CN109963592A

CN109963592A - PD-1 antibody combines the purposes in the drug of preparation treatment tumour with VEGF ligand or vegf receptor inhibitor

Info

Publication number: CN109963592A
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: 2019-07-02
Anticipated expiration: 2038-06-04
Also published as: WO2018223923A1; TW201902514A; CN109963592B

Abstract

It discloses PD-1 antibody and combines the purposes in the drug of preparation treatment tumour with VEGF ligand or vegf receptor inhibitor.

Description

PD-1 antibody combines the purposes in the drug of preparation treatment tumour with VEGF ligand or vegf receptor inhibitor

Technical field

The invention belongs to field of medicaments, it is related to a kind of PD-1 antibody and combines the purposes in the drug of preparation treatment tumour with VEGF ligand inhibitor or vegf receptor inhibitor.

Background technique

Immunotherapy of tumors is one prolonged hot spot of therapeutic field of tumor, and wherein the immunotherapy of tumors of T cell is in its core position again.Immunotherapy of tumors is to make full use of, transfer the intracorporal killer T cell of tumor patient, carries out lethal effect to tumour, and it is also the approach of safest treatment tumour that it, which may be most effective,.At the same time, tumor escape is the huge obstacle that immunotherapy of tumors faces, and tumour cell promotes the fast-growth of tumour using its own to the inhibiting effect of immune system.There is extremely complex relationships between the immune response of tumour for the Immune escaping mechanism and body of tumour.The killer T cell of immunotherapy of tumors infantile tumour specificity has its bioactivity, but as the tumour growth later period loses the function of killing.So immunotherapy of tumors is to improve patient itself to greatest extent to the immune system response of tumour, it will not only activate original immune system response in vivo, duration and the response intensity that more maintain immune system response, are only the key of tumor immunotherapy.

Programmed death 1 (programmed death-l, PD-l) belong to CD28 family, with cytotoxic T lymphocyte epitope (cytotoxic T Iymphocyte antigen 4, CTLA-4) there is 23% amino acid identity, but its expression is different from CTLA, and main expression is in the T cell, B cell and myeloid cell of activation.There are two ligands, respectively PD-L1 and PD-L2 by PD-1.PD-L1's is mainly expressed in T cell, B cell, macrophage and Dendritic Cells (dendritic cell, DC), and the expression on cell is able to carry out up-regulation after activation.New research finds the expression that high PD-L1 albumen is detected in the mankind tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney, melanoma, and the expression of PD-L1 and the clinic of patient and prognosis are closely related.In immunologic test point, PD-1 and its ligand PD-L1 inhibit the activity of T lymphocyte, and the combination of PD-1 and PD-L1 lead to the apoptosis and exhaustion of activating immune cell.Inhibit T cell proliferation since PD-L1 plays the role of second signal access, it is combined into so blocking between PD-L1/PD-1 for the very potential emerging target spot in one, immunotherapy of tumors field, meta-analysis shows that mankind PD-L1 protein expression positive rate mean value is 44.72%, therefore the treatment of PD-L1/PD-1 related immune becomes one of the direction of glioblastoma treatment ([J] .Journal of Hematology & Oncology, 2017, 10 (1): 81), Kathy Boltz reports clinical research of the Pembrolizumab for glioblastoma multiforme, some patientss benefit as the result is shown, but still have 40% patient disease progress (Ne Uro-Oncology.2016；18:abstract ATIM-35), therefore PD-1 antibody list medicine is for the treatment of tumour that there are still many deficiencies.

Although the inhibition for the antibodies on tumor of PD-1 target spot has achieved significant curative effect, to certain patient's unsatisfactory curative effects, or even there is invalid situation, furthermore there is also and immune-related serious adverse reaction.Xia Bu et al. research discovery to the drug resistant melanoma patients of PD-1 immunization therapy show immunosupress, angiogenesis, monocyte and macrophage chemoattractant property, extracellular matrix remodeling and epithelial-mesenchymal conversion etc. gene upregulations feature, therefore the method that PD-1 immunization therapy combines above-mentioned target treatment can produce effective antitumour immune effect ([J] .Trends in molecular medicine, 2016,22 (6): 448-451).The research of S.Yasuda et al., which discloses VEGF monoclonal antibody, can combine anti-PD-1 antibody progress immunization therapy ([J] .Clinical & Experimental Immunology, 2013,172 (3): 500-506)；In addition, because raised VEGF inhibits DC mature, so as to cause immunosupress ([J] .Current treatment options in oncology, 2014,15 (1): 137-146).

Have at present and is carrying out by the clinical test of the anti-tumor drug of target spot of VEGF the PD-1/PD-L1 for grinding or listing is antibody combined, Nivolumab combines with Sunitinib or Pazopanib shows good result (ASCO meeting, (2014): 5010-5010) for treating metastatic renal cell cancer (mRCC)；The clinical effectiveness that Pembrolizumab joint Avastin is used to treat relapsing glioma is shown with therapeutic effect and well-tolerated (ASCO meeting, (2016): 2041-2041), but it is invalid that Blumenthal et al. reports both clinical studies show of the Pembrolizumab joint bevacizumab for progressivity primary brain tumors joint, the two combination therapeutic advance primary brain tumors ([J] .Journal of neuro-oncology is not recommended, 2016, 129 (3): 453-460), therefore PD-1 antibody and VEGF inhibitor combination therapy tumour still have many uncertainties, it is worth further investigation.

WO2013181452 discloses a kind of PD-1 antagonist, oxaliplatin, folinic acid, 5-FU joint or the purposes for not combining Avastin treatment tumour；WO2016100561 discloses the purposes that a kind of PD-1 antibody is used for glioma；WO2016170039, WO2016170040 disclose Avastin and the antibody combined purposes reacted for cancer and mediated immunity of PD-1/PD-L1.

Patent application WO2017054646A provide it is a kind of new there is high-affinity, highly selective, high bioactivity PD-1 antibody, it includes:

Antibody's light chain variable region, the antibody's light chain variable region include that at least one is selected from LCDR:SEQ ID NO:4, the SEQ ID NO:5 or SEQ ID NO:6 as shown in following sequence；With

Antibody heavy chain variable region, the antibody heavy chain variable region include at least one selected from HCDR:SEQ ID NO:1, the SEQ ID NO:2 or SEQ ID NO:3 as described in following sequence.In ASCO meeting in 2017, article " Phase I study of the antiPD-1 antibody SHR-1210 in patients with advanced solid tumors " discloses the experimental result of PD-1 Antybody therapy patients with solid tumor I phase clinic, it has inhibitory effect to kinds of tumors as the result is shown, but the symptom of reactive capillary hemangioma occurs in about 79.3% subject.

Avastin (Bevacizumab, ) it is on 2 26th, 2004 VEGF inhibitors in U.S.'s listing, for treating such as lung cancer, oophoroma kinds of tumors, WO9845331 discloses its sequence and preparation method, and the present invention provides PD-1 monoclonal antibody described in patent application WO2017054646A or its antigen-binding fragment is combined with vegf receptor inhibitor or VEGF ligand inhibitor in the purposes for preparing the drug for treating tumour.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of PD-1 antibody or its antigen-binding fragment to combine the purposes in the drug of preparation treatment tumour with vegf receptor inhibitor or VEGF ligand inhibitor.

Wherein, the PD-1 antibody includes:

Antibody heavy chain variable region, the antibody heavy chain variable region include at least one selected from HCDR:SEQ ID NO:1, the SEQ ID NO:2 or SEQ ID NO:3 as described in following sequence；

It is rich for Buddhist nun, Ponatinib, Nintedanib, Rui Gefeini, Sutent, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib that the vegf receptor inhibitor is selected from Macugen, Vande Thani, Sorafenib, Axitinib, card.

In a preferred embodiment of the present invention scheme, the antibody's light chain variable region includes the LCDR1 as shown in SEQ ID NO:4, the LCDR2 as shown in SEQ ID NO:5, the LCDR3 as shown in SEQ ID NO:6；The antibody heavy chain variable region includes the HCDR1 as shown in SEQ ID NO:1, the HCDR2 as shown in SEQ ID NO:2, the HCDR3 as shown in SEQ ID NO:3.

Wherein, mentioned-above each CDR sequence is as shown in the table:

Title	Sequence	Number
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 present invention scheme, PD-1 antibody of the invention or its antigen-binding fragment are humanized antibody or its segment.

In a preferred embodiment of the present invention scheme, humanized antibody light chain's sequence of PD-1 antibody of the invention or its antigen-binding fragment is the sequence as shown in SEQ ID NO:8 or its variant；The variant preferably has the amino acid of 0-10 to change in light chain variable region；The amino acid of more preferably A43S changes.

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

Particularly preferred humanized antibody light chain's sequence is the sequence as shown in SEQ ID NO:8, and sequence of heavy chain is the sequence as shown in SEQ ID NO:7.

Humanized antibody above-mentioned is heavy, the sequence of light chain is as follows:

Heavy chain

SEQ ID NO:7

Light chain

SEQ ID NO:8

Combine the purposes in the drug of preparation treatment tumour with VEGF ligand inhibitor or vegf receptor inhibitor in PD-1 antibody of the present invention or its antigen-binding fragment, wherein, the VEGF ligand inhibitor is selected from Avastin, Lei Molu monoclonal antibody, Lucentis, VEGF Trap, Compaq is western general, 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 Avastin, Lei Molu monoclonal antibody.

In above scheme, what the PD-1 antibody or its antigen-binding fragment and vegf receptor inhibitor or VEGF ligand inhibitor had a treatment tumour cooperates with drug action；Preferably, what the humanization PD-1 antibody or its antigen-binding fragment and vegf receptor inhibitor or VEGF ligand inhibitor had a treatment tumour cooperates with drug action；It is furthermore preferred that humanization PD-1 antibody or its antigen-binding fragment comprising sequence of light chain and the sequence of heavy chain as shown in SEQ ID NO:7 as shown in sequence SEQ ID NO:8 cooperate with drug action with treatment tumour with vegf receptor inhibitor or VEGF ligand inhibitor.

In the present invention, provide a kind of method for treating tumour, including applying above-mentioned PD-1 antibody or its antigen-binding fragment and vegf receptor inhibitor or VEGF ligand inhibitor to patient, wherein the vegf receptor inhibitor is selected from, Macugen, Vande Thani, Sorafenib, Axitinib, card are rich to replace Buddhist nun, Ponatinib, Nintedanib, Rui Gefeini, Sutent, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib.

Preferably, the tumour is selected from breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney, melanoma, leukaemia, lymthoma, myeloma, the cancer of the esophagus, liver cancer, cancer of bile ducts, cancer of pancreas, head-neck carcinoma, prostate cancer, oophoroma, cervix cancer, carcinoma of endometrium, osteosarcoma, soft tissue sarcoma, neuroblastoma, brain tumor, endocrine organ's tumour, bladder cancer, cutaneum carcinoma, nasopharyngeal carcinoma, rhabdomyosarcoma；It is preferred that breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney, melanoma, cancer of pancreas, cervix cancer, liver cancer, leukaemia, oophoroma, lymthoma, brain tumor, the cancer of the esophagus；Most preferably lung cancer, gastric cancer, intestinal cancer, liver cancer, the cancer of the esophagus, lymthoma, kidney, melanoma, cervix cancer, oophoroma, brain tumor.

Preferably, the lung cancer is selected from non-small cell lung cancer, Small Cell Lung Cancer, preferably non-small cell lung cancer；The intestinal cancer is selected from carcinoma of small intestine, colon and rectum carcinoma, colorectal cancer, preferably colon and rectum carcinoma, colorectal cancer；The lymthoma is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, preferably Hodgkin lymphoma.

Preferably, the brain tumor is selected from neuroepithelial tissue tumour, cranial nerve and spinal nerve tumour, meningeal tissue tumour；The neuroepithelial tissue tumour is selected from astrocytoma, human anaplastic astrocytoma, glioblastoma, Pilocytic Astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, few branch spongiocytoma, ependymocytoma, Mixed Gliomas, tumor of choroid plexus, pinealocytoma, embryonal tumors, most preferably astrocytoma, human anaplastic astrocytoma, glioblastoma.

Preferably, above-mentioned tumour mediates and/or expresses expression PD-L1 by PD-1.

Purposes of the present invention, wherein, the combination weight ratio range of the PD-1 antibody or its antigen-binding fragment and vegf receptor inhibitor or VEGF ligand inhibitor is 0.01-100, is selected from 5:1,3:1,5:2,5:3,2:1,2:3,3:2,4:3,5:4,1:1,5:6,4:5,3:4,3:5,1:2,2:5,1:3,3:10,4:15,1:4,1:5,1:6,2:9,2:15,1:10,2:25,3:8；It is preferred that 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 combination weight ratio range of above-mentioned PD-1 antibody or its antigen-binding fragment and vegf receptor inhibitor or VEGF ligand inhibitor preferably is selected from the combination weight ratio range of PD-1 antibody or its antigen-binding fragment and VEGF ligand inhibitor, more preferably the combination weight ratio range from PD-1 antibody or its antigen-binding fragment and Avastin or Lei Molu monoclonal antibody.

Purposes of the present invention, wherein, the dosage of the PD-1 antibody or its antigen-binding fragment is selected from 0.1-100mg/kg, it is preferred that 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 dosage of Avastin is selected from 0.1-100mg/kg, it is preferred that 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 dosage of Lei Molu monoclonal antibody is selected from 0.1-100mg/kg, it is preferred that 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, most preferably 5mg/kg, 6mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, 15mg/kg, 20mg/kg.

Purposes of the present invention, in another preferred embodiment, the dosage of PD-1 antibody of the present invention or its antigen-binding fragment is selected from 1-2000mg, it is preferred that 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 drug of above-mentioned PD-1 antibody or its antigen-binding fragment joint vegf receptor inhibitor or VEGF ligand inhibitor as treatment tumour is provided, Macugen, Vande Thani, Sorafenib, Axitinib, card are rich to replace Buddhist nun, Ponatinib, Nintedanib, Rui Gefeini, Sutent, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib wherein the vegf receptor inhibitor is selected from.

United administration mode of the present invention is selected from: being administered simultaneously, is independently prepared and be total to administration or independently prepare and be administered in succession.

The preferred Parenteral administration of administration route of PD-1 antibody or its antigen-binding fragment, Avastin or Lei Molu monoclonal antibody of the present invention, more preferable intravenous injection, intramuscular injection, subcutaneous injection.

The invention further relates to above-mentioned PD-1 antibody or its antigen-binding fragment to combine the purposes in preparation tumor with vegf receptor inhibitor or VEGF ligand inhibitor, wherein, the dosage rate of PD-1 antibody or its antigen-binding fragment be once a day, biweekly, three times a week, weekly, two weeks once, once in three weeks, once a month, February is primary, March is primary, June is primary, preferably two weeks once, once in three weeks, once a month, March it is primary；The dosage rate of vegf receptor inhibitor or VEGF ligand inhibitor be once a day, two times a day, three times per day, biweekly, three times a week, weekly, two weeks once, once in three weeks, once a month, February is primary, March is primary, June is primary, preferably once a day, two weeks once, once in three weeks, once a month.

Significantly, the PD-1 antibody or its antigen-binding fragment of the invention has with Avastin or Lei Molu monoclonal antibody use in conjunction cooperates with drug action.

The present invention also provides a kind of methods for reducing adverse reaction caused by anti-PD-1 antibody, including PD-1 antibody will be resisted to be used in combination with vegf receptor inhibitor or VEGF ligand inhibitor.In one embodiment, the adverse reaction is blood vessel related reactions, such as capillary hemangioma.The present invention further provides a kind of pharmaceutical compositions, it contains a effective amount of PD-1 antibody according to the present invention or its antigen-binding fragment, vegf receptor inhibitor or VEGF ligand inhibitor and pharmaceutical excipient, dilution or carrier, wherein the vegf receptor inhibitor is selected from Macugen, Vande Thani, Sorafenib, Axitinib, card is rich to replace Buddhist nun, Ponatinib, Nintedanib, Rui Gefeini, Sutent, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib.

Anti- PD-1 antibody of the invention is used in combination with vegf receptor inhibitor or VEGF ligand inhibitor, collaboration can not only be played and used, antitumous effect is enhanced；The adverse reactions such as capillary hemangioma caused by anti-PD-1 antibody can also be reduced or eliminated.

In the description and claims of this application, unless otherwise stated, Science and Technology noun used herein has the normally understood meaning of those skilled in the art institute.However, for a better understanding of the present invention, the definition and explanation of part relational language is provided below.In addition, with the definition of term provided herein and being construed to quasi- when the definition of term provided herein and explanation and the inconsistent normally understood meaning of those skilled in the art.

Detailed description of the invention

Fig. 1 Avastin and PD-1 antibody A are applied alone or are combined the curative effect to people's glioblastoma U-87MG mice-transplanted tumor

Fig. 2 Avastin and pharmacodynamic result associated with PD-1 antibody A in U87+PBMC tumor model

Fig. 3 Avastin and PD-1 antibody A are applied alone or are combined the influence to people's glioblastoma U-87MG mouse tumor weight

Fig. 4 Avastin and PD-1 antibody A are applied alone or are combined the influence to people's glioblastoma U-87MG mouse weight

Specific embodiment

One, term

In order to be easier to understand the present invention, certain technical and scientific terms are defined in detail below.Except separately explicitly defining at apparent in this document it, otherwise all other technical and scientific term used herein all has the normally understood meaning of those skilled in the art of the art.

Amino acid three-letter codes used in the present invention and single letter code such as J.biol.chem, described in 243, p3558 (1968).

Antibody of the present invention refers to immunoglobulin, is four peptide chain structures being formed by connecting by two identical heavy chains and two identical light chains by interchain disulfide bond.The amino acid of immunoglobulin heavy chain constant region forms and the difference that puts in order, therefore its antigenicity is also different.Accordingly, immunoglobulin can be divided into five classes, or be the isotype of immunoglobulin, i.e. IgM, IgD, IgG, IgA and IgE, corresponding heavy chain is respectively μ chain, δ chain γ, α chain, ε chain.Same class Ig is formed according to its hinge region amino acid and the difference of the number and location of heavy chain disulfide bond, and can be divided into different subclass, as IgG can be divided into IgG1, IgG2, IgG3, IgG4.Light chain is divided into κ chain or λ chain by the difference of constant region.Every class Ig can have κ chain or λ chain in five class Ig.

In the present invention, antibody's light chain variable region of the present invention can further include constant region of light chain, and the constant region of light chain includes κ, λ chain or its variant of source of people or source of mouse.

In the present invention, antibody heavy chain variable region of the present invention can further include heavy chain constant region, and the heavy chain constant region includes the IgG1 of source of people or source of mouse, and 2,3,4 or its variant.

Heavy chain of antibody and light chain are very big close to the sequence variation of about 110 amino acid of N-terminal, are variable region (area V)；Remaining amino acid sequence close to C-terminal is relatively stable, is constant region (area C).The variable region skeleton area (FR) relatively conservative including 3 hypervariable regions (HVR) and 4 sequences.3 hypervariable regions determine the specificity of antibody, also known as complementarity-determining region (CDR).Every light chain variable region (LCVR) and heavy chain variable region (HCVR) are by 3 CDR regions, 4 FR district's groups at the sequence being arranged successively from aminoterminal to c-terminus are as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.3 CDR regions of light chain refer to LCDR1, LCDR2 and LCDR3；3 CDR regions of heavy chain refer to HCDR1, HCDR2 and HCDR3.The cdr amino acid residue in the area LCVR and the area HCVR of the invention antibody or antigen-binding fragment meets known Kabat coding rule (LCDR1-3 in quantity and position, HCDE2-3), or meet the coding rule (HCDR1) of kabat and chothia.

Term " humanized antibody (humanized antibody) ", also referred to as CDR grafted antibody (CDR-grafted antibody), refer to the antibody variable region frame that the CDR sequence of mouse is transplanted to people, i.e., the antibody generated in different types of human germline antibody's frame sequence.Chimeric antibody can be overcome due to carrying a large amount of murine protein ingredients, thus the strong antibody variable antibody response of induction.Such frame sequence can be obtained from the public DNA database or disclosed bibliography for including germline antibody gene sequences.As people's heavy chain and the germline DNA sequence dna of light-chain variable region gene (can get) in " VBase " human germ line sequences database in internet www.mrccpe.com.ac.uk/vbase, and in Kabat, E.A. et al., 1991Sequences of Proteins of Immunological Interest is found in the 5th edition.In a preferred embodiment of the present invention, the CDR sequence of the PD-1 humanized antibody mouse be selected from SEQ ID NO:1,2,3,4,5,6.

Heretofore described " antigen-binding fragment " refers to the Fab segment with antigen-binding activity, Fab ' segment, 2 segment of F (ab '), and the Fv segment sFv segment in conjunction with people PD-1；One or more CDR regions in SEQ ID NO:1 to SEQ ID NO:6 comprising antibody of the present invention.Fv segment contains antibody heavy chain variable region and light chain variable region, but does not have constant region, and has the minimum antibody fragment of whole antigen binding sites.Generally, Fv antibody also includes the peptide linker between VH and VL structural domain, and structure needed for being capable of forming antigen binding.Two antibody variable regions can also be connected into a polypeptide chain, referred to as single-chain antibody (single chain antibody) or scFv (sFv) with different attachments.Term " in conjunction with PD-1 " of the invention, referring to can interact with people PD-1.Term " antigen binding site " of the invention refers to three-dimensional space site discontinuous on antigen, to be identified by antibody of the present invention or antigen-binding fragment.

" giving " and " processing " refers to the contact of exogenous drugs, therapeutic agent, diagnosticum or composition with animal, people, subject, cell, tissue, organ or biofluid when being applied to animal, people, experimental subjects, cell, tissue, organ or biofluid." giving " and " processing " can refer to such as treatment, pharmacokinetics, diagnosis, research and experimental method.The processing of cell includes contact and reagent contact with fluid of the reagent with cell, wherein the fluid is contacted with cell." giving " and " processing " still means that through reagent, diagnosis, combining compositions or passes through another cells in vitro and ex vivo treatment such as cell." processing " refers to treatment processing, prevention or preventive measure, research and diagnostic application when being applied to people, animal medicine or study subject.

" treatment " means to give the interior or topical therapeutic agent of patient, such as composition comprising any binding compounds of the invention, and the patient has one or more disease symptoms, and the known therapeutic agent has therapeutic effect to these symptoms.In general, therapeutic agent is given in subject or group the amount of one or more disease symptoms is effectively relieved, either by inducing this kind of symptom degeneration still to inhibit this kind of symptom development to the degree of any right measurement of clinic.The amount (also referred to as " therapeutically effective amount ") that the therapeutic agent of any disease specific symptom is effectively relieved can change according to many factors, such as morbid state, age and the weight and drug of patient generate the ability for needing curative effect in patient.It is commonly evaluated for the seriousness of the symptom or any clinical testing procedure of development situation by doctor or other professional health care personages, whether evaluable disease symptoms have been mitigated.Embodiment of the present invention (such as treatment method or product) may be invalid in terms of alleviating each target disease symptom suffered from and had to the greatest extent, but examine (H inspection), Jonckheere-Terpstra inspection and Wilcoxon to examine and determine according to any statistical test method known in the art such as Student t inspection, Chi-square Test, according to the U inspection of Mann and Whitney, Kruskal-Wallis, target disease symptom should be mitigated in the patient of statistically significant number.

" effective quantity " includes to be enough to improve or prevent to cure the symptom of word illness or the amount of illness.Effective quantity still means that the amount for being enough to allow or promoting diagnosis.It can change according to following factor for the effective quantity of particular patient or veterinary science subject: such as illness to be treated, the general health of patient, the method and approach of administration and dosage and side effect seriousness.Effective quantity can be the maximum dose or dosage regimen for avoiding significant side effect or toxic effect.

Statement " cell ", " cell line " and " cell culture " used herein is used interchangeably, and all such titles all include offspring.Therefore, word " transformant " and " transformed cells " include primary subject cell and culture as derived from it, shift number without considering.It is to be further understood that all offsprings can not be accurate identical in terms of DNA content due to mutation deliberately or unintentionally.Including having the Mutant progeny with the identical function or biological activity screened in initial transformed cells.It is clearly visible by context in the case where meaning different names.

" optional " or " optionally " mean ground described later event or environment can with but need not occur, which includes the event or environment occurs or not spot occasion.For example, " optionally include 1-3 antibody heavy chain variable region " mean particular sequence antibody heavy chain variable region can with but necessarily exist.

Term " collaboration drug action " includes drug effect summation action, enhancement effect, drug effect sensitization, " collaboration drug action " of the invention includes but is not limited to reduce that PD-1 antibody of the present invention or its antigen-binding fragment is used alone, tolerance phenomenon when vegf receptor inhibitor or VEGF ligand inhibitor, it reduces and PD-1 antibody of the present invention or its antigen-binding fragment is used alone, dosage when vegf receptor inhibitor or VEGF ligand inhibitor, it reduces and PD-1 antibody of the present invention or its antigen-binding fragment is used alone, adverse reaction when vegf receptor inhibitor or VEGF ligand inhibitor, vegf receptor inhibitor or VEGF ligand inhibitor when enhancing is used alone is used in combination in the two, PD-1 antibody of the present invention or it is anti- The curative effect of former binding fragment.

Term " pharmaceutical composition " indicates mixture and other components such as physiology/pharmaceutical carrier and excipient containing one or more compounds described herein or its physiologically/pharmaceutical salt or pro-drug and other chemical constituents.The purpose of pharmaceutical composition is the administration promoted to organism, plays bioactivity in turn conducive to the absorption of active constituent.

PD-1 antibody or its antigen-binding fragment of the present invention and vegf receptor inhibitor or VEGF ligand inhibitor composition can effectively solve Tumor Heterogeneity, play significant inhibition tumour cell effect, effectively inhibit proliferation, migration or the invasion of tumour cell.

Composition of the invention presented below is in the exemplary tests scheme for the treatment of tumour on the way, to show the favorable activity or advantageous effects of the present composition.It is understood that following testing programs are only the example to the content of present invention, rather than limiting the scope of the invention.Those skilled in the art can carry out modifications or changes appropriate to technical solution of the present invention, without departing from the spirit and scope of the invention under the introduction of this specification.

Embodiment 1, PD-1 antibody of the present invention or its antigen-binding fragment combine the curative effect to people's glioblastoma U-87MG mouse subcutaneous transplanting tumor with Avastin

Test sample

PD-1 antibody A (the humanization PD-1 antibody of the present invention being made of the light chain as shown in sequence SEQ ID NO:8 and the heavy chain as shown in SEQ ID NO:7, be defined as PD-1 antibody A, be WO2017054646A in PD-1 antibody), Avastin (according to WO9845331 the method prepare).

Experimental animal

NOD/SCID female mice is purchased from this (lot number: 201703849) of Cavan, quality certification number: SCXK (Soviet Union) 2016-0010,4-6 week old when buying, weight about 19g, the raising of 5/cage, light dark cycles are adjusted within 12/12 hour, 23 ± 1 DEG C of constant temperature of temperature, humidity 50~60%, ad lib water inlet.

Test solution is prepared

PD-1 antibody A is aseptically diluted to 20mg/mL with PBS, is packed as 10 pipes；Aseptic subpackaged after Avastin is opened is 4 pipes.It takes above-mentioned packing antibody 1 pipe to be aseptically diluted to 0.63mg/mL with PBS, and is packed as 2.4mL/ pipe, totally 10 pipe, be placed in 4 DEG C of preservations, when per injection takes 1 pipe.

Experimental method

One, PBMCs is extracted

The used PBMCs of embodiment is extracted from 2 volunteer's new bloods, and extracting method is as follows:

1, the PBS mixed diluting by the venous blood and same volume that are handled with anticoagulant heparin containing 2%FBS；

2, sterile transfer 15mL separating liquid 1077 (gently container inverted makes 1077 to be sufficiently mixed before) into 50mL separating pipe；

3, (room temperature is slowly added to, and is formed an apparent layering in blood and separating liquid 1077, dilute blood is not mixed into separating liquid 1077) is carefully added into the separating pipe containing separating liquid 1077 in 25mL dilute blood；

4,1200g is centrifuged 10 minutes at room temperature, and centrifugation can cause red blood cell and multinuclear leucocyte to precipitate, while one layer of monokaryon lymphocyte is formed in separating liquid 1077；

5, the blood plasma of 4-6cm above lymphocyte is sucked out；

6, it draws buffy coat and the separating liquid 1077 of half is transferred in an other centrifuge tube below.Isometric PBS is added, 300g is centrifuged 8 minutes at room temperature；

7, cell is cleaned with PBS or RPMI-1640 culture medium, cell is resuspended with the RPMI-1640 culture medium containing serum.

Two, CD3 antibody coating and PBMCs activation

1, it will be added in 6 porocyte culture plates with the diluted CD3 antibody (40ng/mL) of PBS, the hole 1mL/, 37 DEG C are incubated for one hour；

2, before adding PBMCs, CD3 antibody diluent is removed, the PBS that 2mL is added in every hole is washed twice；

3, it is separately added into the PBMCs (suspension of RPMI-1640 culture medium) of 2 volunteers: every hole about 2 × 10 ⁶Cell, the hole 2mL/；

4, it is placed in 37 DEG C of incubators and cultivates 4 days.

Three, medication and animal processing

By the U-87MG cell (1.5 × 10 of 100 μ L ⁶A cell/mouse) it is inoculated in that the right flank of NOD/SCID mouse is subcutaneous, the excessive or too small animal of gross tumor volume is removed after 10 days, by mean tumour volume about 65mm ³Mouse is randomly divided into 4 groups: vehicle control group, Avastin 3mg/kg are applied alone group, PD-1 antibody A 3mg/kg that group and Avastin 3mg/kg+PD-1 antibody A 3mg/kg combination group is applied alone.Every group 9, the grouping same day is denoted as the 0th day.The PBMCs of two volunteers stimulated through CD3 antibody was mixed with 1:1 ratio in the grouping same day (the 0th day), with 5 × 10 ⁵A cell/mouse is injected into the tumor tissues of tumor-bearing mice.Remaining PBMCs stops stimulating and continues to cultivate, in the 7th day with 5 × 10 ⁶A cell/mouse peritoneal is injected into mice with tumor body, is operated within the 0th day in repetition in the 11st, 14,17 day.PD-1 antibody A and/or tail vein injection Avastin were injected intraperitoneally respectively in the grouping same day (the 0th day), injects weekly 2 times, is administered 6 times altogether later.Gross tumor volume, the weight of animals are monitored 2 times a week and record data.Animal is euthanized at the end of experiment, strip tumour and weighs tumor weight.

Data representation and statistical procedures

All data are mapped and are statisticallyd analyze using 5 software of Excel and GraphPad Prism.

Gross tumor volume (V) calculation formula are as follows: V=1/2 × a × b ²Wherein a, b respectively indicate length and width.

Relative tumor proliferation rate T/C (%)=(T-T ₀)/(C-C ₀) × 100, wherein T, C are the gross tumor volume for the treatment of group and control group at the end of testing；T ₀、C ₀Gross tumor volume when starting for experiment.

Tumour inhibiting rate TGI (%)=1-T/C (%).

Experimental result

Experimental result is shown: Avastin list medicine group (3mg/kg, I.V., BIW × 6) and Avastin+PD-1 antibody A drug combination group (3+3mg/kg, I.V.+I.P., BIW × 6) growth of people's glioblastoma U-87MG mouse subcutaneous transplanting tumor can be significantly inhibited compared with vehicle control group, tumour inhibiting rate is 66.38% and 78.71% (p < 0.001vs Vehicle controls)；And the 20th day tumour inhibiting rate of PD-1 antibody A list medicine group (3mg/kg, I.P., BIW × 6) is only 9.79%, with vehicle control group without significant difference.Avastin+PD-1 antibody A combination group is compared with group is applied alone in Avastin, and tumor killing effect also has statistical difference (p < 0.05) within the 20th day, shows significant drug effect synergistic effect (table 1 and Fig. 1).

In vitro tumor weight and tumor volume change trend are almost the same, and Avastin+PD-1 antibody A combination group and Avastin are applied alone the tumor weight of group to be all significantly lower than vehicle control group, and have statistical difference (P < 0.001).Although Avastin+PD-1 antibody A combination group and Avastin are applied alone between group tumor weight without statistical difference (p=0.0779), but ratio of two groups of tumor weights in < 0.5g is respectively 67% (6/9) vs.11% (1/9), it is also seen that the advantage (Fig. 2 and Fig. 3) of combination group.

Tumor-bearing mice is applied alone or is combined to Avastin and PD-1 antibody A and can be resistant to well, weight steadily rises in entire administration process, only vehicle control group and PD-1 antibody A the group weight when last measures slightly decreases, it may be to cause mouse constitution to decline mouse subcutaneous growth is too fast due to later period people's glioblastoma U-87MG, no obvious drug causes the symptoms such as weight loss that (Fig. 4) occurs.

1. Avastin of table and PD-1 antibody A are applied alone or are combined the curative effect to people's glioblastoma U-87MG mice-transplanted tumor

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

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

Experiment conclusion

Avastin+PD-1 antibody A drug combination (3mg/kg, I.V.+I.P., BIW × 6) growth (TGI 78.71%) of people's glioblastoma U-87MG mouse subcutaneous transplanting tumor can be significantly inhibited, and tumor killing effect is better than+PD-1 antibody A list medicine group (3mg/kg, I.P., BIW × 6) and Avastin list medicine group (3mg/kg, I.V., BIW × 6), tumor-bearing mice can be resistant to the above drug very well.

Claims

A kind of PD-1 antibody or its antigen-binding fragment are combined with VEGF ligand inhibitor or vegf receptor inhibitor for the purposes in the drug of preparation treatment tumour, which is characterized in that the PD-1 antibody or its antigen-binding fragment include:

Antibody's light chain variable region, the antibody's light chain variable region include that at least one is selected from LCDR:SEQ ID NO:4, the SEQ ID NO:5 or SEQ ID NO:6 as shown in following sequence；With

Antibody heavy chain variable region, the antibody heavy chain variable region include at least one selected from HCDR:SEQ ID NO:1, the SEQ ID NO:2 or SEQ ID NO:3 as described in following sequence；

Wherein, it is rich for Buddhist nun, Ponatinib, Nintedanib, Rui Gefeini, Sutent, pazopanib, Puquitinib, Rebastinib, Lucitanib hydrochloride, Necuparanib, Ningetinib, Altiratinib to be selected from Macugen, Vande Thani, Sorafenib, Axitinib, card for the vegf receptor inhibitor.
Purposes according to claim 1, which is characterized in that the antibody's light chain variable region includes the LCDR1 as shown in SEQ ID NO:4, the LCDR2 as shown in SEQ ID NO:5, the LCDR3 as shown in SEQ ID NO:6；The antibody heavy chain variable region includes the HCDR1 as shown in SEQ ID NO:1, the HCDR2 as shown in SEQ ID NO:2, the HCDR3 as shown in SEQ ID NO:3.
Purposes as described in claim 1-2 any one, which is characterized in that the PD-1 antibody or its antigen-binding fragment is humanized antibody or its segment.
Purposes as claimed in claim 3, which is characterized in that the PD-1 antibody or its antigen-binding fragment, wherein humanized antibody light chain's sequence is the sequence as shown in SEQ ID NO:8 or its variant；The variant preferably has the amino acid of 0-10 to change in light chain variable region；The amino acid of more preferably A43S changes.
Purposes as claimed in claim 3, which is characterized in that the PD-1 antibody or its antigen-binding fragment, wherein humanised antibody heavy chain's sequence is the sequence as shown in SEQ ID NO:7 or its variant；The variant preferably has the amino acid of 0-10 to change in heavy chain variable region；The amino acid of more preferably G44R changes.
Purposes as claimed in claim 3, which is characterized in that the sequence of light chain of the humanization PD-1 antibody or its antigen-binding fragment preferably sequence as shown in SEQ ID NO:8 or its variant, the sequence of heavy chain preferably sequence as shown in SEQ ID NO:7 or its variant.
Purposes as described in claim 1, it is characterized in that, the VEGF ligand inhibitor is selected from Avastin, Lei Molu monoclonal antibody, Lucentis, VEGF Trap, western general, Abicipar pegol of Compaq, 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, it is preferred that Avastin, Lei Molu monoclonal antibody, most preferably Avastin.
Purposes as described in claim 1-7 any one, it is characterized in that, the tumour is selected from breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney, melanoma, leukaemia, lymthoma, myeloma, the cancer of the esophagus, liver cancer, cancer of bile ducts, cancer of pancreas, head-neck carcinoma, prostate cancer, oophoroma, cervix cancer, carcinoma of endometrium, osteosarcoma, soft tissue sarcoma, neuroblastoma, brain tumor, endocrine organ's tumour, bladder cancer, cutaneum carcinoma, nasopharyngeal carcinoma, rhabdomyosarcoma；It is preferred that breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney, melanoma, cancer of pancreas, cervix cancer, liver cancer, leukaemia, oophoroma, lymthoma, brain tumor, the cancer of the esophagus；Most preferably lung cancer, gastric cancer, intestinal cancer, liver cancer, the cancer of the esophagus, lymthoma, kidney, melanoma, cervix cancer, oophoroma, brain tumor.
Purposes as claimed in claim 8, which is characterized in that the lung cancer is selected from non-small cell lung cancer, Small Cell Lung Cancer, preferably non-small cell lung cancer；The intestinal cancer is selected from carcinoma of small intestine, colon and rectum carcinoma, colorectal cancer, preferably colon and rectum carcinoma, colorectal cancer；The lymthoma is selected from non-Hodgkin lymphoma, Hodgkin lymphoma, preferably Hodgkin lymphoma.
Purposes as claimed in claim 8, which is characterized in that the brain tumor is selected from neuroepithelial tissue tumour, cranial nerve and spinal nerve tumour, meningeal tissue tumour；The neuroepithelial tissue tumour is selected from astrocytoma, human anaplastic astrocytoma, glioblastoma, Pilocytic Astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, few branch spongiocytoma, ependymocytoma, Mixed Gliomas, tumor of choroid plexus, pinealocytoma, embryonal tumors, preferably astrocytoma, human anaplastic astrocytoma, glioblastoma.
Such as the described in any item purposes of claim 8-10, which is characterized in that the tumour mediates and/or express expression PD-L1 by PD-1.
A kind of pharmaceutical composition includes a effective amount of PD-1 antibody described in claim 1-7 any one or its antigen-binding fragment and VEGF ligand inhibitor or vegf receptor inhibitor and one or more pharmaceutical excipients, diluent or carrier.