CN110448691A - Purposes of the anti-antibody combined IL-15 albumen composition of PD-1 in the drug of preparation treatment tumour - Google Patents
Purposes of the anti-antibody combined IL-15 albumen composition of PD-1 in the drug of preparation treatment tumour Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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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
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 (21)
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.
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WO2022192259A1 (en) * | 2021-03-09 | 2022-09-15 | Cureimmune Therapeutics Inc. | BIFUNCTIONAL HOMODIMERIC ANTI-PD-1 AND IL-15/IL-15Rα FUSION PROTEINS AND USES THEREOF |
WO2023138573A1 (en) * | 2022-01-21 | 2023-07-27 | 广东菲鹏制药股份有限公司 | Complex of interleukin 21 and receptor thereof |
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CN106459219A (en) * | 2014-12-19 | 2017-02-22 | 江苏恒瑞医药股份有限公司 | Interleukin 15 protein complex and use thereof |
WO2017054646A1 (en) * | 2015-09-28 | 2017-04-06 | 江苏恒瑞医药股份有限公司 | Stable anti-pd-1 antibody pharmaceutical preparation and application thereof in medicine |
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CN106459219A (en) * | 2014-12-19 | 2017-02-22 | 江苏恒瑞医药股份有限公司 | Interleukin 15 protein complex and use thereof |
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WO2022192259A1 (en) * | 2021-03-09 | 2022-09-15 | Cureimmune Therapeutics Inc. | BIFUNCTIONAL HOMODIMERIC ANTI-PD-1 AND IL-15/IL-15Rα FUSION PROTEINS AND USES THEREOF |
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