Combination drug of anti-αvβ3 antibody and anti-PD-1 antibody

Abstract

The present invention relates to a combination drug of an anti-αvβ3 antibody and an anti-PD-1 antibody, a kit, and a method for treating cancer, wherein the combination drug comprises an anti-PD-1 antibody and an anti-αvβ3 antibody.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceutical technology, and specifically to a combination drug of an anti-αvβ3 antibody and an anti-PD-1 antibody, and its use for treating cancer, particularly lung cancer or colon cancer. [Background technology]

[0002] Programmed cell death protein 1 (PD-1) is a protein receptor expressed on the surface of T cells and involved in the cellular apoptosis process. PD-1 has two known ligands: PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273), which are members of the B7 family of cells expressed on the cell surface (Freeman et al., (2000) J Exp Med 192:1027-34; Latchman et al., (2001) Nat Immunol 2:261-8; Carter et al., (2002) Eur J Immunol 32:634-43). The PD-1 immune checkpoint is an inhibitory cell surface receptor, and its corresponding ligand, PD-L1, is upregulated on the surface of tumor cells and immune cells within the tumor environment, allowing tumor cells to evade immune cell attack. The use of anti-PD-1 or PD-L1 antibodies can block this response and produce antitumor effects.

[0003] PD-1 / PD-L1 pathway-based therapy has become a hot topic in the field of oncology immunotherapy. For example, immune checkpoint inhibitors targeting the PD-1 / PD-L1 pathway have significantly improved the prognosis of patients with non-small cell lung cancer, but many patients experience ineffective treatment due to primary resistance. As PD-1 / PD-L1 antibodies are increasingly used in patients with a wide variety of cancers, researchers have discovered that anti-PD pathway therapy is not effective in all cancer patients. PD-1 / PD-L1 antibodies are only effective in a subset of patients for each cancer type. For example, in the case of lung cancer, antibodies are only effective in approximately 30% of patients, but if the patient's tumor tissue is PD-L1 positive, the response rate increases to approximately 50%. Meanwhile, the response rate in patients with Hodgkin lymphoma is approximately 90%, 40-50% in melanoma, 50% in bladder cancer, and approximately 30% in gastrointestinal tumors such as lung cancer, esophageal cancer, and colon cancer. Therefore, there is an urgent need to develop new treatments that improve the therapeutic efficiency of anti-PD-1 / PD-L1, which has high clinical need and innovative value.

[0004] Research has confirmed that integrin αvβ3 plays an important role in the pathological angiogenesis process in lung cancer and other diseases, and that the formation of new blood vessels is a hallmark of tumor invasion and metastasis. Therefore, targeting integrin αvβ3 inhibits tumor angiogenesis, preventing tumor cells from obtaining the necessary blood, oxygen, and other nutrients, thereby promoting cell apoptosis. New monoclonal antibody drugs targeting αvβ3 have already entered clinical trials. Summary of the Invention [Problem to be solved by the invention]

[0005] HY1272 (ABT101) is a novel IgG4 antibody that has been humanized and affinity matured based on the previously reported anti-αvβ3 antibody LM609 (see Patent PCT / US2021 / 028775). It is currently being jointly developed by our company and overseas partners. This study was the first to evaluate the tumor therapeutic efficacy of combination therapy with an anti-αvβ3 antibody and an anti-PD-1 antibody, and found that the anti-αvβ3 antibody HY1272 and anti-PD-1 antibodies have a significant synergistic effect. The combination of these antibodies demonstrated significant tumor suppression effects against lung and colon cancer cells, which are insensitive to anti-PD-1 antibodies. This suggests that the synergistic effect of HY1272 and anti-PD-1 antibodies may further improve tumor therapeutic efficacy based on anti-PD-1 antibody monotherapy. [Means for solving the problem]

[0006] The present invention satisfies the above needs by providing a combination drug comprising an anti-PD-1 antibody and an anti-αvβ3 antibody. In one aspect, the present invention provides a combination drug comprising an anti-αvβ3 antibody and an anti-PD-1 antibody.

[0007] In some embodiments, the anti-αvβ3 antibody is an HCDR1 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1; an HCDR1 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 2; an HCDR2 region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; an HCDR3 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:3; an LCDR1 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:4, and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:5; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:6; and an LCDR3 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:6.

[0008] In some embodiments, the anti-αvβ3 antibody is an HCDR1 region of the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 region of the amino acid sequence shown in SEQ ID NO: 2, and an HCDR3 region of the amino acid sequence shown in SEQ ID NO: 3; an LCDR1 region of the amino acid sequence shown in SEQ ID NO: 4, an LCDR2 region of the amino acid sequence shown in SEQ ID NO: 5, and an LCDR3 region of the amino acid sequence shown in SEQ ID NO: 6; In some specific embodiments, the anti-αvβ3 antibody comprises a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8; In some specific embodiments, the anti-αvβ3 antibody is HY1272.

[0009] In some specific embodiments, the anti-PD-1 antibody may be, for example, purchased from Bioxcell, catalog number BE0146, or Keytruda® (Pembrolizumab), Opdivo® (Nivolumab), or the like.

[0010] In some embodiments, the anti-PD-1 antibody and anti-αvβ3 antibody are each in the form of a pharmaceutical agent and can be administered simultaneously, sequentially, or at intervals.

[0011] In some specific embodiments, the anti-PD-1 antibody and the anti-αvβ3 antibody are each in pharmaceutical form and are administered simultaneously.

[0012] In some embodiments, the route of administration of the combination drug comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, intranasal administration, or transdermal administration.

[0013] In some specific embodiments, the route of administration of the combination drug is intravenous injection.

[0014] In one aspect, the invention provides the use of the above-described combination in the manufacture of a medicament for treating a patient with cancer.

[0015] In some embodiments, the cancer is lung cancer or colon cancer.

[0016] In some specific embodiments, the patient's tumor tissue section is positive for PD-L1 expression, and optionally, positive for αvβ3 expression.

[0017] In one aspect, the present invention provides a method of treating cancer, comprising administering a therapeutically effective amount of the above-described therapeutic combination to a patient with cancer.

[0018] In some embodiments, the cancer is lung cancer or colon cancer.

[0019] In some specific embodiments, the patient's tumor tissue section is positive for PD-L1 expression, and optionally, positive for αvβ3 expression.

[0020] In one aspect, the invention provides the use of an anti-αvβ3 antibody in the manufacture of a combination drug for use in combination with an anti-PD-1 antibody.

[0021] In some embodiments, the anti-αvβ3 antibody is an HCDR1 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1; an HCDR1 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 2; an HCDR2 region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; an HCDR3 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:3; an LCDR1 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:4, and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:5; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:6; and an LCDR3 region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:6.

[0022] In some embodiments, the anti-αvβ3 antibody is an HCDR1 region of the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 region of the amino acid sequence shown in SEQ ID NO: 2, and an HCDR3 region of the amino acid sequence shown in SEQ ID NO: 3; It comprises an LCDR1 region of the amino acid sequence shown in SEQ ID NO:4, an LCDR2 region of the amino acid sequence shown in SEQ ID NO:5, and an LCDR3 region of the amino acid sequence shown in SEQ ID NO:6.

[0023] In some specific embodiments, the anti-αvβ3 antibody comprises a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8.

[0024] In some specific embodiments, the anti-αvβ3 antibody is HY1272.

[0025] In one aspect, the invention provides a combination therapy for treating a patient with cancer, said combination therapy comprising separately administering to said patient a therapeutically effective amount of an anti-αvβ3 antibody as described above, and separately administering a therapeutically effective amount of an anti-PD-1 antibody as described above.

[0026] In some embodiments, the cancer is lung cancer or colon cancer.

[0027] In some specific embodiments, the patient's tumor tissue section is positive for PD-L1 expression, and optionally, positive for αvβ3 expression.

[0028] In some specific embodiments, administration is by administering the anti-αvβ3 antibody within ±5 minutes of administration of the anti-PD-1 antibody. [Effects of the Invention]

[0029] The excellent technical effects of the combination drug, kit and treatment method according to the present invention mainly lie in the following points:

[0030] (1) the combination provides a superior therapeutic effect in terms of suppressing tumor growth or even eliminating tumors compared to the administration of either drug alone;

[0031] (2) allowing for the administration of smaller doses of either drug in the combination compared to administration of either drug alone;

[0032] (3) provide better disease control rates in treated patients;

[0033] (4) Providing longer survival (e.g., median survival, progression-free survival, or overall survival) in treated patients;

[0034] (5) Providing longer survival (e.g., median survival, progression-free survival, or overall survival) in treated patients compared with standard chemotherapy;

[0035] (6) The combination has superior activity in treating tumors or proliferative diseases and exhibits superior anti-tumor synergistic effects compared to administration of either drug alone. [Brief explanation of the drawings]

[0036] [Figure 1] Tumor volume of animals in each group at each time point in the LLC-hITGB3 subcutaneously transplanted tumor model. *: P<0.05, **: P<0.01, compared with the vehicle group. [Figure 2] Tumor volume of animals in each group at each time point in the CT26-hITGB3 subcutaneously implanted tumor model. *: P<0.05, **: P<0.01, compared with the vehicle group. DETAILED DESCRIPTION OF THE INVENTION

[0037] While the present invention may be embodied in many different forms, specific exemplary embodiments illustrating the principles of the present invention are disclosed herein. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0038] Scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art unless otherwise defined herein. Furthermore, unless otherwise required by context, singular terms include the plural, and plural terms include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "one," and "the" include plural referents unless the context clearly dictates otherwise. In this application, "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "comprises" and other forms (e.g., "comprising," "containing") is not limiting. Furthermore, ranges set forth in the specification and appended claims include the endpoints and all values ​​between the endpoints.

[0039] Generally, the terms related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization, and the techniques thereof, described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Abbas et al., Cellular and Molecular Immunology, 6 thed., WB Saunders Company (2010); Harlow and Lane, Using antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terms used in connection with analytical chemistry, synthetic organic chemistry, drugs, and medicinal chemistry, as well as laboratory procedures and techniques described herein, are terms that are well known and commonly used in the art. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. definition

[0040] In order to better understand the present invention, the definitions and interpretations of relevant terms are as follows. Unless otherwise specified, the following terms used in the present invention have the following meanings. Even if a particular term is not specifically defined, it should not be considered as indefinite or unclear, but should be understood according to its ordinary meaning in the art. When a trade name appears in the present invention, it refers to the corresponding product, composition or active ingredient thereof.

[0041] As used herein, the term "antibody" refers to an antigen-binding protein having at least one antigen-binding domain. The antibodies and fragments thereof of the present invention may be whole antibodies or any fragments thereof. Thus, the antibodies and fragments thereof of the present invention include monoclonal antibodies or fragments thereof, antibody variants or fragments thereof, and immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv), and other antibody fragments known in the art. Antibodies and fragments thereof may also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The anti-PD-1 antibodies and anti-αvβ3 antibodies disclosed herein may be of the IgG1, IgG2, IgG3, or IgG4 isotype. The term "isotype" refers to the antibody class encoded by the heavy chain constant region genes. Generally, the term "isotype" is used in conjunction with the antibody class encoded by the heavy chain constant region genes in Fundamental Immunology Ch. 7 (Paul, W., ed., 2002). nd ed., Raven Press, NY (1989).

[0042] As used herein, unless otherwise specified, the term "CDR" or "CDRs" refers to the complementarity determining regions within an immunoglobulin variable region as defined using the Kabat numbering system.

[0043] As used herein, the term "treatment" generally refers to an operation to obtain a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely stabilizes or cures the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a patient, including (a) suppressing the symptoms of the disease, i.e., arresting the progression of the disease, or (b) alleviating the symptoms of the disease, i.e., causing regression of the disease or its symptoms.

[0044] As used herein, the term "administration" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral or topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In certain embodiments, administration is via a parenteral route, and in certain embodiments, administration is via an oral route. Other parenteral routes of administration include topical, epidermal, or mucosal routes of administration, such as intranasal, intravaginal, intrarectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0045] As used herein, "co-administration" of agents, such as a PD-1 antagonist or an αVβ3 antagonist, means that the agents are administered so as to have overlapping therapeutic activity, and does not necessarily mean that the agents are administered to a subject simultaneously. The agents may or may not be physically combined prior to administration. In one embodiment, the agents are administered to a subject simultaneously or nearly simultaneously. For example, anti-PD-1 antibody and anti-αVβ3 antibody drugs in separate vials, when in liquid solution, can be mixed in the same intravenous infusion bag or injection device and administered to a patient simultaneously.

[0046] As used herein, the term "immunotherapy" refers to treating a subject suffering from a disease or at risk of infection or recurrence of a disease by inducing, enhancing, suppressing, or otherwise modifying the immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or disorders, or biochemical markers associated with a disease.

[0047] As used herein, the terms "identity" and "similarity" refer to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. In these calculations, alignment gaps, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M. ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W. ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M. and Griffin, H. G. eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J. eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0048] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In certain embodiments, the subject is a human. "Subject" refers to mammals such as rodents, felines, canines, and primates. Preferably, the subject according to the present invention is a human. As used herein, the terms "subject," "subject," and "patient" are used interchangeably in certain contexts.

[0049] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of drug that, when used alone or in combination with other therapeutic agents, protects a subject from the onset of disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free stages of the disease, or prevention of damage or disability resulting from the disease affliction. It refers to the amount of active compound, or material, composition, or dosage form containing an active compound, effective, when administered in accordance with a desired treatment regimen, to produce a desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0050] As an example of cancer treatment, a therapeutically effective amount of an anti-cancer drug can inhibit cell growth or tumor growth by at least about 10%, at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to untreated subjects, or, in certain embodiments, compared to patients treated with standard therapy. In other embodiments of the invention, tumor regression is observed and persists for at least about 20 days, at least about 40 days, or at least about 60 days. Despite the existence of these ultimate measures of therapeutic efficacy, evaluation of immunotherapeutic drugs must also consider "immune-related" response patterns.

[0051] As used herein, the term "cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Cancer refers to any tumor that causes medical disease, or solid and non-solid tumors through the growth, proliferation, or metastasis of malignant cells. A "recurrent" cancer is one that grows again at the original site or a distant site after responding to initial treatment (e.g., surgery). A "locally recurrent" cancer is one that appears after treatment in the same location as the previously treated cancer. A "metastatic" cancer is one that has spread from one part of the body (e.g., the lungs) to another part of the body.

[0052] As used herein, the term "PD-L1 expression" refers to any detectable expression level of specific PD-L protein on the cell surface or specific PD-L mRNA within cells or tissues. PD-L protein expression can be detected by IHC assay of tumor tissue sections or flow cytometry using a diagnostic PD-L antibody. Alternatively, PD-L protein expression in tumor cells can be detected by PET imaging using a binding agent (e.g., an antibody fragment, affibody, etc.) that specifically binds to the PD-L target of interest (e.g., PD-L1). Techniques for detecting and measuring PD-L mRNA expression include RT-PCR, real-time quantitative RT-PCR, RNA sequencing, and the Nanostring platform (J. Clin. Invest. 2017;127(8):2930-2940).

[0053] Several methods for quantifying PD-L1 protein expression in IHC assays of tumor tissue sections have been described. See, e.g., Thompson, RH, et al., PNAS 101 (49); 17174-17179 (2004); Thompson, RH et al., Cancer Res. 66:3381-3385 ​​(2006); Gadiot, J., et al., Cancer 117:2192-2201 (2011); Taube, JM et al., Sci Transl Med 4, 127ra37 (2012); and Toplian, SL et al., New Eng. J Med. 366 (26):2443-2454 (2012). See also US20170285037, which describes hematoxylin and eosin staining used by pathologists.

[0054] One method uses a simple binary endpoint of positive or negative PD-L1 expression, where a positive result is defined by the percentage of tumor cells that show histological evidence of cell-surface membrane staining. Tumor tissue sections are scored as positive for PD-L1 expression if PD-L1 is present on at least 1% of all tumor cells.

[0055] Another method quantifies PD-L1 expression in tumor tissue sections on tumor cells and infiltrating immune cells, primarily lymphocytes. The percentages of tumor cells and infiltrating immune cells showing membrane staining are quantified as <5%, 5%–9%, ​​and then in 10% increments up to 100%, respectively. PD-L1 expression in immune infiltrates is reported as a semiquantitative measure called the adjusted inflammation score (AIS). This is determined by multiplying the percentage of membrane-stained cells by the intensity of the infiltrate, which is graded as absent (0), mild (score 1, scattered lymphocytes), moderate (score 2, focal infiltration of the tumor with lymphohistiocytic aggregates), or severe (score 3, diffuse infiltration). If the AIS is 5 or greater, the tumor tissue section is counted as positive for PD-L1 expression in the immune infiltrate.

[0056] PD-L mRNA expression levels can be compared to the mRNA expression levels of one or more reference genes frequently used in quantitative RT-PCR.

[0057] As used herein, the term "positive PD-L1 expression" means elevated levels of PD-L1 expression (protein and / or mRNA) on malignant cells and / or infiltrating immune cells within the tumor compared to an appropriate control.

[0058] As used herein, the term "αvβ3 expression" refers to any detectable expression level of specific αvβ3 protein on the cell surface or specific αvβ3 mRNA within cells or tissues. αvβ3 protein expression can be detected by IHC assay of tumor tissue sections or flow cytometry using a diagnostic anti-αvβ3 antibody. Alternatively, αvβ3 protein expression in tumor cells can be detected by PET imaging using a binding agent (e.g., an antibody fragment, an affibody, etc.) that specifically binds to αvβ3. Techniques used to detect and measure αvβ3 mRNA expression include RT-PCR, real-time quantitative RT-PCR, RNA sequencing, and the Nanostring platform (J. Clin. Invest. 2017;127(8):2930-2940).

[0059] As used herein, the term "positive for αvβ3 expression" refers to % αvβ3 positive cells or CPS-like % αvβ3 positive cells being 1% or more.

[0060] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (e.g., tenths and hundredths of integers, etc.), unless otherwise specified.

[0061] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0062] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient known in the art that is pharmacologically and / or physiologically compatible with the subject and the active agent (see, e.g., Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride.

[0063] As used herein, the terms "combination" or "combined use" or "combination therapy" mean that two or more active ingredients are administered to a subject together in an admixture, either simultaneously in a single formulation for each, or sequentially in any order in a single formulation for each.

[0064] As used herein, the term "combination therapy" refers to a combination therapy, a combined therapy, or a combined combination that uses multiple drugs to treat a tumor, i.e., a single disease. In the present invention, "combination therapy" refers to a combination therapy that includes administering an αVβ3 antagonist (e.g., an anti-αVβ3 antibody) and a PD-1 antagonist (e.g., an anti-PD-1 antibody) to treat cancer (e.g., lung cancer or colon cancer). In contrast, "monotherapy" refers to a therapy that uses a single drug (e.g., an anti-αVβ3 antibody or an anti-PD-1 antibody) alone. Administration method

[0065] The following description does not limit the method of administering the combination drug of the present invention.

[0066] The components of the combination of the invention may be formulated separately, or some or all of them may be formulated together, hi one embodiment, the combination of the invention may be formulated as a combination suitable for single or multiple administration.

[0067] The components of the combination of the present invention may be administered separately, or some or all of them may be administered together. The components of the combination of the present invention may be administered non-substantially simultaneously, or some or all of them may be administered substantially simultaneously.

[0068] The components of the combination of the invention, either individually or in whole or in part, may be administered by a variety of suitable routes, including, but not limited to, oral or parenteral (by intravenous, intramuscular, topical, or subcutaneous routes). In some embodiments, the components of the combination of the invention, either individually or in whole or in part, may be administered orally or by injection (e.g., intravenous or intraperitoneal injection).

[0069] The components of the combination drug of the present invention may be individually or in whole or in part made into a suitable dosage form, including, but not limited to, tablets, troches, pills, capsules (e.g., hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions, and sustained-release preparations for oral or parenteral administration.

[0070] The components of the combination drug of the present invention may each independently, or some or all of them together, contain a pharmaceutically acceptable carrier and / or excipient. Sequence Listing Overview

[0071] This application is accompanied by a sequence listing containing a number of nucleic acid and amino acid sequences. Table 1 below provides a summary of the sequences contained in this application.

[0072] [Table 1(1)] [Table 1(2)] [Table 1(3)]

[0073] The present invention will be further described below with reference to specific examples, but these examples are for illustrative purposes only and do not limit the scope of the present invention. Similarly, the present invention is not limited to any specific preferred embodiments described herein. Those skilled in the art should understand that equivalent substitutions or corresponding improvements to the technical features of the present invention still fall within the scope of protection of the present invention. Unless otherwise specified, all reagents used in the following examples are commercially available products, and conventional techniques in the art can be used to prepare solutions. Example 1: Efficacy study of the combination of HY1272 and anti-PD1 antibody in a C57BL / 6 mouse subcutaneously transplanted tumor model of mouse lung cancer LLC-hITGB3 cell line

[0074] 1.1 Establishment of LLC-ITGB3 stable transfected cell lines In this experiment, we established LLC cell lines stably expressing hITGB3 (human integrin β3) by packaging lentivirus using the hITGB3 (human integrin β3) plasmid and infecting LLC mouse Lewis lung carcinoma cells. Flow cytometry analysis demonstrated a positive rate of over 95%, demonstrating the successful establishment of LLC-hITGB3 cell lines. The method is as follows:

[0075] The human ITGB3 gene was constructed into the pLVX-PGK-Puro vector using gene synthesis, packaged using a three-plasmid system, and lentivirus was obtained. A stable overexpressing cell line was established by lentiviral infection. Specifically, 1 × 10 LLC cells in logarithmic growth phase were infected with lentivirus. 5 One mL of cell suspension was added per well to a 12-well plate at a density of 1000 cells / mL. The virus solution was added to the cell culture plate and cultured for 24 hours in a 37°C, 5% CO2 incubator. After incubation, the supernatant was discarded, and complete medium (DMEM + 10% FBS) was added and cultured for an additional 48 hours. After incubation, the cell supernatant was discarded, and the cell pool was subjected to selection pressure using 5 μg / mL puromycin for at least 3 days. After serial dilutions, the cells were inoculated into a 96-well cell culture plate at a dilution of 1 cell per well and cultured until single clones were formed. The cells were then expanded and detected by flow cytometry. If the positive rate of single clones was less than 90%, further screening was performed using increased puromycin concentrations to obtain clonal lines with a positive rate of over 90%. Finally, further screening using two higher puromycin concentrations, 16 μg / mL and 32 μg / mL, resulted in a cell pool with a high positive rate of 98.85%, and the stable transfected cell line LLC-hITGB3 was successfully established.

[0076] The culture supernatant of the established stably transfected cell line LLC-ITGB3 was analyzed by a mycoplasma detection kit (Lonza MycoAlert TM Mycoplasma detection was performed using a mycoplasma detection kit (catalog number LT07-318) according to the manufacturer's instructions. The detection results showed that the cells were mycoplasma-negative. 1.2 Examination of the tumor suppressive efficacy of the test substance in a C57BL / 6 mouse subcutaneously transplanted tumor model of the mouse lung cancer LLC-hITGB3 cell line

[0077] The mouse lung cancer LLC-hITGB3 cell line was cultured in DMEM medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before the 10th passage, the cells were harvested and inoculated into the right axilla of the back of female C57BL / 6 mice at a concentration of 5 × 10 5 The mice were anesthetized with 3-4% isoflurane prior to inoculation. The tumor volume (TV) was approximately 50 mm on average. 3 When the tumors reached 100 μg / kg, the mice were randomly divided into four groups of six mice each according to tumor volume and body weight. The day of grouping and administration was defined as day 0. The grouping and administration schedule are shown in Table 2.

[0078] [Table 2]

[0079] After grouping, the body weight of the mice was measured and recorded twice a week, and the tumor volume was measured at the same time. The tumor volume (TV) was calculated as follows: TV = (length × width) 2 ) / 2. Based on the measured tumor volume, the relative tumor growth inhibition rate (TGI) was calculated using the following formula: TGI% = (1-T / C) × 100%, where T and C are the tumor volumes (TV) of the experimental and vehicle control groups, respectively, at a particular time point.

[0080] The mean TV values ​​for each group are shown as mean ± standard error of the mean (SEM). Comparisons between two groups were performed using Dunnett's multi-comparison test. A P value of <0.05 was considered statistically significant. 1.3 Experimental results

[0081] Under the conditions of this experiment, LLC-hITGB3 cells grew normally, and tumor growth was also normal. During the administration period, no significant differences in animal weight were observed in any of the experimental groups compared with the vehicle control group.

[0082] The tumor growth curves in mice are shown in Figure 1. Compared with the vehicle control group at the same time, the tumor growth inhibition (TGI) rates at day 21 for the anti-PD-1 monotherapy group (10 mg / kg) and the HY1272 monotherapy group (10 mg / kg) were 2.8% and 2.5%, respectively, and no significant difference was observed compared with the vehicle control group. The TGI for the combination therapy group (anti-PD-1 10 mg / kg + HY1272 10 mg / kg) was 47.9%, which was significantly different from the vehicle control group, the anti-PD-1 monotherapy group, or the HY1272 monotherapy group.

[0083] These results demonstrated that in this lung cancer model, administration of either anti-PD-1 or HY1272 alone did not show a clear tumor-suppressing effect, but combined administration of the two drugs showed a significant anti-tumor effect, suggesting that HY1272 and anti-PD-1 have a synergistic effect and can significantly enhance the sensitivity of some lung cancer cells to anti-PD-1 therapy. Example 2: Efficacy study of the combination of HY1272 and anti-PD1 antibody in a C57BL / 6 mouse subcutaneously transplanted tumor model of the mouse colon cancer CT26-hITGB3 cell line

[0084] 2.1 Establishment of CT26-ITGB3 stable transfected cell line In this experiment, we established a CT26 cell line stably expressing hITGB3 by packaging lentivirus using the ITGB3 plasmid and infecting CT26 mouse colon cancer cells. Flow cytometry analysis demonstrated a positive rate of over 95%, demonstrating the successful establishment of a CT26-hITGB3 cell line. The method is as follows:

[0085] The human ITGB3 gene was constructed into the pLVX-PGK-Puro vector using gene synthesis, packaged using a three-plasmid system, and lentivirus was obtained. A stable overexpressing cell line was established by lentiviral infection. Specifically, 1 × 10 CT26 cells in logarithmic growth phase were infected with lentivirus.5 The virus solution was added to a 12-well plate at a density of 1000 cells / mL per well. The virus solution was added to the cell culture plate and incubated for 24 hours in a 37°C, 5% CO2 incubator. After incubation, the supernatant was discarded, and complete medium (DMEM + 10% FBS) was added and incubated for an additional 48 hours. After incubation, the cell supernatant was discarded, and the cell pool was subjected to selection pressure using 5 μg / mL puromycin for at least 3 days. After serial dilutions, the cells were inoculated into a 96-well cell culture plate at a dilution of 1 cell per well and cultured until single clones were formed. The cells were then expanded and detected by flow cytometry. If the detection result indicated a positive rate of single clones below 90%, further selection pressure was applied by increasing the puromycin concentration to obtain clones with a positive rate above 90%. Finally, further screening with 16 μg / mL puromycin yielded a cell pool with a high positive rate of 96.26%, and the cell line CT26-hITGB3 was successfully established.

[0086] The culture supernatant of the established cell line CT26-ITGB3 was analyzed by a mycoplasma detection kit (Lonza MycoAlert TM Mycoplasma detection was performed using a mycoplasma detection kit (catalog number LT07-318) according to the manufacturer's instructions. The detection results showed that the cells were mycoplasma-negative. 2.2 Examination of the tumor suppressive efficacy of the test substance in a BALB / c mouse subcutaneously transplanted tumor model of the mouse lung cancer CT26-hITGB3 cell line

[0087] Mouse colon cancer cells CT26-hITGB3 were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. 2.5 × 10 cells were cultured for 10 passages. 5 The cells were suspended in 100 μL of PBS and inoculated into the right axilla of female BALB / c mice by subcutaneous injection. Before inoculation, the mice were anesthetized with 3-4% isoflurane. The tumor volume was approximately 50-60 mm on average. 3When the tumors reached 100 μg / kg, the mice were randomly assigned to four groups of six mice each based on tumor volume and body weight. The day of group assignment and administration was defined as day 0. The group assignment and administration schedule are shown in Table 1.

[0088] After grouping, the body weight of the mice was measured and recorded twice a week, and the tumor volume was measured at the same time. The tumor volume (TV) was calculated as follows: TV = (length × width) 2 ) / 2. Based on the measured tumor volume, the relative tumor inhibition rate (TGI) was calculated using the following formula: TGI% = (1-T / C) × 100%, where T and C are the tumor volumes (TV) of the experimental and vehicle control groups, respectively, at a particular time point.

[0089] The mean TV values ​​for each group are shown as mean ± standard error of the mean (SEM). Comparisons between two groups were performed using Dunnett's multi-comparison test. A P value of <0.05 was considered statistically significant. 2.3 Experimental results

[0090] Under the conditions of this experiment, CT26-hITGB3 cells grew normally, and tumor growth was also normal. Under the conditions of this experiment, no significant difference in animal weight was observed during the administration period in any of the experimental groups compared with the vehicle control group.

[0091] Figure 2 shows the tumor growth curves in mice. Compared with the vehicle control group at the same time, the tumor growth inhibition rates (TGI) at day 17 for the anti-PD-1 monotherapy group (10 mg / kg) and the HY1272 monotherapy group (10 mg / kg) were 20.2% and 31.7%, respectively. While these groups demonstrated a tendency toward tumor inhibition, this experimental system did not demonstrate a significant difference from the vehicle control group (P<0.05). The TGI for the combination therapy group (anti-PD-1 10 mg / kg + HY1272 10 mg / kg) was 74.8%, demonstrating a significant difference compared with the vehicle control group, the anti-PD-1 monotherapy group, and the HY1272 monotherapy group.

[0092] These results demonstrated that in this colon cancer model, administration of either anti-PD-1 or HY1272 alone did not show a clear tumor-suppressing effect, but the combination of the two showed a significant anti-tumor effect, suggesting that HY1272 and anti-PD-1 have a synergistic effect and can significantly enhance the sensitivity of some colon cancer cells to anti-PD-1 therapy.

[0093] Although the present invention has been described illustratively based on its preferred embodiments, it is not limited to the above embodiments, and it will be understood by those skilled in the art that various modifications and variations are possible. The selection and application of specific antibodies can be adjusted and modified as needed. Therefore, those skilled in the art can make some simple substitutions without departing from the concept and principle of the present invention, and all of them should be included in the scope of protection of the present invention.

Claims

1. A combination drug comprising an anti-PD-1 antibody and an anti-αvβ3 antibody.

2. The anti-αvβ3 antibody an HCDR1 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, and an HCDR3 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3; 2. The combination drug of claim 1, comprising an LCDR1 region having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:4, an LCDR2 region having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:5, and an LCDR3 region having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:

6.

3. The anti-αvβ3 antibody an HCDR1 region of the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 region of the amino acid sequence shown in SEQ ID NO: 2, and an HCDR3 region of the amino acid sequence shown in SEQ ID NO: 3; an LCDR1 region of the amino acid sequence shown in SEQ ID NO: 4, an LCDR2 region of the amino acid sequence shown in SEQ ID NO: 5, and an LCDR3 region of the amino acid sequence shown in SEQ ID NO: 6; Preferably, the anti-αvβ3 antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8; The combination drug of claim 2, wherein the anti-αvβ3 antibody is HY1272.

4. The combination drug according to claims 1 to 3, wherein the anti-PD-1 antibody and the anti-αvβ3 antibody are each in the form of a pharmaceutical agent and can be administered simultaneously, sequentially, or at intervals, and are preferably administered simultaneously.

5. 5. The combination drug of claim 1, wherein the route of administration of the combination drug includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, intranasal administration, or transdermal administration, preferably intravenous injection.

6. 10. Use of the combination drug of any one of claims 1 to 5 in the manufacture of a medicament for treating a patient with cancer.

7. The use according to claim 6, wherein the cancer is lung cancer or colon cancer.

8. The use according to claim 7, wherein the patient's tumor tissue section is positive for PD-L1 expression and, optionally, positive for αvβ3 expression.

9. A method for treating cancer, comprising administering a therapeutically effective amount of the combination drug of any one of claims 1 to 5 to a patient with cancer.

10. 10. The method of treating cancer according to claim 9, wherein the cancer is lung cancer or colon cancer.

11. The method of treating cancer according to claim 10, wherein the patient's tumor tissue section is positive for PD-L1 expression and, optionally, positive for αvβ3 expression.

12. Use of an anti-αvβ3 antibody in the manufacture of a combination drug for use in combination with an anti-PD-1 antibody.

13. The anti-αvβ3 antibody an HCDR1 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, and an HCDR3 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3; The use described in claim 12, comprising an LCDR1 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, an LCDR2 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 5, and an LCDR3 region having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

6.

14. The anti-αvβ3 antibody an HCDR1 region of the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 region of the amino acid sequence shown in SEQ ID NO: 2, and an HCDR3 region of the amino acid sequence shown in SEQ ID NO: 3; an LCDR1 region of the amino acid sequence shown in SEQ ID NO: 4, an LCDR2 region of the amino acid sequence shown in SEQ ID NO: 5, and an LCDR3 region of the amino acid sequence shown in SEQ ID NO: 6; Preferably, the anti-αvβ3 antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8; The use according to claim 13, wherein the anti-αvβ3 antibody is HY1272.

15. 10. A combination therapy for treating a patient with cancer, comprising separately administering to the patient a therapeutically effective amount of the anti-αvβ3 antibody of claim 2 or 3, and separately administering a therapeutically effective amount of an anti-PD-1 antibody.

16. 16. The combination therapy for treating a patient with cancer according to claim 15, wherein the cancer is lung cancer or colon cancer.

17. The combination therapy for treating a patient with cancer according to claim 16, wherein the patient's tumor tissue section is positive for PD-L1 expression and, optionally, positive for αvβ3 expression.

18. The method of any one of claims 15 to 17, wherein the method is administered by administering the anti-αvβ3 antibody within ±5 minutes of administering the anti-PD-1 antibody.

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