WO2018223923A1 - Use of pd-1 antibody combined with vegf ligand or vegf receptor inhibitor in preparing drug for treating tumor - Google Patents

Abstract

Provided is a use of a PD-1 antibody combined with a VEGF ligand or a VEGF receptor inhibitor in preparing a drug for treating a tumor.

Description

Use of PD-1 antibody in combination with VEGF ligand or VEGF receptor inhibitor for the preparation of a medicament for treating tumor Technical field

The invention belongs to the field of medicine and relates to the use of a PD-1 antibody in combination with a VEGF ligand inhibitor or a VEGF receptor inhibitor in the preparation of a medicament for treating tumor.

Background technique

Tumor immunotherapy is a long-term hot spot in the field of cancer therapy, in which T cell tumor immunotherapy is at its core. Tumor immunotherapy is a way to fully utilize and mobilize killer T cells in tumor patients and kill tumors. It may be the most effective and safest way to treat tumors. At the same time, tumor escape is a huge obstacle to tumor immunotherapy. Tumor cells use their own inhibition of the immune system to promote rapid tumor growth. There is an extremely complex relationship between the immune escape mechanism of tumors and the body's immune response to tumors. Early tumor-specific killer T cells in tumor immunotherapy have biological activity, but they lose their killing function at the late stage of tumor growth. Therefore, tumor immunotherapy is to maximize the patient's own immune system response to the tumor. It not only activates the original immune system response in the body, but also maintains the duration of the immune system response and the intensity of the response. key.

Programmed death-l (PD-1) belongs to the CD28 family and has 23% amino acid homology with cytotoxic T Iymphocyte antigen 4 (CTLA-4), but its expression Unlike CTLA, it is mainly expressed on activated T cells, B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DCs), and expression on cells after activation can be up-regulated. The new study found that high PD-L1 protein expression was detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, colon cancer, kidney cancer, and melanoma, and the expression level of PD-L1 was closely related to the clinical and prognosis of patients. . At the immunological checkpoint, PD-1 and its ligand PD-L1 inhibited the activity of T lymphocytes, and binding of PD-1 to PD-L1 resulted in apoptosis and depletion of activated immune cells. Since PD-L1 plays a role in suppressing T cell proliferation by the second signaling pathway, blocking the binding between PD-L1/PD-1 has become a very promising emerging target in the field of tumor immunotherapy. Meta-analysis shows human PD. The mean positive rate of -L1 protein expression was 44.72%, so PD-L1/PD-1 related immunotherapy became one of the directions of treatment of malignant glioma ([J].Journal of Hematology & Oncology,2017,10(1): 81), Kathy Boltz reported the clinical study of Pembrolizumab for glioblastoma multiforme. The results showed that some patients benefited, but 40% of patients still have disease progression (Neuro-Oncology.2016;18: abstract ATIM- 35), so there are still many shortcomings in the treatment of tumors with PD-1 antibody alone.

Although antibodies against the PD-1 target have achieved significant effects on tumor suppression, they are not effective or even ineffective in some patients, and there are serious adverse reactions associated with immunity. Xia Bu et al. found that melanoma patients resistant to PD-1 immunotherapy showed immunosuppression, angiogenesis, monocyte and macrophage chemotaxis, extracellular matrix remodeling, and epithelial-interstitial The characteristics of up-regulation of genes such as mass transfer, therefore, PD-1 immunotherapy combined with the above target treatment can produce effective anti-tumor immunity ([J]. Trends in molecular medicine, 2016, 22(6): 448-451) . A study by S. Yasuda et al. discloses that VEGF monoclonal antibody can be combined with anti-PD-1 antibody for immunotherapy ([J]. Clinical & Experimental Immunology, 2013, 172(3): 500-506); VEGF inhibits DC maturation, leading to immunosuppression ([J]. Current treatment options in oncology, 2014, 15(1): 137-146).

Clinical trials of PD-1/PD-L1 antibodies or VEGF-targeted anti-tumor drugs are currently underway, and Nivolumab is used in combination with Suntinib or Pazopanib for the treatment of metastatic renal cell carcinoma (mRCC). Good results are shown (ASCO meeting, (2014): 5010-5010); clinical results of Pembrolizumab in combination with bevacizumab for the treatment of recurrent gliomas show therapeutic efficacy and good tolerance (ASCO meeting, (2016) :2041-2041), but Blumenthal et al. reported that clinical studies of Pembrolizumab in combination with bevacizumab for progressive primary brain tumors have shown that the combination is ineffective. It is not recommended to use a combination of the two to treat progressive primary brain tumors. ([J]. Journal of neuro-oncology, 2016, 129(3): 453-460), therefore, there is still a lot of uncertainty in the treatment of tumors with PD-1 antibodies in combination with VEGF inhibitors, which deserves further study.

WO2013181452 discloses the use of a PD-1 antagonist, oxaliplatin, folinic acid, 5-FU in combination with or without bevacizumab to treat tumors; WO2016100561 discloses a PD-1 antibody for glioma Uses; WO2016170039, WO2016170040 disclose the use of bevacizumab in combination with a PD-1/PD-L1 antibody for cancer and mediating an immune response.

Patent application WO2017054646A provides a novel PD-1 antibody having high affinity, high selectivity and high biological activity, comprising:

An antibody light chain variable region, said antibody light chain variable region comprising at least one LCDR selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. At the 2017 ASCO meeting, the article "Phase I study of the anti-PD-1 antibody SHR-1210 in patients with advanced solid tumors" disclosed the results of the phase I clinical trial of the PD-1 antibody in the treatment of solid tumor patients, and the results showed that A variety of tumors have inhibitory effects, but about 79.3% of the subjects have symptoms of reactive capillary hemangioma.

Bevacizumab (Bevacizumab,

Is a VEGF inhibitor marketed in the United States on February 26, 2004, for treating various tumors such as lung cancer, ovarian cancer, etc., WO9845331 discloses the sequence and preparation method thereof, and the present invention provides the PD-1 single described in the patent application WO2017054646A. Use of an anti-antigen or antigen-binding fragment thereof in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor for the preparation of a medicament for treating a tumor.

Summary of the invention

The technical problem to be solved by the present invention is to provide a use of a PD-1 antibody or antigen-binding fragment thereof in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor for the preparation of a medicament for treating tumors.

Wherein the PD-1 antibody comprises:

An antibody light chain variable region, said antibody light chain variable region comprising at least one LCDR selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

The VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozantinib, punatinib, nidanib, regolfinib, Sunitinib, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib.

In a preferred embodiment of the invention, the antibody light chain variable region comprises an LCDR1 as shown in SEQ ID NO: 4, an LCDR2 as shown in SEQ ID NO: 5, and a SEQ ID NO: 6 The antibody heavy chain variable region comprises the HCDR 1 as shown in SEQ ID NO: 1, the HCDR 2 as shown in SEQ ID NO: 2, and the HCDR3 as shown in SEQ ID NO: 3.

Among them, the CDR sequences described above are shown in the following table:

name	sequence	Numbering
HCDR1	SYMMS	SEQ ID NO: 1

HCDR2	TISGGGANTYYPDSVKG	SEQ ID NO: 2
HCDR3	QLYYFDY	SEQ ID NO: 3
LCDR1	LASQTIGTWLT	SEQ ID NO: 4
LCDR2	TATSLAD	SEQ ID NO: 5
LCDR3	QQVYSIPWT	SEQ ID NO: 6

In a preferred embodiment of the invention, the PD-1 antibody or antigen-binding fragment thereof of the invention is a humanized antibody or a fragment thereof.

In a preferred embodiment of the invention, the humanized antibody light chain sequence of the PD-1 antibody or antigen-binding fragment thereof of the invention is the sequence set forth in SEQ ID NO: 8 or a variant thereof; The variant preferably has an amino acid change of 0-10 in the light chain variable region; more preferably an amino acid change of A43S.

In a preferred embodiment of the present invention, the humanized antibody light chain sequence of the PD-1 antibody or antigen-binding fragment thereof of the present invention is the sequence shown in SEQ ID NO: 7 or a variant thereof; The body preferably has an amino acid change of 0-10 in the heavy chain variable region; more preferably an amino acid change of G44R.

Particularly preferably, the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 8, and the heavy chain sequence is the sequence set forth in SEQ ID NO: 7.

The sequences of the aforementioned humanized antibody heavy and light chains are as follows:

Heavy chain

SEQ ID NO:7

Light chain

SEQ ID NO:8

Use of a PD-1 antibody or antigen-binding fragment thereof according to the present invention in combination with a VEGF ligand inhibitor or a VEGF receptor inhibitor for the preparation of a medicament for treating a tumor, wherein the VEGF ligand inhibitor is selected from the group consisting of Bevacizumab, Removumab, Ranibizumab, Abbotsep, Composip, Abicipar pegol, Brolucizumab, LMG-324, Nesvacumab, Sevacizumab, Tanibirumab, Navicixizumab, RG-7716, LHA- 510, OPT-302, TK-001, GZ-402663, VGX-100, PG-545, BI-836880, GNR-011, BR-55, OTSGC-A24, PAN-90806, AVA-101, ODM-203, TAS-115, X-82, MP-0250, Sitravatinib, 4SC-203, AL-2846, ABT-165, SIM-010603, BI-836880, HL-217, CS-2164, RGX-314, AMC-303, VXM-01, preferably bevacizumab, remollozumab.

In the above embodiment, the PD-1 antibody or antigen-binding fragment thereof and the VEGF receptor inhibitor or VEGF ligand inhibitor have synergistic pharmacodynamic effects for treating tumors; preferably, the humanized PD-1 antibody Or the antigen-binding fragment thereof and the VEGF receptor inhibitor or VEGF ligand inhibitor have synergistic pharmacodynamic effects for treating tumors; more preferably, the light chain sequence as shown in SEQ ID NO: 8 and SEQ ID NO: The humanized PD-1 antibody or antigen-binding fragment thereof of the heavy chain sequence shown in Figure 7 has a synergistic pharmacological effect on tumor treatment with a VEGF receptor inhibitor or a VEGF ligand inhibitor.

In the present invention, there is provided a method of treating a tumor comprising administering to a patient a PD-1 antibody or antigen-binding fragment thereof and a VEGF receptor inhibitor or a VEGF ligand inhibitor, wherein the VEGF receptor inhibitor is selected From pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozantinib, pentatinib, nidanib, regorafenib, sunitinib, pazodab Ni, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib.

Preferably, the tumor is selected from the group consisting of breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer , ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, brain tumor, endocrine organ tumor, bladder cancer, skin cancer, nasopharyngeal carcinoma, rhabdomyosarcoma; preferably breast cancer, lung cancer, Gastric cancer, intestinal cancer, kidney cancer, melanoma, pancreatic cancer, cervical cancer, liver cancer, leukemia, ovarian cancer, lymphoma, brain tumor, esophageal cancer; most preferred lung cancer, gastric cancer, colon cancer, liver cancer, esophageal cancer, lymphoma , kidney cancer, melanoma, cervical cancer, ovarian cancer, brain tumor.

Preferably, the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, preferably non-small cell lung cancer; and the intestinal cancer is selected from the group consisting of small intestine cancer, colon cancer, rectal cancer, colorectal cancer, preferably colon cancer, rectal cancer, and knot. Rectal cancer; the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, preferably Hodgkin's lymphoma.

Preferably, the brain tumor is selected from the group consisting of a neuroepithelial tissue tumor, a cranial nerve and a spinal cord nerve tumor, and a meningeal tissue tumor; the neuroepithelial tissue tumor is selected from the group consisting of astrocytoma, anaplastic astrocytoma, glioblastoma. , hair cell astrocytoma, pleomorphic yellow astrocytoma, subependymal giant cell astrocytoma, oligodendroglioma, ependymoma, mixed glioma, choroid plexus Tumors, pineal somatic tumors, embryonic tumors, most preferably astrocytoma, anaplastic astrocytoma, glioblastoma.

Preferably, the above tumor is mediated and/or expressed by PD-1 to express PD-L1.

The use according to the present invention, wherein the PD-1 antibody or antigen-binding fragment thereof is used in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor in a weight ratio ranging from 0.01 to 100, selected from 5:1, 3 : 1, 5: 2, 5: 3, 2: 1, 2: 3, 3: 2, 4: 3, 5: 4, 1:1, 5: 6, 4: 5, 3: 4, 3: 5 , 1:2, 2:5, 1:3, 3:10, 4:15, 1:4, 1:5, 1:6, 2:9, 2:15, 1:10, 2:25, 3 :8; preferably 5:3, 4:3, 5:4, 1:1, 3:4, 2:3, 3:5, 1:2, 2:5, 1:3, 3:10, 1: 4, 2:9, 1:5, 1:10, 2:15, 3:8.

The ratio by weight of the above PD-1 antibody or antigen-binding fragment thereof to a VEGF receptor inhibitor or a VEGF ligand inhibitor is preferably in a ratio by weight of a PD-1 antibody or an antigen-binding fragment thereof to a VEGF ligand inhibitor. The range, more preferably, is the range of weight ratios from the combination of the PD-1 antibody or antigen-binding fragment thereof with bevacizumab or remolozumab.

The use according to the invention, wherein the dose of the PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of 0.1-100 mg/kg, preferably 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg. /kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 7.5mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 12.5mg/kg, 15mg/kg, 17.5mg/kg, 20 mg/kg, most preferably 1 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg; bevacizumab The dose is selected from the group consisting of 0.1-100 mg/kg, preferably 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 7.5 mg. /kg, 8mg/kg, 9mg/kg, 10mg/kg, 12.5mg/kg, 12mg/kg, 15mg/kg, 17.5mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, most preferably 5mg/kg 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg; the dose of Remoluzumab is selected from 0.1-100 mg/kg, preferably 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 2.5 mg/ Kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 7.5mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 12mg/kg, 12.5mg/kg, 15mg/ Kg, 18 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, most preferably 5 mg/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 Mg/kg.

In another preferred embodiment, the dose of the PD-1 antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of 1-2000 mg, preferably 25 mg, 40 mg, 50 mg, 60 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, 800 mg, 1000 mg, most preferably 40 mg, 60 mg, 100 mg, 200 mg, 400 mg.

In the present invention, the above PD-1 antibody or antigen-binding fragment thereof is provided in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor as a medicament for treating a tumor, wherein the VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, Vandetanib, sorafenib, axitinib, cabotinib, pentatinib, nidanib, regorafenib, sunitinib, pazopanib, Puquitinib, Rebastinib, Lucitanib Hydrochloride, Necuparanib, Ningetinib, Altiratinib.

The combined modes of administration of the present invention are selected from the group consisting of simultaneous administration, independent formulation and co-administration or independent formulation and sequential administration.

The administration route of the PD-1 antibody or antigen-binding fragment thereof, bevacizumab or remoluzumab according to the present invention is preferably a parenteral administration route, more preferably intravenous injection, intramuscular injection or subcutaneous injection.

The present invention further relates to the use of the above PD-1 antibody or antigen-binding fragment thereof in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor for the preparation of a medicament for treating a tumor, wherein administration of the PD-1 antibody or antigen-binding fragment thereof Frequency is once a day, twice a week, three times a week, once a week, once every two weeks, once every three weeks, once a month, once in February, once in March, once in June, preferably once every two weeks, once every three weeks, one time Once a month, once a month; VEGF receptor inhibitors or VEGF ligand inhibitors are administered once a day, twice a day, three times a day, twice a week, three times a week, once a week, once every two weeks, Once every three weeks, once a month, once in February, once in March, once in June, preferably once a day, once every two weeks, once every three weeks, once a month.

Significantly, the PD-1 antibody or antigen-binding fragment thereof of the present invention has synergistic pharmacological effects in combination with bevacizumab or remolozumab.

The present invention also provides a method of reducing an adverse reaction caused by an anti-PD-1 antibody comprising using an anti-PD-1 antibody in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor. In one embodiment, the adverse reaction is a vascular-related adverse reaction, such as a capillary hemangioma. The invention further provides a pharmaceutical composition comprising an effective amount of a PD-1 antibody or antigen-binding fragment thereof, a VEGF receptor inhibitor or a VEGF ligand inhibitor, and a pharmaceutically acceptable excipient according to the invention , a dilution or carrier, wherein the VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozantinib, punatinib, nidanib , regomafenib, sunitinib, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib.

Combining the anti-PD-1 antibody of the present invention with a VEGF receptor inhibitor or a VEGF ligand inhibitor can not only synergistically enhance the anti-tumor effect, but also reduce or eliminate capillaries caused by anti-PD-1 antibodies Adverse reactions such as tumors.

In the description and claims of the present application, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise indicated. However, for a better understanding of the present invention, definitions and explanations of some related terms are provided below. In addition, when the definitions and explanations of the terms provided by the present application are inconsistent with the meanings generally understood by those skilled in the art, the definitions and explanations of the terms provided by the present application shall prevail.

DRAWINGS

Figure 1 Effect of bevacizumab and PD-1 antibody A alone or in combination on human malignant glioma U-87MG mouse xenografts

Figure 2 Pharmacodynamic results of bevacizumab in combination with PD-1 antibody A in the U87+PBMC tumor model

Figure 3 Effect of bevacizumab and PD-1 antibody A alone or in combination on tumor weight in human malignant glioma U-87MG mice

Figure 4 Effect of bevacizumab and PD-1 antibody A alone or in combination on body weight of human malignant glioma U-87MG mice

detailed description

First, the term

In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter code and the one-letter code for amino acids used in the present invention are as described in J.biol.chem, 243, p3558 (1968).

The antibody of the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, the corresponding heavy chains of which are μ chain, δ chain γ, α, respectively. Chain, ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either a kappa chain or a lambda chain by the constant region. Each of the five types of Ig may have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, 2, 3, 4 or a variant thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or to the kabat and chothia numbering rules ( HCDR1).

The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into human antibody variable region frameworks, ie different types of human germline An antibody produced in an antibody framework sequence. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. The germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc. People, 1991Sequences of Proteins of Immunological Interest, found in the 5th edition. In a preferred embodiment of the invention, the CDR sequence of said PD-1 humanized antibody mouse is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6.

The "antigen-binding fragment" as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment sFv fragment which binds to human PD-1; The antibody is selected from one or more of the CDR regions of SEQ ID NO: 1 to SEQ ID NO: 6. The Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site. Typically, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding. The two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv). The term "binding to PD-1" as used herein refers to the ability to interact with human PD-1. The term "antigen binding site" as used in the present invention refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.

"Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid. "Administration" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells. "Administering" and "treating" also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell. "Treatment", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect. Generally, a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement. The amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. Embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in alleviating the symptoms of the target disease in each of the affected cases, but according to any statistical test methods known in the art such as Student's t test, chi-square test, basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate the diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

As used herein, the expression "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.

"Optional" or "optionally" means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a particular sequence may, but need not, be present.

The term "synergistic pharmacodynamic effect" includes pharmacodynamic additive action, pharmacodynamic enhancing effect, and pharmacodynamic sensitizing effect, and the "synergistic pharmacodynamic effect" of the present invention includes, but is not limited to, reducing the use of the PD-1 antibody of the present invention alone. Tolerance in the case of its antigen-binding fragment, VEGF receptor inhibitor or VEGF ligand inhibitor, reducing the use of the PD-1 antibody or antigen-binding fragment thereof, VEGF receptor inhibitor or VEGF ligand of the present invention alone The dose at the time of the inhibitor reduces the adverse reaction when the PD-1 antibody or antigen-binding fragment thereof, the VEGF receptor inhibitor or the VEGF ligand inhibitor of the present invention is used alone, and the combination of the two is used to enhance the VEGF when used alone. The efficacy of a bulk inhibitor or a VEGF ligand inhibitor, a PD-1 antibody or antigen-binding fragment thereof according to the invention.

The term "pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carrier and excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

The PD-1 antibody or antigen-binding fragment thereof of the invention and the VEGF receptor inhibitor or VEGF ligand inhibitor composition can effectively solve the tumor heterogeneity, play a significant role in inhibiting tumor cells, and effectively inhibit the proliferation and migration of tumor cells or Invasion.

Exemplary experimental protocols for the use of the compositions of the present invention in the treatment of tumors are provided below to demonstrate the beneficial or beneficial technical effects of the compositions of the present invention. However, it should be understood that the following experimental schemes are merely illustrative of the present invention and are not intended to limit the scope of the invention. A person skilled in the art can make appropriate modifications or changes to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Example 1. Therapeutic effect of the PD-1 antibody or antigen-binding fragment thereof of the present invention combined with bevacizumab on subcutaneous xenograft of human malignant glioma U-87MG mice

testing sample

PD-1 antibody A (humanized PD-1 antibody consisting of the light chain as shown in SEQ ID NO: 8 and the heavy chain as shown in SEQ ID NO: 7 of the present invention, defined as PD- 1 Antibody A, which is a PD-1 antibody in WO2017054646A), bevacizumab (prepared according to the method described in WO9845331).

Test animal

NOD/SCID female mice were purchased from Cavans (batch number: 201703849), certificate number: SCXK (Su) 2016-0010, 4-6 weeks old at the time of purchase, body weight about 19g, 5/cage rearing, 12/ 12 hours light / dark cycle adjustment, temperature 23 ± 1 ° C constant temperature, humidity 50 ~ 60%, free to eat water.

Preparation of test solution

PD-1 antibody A was diluted to 20 mg/mL in PBS under sterile conditions, and dispensed into 10 tubes; bevacizumab was opened and then aseptically dispensed into 4 tubes. The above-packed antibody 1 tube was diluted to 0.63 mg/mL with PBS under aseptic conditions, and dispensed into 2.4 mL/tube, a total of 10 tubes, and stored at 4 ° C, and 1 tube was taken for each injection.

experimental method

First, PBMCs extraction

The PBMCs used in the examples were extracted from fresh blood of two volunteers. The extraction method was as follows:

1. Mix venous blood treated with heparin anticoagulation with PBS containing 2% FBS in the same volume;

2. Aseptically transfer 15 mL of the separation solution into the 1077 to 50 mL separation tube (previously gently invert the container to fully mix 1077);

3. Carefully add 25 mL of diluted blood to the separation tube containing the separation solution 1077 (at room temperature, slowly add, form a distinct layer in the blood and separation solution 1077, do not mix the diluted blood into the separation solution 1077);

4, centrifugation at 1200g for 10 minutes at room temperature, centrifugation can lead to the precipitation of red blood cells and multinucleated white blood cells, while forming a layer of mononuclear lymphocytes on the separation solution 1077;

5. Aspirate 4-6 cm of plasma above the lymphocytes;

6. The lymphocyte layer and the lower half of the separation solution 1077 are transferred to another centrifuge tube. Add an equal volume of PBS and centrifuge at room temperature for 300 minutes at room temperature for 8 minutes;

7. Wash the cells with PBS or RPMI-1640 medium and resuspend the cells in serum-containing RPMI-1640 medium.

Second, CD3 antibody coating and PBMCs activation

1. CD3 antibody (40 ng/mL) diluted with PBS was added to a 6-well cell culture plate, 1 mL/well, and incubated at 37 ° C for one hour;

2. Before adding PBMCs, remove the CD3 antibody dilution and wash twice with 2 mL of PBS per well;

3. Add 2 volunteers' PBMCs (RPMI-1640 medium suspension): about 2×10 ⁶ cells per well, 2 mL/well;

4. Incubate in a 37 ° C incubator for 4 days.

Third, the method of administration and animal treatment

100 μL of U-87MG cells (1.5×10 ⁶ cells/mouse) were inoculated subcutaneously into the right flank of NOD/SCID mice, and after 10 days, the animals with excessive or too small tumor volume were removed, with an average tumor volume of about 65 mm ^{3 .} The mice were randomly divided into 4 groups: vehicle control group, bevacizumab 3 mg/kg single use group, PD-1 antibody A 3 mg/kg single use group and bevacizumab 3 mg/kg+PD-1 antibody. A 3 mg/kg combination group. 9 in each group, the day of the group is recorded as the 0th day. PBMCs of two volunteers stimulated with CD3 antibody were mixed in a 1:1 ratio on the day of grouping (Day 0), and injected into tumor tissues of tumor-bearing mice at 5 × 10 ⁵ cells/mouse. The remaining PBMCs were stopped and cultured, and 5 x 10 ⁶ cells/mouse were intraperitoneally injected into tumor-bearing mice on day 7, and the 0th day was repeated on days 11, 14, and 17. On the day of grouping (Day 0), PD-1 antibody A and/or tail vein injection of bevacizumab were injected intraperitoneally, followed by two injections per week for a total of 6 doses. Tumor volume, animal body weight were monitored twice a week and data were recorded. At the end of the experiment, the animals were euthanized, the tumors were stripped and the tumor weight was weighed.

Data representation and statistical processing

All data were plotted and statistically analyzed using Excel and GraphPad Prism 5 software.

The tumor volume (V) is calculated as: V = 1/2 × a × b ² where a and b represent length and width, respectively.

The relative tumor proliferation rate T/C(%)=(TT ₀ )/(CC ₀ )×100, where T and C are the tumor volume of the treatment group and the control group at the end of the experiment; T ₀ and C ₀ are the beginning of the experiment. Tumor volume.

Tumor inhibition rate TGI (%) = 1 - T / C (%).

Experimental result

The results of the experiment showed that the bevacizumab monotherapy group (3 mg/kg, IV, BIW×6) and bevacizumab+PD-1 antibody A combination group (3+3 mg/kg, IV+IP, BIW) ×6) Compared with the vehicle control group, the growth of subcutaneous xenografts in human malignant glioma U-87MG mice was significantly inhibited, and the tumor inhibition rates were 66.38% and 78.71%, respectively (p<0.001 vs vehicle control); The anti-tumor rate of the -1 antibody A single-agent group (3 mg/kg, IP, BIW×6) was only 9.79% on the 20th day, and there was no significant difference from the vehicle control group. Compared with bevacizumab alone, the combination of bevacizumab + PD-1 antibody A showed a statistically significant difference (p<0.05) on the 20th day, showing a significant synergistic effect. (Table 1 and Figure 1).

The tumor weight in vitro was consistent with the trend of tumor volume. The tumor weight of bevacizumab+PD-1 antibody A combination group and bevacizumab alone group was significantly lower than that of vehicle control group, and there were statistics. Learning differences (P < 0.001). There was no significant difference in tumor weight between the bevacizumab+PD-1 antibody A combination group and the bevacizumab monotherapy group (p=0.0779), but the tumor weights in the two groups were <0.5g. For 67% (6/9) vs. 11% (1/9), the advantages of the combined group can also be seen (Figure 2 and Figure 3).

The tumor-bearing mice were well tolerated by bevacizumab and PD-1 antibody A alone or in combination, and the body weight increased steadily throughout the administration, only the vehicle control group and the PD-1 antibody group A. The slight decrease in body weight at the last measurement may be due to the late growth of human malignant glioma U-87MG under the skin of mice, resulting in a decrease in the body of the mouse and no significant drug-induced weight loss (Figure 4).

Table 1. Effect of bevacizumab and PD-1 antibody A alone or in combination on human malignant glioma U-87MG mouse xenografts

D0: first administration time; a: actual number (number of groups); BIW: twice a week; I.P.: intraperitoneal injection: I.V.: intravenous injection

***p<0.001vs vehicle control group; #p<0.05vs bevacizumab 3mg/kg

Experimental results

Bevacizumab + PD-1 antibody A combination (3mg/kg, IV+IP, BIW×6) significantly inhibited the growth of subcutaneous xenografts in human malignant glioma U-87MG mice (TGI 78.71%). And the tumor inhibition effect is better than +PD-1 antibody A single drug group (3mg/kg, IP, BIW×6) and bevacizumab single drug group (3mg/kg, IV, BIW×6), small tumor burden Rats are well tolerated by the above drugs.

Claims (12)

1. Use of a PD-1 antibody or antigen-binding fragment thereof in combination with a VEGF ligand inhibitor or a VEGF receptor inhibitor for the preparation of a medicament for treating a tumor, characterized in that the PD-1 antibody or antigen-binding thereof The fragment contains:

   An antibody light chain variable region, said antibody light chain variable region comprising at least one LCDR selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

   An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

   Wherein the VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozantinib, punatinib, nidanib, regofei Ninet, sunitinib, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib.
2. The use according to claim 1, wherein said antibody light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and SEQ ID NO: The LCDR3 shown in Figure 6; the antibody heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3.
3. The use according to any one of claims 1 to 2, wherein the PD-1 antibody or antigen-binding fragment thereof is a humanized antibody or a fragment thereof.
4. The use according to claim 3, wherein the PD-1 antibody or antigen-binding fragment thereof, wherein the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 8 or Variant; the variant preferably has an amino acid change of 0-10 in the light chain variable region; more preferably an amino acid change of A43S.
5. The use according to claim 3, wherein the PD-1 antibody or antigen-binding fragment thereof, wherein the humanized antibody heavy chain sequence is the sequence shown in SEQ ID NO: 7 or a variant thereof The variant preferably has an amino acid change of 0-10 in the heavy chain variable region; more preferably an amino acid change of G44R.
6. The use according to claim 3, wherein the light chain sequence of the humanized PD-1 antibody or antigen-binding fragment thereof is preferably the sequence shown in SEQ ID NO: 8 or a variant thereof, heavy chain sequence A sequence as shown in SEQ ID NO: 7 or a variant thereof is preferred.
7. The use according to claim 1, wherein the VEGF ligand inhibitor is selected from the group consisting of bevacizumab, remollozumab, ranibizumab, aboxicept, compaqip, Abicipar pegol, Brolucizumab, LMG-324, Nesvacumab, Sevacizumab, Tanibirumab, Navicixizumab, RG-7716, LHA-510, OPT-302, TK-001, GZ-402663, VGX-100, PG-545, BI-836880, GNR -011, BR-55, OTSGC-A24, PAN-90806, AVA-101, ODM-203, TAS-115, X-82, MP-0250, Sitravatinib, 4SC-203, AL-2846, ABT-165, SIM -010603, BI-836880, HL-217, CS-2164, RGX-314, AMC-303, VXM-01, preferably bevacizumab, remollozumab, most preferably bevacizumab.
8. The use according to any one of claims 1 to 7, wherein the tumor is selected from the group consisting of breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, Liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, brain tumor, endocrine organ tumor, bladder cancer, skin Cancer, nasopharyngeal carcinoma, rhabdomyosarcoma; preferably breast cancer, lung cancer, stomach cancer, colon cancer, kidney cancer, melanoma, pancreatic cancer, cervical cancer, liver cancer, leukemia, ovarian cancer, lymphoma, brain tumor, esophageal cancer; Preferred are lung cancer, gastric cancer, intestinal cancer, liver cancer, esophageal cancer, lymphoma, renal cancer, melanoma, cervical cancer, ovarian cancer, and brain tumor.
9. The use according to claim 8, wherein the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, preferably non-small cell lung cancer; and the intestinal cancer is selected from the group consisting of small intestine cancer, colon cancer, rectal cancer, and colorectal Cancer, preferably colon cancer, rectal cancer, colorectal cancer; the lymphoma is selected from the group consisting of non-Hodgkin's lymphoma, Hodgkin's lymphoma, preferably Hodgkin's lymphoma.
10. The use according to claim 8, wherein the brain tumor is selected from the group consisting of a neuroepithelial tissue tumor, a cranial nerve and a spinal cord nerve tumor, and a meningeal tissue tumor; the neuroepithelial tissue tumor is selected from astrocytoma, anaplastic Astrocytoma, glioblastoma, hairy cell astrocytoma, pleomorphic yellow astrocytoma, subependymal giant cell astrocytoma, oligodendroglioma, ependymal cells Tumor, mixed glioma, choroid plexus tumor, pineal somatic cell tumor, embryonic tumor, preferably astrocytoma, anaplastic astrocytoma, glioblastoma.
11. The use according to any one of claims 8 to 10, wherein the tumor is mediated and/or expressed by PD-1 to express PD-L1.
12. A pharmaceutical composition comprising an effective amount of a PD-1 antibody or antigen-binding fragment thereof and a VEGF ligand inhibitor or VEGF receptor inhibitor according to any one of claims 1 to 7, and one or more A pharmaceutically acceptable excipient, diluent or carrier.

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