CN110448691A

CN110448691A - Purposes of the anti-antibody combined IL-15 albumen composition of PD-1 in the drug of preparation treatment tumour

Info

Publication number: CN110448691A
Application number: CN201910370208.5A
Authority: CN
Inventors: 马晨; 孙星; 廖成; 张连山; 杨昌永
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Suzhou Suncadia Biopharmaceuticals Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Suzhou Suncadia Biopharmaceuticals Co Ltd
Priority date: 2018-05-07
Filing date: 2019-05-06
Publication date: 2019-11-15

Abstract

The present invention relates to purposes of the antibody combined IL-15 albumen composition of anti-PD-1 in the drug of preparation treatment tumour.Tumour of the present invention is selected from melanoma, cutaneum carcinoma, lymthoma, clear-cell carcinoma, liver cancer, gastric cancer, breast cancer, colorectal cancer, non-small cell lung cancer, glioblastoma, oophoroma, prostate cancer, leukemia, B cell tumor, Huppert's disease, B cell lymphoma, hodgkin's lymphomas, chronic lymphocytic leukemia, acute casual leukaemia, skin T cell lymphoma, t cell lymphoma, urothelium/bladder cancer, lung cancer, cancer of the esophagus, head and neck cancer, B cell non Hodgkin lymphom and squamous cell head and neck cancer.PD-1 and IL-15 combination therapy tumour can substantially reduce gross tumor volume compared to single therapy.

Description

The anti-antibody combined IL-15 albumen composition of PD-1 is in the drug of preparation treatment tumour Purposes

Technical field

Anti- PD-1 antibody or its antigen-binding fragment and IL-15 compound combine the use in the drug of preparation treatment tumour On the way.

Background technique

Interleukin 15 (IL-15) is Grabstein et al. in a kind of about cell of 12-14kD of discovery in 1994 The factor can play a role in the normal immune response of body, such as promote the increasing of T cell, B cell, natural kill (NK) cell It grows.IL-15 need to be by playing biological activity in conjunction with its receptor.IL-15 receptor is made of three receptor subunits: IL-15 by Body α (IL-15R α), IL-2 receptor β (IL-2R β) and γ c.IL-15R α includes a Sushi structural domain, can in conjunction with IL-15, It and is to play the IL-15 after combining necessary to biological function.In recent years it finds, IL-15 is formed with its receptor IL-15R α After compound, the biological activity of IL-15 can be significantly increased.

Good expection due to IL-15 in immunotherapy of tumors field, have both at home and abroad many companies or research institution from Correlative study in terms of thing IL-15 immunization therapy, such as CN100334112C (Shanghai Haixin Bio-Tech Co., Ltd) patent Related IL-15-hIgG4Fc heterodimeric protein is used for the treatment of anti-microbial infection；CN103370339B is disclosed A kind of IL-15N72D:IL-15R α Su/Fc fusion protein compound, IL-15 polypeptide are mutated comprising N72D, and the mutant is more former Beginning IL-15 display reduce to IL-15 β γ C receptor-binding activity；It is compound that WO2016095642A discloses a kind of IL-15 albumen Object is made of IL-15 polypeptide and IL-15R α/Fc, is introduced disulfide bond between IL-15 and IL-15R α, be can be improved molecule Stability and bioactivity, while can also simplify preparation process.

PD-1 inhibitor is the new class immunotherapy of tumors drug currently to attract attention, has the transnational pharmacy of more families public at present It takes charge of and is directed to the monoclonal antibody of PD-1/PD-L1 signal path in research and development, WO2017054646A discloses a kind of anti-PD-1 Dan Ke The sequence and preparation method of grand antibody, the antibody have been in third stage, and good security, the clinical study results registered are Shown its with certain antitumor action ([J] .Journal of Clinical Oncology 35 (2017): e15572-e15572)。

One of an important factor for drug combination is influence drug effect.When two or more Drug combination, both It can produce synergistic effect, also can produce antagonism.Current anti-PD-1 antibody is clinically controlled with a variety of drug combinations Treat tumour, but still it is not high there is Tumor response rate the problems such as, whether anti-PD-1 antibody and IL-15 albumen composition can be at Function is applied to clinical still worth further investigation.

Summary of the invention

The present invention provides a kind of anti-PD-1 antibody and IL-15 or its albumen composition combines the drug in preparation treatment tumour In purposes, wherein the preferred melanoma of the tumour, cutaneum carcinoma, lymthoma, clear-cell carcinoma, liver cancer, gastric cancer, breast cancer, knot The carcinoma of the rectum, non-small cell lung cancer, glioblastoma, oophoroma, prostate cancer, leukemia, B cell tumor, Huppert's disease, B Cell lymphoma, hodgkin's lymphomas, chronic lymphocytic leukemia, acute casual leukaemia, cutaneous T-cell lymph Tumor, t cell lymphoma, urothelium/bladder cancer, lung cancer, cancer of the esophagus, head and neck cancer, B cell non Hodgkin lymphom and squamous Cell head and neck cancer.

In a preferred embodiment of the present invention, wherein the anti-PD-1 antibody or its antigen-binding fragment are selected from: AMP-224、GLS-010、IBI-308、REGN-2810、PDR-001、BGB-A317、Pidilizumab、PF-06801591、 Genolimzumab、CA-170、MEDI-0680、JS-001、TSR-042、Camrelizumab、Pembrolizumab、LZM- 009, AK-103 and Nivolumab.

In a preferred embodiment of the present invention, wherein the light chain of the anti-PD-1 antibody or its antigen-binding fragment Variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively The heavy chain variable region of the PD-1 antibody includes respectively as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 HCDR1, HCDR2 and HCDR3.

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

Title	Sequence	Number
			HCDR1	SYMMS	SEQID NO:1
HCDR2	TISGGGANTYYPDSVKG	SEQID NO:2
			HCDR3	QLYYFDY	SEQID NO:3
LCDR1	LASQTIGTWLT	SEQID NO:4
			LCDR2	TATSLAD	SEQID NO:5
LCDR3	QQVYSIPWT	SEQID NO:6

Preferably, the anti-PD-1 antibody or its antigen-binding fragment are anti-PD-1 humanized antibody.

Preferably, humanized antibody light chain's variable region sequences are the sequence as shown in SEQ ID NO:10 or its variant；Institute The variant stated preferably has the amino acid of 0-10 to change in light chain variable region；The amino acid of more preferably A43S changes.The source of people Changing antibody heavy chain variable region sequence is the sequence as shown in SEQ ID NO:9 or its variant；The variant is preferably in weight chain variable Area has the amino acid of 0-10 to change；The amino acid of more preferably G44R changes.

Humanized antibody above-mentioned is heavy, the variable region sequences of light chain are as follows:

Heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS

SEQID NO:9

Light chain variable region

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

SEQID NO:10

Preferably, humanized antibody light chain's sequence is the sequence as shown in SEQ ID NO:8 or its variant；The variant is preferred There is the amino acid of 0-10 to change in light chain variable region；The amino acid of more preferably A43S changes.Humanised antibody heavy chain's sequence It is classified as the sequence as shown in SEQ ID NO:7 or its variant；The variant preferably has the amino acid of 0-10 to become in heavy chain variable region Change；The amino acid of more preferably G44R changes.

In a preferred embodiment of the present invention, humanized antibody light chain's sequence is as shown in SEQ ID NO:8 Sequence, sequence of heavy chain are 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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQID NO:7

Light chain

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQID NO:8

In a preferred embodiment of the present invention, wherein the IL-15 albumen composition is by soluble fusion protein (I) it is formed with soluble fusion protein (Π)；Wherein soluble fusion protein (I) includes IL-15 polypeptide or its functional fragment； Soluble fusion protein (Π) includes IL-15R α polypeptide or its functional fragment；Soluble fusion protein (I) or soluble fusion There are one or more amino acid sites to be mutated into Cys, with corresponding soluble fusion protein (Π) or solvable on albumen (Π) The Cys being mutated on property fusion protein (I) amino acid sites matches to form disulfide bond.

In a preferred embodiment of the present invention, wherein soluble fusion protein (Π) also includes Fc segment or it is prominent Variant；Preferably, soluble fusion protein (Π) is connected to the N-terminal group of Fc segment by IL-15R α polypeptide or its functional fragment At；It is highly preferred that the Fc segment is SEQ ID NO:11.

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

SEQ ID NO:11

In a preferred embodiment of the present invention, wherein the sequence of the soluble fusion protein (I) is SEQ ID NO:12.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIIL ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO:12

In a preferred embodiment of the present invention, wherein the amino acid Cys of the IL-15 albumen composition is mutated position Point occurs on L45, Q48, V49, L52, E53, C88 or E89 on IL-15 polypeptide or its functional fragment, preferably takes place On L52, E53 or E89, more preferable L52.

In a preferred embodiment of the present invention, wherein the mutational site the IL-15 albumen composition amino acid Cys Occur on K34, L42, A37, G38 or S40 on IL-15R α polypeptide or its functional fragment, preferably take place A37, G38 or On S40, more preferable S40.

In a preferred embodiment of the present invention, wherein the sequence of soluble fusion protein (Π) is selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, preferably SEQ ID NO:13 or SEQ ID NO:14.

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPAL VHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPS QTTAKNWELTASASHQPPGVYPQGHSDTTGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:13

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTT PSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEI SSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT

SEQ ID NO:14

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPAL VHQRGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

SEQ ID NO:15

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTT PSLKCIRDPALVHQR

SEQ ID NO:16

In a preferred embodiment of the present invention, the IL-15 albumen composition is selected from following soluble fusion egg The combination of white (I) and soluble fusion protein (Π):

In a preferred embodiment of the present invention, wherein the colorectal cancer has undergone surgery.

In a preferred embodiment of the present invention, wherein the colorectal cancer received 5 FU 5 fluorouracil, folinic acid Calcium, capecitabine, Irinotecan, oxaliplatin, bevacizumab, Cetuximab, Victibix, VEGF Trap and Rui Gefeini In one or more combined therapies.

In a preferred embodiment of the present invention, the anti-PD-1 antibody or its antigen-binding fragment dosage are selected from 1-10mg/kg preferably is selected from 1mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 10mg/kg, most preferably 3mg/kg.

In a preferred embodiment of the present invention, the anti-PD-1 antibody or its antigen-binding fragment dosage are selected from 50-600mg preferably is selected from 60mg, 100mg, 200mg, 400mg, 600mg, most preferably 200mg.

In a preferred embodiment of the present invention, wherein the IL-15 or its albumen composition dosage is selected from 1- 100ug/kg preferably is selected from 1ug/kg, 2ug/kg, 3ug/kg, 4ug/kg, 5ug/kg, 6ug/kg, 7ug/kg, 8ug/kg, 9ug/ kg、10ug/kg、11ug/kg、12ug/kg、13ug/kg、14ug/kg、15ug/kg、16ug/kg、17ug/kg、18ug/kg、 19ug/kg、20ug/kg、21ug/kg、22ug/kg、23ug/kg、24ug/kg、25ug/kg、26ug/kg、27ug/kg、28ug/ kg、29ug/kg、30ug/kg、31ug/kg、32ug/kg、33ug/kg、34ug/kg、35ug/kg、36ug/kg、37ug/kg、 38ug/kg、39ug/kg、40ug/kg、41ug/kg、42ug/kg、43ug/kg、44ug/kg、45ug/kg、46ug/kg、47ug/ kg、48ug/kg、49ug/kg、50ug/kg、55ug/kg、60ug/kg、65ug/kg、70ug/kg、75ug/kg、80ug/kg、 85ug/kg、90ug/kg、95ug/kg、100ug/kg。

In a preferred embodiment of the present invention, the anti-PD-1 antibody or its antigen-binding fragment are in a manner of injecting Administration, such as subcutaneous or intravenous injection, need that PD-1 antibody or its antigen-binding fragment will be resisted to be configured to the shape of injectable before injection Formula.The injectable forms of particularly preferred anti-PD-1 antibody or its antigen-binding fragment are injection or freeze-dried powder, it includes Anti- PD-1 antibody or its antigen-binding fragment, buffer, stabilizer, optionally also contain surfactant.Buffer can be selected from One or more of acetate, citrate, succinate and phosphate.Stabilizer can be selected from sugar or amino acid, preferably Disaccharides, such as sucrose, lactose, trehalose, maltose.Surfactant is selected from Crodaret, glycerine fatty acid Ester, polyoxyethylene sorbitan carboxylic ester, the preferably described polyoxyethylene sorbitan carboxylic ester be polysorbate 20,40, 60 or 80, most preferably polysorbate 20.The injectable forms of highly preferred anti-PD-1 antibody or its antigen-binding fragment include Anti- PD-1 antibody or its antigen-binding fragment, acetate buffer, trehalose and polysorbate 20.

A effective amount of anti-PD-1 antibody above-mentioned is applied the present invention also provides a kind of methods for the treatment of, including to patient Or its antigen-binding fragment and IL-15 or its albumen composition.

The present invention also provides a kind of medicine sleeve group or a kind of medicine package boxes, wherein containing above-mentioned a effective amount of Anti- PD-1 antibody or its antigen-binding fragment and IL-15 or its albumen composition.

The present invention also provides a kind of pharmaceutical compositions, include a effective amount of anti-PD-1 antibody above-mentioned or its antigen binding Segment and IL-15 or its albumen composition and one or more pharmaceutical excipients, diluent or carrier.

Detailed description of the invention

One, term

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

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., different types of human germline antibody The antibody generated in frame sequence.Chimeric antibody can be overcome due to carrying a large amount of murine protein ingredients, so that induction is strong Antibody variable antibody response.Such frame sequence can be from public DNA database or public affairs including germline antibody gene sequences The bibliography opened obtains.As people's heavy chain and the germline DNA sequence dna of light-chain variable region gene can be in " VBase " human germ line sequences Database (can get) 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.At the present invention one In preferred embodiment, the CDR sequence of the PD-1 humanized antibody mouse is selected from SEQ ID NO:1, and 2,3,4,5,6.

Term " 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；SEQ ID NO:1 to SEQ ID is selected from comprising antibody of the present invention One or more CDR regions in NO:6.Fv segment contains antibody heavy chain variable region and light chain variable region, but does not have constant region, and Minimum antibody fragment with whole antigen binding sites.Generally, Fv antibody also includes more between VH and VL structural domain Peptide linker, and structure needed for being capable of forming antigen binding.Two antibody variable regions can also be connected with different attachments At a polypeptide chain, referred to as single-chain antibody (single chain antibody) or scFv (sFv).Term of the invention " with PD-1 combination ", referring to can interact with people PD-1.Term " antigen binding site " of the invention refer to it is discontinuous on antigen, by The three-dimensional space site of antibody or antigen-binding fragment identification of the present invention.

" albumen composition " or " compound protein " of the present invention refer to that two different monomeric proteins are combined into Albumen." monomeric protein " (i.e. soluble fusion protein (I), soluble fusion egg of constitutive protein compound in the present invention White (Π)) it can be fusion protein or non pregnant women.

" fusion protein " of the present invention refers to, by with gene recombination method, chemical method or other proper methods The code area of two or more genes is connected, the resulting protein of expression genetic recombination produces under the control of same regulating and controlling sequence Object.In some embodiments of the present invention, soluble fusion protein (I) be IL-15 or its variant and biologically active polypeptide such as The monomeric protein that Fc segment composition or non-fusion expression obtain；Soluble fusion protein (Π) be IL-15R α or its variant with The monomeric protein that biologically active polypeptide such as Fc segment composition or non-fusion expression obtain.In fusion protein of the invention, two Or it can be by the sequence of coding peptide linker in one or several Co-factor propagations between the code area of multiple genes.Peptide linker can also be used for Construct fusion protein of the invention.

IL-15 or functional fragment of the present invention can be any IL-15 (interleukin 15) or its mutant, such as people IL-15 or non-human mammal IL-15 or nonmammalian IL-15.Exemplary non-human mammal for example pig, rabbit, monkey, orangutan, Mouse etc., nonmammalian such as chicken etc..It is preferred that being the IL-15 and its functional fragment of people.Human interleukin 15 maturation molecule is shown in data Library UniProtKB, accession number P40933,49-162aa." IL-15 functional fragment " refers to be replaced by one or more amino acid Change, increase or deletion mutation obtain the mutant having an impact to IL-15 biological function or other properties change, pass through The variation of this amino acid can increase or decrease the interaction energy between IL-15 and its receptor IL-15R α or IL-15R β Power；Or increase or decrease IL-15 biological activity such as its stimulation immune cell propagation activity；Or it is prominent by amino acid Change establishes covalent bond between IL-15 and its receptor or makes to be covalently attached more stable etc..Such as IL-15 (L52C), i.e., 52 Bright base acid L is replaced by cysteine C on position, corresponds to that some amino acid is prominent to be substituted for C and therewith shape by the site and IL-15R α At disulfide bond.

IL-15R α or functional fragment of the present invention can be the IL-15R α or its functional piece of any species Section, such as human IL-15 R α or non-human mammal IL-15R α or nonmammalian IL-15R α.Exemplary non-human mammal is such as Pig, rabbit, monkey, orangutan, mouse etc., nonmammalian such as chicken etc..It is preferred that the IL-15R α of people, the IL-15R α of more preferably people is extracellular Area part, abbreviation IL-15R α ECD are shown in database UniProtKB, accession number Q13261,31-205aa.Term " IL-15R α function Can property segment ", the preferred shortening form of IL-15R α extracellular domain segment, i.e., by one or more amino acid substitutions, insertion or Deletion mutation is resulting to have the active molecule of human interleukin 15 receptor alpha comprising sushi structural domain, such as IL-15R α-sushi+.

Term " Fc segment " refers to human immunoglobulin(HIg) chain constant region, the especially carboxylic of immunoglobulin heavy chain constant region Cardinal extremity or in which a part, nonantigenic combination activity is the position that antibody molecule and effector molecule and cell interact.Example Such as, immunoglobulin fc region may include two or more structural domains and immunoglobulin hinge of heavy chain CH1, CH2, CH3, CH4 The combination in area.According to the amino acid sequence of heavy chain constant region, immunoglobulin can be divided into different types, mainly have 5 classes to exempt from Epidemic disease globulin: IgA, IgD, IgE, IgG and IgM.Some of them can also be further separated into subclass (isotype), such as IgG-1, IgG- 2,IgG-3,IgG-4；IgA-1 and IgA-2 and different genotype.

" Fc segment " preferably includes at least one immunoglobulin hinge region and the area CH2 and CH3 of IgG.More preferably packet Include a CH2 structural domain of IgG1, a CH3 structural domain and an immunoglobulin hinge region, hinge area initial amino acid position Setting can change.

Heretofore described " joint " is a kind of administration mode, refers to and gives at least one agent within the certain time time limit The anti-PD-1 antibody of amount or the IL-15 compound of its antigen-binding fragment and at least one dosage, two of them substance are all shown Pharmacotoxicological effect.The time limit can be in a dosage period, in preferably 4 weeks, in 3 weeks, and in 2 weeks, in 1 week, or Within 24 hours.Anti- PD-1 antibody or its antigen-binding fragment and IL-15 compound can simultaneously or sequentially be given.This time limit Including such treatment, wherein giving anti-PD-1 antibody or its antigen knot by identical administration route or different way of administration Close segment and IL-15 compound.

Detailed description of the invention

The influence to mouse tumor volume is administered in Fig. 1 .IL-15 fusion protein and PD-1 alone or in combination.

The influence to mouse weight is administered in Fig. 2 .IL-15 fusion protein and PD-1 alone or in combination.

Specific embodiment

With reference to embodiments for further describing the present invention, but these embodiments are not intended to limit the scope of the invention.

1, experiment purpose: this experiment inoculates the foundation of mouse MC38 tying colon-cancer cell in people's PD-1 transgenic mice and swells Tumor model evaluates drug combination by the volume and mouse weight of tumour to antitumor action and its safety.Combination medicine Treatment is the anti-PD-1 antibody that doses are given in abdominal cavity, while being injected intravenously the IL-15 fusion protein of doses.

2, experimental material and method:

2.1 experimental animals and material:

People's PD-1 transgenic mice: it 42, is bred by Cephrim company animal center, 3 pairs of original people's PD-1 transgenosis Mouse is purchased from the Isis company of Britain, SPF grades of raisings.

Solvent: 5% glucose, PBS

Human IgG: it comes from serum (Sigma-Aldrich), product number: I4506-50MG

Anti- PD-1 antibody: SEQ ID NO:7 and SEQ ID NO:8 in its heavy, light chain sequence such as present invention, freeze-dried powder, Water for injection dissolution after, then with 5% glucose dilution, ultimate density 0.3mg/ml, lot number P1636.IL-15 fusion protein: It is provided by Jiangsu Hengrui Medicine, sequence is as described in number 3, by soluble fusion protein (I) (SEQ ID NO:12) and solvable Property fusion protein (Π) (SEQ ID NO:15) form, after water for injection dissolution, then diluted with PBS, ultimate density is 25 μ g/ Ml, lot number P1640.

MC38 cell: DMEM culture medium, 10% fetal calf serum, 37 DEG C, 5%CO₂Culture.Cell symbol of the selection for implantation Close following four standard: 1) fast-growth；2) passage number is few；3) inoculation the previous day replaces culture medium and 4) has high existence Power.

2.2 experimental methods:

MC38 cell is harvested on the day of inoculation, by cell (5x10⁵) being inoculated in 42 people PD-1 transgenic mices, (male and female are all Having) right flank is subcutaneous, reach 100mm to mouse mean tumour volume³When left and right, 32 are chosen, is randomly divided into 4 groups, every group 8 Only.It is given after grouping according to scheme and (IP) anti-PD-1 antibody is injected intraperitoneally, and intravenous injection (IV) IL-15 fusion protein.Weekly Gross tumor volume is measured twice, is weighed, data are recorded, and tumor volume change rate is shown in that Fig. 1, mouse weight change rate are shown in Fig. 2.

2.3 experimental groups and dosage regimen

Table 1: experimental group and dosage regimen

	Grouping	Dosage	Dosage regimen	Administration mode
					Group 1	It compares (hIgG and solvent)	3mg/kg	Q2D×7	IP
Group 2	PD-1	3mg/kg	Q2D×7	IP
					Group 3	IL-15 fusion protein	5 μ g/ are only	QW×3	IV
Group 4	PD-1+IL-15 fusion protein	3mg/kg+5 μ g/ is only	Q2D×7+QW×3	IP+IV

Note: it Q2D × 7: once two days, is administered 7 times；QW × 3: it weekly, is administered 3 times；

3, data representation and statistical procedures

Weight, gross tumor volume and the knurl weight of groups of animals, data mean+SD (Mean ± SEM) expression, T- Test carries out statistical analysis to data.Gross tumor volume calculation formula: volume=0.5236 × length × short × high 4, experimental result

2. gross tumor volume (mm of table³)

* p < 0.01vs. group 1；#P < 0.05vs group 3.

Conclusion: compared with the control group, PD-1 and IL-15 are administered in combination gross tumor volume inhibiting rate and significantly rise, mouse weight Change essentially identical.

Sequence table

<110>Hengrui Medicine Co., Ltd., Jiangsu Prov.

Suzhou Sheng Diya biological medicine Co., Ltd

<120>purposes of the antibody combined IL-15 albumen composition of anti-PD-1 in the drug of preparation treatment tumour

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213>source of mouse (Mus musculus)

<400> 1

Ser Tyr Met Met Ser

1 5

<210> 2

<211> 17

<212> PRT

<213>source of mouse (Mus musculus)

<400> 2

Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 7

<212> PRT

<213>source of mouse (Mus musculus)

<400> 3

Gln Leu Tyr Tyr Phe Asp Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213>source of mouse (Mus musculus)

<400> 4

Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Thr

1 5 10

<210> 5

<211> 7

<212> PRT

<213>source of mouse (Mus musculus)

<400> 5

Thr Ala Thr Ser Leu Ala Asp

1 5

<210> 6

<211> 9

<212> PRT

<213>source of mouse (Mus musculus)

<400> 6

Gln Gln Val Tyr Ser Ile Pro Trp Thr

1 5

<210> 7

<211> 443

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(443)

<223>sequence of heavy chain

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 8

<211> 214

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(214)

<223>sequence of light chain

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 116

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(116)

<223>heavy chain variable region

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 10

<211> 107

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223>light chain variable region

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 232

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 12

<211> 114

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 13

<211> 417

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val

65 70 75 80

Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly

85 90 95

Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr

100 105 110

Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro

115 120 125

Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr

130 135 140

Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser

145 150 155 160

His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Gly

165 170 175

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys

180 185 190

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

195 200 205

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

210 215 220

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

225 230 235 240

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

245 250 255

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

260 265 270

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

275 280 285

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

290 295 300

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

305 310 315 320

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

325 330 335

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

340 345 350

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

355 360 365

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

370 375 380

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

385 390 395 400

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

405 410 415

Lys

<210> 14

<211> 417

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

245 250 255

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

260 265 270

Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val

275 280 285

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

290 295 300

Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser

305 310 315 320

Thr Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro

325 330 335

Ser Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala

340 345 350

Ala Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys

355 360 365

Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His

370 375 380

Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser

385 390 395 400

Ala Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr

405 410 415

Thr

<210> 15

<211> 315

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Gly Gly Gly Gly Ser Gly Gly

65 70 75 80

Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

85 90 95

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

100 105 110

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

115 120 125

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

130 135 140

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

145 150 155 160

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

165 170 175

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

180 185 190

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

195 200 205

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

210 215 220

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

225 230 235 240

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

245 250 255

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

260 265 270

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

275 280 285

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

290 295 300

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

305 310 315

<210> 16

<211> 315

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 16

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

245 250 255

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

260 265 270

Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val

275 280 285

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

290 295 300

Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg

305 310 315

Claims

1. anti-PD-1 antibody and IL-15 or its albumen composition combine the purposes in the drug of preparation treatment tumour, wherein institute State the preferred melanoma of tumour, cutaneum carcinoma, lymthoma, clear-cell carcinoma, liver cancer, gastric cancer, breast cancer, colorectal cancer, non-small cell Lung cancer, glioblastoma, oophoroma, prostate cancer, leukemia, B cell tumor, Huppert's disease, B cell lymphoma, Huo Qi Golden lymphomas, chronic lymphocytic leukemia, acute casual leukaemia, skin T cell lymphoma, t cell lymphoma, urine Road skin/bladder cancer, lung cancer, cancer of the esophagus, head and neck cancer, B cell non Hodgkin lymphom and squamous cell head and neck cancer.

2. purposes according to claim 1, wherein the anti-PD-1 antibody or its antigen-binding fragment are selected from: AMP-224, GLS-010、IBI-308、REGN-2810、PDR-001、BGB-A317、Pidilizumab、PF-06801591、 Genolimzumab、CA-170、MEDI-0680、JS-001、TSR-042、Camrelizumab、Pembrolizumab、LZM- 009, AK-103 and Nivolumab.

3. purposes according to claim 1, wherein the light chain variable region of the anti-PD-1 antibody or its antigen-binding fragment Comprising as described in LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively The heavy chain variable region of PD-1 antibody includes respectively as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 HCDR1, HCDR2 and HCDR3.

4. purposes according to claim 3, wherein the anti-PD-1 antibody is humanized antibody.

5. purposes according to claim 4, wherein the light-chain variable sequence of the humanized antibody is such as SEQ ID Sequence shown in NO:10 or its variant, the variant preferably have the amino acid of 0-10 to change in light chain variable region, more preferably The amino acid of A43S changes；Weight chain variabl area sequence is the sequence as shown in SEQ ID NO:9 or its variant, and the variant is preferred There is the amino acid of 0-10 to change in heavy chain variable region, the amino acid variation of more preferably G44R.

6. purposes according to claim 5, wherein humanized antibody light chain's sequence is as shown in SEQ ID NO:8 Sequence or its variant, the variant preferably have the amino acid of 0-10 to change in light chain variable region, more preferably the amino of A43S Acid variation；Sequence of heavy chain is the sequence as shown in SEQ ID NO:7 or its variant, and the variant preferably has 0- in heavy chain variable region 10 amino acid variation；The amino acid of more preferably G44R changes.

7. purposes according to claim 6, wherein humanized antibody light chain's sequence is as shown in SEQ ID NO:8 Sequence, sequence of heavy chain be the sequence as shown in SEQ ID NO:7.

8. purposes according to claim 1, wherein the IL-15 albumen composition is by soluble fusion protein (I) and can Dissolubility fusion protein (Π) composition；Wherein soluble fusion protein (I) includes IL-15 polypeptide or its functional fragment；It is soluble Fusion protein (Π) includes IL-15R α polypeptide or its functional fragment；Soluble fusion protein (I) or soluble fusion protein There are one or more amino acid sites to be mutated into Cys on (Π), melt with corresponding soluble fusion protein (Π) or solubility The Cys being mutated on hop protein (I) amino acid sites matches to form disulfide bond.

9. purposes according to claim 8, wherein soluble fusion protein (Π) also includes Fc segment or its mutant；It is excellent Selection of land, soluble fusion protein (Π) are made of the N-terminal that IL-15R α polypeptide or its functional fragment are connected to Fc segment；It is more excellent Selection of land, the Fc segment are SEQ ID NO:11.

10. purposes according to claim 8, wherein the sequence of the soluble fusion protein (I) is SEQ ID NO: 12。

11. according to the described in any item purposes of claim 8 to 10, wherein the amino acid Cys of the IL-15 albumen composition is prominent Displacement point occurs on L45, Q48, V49, L52, E53, C88 or E89 on IL-15 polypeptide or its functional fragment, preferably sends out Life is on L52, E53 or E89, more preferable L52.

12. according to the described in any item purposes of claim 8 to 10, wherein the IL-15 albumen composition amino acid Cys is mutated Site occur on K34, L42, A37, G38 or S40 on IL-15R α polypeptide or its functional fragment, preferably take place A37, On G38 or S40, more preferable S40.

13. wherein the sequence of soluble fusion protein (Π) is selected from SEQ according to the described in any item purposes of claim 8 to 12 ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, preferably SEQ ID NO:13 or SEQ ID NO: 14。

14. purposes according to claim 8, the IL-15 albumen composition be selected from following soluble fusion protein (I) and The combination of soluble fusion protein (Π):

15. according to claim 1 to 14 described in any item purposes, wherein the colorectal cancer has undergone surgery.

16. according to claim 1 to 14 described in any item purposes, wherein the colorectal cancer received 5 FU 5 fluorouracil, Asia Calcium Folinate-SF, capecitabine, Irinotecan, oxaliplatin, bevacizumab, Cetuximab, Victibix, VEGF Trap and Rui Ge One or more combined therapies in luxuriant and rich with fragrance Buddhist nun.

17. purposes according to any one of claims 1 to 7, wherein the anti-PD-1 antibody or its antigen-binding fragment Dosage is selected from 1-10mg/kg, preferably is selected from 1mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 10mg/kg, most preferably 3mg/ kg。

18. purposes according to any one of claims 1 to 7, wherein the anti-PD-1 antibody or its antigen-binding fragment Dosage is selected from 50-600mg, preferably is selected from 60mg, 100mg, 200mg, 400mg, 600mg, most preferably 200mg.

19. according to claim 1,8 to 14 described in any item purposes, wherein the IL-15 or its albumen composition dosage Selected from 1-100ug/kg, preferably be selected from 1ug/kg, 2ug/kg, 3ug/kg, 4ug/kg, 5ug/kg, 6ug/kg, 7ug/kg, 8ug/kg, 9ug/kg、10ug/kg、11ug/kg、12ug/kg、13ug/kg、14ug/kg、15ug/kg、16ug/kg、17ug/kg、18ug/ kg、19ug/kg、20ug/kg、21ug/kg、22ug/kg、23ug/kg、24ug/kg、25ug/kg、26ug/kg、27ug/kg、 28ug/kg、29ug/kg、30ug/kg、31ug/kg、32ug/kg、33ug/kg、34ug/kg、35ug/kg、36ug/kg、37ug/ kg、38ug/kg、39ug/kg、40ug/kg、41ug/kg、42ug/kg、43ug/kg、44ug/kg、45ug/kg、46ug/kg、 47ug/kg、48ug/kg、49ug/kg、50ug/kg、55ug/kg、60ug/kg、65ug/kg、70ug/kg、75ug/kg、80ug/ kg、85ug/kg、90ug/kg、95ug/kg、100ug/kg。

20. a kind of medicine package box, include a effective amount of anti-PD-1 antibody described in claim 1-19 any one or its Antigen-binding fragment and IL-15 or its albumen composition.

21. a kind of pharmaceutical composition, include a effective amount of anti-PD-1 antibody described in claim 1-19 any one or its Antigen-binding fragment and IL-15 or its albumen composition and one or more pharmaceutical excipients, diluent or carrier.