humanized single variable domain antibody hzF2-m11 variable region amino acid sequence
  • SEQ ID NO.22 Humanized single variable domain antibody hzF2-m12 variable region amino acid sequence
  • SEQ ID NO.23 Humanized single variable domain antibody hzF2-m13 variable region amino acid sequence
  • SEQ ID NO.24 Humanized single variable domain antibody hzF2-m14 variable region amino acid sequence
  • SEQ ID NO.25 Humanized single variable domain antibody hzF2-m15 variable region amino acid sequence
  • SEQ ID NO.26 Humanized single variable domain antibody hzF2-m16 variable region amino acid sequence
  • SEQ ID NO.27 Humanized single variable domain antibody hzF2-m1 variable region nucleotide sequence
  • SEQ ID NO.28 Humanized single variable domain antibody hzF2-m2 variable region nucleotide sequence
  • SEQ ID NO.29 Humanized single variable domain antibody hzF2-m3 variable region nucleotide sequence
  • SEQ ID NO.30 Humanized single variable domain antibody hzF2-m4 variable region nucleotide sequence
  • SEQ ID NO.31 Humanized single variable domain antibody hzF2-m5 variable region nucleotide sequence
  • SEQ ID NO.32 Humanized single variable domain antibody hzF2-m6 variable region nucleotide sequence
  • SEQ ID NO.33 Humanized single variable domain antibody hzF2-m7 variable region nucleotide sequence
  • SEQ ID NO.34 Humanized single variable domain antibody hzF2-m8 variable region nucleotide sequence
  • SEQ ID NO.35 Humanized single variable domain antibody hzF2-m9 variable region nucleotide sequence
  • SEQ ID NO.36 Humanized single variable domain antibody hzF2-m10 variable region nucleotide sequence
  • SEQ ID NO. 37 Humanized single variable domain antibody hzF2-m11 variable region nucleotide sequence
  • SEQ ID NO.38 Humanized single variable domain antibody hzF2-m12 variable region nucleotide sequence
  • SEQ ID NO.39 Humanized single variable domain antibody hzF2-m13 variable region nucleotide sequence
  • SEQ ID NO.40 Humanized single variable domain antibody hzF2-m14 variable region nucleotide sequence
  • SEQ ID NO.41 Humanized single variable domain antibody hzF2-m15 variable region nucleotide sequence
  • SEQ ID NO.42 Humanized single variable domain antibody hzF2-m16 variable region nucleotide sequence

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