CN107973854B - PDL1 monoclonal antibody and application thereof - Google Patents

PDL1 monoclonal antibody and application thereof Download PDF

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CN107973854B
CN107973854B CN201711303454.6A CN201711303454A CN107973854B CN 107973854 B CN107973854 B CN 107973854B CN 201711303454 A CN201711303454 A CN 201711303454A CN 107973854 B CN107973854 B CN 107973854B
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CN107973854A (en
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周宏林
蔡斌
刘杰
陈罡
董欣
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Suzhou Galaxy Biopharma 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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

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Abstract

The invention relates to a PDL1 monoclonal antibody and application thereof, belonging to the technical field of immunology. The present invention provides an isolated human specific binding molecule comprising a) and b); a) three light chain CDRs: a light chain CDR1, a light chain CDR2, and a light chain CDR 3; b) three heavy chain CDRs: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR 3; the isolated human PDL 1-specific binding molecule is an isolated antibody or antigen-binding fragment. The PDL1 monoclonal antibody provided by the invention can effectively inhibit local tumor growth; blocking PD1/PDL1 signals can promote the proliferation of tumor antigen specific T cells and play a role in killing tumor cells; blocking the associated PDL1 signal on tumor cells upregulates infiltrating CD8+Secretion of IFN-. gamma.by T cells.

Description

PDL1 monoclonal antibody and application thereof
Technical Field
The invention belongs to the field of tumor treatment and molecular immunology, and relates to a plurality of anti-PDL 1 antibodies, and a pharmaceutical composition and application thereof. In particular, the invention relates to a plurality of monoclonal antibodies against PDL 1.
Background
T cell-mediated cellular immunity plays an important role in recognizing and killing tumor cells, and T cells recognize tumor cells by binding to Major Histocompatibility Complex (MHC) with specific antigens on the surface of the tumor cells through a T Cell Receptor (TCR). The interaction of TCR and MHC molecules is controlled by a series of immune checkpoints, among which co-stimulatory and co-inhibitory signals, can activate or inhibit T cells. Among them, PD1 and its ligand PDL1 pathway are inhibitory immune checkpoints, which combine to transmit co-inhibitory signals, and can suppress the immune activity of T cells, playing an important role in immune tolerance, and also being an important reason for immune escape of tumor cells.
Programmed death receptor 1 (PD 1), an important immunosuppressive molecule. A type I transmembrane protein which is a member of the CD28 superfamily, originally cloned from the apoptotic mouse T cell hybridoma 2B 4.11. The immunoregulation taking PD1 as a target has important significance for resisting tumor, infection, autoimmune disease, organ transplantation survival and the like. PD1 has two binding ligands, PDL1(B7-H1) and PDL2(B7-DC), the expression of which is different, and PDL2 is relatively limited and is mainly expressed on activated macrophages, dendritic cells and a few tumors; PDL1 is widely expressed in activated T cells, B cells, macrophages, dendritic cells and tumor cells, and is also expressed in some immune-shielding parts of the body, such as placenta, eye and its epithelium, muscle, liver and vascular endothelium, so PDL1 has a far greater effect in vivo than PDL 2.
The Programmed cell death ligand 1 (PDL 1), also known as surface antigen differentiation 274 (CD 274) or B7 homolog (B7 homolog 1, B7-H1), is a protein in the human body and is encoded by the CD274 gene.
PDL1 has IgV and IgC-like regions, a transmembrane region, and a cytoplasmic tail, where the cytoplasmic tail is involved in intracellular signal transduction and the IgV and IgC are involved in intercellular signal transduction. It was found that various cytokines such as TNF, IFN-. gamma.IL-4, granulocyte stimulating factor and IL-10 can up-regulate the expression of PDL1 in various cells.
PDL1 interacts with the receptor PD1 on its T cells and plays an important role in the negative regulation of immune responses; the molecule has a wide tissue expression spectrum and high expression on some tumor cell lines, and a plurality of researches show that the molecule is related to an immune escape mechanism of tumors. The microenvironment of the tumor part can induce the expression of PDL1 on the tumor cells, the expression is wide, and the expressed PDL1 is beneficial to the generation and the growth of tumors and induces the apoptosis of anti-tumor T cells.
PD1 can be combined with PDL1 to transmit inhibitory signal, and can inhibit proliferation and activity of lymphocyte, and inhibit CD4+ T cells differentiate to Th1 and Th17 cells, and release of inflammatory cytokines is inhibited, which play a role in immune negative regulation. Under normal conditions, the combination of PDL1 and PD1 can maintain the immune tolerance of peripheral lymphocytes to self-antigens through the above-mentioned actions, thereby preventing the occurrence of autoimmune diseases. However, in the development of tumor, PDL1 expressed by tumor cells can be combined with PD1 to promote the immune escape of tumor through the inhibitory action on lymphocyte.
Mechanisms of tumor immune escape include: the expression of tumor cell membrane surface compatibility complex (MHC) molecules is down-regulated, immune co-stimulation molecules are lacked, immunosuppressive cytokines are secreted, death ligands or inhibitory ligands are expressed, and the like. Many tumor cell lines and tumor cells highly express PDL1 molecules, which, when combined with PD1 molecules on the surface of lymphocytes, impair the body's anti-tumor immune response, thereby leading to the occurrence of tumor immune escape. In addition, the tumor microenvironment is composed of tumor cells, blood vessels, interstitial fluid, and a small number of infiltrating lymphocytes. Some inflammatory cytokines, such as IFN-alpha, IFN-gamma and the like, also exist in the tumor microenvironment, and the cytokines can promote the expression of PDL1 on the surface of tumor cells and play an important role in the immune escape process of tumors.
At present, although the regulation mechanism of PDL1 and PD1 in tumor immune escape is not completely elucidated, the blocking antibodies against PDL1 and PD1 have achieved better therapeutic effect in clinical trials. With the continuous and intensive research, the roles and the regulation mechanism of PDL1 and PD1 in tumor immune escape are more clear, and PDL1 and PD1 become effective anti-tumor therapeutic targets.
Disclosure of Invention
The invention utilizes the PDL1 which is recombined and expressed by a mammalian cell expression system as an antigen to immunize a mouse, and obtains hybridoma cells by fusing mouse spleen cells and myeloma cells. According to the invention, a plurality of hybridoma cell strains are obtained by screening a large number of samples, a specific monoclonal antibody capable of specifically binding to PDL1 is secreted and generated, the monoclonal antibody is further subjected to a chimeric antibody by means of molecular cloning and the like, and the prepared chimeric antibody can bind to PDL1 on the surface of human T cells (figure 1, table III) and has high affinity (table II). A series of characterization and functional experimental studies were performed, showing that these chimeric antibodies are able to bind to activated T cells and DC cells (fig. 2, 3) and are able to block very effectively the binding between PDL1 and PD1 or CD80 (fig. 4, table IV). Species cross-reactivity experiments showed no or minor cross-reactivity of the chimeric antibody with cynomolgus monkeys and mice (figure 5, table V). The results of in vitro functional assays show that these chimeric antibodies are able to promote immune function of T cell lines or primary T cells (fig. 6, 7); animal experiment results show that the pharmacological antibodies can effectively inhibit the growth of tumors in humanized PDL1 knock-in mice (FIG. 8). Finally, these antibodies have been humanized to reduce immunogenicity and to obtain therapeutic antibodies. The following invention is thus provided:
the present invention provides antibodies that specifically bind to human PDL1, which antibodies are produced from hybridoma cell lines. The hybridoma cell line provided by the invention is derived from mouse immunization, cell fusion, and a series of operations such as screening and the like, so that the monoclonal antibody which specifically blocks the combination of PDL1 and PD1/CD80 can be produced. (see example 1)
The present invention also provides chimeric antibodies that specifically bind to human PDL1, wherein the heavy and light chains of the chimeric antibody are constructed by linking the variable region of murine PDL1 antibody to human IgG1 and the kappa constant region. (see example 2)
The chimeric antibodies provided by the invention have high affinity in kinetic assays, wherein the equilibrium binding constant KDIs less than or equal to 3.89x10-10In one aspect of the invention, kinetic parameters are provided that are measured using the biomolecule interaction system Octet-96 (Pall Life Sciences, S-000959). (see example 3)
One aspect of the invention provides a chimeric antibody that binds to a cell surface antigen with an EC50 in the range of 62.6 ng/mL and 437.7 ng/mL; wherein, the term EC50 refers to half maximum effect concentration (concentration for 50% of maximum effect). (see example 4)
In one aspect, the invention provides chimeric antibodies that bind to activated T cells and DC cells. (see example 5)
According to another aspect of the invention, there is provided a chimeric antibody which blocks the binding of PDL1 to PD1 or CD80 with an IC50 in the range of 119.8 ng/mL and 178.5 ng/mL and 608.5 ng/mL and 1867 ng/mL, respectively. Wherein IC50 (half maximum inhibition concentration) refers to the half inhibitory concentration of the antagonist being measured. (see example 6)
In the present invention, if not specifically stated, the CD80 is B7-1; the specific protein sequence is known in the prior art, and can be referred to the sequences disclosed in the prior literature or GenBank. For example, B7-1 (CD80, NCBI Gene ID: 941).
The chimeric antibody provided by the invention has no species cross reaction with mice and has species cross reaction with cynomolgus monkeys. (see example 7)
The chimeric antibody provided by the invention can enhance the immune function of Jurkat cells. (see example 8)
The chimeric antibody provided by the invention has stronger in vitro biological activity. The antibodies of the invention can enhance secretion of IFN-. gamma.in allogeneic and lymphocytic reactions (MLR) (see example 9).
The patent medicine antibody provided by the invention has stronger in-vivo pharmacological activity. In tumor animal models, the antibodies of the invention can effectively inhibit tumor growth and reduce tumor volume. (see example 10)
In one aspect, the invention provides an isolated human specific binding molecule comprising a) and b), a) three light chain CDRs: a light chain CDR1, a light chain CDR2, and a light chain CDR3,
b) three heavy chain CDRs: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR 3;
the isolated human PDL 1-specific binding molecule is an isolated antibody or antigen-binding fragment;
(i) heavy chain CDR1 is selected from: 37, 43, 49, 55, 61, 67, 73, 79, or 85;
(ii) heavy chain CDR2 is selected from: 38, 44, 50, 56, 62, 68, 74, 80, or 86;
(iii) heavy chain CDR3 is selected from: 39, 45, 51, 57, 63, 69, 75, 81, or 87;
(iv) the light chain CDR1 is selected from: 40, 46, 52, 58, 64, 70, 76, 82, or 88 SEQ ID NO;
(v) the light chain CDR2 is selected from: 41, 47, 53, 59, 65, 71, 77, 83, 9 or 89 SEQ ID NO; and
(vi) the light chain CDR3 is selected from: 42, 48, 54, 60, 66, 72, 78, 84, or 90.
In another aspect of the present invention there is provided an isolated human specific binding molecule comprising a heavy chain variable region and a light chain variable region, wherein:
(i) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 37, heavy chain CDR1 as set forth in SEQ ID NO: 38, and the heavy chain CDR2 as set forth in SEQ ID NO: 39, the heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 40, light chain CDR1 as set forth in SEQ ID NO: 41, and a light chain CDR2 as set forth in SEQ ID NO: 42, a light chain CDR 3; or
(ii) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 43, heavy chain CDR1 as set forth in SEQ ID NO: 44, and a heavy chain CDR2 as set forth in SEQ ID NO: 45, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 46, light chain CDR1 as set forth in SEQ ID NO: 47, and a light chain CDR2 as set forth in SEQ ID NO: 48, a light chain CDR 3; or
(iii) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 49, heavy chain CDR1 as set forth in SEQ ID NO: 50, and the heavy chain CDR2 as set forth in SEQ ID NO: 51, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 52, light chain CDR1 as set forth in SEQ ID NO: 53, and the light chain CDR2 as set forth in SEQ ID NO: 54, a light chain CDR 3; or
(iv) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 55, heavy chain CDR1 as set forth in SEQ ID NO: 56, and the heavy chain CDR2 as set forth in SEQ ID NO: heavy chain CDR3 shown in 57; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 58, light chain CDR1 as set forth in SEQ ID NO: 593, and a light chain CDR2 as set forth in SEQ ID NO: 60, a light chain CDR 3; or
(v) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 61, heavy chain CDR1 as set forth in SEQ ID NO: 62, and the heavy chain CDR2 as set forth in SEQ ID NO: 63, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 64, light chain CDR1 as set forth in SEQ ID NO: 65, and a light chain CDR2 as set forth in SEQ ID NO: 66, a light chain CDR 3; or
(vi) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 67, heavy chain CDR1 as set forth in SEQ ID NO: 68, and a heavy chain CDR2 as set forth in SEQ ID NO: 69, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 70, light chain CDR1 as set forth in SEQ ID NO: 71 and a light chain CDR2 as set forth in SEQ ID NO: 72, the light chain CDR 3; or
(vii) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 73, the heavy chain CDR1 as shown in SEQ ID NO: 74, and a heavy chain CDR2 as set forth in SEQ ID NO: 75 heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 76, light chain CDR1 as set forth in SEQ ID NO: 77, and a light chain CDR2 as set forth in SEQ ID NO: 78, a light chain CDR 3; or
(viii) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 79, as set forth in SEQ ID NO: 80, and the heavy chain CDR2 as set forth in SEQ ID NO: (ii) the heavy chain CDR3 shown in 81; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 82, light chain CDR1 as set forth in SEQ ID NO: 83 and a light chain CDR2 as set forth in SEQ ID NO: 84, the light chain CDR 3; or
(ix) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 85, as shown in SEQ ID NO: 86 and the heavy chain CDR2 as set forth in SEQ ID NO: 87, heavy chain CDR 3; and/or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 88, light chain CDR1 as set forth in SEQ ID NO: 89, and a light chain CDR2 as set forth in SEQ ID NO: 90, light chain CDR 3.
In some embodiments of the invention, the antibody is a full-length antibody.
In some embodiments of the invention, the antigen binding fragment is Fab or F (ab)'2Or a scFv.
In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein:
(i) the heavy chain variable region is selected from: 19, 21, 23, 25; 27, 29, 31, 33, and 35;
(ii) the light chain variable region is selected from: 20, 22, 24, 26; 28, 30, 32, 34, and 36;
(iii) (iii) a sequence having at least 80% homology to the sequences recited in (i) and (ii).
In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein:
(i) the heavy chain variable region is SEQ ID NO 19 and the light chain variable region is SEQ ID NO 20;
(ii) the heavy chain variable region is SEQ ID NO 21 and the light chain variable region is SEQ ID NO 22;
(iii) the heavy chain variable region is SEQ ID NO:23 and the light chain variable region is SEQ ID NO: 24;
(iv) the heavy chain variable region is SEQ ID NO. 25 and the light chain variable region is SEQ ID NO. 26;
(v) the heavy chain variable region is SEQ ID NO 27 and the light chain variable region is SEQ ID NO 28;
(vi) the heavy chain variable region is SEQ ID NO:29 and the light chain variable region is SEQ ID NO: 30;
(vii) the heavy chain variable region is SEQ ID NO 31 and the light chain variable region is SEQ ID NO 32;
(viii) the heavy chain variable region is SEQ ID NO 33 and the light chain variable region is SEQ ID NO 34; and
(ix) the heavy chain variable region is SEQ ID NO 35 and the light chain variable region is SEQ ID NO 36.
In some embodiments of the invention, the antibody, or antigen-binding fragment thereof, specifically binds human PDL1 and exhibits at least one of the following properties:
(i) block binding of PDL1 to PD1 or CD 80;
(ii) PDL1 that binds to the surface of human T cells;
(iii) at 3.89X10-10 K of M or lessDIn conjunction with human PDL 1;
(iv) IFN-gamma production is increased in a Mixed Lymphocyte Reaction (MLR) assay.
In some embodiments, the monoclonal antibody, or antigen binding portion thereof, is used in the preparation of a medicament for inhibiting tumor cell growth in a patient.
In some embodiments, the monoclonal antibody or antigen binding portion thereof for the preparation of a medicament for the treatment of infectious diseases.
In one aspect, the invention provides an isolated nucleic acid encoding one or both of an antibody light chain variable region and an antibody heavy chain variable region, wherein
(i) The fragment encoding the variable region of the antibody heavy chain is selected from the group consisting of: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
(ii) the fragment encoding the variable region of the antibody light chain is selected from the group consisting of: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(iii) (iii) a sequence having at least 80% homology to the sequences recited in (i) and (ii).
The invention also provides a vector containing the nucleic acid molecule.
The invention also provides a host cell containing the nucleic acid molecule or the vector.
The present invention also provides a conjugate comprising an anti-human PDL1 monoclonal antibody covalently linked to an isotope, immunotoxin and/or chemical drug; the anti-human PDL1 monoclonal antibody is the isolated human PDL1 specific binding molecule.
The invention also provides a conjugate formed by coupling the isolated human PDL1 specific binding molecule and/or the conjugate with a solid medium or a semi-solid medium.
The invention also relates to the application of the anti-PDL 1 monoclonal antibody and/or the conjugate in the preparation of drugs for treating diseases;
the disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma or melanoma.
The present invention provides a composition comprising a humanized monoclonal antibody or antigen-binding portion thereof against human PDL1, a nucleic acid molecule, a vector, a host cell, a conjugate, or a conjugate, and optionally a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances.
The invention also relates to a kit comprising said isolated antibody or antigen-binding fragment thereof and a set of reagents for detecting a complex of said antibody or said antigen-binding fragment bound to human PDL 1.
The invention is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).
The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PDL 1). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')2Fragments, i.e. bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) consisting of VH domainsA dAb fragment; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined together using recombinant methods by a synthetic linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule known as single chain Fv (scfv). Such single chain antibodies are also included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained by conventional techniques well known to those skilled in the art and are screened for utility in the same manner as are intact antibodies.
As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PDL1 is substantially free of antibodies that specifically bind to antigens other than PDL 1). However, isolated antibodies that specifically bind to PDL1 may be cross-reactive with other antigens, such as PDL1 molecules from other species. Moreover, the isolated antibody may be substantially free of other cellular material and/or chemicals.
The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.
The term "human antibody" as used herein includes antibodies having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the invention may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity, which has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant methods, for example: (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) for human immunoglobulin genes or hybridomas prepared therefrom (described further below), (b) antibodies isolated from transformed human antibody-expressing host cells, such as transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other method including splicing of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when transgenic animals with human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, while sequences derived from and related to human germline VH and VL sequences, may not naturally occur in vivo in all of the components of the human antibody germline (reportire).
The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Other framework region modifications may also be made within the human framework sequence.
The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, for example, an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
The term "K" as used hereinassoc"or" Ka"refers to the binding rate of a particular antibody-antigen interaction, as used hereinThe term "K" as used hereindis"or" Kd"refers to the off-rate of a particular antibody-antigen interaction. The term "K" as used hereinD"refers to the dissociation constant, which is represented by KdAnd KaObtained by the ratio of (i.e. K)d/Ka) And is expressed as molar concentration (M). K of antibodyDValues may be determined using methods established in the art. Determination of antibody KDA preferred method of (a) is to use a surface plasmon resonance method, preferably using a biosensor system.
"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences when aligned most preferably.
An "isolated nucleic acid molecule" means a genomic DNA or RNA, mRNA, cDNA, or synthetic sources or some combination thereof that is not associated with all or a portion of a polynucleotide, wherein the isolated polynucleotide is found in nature or linked to a polynucleotide not linked in nature.
The PDL1 monoclonal antibody provided by the invention can effectively inhibit local tumor growth; blocking PD1/PDL1 signals can promote the proliferation of tumor antigen specific T cells and play a role in killing tumor cells; blocking the associated PDL1 signal on tumor cells upregulates infiltrating CD8+Secretion of IFN- γ by T cells, indicating that blockade of the PD1/PDL1 signaling pathway plays a role in tumor immune responses for the purpose of inducing immune responses; the anti-PDL 1 monoclonal antibody is selected to be matched with a tumor vaccine for tumor immunotherapy, so that the immune activation of the tumor vaccine can be effectively strengthened. At present, the anti-PD 1/PDL1 treatment is in the front of immunotherapy with good curative effect and safety, and becomes a hot target in the field of lung cancer treatment in recent two years.
Drawings
FIG. 1: FACS detection of binding of PDL1 chimeric antibody to B16-hPDL1 cells;
FIG. 2: FACS detection of binding of PDL1 chimeric antibody to activated T cells;
FIG. 3: FACS detecting binding of PDL1 chimeric antibody to DC cells;
FIG. 4 a: ELISA detection of PDL1 chimeric antibody blocking the interaction between PD1 and PDL 1;
FIG. 4 b: ELISA detection of PDL1 chimeric antibody blocking the interaction between CD80 and PDL 1;
FIG. 5 a: detecting cross reaction of PDL1 chimeric antibody and cynomolgus monkey PDL1 species by ELISA;
FIG. 5 b: FACS detection of PDL1 chimeric antibody cross-reacting with mouse PDL1 species;
FIG. 6: the luciferase detection kit detects the influence of the PDL1 chimeric antibody on the immune response of Jurkat cells;
FIG. 7: ELISA detects that PDL1 chimeric antibody enhances IFN-gamma secretion in MLR experiment;
FIG. 8: PDL1 druggable antibodies inhibited tumor growth in PDL1 humanized transgenic mice.
Fig. 9 a-9 c: table one, sequence numbering for the anti-PDL 1 antibody of the invention;
FIG. 10: table two, kinetic analysis table of human murine PDL1 chimeric antibody;
FIG. 11: table three, cell binding half-effective concentration table for human murine PDL1 chimeric antibody;
FIG. 12: table four, ligand blocking experimental table of human murine PDL1 chimeric antibody;
FIG. 13: table five, cross-reaction table of human murine PDL1 chimeric antibody.
Detailed Description
The invention discloses a monoclonal antibody and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein may be made and equivalents employed without departing from the spirit and scope of the invention.
The monoclonal antibody and the raw materials and reagents used in the application thereof provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: animal immunization and screening for anti-PDL 1 murine antibodies
Balb/c mice with proper age are selected for immune injection. hPDL1-mFc fusion protein was used as antigen mixed with complete Freund's adjuvant (Sigma-Aldrich) and injected subcutaneously into immunized mice to stimulate the corresponding B lymphocyte clones. Subsequently, the immunized mice were boosted by intraperitoneal injection of 100 μ g of 1:1 hPDL1-mFc emulsified in incomplete Freund's adjuvant (Sigma-Aldrich) approximately every two to three weeks. The splenic lymphocytes of the mice are taken out by aseptic technique and are mixed with the prepared SP2/0 myeloma cells according to a certain proportion (splenic cells 1x 10)8Myeloma cell 2x107) Mix and add polyethylene glycol (Sigma, P7181) for cell fusion.
After fusion, adding the fused cells into a 96-well plate, adding 0.1 mL HAT culture medium into each well, putting into a carbon dioxide incubator, and culturing at 37 ℃; on day 4, 0.1 mL of HT medium was added to each well; on day 7, the medium was completely changed to HT medium. Screening was performed on days 8-12, positive cells were subcloned by limiting dilution, expanded and tested by ELISA and FACS as follows:
2 μ g/mL of hPDL1-His were coated onto 96-well plates (Corning, catalog # 9018) and incubated overnight at 4 ℃. Plates were washed three times with PBST (0.05% Tween-20 in PBS) and blocked with 5% skim milk powder (Oxoid, catalog # LP 0031B). After washing the plates three times with PBST, hybridoma supernatants were added and incubated for 2 hours at 37 ℃. After washing, 1/5000 diluted secondary HRP-labeled goat anti-mouse IgG antibody (BioLegend, catalog # 405306) was added and incubated at 37 ℃ for 1 hour. After washing the plate, TMB (Tiangen, catalog # PA 107-02) was added for color development.
Binding of the antibody to B16-hPDL1 cells (endogenous mouse PDL1 gene knockout) was detected by FACS. 2.5X10 after trypsinization was added to each well of a 96-well plate (Corning, catalog # 3799)5B16-hPDL1 cells were incubated with hybridoma supernatants at 4 ℃ for 30 min. After washing the cells twice with buffer (PBS containing 1% BSA and 5 mM EDTA), APC-labeled secondary goat anti-mouse IgG antibody (BD, catalog) was added# 550826), incubated at 4 ℃ for 30 minutes. Cells were washed with buffer and analyzed on an Attune Nxt flow cytometer (Thermo Fisher).
The PDL1 antibody was able to block the binding of PDL1 to PD1, and therefore the hybridoma supernatants were further screened using a ligand blocking assay. A96-well plate was coated with 1.5. mu.g/mL of hPD1-hFc at 4 ℃ overnight. After washing the plates 3 times, they were blocked with 5% skimmed milk powder at 37 ℃ for 2 hours. After washing the plate three times, the pre-mixed hybridoma supernatant and 200 ng/mL biotinylated hPDL1-hFc were added and incubated for 1 hour at room temperature. After washing, HRP-labeled streptavidin was added and incubated at room temperature for 1 hour. After washing the plate, TMB was added for color development.
PDL1 antibody was able to block binding of PDL1 to CD80, and therefore hybridoma supernatants were further screened using a ligand blocking assay. A96-well plate was coated with 2. mu.g/mL of hCD80-hFc at 4 ℃ overnight. After washing the plates 3 times, they were blocked with 5% skimmed milk powder at 37 ℃ for 2 hours. After washing the plate three times, the pre-mixed hybridoma supernatant and 1000 ng/mL biotinylated hPDL1-hFc were added and incubated for 1 hour at room temperature. After washing, HRP-labeled streptavidin was added and incubated at room temperature for 1 hour. After washing the plate, TMB was added for color development.
Cross-reactivity of PDL1 antibody with cynomolgus monkey PDL1 was examined using ELISA methods. mu.g/mL of anti-mouse IgG Fc antibody (Sigma, catalog # M4280) was coated onto 96-well plates overnight at 4 ℃. 5% skim milk powder was used for 2 hours at 37 ℃. After washing the plates with PBST, hybridoma supernatants were added and incubated for 30 minutes at room temperature. After three PBST washes, biotin-labeled cynomolgus monkey PDL1 (SinoBiological, catalog # 90251-C08H) was added to the plates and incubated at room temperature for 1 hour. The plates were washed three times with PBST, 1/1000 diluted HRP-labeled streptavidin was added, and incubated for 1 hour at room temperature. After washing the plate, TMB was added for color development.
Example 2: cloning of murine antibody cDNA and construction of chimeric antibody
The variable region gene sequences of the heavy and light chains of hybridoma antibodies were obtained by degenerate primer PCR. Hybridoma monoclonal cells were lysed using Trizol (Invitrogen, catalog # 15596-. Using the resulting cDNA library as a template, PCR was performed using degenerate primers (Zhou H, et al, Nucleic Acids Research 22: 888-889 (1994), Chardes T et al, FEBS Letters 452: 386-394 (1999)). The PCR products were detected by agarose gel electrophoresis and the PCR amplification products for the heavy and light chain variable regions were predicted to be 400 base pairs in size. The PCR products were cloned into pClone007 vector (Tsingke, catalog # TSV-007 BS) and transformed into E.coli competent Transs 5 α (Transgen, catalog # CD 201-02) and 3-6 E.coli single clones were picked on agar plates for gene sequencing. In some cases, the PCR product can also be used directly for gene sequencing. Through the method, the full-length gene sequences of the variable regions of the heavy chain and the light chain of the antibody are finally obtained. The genes and amino acid Sequences of the Complementarity Determining regions (complementary Determining Region) and Framework regions (Framework Region) of the heavy and light chains of an antibody were obtained after further analysis by NCBI Ig-BLAST (https:// www.ncbi.nlm.nih.gov/projects/igblast /) (Kabat E.A., et al, 1991, Sequences of proteins of immunological interest in NIH Publication number 91-3242, US Department of Health and Human Services, Bethesda, Md.). For specific information see antibody sequence listing and FIGS. 9 a-9 c (Table I).
The heavy and light chains of the chimeric antibody were constructed by linking the heavy and light chain variable regions of murine PDL1 antibody to the human IgG1 (or IgG1 mutant N297A) and kappa constant regions, respectively. Introducing proper enzyme cutting sites into the heavy chain and light chain variable regions by a PCR method, and respectively cloning into corresponding chimeric antibody expression vectors. Plasmids of the heavy and light chains of the chimeric antibody were introduced into 293F cells by lipofection, and culture was continued for 6 days. Antibodies in cell culture supernatants were purified using Protein A columns (GE Healthcare, catalog # 17549112). The Protein A column was washed with 1 mM PBS, and then the antibody was eluted from the column with 50 mM PBS (pH 3.0). The eluate was adjusted to neutral pH with 0.5M sodium hydroxide solution and filtered through a 0.22 μ M filter. The antibody solution was concentrated by Ultra-15 centrifugal concentrators (Millipore, catalog # ACS 500024) and the antibody concentration was quantified by Nanodrop spectrophotometer. Endotoxin content in the purified antibody solution was measured using a Gel Clot TAL kit (Xiaomen Bioendo Technology, catalog # 010250) with a standard of less than 1 EU/mL.
Example 3: kinetic detection of human murine PDL1 chimeric antibody
The kinetic constants (k) for the binding between antibody and antigen were measured using the biomolecular interaction system Octet-96 (Pall Life Sciences, S-000959) assoc And k dissoc ) And further calculating the equilibrium binding constant K D . hPDL1-mFc antigen protein was coupled to the surface of AMC sensor (Pall Life Sciences, PN 18-5099) and binding and dissociation between PDL1 chimeric antibody and sensor surface PDL1-mFc protein was measured by adding different concentrations of antibody. Specifically, AMC sensors were pre-wetted in buffer (PBS containing 0.02% Tween-20 and 0.1% BSA) for 10 minutes, then equilibrated in sample buffer of hPDL1-mFc for 5 minutes, such that PDL1-mFc protein was coupled to the sensor surface. PDL1-mFc coupled AMC sensors were first equilibrated in buffer for 2 minutes and then incubated in buffers containing different concentrations of antibody (3-200 nM) for 5 minutes to measure binding of the antibody to PDL1-mFc protein; finally, the sensor bound to antigen and antibody was replaced in sample buffer and allowed to wait 10 minutes to measure the dissociation of the antibody from the hPDL1-mFc protein. The measured data were obtained using a 1:1 overall fitting the model to obtain the binding and dissociation kinetic constants, and further obtaining the equilibrium binding constant K on the basis of the kinetic constants D . The results of the experiment are shown in FIG. 10 (Table II).
Example 4: binding of human murine PDL1 chimeric antibody to B16-hPDL1 cells
The human murine chimeric antibody was further tested for binding to cell surface hPDL1 using FACS method. B16-hPDL1 cells (endogenous mouse PDL1 gene knockout) were resuspended at 4X10 in FACS buffer (1% BSA in PBS)6cells/mL, 50. mu.L/well were added to a 96-well round bottom plate (Corning, catalog # 3795). The purified antibody to be tested was added to the wells at a concentration (50. mu.L)Perwell), incubated at 4 ℃ for 1 hour. After the cells were washed three times with buffer, PE-labeled fluorescent secondary antibody was added and incubated at 4 ℃ for 0.5 hour. Cells were washed three times with FACS buffer, resuspended to 200 μ L/well, and the fluorescence signal detected using an Attune Nxt flow cytometer (Thermo Fisher). The results are shown in FIG. 1, with the binding half-effective concentrations shown in FIG. 11 (Table III).
Example 5: binding of human murine PDL1 chimeric antibody to activated T cells and DC cells
The human murine chimeric antibody was further tested for binding to activated T cells and hPDL1 on the surface of dendritic cells using FACS method. Peripheral Blood Mononuclear Cells (PBMC) were isolated from human peripheral blood using density gradient centrifugation (Ficoll-Paque Premium, GE Healthcare, catalog # 17-5442-02). Human CD3+ T cells were isolated from PBMC using the Pan T Cell Isolation Kit (Miltenyi, catalog # 130-096-535) and activated for 48 hours by adding an equal amount of CD3/CD28 Dynabeads (Invitrogen 11132D). Activated T cells were resuspended in FACS buffer (1% BSA in PBS) to 4X106cells/mL, 50. mu.L/well were added to a 96-well round bottom plate (Corning, catalog # 3795). The purified antibody to be tested was added to the wells at a certain concentration (50. mu.L/well) and incubated at 4 ℃ for 1 hour. After the cells were washed three times with buffer, PE-labeled fluorescent secondary antibody was added and incubated at 4 ℃ for 0.5 hour. Cells were washed three times with FACS buffer, resuspended to 200 μ L/well, and the fluorescence signal detected using an Attune Nxt flow cytometer (Thermo Fisher). The results are shown in FIG. 2.
Mononuclear cells (monocyte) were obtained from PBMC by further Isolation and purification using CD14 Cell Isolation Kit (Miltenyi, catalog # 130-. Differentiation of monocytes into Dendritic cells (Dendritic cells) was induced by adding the cytokines 1000U/mL GM-CSF (Prospec, catalog # CYT-221) and 1000U/mL IL-4 (Prospec, catalog # CYT 211) to the medium. Cytokines were replenished every 2-3 days and immature dendritic cells were harvested after 5-6 days. Dendritic cells were resuspended to 4 × 10 using FACS buffer (1% BSA in PBS)6cells/mL, 50. mu.L/well were added to a 96-well round bottom plate (Corning, catalog # 3795). The purified antibody to be tested was prepared according toA concentration was added to the wells (50. mu.L/well) and incubated at 4 ℃ for 1 hour. After the cells were washed three times with buffer, PE-labeled fluorescent secondary antibody was added and incubated at 4 ℃ for 0.5 hour. Cells were washed three times with FACS buffer, resuspended to 200 μ L/well, and the fluorescence signal detected using an Attune Nxt flow cytometer (Thermo Fisher). The results are shown in FIG. 3.
Example 6: human murine PDL1 chimeric antibody blocks the interaction between PD1 or CD80 and PDL1
The antibodies were tested for ability to block hPD1 or the binding between CD80 and hPDL1 by ELISA. Use of Biotin Labeling Kit-NH2(Dojindo, catalog # LK 03) kit biotin-labeled hPDL1-hFc protein. hPD1-hFc or hCD80-hFc was coated onto 96-well plates overnight at 4 ℃. 5% skim milk powder was used for 2 hours at 37 ℃. After washing the plate three times, a pre-mix of biotinylated hPDL1-hFc and PDL1 chimeric antibody was added and incubated for 1 hour at room temperature. The plate was washed with PBST, HRP-labeled streptavidin was added, and the incubation was performed at room temperature for 1 hour. After washing the plate, TMB was added for color development. The results are shown in FIGS. 4a and 4b, and a summary of the results is shown in FIG. 12 (Table IV).
Example 7: species cross-reactivity of human murine PDL1 chimeric antibody
The cross-reactivity of the human murine PDL1 chimeric antibody with cynomolgus monkey species was detected using ELISA method. Quantitatively purified antibody was coated onto 96-well plates overnight at 4 ℃. 5% skim milk powder was used for 2 hours at 37 ℃. Biotin-labeled cynomolgus monkey PDL1 (SinoBiological, catalog # 90251-C08H-200) was added to the plate and incubated at room temperature for 1 hour. The plates were washed three times with PBST, 1/1000 diluted HRP-labeled streptavidin was added, and incubated for 1 hour at room temperature. After washing the plate, TMB was added for color development. The results are shown in FIG. 5a, and FIG. 13 (Table V).
Species cross-reactivity of the human murine PDL1 chimeric antibody with the mouse was detected using FACS method. Wild type B16 cells were trypsinized and resuspended to 1.25X10 in FACS buffer (1% BSA in PBS)6cells/mL. 80 μ L of the cell suspension was mixed with 80 μ L of the antibody diluted in a gradient and allowed to stand at room temperature for 30 minutes. After three PBST washes, a fluorescently labeled secondary antibody (BioLegend,catalog # 409304), incubated at 4 ℃ for 1 hour. After PBST washing, the fluorescence signal was detected using an Atture Nxt flow cytometer (Thermo Fisher). The results are shown in FIG. 5b, and FIG. 13 (Table V).
Example 8: human mouse PDL1 chimeric antibody for enhancing immune function of Jurkat cells
Jurkat-PD1 cells containing a luciferase reporter gene were used to test the cellular biological activity of the chimeric human murine PDL1 antibody. In this cell assay, Raji/hPDL1 cells served as antigen presenting cells and Jurkat/hPD 1/NF-. kappa.B-luciferase cells served as effector cells. The addition of the chimeric antibody of human murine PDL1 blocks the interaction between cells PD1/PDL1, and activates Jurkat/hPD1/NF kappa B-Luciferase cells to express Luciferase reporter genes. Specifically, by introducing exogenously expressed hPD1 and a Luciferase reporter gene containing the NF-. kappa.B promoter into Jurkat cells; and exogenously expressed hPDL1 was introduced into Raji cells. Raji/hPDL1 cells and Jurkat/hPD1/NF kappa B-luciferase cells were mixed, and T cell receptor activator and gradient diluted antibody to be detected were added, and detection was performed after 6 to 16 hours using luciferase assay kit (Promega, G7940). The results show that the human murine PDL1 chimeric antibody is able to enhance the fluorescence signal and exhibit a dose-dependent effect, and the results are shown in fig. 6.
Example 9: allogeneic Mixed Lymphocyte experiment (Mixed Lymphocyte cell) of human murine PDL1 chimeric antibody Reaction, MLR）
The in vitro biological activity of the human murine PDL1 chimeric antibody was examined using a mixed lymphocyte reaction. Peripheral Blood Mononuclear Cells (PBMC) were isolated from human peripheral blood using density gradient centrifugation (Ficoll-Paque Premium, GE Healthcare, catalog # 17-5442-02). Mononuclear cells (monocyte) were obtained from PBMC by further Isolation and purification using CD14 Cell Isolation Kit (Miltenyi, catalog # 130-. Differentiation of monocytes into Dendritic cells (Dendritic cells) was induced by adding the cytokines 1000U/mL GM-CSF (Prospec, catalog # CYT-221) and 1000U/mL IL-4 (Prospec, catalog # CYT 211) to the medium. Cytokines were replenished every 2-3 days and dendritic cells were harvested after 5-6 days for subsequent experiments.
Human CD3+T cells were isolated from PBMC using the Pan T Cell Isolation Kit (Miltenyi, catalog # 130-096-535). CD3 was added to a 96-well round bottom plate (Corning, catalog # 3799)+T cells and immature dendritic cells, and varying concentrations of PDL1 antibody. After 5-6 days of cell culture, the supernatant was harvested and the cytokine content was quantitatively determined by ELISA. The results are shown in FIG. 7, that the chimeric human murine PDL1 antibody was able to enhance the secretion of IFN-. gamma.in MLR.
Example 10: human and mouse PDL1 chimeric antibody for inhibiting tumor growth
The tumor animal model is prepared by inoculating genetically modified mouse colon cancer cell MC38/mPDL1 in PDL1 humanized transgenic mouseKOhPDL1, a specific tumor animal model constructed to test in vivo efficacy of the druggable PDL1 antibody in mice.
PDL1 humanized transgenic mice (7-11 weeks old) were injected subcutaneously on the body side with a 0.1 mL volume of 5X105MC38/mPDL1KOhPDL1 tumor cells. 7 days after tumor cell inoculation, the PDL1 antibody, reference antibody, or control PBS was injected twice weekly at a dose of 10 mg/kg (dose volume 10 mL/kg) for a total of 6 doses. During the experiment, subcutaneous tumor volumes were measured twice weekly using a vernier caliper. As shown in FIG. 8, after 6 times of administration, the antibody to be tested significantly inhibited tumor growth, and some tumors almost disappeared after the drug treatment.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Suzhou Galaxy biomedical Co., Ltd
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<211> 355
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.355)
<223> heavy chain variable region DNA sequence
<400> 7
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgttctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggaactggat 660
ccggaaattc 720
ccagggaata 780
aacttgagta 840
catggggttc 900
ataagctaca 960
ctggtagcac 1020
ttactacaat 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacacat 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactac 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
catggcggga 1800
tggttagcct 1860
ggtttgctta 1920
ctggggccaa 1980
gggactctgg 2040
tcactgtctc 2100
tgcag 391
<210> 8
<211> 339
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223> light chain variable region DNA sequence
<400> 8
gacattgtga 60
tgtcacagtc 120
tccatcctcc 180
ctagctgtgt 240
cagttggaga 300
gaaggttact 360
atgagctgca 420
agtccagtca 480
gagcctttta 540
tatagtagca 600
atcaaaagaa 660
ctccttggcc 720
tggtaccagc 780
agaaaccagg 840
gcagtctcct 900
aaactgctga 960
tttactgggc 1020
atccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
ggacagattt 1380
cactctcacc 1440
atcagcagtg 1500
tgaaggctga 1560
agacctggca 1620
gtttattact 1680
gtcagcaata 1740
ttatagctat 1800
ccgtggacgt 1860
tcggtggagg 1920
caccaagctg 1980
gaaatcaaa 373
<210> 9
<211> 355
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.355)
<223> heavy chain variable region DNA sequence
<400> 9
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgttctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggcactggat 660
ccggaaattc 720
cccgggaata 780
aacttgagta 840
catggggtac 900
ataagctaca 960
gtgggagcac 1020
ttacttcatt 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacacat 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactac 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
atcaggggga 1800
tggttactgc 1860
attttgctta 1920
ctggggccaa 1980
gggactctgg 2040
tcactgtctc 2100
tgcag 391
<210> 10
<211> 339
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223> light chain variable region DNA sequence
<400> 10
gacgttgtga 60
tgtcacagtc 120
tccatcctcc 180
ctagctgtgt 240
cagttggaga 300
gaaggttact 360
atgagctgca 420
agtccagtca 480
gagcctttta 540
tatagtagca 600
atcaaaagaa 660
ctccttggcc 720
tggtaccagc 780
agaaaccagg 840
gcagtctcct 900
aaactgctga 960
tttactgggc 1020
atccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
ggacagattt 1380
cactctcacc 1440
atcagcaatg 1500
tgaagactga 1560
agacctggca 1620
gtttattact 1680
gtcagcaata 1740
ttatggctat 1800
ccttacacgt 1860
tcggaggggg 1920
gaccaagctg 1980
gaaataaaa 373
<210> 11
<211> 349
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.349)
<223> heavy chain variable region DNA sequence
<400> 11
gaagtgatgc 60
tggtggagtc 120
tgggggcggc 180
ttagtgaagc 240
ctggagggtc 300
cctgaaactc 360
tcctgtgcag 420
tctctggatt 480
cactttcagt 540
agctatgcca 600
tgtcttgggt 660
tcgccagact 720
ccggaaaaga 780
ggctggagtg 840
ggtcgcaacc 900
attactagtg 960
atggtcatta 1020
cacctactat 1080
ccagacaatg 1140
tgaaggggcg 1200
attcaccatc 1260
tccagagaca 1320
atgccaagaa 1380
caccctctac 1440
ctgcaaatga 1500
gcagtctgag 1560
gtctgaggac 1620
acggccatgt 1680
attactgtac 1740
aagacgtacg 1800
aactactttg 1860
actactgggg 1920
ccaaggcacc 1980
actctcacag 2040
tctcctcag 384
<210> 12
<211> 315
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.315)
<223> light chain variable region DNA sequence
<400> 12
caaattgttc 60
tcacccagtc 120
tccagcactc 180
atgtctgcat 240
atccagggga 300
gaaggtcacc 360
atgacctgca 420
gtgccaactc 480
aagtgttact 540
tacatgtatt 600
ggtaccagca 660
gaagccaaga 720
tcctccccca 780
aaccctggat 840
ttttctcaca 900
tccaacctgg 960
cttctggagt 1020
ccctgctcgc 1080
ttcagtggca 1140
gtgggtctgg 1200
gacctcttac 1260
tctctcacaa 1320
tcagcagcat 1380
ggaggctgaa 1440
gatgctgcca 1500
cttattactg 1560
ccagcagtgg 1620
agaagtaacc 1680
cgacgttcgg 1740
tggaggcacc 1800
aagctggaaa 1860
tcaaa 347
<210> 13
<211> 358
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.358)
<223> heavy chain variable region DNA sequence
<400> 13
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgctctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggaactggat 660
ccggaaattc 720
ccagggaata 780
aacttgagta 840
catggggtac 900
ataagctaca 960
ctggtagcac 1020
ttactacaat 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacacat 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactac 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
atatagagac 1800
tgggtcgtcg 1860
gctactttga 1920
ctactggggc 1980
caaggcacca 2040
ctctcacagt 2100
ctcctcag 394
<210> 14
<211> 321
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.321)
<223> light chain variable region DNA sequence
<400> 14
gactttgtga 60
tgacccagtc 120
tcaaaaattc 180
atgtccacat 240
cagtgggaga 300
cagggtcagc 360
gtcacctgca 420
aggccagtca 480
gaatgtgggt 540
actaatgtag 600
cctggtatca 660
acagaaacca 720
ggacaatctc 780
ctaaagcact 840
gatttactcg 900
gcatcctacc 960
ggtacagtgg 1020
agtccctgat 1080
cgcttcacag 1140
gcagtggatc 1200
tgggacagat 1260
ttcactctca 1320
ccatcagcaa 1380
tgtgcagtct 1440
gaagacttgg 1500
cagaatattt 1560
ctgtcagcaa 1620
tataacagct 1680
atcctctcac 1740
gttcggctcg 1800
gggacaaagt 1860
tggaaataaa 1920
a 354
<210> 15
<211> 355
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.355)
<223> heavy chain variable region DNA sequence
<400> 15
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgttctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggaactggat 660
ccggaaattc 720
ccagggaata 780
aacttgagta 840
catggggtac 900
ataagctaca 960
ctggtagtac 1020
ttactacaat 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacactt 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactgc 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
aaggggggga 1800
tggttactgc 1860
cttttgacta 1920
ctggggccaa 1980
ggcaccactc 2040
tcacagtctc 2100
ctcag 391
<210> 16
<211> 339
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223> light chain variable region DNA sequence
<400> 16
gacattgtga 60
tgacacagtc 120
tccatcctcc 180
ctgactgtga 240
cagcaggaga 300
gaaggtcact 360
atgagctgca 420
agtccagtca 480
gagtctgtta 540
aacagtggaa 600
atcaaaagaa 660
ctgcttgacc 720
tggtaccagc 780
agaaaccagg 840
gcagcctcct 900
aaactgttga 960
tctcctgggc 1020
ttccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
gaacagattt 1380
cactctcacc 1440
atcagcagtg 1500
tgcaggctga 1560
agacctggca 1620
gtttattact 1680
gtcagaatga 1740
ttatggttat 1800
ccgctcacgt 1860
tcggtgctgg 1920
gaccaagctg 1980
gagctgaaa 373
<210> 17
<211> 367
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.367)
<223> heavy chain variable region DNA sequence
<400> 17
gaggttcagc 60
tgcagcagtc 120
tggggcagag 180
cttgtgaagc 240
caggggcctc 300
agtcaaattg 360
tcctgcacag 420
cttctggctt 480
caacattaaa 540
gacacctata 600
tgtactgggt 660
gaagcagagg 720
cctgaacagg 780
gcctggagtg 840
gattggaagg 900
attgatcctg 960
cgaatggtaa 1020
tactaaatat 1080
gacccgaagt 1140
tccagggcaa 1200
ggccactata 1260
acagcagaca 1320
catcctccaa 1380
cacagccttc 1440
ttgcagctca 1500
gcagcctgac 1560
atctgaggac 1620
actgccgtct 1680
attactgtgc 1740
taggagggga 1800
ttatttttta 1860
ctacggtaac 1920
agctattgac 1980
tactggggcc 2040
aaggcaccac 2100
tctcacagtc 2160
tcctcag 404
<210> 18
<211> 321
<212> DNA
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.321)
<223> light chain variable region DNA sequence
<400> 18
gacattgtga 60
tgacccagtc 120
tcacaaattc 180
atgtccacat 240
cagtaggaga 300
cagggtcagc 360
attacctgca 420
aggccagtca 480
ggatgtgagt 540
actgctgtag 600
cctggtatca 660
acaaaaacca 720
gggcaatctc 780
ctaaactact 840
gatttactgg 900
gcatccaccc 960
ggcacactgg 1020
agtccctgat 1080
cgcttcacag 1140
gcagtggatc 1200
tgggacagat 1260
tattctctca 1320
ccatcagcag 1380
tgtgcagtct 1440
gaagacctga 1500
cactttatta 1560
ctgtcagcaa 1620
cattatgaca 1680
ctccgtggac 1740
gttcggtgga 1800
ggcaccaagc 1860
tggaaatcaa 1920
a 354
<210> 19
<211> 115
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.115)
<223> heavy chain variable region amino acid sequence
<400> 19
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Leu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Leu Glu Trp Leu Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Leu Pro Gly Ser Gly Asn Pro Asn Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr
65 70 75 80
Met Gln Leu Ile Ser Leu Ile Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Ala Met Asp Tyr Trp Gly His Gly Thr Ser Val Thr
100 105 110
Val Ser Ser
115
<210> 20
<211> 107
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.107)
<223> light chain variable region amino acid sequence
<400> 20
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 Ser Ala Ser Gln Gly 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 Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 119
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.119)
<223> heavy chain variable region amino acid sequence
<400> 21
Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Lys Asp Tyr Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Lys Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ser Pro Asp Tyr Tyr Gly Ser Ser Leu Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 22
<211> 112
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.112)
<223> light chain variable region amino acid sequence
<400> 22
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ala 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 Ala 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> 23
<211> 119
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.119)
<223> heavy chain variable region amino acid sequence
<400> 23
Glu Val Gln Leu Gln 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 Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Asp 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 Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Gly Thr Asn Trp Asp Gly Arg Tyr Phe Asp Val Trp Gly Ala Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 24
<211> 113
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.113)
<223> light chain variable region amino acid sequence
<400> 24
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
20 25 30
Gly Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Leu 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 Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln
85 90 95
Ser Tyr Asn Leu Met Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 25
<211> 118
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.118)
<223> heavy chain variable region amino acid sequence
<400> 25
Glu Val Gln Leu Gln 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 Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Phe Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser 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
Ser Met Ala Gly Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 26
<211> 113
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.113)
<223> light chain variable region amino acid sequence
<400> 26
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met 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 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 27
<211> 118
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.118)
<223> heavy chain variable region amino acid sequence
<400> 27
Glu Val Gln Leu Gln 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 Thr Ser Gly
20 25 30
Tyr Trp His Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Phe Ile Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser 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 Ser Gly Gly Trp Leu Leu His Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 28
<211> 113
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.113)
<223> light chain variable region amino acid sequence
<400> 28
Asp Val Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met 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 Asn Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 29
<211> 116
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.116)
<223> heavy chain variable region amino acid sequence
<400> 29
Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Thr Ser Asp Gly His Tyr Thr Tyr Tyr Pro Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Arg Arg Thr Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 30
<211> 105
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.105)
<223> light chain variable region amino acid sequence
<400> 30
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Tyr Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Asn Ser Ser Val Thr Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Phe
35 40 45
Leu Thr Ser Asn 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 Arg Ser Asn Pro Thr Phe
85 90 95
Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 31
<211> 119
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.119)
<223> heavy chain variable region amino acid sequence
<400> 31
Glu Val Gln Leu Gln 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 Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys 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 Ile Ser Ile Thr Arg Asp Thr Ser 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 Tyr Arg Asp Trp Val Val Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 32
<211> 113
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.107)
<223> light chain variable region amino acid sequence
<400> 32
Asp Phe Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 33
<211> 118
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.118)
<223> heavy chain variable region amino acid sequence
<400> 33
Glu Val Gln Leu Gln 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 Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys 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 Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Arg Gly Gly Trp Leu Leu Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 34
<211> 164
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.164)
<223> light chain variable region amino acid sequence
<400> 34
Glu Val Gln Leu Gln 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 Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val
50 55 60
Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
65 70 75 80
Leu Asn Ser Gly Asn Gln Lys Asn Cys Leu Thr Trp Tyr Gln Gln Lys
85 90 95
Pro Gly Gln Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu
100 105 110
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
115 120 125
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
130 135 140
Cys Gln Asn Asp Tyr Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
145 150 155 160
Leu Glu Leu Lys
<210> 35
<211> 122
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.122)
<223> heavy chain variable region amino acid sequence
<400> 35
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met Tyr 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 Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Leu Phe Phe Thr Thr Val Thr Ala Ile Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 36
<211> 107
<212> PRT
<213> Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.107)
<223> light chain variable region amino acid sequence
<400> 36
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Ser
65 70 75 80
Glu Asp Leu Thr Leu Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 37
<211> 10
<212> PRT
<213> Artificial sequence ()
<220>
<221> misc_feature

Claims (10)

1. An isolated human PDL 1-specific binding molecule characterized by: the specific binding molecule comprises a light chain variable region and a heavy chain variable region, wherein:
(i) the heavy chain variable region is SEQ ID NO 19 and the light chain variable region is SEQ ID NO 20; or
(ii) The heavy chain variable region is SEQ ID NO 21 and the light chain variable region is SEQ ID NO 22; or
(iii) The heavy chain variable region is SEQ ID NO:23 and the light chain variable region is SEQ ID NO: 24; or
(iv) The heavy chain variable region is SEQ ID NO. 25 and the light chain variable region is SEQ ID NO. 26; or
(v) The heavy chain variable region is SEQ ID NO 27 and the light chain variable region is SEQ ID NO 28; or
(vi) The heavy chain variable region is SEQ ID NO:29 and the light chain variable region is SEQ ID NO: 30; or
(vii) The heavy chain variable region is SEQ ID NO 31 and the light chain variable region is SEQ ID NO 32; or
(viii) The heavy chain variable region is SEQ ID NO 33 and the light chain variable region is SEQ ID NO 34; or
(ix) The heavy chain variable region is SEQ ID NO 35 and the light chain variable region is SEQ ID NO 36.
2. The isolated human PDL 1-specific binding molecule of claim 1, wherein: the specific binding molecule exhibits at least one of the following properties:
(i) block binding of PDL1 to PD1 or CD 80;
(ii) PDL1 that binds to the surface of human T cells;
(iii) at 3.89X10-10K of M or lessDIn conjunction with human PDL 1;
(iv) IFN-gamma production is increased in a Mixed Lymphocyte Reaction (MLR) assay.
3. An isolated nucleic acid encoding an antibody light chain variable region and an antibody heavy chain variable region, characterized in that:
(i) the segment encoding the heavy chain variable region of the antibody is SEQ ID NO:1, and the segment encoding the light chain variable region of the antibody is SEQ ID NO: 2; or
(ii) The segment encoding the heavy chain variable region of the antibody is SEQ ID NO. 3, and the segment encoding the light chain variable region of the antibody is SEQ ID NO. 4; or
(iii) The segment encoding the heavy chain variable region of the antibody is SEQ ID NO:5, and the segment encoding the light chain variable region of the antibody is SEQ ID NO: 6; or
(iv) The segment encoding the heavy chain variable region of the antibody is SEQ ID NO. 7, and the segment encoding the light chain variable region of the antibody is SEQ ID NO. 8; or
(v) The segment encoding the heavy chain variable region of the antibody is SEQ ID NO. 9, and the segment encoding the light chain variable region of the antibody is SEQ ID NO. 10; or
(vi) The segment encoding the heavy chain variable region of the antibody is SEQ ID NO. 11, and the segment encoding the light chain variable region of the antibody is SEQ ID NO. 12; or
(vii) The fragment encoding the antibody heavy chain variable region is SEQ ID NO 13, and the fragment encoding the antibody light chain variable region is SEQ ID NO 14; or
(viii) The fragment encoding the antibody heavy chain variable region is SEQ ID NO. 15, and the fragment encoding the antibody light chain variable region is SEQ ID NO. 16; or
(ix) The fragment encoding the antibody heavy chain variable region is SEQ ID NO 17, and the fragment encoding the antibody light chain variable region is SEQ ID NO 18.
4. A carrier, characterized by: the vector comprising the nucleic acid molecule of claim 3.
5. A host cell, characterized in that: the host cell comprising the nucleic acid molecule of claim 3, or comprising the vector of claim 4.
6. A conjugate, characterized by: the conjugate comprises an anti-human PDL1 monoclonal antibody covalently linked to an isotope, immunotoxin, and/or chemical drug; the anti-human PDL1 monoclonal antibody is the isolated human PDL1 specific binding molecule of any one of claims 1-2.
7. Use of an isolated human PDL 1-specific binding molecule according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a disease; the isolated human PDL1 specific binding molecule is an anti-PDL 1 monoclonal antibody; the disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma or melanoma.
8. Use of a conjugate according to claim 6 for the preparation of a medicament for the treatment of a disease; the disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma or melanoma.
9. A composition characterized by: the composition contains a main substance (M) and an auxiliary substance (N); the major agent (M) is selected from one or more of the group consisting of an isolated human PDL1 specific binding molecule of any one of claims 1-2, an isolated nucleic acid of claim 3, a vector of claim 4, a host cell of claim 5, a conjugate of claim 6; the auxiliary substance (N) is selected from a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances.
10. A kit, characterized in that: the kit comprising an isolated human PDL 1-specific binding molecule according to any one of claims 1-2 and reagents for detecting an immune complex formed upon specific binding to the molecule.
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