CN110156895B

CN110156895B - anti-PD-L1 antibody or functional fragment thereof and application thereof

Info

Publication number: CN110156895B
Application number: CN201910460477.0A
Authority: CN
Inventors: 张明; 原野
Original assignee: Shanghai Jiabei Biomedical Technology Co ltd
Current assignee: Ningbo Jiabei Medical Technology Co ltd; Shanghai Jiabei Biomedical Technology Co ltd; Shanghai Jiabei Pharmaceutical Technology Co ltd
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2020-11-20
Anticipated expiration: 2039-05-30
Also published as: CN110156895A

Abstract

The application belongs to the field of biological medicine and discloses the following aspects: an anti-PD-L1 antibody or functional fragment thereof; nucleic acid molecules encoding the anti-PD-L1 antibody or functional fragment thereof described herein, expression vectors and host cells for expressing the anti-PD-L1 antibody or functional fragment thereof; a method for preparing the anti-PD-L1 antibody or a functional fragment thereof; immunoconjugates comprising the anti-PD-L1 antibody or functional fragments thereof and pharmaceutical compositions thereof; and the use of the anti-PD-L1 antibody or functional fragment thereof for the manufacture of a medicament for the treatment of a disorder of T cell dysfunction.

Description

anti-PD-L1 antibody or functional fragment thereof and application thereof

Technical Field

The application belongs to the field of biological medicine, and relates to the following aspects of an anti-PD-L1 antibody: an anti-PD-L1 antibody or functional fragment thereof; nucleic acid molecules encoding the antibodies or functional fragments thereof described herein, expression vectors and host cells for expressing the antibodies or functional fragments thereof; a method for preparing an anti-PD-L1 antibody or functional fragment thereof as described herein; immunoconjugates comprising an anti-PD-L1 antibody or functional fragment thereof as described herein and pharmaceutical compositions thereof; and the use of an anti-PD-L1 antibody or functional fragment thereof as described herein for the manufacture of a medicament for the treatment of a disorder of T cell dysfunction.

Background

Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD 274) or B7 homologous protein 1 (B7 homolog 1, B7-H1), belongs to the tumor necrosis factor superfamily, is a type I transmembrane glycoprotein consisting of 290 amino acid residues, and comprises an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and an intracellular tail of 30 amino acids, and has a complete molecular weight of 40 kDa. PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is persistently expressed in only a small proportion of tissues, e.g., liver, lung, tonsil, and immune privileged tissues such as eye, placenta, etc. PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, and the like.

The receptor of PD-L1 is Programmed death receptor 1 (PD-1). PD-1 is mainly expressed on the surfaces of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells and activated monocytes. Binding of PD-L1 to PD-1 initiates the phosphorylation of tyrosine residues of PD-1 cytoplasmic Immunoreceptor Tyrosine Inhibitory Motifs (ITIMs), which promotes the binding of tyrosine phospholipases to SHP2, activates SHP2, dephosphorylates downstream Syk and PI3K to transmit termination signals, and restricts the interaction of antigen presenting cells or dendritic cells with T cells. The combination can further inhibit the metabolism of T cells, inhibit the secretion of anti-apoptotic protein Bcl-X2, reduce the secretion of effector cell factor human Interleukin-2 (Interleukin-2, IL-2) and Interferon-gamma (Interferon-r, IFN-r), induce the exhaustion and apoptosis of T cells, thereby reducing the immune response involved by immune T cells and performing the function of negative immune regulation.

PD-L1 is highly expressed in tumor tissues, such as gastric cancer, lung cancer, liver cancer, intrahepatic bile duct cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer, oral squamous cell carcinoma, etc. The expression of PD-L1 at the tumor site can protect tumor cells from being damaged through various ways, and the negative immune regulation function of PD-L1 plays an important role in tumor immunity. Therefore, the immunoregulation taking PD-1/PD-L1 as a target has important significance for resisting tumors, and various chronic and acute viruses also utilize PD-L1 signals to escape human immunodetection.

In recent years, increasing numbers of preclinical and clinical research results indicate that targeting immune checkpoints is becoming the most promising approach to treating cancer patients. PD-1 is one of the immune checkpoint proteins that negatively regulates the immune response and attenuates the expression of anti-tumor immunity PD-L1 on tumors through the interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells, which is associated with decreased survival of esophageal, pancreatic, and other various types of cancers, and this pathway has served as a new promising target for tumor immunotherapy. There are pharmaceutical companies that have developed a variety of drugs directed to the PD-1/PD-L1 pathway, and data from clinical trials indicate long-lasting clinical activity and good safety of such drugs in patients with a variety of tumor types.

However, existing therapeutic approaches are not entirely satisfactory and there is still a need for new human antibodies against PD-L1.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art and the need for clinical applications of anti-PD-L1 antibodies, one aspect of the inventors of the present application is directed to providing an antibody against human PD-L1 and functional fragments thereof; in another aspect, it is intended to provide the use of the antibody or functional fragment thereof for the treatment of a disorder of T cell dysfunction; in another aspect, the invention provides methods for preparing and detecting the antibodies.

To achieve the above objects, in a first aspect, the present application provides an anti-PD-L1 fully human antibody, the anti-PD-L1 antibody being a monoclonal antibody, the anti-PD-L1 antibody comprising at least one or more of the heavy chain hypervariable regions CDR1, CDR2 and CDR3, and/or one or more of the light chain hypervariable regions CDR1, CDR2 and CDR 3; wherein:

the amino acid sequence of the light chain hypervariable region CDR1 is the sequence shown in SEQ ID No.3 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.3, the amino acid sequence of the light chain hypervariable region CDR2 is the sequence shown in SEQ ID No.4 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.4, and the amino acid sequence of the light chain hypervariable region CDR3 is the sequence shown in SEQ ID No.5 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No. 5; and

the amino acid sequence of the heavy chain hypervariable region CDR1 is the sequence shown in SEQ ID No.6 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.6, the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence shown in SEQ ID No.7 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.7, and the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence shown in SEQ ID No.8 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No. 8.

Wherein: the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function with the sequence shown in any one of SEQ ID No.3-8 in the sequence table refers to a polypeptide which is formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with properties which are similar or similar compared with the amino acid sequence of the anti-PD-L1 antibody. The sequence of such variants is at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it was derived.

Furthermore, the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody at least comprises the sequence shown in SEQ ID No.1 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region at least comprises the sequence shown in SEQ ID No.2 or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No. 2.

Further, the light chain Framework Region (FR) of the anti-PD-L1 antibody includes one or more of FR1, FR2 and FR3, wherein the amino acid sequence of the light chain FR1 is the sequence shown in SEQ ID No.9 or an amino acid sequence having the same function as the sequence shown in SEQ ID No.9 formed by substituting, deleting or adding one or more amino acids to the sequence, the amino acid sequence of the light chain FR2 is the sequence shown in SEQ ID No.10 or an amino acid sequence having the same function as the sequence shown in SEQ ID No.10 formed by substituting, deleting or adding one or more amino acids, and the amino acid sequence of the light chain FR3 is the sequence shown in SEQ ID No.11 or an amino acid sequence having the same function as the sequence shown in SEQ ID No.11 formed by substituting, deleting or adding one or more amino acids;

further, the heavy chain framework region comprises one or more of FR1, FR2 and FR3, wherein the amino acid sequence of the heavy chain FR1 is the sequence shown in SEQ ID No.12 or an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.12, the amino acid sequence of the heavy chain FR2 is the sequence shown in SEQ ID No.13 or an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No.13, and the amino acid sequence of the heavy chain FR3 is the sequence shown in SEQ ID No.14 or an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function as the sequence shown in SEQ ID No. 14.

Preferably, the nucleotide sequence encoding the light chain variable region in the anti-PD-L1 antibody is the sequence shown in SEQ ID No.15 or a nucleotide sequence which is formed by replacing, deleting or adding one or more nucleotides to the sequence and has the same function as the sequence shown in SEQ ID No.15, and the nucleotide sequence encoding the heavy chain variable region in the anti-PD-L1 antibody is the sequence shown in SEQ ID No.16 or a nucleotide sequence which is formed by replacing, deleting or adding one or more nucleotides to the sequence and has the same function as the sequence shown in SEQ ID No. 16.

In a second aspect, the present application also provides an expression vector comprising at least one of the following amino acid sequences: the sequence shown in SEQ ID No.15 or the nucleotide sequence which is formed by replacing, deleting or adding one or more nucleotides in the sequence and has the same function with the sequence shown in SEQ ID No. 15; and a sequence shown in SEQ ID No.16 or a nucleotide sequence which is formed by replacing, deleting or adding one or more nucleotides in the sequence and has the same function with the sequence shown in SEQ ID No. 16. Meanwhile, the application also provides a host cell, and the host cell comprises the expression vector. Preferably, the host cell is a mammalian cell.

In a third aspect, the present application also provides a method for preparing the anti-PD-L1 antibody or functional fragment thereof.

In a fourth aspect, the present application also provides immunoconjugates comprising the anti-PD-L1 antibody or a functional fragment thereof and pharmaceutical compositions thereof.

In a fifth aspect, the use of an anti-PD-L1 antibody or functional fragment thereof described herein for the manufacture of a medicament for the treatment of a disorder of T cell dysfunction.

In one embodiment of the first aspect of the present application, the humanized anti-PD-L1 antibody is a monoclonal antibody having the amino acid sequences of hypervariable region CDR1, CDR2 and CDR3 of the light chain of the antibody are the sequences shown in SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, respectively; the amino acid sequences of the heavy chain hypervariable region CDR1, CDR2 and CDR3 of the antibody are respectively the sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No. 8.

Furthermore, the amino acid sequence of the light chain variable region of the antibody is the sequence shown in SEQ ID No.1, or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function with the sequence shown in SEQ ID No. 1; the amino acid sequence of the heavy chain variable region of the antibody is the sequence shown in SEQ ID No.2, or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function with the sequence shown in SEQ ID No. 2.

The amino acid sequences of the light chain framework regions FR1, FR2 and FR3 of the antibody are respectively the sequences shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No. 11; the amino acid sequences of the heavy chain framework regions FR1, FR2 and FR3 of the antibody are respectively the sequences shown in SEQ D No.12, SEQ ID No.13 and SEQ ID No. 14.

In an embodiment of the second aspect of the present application, the present application provides two DNA fragments. These DNA fragments encode an anti-PD-L1 antibody as described above in the first aspect. The DNA fragment comprises a light chain variable region encoding sequence and a heavy chain variable region encoding sequence; the nucleotide sequences for encoding the light chain variable region and the heavy chain variable region are respectively shown as SEQ ID No.15 and SEQ ID No. 16.

In an embodiment of the third aspect of the present application, the present application also provides two expression vectors comprising at least one copy of the DNA fragment as described above in the second aspect. Also, the present application provides a host cell comprising an expression vector as described in the third aspect above.

In embodiments of the fourth aspect of the present application, the present application also provides immunoconjugates comprising the anti-PD-L1 antibody or a functional fragment thereof and pharmaceutical compositions thereof. Preferably, the pharmaceutical composition further comprises a toxin, radioisotope, drug or cytotoxic agent. In other embodiments, the application also relates to a pharmaceutical composition comprising the anti-PD-L1 antibody or functional fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition at least contains an effective dose of the anti-PD-L1 antibody or functional fragment thereof and a pharmaceutically acceptable carrier thereof.

In an embodiment of the fifth aspect of the present application, the present application provides the use of the anti-PD-L1 antibody or functional fragment thereof for the manufacture of a medicament for the treatment of a disorder of T cell dysfunction. Specifically, a method of preventing or treating a disease or alleviating a condition by abolishing, inhibiting or reducing PD-L1 activity comprises administering to a subject in need thereof an effective dose of the anti-PD-L1 antibody or functional fragment thereof, a nucleic acid, an expression vector, a host cell, an immunoconjugate or a pharmaceutical composition. Further, the use described herein includes the use of the anti-PD-L1 antibody or functional fragment thereof, and accordingly a nucleic acid, expression vector, host cell, immunoconjugate or pharmaceutical composition, in the manufacture of a medicament for the treatment of a disease or disorder.

The anti-PD-L1 antibodies described herein are derived from a human having an affinity for PD-L1 with a KD of less than or equal to 1.0x10^-8And M. Specifically, the anti-PD-L1 antibody is at about 9x10^-9M or less, about 8x10^-9M or less, about 7x10^-9M or less, about 6x10^-9M or less, about 5x10^-9M or less, about 4x10^-9M or less, about 3x10^-9M or less, about 2x10^-9M or less, about 10^-9Any one of the KD values of M or less binding affinity KD specifically binds to human PD-L1. In one embodiment, the affinity KD of the anti-PD-L1 antibody and PD-L1 is 7.1nM, the anti-PD-L1 antibody can well inhibit the combination of PD-L1 and PD-1, and the anti-PD-L1 antibody can be used for preventing or treating by eliminating, inhibiting or reducing the activity of PD-L1Diseases or conditions that promote T cell proliferation, secretion of IL-2 and IFN-gamma, and that can enhance T cell function, thereby up-regulating cell-mediated immune responses for the treatment of disorders of T cell dysfunction.

[ description of the drawings ]

FIG. 1a shows FACs detecting the binding reaction of the anti-PD-L1 antibody to HEK293 PD-L1.

FIG. 1b shows the mean fluorescence intensity of FACs detecting different antibody concentrations in the binding reaction of the PD-L1 antibody to HEK293 PD-L1.

FIG. 2 shows AlphaLISA detection the anti-PD-L1 antibody inhibits binding of PD-L1 to PD-1.

FIG. 3 shows the results of the anti-PD-L1 antibody promoting IL-2 secretion by T cells in a mixed lymphocyte reaction.

FIG. 4 shows the results of the anti-PD-L1 antibody promoting IFN- γ secretion by T cells in a mixed lymphocyte reaction.

FIG. 5 shows the results of the anti-PD-L1 antibody promoting T cell proliferation in a mixed lymphocyte reaction.

[ detailed description ] embodiments

For a clearer understanding of the inventive concepts and technical solutions of the present application, the present application will be further explained below by means of specific examples and drawings, wherein the technical solutions in the embodiments are only preferred embodiments and should not be construed as limiting the scope of the present application. It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the technical principles of the present application, and these modifications and variations should also be considered to fall within the scope of the present application.

Unless defined otherwise, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to 20 commonly used L-amino acids.

Reference in this application to a "fully human" or "humanized" antibody, or functional fragment or antigen-binding fragment thereof, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to that of an antibody produced by human or human immune cells or derived from a non-human source, e.g., a transgenic non-human animal utilizing a human antibody repertoire, or other sequence encoding a human antibody. In certain embodiments, a fully human antibody does not comprise amino acid residues (particularly antigen binding residues) derived from a non-human antibody.

Reference herein to a light chain hypervariable region or a heavy chain hypervariable region, wherein "hypervariable region" is also referred to as a complementarity-determining region (CDR).

Reference to "sequence" in this application may refer to a sequence comprising certain biologically functionally equivalent amino acids or "conservative substitutions" and to other sequences may comprise functionally non-equivalent amino acids or "non-conservative substitutions" that have been engineered to improve the properties of the CDRs or CDR-containing antibodies. One skilled in the art can manipulate the sequences in this application, i.e., substitute, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids, to obtain variants of the sequences of the antibodies or functional fragments thereof, without substantially affecting the activity of the antibodies. They are to be considered as included within the scope of protection of the present application. For example, amino acids having similar properties are substituted in the variable region. The sequence of a variant referred to in this application may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it is derived. Sequence identity can be measured using sequence analysis software. For example the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. The various amino acid sequences described herein are detailed in the sequence listing.

The antibodies referred to in the present application are human monoclonal antibodies. The anti-PD-L1 antibody includes a single chain antibody, a diabody, a chimeric antibody, or a derivative thereof. The anti-PD-L1 antibody or the antigen binding fragment thereof can well bind to human PD-L1. The antibodies may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only antigen binding portions (e.g., Fab, F (ab')2, or scFv fragments), or may be modified to affect function. The antibodies described herein also include anti-PD-L1 antibodies with modified glycosylation patterns. In some applications, antibodies may be modified to remove undesired glycosylation sites, e.g., the absence of a fucose moiety on the oligosaccharide chain, to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosylation modifications may be made to alter complement dependent cytotoxic Complement Dependent Cytotoxicity (CDC).

The term "functional fragment" as used herein refers in particular to antibody fragments, such as Fv, scFv (sc refers to single chain), Fab, F (ab ')2, Fab', scFv-Fc fragments, diabodies (diabodies), or any fragment capable of increasing half-life by chemical modification, e.g. addition of poly (alkylene) glycols, such as polyethylene glycol ("pegylation"), or by incorporation into liposomes, which chemically modified fragments are referred to as pegylated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F (ab ')2-PEG or Fab' -PEG, which fragments have Epidermal Growth Factor Receptor (EGFR) binding activity. Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy or light variable chain of the antibody from which it is derived, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. For PD-L1, it is preferably at least equal to or greater than 1/100, and in a more preferred manner at least equal to or greater than 1/10, the affinity of the antibody from which it is derived. Such functional fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

The term "pharmaceutical composition" as used herein includes at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant to achieve a particular purpose. In some embodiments, the pharmaceutical composition further comprises a temporally and/or spatially separated combination of these ingredients, so long as they are capable of acting together to achieve the objectives of the present application. For example, the components contained in the pharmaceutical composition (e.g., the antibodies, nucleic acid molecules, nucleic acid molecule combinations, and/or conjugates according to the present application) may be administered to a subject in whole or separately. When the ingredients contained in the pharmaceutical composition are administered separately to a subject, the ingredients may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. In particular, the type of pharmaceutically acceptable carrier selected will vary depending on whether the compositions of the present application are formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The compositions according to the present application may comprise wetting agents, emulsifiers or buffers as additives. The pharmaceutical compositions of the present application may be administered by any suitable route, for example orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

Reference herein to "preventing or treating a disease or alleviating a condition by eliminating, inhibiting or reducing PD-L1 activity" means a disease or condition caused by expression of PD-L1 or characterized by expression of PD-L1 as a symptom/feature. In some embodiments, the disease or disorder is selected from cancer or an inflammatory disease.

The nucleotide sequence mentioned in the application is detailed in a sequence table. By using the polynucleotides described herein, anti-PD-L1 antibodies or antigen-binding fragments can be efficiently synthesized. The polynucleotides described above can be efficiently synthesized into the anti-PD-L1 antibodies or antigen-binding fragments described in the present application. The method for preparing the antibody may be performed by using techniques well known to those skilled in the art, and is not particularly limited herein.

In view of the degeneracy of codons, for example, the gene sequence encoding the above antibody can be modified in the coding region thereof without changing the amino acid sequence to obtain a gene encoding an antibody having the same function. One skilled in the art can artificially synthesize and modify genes according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

Further, the present application recombines the light chain variable region and the heavy chain variable region of the anti-PD-L1 antibody to obtain a single chain antibody (ScFv) with a smaller molecular weight, which is also capable of binding to PD-L1. The single-chain antibody has strong penetrating power and is easy to enter local tissues to play a role. The antibody and the single-chain antibody can be obtained by cloning the gene encoding the antibody and the ScFv gene into an expression vector, and further transforming or transfecting host cells. In addition, the light chain variable region encoding gene and the heavy chain variable region gene of the aforementioned antibody may be cloned into a whole antibody expression vector and introduced into a host cell to obtain a complete immunoglobulin expressing anti-PD-L1.

In a first aspect, an anti-PD-L1 fully human antibody herein is a monoclonal antibody, the anti-PD-L1 antibody comprising at least one or more of the heavy chain hypervariable regions CDR1, CDR2 and CDR3, and/or one or more of the light chain hypervariable regions CDR1, CDR2 and CDR 3; wherein:

Wherein: the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function with the sequence shown in any one of SEQ ID No.3-8 in the sequence table refers to a polypeptide which is formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with similar or similar properties compared with the amino acid sequence of the anti-PD-L1 antibody. The sequence of such variants is at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it was derived.

Further, the light chain Framework Region (FR) of the anti-PD-L1 antibody includes one or more of FR1, FR2 and FR3, wherein the amino acid sequence of the light chain FR1 is the sequence shown in SEQ ID No.9 or an amino acid sequence having a function equivalent to that of the sequence shown in SEQ ID No.9 formed by substituting, deleting or adding one or more amino acids of the sequence, the amino acid sequence of the light chain FR2 is the sequence shown in SEQ ID No.10 or an amino acid sequence having a function equivalent to that of the sequence shown in SEQ ID No.10 formed by substituting, deleting or adding one or more amino acids, and the amino acid sequence of the light chain FR3 is the sequence shown in SEQ ID No.11 or an amino acid sequence having a function equivalent to that of the sequence shown in SEQ ID No.11 formed by substituting, deleting or adding one or more amino acids;

further, the heavy chain framework region comprises one or more of FR1, FR2 and FR3, wherein the amino acid sequence of the heavy chain FR1 is the sequence shown in SEQ ID No.12 or an amino acid sequence which has the same function as the sequence shown in SEQ ID No.12 and is formed by replacing, deleting or adding one or more amino acids, the amino acid sequence of the heavy chain FR2 is the sequence shown in SEQ ID No.13 or an amino acid sequence which has the same function as the sequence shown in SEQ ID No.13 and is formed by replacing, deleting or adding one or more amino acids, and the amino acid sequence of the heavy chain FR3 is the sequence shown in SEQ ID No.14 or an amino acid sequence which has the same function as the sequence shown in SEQ ID No.14 and is formed by replacing, deleting or adding one or more amino acids.

Preferably, the nucleotide sequence encoding the light chain variable region in the anti-PD-L1 antibody is the sequence shown in SEQ ID No.15 or a nucleotide sequence having the same function as the sequence shown in SEQ ID No.15, which is formed by replacing, deleting or adding one or more nucleotides to the sequence, and the nucleotide sequence encoding the heavy chain variable region in the anti-PD-L1 antibody is the sequence shown in SEQ ID No.16 or a nucleotide sequence having the same function as the sequence shown in SEQ ID No.16, which is formed by replacing, deleting or adding one or more nucleotides to the sequence.

In a third aspect, the application also provides a preparation method of the anti-PD-L1 antibody or the functional fragment thereof correspondingly.

In a fourth aspect, the application also provides immunoconjugates comprising the anti-PD-L1 antibody or functional fragments thereof and pharmaceutical compositions thereof.

In a fifth aspect, the application provides a use of the anti-PD-L1 antibody or functional fragment thereof for the manufacture of a medicament for the treatment of a disorder of T cell dysfunction.

In one embodiment of the first aspect of the application, the humanized anti-PD-L1 antibody is a monoclonal antibody having the amino acid sequences of hypervariable region CDR1, CDR2 and CDR3 of the light chain of the antibody are the sequences shown in SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, respectively; the amino acid sequences of the heavy chain hypervariable region CDR1, CDR2 and CDR3 of the antibody are the sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 respectively.

In an embodiment of the second aspect of the present application, the present application provides two DNA fragments. The DNA fragments encode the anti-PD-L1 antibody of the first aspect as described above. The DNA fragment comprises a light chain variable region encoding sequence and a heavy chain variable region encoding sequence; the nucleotide sequences for encoding the light chain variable region and the heavy chain variable region are respectively shown as SEQ ID No.15 and SEQ ID No. 16.

In an embodiment of the third aspect of the present application, the present application also provides two expression vectors comprising at least one copy of the DNA fragment as in the second aspect above. Also, the present application provides a host cell comprising an expression vector as described in the third aspect above.

In embodiments of the fourth aspect of the present application, the present application also provides immunoconjugates comprising the anti-PD-L1 antibody or a functional fragment thereof and pharmaceutical compositions thereof. Preferably, the pharmaceutical composition further comprises a toxin, radioisotope, drug or cytotoxic agent. In other embodiments, the application also relates to a pharmaceutical composition comprising the anti-PD-L1 antibody or functional fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition at least contains an effective dose of the anti-PD-L1 antibody or the functional fragment thereof and a pharmaceutically acceptable carrier thereof.

In an embodiment of the fifth aspect of the present application, the present application provides the use of the anti-PD-L1 antibody or functional fragment thereof for the manufacture of a medicament for the treatment of a disorder of T cell dysfunction. Specifically, a method of preventing or treating a disease or alleviating a condition by eliminating, inhibiting or reducing PD-L1 activity comprises administering to a subject in need thereof an effective dose of the anti-PD-L1 antibody or functional fragment thereof, a nucleic acid, an expression vector, a host cell, an immunoconjugate or a pharmaceutical composition. Further, the uses described herein include the use of the anti-PD-L1 antibody or functional fragment thereof, and the corresponding nucleic acid, expression vector, host cell, immunoconjugate or pharmaceutical composition in the manufacture of a medicament for the treatment of a disease or disorder.

Example 1 preparation of human PD-L1 extracellular Domain-6 His protein

Example 1.1 Synthesis of human PD-L1 extracellular region-6 His Gene

Human PD-L1 extracellular region-6 His gene (NCBI Reference Sequence: NM-014143.4) was chemically synthesized, cloned into eukaryotic expression plasmid pcDNA3.1, HEK293 cells were transfected with PEI, after 6 days, the culture medium supernatant was collected and the human PD-L1 extracellular region protein was purified by nickel column affinity chromatography.

Human PD-L1 extracellular region-6 His amino acid sequence: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERHHHHHH

Example 1.2 preparation of biotinylated antigen Bio-PD-L1-6 His:

biotinylation of human PD-L1 extracellular domain-6 His molecule was performed using NHS-PEG4-Biotin, resulting in biotinylated antigen Bio-PD-L1-6 His.

Example 2 preparation of human PD-1 extracellular Domain-6 His protein

Example 2.1 Synthesis of human PD-1 extracellular region-6 His Gene

The human PD-1 extracellular region-6 His gene (NCBI Reference Sequence: NM-005018.3) was chemically synthesized, cloned into eukaryotic expression plasmid pcDNA3.1, HEK293 cells were transfected with PEI, after 6 days, the culture medium supernatant was collected and the human PD-1 extracellular region protein was purified by nickel column affinity chromatography.

Human PD-1 extracellular region-6 His amino acid sequence: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVHHHHHH

Example 2.2 preparation of biotinylated protein Bio-PD-1-6 His:

the extracellular region-6 His molecule of PD-L1 was biotinylated using NHS-PEG4-Biotin, resulting in biotinylated antigen Bio-PD-L1-6 His.

Example 3 establishment of human ScFv phage library

Collecting 20 peripheral blood of healthy people, and performing density gradient centrifugal separation to obtain 5 × 10⁹Lymphocytes, Triziol, extract total RNA according to the reference (Andris-Widhopf, j., Steinberger, p., Fuller, r., Rader, c.,&barbas, C.F., 3rd. (2011), Generation of human scFv antibody libraries, PCR amplification and analysis of light-and heavy-chain coding sequences, Cold Spring Harb protocol, 2011(9), doi: 10.1101/pdb.prot065573), constructing human ScFV phage library, wherein the library capacity of the phage primary library is 2 × 10⁹。

Example 4 screening of anti-human PD-L1 ScFv by magnetic bead method

Selection of phage antibody library expressing fully human Single chain antibody 200. mu.L (containing phage 1X 10)¹²one/mL) was mixed with 5. mu.g biotinylated Bio-PD-L1-6His antigen, incubated at room temperature for 30min, 50. mu.L of streptavidin magnetic beads were then added, antigen-bound phage were captured by streptavidin magnetic beads, and unbound phage were subjected to 0.5% TweThe solution of en-20 in PBS (phosphate buffer) was removed after rinsing, and the phage stably bound to the magnetic beads were eluted with a solution of glycine hydrochloride (pH 2.2) until use.

Inoculating XL1-Blue bacteria 200mL, allowing OD600nm (absorbance at 600 nm) to approach 0.6, adding the above eluted phage, standing with XL1-Blue bacteria at 37 deg.C for 30min, spreading the bacterial liquid on ampicillin resistant plate, eluting and collecting bacterial cells on ampicillin resistant plate the next day, and infecting at 1 × 10¹²pfu/ml of VCSM13 helper phage, amplification followed by the next round of selection, for a total of 3 rounds of selection.

XL1-Blue bacterial liquid infecting phage is fully diluted, then the bacterial liquid is coated on LB solid medium plate with the diameter of 15cm ampicillin resistance, each plate is provided with 100-500 clones, monoclonal antibody is picked, and each round of phage library after screening is verified through phage enzyme linked immunosorbent assay.

Phage enzyme-linked immunosorbent assay

XL1-Blue monoclonal bacteria of the transfected phage are inoculated into a 96-well 2mL bacterial culture plate, 500 mu L of SB culture medium containing tetracycline resistance is added, the shaking table is rotated at 200 rpm and at 37 ℃ for 4-6 h, 1 mu L of helper phage is added after the OD600nm value is detected to be close to 0.6, the shaking table is carried out overnight at 30 ℃, and 3000g of supernatant is centrifuged for standby the next day.

Taking an enzyme-linked immunosorbent assay plate, coating antigen overnight at 4 ℃, washing by PBST, then sealing, adding the phage supernatant prepared in the previous step, incubating for 2h at room temperature, adding Anti-M13 HRP antibody into the PBST, incubating for 30min, then washing for 3 times by the PBST, and adding 50 mu L of developing solution ABTS [2, 2' -diazanyl-bis (3-ethylbenzothiopyrroline-6 sulfonic acid) ]. The OD405nm value was detected.

After 3 rounds of screening, phage expressing antibodies that specifically bind to the antigen were enriched. And (3) selecting 700 monoclonals from the 3rd round of screened pools for enzyme-linked immunosorbent assay verification, and finally determining clones with the enzyme-linked immunosorbent assay reading value more than twice that of a control group as positive clones. Sequencing analysis is carried out on 100 positive clones, and the positive clone with the most repetition number is selected through comparison analysis. Sequencing the obtained cloned phagemid, analyzing the nucleotide sequence information of the heavy chain variable region and the light chain variable region, wherein the nucleotide sequences for coding the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID No.15 and SEQ ID No.16 in the sequence table, and the corresponding amino acid sequences are shown as SEQ ID No.1 and SEQ ID No. 2.

Antibody variable region DNA sequences

The variable region nucleic acid sequence of the antibody light chain is shown as SEQ ID No.15 in the sequence table. The heavy chain variable region nucleic acid sequence of the antibody is shown as SEQ ID No.16 in a sequence table.

Antibody variable region amino acid sequence

The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 1. The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID No. 2.

Example 5 expression of full antibodies

Example 5.1 construction of expression vectors for full-Length anti-PD-L1 antibodies

Synthesizing a primer according to a heavy chain variable region sequence, adding restriction enzymes BamH1 and BglII restriction sites at two ends, taking a heavy chain variable region nucleotide coding sequence of the antibody obtained by screening in the experimental example as a template, amplifying by conventional PCR, and then inoculating the fragment into a pFUSEs-CHIg-hG 1 expression vector by an enzyme digestion connection method, thereby obtaining the pFUSE-IgG1 expression vector of the full-length antibody heavy chain.

Synthesizing a primer according to the light chain variable region sequence, carrying EcoRI and AvrII enzyme cutting sites at two ends, taking the light chain variable region nucleotide coding sequence of the antibody obtained by screening in the experimental example as a template, carrying out conventional PCR amplification, and inserting a light chain pFUSE2ss-CLIg-hl2 vector by an enzyme cutting connection method, thereby obtaining the pFUSE-CLIg expression vector of the full-length antibody light chain.

Example 5.2 preparation of fully human monoclonal antibodies against PD-L1

And (2) mixing the 293 Fectin transfection reagent with the 2 eukaryotic antibody expression vectors obtained in the above step according to the volume-to-mass ratio of 30 mu L: 30 μ g: 30 μ g, adding 30 ml 293 Freestyle suspension cells, shaking overnight at 125rpm and 37 ℃, centrifuging, collecting the supernatant, purifying the antibody Protein by HiTrap Protein A HP columns on Ä KTApurifier100 Protein purifier, and finally detecting the antibody concentration according to the instruction of the BCA method Protein quantitative kit.

Example 6 detection of binding of antibodies to PD-L1 by SRP assay

The affinity and kinetic characteristics of the antibody and the antigen are detected by a multi-cycle kinetic method. The antibody immobilization was performed by a capture method: firstly, coupling the anti-PD-L1 fully-human monoclonal antibody on a Protein A chip, then diluting a PD-L1 sample in a gradient manner, flowing the diluted sample on the surface of the chip, wherein the initial concentration of the dilution is 40nM, the dilution multiple is 2 times, and the dilution is totally 6 concentration points, then the PD-L1 is captured by the coupled anti-PD-L1 fully-human monoclonal antibody, the signal is detected and recorded after the antigen is combined with the antibody, and finally, the antibody and the antigen sample on the surface of the Protein A chip are completely eluted by using a regeneration reagent (glycine solution with pH value of 1.5) and a new round of detection is carried out.

The interaction between the anti-PD-L1 fully human monoclonal antibody and the PD-L1 protein is detected by Biacore, and the KD of the anti-PD-L1 fully human monoclonal antibody is 7.1 nM.

Example 7 detection of binding of anti-PD-L1 antibody to PD-L1 at cellular level

The full-length gene of human PD-L1 (NCBI Reference Sequence: NM-014143.4) was chemically synthesized, cloned into eukaryotic expression plasmid in Puromycin (Puromycin) screening system, and PEI was used to transfect HEK293 cells. 24 hours after transfection, selection was performed by puromycin (2. mu.g/ml) until a 293F PD-L1 stable transfectant cell bank was formed. Meanwhile, by a limiting dilution method, laying a 96-well plate according to 0.8 cell per well, selecting a HEK293 PD-L1 monoclonal after 15 days, and carrying out passage to form a HEK293 PD-L1 stable transfer cell strain expressing PD-L1.

The HEK293 PD-L1 stable cell line was digested and resuspended in a FACS buffer by centrifugation at approximately 2.5X 10 cell size⁴Adding 50 mu L of the diluted antibody solution into a 1.5ml EP tube, adding 50 mu L of the diluted antibody solution with different concentrations, mixing uniformly, and incubating at room temperature for 30 min; the initial concentration of the antibody was 0.15ug/ml, and the concentration was diluted in 5-fold concentration gradient for a total of 8 concentrations.

Flow cytometric Fluorescence Sorting (FACS) technology detects the binding reaction of antibodies to PD-L1 expressing cells. The cells were washed twice with FACS buffer, 100. mu.L of Goat Anti-Human IgG-FITC antibody was added, and incubated for 30min in the dark; FACS was performed after washing twice with FACS buffer to detect antibodies at 8 concentration values, as shown in FIGS. 1a and 1 b. Fig. 1a and 1b show that the anti-PD-L1 antibodies of the present application can specifically bind to PD-L1 on HEK293 cells.

Example 8 AlphaLISA detection of anti-PD-L1 fully human monoclonal antibodies inhibits the binding of PD-L1 to PD-1

The anti-PD-L1 fully human monoclonal antibody to be detected is subjected to concentration gradient dilution by 3-fold dilution, the initial concentration value is 5 mug/muL, and 10 concentration values are diluted in total. The detection reaction system respectively comprises 5 mu L of antibody protein or inhibitor of a sample to be detected, 5 mu L of biotinylated PD-15 mu L, PD-L1-6His 5 mu L, and 5 mu L of mixture of streptavidin Donor beads and streptavidin acceptor beads (SA-Donor beads and acceptor beads), and the total amount is 20 mu L. Firstly, preparing 1X diluent, and then preparing each reagent by using the diluent according to the specification, wherein the diluent is a mixed solution of 4X histidine tag PD-L1(20nM), 4X biotinylation PD-1(20nM), 4X Anti-6X histidine AlphaLISA receptor beads (Anti-6 XHis AlphaLISA receptors beads) (40 mu g/mL) and Streptavidin labeled Donor beads (Streptavidin Donor beads) (80 mu g/mL). The above four liquids were mixed together according to a 20. mu.L reaction system, incubated for 90min at room temperature in the dark, and the value at 615nm was read on Envision, and the results are shown in FIG. 2. The results in FIG. 2 show that the anti-PD-L1 antibody of the present application is capable of inhibiting the binding of PD-1/PD-L1 with an IC50 of 37.18 ng/ml.

Example 9 Effect of human anti-PD-L1 antibody on cell proliferation and cytokine production in Mixed lymphocyte reaction

The effect of PD-L1 antibody on the proliferation of CD4+ T lymphocytes and the production of IFN-. gamma.and IL-2 was examined using Mixed Lymphocyte Reaction (MLR).

Example 9.1 isolation, culture and Induction of cells

Human Peripheral Blood Mononuclear Cells (PBMCs) were obtained from fresh blood of healthy volunteers by density gradient centrifugation using Ficoll-Paque. PBMC were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, with the addition of 100U of recombinant human IL-2.

The monocytes were separated from PBMC using human monocyte enrichment kit and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, adjusting the cell density to 2X10⁶And/ml. Meanwhile, recombinant human GM-CSF and IL-4 were added to the culture medium at final concentrations of 800U/ml and 50ug/ml, respectively. Cells were seeded in 6-well plates at 2.5ml per well and induced to become Dendritic Cells (DCs) after 5-7 days of culture. During this period half of the culture medium was discarded every 2-3 days and supplemented with fresh medium containing cytokines. 1ug/ml LPS was added to the culture medium 18 to 24 hours before the mixed lymph reaction, and the culture was carried out to obtain mature dendritic cells (mature DC, mDC).

CD4+ T lymphocytes were isolated from PBMCs using a human CD4+ T cell enrichment kit.

Example 9.2 Mixed lymph reaction

Purified CD4+ T lymphocytes were co-cultured with allogeneic derived mDC cells. Wherein the CD4+ T lymphocytes are 1 × 10⁵Or 2X10⁵Add 50. mu.L per well density (for detection of IFN-. gamma.and IL-2, respectively) followed by 50. mu.L of different concentrations of antibody. Then, 100. mu.L of mDC at various ratios were added, and the 96-well plate was placed in a 37 ℃ 5% CO2 incubator. The initial concentration of the antibody in the culture system was 10. mu.g/mL, and the concentration was diluted in a gradient manner by a dilution factor of 3. Parallel 96-well plates were set, and supernatants from the 96-well plates cultured for 3 days were used for ELISA detection of IL-2, and supernatants from the 96-well plates cultured for 5 days were used for ELISA detection of IFN-. gamma.s. Cells after 5 days of co-culture were examined for proliferation of CD4+ T lymphocytes with 3H-TDR.

Example 9.3 detection of cytokines

The supernatants were tested for human IFN-. gamma.and IL-2 levels by ELISA. Standard products recombinant human IFN-gamma and IL-2 were used for the determination of the standard concentration curve, respectively. The microplate was pre-coated with an antibody specific for human IFN-. gamma.or IL-2, and blocked the next day with a 2% BSA in PBS for 1 hour. mu.L of the standard and the sample to be tested were added to each well and incubated at room temperature for 2 hours. After washing, the biotinylated IFN-. gamma.antibody or IL-2 antibody was added and incubated for 1 hour. After washing, streptavidin-labeled HRP (Horseradish peroxidase) was added and incubated at room temperature for 30 minutes. And (3) adding a TMB substrate after washing again for color development, and stopping the reaction by using 2M HCl after the color of a reaction product is changed from light to dark. The light absorption was read at 450nm in 15 minutes using a microplate Reader (Molecular Devices SpectraMax M5e Plate Reader). The cytokine content in the supernatant to be detected is calculated according to the standard curve, the detection results are shown in fig. 3 and fig. 4, and the results show that the anti-PD-L1 antibody can promote T cells to secrete IL-2 and IFN-gamma.

Example 9.4 detection of cell proliferation

The 3H-thymidine was diluted 20 volumes with 0.9% NaCl solution and added to a 96-well plate to give a final concentration of 0.5 uCi/well. The 96 well plates were further incubated in an incubator at 37 ℃ with 5% CO2 for 16-18 hours. After the completion of the culture, the supernatant was centrifuged and removed from the GF/B plate, which was then washed once with double distilled water. The 96-well plate was placed on a cell harvester (PerkinElmer), vacuum filtered, washed 5 times with pre-chilled double distilled water, and oven dried at 45 ℃. The results of 50. mu.L of scintillation fluid added to each well and counted by a liquid scintillation counter (Top Count NXT HTS Reader) are shown in FIG. 5, and indicate that the anti-PD-L1 antibody of the present application can promote T cell proliferation.

The above results show that the anti-PD-L1 fully human monoclonal antibody of the present application promotes IL-2 secretion, IFN- γ secretion and T cell proliferation in a concentration-dependent manner. Based on the results in the examples, the use of the antibody in the fourth and fifth aspects described above, in particular in the manufacture of a medicament for the treatment of a disorder of T cell dysfunction, can be carried out with certainty by the skilled person.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA techniques, and chemical methodology that are within the capabilities of one of ordinary skill in the art. Although specific embodiments of the present application have been disclosed in detail herein, this has been done by way of example only and is for the purpose of illustration only. The above-described embodiments are not intended to limit the scope of the appended claims, nor is it intended that the application be construed as necessarily dependent upon the particular features described above.

It will be appreciated by those skilled in the art that any equivalent alterations or modifications to the present application, for example, any equivalent alterations or modifications to the methods, sequences, reagents, etc. selected for use in the present application, and additions of auxiliary moieties, selection of particular modes, etc., are within the scope and disclosure of the present application. The various features described in the foregoing detailed description may be combined in any suitable manner without contradiction, and various combinations that may be possible are not described in this application in order to avoid unnecessary repetition.

SEQUENCE LISTING

<110> Shanghai Jiabei biological medicine technology, Inc

<120> an anti-PD-L1 antibody or functional fragment thereof and use thereof

<130> 2018591-1

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> variable region amino acid sequence of antibody light chain

<400> 1

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr

20 25 30

Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser

85 90 95

Asn Asn Leu Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 2

<211> 119

<212> PRT

<213> Artificial Sequence

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<223> variable region heavy chain amino acid sequence of antibody

<400> 2

Gln Val Gln Leu Val Gln Ser Gly Gly Glu Leu Lys Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Tyr Arg His Phe Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

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<223> light chain variable region CDR1 amino acid sequence

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Ser Ser Asp Val Gly Ser Tyr Asn Leu

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<223> light chain variable region CDR2 amino acid sequence

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Glu Val Thr

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<223> light chain variable region CDR3 amino acid sequence

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Ser Ser Tyr Ala Gly Ser Asn Asn Leu Val

1 5 10

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<223> heavy chain variable region CDR1 amino acid sequence

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Gly Gly Thr Phe Ser Ser Tyr Ala

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Ile Ile Pro Ile Phe Gly Thr Ala

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<223> heavy chain variable region CDR3 amino acid sequence

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Ala Ser Ser Ser Tyr Arg His Phe Ala Phe Asp Ile

1 5 10

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<212> PRT

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<220>

<223> light chain variable region FR1 amino acid sequence

<400> 9

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr

20 25

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<213> Artificial Sequence

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<223> light chain variable region FR2 amino acid sequence

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Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile

1 5 10 15

Tyr

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<223> light chain variable region FR3 amino acid sequence

<400> 11

Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly

1 5 10 15

Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala

20 25 30

Asp Tyr Tyr Cys

35

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<223> amino acid sequence of FR1 of heavy chain variable region

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Gln Val Gln Leu Val Gln Ser Gly Gly Glu Leu Lys Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

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<213> Artificial Sequence

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<223> amino acid sequence of FR2 of heavy chain variable region

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Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10 15

Arg

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<223> amino acid sequence of FR3 of heavy chain variable region

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Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu

1 5 10 15

Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 15

<211> 330

<212> DNA

<213> Artificial Sequence

<220>

<223> variable region of antibody in light chain

<400> 15

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgatgttggg agttataacc ttgtctcctg gtaccaacag 120

cacccaggca aagctcccaa actcatgatt tatgaggtca ctaagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agttcatatg caggcagcaa caatttggtc 300

tttggcggag ggaccaaggt caccgtccta 330

<210> 16

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> heavy chain variable region nucleic acid sequence

<400> 16

caggtgcagc tggtgcaatc tggaggtgag ctgaagcggc ctgggtcttc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaagg atcatcccta tctttggtac agcaaactac 180

gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagtagtagt 300

taccggcatt ttgcttttga tatctggggc caagggacca cggtcaccgt ctcctca 357

<210> 17

<211> 244

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<213> Artificial Sequence

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<223> human PD-L1 extracellular region-6 His amino acid sequence

<400> 17

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg His His

225 230 235 240

His His His His

<210> 18

<211> 176

<212> PRT

<213> Artificial Sequence

<220>

<223> human PD-1 extracellular region-6 His amino acid sequence

<400> 18

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val His His His His His His

165 170 175

Claims

1. An anti-PD-L1 fully human antibody, wherein the anti-PD-L1 antibody is a monoclonal antibody, and the affinity dissociation constant KD of the antibody and PD-L1 is less than or equal to 1.0x10^-8M, the anti-PD-L1 antibody comprising heavy chain hypervariable regions CDR1, CDR2 and CDR3, and light chain hypervariable regions CDR1, CDR2 and CDR 3;

wherein:

the amino acid sequence of the light chain hypervariable region CDR1 is the sequence shown in SEQ ID No.3, the amino acid sequence of the light chain hypervariable region CDR2 is the sequence shown in SEQ ID No.4, and the amino acid sequence of the light chain hypervariable region CDR3 is the sequence shown in SEQ ID No. 5;

the amino acid sequence of the heavy chain hypervariable region CDR1 is the sequence shown in SEQ ID No.6, the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence shown in SEQ ID No.7, and the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence shown in SEQ ID No. 8; and

the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID No. 2.

2. The anti-PD-L1 antibody according to claim 1, wherein said anti-PD-L1 antibody further comprises a light chain Framework Region (FR) and a heavy chain Framework region, wherein;

the light chain framework regions of the anti-PD-L1 antibody include one or more of the light chains FR1, FR2, and FR3, wherein: the amino acid sequence of the light chain FR1 is a sequence shown as SEQ ID No. 9; the amino acid sequence of the light chain FR2 is shown as SEQ ID No.10 or; the amino acid sequence of the light chain FR3 is a sequence shown as SEQ ID No. 11; and

the heavy chain framework region of the anti-PD-L1 antibody comprises one or more of heavy chains FR1, FR2, and FR3, wherein: the amino acid sequence of the heavy chain FR1 is a sequence shown as SEQ ID No. 12; the amino acid sequence of the heavy chain FR2 is a sequence shown as SEQ ID No. 13; the amino acid sequence of the heavy chain FR3 is shown as SEQ ID No. 14.

3. The anti-PD-L1 antibody according to claim 2, characterized in that the nucleotide sequence encoding the light chain variable region in the anti-PD-L1 antibody is the sequence shown in SEQ ID No.15 and the nucleotide sequence encoding the heavy chain variable region in the anti-PD-L1 antibody is the sequence shown in SEQ ID No. 16.

4. An expression vector, characterized in that the vector comprises a sequence shown as SEQ ID No.15 and a sequence shown as SEQ ID No. 16.

5. A host cell comprising the vector of claim 4, wherein said host cell is a mammalian cell.

6. An immunoconjugate comprising the anti-PD-L1 antibody or functional fragment thereof of any one of claims 1-3, and pharmaceutical compositions thereof, wherein said pharmaceutical compositions contain at least an effective dose of said anti-PD-L1 antibody or functional fragment thereof and a pharmaceutically acceptable carrier therefor.

7. Use of the anti-PD-L1 antibody or functional fragment thereof of any one of claims 1-3 in the manufacture of a medicament for the treatment of a disorder of T cell dysfunction.