CN109963592B

CN109963592B - Use of PD-1 antibodies in combination with VEGF ligands or VEGF receptor inhibitors for the preparation of a medicament for the treatment of tumors

Info

Publication number: CN109963592B
Application number: CN201880004442.XA
Authority: CN
Inventors: 马晨; 曹国庆; 张蕾; 杨昌永; 张连山
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2017-06-05
Filing date: 2018-06-04
Publication date: 2022-11-22
Anticipated expiration: 2038-06-04
Also published as: TW201902514A; CN109963592A; WO2018223923A1

Abstract

Discloses the use of a PD-1 antibody in combination with a VEGF ligand or a VEGF receptor inhibitor in the preparation of a medicament for the treatment of tumours.

Description

Use of PD-1 antibodies in combination with VEGF ligands or VEGF receptor inhibitors for the preparation of a medicament for the treatment of tumors

Technical Field

The invention belongs to the field of medicines, and relates to an application of a PD-1 antibody and a VEGF ligand inhibitor or VEGF receptor inhibitor in preparation of a medicine for treating tumors.

Background

Tumor immunotherapy is a long-standing hotspot in the field of tumor therapy, where tumor immunotherapy of T cells is in its central position. The tumor immunotherapy is a way for fully utilizing and mobilizing killer T cells in a tumor patient to kill the tumor, and is probably the most effective and the safest way for treating the tumor. Meanwhile, tumor escape is a great obstacle to tumor immunotherapy, and tumor cells promote the rapid growth of tumors by utilizing the inhibition effect of the tumor cells on the immune system. There is a very complex relationship between the immune escape mechanism of tumors and the immune response of the body to the tumors. The tumor specific killer T cells in the early stage of tumor immunotherapy have the bioactivity, but lose the killing function along with the later stage of tumor growth. Therefore, the tumor immunotherapy is to improve the immune system response of the patient to the tumor to the maximum extent, and it is the key of the immunotherapy for the tumor to not only activate the original immune system response in vivo, but also maintain the duration and the response intensity of the immune system response.

Programmed death molecule 1 (PD-l) belongs to the CD28 family, has 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (CTLA-4), but its expression is different from CTLA, and 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 activated cells can be up-regulated. The new research finds that the expression of high PD-L1 protein is detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma and the like, and the expression level of PD-L1 is closely related to the clinic and prognosis of patients. In the immune checkpoint, PD-1 and its ligand PD-L1 inhibit the activity of T lymphocytes, and binding of PD-1 to PD-L1 results in apoptosis and depletion of activated immune cells. Since PD-L1 plays a role in inhibiting T cell proliferation by a second signaling pathway, the PD-L1/PD-1 is blocked to be a very potential emerging target in the field of tumor immunotherapy, meta-analysis shows that the mean value of the positive expression rate of human PD-L1 protein is 44.72%, so PD-L1/PD-1 related immunotherapy becomes one of the directions of malignant glioma treatment (J. Journal of Hematology & Oncology,2017, 10 (1): 81), kathy Boltz reports that Pembrolizumab is used in the clinical research of glioblastoma multiforme, and the result shows that some patients benefit from 40% of the patients' disease progression (neo-Oncology.2016; 18 abstration ATIM-35), so that the PD-1 antibody has a plurality of defects when being used alone for tumor treatment.

Although the antibody aiming at the PD-1 target has remarkable curative effect on tumor inhibition, the antibody has poor curative effect or even ineffective effect on some patients, and in addition, serious adverse reaction related to immunity occurs. Xia Bu et al found that melanoma patients resistant to PD-1 immunotherapy exhibited upregulated characteristics such as immunosuppression, angiogenesis, monocyte and macrophage chemotaxis, extracellular matrix remodeling, and epithelial-mesenchymal transition, and therefore, approaches to PD-1 immunotherapy combined with the above-described target therapy could produce potent anti-tumor immune effects ([ J ] Trends in molecular mediators, 2016, 22 (6): 448-451). S. Yasuda et al, discloses that VEGF monoclonal antibodies can be used in combination with anti-PD-1 antibodies for immunotherapy ([ J ]. Clinical & Experimental Immunology,2013, 172 (3): 500-506); furthermore, DC maturation is inhibited by elevated VEGF, resulting in immunosuppression ([ J ]. Current therapeutic options in oncology,2014, 15 (1): 137-146).

Clinical trials of anti-tumor drugs targeting VEGF in combination with PD-1/PD-L1 antibodies currently under investigation or on the market are under development, and Nivolumab shows good efficacy in the treatment of metastatic renal cell carcinoma (mRCC) in combination with Sunitinib or Pazopanib (ASCO meeting, (2014): 5010-5010); clinical results of Pembrolizumab in combination with bevacizumab for treatment of recurrent glioma showed therapeutic efficacy and was well tolerated (ASCO meeting, (2016): 2041-2041), but clinical studies by Blumenthal et al reported that Pembrolizumab in combination with bevacizumab for advanced primary brain tumors showed that the combination was ineffective and not recommended for treatment of advanced primary brain tumors ([ J ]. Journal of neuro-oncology,2016, 129 (3): 453-460), so PD-1 antibody in combination with VEGF inhibitor treatment of tumors still had much uncertainty, and was worth intensive study.

WO2013181452 discloses the use of a PD-1 antagonist, oxaliplatin, leucovorin, 5-FU in combination or not in combination with bevacizumab for the treatment of tumors; WO2016100561 discloses the use of a PD-1 antibody for glioma; WO2016170039, WO2016170040 disclose the use of bevacizumab in combination with PD-1/PD-L1 antibodies for cancer and for mediating immune responses.

Patent application WO2017054646a provides a novel high affinity, high selectivity, high bioactivity PD-1 antibody comprising:

an antibody light chain variable region comprising at least 1 LCDR selected from the group consisting of seq id nos: SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and

an antibody heavy chain variable region comprising at least 1 HCDR selected from the group consisting of seq id nos: the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. in the ASCO conference of 2017, an article of Phase I study of the anti iPD-1 anti SHR-1210 in drugs with advanced solid tumors discloses the Phase I clinical experiment result of the PD-1 antibody for treating solid tumor patients, and the result shows that the PD-1 antibody has inhibition effect on various tumors, but about 79.3 percent of the subjects have symptoms of reactive capillary hemangioma.

Bevacizumab (Bevacizumab,

) The invention relates to a VEGF inhibitor which is marketed in the United states at 26.2.2004, and is used for treating various tumors such as lung cancer, ovarian cancer and the like, WO9845331 discloses a sequence and a preparation method thereof, and the invention provides application of a PD-1 monoclonal antibody or an antigen binding fragment thereof, described in patent application WO2017054646A, and a VEGF receptor inhibitor or a VEGF ligand inhibitor in combination in preparation of a medicament for treating tumors.

Disclosure of Invention

The invention aims to provide an application of a PD-1 antibody or an antigen binding fragment thereof and a VEGF receptor inhibitor or a VEGF ligand inhibitor in preparation of a medicament for treating tumors.

Wherein the PD-1 antibody comprises:

an antibody heavy chain variable region comprising at least 1 HCDR selected from the group consisting of seq id nos: 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, ponatinib, nintedanib, regorafenib, sunitinib, pazopanib, puquitinib, rebastinib, lucitanib hydrochloride, necopanib, ningitinib, and Altiratinib.

In a preferred embodiment of the invention, the antibody light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, as shown in SEQ ID NO:5, as shown in SEQ ID NO: LCDR3 as shown in 6; the heavy chain variable region of the antibody comprises the amino acid sequence shown as SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, as shown in SEQ ID NO:3 HCDR3 as shown in fig. 3.

Wherein, the CDR sequences are shown in the following table:

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

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 as set forth in SEQ ID NO:7 or a variant thereof; the variant preferably has 0-10 amino acid changes in the heavy chain variable region; more preferably the amino acid of G44R.

Particularly preferred humanized antibody light chain sequences are as set forth in SEQ ID NO:8, and the heavy chain sequence is shown as SEQ ID NO:7, or a sequence shown in the figure.

The sequences of the heavy and light chains of the humanized antibodies are shown below:

heavy chain

Light chain

The PD-1 antibody or an antigen-binding fragment thereof is combined with a VEGF ligand inhibitor or a VEGF receptor inhibitor for preparing a medicament for treating tumors, wherein the VEGF ligand inhibitor is selected from bevacizumab, ramucirumab, ranibizumab, aflibercept, combacept, abipotaglol, 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-3963 zxft AMC, AVA-101, ODM-203, TAS-115, X-82, trauberg-0250, siavib-203, BTAC-2824, PAN-3963 zxft-35314, VXft-35314, preferably VXft-3526, ABXzf-33, and VXf-3526.

In the above scheme, the PD-1 antibody or antigen-binding fragment thereof and VEGF receptor inhibitor or VEGF ligand inhibitor have a synergistic therapeutic effect for treating tumor; preferably, the humanized PD-1 antibody or antigen-binding fragment thereof and the VEGF receptor inhibitor or VEGF ligand inhibitor have a synergistic pharmacodynamic effect in treating tumors; more preferably, the polypeptide comprises the sequence SEQ ID NO:8 and the light chain sequence as set forth in SEQ ID NO:7 or an antigen-binding fragment thereof and a VEGF receptor inhibitor or a VEGF ligand inhibitor have a synergistic pharmacodynamic action in treating a tumor.

In the present invention, there is provided a method of treating a tumor comprising administering to a patient the above PD-1 antibody or antigen-binding fragment thereof and a VEGF receptor inhibitor or VEGF ligand inhibitor, wherein the VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozinib, ponatinib, nintedanib, regoranib, sunitinib, pazopanib, puquitinib, rebastinib hydrochloride, necopananib, ninetinib, and Altiratinib.

Preferably, the tumor is selected from breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney 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 cancer, rhabdomyosarcoma; preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, pancreatic cancer, cervical cancer, liver cancer, leukemia, ovarian cancer, lymphoma, cerebroma, and esophageal cancer; most preferred are lung cancer, gastric cancer, intestinal cancer, liver cancer, esophageal cancer, lymphoma, renal cancer, melanoma, cervical cancer, ovarian cancer, and brain tumor.

Preferably, the lung cancer is selected from non-small cell lung cancer, preferably non-small cell lung cancer; the intestinal cancer is selected from small intestinal cancer, colon cancer, rectal cancer and colorectal cancer, preferably colon cancer, rectal cancer and colorectal cancer; the lymphoma is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, preferably Hodgkin lymphoma.

Preferably, the brain tumor is selected from the group consisting of neuroepithelial tissue tumors, cranial and spinal nerve tumors, meningeal tissue tumors; the neuroepithelial tumors are selected from astrocytomas, anaplastic astrocytomas, glioblastomas, hairy cell astrocytomas, pleomorphic yellow astrocytomas, subendocrine giant cell astrocytomas, oligocolloid cytomas, ependymomas, mixed gliomas, choroid plexus tumors, pineal cytomas, embryonic tumors, and most preferably astrocytomas, anaplastic astrocytomas, and glioblastomas.

Preferably, the tumor is mediated by PD-1 and/or expresses PD-L1.

The application of the invention, wherein the weight ratio range of the PD-1 antibody or the antigen binding fragment thereof to the VEGF receptor inhibitor or the VEGF ligand inhibitor is 0.01-100, and is 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 and 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 combined weight ratio range of the PD-1 antibody or the antigen-binding fragment thereof and the VEGF receptor inhibitor or the VEGF ligand inhibitor is preferably selected from the combined weight ratio range of the PD-1 antibody or the antigen-binding fragment thereof and the VEGF ligand inhibitor, and more preferably from the combined weight ratio range of the PD-1 antibody or the antigen-binding fragment thereof and bevacizumab or Lei Molu monoclonal antibody.

The use of the invention, wherein the dosage of the PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of 0.1-100mg/kg, preferably 0.5mg/kg, 1mg/kg, 2mg/kg, 2.5mg/kg, 3mg/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, 20mg/kg, most preferably 1mg/kg, 2mg/kg, 2.5mg/kg, 3mg/kg, 5mg/kg, 7.5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg; the dose of bevacizumab is selected from 0.1-100mg/kg, preferably 0.5mg/kg, 1mg/kg, 2mg/kg, 2.5mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 7.5mg/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, 7.5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg; the dose of Lei Molu monoclonal antibody is selected from the group consisting of 0.1-100mg/kg, preferably 0.5mg/kg, 1mg/kg, 2mg/kg, 2.5mg/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, 18mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, and most preferably 5mg/kg, 6mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, 15mg/kg, and 20mg/kg.

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

In the present invention, the above-mentioned PD-1 antibody or an antigen-binding fragment thereof is provided in combination with a VEGF receptor inhibitor or a VEGF ligand inhibitor as a medicament for treating tumors, wherein the VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozantinib, ponatinib, nidanib, regorafenib, lucitanib hydrochloride, necopananib, nintib, and altitinib.

The mode of administration of the combination of the invention is selected from: simultaneously, separately formulated and co-administered or separately formulated and administered sequentially.

The administration route of the PD-1 antibody or the antigen-binding fragment thereof, bevacizumab or Lei Molu monoclonal antibody is preferably parenteral, and more preferably intravenous injection, intramuscular injection or subcutaneous injection.

<xnotran> PD-1 VEGF VEGF , , PD-1 , , , , , , , , , , , , , ; </xnotran> The VEGF receptor inhibitor or VEGF ligand inhibitor is administered once a day, twice a day, three times a day, twice a week, three times a week, once a three week, once a month of february, once a month of march, once a month of june, or once a month of june, preferably once a day, once every two weeks, once every three weeks, once a month.

Remarkably, the PD-1 antibody or the antigen binding fragment thereof and bevacizumab or Lei Molu monoclonal antibody have synergistic drug effect when being used in combination.

The present invention also provides a method of reducing adverse effects caused by an anti-PD-1 antibody, comprising administering 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 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 VEGF ligand inhibitor according to the invention, wherein the VEGF receptor inhibitor is selected from the group consisting of pegaptanib sodium, vandetanib, sorafenib, axitinib, cabozantinib, ponatinib, nidanib, regoranib, sunitinib, pazopanib, puquitinib, rebastinib, lucitanib hydrochloride, necopabanib, ninetinib, and Altiratinib, and a pharmaceutically acceptable excipient, diluent or carrier.

The anti-PD-1 antibody and a VEGF receptor inhibitor or a VEGF ligand inhibitor are used in a combined manner, so that the synergistic effect can be achieved, and the anti-tumor effect is enhanced; can also reduce or eliminate adverse reactions such as capillary hemangioma and the like caused by the anti-PD-1 antibody.

In the description and claims of this application, unless otherwise indicated, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. However, for a better understanding of the present invention, the following provides definitions and explanations of some of the relevant terms. In addition, where the definitions and explanations of terms provided herein are inconsistent with the meanings commonly understood by those skilled in the art, the definitions and explanations of terms provided herein shall control.

Drawings

FIG. 1 therapeutic Effect of bevacizumab and PD-1 antibody A alone or in combination on human glioblastoma U-87MG mouse graft

FIG. 2 pharmacodynamic results of bevacizumab in combination with PD-1 antibody A in U87+ PBMC tumor model

FIG. 3 Effect of bevacizumab on the tumor weight of human glioblastoma U-87MG mice with PD-1 antibody A alone or in combination

FIG. 4 Effect of bevacizumab on the body weight of human glioblastoma U-87MG mice with PD-1 antibody A alone or in combination

Detailed Description

1. Term(s)

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j. Diol. Chem,243, p3558 (1968).

The antibody of the invention refers to immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes called immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their corresponding heavy chains being the μ, δ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, for example, igG can be divided into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.

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-derived kappa or 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 sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence-conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in number and position in accordance with known Kabat numbering convention (LCDR 1-3, HCDE2-3), or in accordance with Kabat and chothia numbering convention (HCDR 1).

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the strong antibody variable antibody reaction induced by the chimeric antibody because of carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of genes such as the human heavy and light chain variable regions can be found in the "VBase" human germline sequence database (available at the Internet www.mrccpe.com.ac.uk/VBase), and in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. In a preferred embodiment of the present invention, the CDR sequences of the mouse humanized antibody of PD-1 are selected from the group consisting of SEQ ID NO:1,2,3,4,5,6.

The "antigen-binding fragment" as used herein refers to Fab fragments, fab 'fragments, F (ab') 2 fragments, and Fv fragments sFv fragments that bind to human PD-1, which have antigen-binding activity; an antibody selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 6. The Fv fragment contains the variable regions of the antibody heavy and light chains, but no constant regions, and has the smallest antibody fragment of the entire antigen-binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. Two antibody variable regions can also be joined together with different linkers into a single polypeptide chain, known as single chain antibodies (scFv) or single chain Fv (sFv). The term "binds to PD-1" in the context of the present invention means capable of interacting with human PD-1. The term "antigen binding site" of the present invention refers to a three-dimensional spatial site that is not antigenically contiguous 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 contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, such as a composition comprising any of the binding compounds of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically relevant degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, kruskal-Wallis test (H-test), jonckhere-Terpsra test, and Wilcoxon test.

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 diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clear from the context.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

The term "synergistic pharmacodynamic effect" encompasses additive pharmacodynamic effects, pharmacodynamic enhancing effects, and pharmacodynamic sensitizing effects, and the term "synergistic pharmacodynamic effects" of the present invention includes, but is not limited to, reducing tolerance phenomena when the PD-1 antibody or antigen-binding fragment thereof, VEGF receptor inhibitor, or VEGF ligand inhibitor of the present invention is used alone, reducing the dosage when the PD-1 antibody or antigen-binding fragment thereof, VEGF receptor inhibitor, or VEGF ligand inhibitor of the present invention is used alone, reducing adverse effects when the PD-1 antibody or antigen-binding fragment thereof, VEGF receptor inhibitor, or VEGF ligand inhibitor of the present invention is used alone, and the combination of the two enhances the therapeutic effects of the VEGF receptor inhibitor or VEGF ligand inhibitor, the PD-1 antibody or antigen-binding fragment thereof of the present invention when used alone.

The term "pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and exert biological activity.

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

An exemplary test protocol for the use of the compositions of the invention in the treatment of tumors is provided below to show the advantageous activity or advantageous technical effect of the compositions of the invention. It should be understood, however, that the following experimental protocols are only illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. Those skilled in the art, having the benefit of the teachings of this specification, will be able to make appropriate modifications or alterations to the teachings of this invention without departing from the spirit or scope thereof.

Example 1 therapeutic Effect of the PD-1 antibody or antigen-binding fragment thereof of the present invention in combination with Bevacizumab against human malignant glioma U-87MG mouse subcutaneous transplantation tumor

Test article

PD-1 antibody A (the invention of the sequence SEQ ID NO:8 shows the light chain and SEQ ID NO:7 shows the heavy chain consisting of humanized PD-1 antibody, defined as PD-1 antibody A, WO2017054646A PD-1 antibody), bevacizumab (according to WO9845331 method preparation).

Test animal

NOD/SCID female mice were purchased from Kavens (batch No.: 201703849), and certification No.: SCXK (Su) 2016-0010, purchased 4-6 weeks old, weighing about 19g,5 animals/cage, 12/12 hours light/dark cycle regulation, temperature 23 + -1 deg.C constant temperature, humidity 50-60%, free food intake and water intake.

Preparation of test solution

Diluting the PD-1 antibody A to 20mg/mL by PBS under aseptic condition, and subpackaging into 10 tubes; and opening the bevacizumab, and performing aseptic subpackaging into 4 tubes. Diluting the antibody subpackaged 1 tube with PBS to 0.63mg/mL under aseptic condition, subpackaging to 2.4 mL/tube, and storing at 4 deg.C for 10 tubes, wherein 1 tube is used for each injection.

Experimental methods

1. Extraction of PBMCs

Examples PBMCs were extracted from fresh blood of 2 volunteers by the following method:

1. diluting the heparin-anticoagulated venous blood with the same volume of PBS containing 2% FBS;

2. aseptically transferring 15mL of the separated liquid 1077 into a 50mL separation tube (before gently inverting the container to mix 1077 well);

3. carefully add 25mL of diluted blood to the separation tube containing the separation fluid 1077 (room temperature, slow addition, forming a distinct layer between the blood and the separation fluid 1077, not mixing the diluted blood into the separation fluid 1077);

4. centrifuging at 1200g for 10 min at room temperature to precipitate erythrocytes and polymorphonuclear leukocytes while forming a layer of mononuclear lymphocytes on the separation liquid 1077;

5. sucking out the plasma 4-6cm above the lymphocytes;

6. the lymphocyte layer and the lower half of the separation solution 1077 are aspirated and transferred to another centrifuge tube. Adding PBS with the same volume, and centrifuging for 8 minutes at room temperature at 300 g;

7. the cells were washed with PBS or RPMI-1640 medium and resuspended in serum-containing RPMI-1640 medium.

2. CD3 antibody coating and PBMCs activation

1. CD3 antibody (40 ng/mL) diluted in PBS was added to 6-well cell culture plates at 1 mL/well and incubated at 37 ℃ for one hour;

2. prior to the addition of PBMCs, the CD3 antibody dilutions were removed and washed twice with 2mL PBS per well;

3. PBMCs (RPMI-1640 medium suspension) from 2 volunteers were added separately: about 2X 10 per hole ⁶ Cells, 2 mL/well;

4. the cells were cultured in an incubator at 37 ℃ for 4 days.

3. Methods of administration and animal treatment

100 μ L of U-87MG cells (1.5X 10) ⁶ Individual cell/mouse) was inoculated subcutaneously into the right flank of NOD/SCID mouse, and animals with too large or too small tumor volume were removed 10 days later, based on the average tumor volume of about 65mm ³ Mice were randomly divided into 4 groups: a solvent control group, a single bevacizumab group with the dosage of 3mg/kg, a single PD-1 antibody A group with the dosage of 3mg/kg, and a combined bevacizumab group with the dosage of 3mg/kg and PD-1 antibody A group with the dosage of 3 mg/kg. Each group had 9 individuals, and the day of grouping was recorded as day 0. PBMCs from two volunteers stimulated with CD3 antibody were mixed at a ratio of 1: 1 on the day of grouping (day 0) at 5X 10 ⁵ Individual cells/mouse were injected into tumor tissue of tumor-bearing mice. The remaining PBMCs were stopped from stimulation and continued to culture at 5X 10 on day 7 ⁶ The cells/mice were intraperitoneally injected into tumor-bearing mice, and the procedure was repeated on day 0 on days 11, 14, and 17. On the day of grouping (day 0), the PD-1 antibody a and/or bevacizumab, respectively, were intraperitoneally injected and/or caudal vein injected 2 times per week followed by 6 total administrations. Tumor volume, animal body weight were monitored 2 times per week and data were recorded. At the end of the experiment, the animals were euthanized, the tumors were stripped and weighed.

Data expression and statistical processing

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

Tumor volume (V) was calculated as: v =1/2 × a × b ² Wherein a and b represent length and width, respectively.

Relative tumor proliferation rate T/C (%) = (T-T) ₀ )/(C-C ₀ ) X 100, where T, C is the tumor volume at the end of experiment for treatment and control groups; t is ₀ 、C ₀ Tumor volume at the beginning of the experiment.

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

Results of the experiment

The experimental results show that: compared with a solvent control group, the combination drug combination (3 +3mg/kg, I.V., BIW x 6) of bevacizumab Shan Yaozu (3 MG/kg, I.V., BIW x 6) and bevacizumab + PD-1 antibody A can obviously inhibit the growth of subcutaneous transplanted tumor of a human malignant glioma U-87MG mouse, and the tumor inhibition rates are 66.38% and 78.71% (p is less than 0.001vs solvent control) respectively; whereas the tumor inhibition rate of the PD-1 antibody A single medicine group (3 mg/kg, I.P., BIW x 6) at 20 days is only 9.79 percent, and has no significant difference with the solvent control group. Compared with the bevacizumab single-use group, the bevacizumab + PD-1 antibody A combined group has a statistical difference in tumor inhibition effect (p is less than 0.05) at day 20, and shows a significant pharmacodynamic synergistic effect (Table 1 and FIG. 1).

The in vitro tumor weight and the tumor volume change trend are basically consistent, the tumor weight of the bevacizumab + PD-1 antibody A combined group and the bevacizumab single group is obviously lower than that of the solvent control group, and the statistical difference is realized (P is less than 0.001). Although there was no statistical difference in tumor weight between the bevacizumab + PD-1 antibody a combination group and bevacizumab alone group (p = 0.0779), the ratio of tumor weight in both groups was 67% (6/9) vs.11% (1/9) at < 0.5g, respectively, and the advantages of the combination group can also be seen (fig. 2 and fig. 3).

The tumor-bearing mice can well tolerate the bevacizumab and the PD-1 antibody A singly or jointly, the body weight stably rises in the whole administration process, only the body weight of the solvent control group and the PD-1 antibody A group slightly decreases in the last measurement, and probably the body constitution of the mice is reduced due to the fact that the later-stage human glioblastoma U-87MG grows too fast under the skin of the mice, and no obvious drug-induced symptoms such as weight loss occur (figure 4).

TABLE 1 therapeutic effects of bevacizumab and PD-1 antibody A alone or in combination on human glioblastoma U-87MG mouse transplantable tumors

D0: a first time of administration; a: actual number of packets (number of packets); BIW: twice in mother week; I.P.: and (3) intraperitoneal injection: I.V.: intravenous injection

* P < 0.001vs vehicle control group; # p < 0.05 vs. Bevacizumab 3mg/kg

Conclusion of the experiment

The bevacizumab and PD-1 antibody A combined drug (3 MG/kg, I.V. + I.P., BIW x 6) can obviously inhibit the growth of subcutaneous transplanted tumors of human malignant glioma U-87MG mice (TGI 78.71%), and the tumor inhibition effect is superior to that of a single drug group (3 MG/kg, I.P., BIW x 6) of the bevacizumab Shan Yaozu (3 MG/kg, I.V., BIW x 6), and tumor bearing mice can well tolerate the drugs.

Claims

1. Use of a PD-1 antibody or antigen-binding fragment thereof in combination with bevacizumab for the preparation of a medicament for the treatment of a tumor, wherein said PD-1 antibody or antigen-binding fragment thereof comprises:

an antibody light chain variable region comprising LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5, and LCDR3 as shown in SEQ ID NO. 6; and

an antibody heavy chain variable region comprising HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2, HCDR3 as shown in SEQ ID NO. 3;

wherein the tumor is glioblastoma.

2. The use of claim 1, wherein the PD-1 antibody or antigen-binding fragment thereof is a humanized antibody or fragment thereof.

3. The use of claim 2, 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 a variant of the sequence set forth in SEQ ID No. 8.

4. The use according to claim 3, wherein the variant has an amino acid change of A43S in the light chain variable region.

5. The use of claim 2, wherein the PD-1 antibody or antigen-binding fragment thereof, wherein the humanized antibody heavy chain sequence is the sequence set forth in SEQ ID No. 7 or a variant of the sequence set forth in SEQ ID No. 7.

6. The use of claim 5, wherein the variant has an amino acid change of G44R in the heavy chain variable region.

7. The use as claimed in claim 2 wherein the humanized PD-1 antibody or antigen-binding fragment thereof has a light chain sequence as set forth in SEQ ID NO. 8 or a variant thereof and a heavy chain sequence as set forth in SEQ ID NO. 7 or a variant of the sequence set forth in SEQ ID NO. 7.

8. The use according to claim 1, wherein the tumor is mediated by PD-1 and/or expresses PD-L1.

9. A pharmaceutical composition comprising the PD-1 antibody or antigen-binding fragment thereof and bevacizumab for the use according to any one of claims 1 to 7, together with one or more pharmaceutically acceptable excipients, diluents or carriers.