Anti-PDL1 antibodies and uses thereof

Abstract

The present invention relates to the field of biomedicine, and specifically to an anti-PDL1 antibody or an antigen-binding fragment thereof and uses thereof. The anti-PDL1 antibody or an antigen-binding fragment thereof provided by the present invention contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity therewith: heavy chain variable region CDR sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; light chain variable region CDR sequence: SEQ ID NO: 4, WAS, SEQ ID NO: 5. The antibody has a high binding affinity with PDL1, and the present invention also provides a CD3 and PDL1 bispecific antibody, which has a stronger binding with tumor cells and promotes T cells to play an anti-cancer function.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-PDL1 antibody or an antigen-binding fragment thereof and uses thereof. Background Art

[0002] Cancer is a major disease that threatens human life and health worldwide, and with the intensification of environmental pollution, the incidence of cancer is increasing year by year. At the same time, a variety of cancer therapies have been developed, including traditional surgical resection, radiotherapy, chemotherapy, and targeted therapies that have emerged in recent years, including small molecule targeted drug therapy, macromolecular targeted drug therapy, immune checkpoint therapy, bispecific antibodies, cell and gene therapy, etc.

[0003] In recent years, the most anticipated drugs are immune checkpoint antibody drugs and bispecific antibody drugs. With the approval of CTLA4 antibody, PD-1 / L1 antibody, and LAG3 antibody, immune checkpoint drugs have become a trend in recent years and have achieved miraculous results in a variety of tumors including melanoma, non-small cell lung cancer and other tumors. The approval speed of bispecific antibody drugs has also been significantly accelerated. As of now, Eemovab (CD3×EPCAM), CD3×CD19 (Blincyto), Rybrevant (EGFR×cMet), Lunsumio (CD3×CD20), and Tecvayli (CD3×BCMA) have been approved. Among them, Rybrevant (EGFR×cMet), Lunsumio (CD3×CD20), and Tecvayli (CD3×BCMA) were approved as drugs last year and this year, indicating that these innovative drugs can indeed benefit more patients.

[0004] Radiotherapy and chemotherapy are generally accompanied by strong side effects and immunosuppression in patients, which can easily lead to tumor recurrence. Unlike the cytotoxic effects of traditional radiotherapy and chemotherapy, immune checkpoint therapy and bispecific antibody drugs use the patient's own immune system to fight cancer, with relatively few side effects, and the patient's immune function will not be suppressed.

[0005] PDL1 expression is significantly upregulated in a variety of tumors. It transmits immunosuppressive signals by binding to the PD-1 receptor on the surface of T cells, promotes T cell exhaustion, and reduces the patient's anti-cancer immune function. Using antibodies to block the interaction between PD-1 and PDL1 can reverse the immune exhaustion of T cells and restore anti-cancer function. So far, PD-1 antibodies such as Opdivo and Keytruda, and PDL1 antibodies such as Tecentirq (Atezolizumab) have benefited a lot of patients, but it should be noted that not all patients respond to PD-1 / L1 monoclonal antibody therapy, and its overall efficacy is around 30%, and there are still more patients who cannot benefit from it.

[0006] Since the extracellular segment of the PD-1 receptor or its variants are limited by the diversity of glycosylation modifications and have poor drugability, although there are reports of PDL1 antibodies and related products, they all have the problem of weak binding ability to PDL1 on the surface of tumor cells.

[0007] Therefore, it is urgent to develop an anti-PDL1 antibody with higher affinity to PDL1. Summary of the invention

[0008] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide.

[0009] To this end, the first aspect of the present invention provides an antibody or antigen-binding fragment, comprising:

[0010] A CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto:

[0011] Heavy chain variable region CDR sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

[0012] Light chain variable region CDR sequences: SEQ ID NO: 4, WAS, SEQ ID NO: 5.

[0013] The antibody or antigen-binding fragment provided by the present invention contains the above-mentioned specific CDR sequence, which can specifically bind to PDL1 highly expressed on the surface of tumor cells with high binding affinity, thereby blocking the interaction between PD-1 and PDL1 on the surface of immune cells and promoting the anti-cancer function of immune cells.

[0014] In some embodiments of the present invention, the antibody or antigen-binding fragment comprises:

[0015] The heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 shown in SEQ ID NO:2, the heavy chain variable region CDR3 shown in SEQ ID NO:3, the light chain variable region CDR1 shown in SEQ ID NO:4, the light chain variable region CDR2 shown in SEQ ID NO:WAS, and the light chain variable region CDR3 shown in SEQ ID NO:5.

[0016] In some embodiments of the present invention, the antibody or antigen-binding fragment comprises:

[0017] (a) having a heavy chain variable region shown in SEQ ID NO:6 and a light chain variable region shown in SEQ ID NO:7;

[0018] An amino acid sequence having at least 80% sequence identity with (a);

[0019] (b) having a heavy chain variable region shown in SEQ ID NO: 8 and a light chain variable region shown in SEQ ID NO: 9;

[0020] An amino acid sequence having at least 80% sequence identity compared to (b).

[0021] In some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region as shown in SEQ ID NO:6 or SEQ ID NO:8; and / or a light chain variable region as shown in SEQ ID NO:7 or SEQ ID NO:9.

[0022] In some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region as shown in SEQ ID NO:6, and a light chain variable region as shown in SEQ ID NO:7; or a heavy chain variable region as shown in SEQ ID NO:8, and a light chain variable region as shown in SEQ ID NO:9.

[0023] In some embodiments of the present invention, the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, and a murine antibody or a mutant thereof.

[0024] In some embodiments of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.

[0025] In some embodiments of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0026] In some embodiments of the present invention, the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence shown in any one of SEQ ID NO:10 and SEQ ID NO:12, and a light chain having an amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:13.

[0027] In some embodiments of the present invention, the antibody or antigen-binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO:10 and a light chain with the amino acid sequence shown in SEQ ID NO:11; the antibody or antigen-binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO:12 and a light chain with the amino acid sequence shown in SEQ ID NO:13.

[0028] In some embodiments of the present invention, the antibody or antigen-binding fragment includes a monoclonal antibody or a polyclonal antibody; the monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody and a minimum recognition unit; the antibody or antigen-binding fragment can bind to the amino acid sequence shown in SEQ ID NO:14.

[0029] The second aspect of the present invention provides a bispecific binding molecule, comprising: a first binding region, the first binding region comprising the antibody or antigen-binding fragment described in the first aspect; and a second binding region, the second binding region having CD3 binding activity.

[0030] In some embodiments of the present invention, the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule; the bispecific binding molecule is an asymmetric bispecific binding molecule.

[0031] In some embodiments of the present invention, the first binding region of the bispecific binding molecule comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 comprises the heavy chain variable region SEQ ID NO:6 or SEQ ID NO:8, and peptide chain 2 comprises the light chain variable region SEQ ID NO:7 or SEQ ID NO:9.

[0032] In some embodiments of the present invention, the second binding region comprises at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody and a minimum recognition unit having CD3 binding activity.

[0033] In some embodiments of the invention, the second binding region comprises an anti-CD3 single chain antibody.

[0034] The CD3×PDL1 bispecific antibody provided by the present invention can bind more strongly to tumor cells and promote T cells to exert their anti-cancer function.

[0035] In some embodiments of the present invention, the anti-CD3 single-chain antibody comprises an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, wherein the anti-CD3 antibody heavy chain variable region comprises a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 15, a heavy chain variable region CDR2 shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 shown in SEQ ID NO: 17; the anti-CD3 antibody light chain variable region has a light chain variable region CDR1 shown in SEQ ID NO: 18, a light chain variable region CDR2 shown in GTN, and a light chain variable region CDR3 shown in SEQ ID NO: 19.

[0036] In some embodiments of the present invention, the anti-CD3 single-chain antibody comprises a heavy chain variable region shown in SEQ ID NO:20 and a light chain variable region shown in SEQ ID NO:21.

[0037] In some embodiments of the present invention, the anti-CD3 single-chain antibody further comprises a connecting peptide, wherein the N-terminus of the connecting peptide is connected to the C-terminus of the heavy chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the light chain variable region of the anti-CD3 antibody; or the N-terminus of the connecting peptide is connected to the C-terminus of the light chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the heavy chain variable region of the anti-CD3 antibody.

[0038] In some embodiments of the present invention, the connecting peptide has an amino acid sequence (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0039] In some embodiments of the present invention, the anti-CD3 single-chain antibody has the amino acid sequence shown in SEQ ID NO:22.

[0040] In some embodiments of the present invention, the first binding region further includes at least one of a first heavy chain constant region and a light chain constant region, and at least a portion of at least one of the first heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, and a murine antibody or a mutant thereof.

[0041] In some embodiments of the present invention, the first heavy chain constant region and the light chain constant region are both from human IgG antibodies or mutants thereof.

[0042] In some embodiments of the present invention, the first heavy chain constant region and the light chain constant region are both from human IgG1 antibody or a mutant thereof.

[0043] In some embodiments of the present invention, the N-terminus of the first heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0044] In some embodiments of the present invention, the peptide chain 1 has the amino acid sequence shown in SEQ ID NO:12, and the peptide chain 2 has the amino acid sequence shown in SEQ ID NO:13.

[0045] In some embodiments of the present invention, the peptide chain 1 and the peptide chain 2 are connected via a disulfide bond.

[0046] In some embodiments of the present invention, the second binding region further includes a second heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, and a murine antibody or a mutant thereof.

[0047] In some embodiments of the present invention, the second heavy chain constant region is derived from a human IgG antibody or a mutant thereof.

[0048] In some embodiments of the present invention, the second heavy chain constant region is derived from a human IgG1 antibody or a mutant thereof.

[0049] In some embodiments of the present invention, the N-terminus of the second heavy chain constant region is linked to the C-terminus of the anti-CD3 single chain antibody.

[0050] In some embodiments of the present invention, the first heavy chain constant region and the second heavy chain constant region are connected via a knob-into-hole structure.

[0051] The third aspect of the present invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment of the first aspect or the bispecific binding molecule of the second aspect.

[0052] The fourth aspect of the present invention provides an expression vector, which carries the polynucleotide described in the third aspect.

[0053] The fifth aspect of the present invention provides a recombinant cell carrying the polynucleotide described in the third aspect, the expression vector described in the fourth aspect, or capable of expressing the antibody or antigen-binding fragment described in the first aspect or encoding the bispecific binding molecule described in the second aspect.

[0054] In some embodiments of the present invention, the recombinant cell is obtained by introducing the expression vector of the fourth aspect into a host cell.

[0055] In some embodiments of the invention, the recombinant cell is a eukaryotic cell.

[0056] In some embodiments of the invention, the recombinant cell is a mammalian cell.

[0057] The sixth aspect of the present invention provides a composition, comprising the antibody or antigen-binding fragment described in the first aspect, the bispecific binding molecule described in the second aspect, the polynucleotide described in the third aspect, the expression vector described in the fourth aspect, and the recombinant cell described in the fifth aspect.

[0058] The seventh aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment described in the first aspect, or the bispecific binding molecule described in the second aspect, comprising culturing the recombinant cell described in the fifth aspect.

[0059] The eighth aspect of the present invention provides a drug, comprising the antibody or antigen-binding fragment described in the first aspect, the bispecific binding molecule described in the second aspect, the polynucleotide described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect.

[0060] The ninth aspect of the present invention provides a kit, characterized in that the kit contains the antibody or antigen-binding fragment described in the first aspect, the bispecific binding molecule described in the second aspect, the polynucleotide described in the third aspect, the expression vector described in the fourth aspect, and the recombinant cell described in the fifth aspect.

[0061] The tenth aspect of the present invention provides use of the antibody or antigen-binding fragment described in the first aspect, the polynucleotide described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for preventing and / or treating PDL1-mediated related diseases, including cancer.

[0062] In some embodiments of the present invention, the cancer comprises at least one selected from the following:

[0063] Lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0064] The eleventh aspect of the present invention provides use of the bispecific binding molecule of the second aspect, the polynucleotide of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect or the composition of the sixth aspect in the preparation of a medicament for preventing and / or treating PDL1 and CD3-mediated related diseases.

[0065] In some embodiments of the present invention, the PDL1 and CD3-mediated related diseases include cancer.

[0066] In some embodiments of the invention, the cancer comprises at least one of the following:

[0067] Lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0068] The twelfth aspect of the present invention provides use of the antibody or antigen-binding fragment of the first aspect, the polynucleotide of the third aspect, the expression vector of the fourth aspect, and the recombinant cell of the fifth aspect in preparing a kit for detecting PDL1.

[0069] The thirteenth aspect of the present invention provides use of the bispecific binding molecule of the second aspect, the polynucleotide of the third aspect, the expression vector of the fourth aspect, and the recombinant cell of the fifth aspect in preparing a kit for detecting PDL1 and / or CD3.

[0070] The CD3 antibody provided by the present invention and the PD-1 receptor extracellular segment protein are used to form an antibody-fusion protein, which can bridge T cells and PDL1-positive tumors, thereby promoting T cells to kill tumors. Even if tumor cells lowly express PDL1, T cells can still promote complete tumor killing.

[0071] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0073] Figure 1 This is a diagram showing the ELISA results of the binding of the murine 19B8 antibody to human (left A) and cynomolgus monkey PDL1 (right B) proteins in one embodiment of the present invention;

[0074] Figure 2 This is a diagram showing the ELISA results of the murine 19B8 antibody blocking the binding of PD-1 to PDL1 in one embodiment of the present invention;

[0075] Figure 3 This is a flow cytometry result diagram of the binding of the murine 19B8 antibody in one embodiment of the present invention to human lung cancer A549 cells;

[0076] Figure 4 This is a diagram showing the ELISA results of the binding of the 19B8 chimeric antibody in one embodiment of the present invention to human (left A) and cynomolgus monkey PDL1 (right B) proteins;

[0077] Figure 5 This is a diagram showing the ELISA results of the 19B8 chimeric antibody blocking the binding of PD-1 to PDL1 in one embodiment of the present invention;

[0078] Figure 6 This is a flow cytometry result diagram of the binding of the 19B8 chimeric antibody in one embodiment of the present invention to human melanoma A375 cells (left A) and human lung cancer A549 cells (right B);

[0079] Figure 7 This is a diagram showing the ELISA results of humanized 19B8 antibody blocking the binding of PD-1 to PDL1 in one embodiment of the present invention;

[0080] Figure 8 This is a flow cytometry result diagram of the binding of the humanized 19B8 antibody in one embodiment of the present invention to human melanoma A375 cells (left A) and human lung cancer A549 cells (right B);

[0081] Fig. 9 This is a result diagram showing that the humanized 19B8 antibody in one embodiment of the present invention promotes SEB-induced IL-2 secretion by human PBMC;

[0082] Fig.10 This is a schematic diagram of a CD3×PDL1 bispecific antibody in one embodiment of the present invention;

[0083] Fig.11 This is a diagram showing the result of CD3×PDL1 bispecific antibody binding to PDL1 fusion protein in one embodiment of the present invention;

[0084] Fig.12 This is a diagram showing the ELISA results of the CD3×PDL1 bispecific antibody blocking the binding of PD-1 to PDL1 in one embodiment of the present invention;

[0085] Fig.13 This is a graph showing the result of CD3×PDL1 bispecific antibody in one embodiment of the present invention promoting PBMC to kill human melanoma A375 cells;

[0086] Fig.14 This is a flow cytometry result diagram of the binding of the CD3×PDL1 bispecific antibody in one embodiment of the present invention to human lung adenocarcinoma A549 cells;

[0087] Fig.15 This is a graph showing the result of CD3×PDL1 bispecific antibody in one embodiment of the present invention promoting PBMC to kill human melanoma A375 cells;

[0088] Fig.16 This is a graph showing the results of CD3×PDL1 antibody in one embodiment of the present invention promoting anti-cancer in PBMC-reconstituted mice; DETAILED DESCRIPTION

[0089] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0090] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0091] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0092] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0093] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0094] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0095] The antibody or antigen-binding fragment described in the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence described in the present invention, a person skilled in the art can easily obtain the encoding nucleic acid of the present invention by various known methods. These methods include, but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR. Among them, the antibody or antigen fragment is numbered and defined using the Kabat numbering system. In this article, the term "antibody" is an immunoglobulin molecule that can bind to a specific antigen. It includes two light chains with a lighter molecular weight and two heavy chains with a heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl terminal (C terminal) is relatively stable and varies very little, which is called the constant region (C region). The V regions of the L chain and the H chain are respectively called VL and VH. In the variable region, the amino acid composition and arrangement order of some areas have a higher degree of variation, which is called the hypervariable region (HVR). The hypervariable region is the location where the antigen and antibody bind, so it is also called the complementarity-determining region (CDR). There are three CDR regions in the heavy chain variable region and the light chain variable region.

[0096] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0097] The term "murine antibody" in the present invention refers to a monoclonal antibody against human B7H6 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with an antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine B7H6 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.

[0098] As used herein, the term "antigen-binding fragment" also refers to "antibody fragment". Antibody fragments generally refer to antigen-binding antibody fragments and may include a portion of an intact antibody, generally an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0099] As used herein, the term "monoclonal antibody" refers to an antibody that has a single antigen binding site.

[0100] Herein, the term "double antibody" refers to an antibody having two different antigen binding sites.

[0101] As used herein, the term "mutant" or "variant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0102] As used herein, the term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence in an antibody that is responsible for antigen binding, for example, generally including: amino acid residues around 23-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region, and around 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). of Health, Bethesda, MD. (1991)); and / or from "hypervariable loops" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0103] In the present context, the term "functional fragment" refers in particular to antibody fragments such as Fv, scFv (sc means single chain), Fab, F(ab')2, Fab', scFv-Fc fragments or diabodies, or any fragment that should be able to increase the half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) or by incorporation into liposomes.

[0104] As used herein, the term "chimeric antibody" refers to a recombinant antibody obtained by replacing the constant region amino acid sequence of a monoclonal antibody from one species (such as mouse) with the constant region of an antibody from another species (such as human) using recombinant DNA technology.

[0105] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all non-CDR (Fv framework region (FR)) amino acid sequences of the constant region and variable region of a monoclonal antibody from one species (such as mouse) with non-CDR amino acid sequences of the constant region and variable region of an antibody from another species (such as human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when the non-CDR amino acid sequences of the constant region and variable region are all humanized, it is called a humanized antibody.

[0106] In this article, the "full-length antibody" is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE). The same type of immunoglobulin can also be divided into different subclasses according to the amino acid composition, such as IgG1, IgG2, IgG3, IgG4. Immunoglobulin light chains are divided into κ chains or λ chains according to different constant regions.

[0107] In this article, the "knob into hole structure" refers to a knob and hole mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate the heavy chain biting and form a heterodimer.

[0108] As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0109] Without substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0110] Anti-PDL1 antibodies or antigen-binding fragments

[0111] According to a specific embodiment of the present invention, the present invention provides an antibody (anti-PDL1 antibody) or an antigen-binding fragment that can specifically recognize PDL1, and the antibody or its antigen-binding fragment contains a CDR sequence selected from at least one of the following or an amino acid sequence that has at least 80% identity therewith: heavy chain variable region CDR sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3; light chain variable region CDR sequence: SEQ ID NO: 4, WAS, SEQ ID NO: 5.

[0112] "WAS" in the CDR sequence refers to the amino acid sequence.

[0113] According to a specific embodiment of the present invention, the anti-PDL1 antibody or antigen-binding fragment comprises:

[0114] The heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 shown in SEQ ID NO:2, the heavy chain variable region CDR3 shown in SEQ ID NO:3, the light chain variable region CDR1 shown in SEQ ID NO:4, the light chain variable region CDR2 shown in WAS, and the light chain variable region CDR3 shown in SEQ ID NO:5.

[0115] According to a specific embodiment of the present invention, the anti-PDL1 antibody or antigen-binding fragment comprises: a heavy chain variable region as shown in SEQ ID NO: 6 or SEQ ID NO: 8; and / or

[0116] The light chain variable region is shown in SEQ ID NO:7 or SEQ ID NO:9.

[0117] According to a specific embodiment of the present invention, the anti-PDL1 antibody or antigen-binding fragment comprises:

[0118] The heavy chain variable region shown in SEQ ID NO:6, and the light chain variable region shown in SEQ ID NO:7; or

[0119] The heavy chain variable region is shown in SEQ ID NO:8, and the light chain variable region is shown in SEQ ID NO:9.

[0120] According to a specific embodiment of the present invention, under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain a variant of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The sequence of the variant of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% consistency (or homology) with the reference sequence. The sequence consistency of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the manner of N-terminus to C-terminus. It should be known to those skilled in the art that the CDR sequences analyzed from different databases may be different, but these changes should be included in the scope of protection of the present invention.

[0121] According to a specific embodiment of the present invention, the antibody of the present invention may be full-length (e.g., IgG1 or IgG4 antibody) or may only contain an antigen binding portion (e.g., Fab, F(ab')2 or scFv fragment), or may be modified to affect function. The present invention includes anti-PDL1 antibodies with modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites may be useful, or antibodies without fucose moieties on oligosaccharide chains to, for example, enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC). After a series of modifications, the fragment of the present invention still has PDL1 binding activity, particularly the amino acid sequence shown in SEQ ID NO: 10. Preferably, the functional fragment will be composed of or contain a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least equal to 1 / 100 of the affinity of the antibody from which it is derived, and at least equal to 1 / 10 in a more preferred manner. Such functional fragments will contain a minimum of 5 amino acids, and preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

[0122] According to a specific embodiment of the present invention, in order to further improve the bioacceptability of the antibody, the present invention humanizes the antibody. The humanization method can be performed with reference to conventional antibody engineering technology. The sequence of the heavy chain variable region of the humanized antibody provided by the present invention is shown in SEQ ID NO: 8, and the sequence of the light chain variable region is shown in SEQ ID NO: 9.

[0123] Nucleic acid molecules, recombinant vectors, recombinant cells, immunoconjugates

[0124] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0125] Therefore, the present invention also provides isolated nucleic acids encoding the above-mentioned antibodies or antigen-binding fragments thereof, and recombinant vectors and transformants containing the nucleic acids. The nucleic acid molecules encode the above-mentioned antibodies or antigen-binding fragments thereof, and the nucleic acids are preferably expression cassettes obtained by genetic engineering.

[0126] The recombinant vector may refer to a cloning vector or an expression vector, and can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). Commonly used plasmids include pSeTag2, PEE14, pMH3, and the like.

[0127] In some preferred embodiments, the nucleic acid molecule is species optimized and is more easily expressed in mammalian cells.

[0128] The present invention also provides an expression vector, which comprises the above-mentioned isolated nucleic acid molecule. When the above-mentioned isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself. Of course, the polynucleotide can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode the heavy chain and light chain of the antibody can be inserted into different vectors separately and independently, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, etc.

[0129] The present invention also provides a recombinant cell, which contains the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. The corresponding antibody can be obtained by culturing the recombinant cells. The host cell of the present invention can be a prokaryotic host cell, a eukaryotic host cell or a bacteriophage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis. The eukaryotic host cell can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the host cell of the present invention is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell or COS cell.

[0130] The immunoconjugate provided by the present invention contains a therapeutic agent and the above-mentioned antibody or antigen-binding fragment thereof coupled to the therapeutic agent. The antibody or antigen-binding fragment thereof and the therapeutic agent can be coupled in a conventional manner.

[0131] The composition provided by the present invention contains the antibody or its antigen-binding fragment as described above, and / or the above-mentioned immunoconjugate and a pharmaceutically acceptable carrier. In some embodiments, the composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be applied to the subject as a whole, or separately applied to the subject. When the components contained in the composition are applied to the subject separately, each component can be applied to the subject simultaneously or sequentially.

[0132] Drug, kit and pharmaceutical use and use in preparing kit

[0133] The present invention also provides a drug, which includes the above-mentioned antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, and may also include the above-mentioned immunoconjugate, nucleic acid molecule, expression vector, and recombinant cell.

[0134] In some embodiments, these pharmaceutical compositions further include a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, glidant or lubricant, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0135] The compositions of the present invention may also be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the composition may also contain a chemotherapeutic agent. The antibody or antigen-binding fragment thereof or immunoconjugate of the present invention may also be combined with a second therapeutic agent, exemplary agents of which include but are not limited to other agents that inhibit PDL1 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with PDL1 upstream or downstream signal transduction.

[0136] Typically, the antibody or antigen-binding fragment thereof is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or preventive effect, e.g., an amount that causes the prevention or alleviation of symptoms associated with the disease being treated, e.g., a disease associated with PDL1. The effective amount of the composition administered to the subject will depend on the type and severity of the disease, as well as on the characteristics of the individual, such as general health, age, sex, weight, and tolerance to drugs; it will also depend on the severity and type of the disease, and a person skilled in the art will be able to determine an appropriate dose based on these factors, among others.

[0137] In some embodiments of the present invention, the kit for detecting PDL1 in a sample provided by the present invention contains an antibody or an antigen-binding fragment thereof as described above, a pharmaceutically acceptable carrier, an immunoconjugate, a nucleic acid molecule, an expression vector, and a recombinant cell. In some embodiments, the sample can be a tissue of a patient suffering from a disease mediated by PDL1 (particularly a transplant rejection, an autoimmune disease, an infectious disease, or a cancer patient, more preferably a patient suffering from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer). The kit may also include reagents conventionally used to detect PDL1, such as a coating solution, etc.

[0138] The present invention also provides the antibody or antigen-binding fragment thereof, a pharmaceutically acceptable carrier, an immunoconjugate, a nucleic acid molecule, an expression vector, and a recombinant cell. Use in the preparation of a drug for preventing and / or treating a PDL1-mediated disease. Preferably, the PDL1-mediated disease is transplant rejection, an autoimmune disease, an infectious disease, or a cancer. More preferably, the cancer is a cancer expressing PDL1. Further preferably, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer. Further preferably, the infectious disease includes but is not limited to HIV virus infection and / or hepatitis B virus infection.

[0139] The present invention also relates to a method for preventing and / or treating a PDL1-mediated disease (as described above), comprising administering an effective amount of at least one of the antibodies or antigen-binding fragments thereof, immunoconjugates and compositions of the present invention to a patient. The administration may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous or intraperitoneal.

[0140] Nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention. Based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1.

[0141] Table 1

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0152] Example 1 Preparation of Antibodies

[0153] Mouse monoclonal antibodies against human PDL1 were generated, and purified recombinant PDL1 extracellular domain Strep tag fusion protein (PDL1-strep) was used as an antigen to immunize C57BL / 6 mice (9 weeks old, purchased from Shanghai Lesk, weighing about 20 g).

[0154] Immunized mice were immunized three times with purified antigen and complete Freund's adjuvant, and the immune response was detected after bleeding from the tail vein. The serum was screened by ELISA and flow cytometry to obtain mice with anti-human PDL1 immunoglobulin. The spleen cells of the mice with the highest anti-PDL1 immunoglobulin were taken out and fused with mouse myeloma cells SP2 / 0 cells (ATCC No. CRL-1581). The hybridoma cells after fusion were screened for antibodies to obtain mouse monoclonal antibodies.

[0155] Culture the candidate hybridoma cells to a total number of 10 6, centrifuged at 800 rpm for 10 minutes to collect the cells, and extracted total RNA using Trizol kit (Invitrogen); using total RNA as template, reverse transcribed to synthesize cDNA library (Invitrogen), and using cDNA as template, PCR amplified the variable region nucleic acid sequence corresponding to the hybridoma cells. The primer sequences used in the PCR amplification reaction are complementary to the first framework region or signal peptide region and constant region of the antibody variable region (Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). In a 50μl reaction system, add 2μl of cDNA, 5μl of 10×PCR buffer, 2μl of upstream and downstream primers (5μmol), 2μl of dNTP, 1μl of Taq enzyme (Takara, Ex Taq), and 38μl of H2O; pre-denature at 95℃ for 5min, enter temperature cycle, and perform PCR amplification. The reaction conditions are: denaturation at 94℃ for 30S, annealing at 58℃ for 45S, extension at 72℃ for 50S, a total of 32 cycles, and then extension at 72℃ for 7min. After sequencing the amplified product, the heavy chain and light chain variable region sequences of the mouse monoclonal antibody were obtained.

[0156] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6:

[0157] EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATTYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCVPTGMGYFLMDYWGQGTSVTVSS

[0158] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:7:

[0159] DIVMTQSHKFMSASVGDRVSITCKASQDVGSAVVWYRQKPGQSPKLLIYWASTRHT GVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQFTSYPTFGSGTRLEIK

[0160] Example 2 PDL1 antibody ELISA binding experiment

[0161] ELISA experiments were used to detect the binding properties of PDL1 antibodies. The PDL1 extracellular domain protein was coated into a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and PDL1.

[0162] Antibody production: The heavy chain and light chain nucleotide sequences of the above-mentioned antibodies were transiently transfected into ExpiCHO-S cells (Gibco, Catalog No. A29127) with pcDNA3.4 vectors (synthesized by Nanjing GenScript, mouse 19B8 heavy chain, i.e., 19B8-mIgG1 (amino acid sequence as shown in SEQ ID NO: 10) and mouse h19B8 light chain, i.e., 19B8-mK (amino acid sequence as shown in SEQ ID NO: 11), respectively to prepare antibodies.

[0163] EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATTYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCVPTGMGYFLMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPA VLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLN GKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQID NO:10)

[0164] DIVMTQSHKFMSASVGDRVSITCKASQDVGSAVVWYRQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQFTSYPTFGSGTRLEIKRA DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ IDNO:11)

[0165] One day before transfection, adjust the cell density of ExpiCHO-S cells to (3-4)×10 6 / mL, at 37°C, 8% CO 2The cells were cultured overnight with shaking at 95 rpm. On the day of transfection, the cells grew to 7×10 6 ~1×10 7 / mL, prepare for transfection when the viability is greater than 95%, and dilute the cells to 6×10 using fresh pre-warmed ExpiCHO medium (Gibco, Catalog No. A2910002). 6 / mL, the pcDNA3.4 plasmid carrying the heavy and light chains (the ratio of light and heavy chain plasmids was 1:1) and ExpiFectamine CHO transfection reagent (Gibco, Cat. No. A29129) were transfected into ExpiCHO-S cells and incubated at 37°C and 8% CO 2 18-22h after transfection, mix ExpiFectamine CHO Enhancer and ExpiCHO Feed and add the transfected cells immediately, mix well, and incubate at 32℃, 5% CO 2 and shake culture at 95rpm. On the 5th day after transfection, add 8mL ExpiCHO Feed to the cells, mix and continue to culture. Observe the changes in cell number and cell viability every day, and harvest the cells by centrifugation after the cell viability drops below 80% or after 10-14 days of culture. The expressed supernatant was filtered with a 0.45μm filter membrane, and the antibody with Fc domain was captured from the expression supernatant using MabselectprismAproteinA affinity chromatography column (purchased from Suzhou Nano) . After balancing the chromatography column with pH7.2 phosphate buffer, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100mM citric acid, pH2.7), and finally concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE to have a purity of more than 95%. The results showed that the above-mentioned antibody was finally obtained.

[0166] Dilute human PDL1-Fc fusion protein (purchased from Acro) or monkey PDL1-His tag protein (purchased from Acro) to 1 μg / ml with PBS buffer, add 100 μl / well to a 96-well plate, and place overnight at 4°C. Aspirate the PBS buffer in the 96-well plate, wash the plate 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 hours for blocking. Remove the blocking solution, wash the plate 6 times with PBST, add 100 μl / well of the mouse PDL1 antibody 19B8 to be tested diluted to an appropriate concentration with PBST / 0.05% BSA, and incubate at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled anti-mouse IgG secondary antibody with PBST / 0.05% BSA at 100 μl / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μl / well 4M sulfuric acid to terminate the reaction. Read the absorbance at 450 mm with an enzyme reader. The results are as follows: Figure 1 As shown, it is demonstrated that the PDL1 antibody of the present invention can bind to human and monkey PDL1 proteins.

[0167] Example 3: Mouse PDL1 antibody blocking activity detection experiment

[0168] ELISA experiments were used to detect the effect of antibodies on the binding of PDL1 to its receptor PD-1. The specific experiments are as follows:

[0169] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH7.2 PBS containing 0.1% Tween20) buffer, and then 200 μL / well PBS / 10% BSA was added, and the plate was incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μL / well 19B8 antibody diluted to different concentrations with PBST / 0.05% BSA and PD-1-Avi tag (purchased from Acro) diluted to 2 μg / ml were added, and incubated at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled strreptavidin secondary antibody (purchased from Southernbiotech) with PBST / 0.05% BSA at 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μL / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. mIgG (purchased from Biolegend) was used as a control. Read the absorbance at 450 mm using an enzyme reader. The results are as follows Figure 2 As shown, it is shown that the mouse PDL1 antibody (19B8) of the present invention can block the binding of PDL1 to its receptor PD-1.

[0170] Example 4: Flow cytometry binding experiment of mouse PDL1 antibody

[0171] Human lung adenocarcinoma A549 cells were diluted to 1×10 6 / mL, add it to a 1.5mL EP tube at a volume of 100μL / tube, add 10μL / tube goat serum thereto, and block it at 4℃ for 30min. Add different concentrations of 19B8 antibody (the heavy chain amino acid sequence is shown in SEQ ID NO:10, and the light chain amino acid sequence is shown in SEQ ID NO:11), and incubate at 4℃ for 30min. Add 1mL PBS to the EP tube, centrifuge at 4℃, 3500rpm×5min, discard the supernatant, and wash it again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μL / tube PBS, add 1μL / tube Alexa-647-labeled goat anti-mouse antibody secondary antibody (Biolegend), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, and discard the supernatant after centrifugation. Resuspend the cells with 200μL / tube PBS and detect them by flow cytometry. The results are as follows Figure 3 As shown, it is further demonstrated that the mouse PDL1 antibody of the present invention can bind to PDL1 on the surface of tumor cells.

[0172] Example 5: Human-mouse chimeric PDL1 antibody ELISA binding experiment

[0173] ELISA experiments were used to detect the binding properties of PDL1 antibodies. The PDL1 extracellular domain protein was coated into a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and PDL1.

[0174] Antibody production: The heavy chain and light chain nucleotide sequences of the above antibodies were transiently transfected into ExpiCHO-S cells (Gibco, catalog number A29127) with pcDNA3.4 vectors (synthesized by Nanjing GenScript, mouse 19B8 heavy chain hIgG1LALA, i.e. 19B8-hIgG1LALA (amino acid sequence as shown in SEQ ID NO: 31), mouse h19B8 light chain hK, i.e. 19B8-hK (amino acid sequence as shown in SEQ ID NO: 32), fully human 12A4 antibody heavy chain 12A4-hIgG1LALA (amino acid sequence as shown in SEQ ID NO: 28), and fully human 12A4 antibody light chain 12A4-hK (amino acid sequence as shown in SEQ ID NO: 30) to prepare antibodies.

[0175] EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATTYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCVPTGMGYFLMDYWGQGT SVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 31)

[0176] DIVMTQSHKFMSASVGDRVSITCKASQDVGSAVVWYRQKPGQSPKLLIIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQFTSYPTFGSGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 32)

[0177] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESSTAYMELSSLRSEDTAVIFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKPEKSCDK THTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS(SEQ ID NO:28)

[0178] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 30)

[0179] One day before transfection, adjust the cell density of ExpiCHO-S cells to (3-4)×10 6 / mL, at 37°C, 8% CO 2 The cells were cultured overnight with shaking at 95 rpm. On the day of transfection, the cells grew to 7×10 6 ~1×10 7 / mL, prepare for transfection when the viability is greater than 95%, and dilute the cells to 6×10 using fresh pre-warmed ExpiCHO medium (Gibco, Catalog No. A2910002). 6 / mL, the pcDNA3.4 plasmid carrying the heavy and light chains (the ratio of light and heavy chain plasmids was 1:1) and ExpiFectamine CHO transfection reagent (Gibco, Cat. No. A29129) were transfected into ExpiCHO-S cells and incubated at 37°C and 8% CO 2 18-22h after transfection, mix ExpiFectamine CHO Enhancer and ExpiCHO Feed and add the transfected cells immediately, mix well, and incubate at 32℃, 5% CO 2 and shake culture at 95rpm. On the 5th day after transfection, add 8mL ExpiCHO Feed to the cells, mix and continue to culture. Observe the changes in cell number and cell viability every day, and harvest the cells by centrifugation after the cell viability drops below 80% or after 10-14 days of culture. The expressed supernatant was filtered with a 0.45μm filter membrane, and the antibody with Fc domain was captured from the expression supernatant using MabselectprismAproteinA affinity chromatography column (purchased from Suzhou Nano) . After balancing the chromatography column with pH7.2 phosphate buffer, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100mM citric acid, pH2.7), and finally concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE to have a purity of more than 95%. The results showed that the above-mentioned antibody was finally obtained.

[0180] Human PDL1-Fc fusion protein (purchased from Acro) or monkey PDL1-His tag fusion protein (purchased from Acro) was diluted to 1 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH7.2 PBS containing 0.1% Tween 20) buffer, and then 200 μl / well PBS / 10% BSA was added, and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μl / well of the chimeric antibody 19B8-hIgGLALA to be tested and the control antibody 12A4-hIgG1LALA diluted to an appropriate concentration with PBST / 0.05% BSA were added, and incubated at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled anti-human IgG-Fab secondary antibody with PBST / 0.05% BSA at 100 μl / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μl / well 4M sulfuric acid to terminate the reaction. Read the absorbance at 450 mm with an enzyme reader. The results are as follows: Figure 4 , indicating that the chimeric antibody 19B8-hIgG1LALA of the present invention can bind to human and monkey PDL1 proteins.

[0181] Example 6: Human-mouse chimeric PDL1 antibody blocking activity detection experiment

[0182] ELISA experiments were used to detect the effect of chimeric antibodies on the binding of PDL1 to its receptor PD-1. The specific experiments are as follows:

[0183] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH7.2 PBS containing 0.1% Tween20) buffer, and then 200 μL / well PBS / 10% BSA was added, and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μL / well PBST / 0.05% BSA diluted to different concentrations of 19B8-hIgG1LALA antibody or 12A4-hIgG1LALA antibody, 2 μg / ml PD-1-Avi tag protein (purchased from Acro) was added, and 100 μL / well PBST / 0.05% BSA was added, and incubated at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled strreptavidin secondary antibody (purchased from Southern biotech) with PBST / 0.05% BSA at 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μL / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. mIgG (purchased from Biolegend) was used as a control. Read the absorbance at 450 mm using an enzyme reader. The results are as follows Figure 5 The results show that the chimeric antibody 19B8-hIgG1LALA of the present invention can block the binding of PDL1 to its receptor PD-1.

[0184] Example 7: Flow cytometry binding experiment of human-mouse chimeric PDL1 antibody

[0185] Human melanoma A375 cells and human lung adenocarcinoma A549 cells were diluted to 1×10 6 / mL, add it to a 1.5mL EP tube at a volume of 100μL / tube, add 10μL / tube of rat serum, and block it at 4℃ for 30min. Add different concentrations of chimeric antibody 19B8-hIgG1LALA or control antibody 12A4-hIgG1LALA, and incubate at 4℃ for 30min. Add 1mLPBS to the EP tube, centrifuge at 4℃, 3500rpm×5min, discard the supernatant, and wash it again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μL / tube PBS, add 1μL / tube Alexa-647-labeled rat anti-human antibody secondary antibody (Biolegend), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μL / tube PBS and detect them by flow cytometry. The results are as follows Figure 6As shown, it is further demonstrated that the chimeric antibody of the present invention can bind to the PDL1 protein on the surface of tumor cells, and its binding is stronger than that of the control antibody 12A4.

[0186] Example 8: Mouse antibody humanization experiment

[0187] Referring to the light chain variable region sequence and heavy chain variable region sequence of the PDL1 antibody, a humanized template with the best match to its non-CDR region was selected. The CDR region of the mouse antibody was transplanted to the selected humanized template to replace the CDR region of the human template to obtain a humanized antibody. Then, based on the three-dimensional structure of the mouse antibody, the buried residues, the residues that directly interacted with the CDR region, and the residues that had an important influence on the conformation of VL and VH were reversely mutated to obtain the humanized antibody. The sequence of the heavy chain variable region of the humanized PDL1 antibody is shown in SEQ ID NO: 8, and the sequence of the light chain variable region is shown in SEQ ID NO: 9.

[0188] Example 9: Humanized PDL1 Antibody Blocking Activity Detection Experiment

[0189] ELISA experiments were used to detect the effect of antibodies on the binding of PDL1 to its receptor PD-1. The specific experiments are as follows:

[0190] Antibody production: The heavy chain and light chain nucleotide sequences of the above-mentioned antibodies were transiently transfected into ExpiCHO-S cells (Gibco, Catalog No. A29127) with pcDNA3.4 vectors (synthesized by Nanjing GenScript, humanized 19B8 heavy chain hIgG1LALA, i.e., h19B8-hIgG1LALA (amino acid sequence as shown in SEQ ID NO: 12) and humanized h19B8 light chain hK, i.e., h19B8-hK (amino acid sequence as shown in SEQ ID NO: 13) to prepare antibodies.

[0191] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATTYSQNFKDRATMTVDTSISTAYMELSRLRSDDTAVYYCVPTGMGYFLMDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:12)

[0192] DIQLTQSPSFLSASVGDRVTITCKASQDVGSAVVWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFTSYPTFGQGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 13)

[0193] One day before transfection, adjust the cell density of ExpiCHO-S cells to (3-4)×10 6 / mL, at 37°C, 8% CO 2 The cells were cultured overnight with shaking at 95 rpm. On the day of transfection, the cells grew to 7×10 6 ~1×10 7 / mL, prepare for transfection when the viability is greater than 95%, and dilute the cells to 6×10 using fresh pre-warmed ExpiCHO medium (Gibco, Catalog No. A2910002). 6 / mL, the pcDNA3.4 plasmid carrying the heavy and light chains (the ratio of light and heavy chain plasmids was 1:1) and ExpiFectamine CHO transfection reagent (Gibco, Cat. No. A29129) were transfected into ExpiCHO-S cells and incubated at 37°C and 8% CO 2 18-22h after transfection, mix ExpiFectamine CHO Enhancer and ExpiCHO Feed and add the transfected cells immediately, mix well, and incubate at 32℃, 5% CO 2 and shake culture at 95rpm. On the 5th day after transfection, add 8mL ExpiCHO Feed to the cells, mix and continue to culture. Observe the changes in cell number and cell viability every day, and harvest the cells by centrifugation after the cell viability drops below 80% or after 10-14 days of culture. The expressed supernatant was filtered with a 0.45μm filter membrane, and the antibody with Fc domain was captured from the expression supernatant using MabselectprismAproteinA affinity chromatography column (purchased from Suzhou Nano) . After balancing the chromatography column with pH7.2 phosphate buffer, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100mM citric acid, pH2.7), and finally concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE to have a purity of more than 95%. The results showed that the above-mentioned antibody was finally obtained.

[0194] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH7.2 PBS containing 0.1% Tween20) buffer, and then 200 μL / well PBS / 10% BSA was added, and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μL / well of the humanized PDL1 antibody h19B8-hIgG1LALA or the control antibody 12A4-hIgG1LALA, Atezulizumab analog (purchased from Baiying Bio), and 2 μg / mL PD-1-Avi tag (purchased from Acro) diluted to an appropriate concentration with PBST / 0.05% BSA were added, and incubated at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled strreptavidin secondary antibody (purchased from Southernbiotech) with PBST / 0.05% BSA at 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μL / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. mIgG (purchased from Biolegend) was used as a control. Read the absorbance at 450 mm using an enzyme reader. The results are as follows Figure 7 The results show that the humanized PDL1 antibody h19B8 of the present invention can block the binding of PDL1 to its receptor PD-1, and its blocking ability is comparable to that of 12A4 and Atezulizumab analogs.

[0195] Example 10: Flow cytometry binding experiment of humanized PDL1 antibody

[0196] Human melanoma A375 cells and human lung adenocarcinoma A549 cells were diluted to 1×10 6 / mL, add to a 1.5mL EP tube at a volume of 100μL / tube, add 10μL / tube of rat serum, and block at 4℃ for 30min. Add different concentrations of humanized antibody h19B8-hIgG1LALA (heavy chain and light chain as shown in SEQ ID NO:12-13) or control antibody 12A4-hIgG1LALA, Atezulizumab analog (purchased from Baiying Biology), and incubate at 4℃ for 30min. Add 1mL PBS to the EP tube, centrifuge at 4℃, 3500rpm×5min, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μL / tube PBS, add 1μL / tube Alexa-647 labeled rat anti-human antibody secondary antibody (Biolegend), and incubate at 4℃ for 30min in the dark. Wash twice with PBS, and discard the supernatant after centrifugation. Resuspend the cells with 200 μL / tube PBS and detect them by flow cytometry. The results are as follows: Figure 8 As shown, it is further shown that the chimeric antibody of the present invention can bind to the PDL1 protein on the surface of tumor cells, and its binding is stronger than that of the control antibody 12A4 and Atezulizumab analogs.

[0197] Example 11: Humanized PDL1 antibody promotes SEB-induced IL-2 secretion in human PBMC

[0198] Dilute human PBMC cells to 2 × 10 cells / mL in complete RPMI-1640 medium. 6 / mL, added to a 96-well plate at a volume of 100 μL / well, added 50 μL / well of different concentrations of humanized antibody h19B8-hIgG1LALA or atezulizumab analogs, and blocked at 4°C for 30 minutes. Add different concentrations of humanized antibody h19B8-hIgG1LALA (shown in SEQ ID NO: 12-13) or control antibody atezulizumab analogs (purchased from Bio-Bio), add 50 μL / well of 0.4 μg / ml SEB, and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator for 72 hours. The IL-2 content in the culture supernatant was detected using a human IL-2 ELISA kit. Fig. 9 As shown, it is further shown that the humanized PDL1 antibody of the present invention can promote IL-2 secretion by PBMC (wherein the PBMC cells in Figure A and Figure B are from different human individuals), and the promoting ability is equivalent to or slightly better than that of the Atezulizumab analog.

[0199] Example 12: CD3×PDL1 antibody ELISA binding experiment

[0200] ELISA experiments were used to detect the binding properties of PDL1 antibodies. PDL1 extracellular domain fusion protein was coated into 96-well plates, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and PDL1.

[0201] PDL1-Fc fusion protein was diluted to 1 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH7.2 PBS containing 0.1% Tween 20) buffer, and then 200 μl / well PBS / 10% BSA was added, and the plate was incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μl / well of the humanized antibody h19B8-hIgG1LALA and the bispecific antibody CD3×h19B8 to be tested diluted to an appropriate concentration with PBST / 0.05% BSA were added, and incubated at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled anti-human IgG-Fab antibody secondary antibody with PBST / 0.05% BSA at 100 μl / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μl / well 4M sulfuric acid to terminate the reaction. Read the absorbance at 450 mm using an enzyme reader. Fig.10 Several connection modes of the CD3×PDL1 bispecific antibody of the present invention are shown. Fig.11 This indicates that the CD3×h19B8 antibody of the present invention can bind to the PDL1 protein, and the binding ability is slightly weaker than that of the h19B8 monoclonal antibody.

[0202] The CD3×h19B8 antibody used in the examples of the present invention (schematic diagram as shown in FIG. Fig.10 The specific sequence information of CD3×12A4 antibody is shown in Table 2.

[0203] Table 2

[0204]

[0205] Example 13: CD3×PDL1 antibody blocking activity detection experiment

[0206] ELISA experiments were used to detect the effect of antibodies on the binding of PDL1 to its receptor PD-1. The specific experiments are as follows:

[0207] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH7.2 PBS containing 0.1% Tween20) buffer, and then 200 μL / well PBS / 10% BSA was added, and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μL / well of the humanized antibody h19B8-hIgG1LALA or bispecific antibody CD3×h19B8 to be tested diluted to an appropriate concentration with PBST / 0.05% BSA, 2 μg / mL PD-1-Avi tag (purchased from Acro) was added, and incubated at 37°C for 1 hour. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled strreptavidin secondary antibody (purchased from Southernbiotech) with PBST / 0.05% BSA at 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate with PBST 6 times, add 80 μL / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. Read the absorbance at 450 mm using an ELISA reader. The results are as follows: Fig.12 The results show that the bispecific antibody CD3×h19B8 of the present invention can block the binding of PDL1 to its receptor PD-1, and the blocking ability is slightly weaker than that of the control Atezulizumab.

[0208] Example 14: CD3×PDL1 antibody promotes PBMC to kill tumor cells

[0209] This example detects the killing of PBMC by bispecific antibodies.

[0210] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0211] (2) Human lung adenocarcinoma A375 cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then incubated at 37°C, 5% CO 2 The xCELLigence RTCAMP device was used to detect the cell coefficient for 24 h under the conditions;

[0212] (3) diluting the bispecific antibody into a series of concentration gradients using complete RPMI-1640 medium, and adding the dilution to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0213] (4) PBMC (Sai ​​Li Biotechnology) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with the added volume being 80 μL / well;

[0214] (5) The reaction system obtained in step (4) was heated at 37°C and 5% CO 2 The xCELLigence RTCAMP instrument was used to detect cell proliferation for 24 h.

[0215] The specific experimental results are as follows Fig.13 As shown, it is further shown that the CD3×h19B8(a) bispecific antibody of the present invention can promote PBMC to kill A375 tumor cells, and its killing ability is better than that of (b) and (c) configurations.

[0216] Example 15: CD3×PDL1 antibody flow cytometry binding experiment

[0217] Human lung adenocarcinoma A549 cells were diluted to 1×10 6 / mL, add it to a 1.5mL EP tube at a volume of 100μL / tube, add 10μL / tube of rat serum, and block it at 4℃ for 30min. Add different concentrations of bispecific antibody CD3×h19B8 or control antibody CD3×12A4, and incubate at 4℃ for 30min. Add 1mLPBS to the EP tube, centrifuge at 4℃, 3500rpm×5min, discard the supernatant, and wash it again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μL / tube PBS, add 1μL / tube Alexa-647-labeled rat anti-human antibody secondary antibody (Biolegend), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μL / tube PBS and detect them by flow cytometry. The results are as follows Fig.14 As shown, it is further shown that the bispecific antibody CD3×h19B8 of the present invention can bind to the PDL1 protein on the surface of tumor cells, and its binding is stronger than that of the control antibody CD3×12A4.

[0218] Example 16: CD3×PDL1 antibody promotes PBMC to kill tumor cells

[0219] This example detects the killing of PBMC by bispecific antibodies.

[0220] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0221] (2) Human lung adenocarcinoma A375 cells were diluted to 2×105 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then incubated at 37°C, 5% CO 2 The xCELLigence RTCAMP device was used to detect the cell coefficient for 24 h under the conditions;

[0222] (3) diluting the bispecific antibody into a series of concentration gradients using complete RPMI-1640 medium, and adding the dilution to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0223] (4) PBMC (Sai ​​Li Biotechnology) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with the added volume being 80 μL / well;

[0224] (5) The reaction system obtained in step (4) was heated at 37°C and 5% CO 2 The xCELLigence RTCAMP instrument was used to detect cell proliferation for 24 h.

[0225] The specific experimental results are as follows Fig.15 As shown, it is further shown that the CD3×h19B8 bispecific antibody of the present invention can promote PBMC to kill A375 tumor cells, and its killing ability is equivalent to or slightly better than that of the control antibody CD3×12A4.

[0226] Example 17: Effect of CD3×PDL1 Antibody on Anti-Cancer in Mice

[0227] The in vivo efficacy experiment is used to detect the anti-cancer function of the bispecific antibody CD3×h19B8 in promoting immune reconstitution mice.

[0228] (1) On day -4, human PBMCs were transfused into NCG mice (purchased from Jicui Yaokang) via tail vein at 1E7 per mouse;

[0229] (2) On day 0, NCG mice were subcutaneously injected with 1×10 6 Personal melanoma A375 cells;

[0230] (2) On days 8, 11, 14, and 17, the mice were injected with the CD3×h19B8(a) antibody via tail vein, 25 μg per mouse;

[0231] (3) Tumor volume was measured every 3 days after injection of the above antibodies.

[0232] The results are as follows Fig.16As shown, it can be seen that the CD3×h19B8 bispecific antibody of the present invention can effectively inhibit the growth of tumors and has anti-cancer function.

[0233] It can be seen from the above experimental results that the PDL1 antibody of the present invention has the following characteristics: stronger binding ability with tumor cells; able to bind to human and monkey PDL1; able to block the binding of PDL1 to its receptor PD-1; able to promote the anti-cancer function of immune cells.

[0234] The present invention provides a PDL1 antibody (h19B8), which binds more strongly to PDL1 on the surface of tumor cells than antibodies 12A4 and Atezolizumab. Moreover, when recombinantly expressed as a CD3×PDL1 bispecific antibody, the CD3×h19B8 antibody of the present invention also has a stronger binding ability than the CD3×12A4 antibody, indicating that the bispecific antibody has stronger tumor recognition and thus induces stronger T cell killing. The present invention also compares the functional activities of CD3×PDL1 antibodies of different structures, and obtains a bispecific antibody with stronger functional activity.

[0235] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0236] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment that specifically binds to PD-L1, characterized in that: include: The heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 shown in SEQ ID NO:2, the heavy chain variable region CDR3 shown in SEQ ID NO:3, the light chain variable region CDR1 shown in SEQ ID NO:4, the light chain variable region CDR2 shown in WAS, and the light chain variable region CDR3 shown in SEQ ID NO:

5.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that include: The heavy chain variable region shown in SEQ ID NO:6, and the light chain variable region shown in SEQ ID NO:7; or The heavy chain variable region is shown in SEQ ID NO:8, and the light chain variable region is shown in SEQ ID NO:

9.

3. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 2, characterized in that The monoclonal antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region comes from at least one of a human antibody and a mouse antibody; the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

4. The monoclonal antibody or antigen-binding fragment according to claim 3, characterized in that: The light chain constant region and the heavy chain constant region are both derived from mouse IgG antibody or human IgG antibody.

5. The monoclonal antibody or antigen-binding fragment according to claim 4, characterized in that: The light chain constant region and the heavy chain constant region are both derived from mouse IgG1 antibody or human IgG1 antibody.

6. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 2, characterized in that The monoclonal antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a primate antibody.

7. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO: 10 and a light chain with the amino acid sequence shown in SEQ ID NO: 11; or The monoclonal antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:12 and a light chain with an amino acid sequence shown in SEQ ID NO:

13.

8. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that The monoclonal antibody or antigen-binding fragment includes at least one of a full-length antibody, Fv, a single-chain antibody, and a Fab.

9. A bispecific binding molecule, characterized in that include: a first binding region, the first binding region comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8; and The second binding region has CD3 binding activity.

10. The bispecific binding molecule according to claim 9, characterized in that The bispecific binding molecules include symmetric bispecific binding molecules or asymmetric bispecific binding molecules.

11. The bispecific binding molecule according to claim 9, characterized in that The first binding region comprises peptide chain 1 and peptide chain 2, The peptide chain 1 has the amino acid sequence shown in SEQ ID NO: 12, and the peptide chain 2 has the amino acid sequence shown in SEQ ID NO: 13; The peptide chain 1 and the peptide chain 2 are connected via a disulfide bond.

12. The bispecific binding molecule according to claim 9, characterized in that The second binding region comprises at least one of a full-length antibody, Fv, a single-chain antibody, Fab, and a single-domain antibody having CD3 binding activity.

13. The bispecific binding molecule according to claim 9, characterized in that The second binding region comprises an anti-CD3 single chain antibody.

14. The bispecific binding molecule according to claim 13, characterized in that The anti-CD3 single-chain antibody comprises an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, wherein the anti-CD3 antibody heavy chain variable region CDR1 sequence is as shown in SEQ ID NO: 15, the heavy chain variable region CDR2 sequence is as shown in SEQ ID NO: 16, and the heavy chain variable region CDR3 sequence is as shown in SEQ ID NO: 17; The light chain variable region CDR1 sequence of the anti-CD3 antibody is shown in SEQ ID NO: 18, the light chain variable region CDR2 sequence is shown in GTN, and the light chain variable region CDR3 sequence is shown in SEQ ID NO:

19.

15. The bispecific binding molecule according to claim 13, characterized in that The anti-CD3 single-chain antibody comprises a heavy chain variable region shown in SEQ ID NO: 20 and a light chain variable region shown in SEQ ID NO:

21.

16. The bispecific binding molecule according to claim 13, characterized in that The anti-CD3 single-chain antibody further comprises a connecting peptide, wherein the N-terminus of the connecting peptide is connected to the C-terminus of the heavy chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the light chain variable region of the anti-CD3 antibody; or the N-terminus of the connecting peptide is connected to the C-terminus of the light chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the heavy chain variable region of the anti-CD3 antibody.

17. The bispecific binding molecule according to claim 16, characterized in that The connecting peptide has an amino acid sequence (GGGGS)n, wherein n is an integer greater than or equal to 1.

18. The bispecific binding molecule according to claim 13, characterized in that The anti-CD3 single-chain antibody has the amino acid sequence shown in SEQ ID NO:

22.

19. The bispecific binding molecule according to claim 17, characterized in that n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

20. An isolated polynucleotide, characterized in that The polynucleotide encodes the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8 or encodes the bispecific binding molecule of any one of claims 9 to 19.

21. An expression vector, characterized in that Carrying the polynucleotide of claim 20.

22. A recombinant cell, characterized in that A method of carrying the polynucleotide of claim 20, the expression vector of claim 21, or capable of expressing the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8, or encoding the bispecific binding molecule of any one of claims 9 to 19.

23. The recombinant cell according to claim 22, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 21 into a host cell.

24. The recombinant cell according to claim 23, characterized in that The recombinant cell is a eukaryotic cell.

25. The recombinant cell according to claim 23, characterized in that The recombinant cell is a mammalian cell.

26. A composition, characterized in that The invention comprises the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 8, the bispecific binding molecule according to any one of claims 9 to 19, the polynucleotide according to claim 20, the expression vector according to claim 21, and the recombinant cell according to any one of claims 22 to 25.

27. A method for preparing the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8 or the bispecific binding molecule of any one of claims 9 to 19, characterized in that: The method comprises culturing the recombinant cell according to any one of claims 22 to 25.

28. A drug, characterized in that The invention comprises the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 8, and the bispecific binding molecule according to any one of claims 9 to 19.

29. A kit, characterized in that The kit contains the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 8 and the bispecific binding molecule according to any one of claims 9 to 19.

30. Use of the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8 in the preparation of a medicament for treating melanoma or lung adenocarcinoma.

31. Use of the bispecific binding molecule of claim 14 or 15 in the preparation of a medicament for treating melanoma or lung adenocarcinoma.

32. Use of the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8 in the preparation of a kit for detecting PDL1.

33. Use of the bispecific binding molecule according to any one of claims 9 to 19 in the preparation of a kit for detecting PDL1 and / or CD3.

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