CN112759647A - anti-PD-L1 antibody and pharmaceutical application thereof - Google Patents

anti-PD-L1 antibody and pharmaceutical application thereof Download PDF

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CN112759647A
CN112759647A CN201910999788.4A CN201910999788A CN112759647A CN 112759647 A CN112759647 A CN 112759647A CN 201910999788 A CN201910999788 A CN 201910999788A CN 112759647 A CN112759647 A CN 112759647A
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CN112759647B (en
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宁金鹰
彭浩
郝锋
贺锋
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Shanghai Hongcheng Pharmaceutical Co ltd
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Kangyuan Bochuang Biotechnology Beijing Co ltd
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • C07K2317/24—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
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—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

Abstract

The present invention provides an antibody molecule or antigen-binding fragment thereof that binds to human PD-L1. The invention also provides the use of the antibody molecule or antigen binding fragment thereof in the preparation of a medicament for the treatment of a tumour or cancer. Compared with the existing anti-PD-L1 antibody, the antibody provided by the invention has better affinity and dissociation rate to PD-L1, lower immunogenicity and better tumor inhibition effect.

Description

anti-PD-L1 antibody and pharmaceutical application thereof
Technical Field
The invention belongs to the field of biological medicines, and relates to a novel anti-PD-L1 antibody or a functional fragment thereof. The invention also relates to the use of said antibody or functional fragment thereof.
Background
PD-L1, the gene encoding it, also known as CD274, is a ligand molecule for the programmed death factor PD1, often expressed on hematopoietic and non-hematopoietic cells such as T and B cells, as well as on various types of tumor cells. PD-L1 is classified as a type 1 transmembrane protein with immunoglobulin V-like and C-like domains. When PD-L1 interacts as a ligand with its receptor molecule PD-1, it inhibits T cell activation and cytokine production. This interaction is important to prevent autoimmunity by maintaining the homeostasis of the immune response during infection or inflammation of normal tissues. At the same time, this interaction plays a crucial role in the induction and maintenance of immune tolerance to itself, which in the tumor microenvironment usually leads to the inactivation of toxic T cells, thus providing immune escape for the tumor cells. The expression of the gene in tumor cells is considered to be one of the criteria for whether PD-1 or PD-L1 antibody drugs can be applied to many types of human malignant tumors, including melanoma, non-small cell lung cancer and the like.
A plurality of PD-L1 monoclonal antibody drugs are on the market at present, for example, Atezolizumab (trade name Teentiq) which is the first PD-L1 monoclonal antibody drug developed and produced by GENECECH under the Luo flag is used for treating advanced bladder cancer with failure or progression of platinum chemotherapy. In 2017, the U.S. Food and Drug Administration (FDA) accelerated the approval of a new drug Bavencio for the treatment of Merkel Cell Carcinoma (MCC) in adults and 12 year old children. The main ingredient of the medicine is Aveluab, which has sustained relieving effect on metastatic merkel cell carcinoma (mMCC). In europe, the approval application for PD-L1 antibody biologicals for the treatment of metastatic urothelial cancer is also under FDA priority review. Astrikon, 3 months 2018, also published that its immunotherapy drug, imfinzi (durvalumab), has formally received us Food and Drug Administration (FDA) approval for the treatment of non-small cell lung cancer stage III (NSCLC) patients who cannot be surgically resected.
Then, the existing monoclonal antibody drug aiming at PDL1 has a space for improving affinity and dissociation speed, and clinical test data show that the Atezolizumab drug has higher immunogenicity, so that more anti-antibody is generated, and the treatment effect of the drug is greatly influenced.
Disclosure of Invention
The technical problem to be solved by the invention is to obtain an antibody which specifically binds to human PD-L1, particularly has high affinity to human PD-L1, by hybridoma screening and humanization technology.
In view of the above technical problems, it is an object of the present invention to provide an antibody against human PD-L1 or a fragment thereof, and use thereof based on the antibody or the fragment thereof. "fragments" of the antibody molecules of the invention encompass various functional fragments of antibodies, e.g., antigen-binding portions thereof, e.g., Fab, F (ab')2Or a scFv fragment.
The present invention provides the following technical solutions.
In one aspect, the invention provides an antibody or fragment thereof capable of specifically binding to PD-L1, particularly human PD-L1, the amino acid sequence of which is set forth in SEQ ID NO 3. According to a specific embodiment of the present invention, the antibody of the present invention is a murine antibody obtained using the extracellular region of human PD-L1 shown in SEQ ID NO. 3 as an immunogen, and a chimeric antibody and a humanized antibody obtained based on the murine antibody.
Specifically, the antibody or fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) comprise a CDR combination (H-CDR1, H-CDR2, H-CDR 3; L-CDR1, L-CDR2, L-CDR3) selected from:
(1) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:35(EYTFTNNWIA), SEQ ID NO:36(DIHPGGGFTNYNEKFKV), SEQ ID NO:37 (SKTRDYDAWFAY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:38(KSSQSLLYTGNQKNYLA), SEQ ID NO:39(WASTRES), SEQ ID NO:40 (QQYYTYRT);
(2) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:41(GYTFTNYVVH), SEQ ID NO:42(YVNPNNDGTIFNEKFKD), SEQ ID NO:43 (SPBAH); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:44(SASESVDFYGTSLMQ), SEQ ID NO:45(TASNVDS), SEQ ID NO:46 (HQTRKVPYT);
(3) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO:47(GYIFTEYIIH), SEQ ID NO:48(WFYPGSDNIKYNEKFKD), SEQ ID NO:49(HETGYFFDY) in that order; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:50(SASSSVSKMN), SEQ ID NO:51(DTSKLAS), SEQ ID NO:52 (QQWSSNPLT);
(4) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:53(GYSFTGYNMN), SEQ ID NO:54(NIDPYYGVTHYNQKFKG), SEQ ID NO:55 (GIPFYGLDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:56(GASSSVSFMH), SEQ ID NO:51(DTSKLAS), SEQ ID NO:57 (QQWNTNPFT);
(5) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:58(AFNIDDTYIH), SEQ ID NO:59(RIDPANGNTDYDPECQG), SEQ ID NO:60 (GLRLPGLVY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:61(RASQDISNYLN), SEQ ID NO:62(YTSILYS), SEQ ID NO:63 (QQGNTLPWT);
(6) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:64(GFNIEDTYLH), SEQ ID NO:65(RIDPANGNTYYDPKFQG), SEQ ID NO:66 (GLRLPGFPY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:67(RASQDISNYLS), SEQ ID NO:68(YTSILHS), SEQ ID NO:63 (QQGNTLPWT);
(7) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO:69(GDSIISGYWN), SEQ ID NO:70(YISYTGSTYYNPSLKS), SEQ ID NO:71(RGEWLLHFDV) in that order; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:72(KSSQSLLYSSNQKNSLA), SEQ ID NO:39(WASTRES), SEQ ID NO:73 (QQYYSYPLT);
(8) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:74(GFSLTGYGVN), SEQ ID NO:75(KIWGDGITDYNSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:77(SASSSISYMH), SEQ ID NO:51(DTSKLAS), SEQ ID NO:78 (HHRSPYPT);
(9) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:74(GFSLTGYGVN), SEQ ID NO:79(KIWGDGSTDYTSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:77(SASSSISYMH), SEQ ID NO:51(DTSKLAS), SEQ ID NO:80 (HQRSPYPT);
(10) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:74(GFSLTGYGVN), SEQ ID NO:75(KIWGDGITDYNSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:81(RSSQSIEQSNGNTYLE), SEQ ID NO:82(KVSNRFS), SEQ ID NO:83 (FQGSHVPYT);
(11) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:74(GFSLTGYGVN), SEQ ID NO:75(KIWGDGITDYNSALKS), SEQ ID NO:76 (DVMDY); and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:84(SASSSINYMH), SEQ ID NO:51(DTSKLAS), SEQ ID NO:78 (HHRSPYPT);
(12) H-CDR1, H-CDR2, H-CDR3 shown in sequence in SEQ ID NO:35(EYTFTNNWIA), SEQ ID NO:85(DIHPGGGYTNYNEKFKG), SEQ ID NO:86 (SKTRDYDSWFAY); and, L-CDR1, L-CDR2, and L-CDR3 shown in sequence in SEQ ID NO:50(SASSSVSKMN), SEQ ID NO:51(DTSKLAS), and SEQ ID NO:52 (QQWSSNPLT).
The light and heavy chain CDR combinations provided herein are derived from an antibody or fragment thereof of the invention, and the CDRs contained therein can be routinely determined by one of skill in the art based on the variable region amino acid sequences contained in a given antibody or fragment thereof. For example, according to a specific embodiment of the present invention, the CDRs in the variable region amino acid sequences are defined using the AbM definition method.
In the antibody or fragment thereof provided by the present invention, preferably, the heavy chain variable region comprises a sequence selected from the group consisting of:
the amino acid sequence shown in SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31 or SEQ ID NO 33 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or
In the antibody or fragment thereof provided by the present invention, preferably, the light chain variable region comprises a sequence selected from the group consisting of:
the amino acid sequence shown in SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32 or SEQ ID NO 34 or an amino acid sequence having at least 75% identity to said amino acid sequence.
More preferably, the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 9; and, an amino acid sequence as set forth in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 10;
(2) an amino acid sequence as shown in SEQ ID NO. 11 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 11; and, an amino acid sequence as set forth in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 12;
(3) an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 13; and, an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 14;
(4) an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 15; and, an amino acid sequence as set forth in SEQ ID NO 16 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO 16;
(5) an amino acid sequence as shown in SEQ ID NO. 17 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 17; and, the amino acid sequence as set forth in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 18;
(6) an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 19; and, an amino acid sequence as set forth in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 20;
(7) an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 21; and, the amino acid sequence as set forth in SEQ ID NO. 22 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 22;
(8) an amino acid sequence as shown in SEQ ID NO. 23 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 23; and, an amino acid sequence as set forth in SEQ ID NO. 24 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 24;
(9) an amino acid sequence as set forth in SEQ ID NO. 25 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 25; and, the amino acid sequence as set forth in SEQ ID NO. 26 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 26;
(10) an amino acid sequence as set forth in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 27; and, the amino acid sequence as set forth in SEQ ID NO 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 28;
(11) an amino acid sequence as shown in SEQ ID NO. 29 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 29; and, an amino acid sequence as set forth in SEQ ID NO. 30 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 30;
(12) an amino acid sequence as shown in SEQ ID NO. 31 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 31; and, an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 32;
(13) the amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 33; and, the amino acid sequence shown in SEQ ID NO. 34 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 34.
In the context of the present invention, "at least 75% identity" is any percent numerical identity between 75% and 100%, such as 75%, 80%, 85%, 90%, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
In particular, the antibody or fragment thereof of the invention comprises at least a heavy chain variable region and a light chain variable region, both comprising the CDRs and a framework region (framework work) in such a way that the domains are arranged: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Further alternatively, the up to 25% difference in amino acid sequence resulting from the "at least 75% identity" may be present in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence in the antibody or fragment thereof of the invention other than the heavy chain variable region and the light chain variable region. The difference may result from amino acid deletion, addition or substitution at any position.
With respect to the antigen, the antibody or fragment thereof of the present invention is an antibody or fragment thereof against PD-L1; preferably, the antibody is in any form of a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or the antigen-binding fragment is a half-antibody or an antigen-binding fragment of an antibody or half-antibody, such as scFv, BsFv, dsFv, (dsFv)2、Fab、Fab'、F(ab')2Or Fv; more preferably, the antibody is an IgG.
In addition to the variable region, the antibody or fragment thereof comprises a constant region of human or murine origin, preferably a heavy chain constant region (CH) and/or a light chain constant region (CL) of human or murine origin; preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.
According to a particular embodiment of the invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;
preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 1;
preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 2.
In another aspect, the invention also provides a nucleic acid molecule encoding the antibody or fragment thereof of the invention or encoding a heavy chain CDR, a light chain variable region, a heavy chain or a light chain comprised in the antibody or fragment thereof.
The nucleic acid molecules of the invention may be cloned into vectors for transformation or transfection of host cells. Thus in a further aspect, the invention also provides a vector comprising a nucleic acid molecule of the invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like. The vectors or nucleic acid molecules of the invention may be used to transform or transfect host cells for purposes of preservation or antibody expression, etc. Thus, in a further aspect, the invention provides a host cell comprising or transformed or transfected with a nucleic acid molecule and/or vector of the invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
The antibodies or fragments thereof provided by the present invention can be obtained using any method known in the art. For example, the heavy chain variable region and/or the light chain variable region of the antibody may be obtained from the nucleic acid molecule provided by the present invention, or the heavy chain and/or the light chain of the antibody may be obtained and then assembled into an antibody with optional other domains of the antibody; alternatively, the host cell provided by the present invention is cultured under conditions that allow the host cell to express the heavy chain variable region and/or the light chain variable region of the antibody or the heavy chain and/or the light chain of the antibody to assemble into the antibody. Optionally, the method further comprises the step of recovering the produced antibody.
The antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention may be comprised in a pharmaceutical composition, more particularly in a pharmaceutical preparation, for use for various purposes according to the actual needs. Thus, in a further aspect, the invention also provides a pharmaceutical composition comprising an antibody or fragment thereof, a nucleic acid molecule, a vector and/or a host cell according to the invention, and optionally a pharmaceutically acceptable excipient.
In yet another aspect, the present invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of a disease including a tumor or cancer, such as non-small cell lung cancer, melanoma, bladder cancer, merkel lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, stomach cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, ear-eye tumor, biliary tract cancer, gall bladder cancer, adrenal cancer, reproductive system tumor, multiple myeloma, nervous system tumor, urothelial cell cancer.
Accordingly, the present invention also provides a method of preventing or treating a disease comprising a tumor or cancer, such as non-small cell lung cancer, melanoma, bladder cancer, mercker's lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin's lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin's lymphoma, breast cancer, thyroid cancer, stomach cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, ear and eye tumor, biliary tract cancer, gallbladder cancer, adrenal cancer, reproductive system tumor, multiple myeloma, nervous system tumor, urothelial cell cancer, comprising administering the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the invention to a subject in need thereof. Preferably, the subject is a mammal; more preferably, the subject is a human.
In yet another aspect, the invention provides a kit comprising an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell and/or a pharmaceutical composition of the invention. The kit may be for therapeutic or diagnostic use.
In contrast to the prior art, the present invention provides a novel antibody capable of binding to PD-L1, in particular human PD-L1, with high affinity. Compared with the existing anti-PD-L1 antibody, the antibody of the invention has the following characteristics: compared with the existing antibody (taking Atezolizumab (Tecnriq) of Genetech as an example), the antibody screened by the invention has improved affinity and dissociation rate to PD-L1; meanwhile, based on the mouse antibody obtained by initial screening, the immunogenicity of the antibody is reduced by performing humanized transformation to the maximum extent; in addition, pharmacodynamic experiments prove that the antibody provided by the invention has a better tumor inhibition effect.
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Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the results of FACS detection of the binding of the culture supernatant of the hybridoma cell line of the present invention to the antigen PD-L1.
FIG. 2 shows the results of FACS detection of the culture supernatant of the hybridoma cells of the invention blocking the binding of PD-L1 to the cells.
FIG. 3 shows the results of FACS detection of the binding of the antibody of the invention to the antigen PD-L1.
FIG. 4 shows the results of FACS detection of the antibody of the invention blocking the binding of PD-L1 to cells.
Fig. 5 shows ForteBio Octet assay results for binding of the antibody of the invention to antigen PD-L1, wherein 5A: 14a7 (hz); 5B: reference; 5C: 14a7 (chi); 5D: 14a 7.
FIG. 6 shows the results of the in vivo efficacy test of the antibody of the present invention in animals.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
Atezolizumab (reference): manufactured by Genetech under the trade name Tecntriq, disclosed in US2016/0319022A1, for light and heavy chains as shown in SEQ ID NO 6 and SEQ ID NO 7, respectively.
Example 1Preparation of hybridoma cells of the invention
Mice were immunized with a fusion protein comprising the extracellular region (position 19 to position 238 of SEQ ID NO:3) of human PD-L1 protein (from Genbank accession NP-054862.1, SEQ ID NO:3) and mouse IgG2a-FC (from Genbank accession AAH31470.1) as immunogen, 5 mice were immunized subcutaneously, 5 mice were immunized intramuscularly, the adjuvant was quick anti body 5W water soluble adjuvant, the titer was measured 2 weeks after boosting, two mice with high titer were selected for immunization shock, and 3 days later cell fusion described below was performed.
Taking 2 mice to be fused, taking serum, dissecting, taking spleen, separating splenocytes, fusing with cultured myeloma cells, laying a 96-well plate, adding a selective culture medium for screening, changing liquid after 7 days, performing ELISA detection after 10 days, and performing flow cytometry detection after the OD value is more than 10 times that of a negative control.
Selecting double positive cells, performing subclone plating by a cell limiting dilution method, and selecting monoclonal cells. And (3) taking the selected monoclonal cells, taking culture supernatant, performing ELISA detection and flow cytometry detection, and selecting the cells of double positive for amplification culture.
Example 2ELISA detection of binding of culture supernatant of hybridoma cells of the invention to PD-L1
The fusion protein comprising the extracellular region (position 19 to 238 of SEQ ID NO:3) of human PD-L1 protein (from Genbank accession NP-054862.1, SEQ ID NO:3) and human IgG1-FC (from Genbank accession CAC20454.1) was diluted to 1-2. mu.g/ml with the coating solution, and then added to the wells of the microplate at 50-100. mu.l/well and allowed to adsorb at 4 ℃ overnight or 37 ℃ for 2 hours. The liquid in the wells was discarded while washing 3 times with wash solution for 3-5 minutes each time, and patted dry. Add 200. mu.l of blocking solution to each well overnight at 4 ℃ or blocked for 2 hours at 37 ℃. Washing with washing solution for 3 times, and storing at-20 deg.C or 4 deg.C.
50-100. mu.l of culture supernatant of the hybridoma to be tested was added to each well, and a positive control (added with serum from the fusion mouse), a negative control (added with serum from the normal mouse), and a blank control (added with the medium) were set up at the same time. Incubate at 37 ℃ for 1-2 hours, wash, and pat dry. Then adding enzyme-labeled secondary antibody, namely horse radish peroxidase-labeled goat anti-mouse IgG (SIGMA, A9044-2ml) diluted at a ratio of 1:10000 into each well, incubating for 1-2 hours at 37 ℃ in each well for 50-100 mu l, washing, and patting to dry. 50-100. mu.l of a freshly prepared substrate developing solution TMB was added to each well, and incubated at 37 ℃ for 10-30 minutes.
2mol/L H was added2SO4The reaction was stopped and the OD read on an ELISA reader.
And (4) judging a result: positive was obtained with P/N >2:1(P for positive values and N for normal mouse serum values). If the negative control hole is colorless or nearly colorless, and the positive control hole is clearly colored, the result can be directly observed by naked eyes.
Example 3FACS detection of binding of culture supernatant of hybridoma cells of the present invention to PD-L1
Synthesizing the extracellular region (position 19 to position 238 of SEQ ID NO:3) gene fragment of human PD-L1 protein (from Genbank accession number NP-054862.1, SEQ ID NO:3), constructing a PLVX virus packaging vector (Clontech, virus package mix, cat. No. 631275), transfecting 293T cell packaging virus, infecting HEK293 cells with the virus, screening with puromycin (puromycin) to obtain a drug-resistant cell strain, namely HEK293 stably expressing PD-L1, named 293T-h-PDL1, and preparing the cell strain in PBS containing 2% FBS to a cell concentration of 1 × 107Individual cells/ml of cell suspension.
50. mu.l of the cell suspension was put into each flow tube (sample tube), and then 50. mu.l of the culture supernatant of the hybridoma to be tested or positive control antibody Atezolizumab (reference) was added thereto, and incubated at 4 ℃ for 60 minutes. To each flow tube, 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes, the supernatant was discarded, and the washing was repeated three times. Control tubes 1 (without addition of culture supernatant and secondary antibody below, only cell suspension) and 2 (without addition of culture supernatant, only cell suspension and secondary antibody below) were set simultaneously.
Then 100. mu.l of flow buffer was added to each flow tube for resuspension, 5. mu.l of PE-labeled anti-mouse Fc-labeled secondary antibody (Biolegend, cat. No. 409304) was added according to the experimental requirements, incubated at 4 ℃ for 30 minutes in the dark, then 1ml of flow buffer was added, centrifuged at 1200rpm for 5 minutes at room temperature, the supernatant was discarded, and the washing was repeated three times.
Add 250. mu.l of flow buffer to each flow tube again, resuspend and mix well, and test on the machine.
The results of the FACS binding measurements are shown in table 1 and figure 1.
TABLE 1 FACS detection of hybridoma culture supernatants by binding to PD-L1
EC50(μg/mL)
13C9-1 0.4401
4G7-3 0.543
8A2-2 0.336
15G10-3 1.45
14A7-4 0.5383
28B1-2 0.3904
22B10-1 0.5172
18B4-2 0.1249
Reference 1.33
The left column in the table shows the number of hybridoma cell lines and the numbers of subclones thereof, and for example "13C 9-1" is the first clone of a subclone numbered hybridoma cell line 13C 9.
Example 4FACS detection of blocking of cell binding by PD-L1 in culture supernatant of hybridoma cells of the invention
An extracellular region (from position 24 to position 170 of SEQ ID NO:8) gene fragment of human PD-1 protein (SEQ ID NO:8) was synthesized, constructed into PLVX-IRES-PURO (Clontech), transformed into DH 5. alpha. competent cells, plated, and cultured overnight at 37 ℃. Selecting a monoclonal colony, shaking bacteria, carrying out enzyme digestion identification, extracting plasmids, selecting correct clones to extract plasmids through sequencing verification, and storing for later use.
Recovering 293T cells, after 2 serial passages, according to 2X 106Plating each cell/10 ml/dish, transfecting the cell with the eukaryotic expression plasmid after 16-24 h until the cell density reaches 70-80%, placing at 37 ℃ and 5% CO2Culturing in an incubator. Culturing for 12-16 h, removing the culture medium, changing the culture medium, and adding a corresponding amount of Puromycin (the final concentration is 10 mu g/ml) after 48-56 h. The cells were cultured continuously for 10 days in DMEM containing 10% FBS + 10. mu.g/ml Puromycin if the growth state of the cells was good. And (5) initially judging to take the polyclonal cell and carrying out flow detection.
Taking positive polyclonal cells, plating each well according to 0.5 cell, picking out monoclonal cells after 7 days, and carrying out expanded culture. And performing FACS detection on the selected monoclonal, selecting positive cells, namely 293T cells stably expressing h-PD1, and naming the cells as 293T-h-PD 1.
The cells were washed once with FACS buffer at 2-5X 105Cells/well were plated in 96-well plates, and 50. mu.L of a premix of hybridoma supernatant or positive control antibody Atezolizumab (reference) and 500ng of fusion protein PD-L1(hFC tag) from example 2 was added to each well and incubated for 2 hours. Then, 400. mu.l of FACS buffer was added to each well and washed twice, a PE anti-human IgG Fc secondary antibody was added thereto and incubated for 1 hour, and then 400. mu.l of FACS buffer was added to each well and washed twice. And (6) performing detection on the machine.
The results of the FACS blockade detection are shown in table 2 and fig. 2.
TABLE 2 FACS detection of hybridoma culture supernatants for blocking the binding of PD-L1 to cells
IC50(μg/mL)
13C9-1 1.95
4G7-3 3.42
8A2-2 5.17
15G10-3 4.00
14A7-4 2.31
28B1-2 2.31
22B10-1 4.31
18B4-2 2.89
Reference 2.97
Example 5Affinity detection of binding of culture supernatant of hybridoma cells of the present invention to PD-L1
The experimental method comprises the following steps:
1. selecting the same kind of Capture Surface Dip.
2. A96-well plate was taken and 200. mu.l SD buffer (1 XPBS + 0.02% Tween20+ 0.1% BSA) was added to each well and placed in a ForteBio Octet for pre-cycling.
3. The antibody was immobilized, and the antibody concentration of the supernatant after purification was 10. mu.g/ml.
4. The extracellular region of human PD-L1 protein (position 19 to position 238 of SEQ ID NO:3) was diluted with 6 gradients of 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, respectively, and added to the corresponding wells.
5. And (6) performing detection on the machine.
The results are shown in Table 3.
TABLE 3 affinity assay of hybridoma culture supernatants for binding to PD-L1
Figure BDA0002240929810000131
And selecting corresponding hybridoma cell strains based on the experiment, extracting RNA from the monoclonal cells, performing reverse transcription to form cDNA, and connecting the cDNA into a sequencing vector for sequencing analysis.
The sequences of exemplary murine antibodies are as follows, with the CDRs (obtained according to the AbM definition method) underlined. The nomenclature of the murine antibody depends on the hybridoma cell line from which it is derived.
A1 (murine antibody 14A7)
>14A7-VH
QVQLKQSGAELVRPGTSVKMSCKATEYTFTNNWIAWVKQRPGHGLEWVGDIHPGGGFTNYNEKFKVKATLTADTSSSTAYMQLRSLTSEDSAIYYCAGSKTRDYDAWFAYWGQGTLVTVSA
>14A7-VLDIVMTQAPSSLAVSVGEKVTLNCKSSQSLLYTGNQKNYLAWYQQKPGQSPKLLIYWASTR ESGVPDRFTGSGSGTDFTLTISNVKAEDLAVYFCQQYYTYRTFGGGTKLEIK
A2 (murine antibody 15G10)
>15G10-VH
QVQLKQSGPELVKPGASVKTSCKASGYTFTNYVVHWVKQNPGQGLEWIGYVNPNNDGTIFNEKFKDKAILTSDKSSSTAYMELSSLTSEDSAVYYCARSPFAHWGQGTLVTVSA
>15G10-VL
QIVLTQSPASLTVSLGQRATISCSASESVDFYGTSLMQWFQQKPGQPPKLLVYTASNVDSEVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCHQTRKVPYTFGGGTKLEIK
A3 (murine antibody 13C9)
>13C9-VH
QVQLKQSGAGLVKPGASVKLSCKASGYIFTEYIIHWVKQRSGQGLEWIGWFYPGSDNIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHETGYFFDYWGQGTTLTVSS
>13C9-VL
DIVITQSPAIMSASPGEKVTMTCSASSSVSKMNWYQQKSGTSPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLELKRA
A4 (murine antibody 18B4)
>18B4-VH
QVQLKQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGVTHYNQKFKGKATLTVDESSSTASMQLKSLTSEDSAIYYCARGIPFYGLDYWGQGTSVTVSS
>18B4-VL
QIVLTQSPAIMSASPGEKVTMTCGASSSVSFMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNTNPFTFGSGTKLEIKRA
A5 (murine antibody 28B1)
>28B1-VH
QVQLKQSGAELVKPGASVMLSCTASAFNIDDTYIHWVKQRPEQGLEWIGRIDPANGNTDYDPECQGKATITTDMSSNTAYLQLSSLTSEDTAVYFCARGLRLPGLVYWGRGTLVTVSA
>28B1-VL
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILYSGVPSRFSGSGSGTDYSLTINTLEEEDIATYFCQQGNTLPWTFGGGTKLEIIRA
A6 (murine antibody 22B10)
>22B10-VH
QVQLKQSGAELVKPGASVELSCTASGFNIEDTYLHWVNQRPEQGLEWIGRIDPANGNTYYDPKFQGKATITTDTSSNTAYLQLSRLTSEDTAVYYCARGLRLPGFPYWGQGTLVTVSA
>22B10-VL
DIQMTQTPSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLAISNLDQEDIATYFCQQGNTLPWTFGGGTKLEIKRA
A7 (murine antibody 4G7)
>4G7-VH
QVQLKESGPSLVKPSQTLSLTCSVTGDSIISGYWNWIRKFPGNELEYMGYISYTGSTYYNPSLKSRVSIIRDTFKNQYYLQLNSVTTEDTATYYCARRGEWLLHFDVWGAGTTVTVSS
>4G7-VL
DIVMTQAPSSLAVSVGEKVTVSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK
A8 (murine antibody 8A2)
>8A2-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGITDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARDVMDYWGQGTSVTVSS
>8A2-VL
QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSESGTSYSLTISSMEAEDAATYYCHHRSPYPTFGAGTKLELK
A9 (murine antibody 7E5)
>7E5-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGSTDYTSALKSRLSISKDNSKSQVFLKVNSLQTDDTARYYCARDVMDYWGQGTSVTVSS
>7E5-VL
QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSESGTSYSLTISSMEAEDAATYYCHQRSPYPTFGAGTKLELK
A10 (murine antibody 2E5)
>2E5-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGITDYNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCARDVMDYWGQGTSVTVSS
>2E5-VL
DVLMTQIPLSLPVSLGDQASISCRSSQSIEQSNGNTYLEWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVESEDLGVYYCFQGSHVPYTFGGGTKLEIK
A11 (murine antibody 10A3)
>10A3-VH
QVQLKQSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGKIWGDGITDYNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCARDVMDYWGQGTSVTVSS
>10A3-VL
QIVLTQSPAIMSASPGEKVTMTCSASSSINYMHWFQQKPGTSPKRWIYDTSKLASGVPARFSGSESGTSYSLTISSMEAEDAATYYCHHRSPYPTFGAGTKLELKRA
A12 (murine antibody 6G5)
>6G5-VH
QVQLKQSGAELVRPGTSVKMSCKATEYTFTNNWIAWVKQRPGHGLEWIGDIHPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAIYYCAGSKTRDYDSWFAYWGQGTLVTVSA
>6G5-VL
DIVITQSPAIMSASPGEKVTMTCSASSSVSKMNWYQQKSGTSPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLELKRA
Example 6Humanization engineering of antibodies of the invention
The a1 antibody (murine antibody 14a7) was selected for humanization of the antibody. Comprehensively considers the sequence similarity, expression quantity and whether the light and heavy chain combination of the antibody and different humanized templates is used by the antibody which is already prepared, and the like. According to the factors that the similarity value of the light chain and the heavy chain is more than 200, the expression amount is more than 50mg/ml, the antibody of the light chain and the heavy chain is used in combination with the drug and the like, finally, the humanized sequences are respectively selected as templates of the heavy chain and the light chain: IGHV1-46 x 01(SEQ ID NO:4) and IGKV1-5 x 01(SEQ ID NO: 5). Homology modeling was performed on a1 murine mab and structural simulation of the Fab region was performed. Through homologous modeling calculation, the predicted Fab structure of the A1 antibody is finally obtained.
By comparing the predicted Fab structure and heavy chain with IGHV1-46 x 01 sequence, CDR regions were replaced by human-derived templates, and structure simulation and kinetic calculations were performed. Through structural simulation and kinetic analysis, several amino acids affecting the CDR structure were retained, and the final heavy chain version except the CDR region and 68A, 70L, etc. are original murine amino acids and all other murine amino acids were replaced with corresponding IGHV1-46 x 01 template human amino acids.
Through the comparison analysis of the predicted Fab structure and light chain with IGKV 1-5X 01 sequence, CDR area is replaced with adult template, and structure simulation and kinetic calculation are performed. Through structural simulation and kinetic analysis, several amino acids affecting the CDR structure were retained murine, and the final light chain version was replaced with all the other murine amino acids except the original murine amino acids in the CDR region by the corresponding IGKV1-5 × 05 template human amino acids.
The sequence of a humanized antibody of the a1 antibody is as follows:
VH humanized sequence 14A7-VH-hz
QVQLVQSGAEVKKPGASVKVSCKASEYTFTNNWIAWVRQAPGQGLEWVGDIHPGGGFTNYNEKFKVRATLTADTSTSTAYMELSSLRSEDTAVYYCAGSKTRDYDAWFAYWGQGTTVTVSS
' VL humanized sequence 14A7-VL-hz
DIQMTQSPSTLSASVGDRVTITCKSSQSLLYTGNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYTYRTFGQGTKVEIK
The sequence shown in SEQ ID NO. 1 is used as a heavy chain constant region, the sequence shown in SEQ ID NO. 2 is used as a light chain constant region, a primer is redesigned for the DNA sequence of an antibody aiming at the PD-L1 target obtained by sequencing, the genes of a corresponding chimeric antibody and a humanized antibody are synthesized and connected into a eukaryotic expression vector, DH5alpha competent cells are transformed, the cells are cultured in a constant temperature incubator at 37 ℃ for overnight, and monoclonal strains are selected for sequencing and identification. Selecting strains with correct sequences, shaking, extracting plasmids, transfecting mammalian expression cells 293F, placing in an incubator with 37 ℃ and 5% CO2, and performing expression culture for 7 days.
Collecting the expression supernatant, centrifuging, filtering, selecting protein G affinity chromatography column, purifying, detecting purity of the purified antibody by SDS-PAGE electrophoresis, detecting antibody concentration by using BCA protein detection kit, subpackaging, and storing in a refrigerator at-80 ℃ for later use. Among them, the chimeric antibody was named "murine antibody abbreviated (chi)", and the humanized antibody was named "murine antibody abbreviated (hz)".
Example 7FACS detection of binding of the antibodies of the invention to PD-L1
The experimental procedure was as in example 3, except that after 50. mu.l of cell suspension was put into each flow tube, the test antibody, the positive control antibody (Reference) or the negative Reference antibody (hIgG1) was added at the concentrations shown in FIG. 3. Control tubes 1 (no antibody and secondary antibody added, only cell suspension added) and 2 (no antibody added, only cell suspension and secondary antibody added) were set simultaneously.
The results of the FACS binding measurements are shown in table 4 and figure 3.
TABLE 4 FACS detection of antibody binding to PD-L1
EC50(μg/mL)
14A7-4(hz) 0.77
14A7-4(chi) 1.32
Reference 1.32
14A7-4 0.80
13C9-1(chi) 1.42
13C9-1 0.49
Example 8FACS detection of the blocking of PD-L1 binding to cells by the antibodies of the invention
The experimental procedure is as in example 4, except that the test antibody, the positive control antibody Atezolizumab (reference) or the negative reference antibody (hIgG1) is added to each well, and the concentrations are shown in FIG. 4.
The results of the FACS blockade detection are shown in table 5 and fig. 4.
TABLE 5 FACS detection of antibody blocking the binding of PD-L1 to cells
Figure BDA0002240929810000181
Figure BDA0002240929810000191
Example 9Affinity detection of binding of the antibodies of the invention to PD-L1
The extracellular region of human PD-L1 protein (position 19 to position 238 of SEQ ID NO:3) was prepared in PBS buffer at a maximum concentration of 30nM and 3-fold diluted to 6 concentrations and a 0 concentration control was set. The murine, chimeric and humanized antibodies of the invention were prepared as 20nM solutions in PBS.
The affinity of the antibody and antigen was determined using ForteBio Octet as an affinity detection instrument. The specific procedure was as in example 5.
The results are shown in table 6 and fig. 5.
TABLE 6 results of affinity assay of antibody binding to antigen PD-L1
Antibodies Antigens KD(M) kdis(1/s)
Reference Human PD-L1-his 2.23E-09 7.96E-04
14A7(hz) Human PD-L1-his 8.84E-10 3.58E-04
14A7(chi) Human PD-L1-his 4.56E-09 1.73E-03
14A7 Human PD-L1-his 5.45E-10 3.59E-04
Example 10Test of efficacy of the humanized PD-L1 antibody of the present invention in animals
Referring to example 4, the extracellular region (19 th to 238 th of SEQ ID NO:3) gene fragment of human PD-L1 protein (SEQ ID NO:3) was synthesized and constructed into PLVX-IRES-PURO
(Clontech), a eukaryotic expression vector carrying h-PDL1 was obtained, and plasmids were extracted.
Referring also to example 4, except for replacing 293T cells with MC38 cells, MC38 cells stably expressing h-PDL1 were obtained and designated MC38-h-PDL1 cells.
Recovering MC38-h-PDL1 cells, and culturing in vitro to obtain 0.3X10E8 cells. 30 male BALB/C mice at 6-8 weeks were acclimatized for 1 week and weighed. Cells were seeded according to the seeding conditions shown in table 7.
TABLE 7 mouse inoculation information
Animal number (only) Inoculation site Amount of cells seeded/ Inoculation cell suspension volume/body (mL)
30 Under the skin 1 X 106 0.1mL
After inoculation, tumor volume and body weight were measured 2 times a week, when the mean tumor volume reached about 80-120mm3The groups were randomized into 3 groups of 10 according to tumor volume and body weight. Administration was started immediately after the grouping. The administration start date was regarded as day 0. The administration and grouping information is shown in table 8.
TABLE 8 grouping and administration information
Figure BDA0002240929810000201
After the start of administration, the body weight and tumor volume of the mice were measured. The results are shown in FIG. 6.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg
<210> 5
<211> 95
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 1-5.01 stencil
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser
85 90 95
<210> 6
<211> 448
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Atezolizumab heavy chain
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 7
<211> 214
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> Atezolizumab light chain
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 8
<211> 288
<212> PRT
<213> Homo sapiens
<400> 8
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 9
<211> 121
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VH
<400> 9
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Thr Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Val Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 10
<211> 112
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VL
<400> 10
Asp Ile Val Met Thr Gln Ala Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Leu Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Asn Val Lys Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 11
<211> 114
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 15G10-VH
<400> 11
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Thr Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Val Val His Trp Val Lys Gln Asn Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Val Asn Pro Asn Asn Asp Gly Thr Ile Phe Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Pro Phe Ala His Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ala
<210> 12
<211> 111
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 15G10-VL
<400> 12
Gln Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Ser Ala Ser Glu Ser Val Asp Phe Tyr
20 25 30
Gly Thr Ser Leu Met Gln Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Val Tyr Thr Ala Ser Asn Val Asp Ser Glu Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys His Gln Thr Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 13
<211> 118
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 13C9-VH
<400> 13
Gln Val Gln Leu Lys Gln Ser Gly Ala Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Glu Tyr
20 25 30
Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 14
<211> 108
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 13C9-VL
<400> 14
Asp Ile Val Ile Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 105
<210> 15
<211> 118
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 18B4-VH
<400> 15
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Val Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Ser
65 70 75 80
Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Pro Phe Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 16
<211> 108
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 18B4-VL
<400> 16
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Gly Ala Ser Ser Ser Val Ser Phe Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala
100 105
<210> 17
<211> 118
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 28B1-VH
<400> 17
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Met Leu Ser Cys Thr Ala Ser Ala Phe Asn Ile Asp Asp Thr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Asp Tyr Asp Pro Glu Cys
50 55 60
Gln Gly Lys Ala Thr Ile Thr Thr Asp Met Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Leu Arg Leu Pro Gly Leu Val Tyr Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 18
<211> 109
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 28B1-VL
<400> 18
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ile Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Thr Leu Glu Glu
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ile Arg Ala
100 105
<210> 19
<211> 118
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 22B10-VH
<400> 19
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Glu Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr
20 25 30
Tyr Leu His Trp Val Asn Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Tyr Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Arg Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Leu Arg Leu Pro Gly Phe Pro Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 20
<211> 109
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 22B10-VL
<400> 20
Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Ala Ile Ser Asn Leu Asp Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
100 105
<210> 21
<211> 118
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 4G7-VH
<400> 21
Gln Val Gln Leu Lys Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Ile Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Glu Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Ser Ile Ile Arg Asp Thr Phe Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Arg Gly Glu Trp Leu Leu His Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 22
<211> 113
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 4G7-VL
<400> 22
Asp Ile Val Met Thr Gln Ala Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Val Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 23
<211> 113
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 8A2-VH
<400> 23
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 24
<211> 105
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 8A2-VL
<400> 24
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His His Arg Ser Pro Tyr Pro Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 25
<211> 113
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 7E5-VH
<400> 25
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ser Thr Asp Tyr Thr Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Val Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 26
<211> 105
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 7E5-VL
<400> 26
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Pro Tyr Pro Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 27
<211> 113
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 2E5-VH
<400> 27
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 28
<211> 112
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 2E5-VL
<400> 28
Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Glu Gln Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ser Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 29
<211> 113
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 10A3-VH
<400> 29
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 30
<211> 107
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 10A3-VL
<400> 30
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Asn Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His His Arg Ser Pro Tyr Pro Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 105
<210> 31
<211> 121
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 6G5-VH
<400> 31
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Thr Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ser Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 32
<211> 108
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 6G5-VL
<400> 32
Asp Ile Val Ile Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 105
<210> 33
<211> 121
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VH-hz
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Val Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 34
<211> 112
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> 14A7-VL-hz
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 35
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 35
Glu Tyr Thr Phe Thr Asn Asn Trp Ile Ala
1 5 10
<210> 36
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 36
Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Val
<210> 37
<211> 12
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 37
Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr
1 5 10
<210> 38
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 38
Lys Ser Ser Gln Ser Leu Leu Tyr Thr Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 39
<211> 7
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 39
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 40
Gln Gln Tyr Tyr Thr Tyr Arg Thr
1 5
<210> 41
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 41
Gly Tyr Thr Phe Thr Asn Tyr Val Val His
1 5 10
<210> 42
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 42
Tyr Val Asn Pro Asn Asn Asp Gly Thr Ile Phe Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 43
<211> 5
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 43
Ser Pro Phe Ala His
1 5
<210> 44
<211> 15
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 44
Ser Ala Ser Glu Ser Val Asp Phe Tyr Gly Thr Ser Leu Met Gln
1 5 10 15
<210> 45
<211> 7
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 45
Thr Ala Ser Asn Val Asp Ser
1 5
<210> 46
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 46
His Gln Thr Arg Lys Val Pro Tyr Thr
1 5
<210> 47
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 47
Gly Tyr Ile Phe Thr Glu Tyr Ile Ile His
1 5 10
<210> 48
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 48
Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 49
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 49
His Glu Thr Gly Tyr Phe Phe Asp Tyr
1 5
<210> 50
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 50
Ser Ala Ser Ser Ser Val Ser Lys Met Asn
1 5 10
<210> 51
<211> 7
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 51
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 52
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 52
Gln Gln Trp Ser Ser Asn Pro Leu Thr
1 5
<210> 53
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 53
Gly Tyr Ser Phe Thr Gly Tyr Asn Met Asn
1 5 10
<210> 54
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 54
Asn Ile Asp Pro Tyr Tyr Gly Val Thr His Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 55
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 55
Gly Ile Pro Phe Tyr Gly Leu Asp Tyr
1 5
<210> 56
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 56
Gly Ala Ser Ser Ser Val Ser Phe Met His
1 5 10
<210> 57
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 57
Gln Gln Trp Asn Thr Asn Pro Phe Thr
1 5
<210> 58
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 58
Ala Phe Asn Ile Asp Asp Thr Tyr Ile His
1 5 10
<210> 59
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 59
Arg Ile Asp Pro Ala Asn Gly Asn Thr Asp Tyr Asp Pro Glu Cys Gln
1 5 10 15
Gly
<210> 60
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 60
Gly Leu Arg Leu Pro Gly Leu Val Tyr
1 5
<210> 61
<211> 11
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 61
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 62
<211> 7
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 62
Tyr Thr Ser Ile Leu Tyr Ser
1 5
<210> 63
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 63
Gln Gln Gly Asn Thr Leu Pro Trp Thr
1 5
<210> 64
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 64
Gly Phe Asn Ile Glu Asp Thr Tyr Leu His
1 5 10
<210> 65
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 65
Arg Ile Asp Pro Ala Asn Gly Asn Thr Tyr Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 66
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 66
Gly Leu Arg Leu Pro Gly Phe Pro Tyr
1 5
<210> 67
<211> 11
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 67
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ser
1 5 10
<210> 68
<211> 7
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 68
Tyr Thr Ser Ile Leu His Ser
1 5
<210> 69
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 69
Gly Asp Ser Ile Ile Ser Gly Tyr Trp Asn
1 5 10
<210> 70
<211> 16
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 70
Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 71
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 71
Arg Gly Glu Trp Leu Leu His Phe Asp Val
1 5 10
<210> 72
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 72
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Ser Leu
1 5 10 15
Ala
<210> 73
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 73
Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr
1 5
<210> 74
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR1
<400> 74
Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn
1 5 10
<210> 75
<211> 16
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 75
Lys Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys Ser
1 5 10 15
<210> 76
<211> 5
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 76
Asp Val Met Asp Tyr
1 5
<210> 77
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 77
Ser Ala Ser Ser Ser Ile Ser Tyr Met His
1 5 10
<210> 78
<211> 8
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 78
His His Arg Ser Pro Tyr Pro Thr
1 5
<210> 79
<211> 16
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 79
Lys Ile Trp Gly Asp Gly Ser Thr Asp Tyr Thr Ser Ala Leu Lys Ser
1 5 10 15
<210> 80
<211> 8
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 80
His Gln Arg Ser Pro Tyr Pro Thr
1 5
<210> 81
<211> 16
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 81
Arg Ser Ser Gln Ser Ile Glu Gln Ser Asn Gly Asn Thr Tyr Leu Glu
1 5 10 15
<210> 82
<211> 7
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR2
<400> 82
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 83
<211> 9
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR3
<400> 83
Phe Gln Gly Ser His Val Pro Tyr Thr
1 5
<210> 84
<211> 10
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> L-CDR1
<400> 84
Ser Ala Ser Ser Ser Ile Asn Tyr Met His
1 5 10
<210> 85
<211> 17
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR2
<400> 85
Asp Ile His Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 86
<211> 12
<212> PRT
<213> Artificial (Artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> H-CDR3
<400> 86
Ser Lys Thr Arg Asp Tyr Asp Ser Trp Phe Ala Tyr
1 5 10

Claims (12)

1. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) comprise a combination of CDRs selected from:
(1) 35, 36, 37, H-CDR1, H-CDR2, H-CDR 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40;
(2) 41, 42, 43, H-CDR1, 2, H-CDR 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46;
(3) 47, 48, 49, H-CDR1, 2, H-CDR 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 52;
(4) 53, 54, 55H-CDR 1, 2, H-CDR 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 56, SEQ ID NO 51, SEQ ID NO 57;
(5) 58, 59, 60H-CDR 1, 2, 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63;
(6) is shown in sequence in SEQ ID NO 64, SEQ ID NO 65, H-CDR1, H-CDR2, H-CDR3 of SEQ ID NO 66; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 63;
(7) 69, 70, 71, H-CDR1, H-CDR2, H-CDR 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 72, SEQ ID NO 39, SEQ ID NO 73;
(8) is shown in sequence in SEQ ID NO 74, SEQ ID NO 75, H-CDR1, H-CDR2, H-CDR3 of SEQ ID NO 76; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 77, SEQ ID NO 51, SEQ ID NO 78;
(9) is shown in sequence in SEQ ID NO 74, SEQ ID NO 79, H-CDR1, H-CDR2 and H-CDR3 of SEQ ID NO 76; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 77, SEQ ID NO 51, SEQ ID NO 80;
(10) is shown in sequence in SEQ ID NO 74, SEQ ID NO 75, H-CDR1, H-CDR2, H-CDR3 of SEQ ID NO 76; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83;
(11) is shown in sequence in SEQ ID NO 74, SEQ ID NO 75, H-CDR1, H-CDR2, H-CDR3 of SEQ ID NO 76; and, shown sequentially in SEQ ID NO:84, SEQ ID NO:51, L-CDR1, L-CDR2, L-CDR3 of SEQ ID NO: 78;
(12) 35, 85, 86, H-CDR1, 2, H-CDR 3; and, L-CDR1, L-CDR2, L-CDR3 shown in sequence in SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 52.
2. The antibody or fragment thereof of claim 1, wherein the heavy chain variable region comprises a sequence selected from the group consisting of:
the amino acid sequence shown in SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31 or SEQ ID NO 33 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or
The light chain variable region comprises a sequence selected from:
the amino acid sequence shown in SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32 or SEQ ID NO 34 or an amino acid sequence having at least 75% identity to said amino acid sequence.
3. The antibody or fragment thereof of claim 1 or 2, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) an amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 9; and, the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 10;
(2) an amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 11; and, the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 12;
(3) an amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 13; and, the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 14;
(4) an amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 15; and, the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 16;
(5) 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID No. 17; and, the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 18;
(6) an amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 19; and, the amino acid sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 20;
(7) an amino acid sequence shown in SEQ ID NO. 21 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 21; and, the amino acid sequence shown in SEQ ID NO. 22 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 22;
(8) an amino acid sequence shown in SEQ ID NO. 23 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 23; and, the amino acid sequence shown in SEQ ID NO. 24 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 24;
(9) an amino acid sequence shown in SEQ ID NO. 25 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 25; and, the amino acid sequence shown in SEQ ID NO. 26 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 26;
(10) an amino acid sequence shown in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 27; and, the amino acid sequence shown in SEQ ID NO 28 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO 28;
(11) 29 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID No. 29; and, the amino acid sequence shown in SEQ ID NO. 30 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 30;
(12) 31 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID No. 31; and, the amino acid sequence shown in SEQ ID NO. 32 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 32;
(13) the amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 33; and, the amino acid sequence shown in SEQ ID NO. 34 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 34.
4. The antibody or fragment thereof according to any one of claims 1 to 3, wherein the antibody or fragment thereof is an anti-PD-L1 antibody or fragment thereof;
preferably, the antibody is in any form of a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or the antigen-binding fragment is a half-antibody or an antigen-binding fragment of an antibody or half-antibody, such as scFv, BsFv, dsFv, (dsFv)2、Fab、Fab'、F(ab')2Or Fv; more preferably, the antibody is an IgG.
5. The antibody or fragment thereof according to any one of claims 1 to 4, wherein the antibody or fragment thereof further comprises a constant region of human or murine origin, preferably a heavy chain constant region (CH) and/or a light chain constant region (CL) of human or murine origin; preferably, the antibody or fragment thereof comprises a heavy chain and a light chain;
more preferably, the antibody or fragment thereof comprises a heavy chain constant region of an IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.
6. The antibody or fragment thereof according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;
preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 1;
preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO. 2.
7. A nucleic acid molecule encoding the antibody or fragment thereof of any one of claims 1 to 6 or encoding a heavy chain CDR, a light chain variable region, a heavy chain or a light chain comprised in said antibody or fragment thereof.
8. A vector comprising the nucleic acid molecule of claim 7.
9. A host cell comprising or transformed or transfected with the nucleic acid molecule of claim 7 and/or the vector of claim 8.
10. A pharmaceutical composition comprising the antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, or the host cell of claim 9, and optionally a pharmaceutically acceptable excipient.
11. Use of the antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10 in the manufacture of a medicament for the prevention or treatment of a disease comprising a tumor or cancer, such as non-small cell lung cancer, melanoma, bladder cancer, merkel lymphoma, cutaneous squamous cell carcinoma, lung cancer, hodgkin lymphoma, kidney cancer, liver cancer, esophageal cancer, non-hodgkin lymphoma, breast cancer, thyroid cancer, gastric cancer, intestinal cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, leukemia, laryngeal cancer, oral cancer, otic eye tumor, biliary tract cancer, gallbladder cancer, adrenal cancer, reproductive system tumor, multiple myeloma, nervous system tumor, urothelial cell cancer.
12. A kit comprising the antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10.
CN201910999788.4A 2019-10-21 2019-10-21 anti-PD-L1 antibody and pharmaceutical application thereof Active CN112759647B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925937A (en) * 2022-08-05 2023-04-07 赛灵药业科技集团股份有限公司北京分公司 Antibody or fragment thereof and pharmaceutical application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108250296A (en) * 2018-01-17 2018-07-06 长春金赛药业股份有限公司 Human anti-human PD-L1 monoclonal antibodies and its application
CN108456251A (en) * 2017-02-21 2018-08-28 上海君实生物医药科技股份有限公司 Anti- PD-L1 antibody and its application
CN108699146A (en) * 2016-01-04 2018-10-23 江苏怀瑜药业有限公司 Anti- PD-L1 antibody and application thereof
CN109608544A (en) * 2015-11-17 2019-04-12 苏州盛迪亚生物医药有限公司 PD-L1 antibody, its antigen-binding fragment and its medical usage

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109608544A (en) * 2015-11-17 2019-04-12 苏州盛迪亚生物医药有限公司 PD-L1 antibody, its antigen-binding fragment and its medical usage
CN108699146A (en) * 2016-01-04 2018-10-23 江苏怀瑜药业有限公司 Anti- PD-L1 antibody and application thereof
CN108456251A (en) * 2017-02-21 2018-08-28 上海君实生物医药科技股份有限公司 Anti- PD-L1 antibody and its application
CN108250296A (en) * 2018-01-17 2018-07-06 长春金赛药业股份有限公司 Human anti-human PD-L1 monoclonal antibodies and its application

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925937A (en) * 2022-08-05 2023-04-07 赛灵药业科技集团股份有限公司北京分公司 Antibody or fragment thereof and pharmaceutical application thereof
CN116789833A (en) * 2022-08-05 2023-09-22 赛灵药业科技集团股份有限公司北京分公司 Antibody or fragment thereof and pharmaceutical application thereof
CN116789833B (en) * 2022-08-05 2023-12-12 赛灵药业科技集团股份有限公司北京分公司 Antibody or fragment thereof and pharmaceutical application thereof

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