TWI816616B - Anti-human programmed death ligand-1 (pd-l1) antibodies and uses thereof - Google Patents

Abstract

Disclosed are antibodies capable of specifically binding to human programmed death ligand-1 (PD-L1) or antigen-binding fragments thereof. The antibodies or antigen-binding fragments can enhance the function of T cells, up-regulate the immune response mediated by T cells, and is used for treating diseases related to abnormal PD-L1 expression and/or abnormal T cell function, such as tumors.

Description

Antibodies against human programmed death ligand-1 (PD-L1) and their uses

The present invention relates to antibodies against human programmed death ligand-1 (PD-L1) or antigen-binding fragments thereof, their encoding nucleic acids, expression vectors and expression cells, preparation methods, pharmaceutical compositions, and their use for enhancing T cells. Function, upregulates T cell-mediated immune responses and is used to treat diseases related to abnormal PD-L1 expression and abnormal T cell function, such as tumors.

Immunotherapy has become one of the most rapidly developing and promising research fields in tumor treatment, and the use of immune checkpoint inhibitors, such as PD-1/PD-L1 monoclonal antibodies and CTLA-4 monoclonal antibodies, is a revolution in tumor immunotherapy. This new treatment method has greatly improved the survival time of patients with malignant tumors.

T cell-mediated immune responses are strictly regulated by co-stimulatory and co-inhibitory mechanisms, maintaining an optimal balance between antigen immune response and maintenance of self-tolerance. This balance is involved in a variety of initiating and inhibitory proteins. Inhibitory proteins, also known as immune checkpoint proteins, regulate the priming and effector functions of killer T cells (Cytotoxic T Lymphocytes, CTL) to maintain self-tolerance. Immune checkpoint inhibitory proteins play a key role in the regulatory pathways of tumors. One of the important immune checkpoint proteins, PD-1, binds to its ligand PD-L1, which transmits immunosuppressive signals and reduces the activity of T cells. At the same time, tumor cells can also inhibit the initiation and proliferation of T cells by expressing PD-L1 on the cell surface, thereby evading CTL attack and killing. Using PD-1 or PD-L1 monoclonal antibodies to prevent the binding and interaction of PD-1/PD-L1 can partially restore the function of T cells, thereby enhancing the ability to kill tumor cells. In 2011, the first immune checkpoint inhibitor, ipilimumab, was developed as an anti-CTLA-4 monoclonal antibody Longan antibody has become a tumor immunotherapy successfully used to treat melanoma. Many patients treated so far have achieved a better 5-year survival compared with traditional treatment methods. After that, the FDA approved 3 PD-1 monoclonal antibodies and 3 PD-L1 monoclonal antibodies, which were successfully used in immunotherapy for more than a dozen tumors except melanoma, and became the first-line treatment for various cancers, such as Non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC) and bladder or urothelial cancer, etc. China has so far approved 2 imported PD-1 antibodies and 3 domestic PD-1 antibodies for marketing, but no PD-L1 antibody has yet been approved, and in view of the therapeutic mechanisms of PD-L1 antibodies and PD-1 antibodies and current clinical trials Depending on the combination of drugs and applicable indications, the development of new PD-L1 monoclonal antibodies and PD-L1-based dual antibodies still has great social and economic significance.

The present invention provides antibodies and antigen-binding fragments that specifically bind to human programmed death ligand-1 (PD-L1), nucleic acids encoding these antibodies and antigen-binding fragments, including the antibodies and antigen-binding fragments and pharmaceutical compositions and reagents. cassettes, and their use for enhancing the function of T cells, upregulating T cell-mediated immune responses and for treating conditions associated with abnormal PD-L1 expression and abnormal T cell function, such as tumor immunity. The antibody not only binds to human and cynomolgus PD-L1 proteins, but also blocks the interaction between human PD-L1 and human PD-1.

In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds human programmed death ligand-1 (PD-L1) includes a combination of heavy chain CDRs and a combination of light chain CDRs: (1) the heavy chain CDRs The combination includes: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or have 1, 2 or 3 compared with the sequence combination. or more sequence combinations of amino acid insertions, deletions and/or substitutions:

And, (2) the light chain CDRs combination includes: CDR1-VL, CDR2-VL and CDR3-VL, the CDR1-VL, CDR2-VL and CDR3-VL have any sequence selected from the following or are combined with the sequence Combinations compared to sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:

Each CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL, and CDR3-VL is coded according to popular analysis methods of KABAT, Chothia, or IMGT.

In some embodiments, an isolated humanized antibody or antigen-binding fragment that specifically binds human programmed death ligand-1 (PD-L1) includes a combination of heavy chain CDRs and a combination of light chain CDRs: (1) The heavy chain CDRs combination includes: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or have 1, Sequence combinations of 2, 3 or more amino acid insertions, deletions and/or substitutions:

And, (2) the light chain CDRs combination includes: CDR1-VL, CDR2-VL and CDR3-VL, the CDR1-VL, CDR2-VL and CDR3-VL have any sequence selected from the following or are combined with the sequence Combinations compared to sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:

Each CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL, and CDR3-VL is coded according to popular analysis methods of KABAT, Chothia, or IMGT.

In particular, for example, the antibody or antigen-binding fragment thereof of the invention comprises a combination of heavy chain CDRs and light chain CDRs selected from the following: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6 , VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19 +VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+VL23, VH24+VL24, VH25+VL25, or VH26+VL26, and compared with the sequence of the heavy chain and light chain CDRs combination, it has 1, CDRs combinations of 2, 3 or more amino acid insertions, deletions and/or substitutions.

In another specific embodiment, the present invention provides such an antibody or an antigen-binding fragment thereof, wherein: 1) the heavy chain variable region and the light chain variable region have SEQ ID NO: 1 and SEQ ID NO: 2 respectively sequence, or a sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical to the sequence shown; 2) the heavy chain can The variable region and the light chain variable region have the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively, or are 70%, 75%, 80%, 85%, 90%, 95%, 96% identical to the sequences shown. %, 97%, 98%, 99% or higher identity sequences; 3) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively, or are identical to The sequence shown has a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher; 4) Heavy chain variable region and light chain The chain variable region has the sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively, or has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% with the sequence shown. , 98%, 99% or higher identity sequences; 5) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively, or have 70%, 75%, 80%, 85% or 90% of the sequences shown. , 95%, 96%, 97%, 98%, 99% or higher identity sequences; 6) The heavy chain variable region and the light chain variable region have SEQ ID NO: 11 and SEQ ID NO: 12 respectively. The sequence shown, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the sequence shown; 7) Heavy chain The variable region and the light chain variable region have the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 respectively, or have 70%, 75%, 80%, 85%, 90%, 95%, Sequences with 96%, 97%, 98%, 99% or higher identity; 8) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 respectively, or Sequences that are 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical to the sequence shown; 9) Heavy chain variable region and The light chain variable region has the sequence shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively, or is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% identical to the sequence shown. %, 98%, 99% or higher identity sequences; 10) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 127 and SEQ ID NO: 128 respectively, or are identical to the sequences shown Sequences with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity; 11) Heavy chain variable region and light chain variable region The regions have the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130 respectively, or are 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% identical to the sequences shown. , 99% or higher identity sequence; 12) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 131 and SEQ ID NO: 132 respectively, or have 70%, Sequences with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity; or, 13) heavy chain variable region and light chain variable region respectively Have the sequence shown in SEQ ID NO: 133 and SEQ ID NO: 134, or have 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 with the sequence shown % or higher identity sequence.

In a preferred embodiment, the antibodies of the invention or antigen-binding fragments thereof are chimeric or humanized or fully human.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention, which binds to human programmed death ligand-1 (PD-L1) with a dissociation constant (KD) of not greater than 10 nM, and cynomolgus monkey programmed death The dissociation constant (KD) of ligand-1 (PD-L1) binding is not greater than 100 nM.

In a preferred embodiment, the antibody of the present invention or its antigen-binding fragment comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably it contains human or The sequence of the constant region of a murine antibody IgG1, IgG2, IgG3 or IgG4; or the sequence of the constant region of a human or murine antibody IgG1, IgG2, IgG3 or IgG4 carrying a mutation.

In a preferred embodiment, the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, Nanobodies and minimal recognition units of antibodies.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention can competitively bind to PD-L1 with an antibody selected from the group consisting of No. 34, 50, 90, 130, 156, 370, 373, 413, or 794, and It has the following characteristics: 1) specifically binds to PD-L1 recombinant protein and cells expressing PD-L1; 2) blocks the binding of PD-L1 to PD-1 protein; 3) inhibits PD-1 and PD- expressed on the cell surface Binding of L1; 4) enhances T cell activity; 5) mediates antibody-dependent cell killing (ADCC) activity; or/and 6) inhibits tumor growth.

In some embodiments, the invention provides an isolated nucleic acid molecule encoding an antibody, an antigen-binding fragment, or any combination thereof as described above.

In some embodiments, the invention provides an expression vector comprising an isolated nucleic acid molecule as described above.

In some embodiments, the invention provides a host cell comprising the isolated nucleic acid molecule or expression vector of the invention as described above.

In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cell The host cells were selected from Chinese hamster ovary cells (CHO).

In some embodiments, the present invention provides a method for preparing antibodies or antigen-binding fragments, culturing the above-described host cells of the present invention under appropriate conditions, and isolating the antibodies or antigen-binding fragments.

In some embodiments, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, the above-mentioned expression vector of the present invention, The cells, or products (such as antibodies and antigen-binding fragments) prepared by the above-described method of the present invention, and pharmaceutically acceptable carriers.

In a preferred embodiment, the pharmaceutical composition further comprises an additional anti-tumor agent.

In some embodiments, the present invention provides a method for preventing and/or treating diseases related to abnormal PD-L1 expression and/or abnormal T cell function, comprising administering the above-described antibody or antigen of the present invention to a patient in need thereof. Binding fragments, the isolated nucleic acid molecules described above in the present invention, the expression vectors described above in the present invention, the cells described above in the present invention, products prepared by the methods described above in the present invention (such as antibodies and antigen-binding fragments), Or the above-mentioned pharmaceutical composition of the present invention; the disease is preferably a tumor; the tumor is preferably colorectal cancer.

In some embodiments, the present invention provides the above-described antibody or antigen-binding fragment, the above-described isolated nucleic acid molecule of the present invention, the above-described expression vector of the present invention, the above-described cell of the present invention, the above-described cell of the present invention. The products prepared by the method (such as antibodies and antigen-binding fragments), or the use of the above-mentioned pharmaceutical composition of the present invention in the preparation of drugs for preventing and/or treating diseases related to abnormal expression of PD-L1, the diseases are preferably tumor; said tumor Colorectal cancer is preferred.

In some embodiments, the present invention provides a kit comprising the above-described antibody or antigen-binding fragment of the present invention, the above-described isolated nucleic acid molecule of the present invention, the above-described expression vector of the present invention, the above-described The cells, or products (such as antibodies and antigen-binding fragments) prepared by the above-described method of the present invention, and instructions for use.

Terms and definitions:

Unless otherwise stated, terms used herein have the meanings commonly understood by those of ordinary skill in the art. For terms that are expressly defined herein, the meaning of that term shall be governed by the stated definition.

As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds or is immunoreactive to a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugated antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies), antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments). Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding to a target protein as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack The Fc fragment of the intact antibody (clears more quickly from the animal circulation) and therefore lacks Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; cited in Join this article)).

As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind a target antigen. The antigen-binding function of an antibody can be performed by fragments of the full-length antibody. The antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, tribody, affibody, Nanobody, aptamer or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 fragments , a bivalent fragment containing two Fab fragments connected by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) consisting of the VL and VH domains of a single arm of the antibody Fv fragment; (V) dAb containing VH and VL domains; (vi) dAb fragment consisting of VH domain (Ward et al., Nature 341:544-546, 1989); (vii) consisting of VH or VL domain A dAb consisting of; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (termed a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 ,1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

As used herein, the term "PD-L1" refers to programmed death ligand-1, also known as CD279 (cluster of differentiation 279), an important immunosuppressive molecule. The PD-L1 is preferably human PD-L1.

As used herein, the terms "anti-programmed death ligand-1 antibody", "programmed death ligand-1 antibody", "anti-PD-L1 antibody", "PD-L1 antibody", "anti-PD-L1 "Antibody portion" and/or "anti-PD-L1 antibody fragment" and the like refers to any protein containing at least a portion of an immunoglobulin molecule capable of specifically binding to PD-L1 (such as, but not limited to, at least one complementary determinant of a heavy chain or a light chain). (CDR) or ligand-binding portion thereof, heavy or light chain variable region, heavy or light chain constant region, framework region or any part thereof). PD-L1 antibodies also include antibody-like protein scaffolds (such as the tenth fibronectin type III domain (10Fn3)), which contain BC, DE and FG structural loops similar in structure and solvent accessibility to the antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and was modified by replacing the residues of the BC, DE and FG loops of 10Fn3 with CDR-H1, CDR-H2 from the PD-L1 monoclonal antibody. Or the residue replacement of the CDR-H3 region, those skilled in the art can, for example, replace the CDR of the PD-L1 monoclonal antibody Grafted onto fibronectin scaffold.

As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, with monoclonal binding specificity for at least two different antigens. In the present invention, one of the binding specificities can be detected against an epitope of PD-L1, and the other can be detected against another epitope of PD-L1 or any other antigen, for example, against a cell surface protein, receptor, Receptor subunits, tissue-specific antigens, viral-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins are detected.

As used herein, the term "chimeric" antibody refers to an antibody that has variable sequences of immunoglobulins derived from one source organism (e.g., rat or mouse) and immunoglobulins derived from a different organism (e.g., human). The constant region of a globulin. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; the above incorporated by reference. Enter this article.

As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable regions found in both light and heavy chain variable domains. The more conserved portion of the variable domain is called the framework region (FR). As is understood in the art, the amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT) and within a different set of Outside the hypervariable region under standards such as KABAT or IMGT. One or more of these locations may also be found in extended hypervariable zones. The invention includes antibodies containing modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each contain four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops that connect the sheet structure , and in some cases form part of the lamellar structure. The CDRs in each chain are held closely together by the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Protein sofImmunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, in this article, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, second CDR and third CDR of the heavy chain variable region (VH) respectively. These three CDRs constitute the heavy chain variable region (VH). The CDR combination of the light chain (or its variable region) (VHCDR combination); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and third CDR of the light chain variable region (VL) respectively. Three CDRs, these three CDRs constitute the CDR combination of the light chain (or its variable region) (VLCDR combination).

As used herein, the term "antibody conjugate" refers to a conjugate/conjugate in which an antibody molecule is chemically bonded to another molecule, either directly or through a linker. An example is an antibody-drug conjugate (ADC), where the drug molecule is the other molecule.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method of production of the antibody.

As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody (including the heavy chain of a Fv, scFv, or Fab). The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).

As used herein, the term "percent (%) sequence identity" refers to the alignment of sequences and the introduction of gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, the candidate and reference After introducing gaps in one or both of the sequences, and for comparison purposes, non-homologous sequences can be ignored), the amino acid (or nucleotide) residues of the candidate sequence are compared with the amino acids (or nucleotides) of the reference sequence. or nucleotide) residues are identical. For the purpose of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, such as using publicly available computer software, such as BLAST, ALIGN or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50%/( to 100% sequence identity. The length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, e.g., by antibodies or antigen-binding fragments thereof Specific recognition. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that is not shown to specifically bind to a particular antigen or epitope thereof Antigen-binding fragments will exhibit a KD greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay modalities can be used to select specificity for a particular protein or carbohydrate Immunoreactive antibodies. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988 ) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describes immunoassay formats and conditions that can be used to determine specific immunoreactivity.

As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Expression vectors of the present invention contain polynucleotide sequences as well as additional sequence elements for, for example, expression of proteins and/or integration of these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences that direct gene transcription, such as promoter and enhancer regions. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites, to This will guide the efficient transcription of the gene carried on the expression vector. Expression vectors of the invention may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

As used herein, the terms "subject," "subject," and "patient" refer to an organism undergoing treatment for a particular disease or condition (eg, cancer or infectious disease) as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.

As used herein, the term "treatment" refers to surgical or therapeutic treatment with the purpose of preventing, slowing down (reducing) undesirable physiological changes or pathologies in the subject of treatment, such as cell proliferative disorders (e.g., cancer). or infectious disease) progression. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable. Objects in need of treatment include those already suffering from a condition or disease as well as those susceptible to the condition or disease or those intended to prevent the condition or disease. When referring to terms such as slow down, alleviation, weakening, alleviation, alleviation, their meanings also include elimination, disappearance, non-occurrence, etc.

The previously described aspects and other aspects of the present invention will be clearly explained below through the detailed description and drawings of the present invention. The drawings herein are intended to illustrate certain preferred embodiments of the invention, however, it is to be understood that the invention is not limited to the specific embodiments disclosed.

Figure 1. Titers of mouse serum binding to human PD-L1-mFc (A) and PD-L1-His (B) recombinant proteins measured after the final immunization.

Figure 2. Flow cytometry staining and sorting (FACS) and gating strategy of PD-L1-specific B cells Figure.

Figure 3. Competitive ELISA method determines that anti-PD-L1 antibodies block the binding of PD-L1 protein to PD-1 protein.

Figure 4. FACS determination of EC50 of anti-PD-L1 antibody binding to cell surface PD-L1 protein.

Figure 5. Anti-PD-L1 antibodies increase the expression and activity of reporter genes in the Jurkat-PD-1-CHO-PD-L1-NFAT system.

Figure 6. Anti-PD-L1 antibody promotes the secretion of IFN-γ in mixed lymphocyte reaction.

Figure 7. Antibody-dependent cell killing (ADCC) activity of anti-PD-L1 antibodies on A431 cells.

Figure 8. Murine anti-PD-L1 antibodies inhibit the growth of MC38-hPD-L1 colon cancer tumors in human PD-1/PD-L1 transgenic mice.

Figure 9. Humanized anti-PD-L1 antibodies inhibit MC38-hPD-L1 colon cancer tumor growth in human PD-L1 transgenic mice.

The present invention will be described in detail below with reference to the examples and drawings. The drawings herein are intended to illustrate some preferred embodiments of the present invention. However, it can be understood that the present invention is not limited to the specific embodiments disclosed or regarded as limiting the invention. Limitations on the Scope of the Invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1 Immunization of mice to produce PD-L1 antibodies

For 6-8 week old female SJL mice (purchased from Beijing Vitong Lever Laboratory Animal Technology Co., Ltd.) or Balb/c mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.), human Fc-fused human PD-L1 protein (PD-L1-mFc, Novoprotein, Cat. CM06, or Sino Biological, Cat. 10084-H05H) or human PD-L1-His (Novoprotein, Cat. C315 or Sino Biological, Cat: .10084-H08H ) and Freund's complete adjuvant (complete freund's adjuvant, CFA, Sigma, Cat. F5881) for the first immunization; use the above-mentioned PD-L1-mFc or human PD-L1-His and Freund's incomplete adjuvant (incomplete freund's adjuvant, IFA, Sigma, Cat. F5506) and unmethylated cytosine guanine dinucleotide (CpGODN1826, synthesized from Shanghai Sangon Biotechnology) were used for three subsequent immunizations. During immunization, 50 μg/animal/time was injected through emulsification operation. A uniform and stable emulsion is formed. In particular, the first and second immunization injections are performed on the foot pads and back, and the third and fourth immunization injections are performed on the tail subcutaneously and on the back to obtain high titer, high affinity, high specificity antiserum and specific immunity. cells. On the 5th to 7th day after the last immunization (the fourth immunization), the mice were euthanized and the spleens were aseptically removed. Mouse spleen lymphocytes were aseptically isolated and extracted, aliquoted into cryovials, and frozen in liquid nitrogen. Blood was collected from the mice 10 days after the second immunization, three days after the third immunization and on the day of euthanasia, the serum was separated, and the titer of anti-PD-L1 specific antibodies in the serum was measured using the enzyme-linked immunosorbent (ELISA) method.

The experimental results are shown in Figure 1, which shows that after four immunizations, the serum of the immunized mice had high binding titers to human PD-L1-mFc and PD-L1-His. It shows that using this method to immunize mice can produce high titers of anti-PD-L1 antibodies.

Example 2 Fluorescence-activated cell sorting (FACS) of PD-L1-specific single B cells

Spleen cells of mice immunized with PD-L1 protein were labeled with the antigen PD-L1-His protein (Novoprotein, Cat. C315 or Sino Biological, Cat. 10084-H08H) and the indirect labeled antibody anti-His-APC (R&D Systems, Cat. IC050A) and antibodies against specific markers on the surface of mouse B cells (anti-mouse B220-Pacfic Blue, R&D Systems, Cat.553089; anti-mouse IgD-PE, R&D Systems, Cat.558597; anti-mouse IgM-PE Cy7 , R&D Systems, Cat. 552867) staining, and before sorting, add the dye 7-AAD (R&D Systems, Cat. 51-68981E) to distinguish between dead cells and living cells, and use AriaIII (BD Company) flow cytometry sorting The instrument sorted PD-L1-specific single B cells (7AAD ^- B220 ⁺ IgD ^- IgM ^- PDL1-His ⁺ ) into PCR wells containing cell lysate and RNase inhibitor, and collected one cell from each well. The results show (Figure 2) that no obvious PD- was detected in the spleen of the blank control unimmunized mouse (A) or the spleen of the mouse immunized with PD-L1 stained with CREG-His, an unrelated protein with His (B). L1 ⁺ antigen-specific B cell subsets, whereas PD-L1-immunized mouse spleens stained with PD-L1-His (C) detected a population of PD-L1 ⁺ B cells, approximately per ¹⁰ spleen cells. 114 PD-L1 ⁺ B cells.

Example 3 Amplification and high-throughput expression of monoclonal antibodies

The single-cell mRNA was reverse-transcribed into cDNA using the method in Example 1 of the patent "A combination primer for nested amplification and its application" patent application number: 201811618134.4. Then use cDNA as a template to perform nested PCR to amplify the antibody heavy chain and light chain respectively. The antibody heavy chain variable region and light chain variable region were amplified and cloned into heavy chain expression vector and light chain expression vector respectively through homologous recombination method. The constant regions of both the heavy chain expression vector and the light chain expression vector are derived from human IgG1. The complete heavy chain expression sequence is signal peptide-VH-CH1-hinge region-CH2-CH3, and the complete light chain expression sequence is signal peptide-Vκ-Cκ. The above-mentioned single B cell antibody cloning and expression are achieved in a high-throughput manner in a 96-well plate to achieve rapid identification and discovery of antibodies. After a series of physical, chemical and functional screening of 324 pairs of clonally expressed antibody heavy chains and light chains, a total of 9 candidate mouse antibody molecules were obtained that were equivalent to or better than the already marketed PD-L1 antibodies Avelumab or Atezolizumab in terms of physical, chemical and functional activity. The CDRs of the sequence were analyzed using IMGT and KABAT software respectively. The corresponding sequence information is shown in Tables 1 to 2 below. Table 1 shows the VH and VL sequences of the mouse antibody molecule, and Table 2 shows the IMGT and VL sequences of the mouse antibody molecule. KABAT analysis results.

KABAT and IMGT software were used to analyze the CDRs of each antibody. The specific sequence information is as follows in Table 2:

Example 4 Antibody humanization

First, the classic "CDRs transplantation" method is used to humanize the antibody, that is, the humanized antibody with the highest homology is selected through the sequence to provide the antibody framework region (FRs), and the antigen-binding fragment complementarity determining region of the target antibody based on the Kabat naming method is (CDRs), transplanted into the former to form humanized antibodies. Secondly, in order to better maintain the activity and affinity of the antibody, based on the antibody structure modeling analysis (MOE software): 1). Select the amino acid residues in the antibody backbone region that are located at the VH-VL interface, close to or have direct interactions with CDRs. Back mutation, this type of amino acid residues is important for maintaining the conformation of the CDRs region; 2). Considering immunogenicity, try to select amino acids embedded within the protein for reverse mutation; 3). Taking into account the stability of the antibody properties and expression levels, giving priority to mutations that reduce molecular energy; 4). During the humanization process, try to further improve the affinity of humanized antibodies through amino acid site-directed mutations in the CDRs region. By testing the affinity of humanized antibodies containing different mutations to human PD-L1 and their binding to cells expressing PD-L1 on their surface, we screen those with equivalent or better affinity, antibody characterization, and activity functions than murine PD-L1 antibodies. sourced antibodies.

Among them, the CDRs of the preferred candidate antibody molecule sequences after humanization of the PDL1-156 antibody are as follows. They were analyzed using IMGT and KABAT software respectively. The corresponding sequence information is shown in Table 3 and Table 4 below. Table 3 shows the human source. VH and VL sequences of humanized antibody molecules, Table 4 shows the results of IMGT and KABAT analysis of humanized antibody molecules).

Use KABAT and IMGT software to analyze the CDRs of each humanized antibody. The specific sequence information is as follows:

Example 5 Determination of antibody purity by size exclusion chromatography

Use TSKgel G3000SWXL column (TOSOH, Cat.0008541) and pre-column Tskgel guard column SWXL (TOSOH, Cat.0008543) to determine the antibody purity by size exclusion chromatography. The mobile phase is phosphate buffer (NaH ₂ PO ₄ -Na ₂ HPO ₄ ). Preparation: weigh 8.88g of NaH ₂ PO ₄ ˙2H ₂ O and 33.33g of Na ₂ HPO ₄ ˙12H ₂ O. The mobile phase was used to equilibrate the chromatographic column with a flow rate of 1mL/min. After the baseline has flattened, inject the sample with a sample volume of 10 μL, a UV detection wavelength of 280 nm, a bandwidth of 16 nm, and the reference wavelength is turned off. The measurement results are shown in Table 5.

Example 6 KD determination of antibody binding to human and cynomolgus monkey PD-L1 recombinant proteins

Biacore T200 (GE Healthcare) was used to determine the binding affinity of PD-L1 antibodies to human and cynomolgus monkey PD-L1-His proteins. Anti-human IgG Fc (Genway, Cat. GWB-20A705) was immobilized on a CM5 wafer (GE Healthcare, Cat. BR-1005-30) at 25°C. Anti-human Fc (Genway, Cat. GWB-20A705) was diluted to 20 μg/mL with Acetate pH5.0 (GE Healthcare, BR-1003-51). Use the Amine method in Immobilization method for immobilization. Or use commercial Protein A (GE Healthcare, Cat. 29127556) wafer for detection. The affinity between the antibody and the antigen is measured using a multi-cycle kinetic method at 25°C. In each cycle, the antibody to be tested is first captured on a fixed CM5 chip, and then recombinant human PD-L1-His (Novoprotein, Cat .315) and cynomolgus monkey PD-L1-His protein (Sino Biological, Cat.90251-C08H), and finally regenerated with Glycine pH1.5. The mobile phase was HBS-EP+Buffer (GE Healthcare, Cat. BR-1006-69), the flow rate was 30 μL/min, and the binding time was 300 seconds. The regeneration flow rate is 30 μL/min and the time is 30 seconds. Use Biacore T200 Evaluation Software (version 3.0) to analyze the experimental data with a 1:1 binding model, fit the equilibrium dissociation constant KD of the antibody antigen, and determine the binding rate constant ka and the dissociation rate constant kd.

It can be seen from the results that the tested PD-L1 antibodies all showed an affinity of nM or higher for binding to the human PD-L1 recombinant protein, and their affinity to the cynomolgus monkey PD-L1 recombinant protein ranged from 62.5nM to 0.375nM. Please see Table 6 below for details.

Table 7 shows that the humanized antibodies 156-1H, 156-7H, 156-10H and 156-11H derived from the murine antibody PDL1-156 bind to the human PD-L1 protein and show an affinity comparable to that of PDL1-156.

Example 7 IC50 determination of antibodies blocking the interaction between PD-L1 and PD-1

The IC50 of anti-PD-L1 antibody blocking the binding of PD-L1 protein to PD-1 protein was determined by competitive ELISA method.

Use carbonate buffer to dilute human PD-L1 recombinant protein (Sino Biological, Cat. 10084-H05H) and add it to a 96-well enzyme plate to a final concentration of 1 μg/ml. Block with PBS solution containing 3% BSA, add serially diluted anti-PD-L1 antibody (6000ng/ml~2ng/ml) and human PD-1-His recombinant protein (Sino Biological, Cat.10377-H08H) for co-incubation Finally, add HRP-labeled anti-His tag antibody (MBL, Cat.D291-7), develop color with TMB, and read the OD value (dual wavelength 450nm-630nm) after termination with 1M sulfuric acid. Corresponding the antibody concentration to the OD value can draw the competition binding curve of the test antibody and calculate the IC50 value. Figure 3 shows the competition binding curve of anti-PD-L1 antibody and human PD-L1 recombinant protein. The results show that compared with the antibody isotype negative control anti-Hel (Biying Biological Preparation) without any blocking effect, the 9 mouse anti-PD-L1 antibodies tested (A) and 4 humanized Antibodies (B) can effectively block the interaction between human PD-L1 protein and human PD-1 protein, and the inhibitory activity of humanized antibodies is equivalent to that of mouse PDL1-156 before humanization (B), IC50 They are 197.0ng/mL (156-1H), 230.5ng/mL (156-7H), 250.1ng/mL (156-10H), and 207.2ng/mL (156-11H) respectively. The IC50 of mouse-derived PD-L1-156 is 221.3ng/ml, the positive control Atezolizumab (Biying Biotechnology) is 446.4ng/ml, and Avelumab (Pfizer, lot AU020322) is 190.3ng/ml.

Example 8 FACS determination of EC of PD-L1 antibody binding to cell surface PD-L1 ₅₀

The gradient concentration of the antibody to be detected (final antibody concentration: 10000ng/ml-0.1ng/ml, 10-fold serial dilution) was mixed with CHO-PD-L1 cells that highly express PD-L1 on the cell surface (Nanjing Yongshan Biotechnology Co., Ltd., 10 ⁵ /well) and incubate at 4°C for 30 minutes. After the incubation, add anti-human IgG PE fluorescent antibody (eBioscience, Cat. 12-4998-8) diluted at 1:250, and incubate for 30 minutes at 4°C. The fluorescent antibody will produce specificity with the Fc segment of the antibody to be detected. Binding, the PE fluorescence intensity is detected by FACS and the ability of the antibody to be detected to bind to the highly expressed PD-L1 protein on the cell surface is analyzed. The results in Figure 4 show that the EC50 of the mouse-derived PD-L1 antibodies to be tested are similar to the positive controls Avelumab (EC50 of 58.2ng/ml) and Atezolizumab (~99.73ng/ml) in this experiment, among which PDL1-156 and PDL1- 370 has the lowest EC50, and the EC50 of the tested humanized antibodies 156-1H, 156-7H, 156-10H and 156-11H are 49.42ng/ml, 78.37ng/ml, 63.5ng/ml and 49.97ng/ml respectively. , similar to the positive control Avelumab (EC50 of 56.88ng/ml) in this experiment. This assay quantitatively confirms the ability of each PD-L1 antibody to be tested to bind to the PD-L1 target in a dose-dependent manner on the cell surface. MFI fold = MFI value of the experimental group / MFI value of the control group without drugs.

Example 9 PD-1/PD-L1-NFAT reporter gene testing anti-PD-L1 antibodies Blocking PD-1: PD-L1 binding and signaling

To compare the effects of PD-L1 antibodies on PD-1/PD-L1 protein using Jurkat cell lines stably transfected with PD-1 (GenScript, Cat.00612) and CHO cell lines stably transfected with PD-L1 (GenScript, Cat.M00613). interactions and antagonism of signaling pathways. When the inhibitory signaling pathway is inhibited, the expression of the luminescent reporter gene controlled by NFAT is enhanced, and the luminescent signal value increases. The intensity of the antibody's blocking effect on PD-L1 is reflected by the relative light units (RLU) of the luminescence reading.

Seed the CHO cell line stably transduced with PD-L1 on a 96-well white bottom plate, with 40,000 cells per well, 100 μl/well, and place it back in the incubator overnight; the next day, take out the well plate, aspirate the culture medium, and add stably transduced PD -1 cell line and the PD-L1 antibody to be tested were co-incubated. The PD-1 cells were added to each well at a volume of 16,000 cells/well, and the antibody was serially diluted. Each dose was 3 replicate wells, and the incubation volume was 100 μl/well. , the incubation time is 6 hours. When the incubation is completed, take out the well plate, add the luminescence detection reagent in an equal volume (100 μl), and read the value. Graphpad was used to perform a 4-parameter analysis based on the detection values to make a regression curve, and the EC50 values of each antibody were obtained. The results are shown in Figure 5A. The EC50 values of the tested mouse PD-L1 antibodies were all the same as the EC50 values of the positive control antibodies Avelumab and Atezolizumab ( 222.9ng/ml, 321.6ng/ml) are similar. Figures 5B and 5C show that the EC50 of the four humanized PD-L1 antibodies 156-1H, 156-7H and 156-10H, and 156-11H are: 342ng/ml, respectively. 313.7ng/ml, 357.1ng/ml and 282.2ng/ml, which are similar to the EC50 of the positive control Avelumab. This assay quantitatively confirmed that murine and humanized anti-PD-L1 antibodies showed a dose-dependent inhibition of T cell activity inhibition caused by cell surface PD-1:PD-L1 interaction, thereby enhancing dose-dependently. Reporter gene activity in Jurkat cells.

Example 10 ELISA detection of IFN-γ secreted by T cells in mixed lymphocyte reaction

The activity of PD-L1 monoclonal antibodies in enhancing T cells was determined by mixed lymphocyte reaction (MLR). CD14 ⁺ monocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy human donor 1, using recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Cat.300 - 03) and recombinant human interleukin 4 (rhIL-4, Peprotech, Cat. 200-04) were induced to differentiate into dendritic cells (DC) in vitro, and LPS (Sigma, Cat: L4516) was added on the 6th day of culture. ) to stimulate mature DC. On the 7th day, DC cells from donor 1 were mixed and co-cultured with CD4 ⁺ T cells enriched from PBMC of healthy donor 2. The ratio of DC: CD4 ⁺ T cells was 1:10, and the cells to be tested were added. Antibody or positive control antibody Avelumab or Atezolizumab (antibody concentration is 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml), cultured for 4 days. The cell culture supernatant was collected after 4 days, and the IFN-γ content in the supernatant was detected by ELISA. Figure 6 shows that all tested mouse antibodies and positive control antibodies Avelumab and Atezolizumab were compared with anti-HEL monoclonal antibodies and no treatment (i.e. No administration) negative control group can significantly enhance the ability of CD4 ⁺ T cells to secrete IFN-γ in the MLR experiment, and as the PD-L1 antibody drug concentration decreases, the increased activity of IFN-γ secretion also decreases. This result shows that PD-L1 antibodies can enhance the function of T cells in a dose-dependent manner. T-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Example 11 Detection of Antibody Dependent Cell-mediated Cytotoxicit (ADCC) activity of anti-PD-L1 antibodies

The antibody-dependent cell killing activity mediated by anti-PD-L1 monoclonal antibodies was measured through the killing experiment of isolated and purified natural killer (NK) cells in normal human PBMC against A431 cells that highly express human PD-L1.

Preparation of effector cells: After resuscitation of frozen human PBMC cells (Stemexpress Company), RPMI1640 medium (Invitrogen, Cat.11835030) supplemented with 100IU/ml recombinant human interleukin 2 (rhIL-2, Peprotech, Cat.200-02) ) and incubate overnight. Thereafter, cells were collected and viable cells were counted. Then, NK cells were purified from PBMC using NK cell magnetic bead isolation kit (Stemcell, Cat. 17955), and the NK cells were resuspended in DMEM (Invitrogen, Cat. 11965084) supplemented with 10% inactivated fetal calf serum. Counted and used as effector cells.

Preparation of target cells: The target cells A431 were cultured in DMEM medium containing 10% fetal calf serum. One day before the ADCC experiment, a final concentration of 500IU interferon IFN-γ was added. (Peprotech, Cat. 300-02) for stimulation and culture overnight and set aside for use.

The anti-PD-L1 antibody was diluted with DMEM medium containing 10% fetal calf serum. The diluted antibody was dispensed into a white-walled 96-well plate (Corning, Cat. 3610) at 25 μl/well, and target cells were added to the well (25 μl/well). well, 10,000 cells/well). Allow the plate to incubate in a 37 °C, 5% _CO2 incubator for 30 min. Subsequently, effector cells were added to the wells (25 μl/well, 40,000 cells/well, immediate target ratio NK:A431 is 4:1), with a total volume of 100 μl, and the plate was incubated in a 37°C, 5% _CO2 incubator 4 hours.

It is necessary to set up the culture medium background control well (that is, add 100 μl of culture medium to the well), the target cell spontaneous death release hole (that is, there are only target cells in the well), the effector cell spontaneous death release hole (that is, there are only NK cells in the well), and the target cell's maximum death. Release wells (that is, the target cells plus the lysis buffer provided in the CytoTox-Glo ^TM Cytotoxicity Kit (Promega, Cat. G9291)), and the volume of all control wells is finally uniformly replenished with culture medium to 100 μl. After 4 hours of culture, the plate is taken out, and each Add 50 μl of CytoTox-Glo ^TM Cytotoxicity Assay Reagent to the well, mix on a shaker, and place at room temperature for 15 minutes before reading the chemiluminescence (RLU).

Calculate the cell lysis rate caused by ADCC activity according to the following steps: first subtract the average reading value of the medium background control well from the reading value of all wells, and then calculate the lysis rate % = (experimental well reading value - target cell spontaneous death release well Reading value - effector cell spontaneous death release hole) / (target cell maximum death release hole reading - target cell spontaneous death release hole reading) * 100

The results are shown in Figure 7. The experimental results show that the three murine PD-L1 antibodies tested and the FDA-approved positive control anti-PD-L1 drug Avelumab all showed antibody concentration-dependent lysis of the target cell A431 cells. Killing activity and similar EC50 values, while the negative isotype control antibody hIgG1,K (Abdserotec, PHP010) showed no ADCC activity even at the highest concentration.

Example 12 In vivo efficacy testing of murine PD-L1 antibody PDL1-156 in mice

B-hPD-1/hPD-L1 transgenic mice (Beijing Biocytogen Biotechnology Co., Ltd.) were used to establish the MC38-hPD-L1 colon cancer animal model and conduct PD-L1 antibody efficacy experiments. The mice were subcutaneously inoculated with 5×10 ⁵ MC38-hPD-L1 colon cancer cells on the right side. When the tumor volume reaches ~110 mm ³ , mice with moderate individual tumor volumes are selected into the group, and the animals are assigned to three experimental groups according to tumor volume using random grouping software: anti-Hel hIgG isotype control group, and anti-PDL1-156 antibody group , positive drug Avelumab group (Pfizer, lot AU020322), 8 animals in each group, administration started on the day of grouping (defined as study day 0). Each group was administered intraperitoneally twice a week at a dose of 10 mg/kg for a total of 7 times. Measure the tumor volume and mouse weight twice a week, record the measurement values, and calculate the tumor volume (long diameter x short diameter 2/2) and growth inhibition rate (tumor growth inhibition %, TGITV (%) = [1-(Ti -T0)/(Vi-V0)]×100%; Ti: the mean tumor volume of the treatment group on the i-th day of administration, T0: the mean tumor volume of the treatment group on the 0th day of administration; Vi: the solvent control group on The mean tumor volume on the i-th day of administration, V0: the mean tumor volume of the solvent control group on the 0th day of administration), and statistical analysis was performed on the tumor volume. P<0.05 was considered to have a significant difference.

On the 21st day of group administration, compared with the control group, the positive drug Avelumab group and the PDL1-156 mouse anti-PD-L1 antibody group had significant and similar inhibitory effects on tumor volume, and there was a statistical difference (P< 0.05). See Figure 8A, B, C, Table 8.

In addition, during the experiment, one mouse in the hIgG control group died prematurely and abnormally. The rest of the experimental animals were active and eating well during the administration period, and their weights increased to a certain extent. The results show that the antibody is safe, as shown in Figure 8D and Table 9.

Example 13 In vivo efficacy testing of humanized anti-PD-L1 antibodies in mice

MC38-hPD-L1 cells were inoculated subcutaneously on the right side of female 6-8 week old B-hPD-L1 transgenic mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) at a concentration of 5 × 10 ⁵ cells/0.1 mL. Wait until the tumor grows to At about ^108mm3 , 24 were randomly selected according to tumor volume, with 8 in each group, a total of 3 groups, namely: normal saline, Avelumab (5mg/kg), and 156-10H (5mg/kg). The administration route for all groups was intraperitoneal injection, twice a week, for 6 consecutive times, and the experiment ended 5 days after the last administration. During the administration and observation period, the mouse body weight and tumor volume were measured three times a week, and the measurement values were recorded to calculate the tumor volume (long diameter x short diameter ^2/2 ) and growth inhibition rate (TGI _TV (%)). On day 21, compared with the normal saline control group, the positive drug Avelumab group and the PD-L1 antibody 156-10H group had significant and similar inhibitory effects on tumor volume, and there was a statistical difference (P<0.05). See the figure 9A, B, C, Table 10.

During the administration period, the experimental animals were active and eating well, and their body weight increased to a certain extent, as shown in Figure 9D and Table 11.

The above results indicate that the humanized PD-L1 antibody 156-10H has a significant inhibitory effect on the growth of subcutaneous transplanted MC38-hPD-L1 tumors and exhibits high safety. Compared with the positive control antibody Avelumab, the TGI levels of the two are similar, and 156-10H shows a more uniform anti-tumor effect.

The antibodies against human programmed death ligand-1 (PD-L1) provided by the present invention and their uses are described in detail above. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the methods and implementation methods of the present invention. its core idea. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (20)

An isolated antibody or antigen-binding fragment that specifically binds to human programmed death ligand-1 (PD-L1), characterized in that the antibody or antigen-binding fragment contains a combination of heavy chain CDRs and a combination of light chain CDRs: (1 ) The heavy chain CDRs combination includes: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have a sequence combination selected from the following:

And, (2) the light chain CDRs combination includes: CDR1-VL, CDR2-VL and CDR3-VL, and the CDR1-VL, CDR2-VL and CDR3-VL have a sequence combination selected from the following:

Each CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is encoded according to the popular analysis method of KABAT or IMGT, wherein the heavy chain CDRs of the antibody or antigen-binding fragment The combination with light chain CDRs is VH9+VL9 or VH18+VL18. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively. The antibody or antigen-binding fragment as described in claim 1, wherein it is combined with human The dissociation constant (KD) of binding to programmed death ligand-1 (PD-L1) is not greater than 10nM, and the dissociation constant (KD) of binding to cynomolgus monkey programmed death ligand-1 (PD-L1) is not greater than 100nM. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; (3) a fully human source antibody or fragment thereof. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. The antibody or antigen-binding fragment of claim 5, wherein the antibody comprises the sequence of the constant region of human or murine antibody IgG1, IgG2, IgG3 or IgG4. The antibody or antigen-binding fragment of claim 1, wherein the antigen-binding fragment is selected from the group consisting of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, Nanobodies and minimal recognition units of antibodies. one or more. An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the antibody, antigen-binding fragment, or any combination thereof as described in any one of claims 1 to 7. An expression vector comprising an isolated nucleic acid molecule as described in claim 8. An isolated host cell comprising the isolated nucleic acid molecule of claim 8 or the expression vector of claim 11. The host cell according to claim 10, wherein the host cell is a eukaryotic cell or a prokaryotic cell. The host cell of claim 11, wherein the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis. The host cell according to claim 11, wherein the host cell is Chinese hamster ovary cells (CHO). A method for preparing an antibody or antigen-binding fragment, characterized in that: The host cell as described in any one of claims 10 to 13 is cultured under appropriate conditions, and the antibody or antigen-binding fragment is isolated. A pharmaceutical composition, characterized in that the composition contains the antibody or antigen-binding fragment as described in any one of claims 1 to 7, the isolated nucleic acid molecule as described in claim 8, or the isolated nucleic acid molecule as described in claim 9 The expression vector, the cell as described in claim 10, or the product prepared by the method as described in claim 14, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition further contains an additional anti-tumor agent. The antibody or antigen-binding fragment as described in any one of claims 1 to 7, the isolated nucleic acid molecule as described in claim 8, the expression vector as described in claim 9, the cell as described in claim 10, as The use of the product prepared by the method described in claim 14 or the pharmaceutical composition described in claim 15 in the preparation of drugs for preventing and/or treating diseases related to abnormal expression of PD-L1. For the use described in claim 17, the disease is tumor. For the use described in claim 18, the tumor is colorectal cancer. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, the isolated nucleic acid molecule as described in claim 8, the expression vector as described in claim 9, as claimed in claim 9 The cell described in claim 10, the product prepared by the method described in claim 14, and instructions for use.

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