A human PD-L1 antibody based on fully human antibody mice and its preparation method and application

Abstract

The present invention discloses an anti-human PD-L1 antibody or antigen-binding fragment thereof based on a fully humanized antibody mouse, a nucleic acid molecule encoding them, and a preparation method and application thereof. The anti-human PD-L1 antibody or antigen-binding fragment thereof is a fully humanized sequence, has good specificity and affinity for PD-L1, and can effectively promote the secretion of IL2 and IFNγ of T cells. It has lower immunogenicity. It further relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and its use in the preparation of a drug for preventing and / or treating PD-L1-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of fully human antibody development, and specifically relates to an anti-human PD-L1 monoclonal antibody and its application, and in particular to a fully humanized monoclonal antibody and its application that has high affinity for human PD-L1, cross-binding with monkey PD-L1, and does not bind to mouse PD-L1. Technical Background

[0002] Tumor immunotherapy has been a long-term hot topic in the field of tumor treatment, and T cell tumor immunotherapy is at its core. Tumor immunotherapy is to fully utilize and mobilize the killer T cells in the body of tumor patients to kill tumors. It may be the most effective and safest way to treat tumors. At the same time, tumor escape is a huge obstacle faced by tumor immunotherapy. Tumor cells use their own inhibitory effect on the immune system to promote the rapid growth of tumors.

[0003] PD-L1, also known as B7-H1 or CD274, is the first characterized functional ligand of coinhibitory programmed death receptor 1 (PD-1). PD-L1 and its cognate ligand PD-L2 bind to the PD-1 receptor and play a key role in maintaining the tolerance of peripheral and central immune cells. Studies have found that PD-L1 is highly expressed in different types of tumors, including melanoma and ovarian cancer, lung cancer, and renal cancer. During the carcinogenic process, PD-L1 may be overexpressed due to carcinogenic events caused by driver genes.

[0004] In addition, the PD-1 / PD-L1 pathway is essential for the development of immune tolerance, a process by which autoreactive lymphocytes are negatively selected in primary (central tolerance) and secondary lymphoid organs (peripheral tolerance). In fact, high expression of PD-L1 has been demonstrated in the thymus and on dendritic cells, where the PD-L1 / PD-1 interaction prevents the proliferation and differentiation of naive T cells. Therefore, PD-L1 plays a key role in preventing autoimmunity in normal tissues and maintaining peripheral tolerance, and tumor cells can use the PD-1 / PD-L1 signaling pathway to evade antitumor immune responses and ultimately spread and metastasize. Therefore, blocking the PD-1 / PD-L1 signaling pathway can activate endogenous antitumor immune responses, thereby exerting a therapeutic effect on tumors.

[0005] At present, most PD-L1 antibodies in the field are traditional IgG heavy and light chain antibodies and fully human antibodies (nanobodies). However, fully humanized PD-L1 antibodies are closer to natural human sequences, have lower immunogenicity, and less drug toxicity. Therefore, there is a need to develop new specific fully human antibodies that are effective against human PD-L1. Summary of the invention

[0006] The present invention provides a novel anti-human PD-L1 antibody produced based on fully human antibody mice and its use.

[0007] In one aspect, the present invention provides a fully human antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) amino acid sequence and a light chain variable region (VL) amino acid sequence; the heavy chain variable region (VH) comprises the following CDR1, CDR2 and CDR3, and the light chain variable region comprises the following CDR1, CDR2 and CDR3; (1) the amino acid sequences of the selected VH CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 7, 8 and 9, respectively, and the amino acid sequences of the selected VL CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 10, 11 and 12, respectively; (2) the amino acid sequences of the selected VH CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 13, 14 and 15, respectively, and the amino acid sequences of the selected VL CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: NO: 16, 17 and 18; (3) the amino acid sequences of the selected VHCDR1, CDR2 and CDR3 are shown in SEQ ID NO: 19, 20 and 21, respectively, and the amino acid sequences of the selected VL CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 22, 23 and 24, respectively.

[0008] On the one hand, the fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 described in the present invention is characterized in that the VH comprises an amino acid sequence with at least 75% identity to SEQ ID NO: 1, 3, 5, and the VL comprises an amino acid sequence with at least 75% identity to SEQ ID NO: 2, 4, 6.

[0009] In one aspect, the present invention provides a fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1, wherein the VH comprises one or more amino acid substitutions, deletions or additions compared to the sequences shown in SEQ ID NOs: 1, 3 and 5, and the VL comprises one or more amino acid substitutions, deletions or additions compared to the sequences shown in SEQ ID NOs: 2, 4 and 6.

[0010] In one aspect, the fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 of the present invention, wherein the antibody or antigen-binding fragment thereof is a fully human antibody based on a fully human antibody mouse.

[0011] In one aspect, the fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 described in the present invention, wherein the antibody or antigen-binding fragment thereof further comprises heavy chain and light chain constant regions or variants thereof, the heavy chain constant region is selected from IgG, IgM, IgE, IgD or IgA, preferably, the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4, preferably, the heavy chain constant region is a human IgG1 heavy chain constant region, more preferably, the human IgG1 heavy chain constant region comprises the sequence shown in SEQ ID NO: 25; and the light chain constant region is selected from kappa and lambda, preferably, the light chain constant region is a human kappa light chain constant region, more preferably, the human kappa light chain constant region comprises the sequence shown in SEQ ID NO: 26.

[0012] In one aspect, the fully human anti-human PD-L1 antibody or antigen-binding fragment thereof described in the present invention can specifically bind to the extracellular region of human PD-L1, and the affinity constant (KD) with the extracellular region of human PD-L1 is less than 1E-10.

[0013] In one aspect, the present invention provides a nucleic acid molecule encoding the aforementioned fully human antibody or antigen-binding fragment thereof.

[0014] In one aspect, the present invention provides a vector comprising the aforementioned nucleic acid molecule; preferably, the vector is a cloning vector or an expression vector.

[0015] In one aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or vector.

[0016] In one aspect, the present invention provides a method for preparing the aforementioned fully human antibody or its antigen-binding fragment; the method comprises culturing the host cell of claim 10 under conditions allowing protein expression, and collecting the antibody or its antigen-binding fragment or the multispecific antibody from the cultured host cell culture.

[0017] In one aspect, the present invention provides a conjugate comprising the aforementioned fully human antibody or its antigen-binding fragment and a coupling portion; preferably, the coupling portion is selected from any one of the following: a detectable label, a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, polyethylene glycol (PEG), a nuclide, a nucleic acid, an immune agonist, a cytokine, a small molecule toxin, a polypeptide with binding activity, a protein, a receptor, a ligand, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0018] In one aspect, the present invention provides a pharmaceutical composition comprising the aforementioned fully human antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, mammalian cell or conjugate, and a pharmaceutically acceptable carrier and / or excipient. Furthermore, the pharmaceutical composition also includes another pharmaceutically active agent; preferably, the other pharmaceutically active agent is a drug with anti-tumor activity, an immunotherapy-related drug, an immunity-enhancing drug or an immune system activator, a radioactive substance or a colorimetric agent.

[0019] In one aspect, the present invention provides a kit comprising the aforementioned fully human antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, mammalian cell, conjugate or pharmaceutical composition.

[0020] In one aspect, the present invention provides the use of the aforementioned fully human antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, mammalian cells, conjugates or pharmaceutical compositions in the preparation of drugs for the prevention and / or treatment and / or adjuvant treatment of diseases associated with PD-L1. Further, the diseases associated with PD-L1 are tumors, pulmonary vascular diseases, autoimmune diseases, neurodegenerative diseases, viral infections, systemic inflammatory response syndrome, immune disorders, pulmonary fibrosis or immune responses to transplants.

[0021] Furthermore, in the above-mentioned use, the tumor is selected from solid tumors and blood tumors; the pulmonary vascular disease is selected from pulmonary hypertension; the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease or Huntington's disease; the viral infection is selected from HIV infection, hepatitis B infection or papillomavirus infection; the systemic inflammatory response syndrome is selected from sepsis or septicemia; the immune disorder is selected from allergic reaction; preferably, the solid tumor is selected from melanoma, lung cancer, kidney cancer, bladder cancer, head and neck tumors, gastric cancer, esophageal cancer, liver cancer, skin cancer or lymphoma.

[0022] Furthermore, in the above-mentioned use, the antibody or its antigen-binding fragment, the multispecific antibody, the isolated nucleic acid, the vector, the host cell, the conjugate or the pharmaceutical composition is administered separately, in combination, simultaneously or sequentially with another pharmaceutically active agent; wherein the other pharmaceutically active agent is a drug with anti-tumor activity, an immunotherapy-related drug, an immunity-enhancing drug or an immune system activator, a radioactive substance or a colorimetric agent.

[0023] In one aspect, the present invention discloses a method for preventing and / or treating a disease associated with PD-L1, and / or delaying the progression of a disease associated with PD-L1, and / or reducing or inhibiting the recurrence of a disease associated with PD-L1 in a subject, comprising administering an effective amount of the above-mentioned pharmaceutical composition to a subject in need thereof.

[0024] Furthermore, in the above method, the disease associated with PD-L1 is a tumor, a pulmonary vascular disease, an autoimmune disease, a neurodegenerative disease, a viral infection, a systemic inflammatory response syndrome, an immune disorder, a pulmonary fibrosis or an immune response to a transplant.

[0025] Furthermore, in the above method, the tumor is selected from solid tumors and blood tumors; the pulmonary vascular disease is selected from pulmonary hypertension; the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease or Huntington's disease; the viral infection is selected from HIV infection, hepatitis B infection or papillomavirus infection; the systemic inflammatory response syndrome is selected from sepsis or septicemia; the immune disorder is selected from allergic reaction; preferably, the solid tumor is selected from melanoma, lung cancer, kidney cancer, bladder cancer, head and neck tumors, gastric cancer, esophageal cancer, liver cancer, skin cancer or lymphoma.

[0026] Furthermore, the above method also includes administering a second therapy to the subject, wherein the second therapy is selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, therapy, viral therapy, adjuvant therapy, cell therapy and any combination thereof; preferably, the method is applied simultaneously or sequentially with the second therapy.

[0027] In one aspect, the present invention also discloses a method for detecting the presence or level of PD-L1 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of the present invention under conditions allowing formation of a complex between the antibody or antigen-binding fragment thereof and PD-L1; and detecting the formation of the complex.

[0028] In one aspect, the present invention also discloses the use of the antibody or antigen-binding fragment or conjugate of the present invention in the preparation of a diagnostic kit for diagnosing or distinguishing tumors, pulmonary vascular diseases, autoimmune diseases, neurodegenerative diseases, viral infections, systemic inflammatory response syndrome, immune disorders, pulmonary fibrosis or immune responses to transplants.

[0029] Furthermore, in the above-mentioned use, the tumor is selected from solid tumors and blood tumors; the pulmonary vascular disease is selected from pulmonary hypertension; the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease or Huntington's disease; the viral infection is selected from HIV infection, hepatitis B infection or papillomavirus infection; the systemic inflammatory response syndrome is selected from sepsis or septicemia; the immune disorder is selected from allergic reaction; preferably, the solid tumor is selected from melanoma, lung cancer, kidney cancer, bladder cancer, head and neck tumors, gastric cancer, esophageal cancer, liver cancer, skin cancer or lymphoma.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] In the present invention, we obtained multiple positive candidate antibody molecules by immunizing fully human antibody mice based on phage display technology. The results of sequence analysis showed that the variable region sequences of the candidate antibodies were all fully human sequences. Therefore, the present invention provides a method for developing fully human antibodies based on the immunization of fully human antibody mice. Compared with the natural human phage library display method, this method can theoretically obtain high-affinity antibodies without the need for further affinity maturation; compared with the antibody development method of ordinary mouse immunization, the antibody sequence obtained based on the immunization of fully human antibody mice has a comparable affinity, but does not require further humanization. Therefore, antibody development based on fully human antibody mice will greatly accelerate the development process of antibody drugs and eliminate the cumbersome steps of affinity maturation and humanization. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The binding activity of anti-human PD-L1 fully human antibody to PDL1-His;

[0033] Figure 2 Anti-human PD-L1 fully human antibody and PD1 blocking activity;

[0034] Figure 3 Cross-reactivity of fully human anti-human PD-L1 antibodies;

[0035] Figure 4 Binding activity of anti-human PD-L1 fully human antibody to CHO-PDL1;

[0036] Figure 5 The blocking activity of anti-human PD-L1 fully human antibody in the PD-1 / PD-L1 signaling pathway;

[0037] Figure 6 Anti-human PD-L1 fully human antibodies activate T cells to produce cytokines; Figure 6 a represents the detection results of 6-B11-A2, 131-4-G9 and Atezolizumab activating T cells to produce IL2; Figure 6 b shows the detection results of 6-B11-A2, 131-4-G9 and Atezolizumab activating T cells to produce IFNγ;

[0038] Figure 7 Tumor inhibition rate. DETAILED DESCRIPTION

[0039] The information of the sequences involved in the present invention is described in the following Table 1 (Kabat numbering scheme):

[0040] Table 1 Sequence information

[0041]

[0042]

[0043] Definition of terms:

[0044] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0045] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0046] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0047] In the present invention, the "fully human antibody" described in the present invention refers to the antibody produced by fully human antibody mice immunized or vaccinated with an antigen against the antigen. Fully human antibody mice are fully human antibody mouse transgenic models established by replacing the variable region germline antibody genes of mice with human germline antibody genes. This antibody mouse model contains all human heavy chain variable region genes and all light chain variable region genes, and the antibody sequence produced by it is a completely human sequence. Compared with traditional chimeric antibodies, humanized antibodies have lower immunogenicity and are a better choice in antibody drug production.

[0048] The "antibody" described in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or called the same subtypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0049] The term "complementarity determining region" or "CDR" as used herein refers to the amino acid residues in the variable region of a fully human antibody that are responsible for antigen binding. There are three CDRs in the variable region of the heavy chain of a fully human antibody, named VH CDR1, CDR2 and CDR3. There are three CDRs in the variable region of the light chain of a fully human antibody, named VL CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the definition in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). For a given fully human antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system.

[0050] The term "framework region" or "FR" residues as used herein refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0051] The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction. The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the speed of formation and dissociation of an antigen binding site or antigen complex. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis or kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method.

[0052] The term "vector" as used in the present invention refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); 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, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0053] The term "host cell" as used in the present invention refers to cells that can be used to introduce vectors, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0054] The term "germline antibody gene" or "germline antibody gene segment" described in the present invention refers to a sequence encoding an immunoglobulin present in the genome of an organism, which has not undergone a maturation process that can lead to genetic rearrangement and mutation that can express specific immunoglobulins. In the present invention, the expression "heavy chain germline gene" refers to a germline antibody gene or gene segment encoding an immunoglobulin heavy chain, which includes a V gene (variable), a D gene (diversity), a J gene (joining) and a C gene (constant). Germline antibody genes or germline antibody gene segments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from professional databases (e.g., IMGT, UNSWIg, NCBI or VBASE2). It is generally believed that germline antibody genes are more likely to retain key amino acid sequence structures that are unique to individuals in a species than mature antibody genes, and therefore are relatively less likely to be considered exogenous when used for treatment in the species.

[0055] The term "identity" as used herein refers to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, a gap may be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions, total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0056] The determination of the percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as improved in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0057] The term "variant" of the present invention also refers to a polypeptide or peptide comprising an amino acid sequence that has been changed by introducing amino acid residue substitution, deletion or addition in the context of a polypeptide (including a polypeptide). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limiting, a polypeptide can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection, blocking groups, proteolytic cleavage, connection to a cell ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function similar, identical or improved to the polypeptide or peptide from which it is derived.

[0058] The term "conservative substitution" of the present invention means an amino acid substitution that does not adversely affect or change the expected properties of a protein or polypeptide comprising an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0059] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0060] The term "pharmaceutically acceptable carrier and / or excipient" as used herein refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed.

[0061] Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.

[0062] The term "prevention" as used herein refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

[0063] The term "subject" as used herein refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a tumor (e.g., a tumor expressing PD-L1), an inflammatory disease, or an autoimmune disease, or is at risk of suffering from the above diseases.

[0064] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0065] The "combination" described in the present invention includes therapies that can be administered separately, such as separately formulated for separate administration (for example, they can be provided in a kit), and therapies that can be administered together in a single formulation (ie, a "co-formulation"). In certain embodiments, the anti-human PD-L1 fully human antibodies or antigen-binding fragments thereof of the present invention can be administered sequentially. In other embodiments, the anti-human PD-L1 fully human antibodies or antigen-binding fragments thereof can be administered simultaneously. The anti-human PD-L1 fully human antibodies or antigen-binding fragments thereof of the present invention can be used in combination with at least one other (active) agent in any manner.

[0066] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0067] Example 1

[0068] Fully human antibody mouse immunization and titer determination

[0069] In order to generate humoral immune response against PD-L1 in fully human antibody mice, 5 fully human antibody mice (from Saiye (Suzhou) Biotechnology Co., Ltd.) were selected for immunization. The mice were first immunized with Freund's complete adjuvant (CFA) mixed with 0.05 mg PD-L1 His (Kaixia Bio, PDL-HM110) protein and injected subcutaneously. After that, Freund's incomplete adjuvant (IFA) mixed with 0.025 mg PD-L1 His protein was injected subcutaneously, and immunization was performed every 2 weeks.

[0070] The fully human antibody mouse serum after the third immunization was taken for serum titer monitoring. 1 μg / ml antigen was coated overnight with CBS (carbonate buffer solution), and 2% BSA was added after washing with PBST (phosphate buffer solution), and blocked at 37°C for 2h. After washing with PBST, serum diluent (diluted from 1:2000, 8 gradients) was added and blocked at 37°C for 2h. After washing with PBST, horseradish peroxidase-labeled goat anti-mouse secondary antibody (diluted with PBS at 1:10000) was added and incubated at 37°C for 1h. After washing with PBST, TMB colorimetric solution (Biyuntian, P0209-500ml) was added for color development, 37°C, 5-10min, and the reaction was terminated by adding stop solution. The optical density was measured at 450nm to detect serum titer.

[0071] Example 2

[0072] Establishment of phage library

[0073] The spleen of the mouse with the highest titer was collected and the cells were collected after grinding. Total RNA of spleen cells was extracted using an RNA extraction kit (Fuji Bio, RE-03011). cDNA was synthesized and the antibody VH and VL sequences were amplified using slot PCR. The target fragments were connected to the phage vector, and the ligation products were electrotransformed into TG1 competent cells to construct a PD-L1 scFv antibody library. 40 clones were randomly selected from the library transformant number titer plate for sequencing identification, and the results showed that the insertion rate was 100%. The helper phage M13KO7 (NEB, N0315S) was used for library packaging, and the phage library titer was detected to be 1.2×10 13 cfu / mL.

[0074] Example 3

[0075] Screening of fully human anti-human PD-L1 antibodies

[0076] 3.1 Phage panning

[0077] Using the magnetic bead selection method, streptavidin magnetic beads (Dynabeads TM M-280Streptavidin) was added to PBS buffer prepared with 3% skim milk powder and rotated at room temperature for 1 hour, and then 5 μg of biotinylated PD-L1 (ACRO, PD1-H82E5) antigen was added and rotated at room temperature for 1 hour to form a magnetic bead protein complex. Then 50 μl of phage library was added to the magnetic bead protein complex, and it was rotated and incubated at room temperature for 1-2 hours, and then washed 8 times with 0.05% PBST to remove non-specifically bound phages, and then infected with OD 600 The TG1 bacteria were 0.6-0.8, and then the 2YT medium plates were coated overnight at 37°C. The same screening operation was performed for 2 rounds to complete the enrichment of positive phages.

[0078] 3.2 Prokaryotic expression and screening

[0079] (1) Library monoclonal expression

[0080] The plasmids of the TG1 library bacteria after the above two rounds of panning were extracted and constructed into the Pet28a vector, and then single clones were picked and expressed in 96 deep-well plates for IPTG induction. The supernatant was used for ELISA screening.

[0081] (2) Enzyme-linked immunosorbent assay (ELISA) to screen binding active clones

[0082] Use CBS coating solution to coat 2 μg / ml human PD-L1 Fc protein, add 50 μl of the supernatant protein prepared above, incubate for 2 hours, wash away unbound nonspecific clones with 0.05% PBST, add anti-Flag-HRP secondary antibody (proteintech) and incubate for 1 hour, wash away unbound nonspecific clones with 0.05% PBST, add TMB colorimetric solution and incubate for 10-15 minutes, and read at a wavelength of 450 nm using an enzyme reader.

[0083] (3) Enzyme-linked immunosorbent assay (ELISA) screening of blocking active clones

[0084] Use CBS coating solution to coat 2 μg / ml human PD-L1 Fc protein, add 50 μl of the supernatant protein prepared above and 5 nM PD1 mFc protein, incubate for 2 h, wash away unbound nonspecific clones with 0.05% PBST, add Goat anti-Mouse IgG2a, HRP secondary antibody (Thermo) and incubate for 1 h, wash away unbound nonspecific clones with 0.05% PBST, add TMB colorimetric solution and incubate for 10-15 min, and read at a wavelength of 450 nm using an enzyme reader.

[0085] Example 4

[0086] Anti-human PD-L1 fully human antibody expression

[0087] The VH and VL sequences of the positive clones with both binding and blocking activities were constructed into the PTT5 eukaryotic expression vector, and the constant region was the human IgG1 constant region sequence. The plasmids with VH and VL sequences were transferred into HEK293F cells for expression by transient expression, and the antibodies were purified by protein A filler after expression. The antibody sequence was analyzed, and the CDR region was defined by IMGT. The variable region and CDR sequences of the obtained anti-human PD-L1 fully human antibody are shown in Table 2:

[0088] Table 2 Variable region and CDR amino acid sequences of anti-human PD-L1 fully human antibody

[0089]

[0090] Example 5

[0091] Anti-human PD-L1 fully human antibody binding and blocking activity detection

[0092] 5.1ELISA to detect the binding activity of anti-human PD-L1 fully human antibody

[0093] Use CBS coating solution to coat 2μg / ml human PD-L1 His protein, then add gradient diluted anti-human PD-L1 fully human antibody (starting at 33.33nM, 3-fold gradient dilution, a total of 10 gradients), incubate for 2h, wash off unbound nonspecific clones with 0.05% PBST, add Goat anti-Human IgG Fc, HRP secondary antibody (Thermo) for 1h, wash off unbound nonspecific clones with 0.05% PBST, add TMB colorimetric solution for 10-15min, and read at 450nm using an enzyme reader. The experimental data was fitted with EC50 by GraphPad Prism, and the results are as follows: Figure 1 Table 3 shows the data of binding affinity of anti-human PD-L1 fully humanized antibodies, and the results show that the antibodies of the present invention can bind to human PD-L1 protein.

[0094] Table 3 Binding affinity of anti-human PD-L1 fully human antibody to PD-L1 His

[0095] Antibody Name <![CDATA[EC 50 (nM)]]> 6-B11-A2 0.03304 131-4-G9 0.03357 131-2-C3 0.02829

[0096] 5.2ELISA detection of blocking activity of anti-human PD-L1 fully human antibody

[0097] Use CBS coating solution to coat 2μg / ml human PD-L1 His protein, then add gradient diluted anti-human PD-L1 fully human antibody (starting at 33.33nM, 3-fold gradient dilution, a total of 10 gradients) and a constant concentration of 5nM PD1 mFc protein, incubate for 2h, wash off unbound nonspecific clones with 0.05% PBST, add Goat anti-Mouse IgG2a, HRP secondary antibody (Thermo) for 1h, wash off unbound nonspecific clones with 0.05% PBST, add TMB colorimetric solution for 10-15min, and read at 450nm using an enzyme reader. The experimental data was fitted with IC50 by GraphPad Prism, and the results are as follows Figure 2 Table 4 shows the activity data of the anti-human PD-L1 fully human antibody in blocking the binding of human PD-L1 to PD-1. The results show that the anti-human PD-L1 fully human antibody of the present invention can block the binding of human PD-L1 to PD-1.

[0098] Table 4 Anti-human PD-L1 fully human antibody blocks the binding affinity between human PD-1 and PD1

[0099] Antibody Name <![CDATA[IC 50 (nM)]]> 6-B11-A2 1.811 131-4-G9 1.324 131-2-C3 1.452

[0100] Example 6

[0101] Cross-reactivity detection of anti-human PD-L1 fully human antibody

[0102] Use CBS coating solution to coat 2μg / ml monkey and mouse PD-L1 His protein, then add gradient diluted anti-human PD-L1 fully human antibody (starting at 33.33nM, 3-fold gradient dilution, a total of 10 gradients), incubate for 2h, wash off unbound non-specific clones with 0.05% PBST, add Goat anti-Human IgG Fc, HRP secondary antibody (Thermo) for 1h, wash off unbound non-specific clones with 0.05% PBST, add TMB colorimetric solution for 10-15min, and read at 450nm using an enzyme reader. The experimental data was fitted with EC50 by GraphPad Prism, and the results are as follows: Figure 3 Table 5 shows the data of binding affinity of fully humanized anti-human PD-L1 antibodies, and the results show that the antibodies of the present invention can bind to human PD-L1 protein.

[0103] Table 5 Cross-binding activity of monkey and mouse anti-human PD-L1 fully human antibodies

[0104] Antibody Name <![CDATA[Cyno PD-L1 EC 50 (nM)]]> <![CDATA[Mouse PD-L1 EC 50 (nM)]]> 6-B11-A2 0.1462 N / A 131-4-G9 0.5032 N / A 131-2-C3 0.1754 N / A

[0105] Example 7

[0106] Flow cytometry detection of anti-human PD-L1 fully human antibody binding activity to cells

[0107] CHO-PDL1 overexpressing cells were taken, washed with 1xPBC, and resuspended in 1% BSA. 1×10 5 Cells were added to a 96-well V-bottom plate, and then gradiently diluted anti-human PD-L1 fully human antibody (starting at 30ug / ml, 3-fold gradient dilution, a total of 10 gradients) was added, incubated on ice for 1h, washed with 1xPBS, and then PE-labeled goat anti-human Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30min, washed with 1xPBS, and detected using a flow cytometer. The experimental data was fitted with EC50 by GraphPad Prism, and the results are shown in the figure. Figure 4 Table 6 shows the data of affinity of anti-human PD-L1 fully human antibody cell binding, and the results show that the antibody of the present invention can bind to CHO cells overexpressing human PD-L1.

[0108] Table 6 Binding affinity of anti-human PD-L1 fully human antibodies to CHO cells overexpressing human PD-L1

[0109] Antibody Name <![CDATA[EC 50 (ug / ml)]]> 6-B11-A2 1.064 131-4-G9 1.387 131-2-C3 1.659

[0110] Example 8

[0111] Detection of blocking activity of anti-human PD-L1 fully human antibody in T cell PD-1 / PD-L1 signaling pathway

[0112] In a 96-well plate, 100 μL of 2x10 4 Effector cells Jurkat-PD1-NFAT-Luc monoclonal cells and 2x10 4 Target cells CHO-PDL1. 100 μL of gradient diluted antibody (starting at 30ug / ml, 3-fold gradient dilution, 10 points in total) was added to the corresponding wells, and Atezolizumab was used as a positive control. Incubate for 6 hours, add 100 μL Bright-Glo reagent to each well, and use Tecan M1000pro microplate reader to detect the luciferase activity value in the well. The results are shown in Figure 5 Table 7 shows the blocking activity of anti-human PD-L1 fully human antibodies on the T cell PD-1 / PD-L1 signaling pathway. The results showed that the anti-human PD-L1 fully human antibodies 131-4-G9, 131-15-2C3 and Atezolizumab had comparable blocking activity on the T cell PD-1 / PD-L1 signaling pathway.

[0113] Table 7 Blocking activity of anti-human PD-L1 fully human antibodies in the T cell PD-1 / PD-L1 signaling pathway

[0114] Antibody Name <![CDATA[EC 50 (nM)]]> 6-B11-A2 3.79 131-4-G9 1.4 131-2-C3 0.97 Atezolizumab 1.74

[0115] Example 9

[0116] Dynamic affinity determination of fully human anti-human PD-L1 antibody

[0117] The binding kinetics of anti-human PD-L1 fully human antibody to human PD-L1 was characterized based on the ForteBio molecular interaction system. In short, the human PD-L1 His protein was solidified using the HIS1K sensor, the solidification concentration was 5ug / ml, the solidification time was 250s, the antibody to be tested was diluted according to a certain concentration (diluted according to 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, 0nM), and Atezolizumab was used as a positive control. The affinity detection procedure was equilibrium for 60s, binding for 180s, dissociation for 180s, and detection temperature of 25°C. The results were then processed according to the corresponding analytical method. The results are shown in Table 8, and the dynamic affinity of the anti-human PD-L1 fully human antibody is equivalent to that of Atezolizumab.

[0118] Table 8 Dynamic affinity of fully human anti-human PD-L1 antibodies

[0119] Antibody Name KD(M) 6-B11-A2 <![CDATA[4.230x10 -10 ]]> 131-2-C3 <![CDATA[4.319x10 -11 ]]> 131-4-G9 <![CDATA[7.757x10 -11 ]]> Atezolizumab <![CDATA[9.605x10 -11 ]]>

[0120] Example 10

[0121] Effect of fully human anti-human PD-L1 antibody on T cell activation in lymphocyte response

[0122] DC cells (Aoneng Biotech, FPB-DC002F-C) and PBMC cells (Aoneng Biotech, FPB004F-C-MLR) were revived. DC cells were 1×10 4 PBMC cells per well: 1×10 5 Mix, 200 μl per well, and add to 96-well flat-bottom plates. Two parallel experiments were set up for each group, and different concentrations of anti-human PD-L1 fully human antibody and Atezolizumab (111.1nM, 44.44nM, 4.444nM, 0nM) were added respectively. The 96-well plate was placed at 37°C, 5% CO2 for 5 days, and the IL2 Elisa Kit (Biolegend, CAT#431804) and IFNgamma Elisa Kit (Biolegend, CAT#430104) were used to detect the concentrations of IL2 and IFNγ. The results are shown in the figure. Figure 6 a, 6b, Figure 6 a represents the detection results of 6-B11-A2, 131-4-G9 and Atezolizumab activating T cells to produce IL2; Figure 6 b shows the detection results of 6-B11-A2, 131-4-G9 and Atezolizumab activating T cells to produce IFNγ. The results show that the anti-human PD-L1 fully human antibody

[0123] 6-B11-A2, 131-4-G9 and Atezolizumab can all stimulate the donor to produce cytokines, indicating that the anti-human PD-L1 fully human antibody 131-4-G9 of the present invention can effectively activate T cells.

[0124] Embodiment 11

[0125] Anti-tumor effect of fully human anti-human PD-L1 antibody

[0126] Experimental animals: NOG mice, female, 6-8 weeks old (Shanghai Weitong Lihua Experimental Animal Co., Ltd.). Tumor cell inoculation: 0.2 mL (5×10 6 The NCI-H358 cell suspension resuspended in DPBS (containing 50% BD Matrigel) was subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached about 80mm 3 The drug was administered in groups at 1:00 pm. One day before inoculation, each animal was injected with 0.2 mL (5×10 6 )PBMC (Rubai Bio, 230520077) cells. Animal grouping: Weigh the animals before administration and measure the tumor volume. Randomly group the animals according to the tumor volume (randomized block design). The experimental grouping and dosing regimen are shown in Table 9. Observation: Monitor the health status and mortality of the animals every day. Routine examinations include observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (visual inspection only), weight changes (measured every three days), external signs or other abnormal conditions. Experimental indicators: The experimental indicators are to examine whether tumor growth is inhibited, delayed or cured. The tumor diameter is measured every three days. The calculation formula for tumor volume is: V = 0.5L × W2, L and W represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound is evaluated by TGI (%). Calculation of TGI (%): TGI (%) = [(1-(average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the beginning of drug administration)) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the beginning of treatment)] × 100.

[0127] Tumor suppression rate Figure 7 As shown in Table 10, on the 21st day after inoculation, compared with the vehicle control group, the tumor inhibition rate of 131-4-G9 antibody at a single dose of 10 mg / kg was about 38.28%, and the tumor inhibition rate of the positive control drug Tecentriq at a single dose of 10 mg / kg was about 23.91%. This shows that the anti-human PD-L1 fully human antibody 131-4-G9 of the present invention has excellent anti-tumor activity.

[0128] Table 9 Experimental groups and dosing regimen

[0129] Group Test substance Dosage (mg / kg) Route of administration Dosing frequency 1 Solvent control N / A ip BIW×6 2 131-4-G9 5 ip BIW×6 3 131-4-G9 10 ip BIW×6 4 Tecentriq 10 ip BIW×6

[0130] Table 10 Tumor inhibition rate on day 21

[0131] Group Test substance Dosage (mg / kg) <![CDATA[Tumor volume (mm 3 )]]> Tumor inhibition rate (TGI%) 1 Solvent control N / A 1307.65 N / A 2 131-4-G9 5 1260.68 3.87％ 3 131-4-G9 10 842.72 38.28％ 4 Tecentriq 10 1017.28 23.91％

[0132] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A fully human antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) amino acid sequence and a light chain variable region (VL) amino acid sequence; The heavy chain variable region (VH) comprises the following CDR1, CDR2 and CDR3, and the light chain variable region (VL) comprises the following CDR1, CDR2 and CDR3; The amino acid sequences of VH CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 13, 14 and 15, respectively; the amino acid sequences of VL CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 16, 17 and 18, respectively.

2. The fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 according to claim 1, characterized in that: The VH comprises an amino acid sequence at least 75% identical to SEQ ID NO:3, and the VL comprises an amino acid sequence at least 75% identical to SEQ ID NO:

4.

3. The fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 according to claim 1 or 2, characterized in that: The VH comprises one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:3, and the VL comprises one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:

4.

4. The fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 according to any one of claims 1 to 2, characterized in that: The antibody or antigen-binding fragment thereof is a fully human antibody based on a fully human antibody mouse.

5. The fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 according to any one of claims 1 to 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises heavy and light chain constant regions or variants thereof, the heavy chain constant region being selected from IgG, IgM, IgE, IgD or IgA and the light chain constant region being selected from kappa or lambda.

6. The fully human antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 according to claim 5, characterized in that: The heavy chain constant region comprises the sequence shown in SEQ ID NO: 25; the light chain constant region comprises the sequence shown in SEQ ID NO:

26.

7. An isolated nucleic acid molecule, characterized in that Encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A carrier, characterized in that Comprising the nucleic acid molecule of claim 7.

9. The carrier according to claim 8, characterized in that The vector is a cloning vector or an expression vector. 10 . A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8 or 9 .

11. A method for preparing an antibody or an antigen-binding fragment thereof, characterized in that: The host cell of claim 10 is cultured under conditions that allow protein expression, and the antibody or antigen-binding fragment thereof is collected from the cultured host cell.

12. A pharmaceutical composition, characterized in that It contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the isolated nucleic acid according to claim 7, or the vector according to claim 8 or 9, or the host cell according to claim 10, and a pharmaceutically acceptable carrier and / or excipient.

13. The pharmaceutical composition according to claim 12, characterized in that It also includes another pharmaceutically active agent, which is a drug with anti-tumor activity, an immunotherapy-related drug, an immunity-enhancing drug or an immune system activator, a radioactive substance or a color developing agent.

14. A diagnostic or therapeutic kit, characterized in that A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the isolated nucleic acid according to claim 7, or the vector according to claim 8 or 9, or the host cell according to claim 10, or the pharmaceutical composition according to claim 12 or 13.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the isolated nucleic acid according to claim 7, or the vector according to claim 8 or 9, or the host cell according to claim 10, or the pharmaceutical composition according to claim 12 or 13 in the preparation of a drug for the treatment and / or adjuvant treatment of lung cancer.

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