WO2023046113A1 - Anti-human pd-l1 humanized antibody or antigen-binding fragment thereof, and application thereof - Google Patents

Abstract

Provided are an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof, and an application thereof, which relate to the technical field of biomedicine. The antibody or the antigen-binding fragment thereof has high affinity and strong binding specificity with PD-L1, can effectively block the binding of PD-L1 with PD-1 and CD80, stimulates the production of cytokines, has a remarkable anti-tumor effect, and can be used alone or in combination with other reagents to prevent or treat immune diseases or tumor-related diseases.

Description

A kind of anti-human PD-L1 humanized antibody or its antigen-binding fragment and application thereof

priority statement

This application claims the priority of the Chinese invention patent application with the application number 202111123520.8, the application date is September 24, 2021, and the invention name is an anti-human PD-L1 humanized antibody or its antigen-binding fragment and its application. The entire contents are incorporated herein by reference.

technical field

The present invention relates to the technical field of biomedicine, and more specifically, to an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof and applications thereof.

Background technique

Programmed cell death protein 1 (PD-1, also known as CD279) is a costimulatory receptor expressed on the surface of antigen-stimulated T cells. PD-L1 (CD274) and PD-L2 (CD273) are two ligands of PD-1. PD-L1 is expressed in hematopoietic cells including T cells, B cells, macrophages, dendritic cells, giant cells, and vascular endothelial cells, keratinocytes, islet cells, astrocytes, placental syncytocyte trophoblast, Non-hematopoietic cells including corneal epidermis and endothelial cells. PD-L1 and PD-L2 are expressed in a variety of tumor cells and tumor stroma. Both PD-1 and PD-L1 belong to type I transmembrane proteins in the immune superfamily proteins, which consist of Ig-V and Ig-C-like extracellular domains, transmembrane domains, and short-sequence intracellular domains. The interaction between PD-L1 and the extracellular region of PD-1 can induce conformational changes in PD-1 protein, thereby inducing the intracellular immunoreceptor tyrosine inhibition motif (ITIM) and immunoreceptor tyrosine conversion motif (ITSM). ) is phosphorylated by Src family kinases. The phosphorylated tyrosine motif subsequently recruits SHP-2 and SHP-1 protein tyrosine phosphatases to downregulate T-cell activation signaling. In addition to PD-1, PD-L1 interacts with CD80, thereby transmitting signals that inhibit T cell activity. PD-1 and PD-L1 interactions can downregulate T cell activity in multiple ways, including inhibition of T cell proliferation, cytokine release, and other effector functions.

The interaction of PD-1 and PD-L1 is critical for the homeostasis of the immune system. Different genotypes of PD-1-deficient mice are prone to lupus-like autoimmune disease or fatal autoimmune cardiomyopathy. PD-1-deficient mice have altered thymic T-cell domestication, and PD-L1 blockade breaks tolerance between fetus and mother. At the same time, inhibiting the interaction of PD-1 and PD-L1 enhanced host immunity against pathogens. PD-L1 is expressed in a variety of tumors with poor prognosis (eg, renal cancer, gastric cancer, urothelial cancer, ovarian cancer, and melanoma), and the use of PD-L1 antibodies can kill tumor cells or inhibit the activity of killer T cell CTLs .

Given the importance of PD-1, CD80, and PD-L1 in downregulating immune responses, anti-PD-L1 antibodies that block the interaction of PD-L1 protein with PD-1, CD80 and have a low risk of immunogenicity should be developed to It is of great significance for tumor immunotherapy.

In view of this, the present invention is proposed.

Contents of the invention

The present invention provides an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof. The antibody comprises a heavy chain CDR region and a light chain CDR region. The heavy chain CDR region consists of HCDR1, HCDR2, and HCDR3. The light chain CDR The region is composed of LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NO: 14-16 in sequence, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NO: 17-19 in sequence. The antibody The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 3～7.

The present invention also provides nucleic acids, vectors, and cells related to the antibodies or antigen-binding fragments thereof.

The present invention also provides a method of producing the antibody or antigen-binding fragment thereof.

The present invention also provides pharmaceutical compositions related to the antibodies or antigen-binding fragments thereof.

The present invention also provides the use of the antibody or its antigen-binding fragment, the nucleic acid, the carrier, the cell, and the pharmaceutical composition in the preparation of drugs for preventing or treating immune diseases or tumor-related diseases application.

The present invention also provides a method for preventing, treating or diagnosing tumor or cancer.

Description of drawings

Figure 1 is a diagram of the binding activity of 13 anti-human PD-L1 humanized antibodies to CHO-FP1 empty cells.

Figure 2 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to CHO-mPD-L1 stable cell lines.

Figure 3 is a diagram of the binding activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H2, 176L2H3 to CHO-hPD-L1 stable cell lines.

Figure 4 is a diagram of the binding activity of humanized anti-human PD-L1 antibodies 176L3H2, 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, 176L6H5 to CHO-hPD-L1 stable cell lines.

Figure 5 is a diagram of the binding activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H2, 176L2H3 to CHO-cynoPD-L1 stable cell lines.

Figure 6 is a diagram of the binding activity of anti-human PD-L1 humanized antibodies 176L3H2, 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, 176L6H5 to CHO-cynoPD-L1 stable cell lines.

Figure 7 is a graph showing the binding activity of humanized anti-human PD-L1 antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H3, 176L3H2 to hIFNγ-stimulated A375 cells.

Figure 8 is a graph showing the binding activity of humanized anti-human PD-L1 antibodies 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, 176L6H5 to hIFNγ-stimulated A375 cells.

Figure 9 is a graph showing the blocking activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H3, and 176L3H2 blocking the binding of PD-1 to PD-L1.

Figure 10 is a graph showing the blocking activity of anti-human PD-L1 humanized antibodies 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5 blocking the binding of PD-1 to PD-L1. Figure 11 is a graph showing the blocking activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H3, and 176L3H2 blocking the binding of CD80 to PD-L1.

Figure 12 is a graph showing the activity of blocking the binding of CD80 and PD-L1 by humanized anti-human PD-L1 antibodies 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5.

Figure 13 is a diagram of the in vitro activity of anti-human PD-L1 humanized antibodies 176L2H3, 176L3H2, 176L4H2, and 176L4H4 analyzed by luciferase reporter gene method.

Figure 14 is a graph showing the in vitro activity of anti-human PD-L1 humanized antibodies 176L4H1, 176L4H3, 176L5H2, and 176L6H5 analyzed by luciferase reporter gene method.

Figure 15 is a graph showing the results of IL-2 secretion in mixed lymphocyte reactions mediated by anti-human PD-L1 humanized antibodies 176L4H1 and 176L4H3.

Figure 16 is a graph showing the results of IL-2 secretion in mixed lymphocyte reactions mediated by anti-human PD-L1 humanized antibodies 176L4H2 and 176L6H5.

Figure 17 is a graph showing the results of IL-2 secretion in mixed lymphocyte reactions mediated by anti-human PD-L1 humanized antibodies 176L4H4 and 176L5H2.

Figure 18 is a graph showing the results of secretion of IFNγ in the mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H1 and 176L4H3.

Figure 19 is a graph showing the results of IFNγ secretion in mixed lymphocyte reactions mediated by anti-human PD-L1 humanized antibodies 176L4H2 and 176L6H5.

Figure 20 is a graph showing the results of secretion of IFNγ in the mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H4 and 176L5H2.

Figure 21 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human PD-L1 protein.

Figure 22 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human PD-L2 protein.

Figure 23 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human PD-1 protein.

Figure 24 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human ICOS protein.

Figure 25 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human ICOSLG protein.

Figure 26 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human CD276 protein.

Figure 27 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human CD86 protein.

Figure 28 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human CD28 protein.

Figure 29 is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human CTLA-4 protein.

Figure 30 is a graph showing the in vivo anti-tumor activity results of anti-human PD-L1 humanized antibodies 176L6H5, 176L5H2 and 176L4H1; among them, (a) is the effect of anti-human PD-L1 humanized antibodies on the tumor volume of tumor-bearing mice Result graph; (b) graph is the result graph of the effect of anti-human PD-L1 humanized antibody on the survival rate of tumor-bearing mice; (c) graph is the effect of anti-human PD-L1 humanized antibody on the body weight of tumor-bearing mice Affect the result graph.

Detailed ways

The present invention relates to an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof, the antibody comprises a heavy chain CDR region and a light chain CDR region, the heavy chain CDR region consists of HCDR1, HCDR2, and HCDR3, and the light chain CDR region Composed of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are sequentially selected from SEQ ID NO: 14-16, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are sequentially selected from SEQ ID NO: 17-19, the antibody The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 3-7.

The antibody or antigen-binding fragment thereof of the present invention has at least one of the following characteristics: 1. Blocking the binding of PD-1, CD80 and PD-L1, and CHO stably expressing human PD-L1, and stably expressing rhesus monkey PD-L1 CHO has high binding activity, and the binding kinetic constant with human PD-L1 is at the pM level; 2. It can mediate the production of IL-2 and IFNγ cytokines; 3. It has high specificity and does not bind to other proteins except PD-L1 Binding to B7 family proteins and other related proteins; 4. Has significant anti-tumor activity. Therefore, the antibody or antigen-binding fragment thereof of the present invention can be used alone or in combination with other agents for the prevention or treatment of immune diseases or tumor-related diseases.

In the present invention, the term "antibody or antigen-binding fragment thereof" refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments including complementarity determining regions (CDR regions). "Antibody" specifically refers to a full-length antibody. The term "full-length antibody" includes polyclonal as well as monoclonal antibodies, and the term "antigen-binding fragment" is a substance comprising part or all of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specificity. Bind to antigen. Such fragments are biologically active and thus can bind to the target antigen and can compete with other antigen binding molecules, including intact antibodies, for binding to a given epitope.

In the present invention, the term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the circumstances, the terms "CDR" and "CDRs" are used to mean comprising one or more or even all of the major amino acids that contribute to the binding affinity of an antibody or antigen-binding fragment thereof to the antigen or epitope it recognizes. region of residues.

In the present invention, the complementarity determining region of the heavy chain is represented by HCDR, and the complementarity determining region of the light chain is represented by LCDR. CDR notation methods commonly used in the field include: Kabat numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. In their compilation "Sequences of Proteins of Immunological Interest", the amino acid sequences of the light chain (λ, κ) variable regions and antibody heavy chains, and the variable region of the T cell receptor (α , β, γ, δ) are aligned and numbered. The accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but the CDR region marked by other methods also belongs to the protection scope of the present invention.

In some embodiments, the amino acid sequence of the light chain variable region of the antibody is as shown in any one of SEQ ID NO: 8-13.

In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are shown in any one of the following tables:

Anti-human PD-L1 humanized antibody	heavy chain variable region	light chain variable region
176L1H1	SEQ ID NO.3	SEQ ID NO.8
176L1H2	SEQ ID NO.4	SEQ ID NO.8
176L1H3	SEQ ID NO.5	SEQ ID NO.8
176L2H1	SEQ ID NO.3	SEQ ID NO.9
176L2H2	SEQ ID NO.4	SEQ ID NO.9
176L2H3	SEQ ID NO.5	SEQ ID NO.9
176L3H2	SEQ ID NO.4	SEQ ID NO.10
176L4H1	SEQ ID NO.3	SEQ ID NO.11
176L4H2	SEQ ID NO.4	SEQ ID NO.11
176L4H3	SEQ ID NO.5	SEQ ID NO.11
176L4H4	SEQ ID NO.6	SEQ ID NO.11
176L5H2	SEQ ID NO.4	SEQ ID NO.12
176L6H5	SEQ ID NO.7	SEQ ID NO.13

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is as shown in any one of SEQ ID NO.3-6, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.11, or the antibody The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.4, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.12, or the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO .7. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.13.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.11, or the heavy chain of the antibody can be The amino acid sequence of the variable region is shown in SEQ ID NO.4, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.12, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.7, the light chain variable region The amino acid sequence of the chain variable region is shown in SEQ ID NO.13.

In some embodiments, the antibody contains a heavy chain constant region and a light chain constant region, and the heavy chain constant region sequence is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD Constant region sequence.

In some embodiments, the light chain constant region is a kappa or lambda chain.

In some embodiments, the species sources of the heavy chain constant region and the light chain constant region are selected from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer , mink, chicken, duck, goose or human.

In some embodiments, the antibody is any one or more of bispecific antibodies, CDR-grafted antibodies or multimeric antibodies.

In the present invention, the term "bispecific antibody" is a multispecific antigen binding protein or multispecific antibody, and can be produced by various methods including, but not limited to fusion of hybridomas or linking of Fab' fragments . The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes, present on the same or different protein targets.

In the present invention, the term "CDR-grafted antibody", also referred to as "humanized antibody", specifically refers to an antibody produced by grafting mouse CDR region sequences into human antibody variable region frameworks. The purpose is to overcome the strong immune side effects induced by chimeric antibodies in humans due to carrying a large number of protein components from other species such as mice.

In some embodiments, the antigen-binding fragment is any one or more of scFv, Fab, Fab', F(ab') ₂ and Fv.

In the present invention, the term "F(ab') ₂ " contains two light chains and two heavy chains containing the part between the CH1 and VH domains, so that an interchain disulfide bond is formed between the two heavy chains . The F(ab') ₂ fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker; the term "Fab" refers to an antibody consisting of VL, VH, CL and CH1 domains Fragment; the term "Fab' fragment" means the fragment obtained after reduction of the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, consisting of an intact light chain and the Fd fragment of the heavy chain (consisting of VH and The term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, which is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site.

The invention also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof.

In the present invention, the nucleic acid is usually RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence. DNA is preferably used when it is ligated into a vector. In addition, since antibodies are membrane proteins, the nucleic acid usually carries a signal peptide sequence.

The invention also relates to a vector comprising said nucleic acid.

In the present invention, the term "vector" is a nucleic acid delivery tool into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).

The invention also relates to cells carrying said nucleic acid, containing said vector or expressing said antibody or antigen-binding fragment thereof.

In some embodiments, the cell is a eukaryotic mammalian cell.

In some embodiments, the cells are Chinese hamster CHO cells.

The present invention also relates to a method of producing the antibody or antigen-binding fragment thereof, comprising: culturing the cells in a culture medium; and recovering the antibody or antigen-binding antibody thus produced from the culture medium or from the cultured cells fragment.

The present invention also relates to a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, or said nucleic acid, or said vector or said cell.

In the present invention, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredients to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered.

In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients.

In the present invention, the term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible, used for Extend the shelf life or potency of antibodies.

The present invention also relates to the application of the antibody or its antigen-binding fragment, the nucleic acid, the carrier, the cell, and the pharmaceutical composition in the preparation of drugs for preventing or treating immune diseases or tumor-related diseases .

The present invention also relates to a method for preventing, treating or diagnosing tumors or cancers, the method comprising administering an effective amount of the antibody or antigen-binding fragment thereof, or the pharmaceutical composition to a subject in need.

In some embodiments, the cancer is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, colon cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, gastric cancer, rectal cancer , testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head and neck cancer, gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, or combinations thereof.

The present invention comprises following beneficial effect:

The anti-human PD-L1 humanized antibody or antigen-binding fragment thereof of the present invention has high binding activity to CHO stably expressing human PD-L1 and CHO stably expressing rhesus monkey PD-L1, and has a binding kinetic constant to human PD-L1 At the pM level, it has very good binding activity of blocking PD-1, CD80 and PD-L1, can mediate the production of IL-2 and IFNγ cytokines, has high specificity, and does not bind to other B7 except PD-L1 Combined with family proteins and other related proteins, it has significant anti-tumor activity, which is equivalent to or better than the anti-tumor effect of the positive control antibody; and the immune source of the anti-human PD-L1 humanized antibody obtained after humanization of the present invention Therefore, the antibody or antigen-binding fragment thereof of the present invention can be used alone or in combination with other agents for the prevention or treatment of immune diseases or tumor-related diseases.

Example

Example 1 Preparation of anti-human PD-L1 humanized antibody

1. Humanized antibody design

Anti-human PD-L1 mouse monoclonal antibody in the patent application number 202011541107.9 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2) It has high affinity and specificity to PD-L1 protein, can block the interaction between PD-L1 expressed on the cell surface and PD-1, and shows the biological function activity of stimulating cytokine production; The anti-human PD-L1 mouse monoclonal antibody was humanized, and a total of 5 heavy chain variable regions and 6 light chain variable region sequences were designed, and their amino acid sequences are shown in Table 1 below. Among them, the anti-human PD-L1 mouse monoclonal antibody is hereinafter referred to as 176F9.

Table 1 Amino acid sequence of anti-human PD-L1 humanized antibody

Anti-human PD-L1 humanized antibody	heavy chain variable region	light chain variable region
176L1H1	SEQ ID NO.3	SEQ ID NO.8
176L1H2	SEQ ID NO.4	SEQ ID NO.8
176L1H3	SEQ ID NO.5	SEQ ID NO.8
176L2H1	SEQ ID NO.3	SEQ ID NO.9
176L2H2	SEQ ID NO.4	SEQ ID NO.9
176L2H3	SEQ ID NO.5	SEQ ID NO.9
176L3H2	SEQ ID NO.4	SEQ ID NO.10
176L4H1	SEQ ID NO.3	SEQ ID NO.11
176L4H2	SEQ ID NO.4	SEQ ID NO.11
176L4H3	SEQ ID NO.5	SEQ ID NO.11
176L4H4	SEQ ID NO.6	SEQ ID NO.11
176L5H2	SEQ ID NO.4	SEQ ID NO.12
176L6H5	SEQ ID NO.7	SEQ ID NO.13

2. Production of anti-human PD-L1 humanized antibody

The chimeric antibodies disclosed herein are composed of human IgGl constant domains combined with mouse heavy and light chain variable regions. A secretion signal peptide expression sequence was added to the front of the chimeric antibody expression sequence, cloned into a mammalian expression vector, and transfected into Expi293 cells to generate chimeric (murine-human) antibodies. The culture supernatant containing the chimeric antibody was collected and purified using protein A.

The design of the humanized antibody light and heavy chains disclosed herein follows the generally accepted rules disclosed in: "Protein Sequence and Structure Analysis of Antibody Variable Domains [antibody variable domain protein sequence and structure analysis]", in the following : Antibody Engineering Lab Manual, eds.S.Duebel and R.Kontermann, Springer-Verlag, Heidelberg (2001) (2001)]). Add the secretory signal peptide expression sequence at the front end of the heavy chain expression sequence and light chain expression sequence of the humanized antibody, clone the heavy chain expression sequence and light chain expression sequence connected with the signal peptide sequence into a mammalian expression vector, and transfect Transfected into 293 cells to generate humanized monoclonal antibodies (mAbs). The culture supernatant containing the humanized antibody was collected and purified using protein A.

Expression and purification in Expi293 cells The avelumab positive control antibody used in the examples refers to the human PD-L1 antibody transiently expressed in Expi293 cells (International invention patent WO 2013/079174 A1).

The antibody was transiently expressed in Expi293 cells using vector pCDNA3.4. The heavy and light chains of the antibody were first cloned into separate pCDNA3.4 vectors. Then, the pCDNA3.4 vector carrying the heavy and light chains of antibody molecules was transferred into Expi293 cells by chemical transfection. The chemical transfection reagent used was PEI (purchased from Polysciences), and Expi293 was transiently transfected and cultured according to the protocol provided by the manufacturer.

The day before transient transfection, Expi293 (ThermoFisher Scientific, Cat.A14635) cells were subcultured, Dynamis medium (Gibco, Cat.A2617502) was used to inoculate 1L shake flasks (Corning, Cat.431147) at a density of 2E6, and placed in cell culture Culture in a shaker (Adolf Kuhner, Cat.ISF4-XC) at 37°C, 8% CO ₂ , 120rpm; on the day of transfection, count Expi293 cells with a cell counter (Countstar, Cat.IC1000), and use fresh Dynamis medium Dilute and adjust the cell density to 2.9E6; prepare for transfection; PEI:DNA=3:1; mix for 5 minutes, mix the two gently for 20 times, let stand for 15-30 minutes, not more than 30 minutes. The DNA-PEI mixture was added to Expi293 cells, mixed evenly, and placed in a cell culture shaker (Adolf Kuhner, Cat.ISF4-XC) at 37°C, 8% CO ₂ , 120rpm culture; 4h after transfection, double antibody ( Gibco, Cat.15140122) and anticoagulant (Gibco, Cat.0010057); the supernatant was harvested and purified, and the transfection was continuously cultured for 7 days. After collecting the samples, first centrifuge at low speed 1000rpm, 10min, 4°C (Xiangyi, Cat.H2050R), then high speed 12000rpm, 30min, 4°C; collect the cell culture supernatant and filter at 0.22μm. The culture supernatant was applied to a Protein A Sepharose column (GE Healthcare). The column was washed with PBS, and then the protein was eluted with elution buffer (0.1M sodium citrate buffer, pH 3.0), and the collected fractions were neutralized with 1M Tris pH 9.0. Finally, the purified sample was filtered and sterilized using a 0.22 μm filter membrane (Pall, Cat.4612) to obtain an anti-human PD-L1 humanized antibody.

Example 2 Analysis of Binding Activity of Anti-human PD-L1 Humanized Antibody

1. Binding activity of anti-human PD-L1 humanized antibody to Chinese hamster cells (CHO) and CHO stably expressing full-length mouse PD-L1

The binding activity of the 13 anti-human PD-L1 humanized antibodies prepared in Example 1 to CHO-FP1 empty cells and CHO (CHO-mPD-L1 stable cell line) stably expressing full-length mouse PD-L1 was determined, And set avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-human IgG control antibody, the process is as follows:

Resuspend CHO-FP1 empty cells and CHO-mPD-L1 stable cell lines with 1×FCM buffer (1×PBS+3% BSA) to 2×10E6/ml, and add 100 μl/well into 96-well V-bottom plates. Dilute the anti-human PD-L1 humanized antibody with 1×FCM buffer to a concentration of 20 μg/ml. Add 100 μl/well to the cells, incubate on ice for 30 minutes, centrifuge at 250×g for 5 minutes, and discard the supernatant. After washing twice with 1×FCM, add 100 μl/well of PE antibody human IgG fluorescent secondary antibody diluted with 1×FCM buffer (1:500 dilution factor) (Biolegend, Cat.409304), and incubate on ice for 30 minutes. After centrifugation at 250×g for 5 min, the supernatant was discarded, washed twice with 1×FCM, and 100 μl/well of 1×PBS was added to resuspend the cells, and a flow cytometer (Beckman, CytoFLEX) was used for detection.

The binding activities of anti-human PD-L1 humanized antibodies to CHO-FP1 empty cells and CHO-mPD-L1 stable cell lines are shown in Figure 1 and Figure 2. The results show that: 13 strains of anti-human PD-L1 humanized antibodies Neither binds to CHO-FP1 empty cells nor CHO-mPD-L1 stable cell lines.

2. Binding activity of anti-human PD-L1 humanized antibody to CHO stably expressing human PD-L1

In order to characterize the binding affinity of anti-human PD-L1 humanized antibody to CHO (CHO-hPD-L1 stable cell line) stably expressing human PD-L1, the The 13 strains of anti-human PD-L1 humanized antibodies prepared in Example 1 were diluted 3-fold, with an initial concentration of 30 μg/ml, and then co-incubated with CHO-hPD-L1 stable cell lines. And set avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-human IgG control antibody, the specific method flow is the same as above.

The binding activity of anti-human PD-L1 humanized antibodies to CHO-hPD-L1 stable cell lines is shown in Figure 3 and Figure 4. The results show that: 13 strains of anti-human PD-L1 humanized The binding activity EC50 of stable cell lines is 2-8nM.

3. Binding activity of anti-human PD-L1 humanized antibody to CHO stably expressing rhesus monkey PD-L1

In order to characterize the binding affinity of anti-human PD-L1 humanized antibody to CHO (CHO-cynoPD-L1 stable cell line) stably expressing macaque PD-L1, 1×FCM buffer (1×PBS+3%BSA) was used to The 13 strains of anti-human PD-L1 humanized antibodies prepared in Example 1 were diluted 3-fold, with an initial concentration of 30 μg/ml, and then co-incubated with CHO-cynoPD-L1 stable cell lines. And set avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-human IgG control antibody, the specific method flow is the same as above.

The binding activity of anti-human PD-L1 humanized antibodies to CHO-cynoPD-L1 stable cell lines is shown in Figure 5 and Figure 6. The results show that: 13 strains of anti-human PD-L1 humanized The binding activity EC50 of stable cell lines is 1-12nM.

4. Binding activity of anti-human PD-L1 humanized antibody to A375 cells stimulated by IFNγ

Human IFNγ was used to stimulate the expression of PD-L1 on the surface of tumor cells, and the binding activity of the 13 anti-human PD-L1 humanized antibodies prepared in Example 1 to PD-L1 membrane protein was detected. And set avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-human IgG control antibody, the experimental process is as follows:

Use 50ng/ml human IFNγ (R&D, Cat.285-IF-100) to stimulate the expression of human PD-L1 on human melanoma cells A375 (Shanghai Cell Bank, Cat.SCSP-533) overnight, and the next day, use 0.25% pancreatic A375 cells were digested with enzymes (Gibco, Cat. 25200-056), washed once with DPBS, the density of viable cells was adjusted to 2E6/ml, and 100 μl/well was added to a 96-well V-bottom plate. Dilute the anti-human PD-L1 humanized antibody with 1×FCM buffer to a concentration of 20 μg/ml. Add 100 μl/well to the cells, incubate on ice for 30 minutes, centrifuge at 250×g for 5 minutes, and discard the supernatant. After washing twice with 1×FCM, add 100 μl/well of PE antibody human IgG fluorescent secondary antibody diluted with 1×FCM buffer (1:500 dilution factor) (Biolegend, Cat.409304), and incubate on ice for 30 minutes. After centrifugation at 250×g for 5 minutes, the supernatant was discarded, washed twice with 1×FCM, and 1×PBS was added at 100 μl/well to resuspend the cells, and detected by flow cytometry (Beckman, CytoFLEX).

The binding activity of anti-human PD-L1 humanized antibodies to IFNγ-stimulated A375 cells is shown in Figure 7 and Figure 8. The results show that: 13 anti-human PD-L1 humanized antibodies and IFNγ-stimulated A375 cells expressed PD The binding EC50 of -L1 membrane protein is about 0.2 nM.

5. Binding kinetics analysis of anti-human PD-L1 humanized antibody

The MD ForteBIO QKe platform was used to analyze the binding kinetic constant of anti-human PD-L1 humanized antibody to human PD-L1. The experimental method is as follows:

The human PD-L1 extracellular domain recombinant protein hPD-L1-his carrying his tag was diluted with equilibration buffer (1×PBS+0.02% Tween 20) to a final concentration of 5 μg/ml. The anti-human PD-L1 humanized antibody was diluted 2-fold with equilibration buffer (1×PBS+0.02% Tween 20), the initial concentration was 10 μg/ml, and there were 7 concentrations in total. After the anti-Penta-HIS biosensor (ForteBIO, Cat.18-5122) is fully hydrated, first solidify hPD-L1-his recombinant protein for 150 seconds, and after equilibrating for 90 seconds, combine with anti-human PD-L1 mouse antibody, in which The association time was 180 seconds and the dissociation time was 600 seconds. The whole reaction was carried out at 25,1000rpm. Finally, the Octet analysis software was used for curve fitting to obtain the binding kinetic constant KD of the anti-human PD-L1 humanized antibody.

The analysis results of the binding kinetic constants of 13 anti-human PD-L1 humanized antibodies to human PD-L1 are shown in Table 2. The results show that the binding kinetic constants are all at the pM level.

Table 2

Antibody name	KD, (M)
176L4H1	8.40E-12
176L4H2	8.48E-11
176L4H3	8.00E-11
176L4H4	8.81E-11
176L5H2	1.09E-10
176L6H5	3.02E-11
F016-870-hIgG1	<1.0E-12
Avelumab-hIgG1	<1.0E-12

Example 3 Ligand blocking experiment mediated by anti-human PD-L1 humanized antibody

1. Anti-human PD-L1 humanized antibody binding blocking activity to PD-1

The PD-L1 protein expressed by tumor cells or antigen-presenting cells inhibits the stimulatory activity of lymphocytes by binding to the PD-1 protein expressed on the surface of lymphocytes. In this example, the blocking activity of anti-human PD-L1 humanized antibody to block the combination of PD-1 and PD-L1 was evaluated, and avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-mouse IgG control antibody were set. The experimental method is as follows:

Dilute the anti-human PD-L1 humanized antibody 3-fold with 1×FCM buffer, and the initial concentration is 200 μg/ml. The self-produced recombinant human PD-1 protein carrying mFc was diluted with 1×FCM buffer to a concentration of 4 μg/ml. Resuspend CHO-hPD-L1 cells with 1×FCM buffer to a cell density of 2×10E6/ml, and distribute 100 μl/well in a 96-well V-bottom plate. Antibody was added to CHO-hPD-L1 cells at 50 μl/well, and after incubation on ice for 30 minutes, recombinant human PD-1-mFc protein was added at 50 μl/well, and incubated on ice for 30 minutes. After centrifugation at 250×g for 5 min, discard the supernatant, wash once with 1×FCM, add PE anti-mouse IgG fluorescent secondary antibody diluted with 1×FCM buffer (1:500) at 100 μl/well ( Biolegend, Cat.405307), incubated on ice for 30min. After centrifugation at 250×g for 5 min, the supernatant was discarded, washed twice with 1×FCM, and 100 μl/well of 1×PBS was added to resuspend the cells, and a flow cytometer (Beckman, CytoFLEX) was used for detection.

The blocking activity of anti-human PD-L1 humanized antibody binding to PD-1 is shown in Figure 9 and Figure 10. The results show that all 13 anti-human PD-L1 humanized antibodies can block the binding of PD-1 and PD-1. Binding of L1.

2. Anti-human PD-L1 humanized antibody binding blocking activity to CD80

The PD-L1 protein expressed by tumor cells or antigen-presenting cells inhibits the stimulatory activity of lymphocytes by binding to the CD80 protein expressed on the surface of lymphocytes. In this example, the blocking activity of anti-human PD-L1 humanized antibody to block the binding of CD80 and PD-L1 was evaluated, and avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-mouse IgG control antibody were set. Experimental method as follows:

Dilute the anti-human PD-L1 humanized antibody 3-fold with 1×FCM buffer, and the initial concentration is 200 μg/ml. The self-produced recombinant human CD80 protein carrying mFc was diluted with 1×FCM buffer to a concentration of 24 μg/ml. Resuspend CHO-hPD-L1 cells with 1×FCM buffer to a cell density of 2×10E6/ml, and distribute 100 μl/well in a 96-well V-bottom plate. Antibody was added to CHO-hPD-L1 cells at 50 μl/well, and after incubation on ice for 30 minutes, recombinant human CD80-mFc protein was added at 50 μl/well, and incubated on ice for 30 minutes. After centrifugation at 250×g for 5 min, discard the supernatant, wash once with 1×FCM, add PE anti-mouse IgG fluorescent secondary antibody diluted with 1×FCM buffer (1:500) at 100 μl/well ( Biolegend, Cat.405307), incubated on ice for 30min. After centrifugation at 250×g for 5 min, the supernatant was discarded, washed twice with 1×FCM, and 100 μl/well of 1×PBS was added to resuspend the cells, and a flow cytometer (Beckman, CytoFLEX) was used for detection.

The blocking activity of anti-human PD-L1 humanized antibodies binding to CD80 is shown in Figure 11 and Figure 12. The results showed that all 13 anti-human PD-L1 humanized antibodies could block the binding of PD-L1 and CD80.

Example 4 In vitro activity characterization of anti-human PD-L1 humanized antibody

1. Detection of luciferase reporter gene system under co-incubation of Jurkat-GL4.30-hPD-1 and CHO-hPD-L1-OKT3

In order to detect the in vitro functional activity of anti-human PD-L1 humanized antibody using a luciferase reporter gene system, a Jurkat-GL4.30-hPD-1 effector stably expressing hPD-1 and luciferase genes was constructed by a lentiviral packaging system cell line. At the same time, PD-L1-presenting cells CHO-hPD-L1-OKT3 were constructed, that is, Chinese hamster CHO cells were used to stably express hPD-L1 and antigen-independent TCR cell surface kinesin. On the day of the experiment, resuspend CHO-hPD-L1-OKT3 cells and Jurkat-GL4.30-hPD-1 cells in RPMI1640 complete medium containing 1% FBS, and the viable cell densities were 1.6×10E6/ml and 8.0×10E6/ml, respectively. ml, respectively divided into 96-well fluorescent microplates according to 25 μl/well. Dilute the anti-human PD-L1 humanized antibody with RPMI1640 complete medium containing 1% FBS, the initial concentration is 20 μg/ml, pipette the antibody to Jurkat-GL4.30-hPD-1 and CHO-hPD-1 at 50 μl/well L1-OKT3 mixed cells were mixed, and cultured in a 37° C., 5% CO ₂ cell incubator for 6 hours. Add pre-thawed Bright-LumiTM solution (Biyuntian, Cat.RG051M) at 100 μl/well, and after 5 minutes of resting in the dark, use a multi-functional microplate reader (Molecular Device, SpectraMax i3x multi-functional microplate reader) in Lumi mode. detection. And set hIgG1 isotype control antibody.

The in vitro activity of the anti-human PD-L1 humanized antibody analyzed by the luciferase reporter gene method is shown in Figure 13 and Figure 14. The results show that the anti-human PD-L1 humanized antibody can mediate the luciferase reporter gene signal callback, and was antibody dose-dependent.

2. Stimulate the production of IL-2 and IFN gamma in T-DC allogeneic mixed lymphatic reaction system

The activity of anti-human PD-L1 humanized antibody to stimulate cytokines IL-2 and IFN gamma was evaluated by allogeneic T-DC MLR assay. The experimental procedure is as follows:

LymphoprepTM (Axis-Shield, Cat. 07851) was used to separate peripheral blood lymphocyte PBMC from healthy human peripheral blood. Among them, the PBMC of donor 1 were screened by human CD14 Microbeads (Miltenyi, Cat. 130-050-201) to obtain CD14+ monocytes. Inoculate monocytes into T75 culture flasks at 5×10E5/ml, add cytokines human GM-CSF and IL-4 to a final concentration of 50ng/ml, and after continuous stimulation for 6 days, add human TNF alpha to a final concentration At 50ng/ml, continue to induce differentiation for 3 days to obtain mature DC cells. PBMC from donor 2 were negatively screened by EasySepTM Human T Cell Enrichment Kit (Stemcell, Cat.19051) to obtain CD3+ T cells. According to the DC:T cell ratio of 1:5, the amount of DC cells is 2×10E4/well, the DC and T cells are evenly mixed and distributed in 96-well U-shaped plates, a total of 150 μl/well system. Dilute the anti-human PD-L1 humanized antibody with X-VIVO 15 complete medium, the initial concentration is 20 μg/ml, dilute it 4 times, and add it to the cells at 50 μl/well. After 3-5 days of reaction in the mixed lymphatic experiment, the expression of IL-2 and IFN gamma in the cell supernatant was detected. And set hIgG1 isotype control antibody.

The secretion results of IL-2 in the mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibody are shown in Figure 15, Figure 16 and Figure 17, and the mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibody The secretion results of IFNγ in the reaction are shown in Figure 18, Figure 19 and Figure 20. The results show that: in the T-DC allogeneic mixed lymphoid reaction experiment, the anti-human PD-L1 humanized antibody can mediate the cytokines IL-2 and The up-regulation of IFNγ was positively correlated with the amount of antibody.

Example 5 Analysis of binding specificity of anti-human PD-L1 humanized antibody

PD-L1 protein belongs to the B7 family of proteins, and its same family proteins include PD-L2 (B7-DC), ICOSL (B7-H2), B7-H3, CD80 (B7-1), CD86 (B7-2) and so on. In order to detect the binding specificity of the anti-human PD-L1 humanized antibody, avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and HRP antibody human IgG Fab control antibody were set up. The experimental procedure is as follows:

Dilute recombinant human PD-L1 protein carrying hFc tag with 50mM CB buffer, recombinant human PD-L2 protein (Acrobiosystem, Cat.PD2-H5251), recombinant human PD-1 protein (self-produced), recombinant human ICOS protein (Acrobiosystem , Cat.ICS-H5250), recombinant human ICOSLG protein (Acrobiosystem, Cat.B72-H5254), recombinant human CD276 protein (Acrobiosystem, Cat.B73-H5253), recombinant human CD86 protein (Acrobiosystem, Cat.CD6-H5257), Recombinant human CD28 protein (Acrobiosystem, Cat.CD8-H525a) and recombinant human CTLA-4 protein (Acrobiosystem, Cat.CT4-H5255) to 1 μg/ml, add 100 μl/well into 96-well ELISA detection plate, 2-4°C Pack overnight. Discard the supernatant, add blocking solution (1xPBS+1%BSA) at 200μl/well, and block at 37°C for 1h. Dilute anti-human PD-L1 humanized antibody to 10 μg/ml with PBS containing 1% BSA, and positive control antibodies anti-hPD-1 commercial antibody (Opdivo), anti-ICOS-hIgG4 and anti-hCD28-mIgG2a To 10μg/ml, add 100μl/well, and incubate at 37°C for 30min. Wash 3 times with 1×PBS. Add HRP-labeled goat anti-human IgG Fab (Sigma A0293-1ML) or HRP-labeled goat anti-mouse IgG (Sigma, AP113P) at 100 μl/well, incubate at 37°C for 30 min, wash 3 times with 1×PBS, and carry out Color reaction.

Anti-human PD-L1 humanized antibody and recombinant human PD-L1 protein, recombinant human PD-L2 protein, recombinant human PD-1 protein, recombinant human ICOS protein, recombinant human ICOSLG protein, recombinant human CD276 protein, recombinant human CD86 protein , the binding activities of recombinant human CD28 protein and recombinant human CTLA-4 protein are shown in Figure 21-Figure 29, and the results show that: 13 strains of humanized anti-human PD-L1 antibodies have high specificity, and none of them are compatible with PD-L1 Binding to other B7 family proteins other than L1 and other related proteins.

Example 6 In vivo anti-tumor activity of anti-human PD-L1 humanized antibody

Since the mouse monoclonal antibody of the present invention does not cross with mouse PD-L1, hPD-L1 knock in transgenic mice were used to determine the in vivo anti-tumor effects of anti-human PD-L1 humanized antibodies 176L6H5, 176L5H2 and 176L4H1, and hIgG1 Isotype control antibody, positive control antibody avelumab-hIgG1 and Tecentriq (Roche), the specific method is as follows:

hPD-L1 knock in transgenic mice:

Female C57BL/6 background hPD-L1 knock in transgenic mice (6-8 weeks old) were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., the certificate number is NO.320726210100112386.

To grow cells and inoculate mice:

After knocking out the endogenous mouse PD-L1 gene of MC38 colorectal cancer cells (National Experimental Cell Sharing Resource Platform, resource number: 3111C0001CCC000523) by using sgRNA through CRISPR CAS 9 technology, the mouse PD-L1 completely knocked out MC38 was screened cell. Then, the human PD-L1 cDNA carrying the multi-cloning site MCS was integrated into the MC38 cell line after MC38mPD-L1 knockout by lentiviral transduction, and the MC38 cells (MC38-hPD) stably expressing human PD-L1 were obtained after screening. -L1 cells). MC38-hPD-L1 cells were routinely subcultured for subsequent experiments in mice.

MC38-hPD-L1 cells in the logarithmic growth phase were collected, washed twice with DPBS (Hyclone, Cat.SH30028.02), and the cell concentration was adjusted to 1×10E7 cells/ml with DPBS (Hyclone, Cat.SH30028.02). Female hPD-L1 knock in transgenic mice were taken, and MC38-hPDP-L1 cells were subcutaneously inoculated on the right side with an inoculation volume of 0.1ml/mouse, that is, 1×10E6 cells/mouse. The day of MC38-hPD-L1 cell inoculation was defined as study day 0.

Grouping and administration of tumor-bearing mice:

When the average tumor volume reaches 60-80 mm ³ , the mice can be randomly divided into groups according to the tumor volume. In this study, on the seventh day (D7), the mice were randomly divided into 7 groups according to the tumor volume, with 8 mice in each group. Intraperitoneal administration (ip) was performed. The dosage, method and frequency of administration of MC38-hPD-L1 tumor model are shown in Table 3, where Q3Dx3 means administration once every 3 days, and a total of 3 administrations

table 3

group	Dose (mg/kg)	Dosing volume (μl/g)	Route of administration	Dosing frequency
hIgG1 isotype control	10	10	i.p.	Q3Dx3
176L4H1	10	10	i.p.	Q3Dx3
176L5H2	10	10	i.p.	Q3Dx3
176L6H5	10	10	i.p.	Q3Dx3
avelumab-hIgG1	10	10	i.p.	Q3Dx3
Tecentriq	10	10	i.p.	Q3Dx3
PBS	-	10	i.p.	Q3Dx3

Tumor volumes were measured and recorded from day 7, and tumor volumes and body weights of mice were monitored twice a week for the duration of the study. The long and short diameters of tumors were measured with a vernier caliper. The tumor volume was calculated according to (1/2)×long diameter×(short diameter) ² . The benevolent endpoint was reached when the mouse volume decreased by 20% or the tumor volume exceeded 2000 mm ³ , and the mice were sacrificed by CO ₂ asphyxiation.

The independent sample T test was used for comparison between two groups. One-Way ANOVA was used for comparison of more than 3 groups. If the F value shows a significant difference, time analysis among multiple groups can be performed. Data were processed using GraphPad Prism, when p<0.05 indicated a statistically significant difference. Tumor growth inhibition rate TGI (%)=[1-(Ti-T0)/(Vi-V0)]×100, Ti is the average tumor volume of the treatment group on day i, and T0 is the average tumor volume of the treatment group at the beginning of treatment , Vi is the average tumor volume of the vehicle control group on day i, and V0 is the average tumor volume of the vehicle control group at the beginning of treatment.

The effect of anti-human PD-L1 humanized antibody on the tumor volume of mice is shown in Table 4, and the in vivo anti-tumor activity results of anti-human PD-L1 humanized antibody are shown in Figure 30, where (a) is The effect of anti-human PD-L1 humanized antibody on the tumor volume of mice; (b) is the effect of anti-human PD-L1 humanized antibody on the survival rate of mice; (c) is the effect of anti-human PD-L1 The effect of L1 humanized antibody on the body weight of mice; the results show that the anti-human PD-L1 humanized antibody in this example has a significant anti-tumor effect, and the TGI on day 32 are 104.86%, 102.16% and 85.08 respectively %. Among the 8 mice, the tumor eradication (CR) mice were 8, 5 and 5 respectively (ie, CR were 8/8, 5/8 and 5/8, respectively). The TGIs of the positive control antibodies Avelumab-hIgG1 and Tecentriq (Roche) were 100.42% and 75.22%, and the CRs were 7/8 and 4/8, respectively. Anti-human PD-L1 humanized antibody significantly prolongs the survival period of mice, and each administration group has no significant effect on the body weight of mice.

Table 4

Note: The unit of tumor volume in the table is mm ³ ; "N/A" means that the mouse was killed by CO ₂ asphyxiation because the tumor volume of the mouse exceeded 2000 mm ³ and reached the benevolent end point.

Claims (15)

1. An anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof, characterized in that the antibody comprises a heavy chain CDR region and a light chain CDR region, the heavy chain CDR region is composed of HCDR1, HCDR2, and HCDR3, and the light chain CDR region is composed of HCDR1, HCDR2, and HCDR3. The region is composed of LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NO: 14-16 in sequence, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NO: 17-19 in sequence. The antibody The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 3～7.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain variable region of the antibody is as shown in any one of SEQ ID NO: 8-13.
3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are shown in any one of the following tables:

   Anti-human PD-L1 humanized antibody heavy chain variable region light chain variable region 176L1H1 SEQ ID NO.3 SEQ ID NO.8 176L1H2 SEQ ID NO.4 SEQ ID NO.8 176L1H3 SEQ ID NO.5 SEQ ID NO.8 176L2H1 SEQ ID NO.3 SEQ ID NO.9 176L2H2 SEQ ID NO.4 SEQ ID NO.9 176L2H3 SEQ ID NO.5 SEQ ID NO.9 176L3H2 SEQ ID NO.4 SEQ ID NO.10 176L4H1 SEQ ID NO.3 SEQ ID NO.11 176L4H2 SEQ ID NO.4 SEQ ID NO.11 176L4H3 SEQ ID NO.5 SEQ ID NO.11 176L4H4 SEQ ID NO.6 SEQ ID NO.11 176L5H2 SEQ ID NO.4 SEQ ID NO.12 176L6H5 SEQ ID NO.7 SEQ ID NO.13
4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the amino acid sequence of the heavy chain variable region of the antibody is as shown in any of SEQ ID NO.3-6, and the light The amino acid sequence of the chain variable region is shown in SEQ ID NO.11, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.12 Shown, or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.13.
5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the amino acid sequence of the heavy chain variable region of the antibody is such as SEQ ID NO.3, the amino acid sequence of the light chain variable region The sequence is as shown in SEQ ID NO.11, or the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.12, or the described The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.13.
6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody contains a heavy chain constant region and a light chain constant region, and the sequence of the heavy chain constant region is selected from IgG1, IgG2 , IgG3, IgG4, IgA, IgM, IgE, IgD any one of the constant region sequence;

   The light chain constant region is a kappa or lambda chain;

   The species sources of the heavy chain constant region and the light chain constant region are selected from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks , goose or man.
7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody is any one or more of bispecific antibodies, CDR-grafted antibodies or multimeric antibodies; The antigen-binding fragment is any one or more of scFv, Fab, Fab', F(ab') ₂ and Fv.
8. A nucleic acid, characterized in that the nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-7.
9. A vector, characterized in that the vector comprises the nucleic acid according to claim 8.
10. The cell, characterized in that the cell carries the nucleic acid according to claim 8, contains the vector according to claim 8, or expresses the antibody or antigen-binding fragment thereof according to any one of claims 1-7.
11. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-7, comprising: cultivating the cell according to claim 10 in a culture medium; and recovering such from the culture medium or from the cultured cells Antibodies or antigen-binding fragments thereof are produced.
12. A pharmaceutical composition, characterized in that the composition contains the antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the nucleic acid according to claim 8, or the carrier according to claim 9, or the vector according to claim 10. said cells.
13. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid according to claim 8, the carrier according to claim 9, the cell according to claim 10, and the pharmaceutical composition according to claim 12 are used in the preparation of Application in drugs for preventing or treating immune diseases or tumor-related diseases.
14. A method for preventing, treating or diagnosing tumors or cancers, characterized in that an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the drug according to claim 12 is administered to a subject in need combination.
15. The method according to claim 14, wherein the cancer is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, colon cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer , liver cancer, gastric cancer, rectal cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head and neck cancer, gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, or combinations thereof.

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