An anti-human PD-L1 humanized antibody or its antigen-binding fragment and its application

Abstract

The present invention relates to the field of biomedicine, and more specifically, to a humanized anti-human PD-L1 antibody or antigen-binding fragment thereof and its use. The antibody or antigen-binding fragment thereof of the present invention has high affinity and strong binding specificity for PD-L1, effectively blocks the binding of PD-L1 to PD-1 and CD80, stimulates cytokine production, and exhibits significant anti-tumor effects. It can be used alone or in combination with other agents to prevent or treat immune disorders or tumor-related diseases.

Description

[0001] Priority Declaration

[0002] This application claims priority to the Chinese invention patent application with application number 202111123520.8, filing date September 24, 2021, entitled "An anti-human PD-L1 humanized antibody or antigen-binding fragment thereof and its application," the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine technology, and more specifically, to an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0004] Programmed cell death protein 1 (PD-1, also known as CD279) is a co-stimulatory receptor expressed on the surface of antigen-stimulated T cells. PD-L1 (CD274) and PD-L2 (CD273) are the two ligands for PD-1. PD-L1 is expressed on hematopoietic cells, including T cells, B cells, macrophages, dendritic cells, and giant cells, as well as non-hematopoietic cells, including vascular endothelial cells, keratinocytes, pancreatic islet cells, astrocytes, placental syncytial trophoblasts, corneal epithelium, and endothelial cells. PD-L1 and PD-L2 are expressed on a variety of tumor cells and tumor stroma. Both PD-1 and PD-L1 belong to the immune superfamily of proteins and are type I transmembrane proteins. They are composed of Ig-V and Ig-C-like extracellular domains, a transmembrane domain, and a short intracellular domain. The interaction between PD-L1 and the PD-1 extracellular domain induces conformational changes in the PD-1 protein, leading to phosphorylation of the intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based translation motif (ITSM) by Src family kinases. The phosphorylated tyrosine motifs subsequently recruit SHP-2 and SHP-1 protein tyrosine phosphatases to downregulate T cell activation signals. In addition to PD-1, PD-L1 interacts with CD80, thereby transmitting signals that inhibit T cell activity. The interaction between PD-1 and PD-L1 can downregulate T cell activity in multiple ways, including inhibiting T cell proliferation, cytokine release, and other effector functions.

[0005] The interaction between PD-1 and PD-L1 is crucial for the homeostasis of the immune system. PD-1-deficient mice of different genotypes tend to develop lupus-like autoimmune diseases or fatal autoimmune cardiomyopathy. PD-1-deficient mice have altered thymic T cell domestication, and PD-L1 blockade can disrupt tolerance between the fetus and the mother. At the same time, inhibiting the interaction between PD-1 and PD-L1 enhances host immunity against pathogens. PD-L1 is expressed on a variety of tumors with poor prognosis (e.g., renal cancer, gastric cancer, urothelial carcinoma, ovarian cancer, and melanoma), and the use of PD-L1 antibodies can kill tumor cells or inhibit the activity of cytotoxic T cells (CTLs).

[0006] Given the importance of PD-1, CD80, and PD-L1 in downregulating immune responses, it is of great significance to develop anti-PD-L1 antibodies that block the interaction between PD-L1 protein and PD-1 and CD80 while having a low risk of immunogenicity, so that they can be used in tumor immunotherapy.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The present invention provides an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof, wherein 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 consists of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 14 to 16, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 17 to 19, respectively. The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs: 3 to 7.

[0009] GDSITSGYWN (SEQ ID NO: 14)

[0010] YISYTGSTYQNPSLKS(SEQ ID NO:15)

[0011] SRAWIRTYFDY (SEQ ID NO: 16)

[0012] SVSSSISSSNLH (SEQ ID NO: 17)

[0013] GTSNLAS (SEQ ID NO: 18)

[0014] QQWSSYPLT (SEQ ID NO: 19)

[0015] QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:3)

[0016] QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKKLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:4)

[0017] QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKKLEYIGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:5)

[0018] QVQLQESGPGLVKPSETLSLTCSVYGDSITSGYWNWIRKPPGKKLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:6)

[0019] EVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTATYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:7)

[0020] The present invention also provides nucleic acids, vectors, and cells related to the antibody or antigen-binding fragment thereof.

[0021] The present invention also provides a method for producing the antibody or antigen-binding fragment thereof.

[0022] The present invention also provides pharmaceutical compositions related to the antibody or antigen-binding fragment thereof.

[0023] The present invention also provides use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell, and the pharmaceutical composition in preparing a drug for preventing or treating immune diseases or tumor-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to CHO-FP1 empty cells.

[0025] Figure 2 This is a graph of the binding activity of 13 anti-human PD-L1 humanized antibodies to the CHO-mPD-L1 stable cell line.

[0026] Figure 3 This is a graph showing the binding activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H2, and 176L2H3 to CHO-hPD-L1 stable cell lines.

[0027] Figure 4 This is a graph showing the binding activity of anti-human PD-L1 humanized antibodies 176L3H2, 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5 to CHO-hPD-L1 stable cell lines.

[0028] Figure 5 This is a graph of the binding activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H2, and 176L2H3 to the CHO-cynoPD-L1 stable cell line.

[0029] Figure 6 This is a graph showing the binding activity of anti-human PD-L1 humanized antibodies 176L3H2, 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5 to CHO-cynoPD-L1 stable cell lines.

[0030] Figure 7 This is a graph showing the binding activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H3, and 176L3H2 to A375 cells stimulated with hIFNγ.

[0031] Figure 8 This is a graph showing the binding activity of anti-human PD-L1 humanized antibodies 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5 to hIFNγ-stimulated A375 cells.

[0032] Figure 9 This is a graph showing the blocking activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H3, and 176L3H2 in blocking the binding of PD-1 to PD-L1.

[0033] Figure 10 This is a graph showing the blocking activity of anti-human PD-L1 humanized antibodies 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5 in blocking the binding of PD-1 to PD-L1. Figure 11 This is a graph showing the blocking activity of anti-human PD-L1 humanized antibodies 176L1H1, 176L1H2, 176L1H3, 176L2H1, 176L2H3, and 176L3H2 in blocking the binding of CD80 to PD-L1.

[0034] Figure 12 This is a diagram of the blocking activity of anti-human PD-L1 humanized antibodies 176L4H1, 176L4H2, 176L4H3, 176L4H4, 176L5H2, and 176L6H5 in blocking the binding of CD80 to PD-L1.

[0035] Figure 13 This figure shows the in vitro activity of anti-human PD-L1 humanized antibodies 176L2H3, 176L3H2, 176L4H2, and 176L4H4 analyzed using the luciferase reporter gene assay.

[0036] Figure 14 This figure shows the in vitro activity of anti-human PD-L1 humanized antibodies 176L4H1, 176L4H3, 176L5H2, and 176L6H5 analyzed using the luciferase reporter gene assay.

[0037] Figure 15 This figure shows the results of IL-2 secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H1 and 176L4H3.

[0038] Figure 16 This figure shows the results of IL-2 secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H2 and 176L6H5.

[0039] Figure 17 This figure shows the results of IL-2 secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H4 and 176L5H2.

[0040] Figure 18This figure shows the results of IFNγ secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H1 and 176L4H3.

[0041] Figure 19 This figure shows the results of IFNγ secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H2 and 176L6H5.

[0042] Figure 20 This figure shows the results of IFNγ secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibodies 176L4H4 and 176L5H2.

[0043] Figure 21 This is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human PD-L1 protein.

[0044] Figure 22 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human PD-L2 protein.

[0045] Figure 23 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human PD-1 protein.

[0046] Figure 24 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human ICOS protein.

[0047] Figure 25 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human ICOSLG protein.

[0048] Figure 26 This is a graph showing the binding activity of 13 anti-human PD-L1 humanized antibodies to recombinant human CD276 protein.

[0049] Figure 27 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human CD86 protein.

[0050] Figure 28 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human CD28 protein.

[0051] Figure 29 This is a graph showing the binding activity of 13 humanized anti-human PD-L1 antibodies to recombinant human CTLA-4 protein.

[0052] Figure 30These are the results of the in vivo anti-tumor activity of the anti-human PD-L1 humanized antibodies 176L6H5, 176L5H2 and 176L4H1; among them, (a) is the result of the effect of the anti-human PD-L1 humanized antibodies on the tumor volume of tumor-bearing mice; (b) is the result of the effect of the anti-human PD-L1 humanized antibodies on the survival rate of tumor-bearing mice; (c) is the result of the effect of the anti-human PD-L1 humanized antibodies on the body weight of tumor-bearing mice. DETAILED DESCRIPTION

[0053] The present invention relates to an anti-human PD-L1 humanized antibody or an antigen-binding fragment thereof, wherein 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 consists of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 14 to 16, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 17 to 19, respectively. The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs: 3 to 7.

[0054] GDSITSGYWN (SEQ ID NO: 14)

[0055] YISYTGSTYQNPSLKS(SEQ ID NO:15)

[0056] SRAWIRTYFDY (SEQ ID NO: 16)

[0057] SVSSSISSSNLH (SEQ ID NO: 17)

[0058] GTSNLAS (SEQ ID NO: 18)

[0059] QQWSSYPLT (SEQ ID NO: 19)

[0060] QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:3)

[0061] QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKKLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:4)

[0062] QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKKLEYIGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:5)

[0063] QVQLQESGPGLVKPSETLSLTCSVYGDSITSGYWNWIRKPPGKKLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:6)

[0064] EVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTATYYCARSRAWIRTYFDYWGQGTLVTVSS(SEQ ID NO:7)

[0065] The antibodies or antigen-binding fragments of the present invention possess at least one of the following properties: 1. Blocking the binding of PD-1, CD80, and PD-L1, with high binding activity to CHO cells stably expressing human PD-L1 and CHO cells stably expressing macaque PD-L1, with binding kinetic constants for human PD-L1 in the pM range; 2. Mediating the production of IL-2 and IFNγ cytokines; 3. High specificity, not binding to other B7 family proteins and related proteins besides PD-L1; and 4. Significant anti-tumor activity. Therefore, the antibodies or antigen-binding fragments of the present invention can be used alone or in combination with other agents to prevent or treat immune diseases or tumor-related diseases.

[0066] 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 that contain a complementary determining region (CDR region). "Antibody" refers specifically to a full-length antibody. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antigen-binding fragment" refers to a substance that contains part or all of the antibody CDRs, which lacks at least some of the amino acids present in the full-length chain but is still able to specifically bind to the antigen. Such fragments are biologically active and can therefore bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope.

[0067] In the present invention, the term "complementarity determining region" or "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to the region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment thereof to its recognized antigen or epitope.

[0068] In the present invention, the complementarity determining regions of the heavy chain are represented by HCDRs, and the complementarity determining regions of the light chain are represented by LCDRs. Commonly used CDR labeling methods in the art include: the Kabat numbering scheme, the Chothia and Lesk numbering scheme, and the 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 of "Sequences of Proteins of Immunological Interest", the amino acid sequences of light chain (λ, κ) variable regions and antibody heavy chains, as well as the variable regions of T cell receptors (α, β, γ, δ) were aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be the widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0069] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in any one of SEQ ID NOs: 8 to 13.

[0070] EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKLWIYGTSNLASGVPSRFSGSGSGTDYTLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKR(SEQ ID NO:8)

[0071] EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKPWIYGTSNLASGVPSRFSGSGSGTDYTLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKR(SEQ ID NO:9)

[0072] EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKPWIYGTSNLASGVPSRFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKR(SEQ ID NO:10)

[0073] DIQMTQSPSSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSYPLTFGQGTKLEIKR(SEQ ID NO:11)

[0074] EIVLTQSPSSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSYPLTFGQGTKLEIKR(SEQ ID NO:12)

[0075] EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKLLIYGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKR(SEQ ID NO:13)

[0076] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are as shown in any one of the following tables:

[0077] Humanized anti-human PD-L1 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

[0078] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs. 3 to 6, and the amino acid sequence of the light 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, 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.

[0079] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.11, or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.12, or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13.

[0080] 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 the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0081] In some embodiments, the light chain constant region is a kappa or lambda chain.

[0082] In some embodiments, the species origin of the heavy chain constant region and the light chain constant region is selected from any one of cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

[0083] In some embodiments, the antibody is any one or more of a bispecific antibody, a CDR-grafted antibody, or a multimeric antibody.

[0084] As used herein, the term "bispecific antibody" refers to a multispecific antigen-binding protein or antibody that can be produced by a variety of 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 to two different epitopes, either on the same or different protein targets.

[0085] In this invention, the term "CDR-grafted antibody," also known as "humanized antibody," specifically refers to an antibody produced by transplanting mouse CDR sequences into a human antibody variable region framework. This is done to overcome the strong immune side effects that chimeric antibodies induce in humans due to the presence of a large number of protein components from other species, such as mouse.

[0086] In some embodiments, the antigen-binding fragment is any one or more of scFv, Fab, Fab', F(ab')2 and Fv.

[0087] In the present invention, the term "F(ab')2" contains two light chains and two heavy chains containing the portion between the CH1 and VH domains, so that an interchain disulfide bond is formed between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.

[0088] The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker; the term "Fab" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains); 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 minimum antibody fragment that can form a complete antigen-binding site.

[0089] The present invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof.

[0090] In the present invention, nucleic acids are typically RNA or DNA. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. When incorporated into a vector, DNA is preferably used. In addition, since antibodies are membrane proteins, nucleic acids typically carry a signal peptide sequence.

[0091] The present invention also relates to a vector comprising the nucleic acid.

[0092] In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as 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 are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0093] The present invention also relates to cells, which carry the nucleic acid, contain the vector, or express the antibody or antigen-binding fragment thereof.

[0094] In some embodiments, the cell is a eukaryotic mammalian cell.

[0095] In some embodiments, the cells are Chinese hamster CHO cells.

[0096] The present invention also relates to a method for producing the antibody or antigen-binding fragment thereof, comprising: culturing the cells in a culture medium; and recovering the antibody or antigen-binding fragment thereof produced from the culture medium or from the cultured cells.

[0097] The present invention also relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector or the cell.

[0098] In the present invention, the term "pharmaceutical composition" is in a form that permits the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition would be administered.

[0099] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0100] 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 and serve to prolong the shelf life or effectiveness of the antibody.

[0101] The present invention also relates to the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell, and the pharmaceutical composition in the preparation of drugs for preventing or treating immune diseases or tumor-related diseases.

[0102] The present invention includes the following beneficial effects:

[0103] The anti-human PD-L1 humanized antibody or its antigen-binding fragment of the present invention has high binding activity to CHO cells stably expressing human PD-L1 and CHO cells stably expressing macaque PD-L1, and the binding kinetic constant to human PD-L1 is at the pM level. It has excellent blocking activity for the binding of PD-1, CD80 and PD-L1, can mediate the production of IL-2 and IFNγ cytokines, has high specificity, does not bind to other B7 family proteins and other related proteins except PD-L1, has significant anti-tumor activity, and the anti-tumor effect is equivalent to or better than that of the positive control antibody; and the anti-human PD-L1 humanized antibody obtained after humanization of the present invention has a low immunogenic risk; therefore, the antibody or its antigen-binding fragment of the present invention can be used alone or in combination with other agents to prevent or treat immune diseases or tumor-related diseases.

[0104] Example

[0105] Example 1 Preparation of humanized anti-human PD-L1 antibody

[0106] 1. Humanized Antibody Design

[0107] The anti-human PD-L1 mouse monoclonal antibody in 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) has high affinity and specificity for the PD-L1 protein, can block the interaction between PD-L1 expressed on the cell surface and PD-1, and exhibits biological functional activity of stimulating cytokine production; therefore, in this example, the above anti-human PD-L1 mouse monoclonal antibody was humanized, and a total of 5 heavy chain variable region and 6 light chain variable region sequences were designed, whose 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.

[0108] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYTGSTYQNPSLKSRISFTRDTSKNQYYLQLSSVTTEDTATYYCARSRAWIRTYFDYWGQGTTLTVSS(SEQ ID NO:1)

[0109] EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGAGTKLELKR(SEQ ID NO:2)

[0110] Table 1 Amino acid sequences of humanized anti-human PD-L1 antibodies

[0111] Humanized anti-human PD-L1 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

[0112] 2. Production of humanized anti-human PD-L1 antibodies

[0113] The chimeric antibodies disclosed herein are composed of a human IgG1 constant domain combined with mouse heavy and light chain variable regions. A secretion signal peptide expression sequence is added to the front of the chimeric antibody expression sequence, cloned into a mammalian expression vector, and transfected into Expi293 cells to produce chimeric (murine-human) antibodies. The culture supernatant containing the chimeric antibody is collected and purified using Protein A.

[0114] 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 Engineering Lab Manual, eds. S. Duebel and R. Kontermann, Springer-Verlag, Heidelberg (2001)). A secretory signal peptide expression sequence is added to the front end of the heavy and light chain expression sequences of the humanized antibody. The heavy and light chain expression sequences, followed by the signal peptide sequences, are cloned into a mammalian expression vector and transfected into 293 cells to produce humanized monoclonal antibodies (mAbs). The culture supernatant containing the humanized antibody is collected and purified using protein A.

[0115] Expression in Expi293 Cells and Purification The avelumab positive control antibody used in the Examples refers to a human PD-L1 antibody transiently expressed in Expi293 cells (International Invention Patent WO 2013 / 079174 A1).

[0116] The antibody was transiently expressed in Expi293 cells using the pCDNA3.4 vector. The heavy and light chains of the antibody were first cloned into separate pCDNA3.4 vectors. The pCDNA3.4 vectors carrying the heavy and light chains of the antibody were then chemically transfected into Expi293 cells using PEI (purchased from Polysciences). Transient transfection was performed using the manufacturer's protocol.

[0117] The day before transient transfection, Expi293 cells (ThermoFisher Scientific, Cat. A14635) were passaged and inoculated at a density of 2E6 in a 1L shake flask (Corning, Cat. 431147) with Dynamis medium (Gibco, Cat. A2617502). The cells were then cultured in a cell culture shaker (Adolf Kuhner, Cat. ISF4-XC) at 37°C, 8% CO2, and 120 rpm. On the day of transfection, Expi293 cells were counted using a cell counter (Countstar, Cat. IC1000) and diluted with fresh Dynamis medium to adjust the cell density to 2.9E6. Prepare for transfection using a PEI:DNA ratio of 3:1. Mix for 5 minutes, gently mix 20 times, and let stand for 15-30 minutes (no more than 30 minutes). The DNA-PEI mixture was added to Expi293 cells, mixed thoroughly, and incubated in a cell culture shaker (Adolf Kuhner, Cat. ISF4-XC) at 37°C, 8% CO2, and 120 rpm. Four hours after transfection, a double-antibody (Gibco, Cat. 15140122) and anticoagulant (Gibco, Cat. 0010057) were added. The supernatant was harvested and purified, and the cells were cultured continuously for 7 days. The cells were then harvested and centrifuged at a low speed of 1000 rpm for 10 minutes at 4°C (Xiangyi, Cat. H2050R), followed by a high speed of 12000 rpm for 30 minutes at 4°C. The cell culture supernatant was collected and filtered through a 0.22 μm filter. The culture supernatant was applied to a Protein A Sepharose column (GE Healthcare). The column was washed with PBS, and the protein was eluted with elution buffer (0.1 M sodium citrate buffer, pH 3.0). The collected fractions were neutralized with 1 M Tris pH 9.0. Finally, the purified sample was sterilized by filtration using a 0.22 μm filter membrane (Pall, Cat. 4612) to obtain an anti-human PD-L1 humanized antibody.

[0118] Example 2 Binding Activity Analysis of Anti-Human PD-L1 Humanized Antibody

[0119] 1. Binding activity of humanized anti-human PD-L1 antibodies to Chinese hamster cells (CHO) and CHO cells stably expressing full-length mouse PD-L1

[0120] The binding activity of the 13 anti-human PD-L1 humanized antibodies prepared in Example 1 to CHO-FP1 empty cells and CHO stably expressing full-length mouse PD-L1 (CHO-mPD-L1 stable cell line) was determined, and avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody and PE anti-human IgG control antibody were set. The process is as follows:

[0121] Resuspend CHO-FP1 cells and CHO-mPD-L1 stable cell lines in 1× FCM buffer (1× PBS + 3% BSA) to 2×10E6 / ml and add 100 μl / well to a 96-well V-bottom plate. Dilute anti-human PD-L1 humanized antibody in 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 (1:500 dilution) (Biolegend, Cat. 409304) diluted in 1× FCM buffer and incubate on ice for 30 minutes. After centrifugation at 250×g for 5 min, the supernatant was discarded and the cells were washed twice with 1×FCM. 1×PBS was added at 100 μl / well to resuspend the cells and detected by flow cytometry (Beckman, CytoFLEX).

[0122] The binding activity of anti-human PD-L1 humanized antibody to CHO-FP1 empty cells and CHO-mPD-L1 stable cell lines is as follows Figure 1 and Figure 2 As shown, the results showed that none of the 13 anti-human PD-L1 humanized antibodies bound to CHO-FP1 empty cells and CHO-mPD-L1 stable cell lines.

[0123] 2. Binding activity of anti-human PD-L1 humanized antibody to CHO cells stably expressing human PD-L1

[0124] To characterize the binding affinity of the anti-human PD-L1 humanized antibodies to CHO cells stably expressing human PD-L1 (CHO-hPD-L1 stable cell line), the 13 anti-human PD-L1 humanized antibodies prepared in Example 1 were diluted three-fold with 1× FCM buffer (1× PBS + 3% BSA) to a starting concentration of 30 μg / ml and then co-incubated with the CHO-hPD-L1 stable cell line. Avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody, and PE anti-human IgG control antibody were also set up, and the specific method and process were the same as above.

[0125] The binding activity of anti-human PD-L1 humanized antibody to CHO-hPD-L1 stable cell line is as follows Figure 3 and Figure 4 As shown, the results showed that the binding activity EC50 of 13 anti-human PD-L1 humanized antibodies to the CHO-hPD-L1 stable cell line was 2-8 nM.

[0126] 3. Binding activity of humanized anti-human PD-L1 antibody to CHO cells stably expressing macaque PD-L1

[0127] To characterize the binding affinity of the anti-human PD-L1 humanized antibodies to CHO cells stably expressing macaque PD-L1 (CHO-cynoPD-L1 stable cell line), the 13 anti-human PD-L1 humanized antibodies prepared in Example 1 were diluted three-fold with 1× FCM buffer (1× PBS + 3% BSA) to a starting concentration of 30 μg / ml and then co-incubated with the CHO-cynoPD-L1 stable cell line. Avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody, and PE anti-human IgG control antibody were also set up, and the specific method and process were the same as above.

[0128] The binding activity of anti-human PD-L1 humanized antibody to CHO-cynoPD-L1 stable cell line is as follows Figure 5 and Figure 6 As shown, the results showed that the binding activity EC50 of 13 anti-human PD-L1 humanized antibodies to the CHO-cynoPD-L1 stable cell line was 1-12 nM.

[0129] 4. Binding activity of anti-human PD-L1 humanized antibody to IFNγ-stimulated A375 cells

[0130] 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 the PD-L1 membrane protein was detected. Avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody, and PE anti-human IgG control antibody were set up. The experimental process is as follows:

[0131] Human PD-L1 expression on human melanoma A375 cells (Shanghai Cell Bank, Cat. SCSP-533) was stimulated overnight with 50 ng / ml human IFNγ (R&D, Cat. 285-IF-100). The next day, A375 cells were digested with 0.25% trypsin (Gibco, Cat. 25200-056), washed once with DPBS, and the viable cell density was adjusted to 2E6 / ml. 100 μl / well was added to a 96-well V-bottom plate. Humanized anti-human PD-L1 antibody was diluted in 1× FCM buffer to a concentration of 20 μg / ml. 100 μl / well was added to the cells, incubated on ice for 30 minutes, centrifuged at 250×g for 5 minutes, and the supernatant was discarded. After washing twice with 1× FCM, 100 μl / well of a PE-antibody human IgG fluorescent secondary antibody (1:500) diluted in 1× FCM buffer (Biolegend, Cat. 409304) was added and incubated on ice for 30 min. After centrifugation at 250×g for 5 min, the supernatant was discarded, and the cells were washed twice with 1× FCM. Cells were then resuspended in 1× PBS at 100 μl / well and analyzed using a flow cytometer (Beckman, CytoFLEX).

[0132] The binding activity of anti-human PD-L1 humanized antibody to IFNγ-stimulated A375 cells is shown in Figure 2 Figure 7 and Figure 8 As shown, the results showed that the EC50 of the binding of 13 anti-human PD-L1 humanized antibodies to the PD-L1 membrane protein expressed in IFNγ-stimulated A375 cells was approximately 0.2 nM.

[0133] 5. Binding kinetics analysis of humanized anti-human PD-L1 antibodies

[0134] The MD ForteBIO QKe platform was used to analyze the binding kinetic constants of the humanized anti-human PD-L1 antibody to human PD-L1. The experimental method is as follows:

[0135] The His-tagged human PD-L1 extracellular domain recombinant protein, hPD-L1-his, was diluted in equilibration buffer (1×PBS + 0.02% Tween 20) to a final concentration of 5 μg / ml. Seven concentrations of anti-human PD-L1 humanized antibody were diluted two-fold in equilibration buffer (1×PBS + 0.02% Tween 20) to a starting concentration of 10 μg / ml. After fully hydrating the anti-Penta-HIS biosensor (ForteBIO, Cat. 18-5122), the hPD-L1-his recombinant protein was immobilized for 150 seconds. After equilibration for 90 seconds, the anti-human PD-L1 mouse antibody was bound with an association time of 180 seconds and a dissociation time of 600 seconds. The entire reaction was performed at 25, 1000 rpm. Curve fitting was performed using Octet analysis software to obtain the binding kinetic constant, KD, of the anti-human PD-L1 humanized antibody.

[0136] The results of the binding kinetic constant analysis 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.

[0137] Table 2

[0138] 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

[0139] Example 3 Anti-human PD-L1 humanized antibody mediated ligand blocking experiment

[0140] 1. Anti-human PD-L1 humanized antibody binding to PD-1 blocking activity

[0141] PD-L1 protein expressed by tumor cells or antigen-presenting cells inhibits the stimulatory activity of lymphocytes by binding to PD-1 protein expressed on the surface of lymphocytes. In this example, the blocking activity of anti-human PD-L1 humanized antibodies in blocking the binding of PD-1 to PD-L1 was evaluated. Avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody, and PE anti-mouse IgG control antibody were set up. The experimental method is as follows:

[0142] Dilute the anti-human PD-L1 humanized antibody three-fold in 1× FCM buffer to a starting concentration of 200 μg / ml. Dilute the self-produced recombinant human PD-1 protein carrying mFc to a concentration of 4 μg / ml in 1× FCM buffer. Resuspend CHO-hPD-L1 cells to a cell density of 2×10E6 / ml in 1× FCM buffer and aliquot 100 μl / well into a 96-well V-bottom plate. Add 50 μl / well of the antibody to the CHO-hPD-L1 cells and incubate on ice for 30 minutes. Then, add 50 μl / well of recombinant human PD-1-mFc protein and incubate on ice for 30 minutes. After centrifugation at 250 × g for 5 min, the supernatant was discarded, and the cells were washed once with 1× FCM. 100 μl / well of PE anti-mouse IgG fluorescent secondary antibody (1:500) (Biolegend, Cat. 405307) diluted in 1× FCM buffer was added and incubated on ice for 30 min. After centrifugation at 250 × g for 5 min, the supernatant was discarded, and the cells were washed twice with 1× FCM. 100 μl / well of 1× PBS was added to resuspend the cells and analyzed using a flow cytometer (Beckman, CytoFLEX).

[0143] Anti-human PD-L1 humanized antibody binds to PD-1 and blocks its activity Figure 9 and Figure 10 As shown, the results showed that all 13 anti-human PD-L1 humanized antibodies could block the binding of PD-1 and PD-L1.

[0144] 2. Anti-human PD-L1 humanized antibody binds to CD80 and blocks its activity

[0145] PD-L1 protein expressed by tumor cells or antigen-presenting cells inhibits the stimulatory activity of lymphocytes by binding to CD80 protein expressed on the surface of lymphocytes. In this example, the blocking activity of anti-human PD-L1 humanized antibodies in blocking the binding of CD80 to PD-L1 was evaluated. Avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody, and PE anti-mouse IgG control antibody were set up. The experimental method is as follows:

[0146] Dilute the anti-human PD-L1 humanized antibody three-fold in 1× FCM buffer to a starting concentration of 200 μg / ml. Dilute the self-produced recombinant human CD80 protein carrying mFc to a concentration of 24 μg / ml in 1× FCM buffer. Resuspend CHO-hPD-L1 cells to a cell density of 2×10E6 / ml in 1× FCM buffer and aliquot 100 μl / well into a 96-well V-bottom plate. Add 50 μl / well of the antibody to the CHO-hPD-L1 cells and incubate on ice for 30 minutes. Then, add 50 μl / well of recombinant human CD80-mFc protein and incubate on ice for 30 minutes. After centrifugation at 250 × g for 5 min, the supernatant was discarded, and the cells were washed once with 1× FCM. 100 μl / well of PE anti-mouse IgG fluorescent secondary antibody (1:500) (Biolegend, Cat. 405307) diluted in 1× FCM buffer was added and incubated on ice for 30 min. After centrifugation at 250 × g for 5 min, the supernatant was discarded, and the cells were washed twice with 1× FCM. 100 μl / well of 1× PBS was added to resuspend the cells and analyzed using a flow cytometer (Beckman, CytoFLEX).

[0147] Anti-human PD-L1 humanized antibody binds to CD80 and blocks its activity Figure 11 and Figure 12 As shown, the results showed that all 13 anti-human PD-L1 humanized antibodies could block the binding of PD-L1 to CD80.

[0148] Example 4 Characterization of in vitro activity of anti-human PD-L1 humanized antibodies

[0149] 1. Detection of luciferase reporter gene system under co-incubation of Jurkat-GL4.30-hPD-1 and CHO-hPD-L1-OKT3

[0150] To test the in vitro functional activity of a humanized anti-human PD-L1 antibody using a luciferase reporter gene system, a Jurkat-GL4.30-hPD-1 effector cell line stably expressing hPD-1 and luciferase genes was constructed using a lentiviral packaging system. Simultaneously, a PD-L1-presenting cell line, CHO-hPD-L1-OKT3, was constructed using Chinese hamster hamster (CHO) cells stably expressing hPD-L1 and an antigen-independent TCR cell surface driver. On the day of the experiment, CHO-hPD-L1-OKT3 and Jurkat-GL4.30-hPD-1 cells were resuspended in RPMI1640 complete medium supplemented with 1% FBS to viable cell densities of 1.6 × 106 cells / ml and 8.0 × 106 cells / ml, respectively, and plated at 25 μl / well in a 96-well fluorescence microscopy plate. Dilute the anti-human PD-L1 humanized antibody in RPMI1640 complete medium containing 1% FBS to a starting concentration of 20 μg / ml. Pipette 50 μl / well of the antibody into a mixed Jurkat-GL4.30-hPD-1 and CHO-hPD-L1-OKT3 cell culture medium. Mix thoroughly and incubate in a 37°C, 5% CO2 cell culture incubator for 6 hours. Add 100 μl / well of pre-thawed Bright-Lumi™ solution (Biyuntian, Cat.RG051M) and keep the cells in the dark for 5 minutes before detection using a multi-function microplate reader (Molecular Device, SpectraMax i3x multi-function microplate reader) in Lumi mode. Set up an hIgG1 isotype control antibody.

[0151] Luciferase reporter gene assay to analyze the in vitro activity of humanized anti-human PD-L1 antibodies Figure 13 and Figure 14 As shown, the results showed that the humanized anti-human PD-L1 antibody can mediate the callback of the luciferase reporter gene signal, and is antibody dose-dependent.

[0152] 2. Stimulation of IL-2 and IFN gamma production in T-DC allogeneic mixed lymphoid reaction system

[0153] The allogeneic T-DC MLR assay was used to evaluate the activity of anti-human PD-L1 humanized antibodies in stimulating the production of cytokines IL-2 and IFNgamma. The experimental procedure is as follows:

[0154] Peripheral blood lymphocytes (PBMCs) were isolated from healthy human peripheral blood using the human lymphocyte separation medium Lymphoprep™ (Axis-Shield, Cat. 07851). PBMCs from donor 1 were screened using human CD14 Microbeads (Miltenyi, Cat. 130-050-201) to obtain CD14+ monocytes. Monocytes were seeded at 5×105 / ml in a T75 culture flask and supplemented with human GM-CSF and IL-4 to a final concentration of 50 ng / ml. After six days of continuous stimulation, human TNF-alpha was added to a final concentration of 50 ng / ml and induced to differentiate for another three days to obtain mature DCs. PBMCs from donor 2 were negatively screened using the EasySep™ Human T Cell Enrichment Kit (Stemcell, Cat. 19051) to obtain CD3+ T cells. At a DC:T cell ratio of 1:5 and a DC cell count of 2 × 104 cells / well, DCs and T cells were mixed evenly and then plated in a 96-well U-shaped plate, with a total volume of 150 μl / well. Humanized anti-human PD-L1 antibody was diluted in X-VIVO 15 complete medium at a starting concentration of 20 μg / ml, diluted fourfold, and added to the cells at 50 μl / well. After 3-5 days of mixed lymphocyte assay reaction, the expression of IL-2 and IFNgamma in the cell supernatant was measured. An hIgG1 isotype control antibody was also used.

[0155] The results of IL-2 secretion in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibody are as follows Figure 15 、 Figure 16 and Figure 17 As shown in Figure 2, the secretion of IFNγ in mixed lymphocyte reaction mediated by anti-human PD-L1 humanized antibody is shown in Figure 2. Figure 18 、 Figure 19 and Figure 20 As shown, the results showed that in the T-DC allogeneic mixed lymphocyte reaction experiment, anti-human PD-L1 humanized antibody could mediate the upregulation of cytokines IL-2 and IFNγ, and was positively correlated with the antibody dosage.

[0156] Example 5 Binding Specificity Analysis of Anti-Human PD-L1 Humanized Antibodies

[0157] The PD-L1 protein belongs to the B7 family of proteins, and its homologous family proteins include PD-L2 (B7-DC), ICOSL (B7-H2), B7-H3, CD80 (B7-1), CD86 (B7-2), etc. To test the binding specificity of the humanized anti-human PD-L1 antibody, avelumab-hIgG1 positive control antibody, hIgG1 isotype control antibody, and HRP antibody human IgG Fab control antibody were set up. The experimental process is as follows:

[0158] Recombinant human PD-L1 protein carrying an hFc tag, 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) were diluted to 1 μg / ml with 50 mM CB buffer. 100 μl / well was added to a 96-well ELISA test plate and coated overnight at 2-4°C. Discard the supernatant and add 200 μl / well of blocking buffer (1xPBS + 1% BSA) and block at 37°C for 1 hour. Dilute the humanized anti-human PD-L1 antibody to 10 μg / ml in PBS containing 1% BSA, as well as the commercially available positive control antibodies anti-hPD-1 (Opdivo), anti-ICOS-hIgG4, and anti-hCD28-mIgG2a to 10 μg / ml. Add 100 μl / well and incubate at 37°C for 30 minutes. Wash three times with 1x PBS. Add 100 μl / well of HRP-conjugated goat anti-human IgG Fab (Sigma A0293-1ML) or HRP-conjugated goat anti-mouse IgG (Sigma, AP113P) and incubate at 37°C for 30 minutes. Wash three times with 1x PBS before developing the color reaction.

[0159] The binding activity of anti-human PD-L1 humanized antibody to 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, recombinant human CD28 protein and recombinant human CTLA-4 protein is as follows: Figure 21-29 As shown, the results showed that the 13 anti-human PD-L1 humanized antibodies had high specificity and did not bind to other B7 family proteins and other related proteins except PD-L1.

[0160] Example 6 In vivo anti-tumor activity of humanized anti-human PD-L1 antibodies

[0161] Since the murine monoclonal antibody of the present invention does not cross-react with murine PD-L1, the in vivo anti-tumor efficacy of the anti-human PD-L1 humanized antibodies 176L6H5, 176L5H2, and 176L4H1 was determined using hPD-L1 knock-in transgenic mice. An hIgG1 isotype control antibody, a positive control antibody avelumab-hIgG1, and Tecentriq (Roche) were also used. The specific method is as follows:

[0162] hPD-L1 knock-in transgenic mice:

[0163] Female hPD-L1 knock-in transgenic mice (6-8 weeks old) on a C57BL / 6 background were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. with a certificate number of NO.320726210100112386.

[0164] Culturing cells and inoculating mice:

[0165] By using CRISPR CAS 9 technology to knock out the endogenous mouse PD-L1 gene in MC38 colorectal cancer cells (National Laboratory Cell Sharing Resource Platform, Resource Number: 3111C0001CCC000523), MC38 cells with complete mouse PD-L1 knockout were screened. Human PD-L1 cDNA carrying the multicloning site MCS was then integrated into the MC38 mPD-L1 knockout MC38 cell line via lentiviral transduction. MC38 cells stably expressing human PD-L1 (MC38-hPD-L1 cells) were screened and obtained. MC38-hPD-L1 cells were routinely subcultured for subsequent in vivo mouse experiments.

[0166] MC38-hPD-L1 cells were harvested during the logarithmic growth phase and washed twice with DPBS (Hyclone, Cat. SH30028.02). The cell concentration was adjusted to 1 × 10E7 cells / ml using DPBS (Hyclone, Cat. SH30028.02). Female hPD-L1 knock-in transgenic mice were subcutaneously inoculated with MC38-hPDP-L1 cells on the right flank at a volume of 0.1 ml / mouse, yielding 1 × 10E6 cells / mouse. The day of MC38-hPD-L1 cell inoculation was defined as study day 0.

[0167] Grouping and dosing of tumor-bearing mice:

[0168] When the average tumor volume reaches 60-80 mm 3Mice can be randomly divided into groups based on tumor volume. In this study, mice were randomly divided into seven groups of eight mice each on day 7 (D7) based on tumor volume. Intraperitoneal (ip) administration began. The dosage, method, and frequency of administration in the MC38-hPD-L1 tumor model are shown in Table 3. Q3Dx3 indicates dosing every three days for a total of three doses.

[0169] Table 3

[0170] Group Dosage (mg / kg) Dosage volume (μl / g) Route of administration Dosing frequency hIgG1 isotype control 10 10 ip Q3Dx3 176L4H1 10 10 ip Q3Dx3 176L5H2 10 10 ip Q3Dx3 176L6H5 10 10 ip Q3Dx3 avelumab-hIgG1 10 10 ip Q3Dx3 Tecentriq 10 10 ip Q3Dx3 PBS - 10 ip Q3Dx3

[0171] Tumor volume was measured and recorded starting from day 7. Tumor volume and weight of mice were monitored twice a week during the duration of the study. The long and short diameters of the tumor were measured with a vernier caliper. The formula was (1 / 2) × long diameter × (short diameter). 2 Calculate the tumor volume. When the mouse body volume decreased by 20% or the tumor volume exceeded 2000mm 3 When the end point was reached, the mice were killed by CO2 asphyxiation.

[0172] Comparisons between two groups can be performed using the independent sample T test. Comparisons between more than three groups should be performed using a one-way ANOVA. If the F value indicates a significant difference, multiple group analyses can be performed. Data were processed using GraphPad Prism, and a p value < 0.05 indicated a statistically significant difference. Tumor growth inhibition rate (TGI) (%) = [1-(Ti-T0) / (Vi-V0)] × 100, where Ti is the mean tumor volume of the treatment group on day i, T0 is the mean tumor volume of the treatment group at the start of treatment, Vi is the mean tumor volume of the vehicle control group on day i, and V0 is the mean tumor volume of the vehicle control group at the start of treatment.

[0173] The effects of anti-human PD-L1 humanized antibodies on mouse tumor volume are shown in Table 4 , and the in vivo anti-tumor activity results of anti-human PD-L1 humanized antibodies are shown in Table 4 Figure 30As shown, (a) shows the effect of anti-human PD-L1 humanized antibody on mouse tumor volume; (b) shows the effect of anti-human PD-L1 humanized antibody on mouse survival rate; (c) shows the effect of anti-human PD-L1 humanized antibody on mouse body weight. The results show that the anti-human PD-L1 humanized antibody in this example has a significant anti-tumor effect, with TGIs of 104.86%, 102.16%, and 85.08% on day 32, respectively. Among the 8 mice, 8, 5, and 5 mice achieved complete tumor elimination (CR), respectively (i.e., CRs 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%, respectively, and CRs were 7 / 8 and 4 / 8, respectively. The anti-human PD-L1 humanized antibody significantly prolonged the survival of mice, and there was no significant effect on the weight of mice in each administration group.

[0174] Table 4

[0175]

[0176] Note: The unit of tumor volume in the table is mm 3 "N / A" means the tumor volume of the mouse is more than 2000mm 3 When the merciful endpoint was reached, the mice were killed by CO2 asphyxiation.

Claims

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 consists of HCDR1, HCDR2, and HCDR3, and the light chain CDR region consists of LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 14 to 16, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 17 to 19, respectively. The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs: 3 to 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 shown in any one of SEQ ID NOs: 8 to 13.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are as shown in any one of the following: The heavy chain variable region of antibody 176L1H1 is shown in SEQ ID NO. 3, and the light chain variable region is shown in SEQ ID NO. 8; The heavy chain variable region of antibody 176L1H2 is shown in SEQ ID NO. 4, and the light chain variable region is shown in SEQ ID NO. 8; The heavy chain variable region of antibody 176L1H3 is shown in SEQ ID NO. 5, and the light chain variable region is shown in SEQ ID NO. 8; The heavy chain variable region of antibody 176L2H1 is shown in SEQ ID NO. 3, and the light chain variable region is shown in SEQ ID NO. 9; The heavy chain variable region of antibody 176L2H2 is shown in SEQ ID NO. 4, and the light chain variable region is shown in SEQ ID NO. 9; The heavy chain variable region of antibody 176L2H3 is shown in SEQ ID NO. 5, and the light chain variable region is shown in SEQ ID NO. 9; The heavy chain variable region of antibody 176L3H2 is shown in SEQ ID NO. 4, and the light chain variable region is shown in SEQ ID NO. 10; The heavy chain variable region of antibody 176L4H1 is shown in SEQ ID NO. 3, and the light chain variable region is shown in SEQ ID NO. 11; The heavy chain variable region of antibody 176L4H2 is shown in SEQ ID NO. 4, and the light chain variable region is shown in SEQ ID NO. 11; The heavy chain variable region of antibody 176L4H3 is shown in SEQ ID NO. 5, and the light chain variable region is shown in SEQ ID NO. 11; The heavy chain variable region of antibody 176L4H4 is shown in SEQ ID NO. 6, and the light chain variable region is shown in SEQ ID NO. 11; The heavy chain variable region of antibody 176L5H2 is shown in SEQ ID NO. 4, and the light chain variable region is shown in SEQ ID NO. 12; The heavy chain variable region of antibody 176L6H5 is shown in SEQ ID NO.7, and the light chain variable region is shown in SEQ ID NO.

13.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NOs. 3 to 6, and the amino acid sequence of the light 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, 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 claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light 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, 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.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody contains a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region sequence is selected from the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; The light chain constant region is a kappa or lambda chain; The species origin of the heavy chain constant region and the light chain constant region is selected from any one of cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, characterized in that The antibody is any one or more of a bispecific antibody, a CDR-grafted antibody or a multimeric antibody; 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 to 7.

9. A carrier, characterized in that The vector comprises the nucleic acid according to claim 8.

10. A cell, characterized in that The cell carries the nucleic acid of claim 8, contains the vector of claim 9, or expresses the antibody or antigen-binding fragment thereof of any one of claims 1 to 7.

11. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising: Cultivating the cell according to claim 10 in a culture medium; and recovering the produced antibodies or antigen-binding fragments thereof from the culture medium or from the cultured cells.

12. A pharmaceutical composition, characterized in that The composition contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the nucleic acid according to claim 8, or the vector according to claim 9, or the cell according to claim 10.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the nucleic acid according to claim 8, the vector according to claim 9, the cell according to claim 10, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing or treating colorectal cancer, renal cancer, gastric cancer, urothelial carcinoma, ovarian cancer, or melanoma.

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