CN108440673A - Fc fusion proteins PD1/FGFR1 and its application - Google Patents
Fc fusion proteins PD1/FGFR1 and its application Download PDFInfo
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Abstract
The present invention relates to antitumor drug technical field, provides a kind of encoding gene by PD1, FGFR1 and mouse source antibody Fc section and encode to obtain Fc fusion proteins PD1/FGFR1.Fc fusion proteins PD1/FGFR1 of the present invention has significant tumor vascular targeting effect, so that enrichment of the active principle in tumor tissues and/or tumor neogenetic blood vessels middle and high concentration, fusion protein of the present invention contains PD1 albumen, it is combined with the PDL1 in tumor microenvironment, by competitive binding come reduce T cell surface PD1 and tumor cell surface ligand PDL1, PDL2 combination by block PD1 signal paths sustained activation, immunosuppressive action is removed to give full play to fusion protein, efficient immunotherapy of tumors effect is provided.
Description
Technical field
The present invention relates to field of antineoplastic medicaments, and in particular to a kind of Fc fusion proteins PD1/ of coding specificity antineoplastic
FGFR1 and its nucleotide sequence, recombinant expression carrier, expression cell and application further relate to a kind of include Fc fusion proteins PD1/
The antitumor drug of FGFR1.
Background technology
Apoptosis albumen 1 (Programmed Cell DeathProtein 1, PD1), is a kind of immune ball
Superfamily protein I type transmembrane glycoproteins, belong to T cell co-suppression receptor.Express to its being induced property T cell in activation, B
Cell, NK cells, monocyte and surface of dendritic cells, (it is micro- that the ligand is mainly expressed in tumour with its ligand PDL1, PDL2
In environment, it is prolonged exposure to antigen and mediate T cell inhibiting effect) it is combined and inhibition is generated to the activation of lymphocyte
Effect, to inhibit the immune response of immunocyte in tumor microenvironment.
Recently studies have shown that the immunization therapy for PD1 is effective to kinds of tumors, by blocking PD1 and PDL1/2 signals
Access can effectively remove the immunosuppressive action of tumor microenvironment, significantly improve T cell antineoplastic immune activity, this be because
After once tumour cell by PD1 antibodies attacks, immunocyte can each show his special prowess and is defeated in detail according to battle plan swollen
Oncocyte.For the immunization therapy of PD1/PDL1, PDL2 access, patient even invalid to other treatment still induces very
Lasting immune response is most successful Immunotherapy Strategy so far in all types tumour.
Currently, the immunization therapy somewhat expensive of PD1, inefficient for the immunization therapy and generally existing of oncotherapy
Problem, thus exist for clinical further genralrlization and limit.
Invention content
In order to further increase the antitumor action of PD1, the present invention provides a kind of specificity antineoplastic Fc fusion proteins
PD1/FGFR1, the fusion protein have tumour cell and tumor neogenetic blood vessels selectively targeted, obviously tumour can be inhibited to give birth to
It is long.
The present invention also aims to provide a kind of specificity antineoplastic drug including Fc fusion proteins PD1/FGFR1.
In order to solve the above technical problem, the present invention provides following technical schemes：
The present invention provides a kind of Fc fusion proteins PD1/FGFR1, by the encoding gene of PD1, FGFR1 and mouse source antibody section
Coding obtains.
Preferably, the sequence of described PD1, FGFR1 and mouse source antibody Fc section encoding gene is followed successively by by 5 ' ends to 3 ' ends
PD1-FGFR1-Fc。
Preferably, the encoding gene of the PD1 is selected from SEQ ID NO:Nucleotide sequence shown in 2 or with SEQ ID NO:2
Shown nucleotide sequence has the nucleotide sequence of 99% or more homology；
The encoding gene of the FGFR1 is selected from SEQ ID NO:Nucleotide sequence shown in 3 or with SEQ ID NO:Core shown in 3
Nucleotide sequence has the nucleotide sequence of 99% homology；
The encoding gene of shown mouse source antibody Fc section is selected from SEQ ID NO:Nucleotide sequence shown in 4 or with SEQ ID NO:
Nucleotide sequence shown in 4 has the nucleotide sequence of 99% or more homology.
Preferably, the nucleotide sequence such as SEQ ID NO of the coding Fc fusion proteins PD1/FGFR1:Shown in 1.
Preferably, the amino acid sequence of the fusion protein is selected from SEQ ID NO:Amino acid sequence shown in 7 or and SEQ
ID NO:Amino acid sequence shown in 7 has the amino acid sequence of 99% homology.
The present invention provides a kind of nucleic acid molecules, for Fc fusion proteins PD1/FGFR1 described in coding above-mentioned technical proposal
Nucleotide sequence.
The present invention provides a kind of recombinant expression carriers, including the nucleic acid molecules described in above-mentioned technical proposal.
The present invention provides a kind of expression cells, contain the recombinant expression carrier described in above-mentioned technical proposal.
Fc fusion proteins PD1/FGFR1, nucleic acid molecules, recombinant expression carrier or table described in preceding solution of the present invention
Up to application of the cell in preparing specificity antineoplastic drug.
The present invention also provides a kind of specificity antineoplastic drugs, including the Fc fusion proteins described in preceding solution
PD1/FGFR1 and pharmaceutically acceptable auxiliary material.
Compared with prior art, technical solution provided by the invention has the advantage that：
The present invention provides a kind of encoding genes by PD1, FGFR1 and mouse source antibody Fc section to encode to obtain Fc fusion proteins
PD1/FGFR1.Fc fusion proteins PD1/FGFR1 of the present invention has significant tumor vascular targeting effect so that working substance
Matter contains in the enrichment of tumor tissues and/or tumor neogenetic blood vessels middle and high concentration in Fc fusion proteins PD1/FGFR1 of the present invention
Some PD1 albumen, is combined with the PDL1 in tumor microenvironment, and the PD1 and tumour on T cell surface are reduced by competitive binding
The combination of cell surface ligand PDL1, PDL2 and then the sustained activation for blocking PD1 signal paths are immunized to give full play to remove
Inhibiting effect provides efficient immunotherapy of tumors effect.
Meanwhile Fc fusion proteins PD1/FGFR1 of the present invention includes mouse source antibody Fc section, mouse source antibody Fc section is not
The Half-life in vivo that fusion protein can only be extended also has enhancement antigen presenting cells to handle to extend action time
Antigen offers ability, and mouse source antibody Fc section is combined with FGFR1/PD1 can significantly increase HLA-II antigen, and then increases this
Invent killing abilities of the Fc fusion proteins PD1/FGFR1 to tumour cell.Fc fusion proteins PD1/FGFR1 of the present invention
Energy is offered in the selectively targeted effect of the ingenious tumor vessel by FGFR1, mouse source Fc elongated segments Half-life in vivo and enhancement antigen
The effect of power so that PD1 can more accurately target tumor tissue induces more strength and lasting antitumor work in turn
With.Studies have shown that Fc fusion proteins PD1/FGFR1 of the present invention has higher tumor vascular targeting characteristic, and can show
It writes and inhibits the growth of the tumours such as breast cancer, reduces gross tumor volume, tumour inhibiting rate can reach 70% or more.
Description of the drawings
Fig. 1 is the recombinant expression carrier structural schematic diagram of Fc fusion proteins PD1/FGFR1 of the present invention；
Fig. 2 is the electrophoretogram of 1 step 3) pcr amplification product of embodiment,
Wherein, M is DNA standard molecular weights, and 1~3 is the amplified production obtained in step 3)；
Fig. 3 is the electrophoretogram of 1 step 7) PCR identifications of embodiment,
Wherein, M is DNA standard molecular weights, and 1~5 is the transformed bacteria amplified production selected at random；
Fig. 4 is the electrophoretogram of Fc fusion proteins PD1/FGFR1 electrophoretic separation identification in embodiment 2,
Wherein, M is protein standard molecular weight, and swimming lane 1 is the denaturing electrophoretic figure of 1 μ g Fc fusion proteins PD1/FGFR1, swimming
Road 2 is the non-denatured electrophoretogram of 1 μ g Fc fusion proteins PD1/FGFR1；
Fig. 5 is immunofluorescence dyeing figure in embodiment 3,
Wherein, A is the tumor tissues immunofluorescence dyeing figure of test group mouse, and B is that the tumor tissues of control group mice are glimmering
Light immunostaining figure；
Fig. 6 is the administration time of each group mouse and the curve graph of gross tumor volume in embodiment 4；
Fig. 7 is influence block diagram of the fusion protein drug to mouse breast cancer 4T1 tumour knurl weights；
* indicates that there are pole significant differences relative to saline control group.
Specific implementation mode
The present invention provides a kind of Fc that the encoding gene by PD1, FGFR1 and mouse source antibody Fc section encodes to merge egg
White PD1/FGFR1.The molecular weight of the Fc fusion proteins PD1/FGFR1 in 83KDa, have tumor vascular targeting specificity and
Significant antitumor action.
In the present invention, in the nucleotide sequence of the coding Fc fusion proteins PD1/FGFR1, the sequence of encoding gene is excellent
It is selected as：It is followed successively by PD1-FGFR1-Fc from 5 ' ends to 3 ' ends.
In the present invention, the encoding gene of the PD1 is any nucleotide sequence that can encode PD1 albumen, preferably
Selected from SEQ ID NO:Nucleotide sequence shown in 2 or with SEQ ID NO:Nucleotide sequence shown in 2 has 99% or more homology
Nucleotide sequence, the encoding gene of PD1 is more preferably SEQ ID NO in certain specific embodiments of the invention:Shown in 2
Nucleotide sequence.Described and SEQ ID NO:2 nucleotide sequences have the nucleotide sequence of 99% or more homology can
To be obtained from PD1 coded sequences known in the art, details are not described herein.
The SEQ ID NO:Nucleotide sequence shown in 2 is as follows：
GAGGTCCCCAATGGGCCCTGGAGGTCCCTCACCTTCTACCCAGCCTGGCTCACAGTGTCAGAGGGAGCAAATGCCAC
CTTCACCTGCAGCTTGTCCAACTGGTCGGAGGATCTTATGCTGAACTGGAACCGCCTGAGTCCCAGCAACCAGACTG
AAAAACAGGCCGCCTTCTGTAATGGTTTGAGCCAACCCGTCCAGGATGCCCGCTTCCAGATCATACAGCTGCCCAAC
AGGCATGACTTCCACATGAACATCCTTGACACACGGCGCAATGACAGTGGCATCTACCTCTGTGGGGCCATCTCCCT
GCACCCCAAGGCAAAAATCGAGGAGAGCCCTGGAGCAGAGCTCGTGGTAACAGAGAGAATCCTGGAGACCTCAACAA
GATATCCCAGCCCCTCGCCCAAACCAGAAGGCCGGTTTCAAGGCATG。
Apoptosis albumen 1 (PD1) belongs to T cell co-suppression receptor, be combined with ligand PDL1, PDL2 and
Inhibiting effect is generated to the activation of lymphocyte.The ligand PDL1 and PDL2 of the high expression PD1 albumen of meeting, tumour are thin in tumour cell
The PDL1 and PDL2 that born of the same parents generate are capable of being combined with PD1 in microenvironment for specificity so that PD1 access sustained activations, and then T cell
Function is suppressed, and can not pass through immunization normal killer tumour cell.Nucleotide sequence coded fusion egg of the present invention
In vain contain PD1 albumen, is combined with PDL1 in tumor microenvironment or PDL2, by competitive binding come reduce T cell surface PD1 and
The combination of tumor cell surface ligand PDL1 and PDL2, and then the sustained activation of PD1 signal paths is blocked, reach the immune suppression of removing
The effect of system activates normal body immune system killing tumor cell.
In the present invention, the encoding gene of the FGFR1 is any nucleotide sequence that can encode FGFR1 albumen, excellent
Choosing is selected from SEQ ID NO:Nucleotide sequence shown in 3 or with SEQ ID NO:Nucleotide sequence shown in 3 is same with 99% or more
The nucleotide sequence of source property.In some embodiments of the invention, the encoding gene of FGFR1 is more preferably SEQ ID NO:3 institutes
The nucleotide sequence shown.Described and SEQ ID NO:3 nucleotide sequences have the nucleotide sequence of 99% or more homology
It can be obtained from FGFR1 coded sequences known in the art, details are not described herein.
The SEQ ID NO:Nucleotide sequence shown in 3 is as follows：
GGTGGAGGTTCGGGTGGAGGTTCAGGTGGAGGTTCTAGGCCAGCCCCAACCTTGCCTGAACAAGCTCAGCCCTGGGG
AGTCCCTGTGGAAGTGGAGTCTCTCCTGGTCCACCCTGGCGACCTGCTACAGCTTCGCTGTCGGCTTCGCGATGATG
TGCAGAGCATCAACTGGCTGCGGGATGGGGTGCAGCTGGTGGAGAGCAACCGTACCCGCATCACAGGGGAGGAGGTG
GAGGTGCGGGACTCCATCCCCGCTGACTCTGGCCTCTACGCTTGCGTGACCAGCAGCCCCTCTGGCAGCGATACCAC
CTACTTCTCCGTCAATGTCTCAGATGCACTCCCATCCTCGGAAGATGATGACGACGACGATGACTCCTCCTCGGAGG
AGAAAGAGACGGACAACACCAAACCAAACCGTAGGCCTGTAGCTCCCTACTGGACATCCCCAGAGAAAATGGAGAAG
AAACTGCATGCGGTGCCCGCTGCCAAGACGGTGAAGTTCAAGTGCCCGTCGAGTGGGACACCCAACCCCACTCTGCG
CTGGTTGAAAAATGGCAAAGAGTTTAAGCCTGACCACCGAATTGGAGGCTACAAGGTTCGCTATGCCACCTGGAGCA
TCATAATGGATTCTGTGGTGCCTTCTGACAAGGGCAACTACACCTGCATCGTGGAGAATGAGTATGGGAGCATCAAC
CACACCTACCAGCTTGACGTC
GTGGAACGATCTCCGCACCGACCCATCCTTCAGGCAGGGCTGCCTGCCAACAAGACAGTGGCCCTGGGCAGCAATGT
GGAGTTCATGTGTAAGGTGTACAGCGATCCGCAGCCTCACATTCAGTGGCTGAAGCACATCGAGGTGAACGGGAGTA
AGATCGGGCCAGACAACTTGCCGTATGTCCAGATCCTGAAGACTGCTGGAGTTAATACCACCGACAAGGAAATGGAG
GTGCTTCATCTACGGAATGTCTCCTTTGAGGATGCGGGGGAGTATACGTGCTTGGCGGGTAACTCTATCGGACTCTC
CCATCACTCTGCATGGTTGACCGTTCTGGAAGCCCTGGAAGAGAGACCAGCTGTGATGACCTCACCGCTCTACCTGG
A。
Basic fibroblast growth factor receptor 1 (FGFR1) be by with high-affinity part bFGF in conjunction with by play
Biological activity promotes tumor angiogenesis, and the present invention is based on following reason selection FGFR1 to form fusion protein：(1) special
Anisotropic and natural targeting, FGFR1 high efficient expressions in tumor neovasculature endothelial cell, and in normal cell and blood vessel
Expression is had no in endothelial cell, it is extremely strong to the specificity of tumour cell；(2) genetic stability, vascular endothelial cell heredity are steady
Fixed, mutation rate is low, therefore will not or can only cause very little drug resistance for the therapeutic strategy of tumor vascular endothelial cell.Institute
With, select FGFR1 and PD1, mouse source antibody Fc section composition Fc fusion proteins PD1/FGFR1 to be capable of providing accurately targeting,
So that anti-tumor active ingredient concentrates on tumour cell and/or tumor neogenetic blood vessels to give full play to antitumor efficacy.
Fc fusion proteins PD1/FGFR1 of the present invention contains FGFR1 albumen, into after in vivo can specifically with it is swollen
The bFGF of high expression is combined in oncocyte, is realized the selectively targeted tumour cells of Fc fusion proteins PD1/FGFR1 and/or tumour
The purpose of new vessels.Meanwhile FGFR1 and the ligand in tumour cell in Fc fusion proteins PD1/FGFR1 of the present invention
BFGF can also be interfered after combining bFGF/FGFR1 signal paths and then inhibits tumor angiogenesis, reach inhibit tumour after
Continuous growth.
In the present invention, the encoding gene of mouse source antibody Fc section can encode to obtain mouse source antibody Fc section to be any
Nucleotide sequence is preferably selected from SEQ ID NO:Nucleotide sequence shown in 4 or with SEQ ID NO:Nucleotide sequence shown in 4 has
There is the nucleotide sequence of 99% or more homology.The encoding gene of mouse source antibody Fc section is preferred in some embodiments of the invention
For SEQ ID NO:Nucleotide sequence shown in 4.Described and SEQ ID NO:4 nucleotide sequences are homologous with 99% or more
The nucleotide sequence of property can be obtained from the coded sequence of mouse source known in the art antibody Fc section, and details are not described herein.
The SEQ ID NO:Nucleotide sequence shown in 4 is as follows：
CAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCA
TCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGC
GAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAG
AGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGT
TCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGA
GCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCAC
AGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAAC
CAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAAT
AGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAA
ATGA。
Fc fusion proteins are fusion proteins caused by fusion protein molecule is merged with antibody Fc section, are not only able to retain former
The all biological activity of functional protein can also extend Half-life in vivo, and replace antibody function completely and immunogenicity pole
It is low.The present invention is using the antibody Fc section in mouse source other than above-mentioned advantage, additionally it is possible to which enhancement antigen presenting cells carry target antigen
In ability.Fc fusion proteins PD1/FGFR1 of the present invention by PD1 in vivo with the PD1 Ligand Competitions of tumor cell surface
Property combine, remove immunosuppressive purpose to block PD1 accesses to realize.The effect of ability is offered in the antibody Fc section enhancing of mouse source
Immunity of organism lethal effect can be then further enhanced, collaboration PD1 enhances antitumous effect, so that Fc of the present invention melts
Hop protein PD1/FGFR1 has significant tumor cytotoxicity effect.
The present invention will have cancer target (FGFR1), Antineoplastic angiogenesis (FGFR1), remove immunosuppressive action
(PD1) and enhancement antigen offer act on (mouse source antibody Fc section) combine structure nucleotide sequence, to coding obtain
Each section is mutual in a kind of Fc fusion proteins PD1/FGFR1, the Fc fusion protein PD1/FGFR1 with specificity antineoplastic effect
Promote, to cooperate with performance antitumor action, achievees the effect that inhibit tumour growth, extends cancer patient's life span.
Existing research mainly blocks PD1/PDL1, PDL2 access by the way of being directly injected in vivo PD1 antibody, however
On the one hand this mode can have partial loss after PD1 antibody enters body；Another aspect PD1 antibody enters also difficult after body
It is caused with through tumor tissues, distribution is difficult to effectively control, to also be difficult to form effective therapeutic agent in tumor tissues
Amount.Fusion protein provided by the invention can preferably carry out administration in local tumor using Fc fusion proteins PD1/FGFR1, directly
At tumour cell site targeting obtain high concentration PD1, solve routine PD1 antibody be directly administered existing for loss of activity and
It is difficult to the problem of tumor tissues part obtains effective concentration.
In the present invention, the amino acid sequence of the Fc fusion proteins PD1/FGFR1 is selected from SEQ ID NO:Amino shown in 7
Acid sequence or with SEQ ID NO:Amino acid sequence shown in 7 has the amino acid sequence of 99% homology.
The SEQ ID NO:Amino acid sequence shown in 7 is as follows：
EVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDAR
FQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMGG
GSGGGSGGGSRPAPTLPEQAQPWGVPVEVESLLVHPGDLLQLRCRLRDDVQSINWLRDGVQLVESNRTRITGEEVEV
RDSIPADSGLYACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNRRPVAPYWTSPEKMEKKL
HAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHT
YQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTT
DKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLYLEDPRGPTIKPCPPCKCPAPNL
LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ
DWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE
LNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG
The present invention provides the nucleic acid molecules of Fc fusion proteins PD1/FGFR1 described in coding above-mentioned technical proposal a kind of.Institute
The nucleotide sequence of coding Fc fusion proteins PD1/FGFR1 is stated preferably such as SEQ ID NO:Shown in 1.
The present invention provides a kind of recombinant expression carriers containing nucleic acid molecules described in preceding solution, for expressing Fc
Fusion protein PD1/FGFR1.Recombinant expression carrier of the present invention is preferably mammalian expression vector, in mammals table
Up to the bioactivity for being conducive to keep Fc fusion proteins PD1/FGFR1.The collection of illustrative plates of recombinant expression carrier of the present invention is preferred
As shown in Figure 1.
The present invention provides a kind of expression cells containing recombinant expression carrier described in above-mentioned technical proposal, for expressing Fc
Fusion protein PD1/FGFR1.It is currently preferred using HEK293 cells as the expression cell of the recombinant expression carrier.
In the present invention, nucleotide sequence, the Fc fusion proteins PD1/ of the coding Fc fusion proteins PD1/FGFR1
FGFR1, recombinant expression carrier prepare and structure is all made of method commonly used in the art, and the present invention is not particularly limited.
The present invention also provides applications of the Fc fusion proteins PD1/FGFR1 in preparing specificity antineoplastic drug.
The present invention also provides the nucleotides sequence of the coding Fc fusion proteins PD1/FGFR1, to be listed in preparation specificity anti-swollen
Application in tumor medicine.
The present invention also provides the recombination tables of the nucleotide sequence containing the coding Fc fusion proteins PD1/FGFR1
Up to application of the carrier in preparing specificity antineoplastic drug.
The present invention also provides the expression cells of the recombinant expression carrier containing Fc fusion proteins PD1/FGFR1 to make
Application in standby specificity antineoplastic drug.
Currently preferred, tumour described in above application includes but not limited to breast cancer, malignant mela noma, non-small thin
Born of the same parents' lung cancer, liver cancer and clear-cell carcinoma etc..
The present invention also provides a kind of specificity antineoplastic drugs, including the Fc fusion proteins PD1/FGFR1 and pharmacy
Upper acceptable auxiliary material.Preferably, in the specificity antineoplastic drug, Fc fusion protein PD1/FGFR1 contents be 1~
99%, more preferably 50~99%.
In the present invention, the specificity antineoplastic drug is preferably anti-tumor vaccine, which passes through tumor
Interior injection makes internal local tumor tissue enrichment high concentration PD1, and then plays effective immune clearance effect.
Further, further include pharmaceutic adjuvant in specificity antineoplastic drug of the present invention；The pharmaceutic adjuvant includes
But it is not limited to one or more in pharmaceutical carrier, diluent, adjuvant and excipient.Wherein, pharmaceutical carrier includes but not limited to
Liposome, alcohol plastid, polymer micelle, nano structured lipid carrier, solid lipid nano carrier, mesoporous silicon dioxide nano particle
Deng；The excipient substances such as the diluent, adjuvant and excipient may include specifically pharmaceutical preservative, antioxidant, filler, collapse
Solution agent, wetting agent, emulsifier, suspending agent, solvent, decentralized medium, coating, antiseptic or wait, which blend, absorbs delayer etc..
Further, the dosage form of specificity antineoplastic drug of the present invention includes but not limited to tablet, capsule, solution
Agent, granule, pill, powder, paste, sublimed preparation, suspension, pulvis, injection, suppository, creme, spray, patch, sustained release system
Agent, controlled release preparation or targeting preparation；Specifically, the tablet includes but not limited to sugar coated tablet, film coated tablet, Enteric coated tablets
Agent, buccal tablet etc.；The solution includes but not limited to injection, oral solution；The capsule includes but not limited to hard capsule
Agent, soft capsule etc..Specifically, when the present invention needs antitumor drug particular dosage form is made, it is suitable according to dosage form selection
Pharmaceutic adjuvant.
Further, the administration route of specificity antineoplastic drug of the present invention includes but not limited to drug administration by injection, mouth
Take administration, rectally etc..
In order to further illustrate the present invention, technical solution provided by the invention is retouched in detail with reference to embodiment
It states, but they cannot be interpreted as limiting the scope of the present invention.
1 construction recombination plasmid of embodiment
1) according to document《Clone, eukaryotic expression and the biological activity detection of mouse soluble PD1》The method system of record
Standby PD1/Fc-pCDNA3.1 plasmids (Qin Xiaolin, Liu Chaoqi, Tang Guohui, wait the clone of mouse solubles PD1, eukaryotic expression and
Biological activity detects [J] biotechnologys notification, 2011 (2):121-125.).The PD1/Fc- being prepared
Containing such as SEQ ID NO in pCDNA3.1 plasmids:PD1 encoding genes shown in 2.
2) according to document《The structure of mouse FGFR1/MIP3a-Fc fusion carrier for expression of eukaryon and expression》Record,
Preparation MIP3a/FGFR1-Fc plasmids (Huang Yonghao, and strengthen forever, dawn coin waits mouse FGFR1/MIP3a-Fc fusion eukaryons
The structure of expression vector and expression [J] Tropical China medicine, 2017,17 (4):340-343.), the MIP3a/ being prepared
Containing such as SEQ ID NO in FGFR1-Fc plasmids:FGFR1 coded sequences shown in 3 and such as SEQ ID NO:Mouse source shown in 4 is anti-
Fc sections of coded sequences of body.
3) the MIP3a/FGFR1-Fc plasmids being prepared using step 2) carry out PCR expansions as template using upstream and downstream primer
Increasing obtains amplified production, carries out electrophoresis to amplified production, the results are shown in Figure 2, and PCR amplification obtains the spy that size is about 1146bp
Specific fragment is consistent with theoretical value to get the amplified production containing target fragment Linker-FGFR1-Fc.
Wherein, the nucleotide sequence of sense primer such as SEQ ID NO:Shown in 5, i.e.,：
AAGGCCGGTTTCAAGGCATGGGTGGAGGTTCGGGTGGAGGTTCAGGTGGAGGTTCTAG；
The nucleotide sequence of downstream primer such as SEQ ID NO:Shown in 6, i.e.,：
GCTTGATTGTGGGCCCTCTGGGATCCTCCAGGTAGAGCGGTGAGGT。
The PCR amplification system is 50 μ L systems, is contained：ddH2O 41μL、10PCR Buffer5μL、dNTP(2.5mM
Each) 1 μ L, each 0.5 μ L of upstream and downstream primer (20mM), 1 μ L of Pfu high-fidelity DNA polymerases (2.5U/L), 1 μ L of template (20ng).
The PCR reaction conditions are：95 DEG C of pre-degeneration 5min → 94 DEG C are denaturalized 30s, 56 DEG C of annealing 30s, 72 DEG C of extensions
1min recycles 30 times → 72 DEG C and fully extends 5min.
4) amplified production that step 3) obtains is separated by electrophoresis with 1.8% Ago-Gel, in ultraviolet transilluminator
It is middle that the Ago-Gel for detaching purposeful segment Linker-FGFR1-Fc is cut with scalpel, it is solidifying with Axygen agaroses to it
Plastic recovery kit purifies target fragment (detailed step is shown in instructions book).
5) the PD1/Fc-pCDNA3.1 plasmids being prepared using step 1) is carriers, carrier restriction enzyme BamHI
Digestion is linear；Take 3 μ L of target fragment (50ng), 3 μ L of carrier (50ng), 10 μ of Gibson Mix after digestion that step 4) purifies
L and ddH24 μ L of O are mixed, and water-bath 15min at 50 DEG C obtains recombining reaction liquid.
6) 5 μ l of recombining reaction liquid is taken to add in the competent cell of 50 μ l DH5a, mixing, ice bath 30min；42 DEG C of water-baths
90s, then ice bath 2min；After adding 600 μ l LB, 37 DEG C of 1h, 100 μ l is taken to be coated on the LB plates containing 100 μ g/mLAmp, 37 DEG C of trainings
It supports overnight, obtains transformed bacteria.
7) recombinant plasmid PCR is identified：
The random consistent clone uniform in size of picking 8 carries out PCR identifications on the transformed bacteria tablet of culture.PCR reacts
System totally 25 μ L, contain：ddH218.75 μ L of O, 2.5 10PCRbuffer μ L, 0.5 μ L of 10mmol dNTPs, step 3) are described
2 μ L of each 0.5 μ L of upstream and downstream primer, 0.25 μ L of Taq DNApolymerase (12U/L) and transformed bacteria.
Then the first 94 DEG C of pre-degeneration 5min of the above system are denaturalized 30sec, 56 DEG C of annealing 30sec, 72 DEG C of extensions for 94 DEG C
1min, 30 cycles, last 72 DEG C of extensions 5min.Products therefrom is detected with 1.2% agarose electrophoresis.
The results are shown in Figure 3 for electrophoresis detection, and it is positive colony to have 4 clones in 5 clones.1st~3 and 5 swimming lanes expand
Increasing has obtained the specific DNA molecule fragment that size is about 1146bp, in the same size with FGFR1 genetic fragments, shows at these
FGFR1 genetic fragments have been successfully plugged into vector plasmid in clone.
8) positive colony identified in step 7) is sent to the Suzhou bio tech ltd Jin Weizhi and is sequenced, through ratio is sequenced
It is pair completely the same with expected sequence.Recombinant plasmid in positive colony contains such as SEQ ID NO:Nucleotide sequence shown in 1.
The expression and purifying of embodiment 2Fc fusion proteins PD1/FGFR1
(1) Fc fusion proteins PD1/FGFR1 transient expressions in HEK293 cells
It is diluted to 6 × 10 after HEK293 cells in exponential phase are digested with pancreatin5The density of/mL, is inoculated into
In 15cm culture dishes, each culture dish adds 20mL serum free mediums, culture to be transfected for 24 hours.It utilizes
The Transfected Recombinant Plasmid that 2000 transfection reagents of Lipofectamine (Invitrogen companies) obtain embodiment 1 is to HEK293
Cell, plasmid used extract (Sigma companies) by endotoxin-free plasmid extraction agent box.Titanium dioxide of the cell of transfection 5%
After being cultivated 5 days in carbon incubator, supernatant is obtained.The fusion protein production in supernatant is measured with enzyme linked immunosorbent assay (ELISA)
Amount, expresses a concentration of 3.3mg/L.
(2) purifying of Fc fusion proteins PD1/FGFR1
By ProteinA affinity chromatographic columns (the being purchased from GE companies) purifying of the culture supernatant of collection.With 1X PBS (pH=
7.4) purification column is cleaned and balances, loading speed 2mL/min crosses 15 times of column volumes.Loading flow velocity is adjusted to carry out to 1mL/min
Sample.After end of the sample, purification column is cleaned with 1X PBS (pH=7.4), loading speed 2mL/min crosses 15 times of column volumes.Liquid feeding end
Glycine solution (pH=2.5) is changed, adjusts flow velocity to 1mL/min elution fusion proteins.It is slow that Tris8.0 is added into collecting pipe
Albumen is adjusted to pH value neutrality by fliud flushing.Last fusion protein is dialysed into 1X PBS (pH=7.4).Purified Fc fusion proteins
PD1/FGFR1 is detected through SDS-PAGE.
SDS-PAGE testing results as shown in figure 4, swimming lane 1 be FGFR1-PD1/Fc fusion proteins denaturing electrophoretic figure, melt
The molecular weight of hop protein is 130KDa；Swimming lane 2 is the non-denatured electrophoretogram of Fc fusion proteins PD1/FGFR1, point of fusion protein
Son amount is 83KDa.It can be seen that the protein of purifying is occurring relatively single band positioned at the places 83kDa or so, illustrate to purify
The protein being collected into is restructuring destination protein.
Comparative example 1
1) according to document《Clone, eukaryotic expression and the biological activity detection of mouse soluble PD1》The method system of record
Standby PD1/Fc-pCDNA3.1 plasmids (Qin Xiaolin, Liu Chaoqi, Tang Guohui, wait the clone of mouse solubles PD1, eukaryotic expression and
Biological activity detects [J] biotechnologys notification, 2011 (2):121-125).The PD1/Fc-pCDNA3.1 being prepared
Containing such as SEQ ID NO in plasmid:PD1 encoding genes shown in 2.
2) PD1/Fc-pCDNA3.1 plasmids are transferred in HEK293 cells according to 2 the method for embodiment and express and purifies,
Obtain PD1/Fc fusion proteins.
Comparative example 2
1) according to the method for Gibson clones by the coded sequence of FGFR1 albumen, the encoding gene gram of mouse source antibody Fc section
It is grand enter pCDNA3.1 (+) plasmid (purchase in Invitrogen), prepare FGFR1/Fc recombinant expression carriers；Wherein FGFR1 albumen
Coded sequence such as SEQ ID NO:Shown in 3, the coding gene sequence such as SEQ ID NO of mouse source antibody Fc section:Shown in 4；
2) the FGFR1/Fc recombinant expression carriers that step 1) obtains are transferred to HEK293 cells according to 2 the method for embodiment
Middle expression simultaneously purifies, and obtains FGFR1/Fc fusion proteins.
The fluorescent dye with tumour-specific targeting of embodiment 3Fc fusion proteins PD1/FGFR1 acts on
The preparation of 1 model
Purchase mouse mastopathy cell strain 4T1 is simultaneously cultivated, and the cell liquid of the mouse mastopathy cell 4T1 of culture is dense
Degree is adjusted to 1 × 107It is subcutaneous to be inoculated in right side of mice armpit with every 0.1mL by a/mL.
2 groupings and administration
The mouse of inoculation is randomly divided into test group and control group, every group 8, waits for that tumour is grown to volume about 110mm3Shi Jing
It is injected according to the ratio of 500 μ g/kg mouse weights in tumor, the Fc fusions that wherein test group injection embodiment 2 is prepared
The fusion protein PD1/Fc that albumen PD1/FGFR1, control group injection comparative example 1 are prepared.It is put to death after 30 days after each group administration
Mouse takes out tumor tissues and carries out frozen section and carry out immunofluorescence dyeing observation.
The results are shown in Figure 5, and A is the tumor biopsy colored graph of test group mouse, it can be seen that tumor-microvessel surface is seen
Green florescent signal deposits, and without apparent fluorescence signal in other structural constituents；B is the mouse slice dyeing of control group
Figure, and control group tumor-microvessel surface has no that green florescent signal deposits.
This shows that Fc fusion proteins PD1/FGFR1 prepared by the present invention has higher tumor tissue specificity and blood vessel target
Tropism can effectively be enriched in tumor tissues and tumor neogenetic blood vessels, can also reduce drug dose.
Embodiment 4
1, the preparation of model
Purchase mouse mastopathy cell strain 4T1 is simultaneously cultivated, and the cell liquid of the mouse mastopathy cell 4T1 of culture is dense
Degree is adjusted to 1 × 107It is subcutaneous to be inoculated in right side of mice armpit with every 0.1mL by a/mL.
2, grouping and administration
Wait for that tumour is grown to 100~150mm3When animal is randomly divided into 5 groups, every group 8.Specifically it is grouped into：
(1) PD1/FGFR1/Fc groups：According to 500 μ g/kg mouse weights, the Fc fusion proteins that embodiment 2 is prepared
PD1/FGFR1 and adjuvant (using Freund's complete adjuvant for the first time, remaining uses incomplete Freund's adjuvant) 1：1 emulsification after to mouse into
Row intratumor injection is administered once for three days；
(2) PD1/Fc groups：According to 500 μ g/kg mouse weights, the PD1/Fc fusion proteins that comparative example 1 is prepared with
Adjuvant (using Freund's complete adjuvant for the first time, remaining uses incomplete Freund's adjuvant) 1：Intratumor injection is carried out to mouse after 1 emulsification,
It is administered once within three days；
(3) FGFR1/Fc groups：According to 500 μ g/kg mouse weights, the FGFR1/Fc that comparative example 2 is prepared merges egg
In vain with adjuvant (for the first time use Freund's complete adjuvant, remaining use incomplete Freund's adjuvant) 1：Mouse is carried out in tumor after 1 emulsification
Injection, is administered once for three days；
(4) adjuvant physiological saline group：Isometric physiological saline (uses Freund's complete adjuvant, remaining is for the first time with adjuvant
With incomplete Freund's adjuvant) 1：Intratumor injection is carried out to mouse after 1 emulsification, is administered once within three days；
(5) physiological saline group：The physiological saline that same volume will be organized with (1) is administered once for three days through intratumor injection.
Use the method for measuring knurl footpath, the anti-tumor effect of dynamic observation given the test agent.It observes animal administration process and gives
Variation (activity, appearance, spirit) after medicine, after being even administered 30 days, puts to death mouse, operation strips tumor mass and weighs immediately.
Specific fusion protein and adjuvant preparation method are as follows：
Drug dosage is 500ug/kg in this experiment, and fusion protein concentration is 0.1mg/ml, and administered volume is
100ul/20g mouse weights.It first uses normal saline dilution albumen to the concentration of 0.2mg/ml when preparation, presses and adjuvant volume ratio
1:1 grinds, a concentration of 0.1mg/ml of gained, according to 100ul/20g intratumor injections.
Physiological saline adjuvant group is with 1:1 physiological saline and adjuvant grinding emulsification, according to 100ul/20g intratumor injections.
The emulsification method of wherein adjuvant and fusion protein solution is：The adjuvant of certain volume is taken to be put into sterile glass mortar
It is interior, then isometric fusion protein solution is slowly instilled, it is ground at same direction in drop.After albumen is all added, continue
Grinding a period of time, make the sticky water-in-oil emulsion of milky.
The Freund's complete adjuvant used in this experiment is purchased from Sigma companies, article No. F5881；The Freund of use is incomplete
Adjuvant is purchased from Sigma companies, article No. F5506.
Use the method for measuring knurl footpath, the anti-tumor effect of dynamic observation given the test agent.It observes animal administration process and gives
Variation (activity, appearance, spirit) after medicine, after being even administered 30 days, puts to death mouse, operation strips tumor mass and weighs immediately.
3, observation index
1) calculation formula of gross tumor volume (tumorvolume, TV) is：
TV=1/2 × a × b2
Wherein a, b indicate length and width respectively.
2) relative tumour volume (relative tumorvolume, RTV), calculation formula are calculated according to the result of measurement
For：
RTV=Vt/V0
Wherein V0For sub-cage administration when (d0) measure gained gross tumor volume, VtGross tumor volume when to measure each time.
3) evaluation index of antitumor activity：Relative tumor proliferation rate T/C (%), calculation formula is as follows：
TRTV：Treatment group RTV (relative tumour volume)；CRTV：Model group (physiological saline group) RTV (relative tumour volume)；
4) evaluation index of antitumor activity：Inhibition rate of tumor growth (%), calculation formula is as follows：
4, observed result：
(1) influences of the PD1/FGFR1/Fc to gross tumor volume
Tumor volume change (cm in 1 each group successive administration 30d of table3)
| Group | Physiological saline group | Adjuvant physiological saline group | FGFR1/Fc groups | PD1/Fc groups | PD1/FGFR1/Fc groups |
| 1d | 0.112 | 0.109 | 0.112 | 0.111 | 0.112 |
| 4d | 0.204 | 0.191 | 0.181 | 0.174 | 0.166 |
| 7d | 0.327 | 0.319 | 0.265 | 0.254 | 0.230 |
| 10d | 0.523 | 0.479 | 0.379 | 0.360 | 0.337 |
| 13d | 0.771 | 0.747 | 0.498 | 0.494 | 0.413 |
| 16d | 1.035 | 0.966 | 0.594 | 0.571 | 0.475 |
| 19d | 1.305 | 1.189 | 0.665 | 0.646 | 0.541 |
| 22d | 1.584 | 1.467 | 0.742 | 0.713 | 0.587 |
| 25d | 1.825 | 1.701 | 0.791 | 0.739 | 0.635 |
| 28d | 2.192 | 1.969 | 0.871 | 0.786 | 0.677 |
| 31d | 2.550 | 2.348 | 0.937 | 0.845 | 0.730 |
The tumor Volume Changes figure that each group mouse successive administration 30d is observed is as shown in fig. 6, concrete outcome is as shown in table 1.
As shown in the data of Fig. 6 and table 1, PD1/FGFR1/Fc groups, FGFR1/Fc groups, PD1/Fc groups and physiological saline group or
Adjuvant physiological saline group is compared, and gross tumor volume has significant decrease, and will not significantly increase with the extension gross tumor volume of administration
Add, i.e., Fc fusion proteins PD1/FGFR1, FGFR1/Fc fusion proteins and PD1/Fc fusion proteins all have certain inhibition tumour
The effect of growth.
It can also be seen that PD1/FGFR1/Fc groups are compared with FGFR1/Fc groups, PD1/Fc groups, gross tumor volume smaller, and
There is significant difference.This show Fc fusion proteins PD1/FGFR1 provided by the invention relative to PD1/Fc fusion proteins,
FGFR1/Fc fusion proteins have the function of significantly more inhibition tumour growth, reduce gross tumor volume.
(2) influences of the Fc fusion proteins PD1/FGFR1 to knurl weight
The knurl weight (g) after 30d is administered in 2 each group of table
| Group | Physiological saline | Adjuvant physiological saline group | FGFR1/Fc groups | PD1-Fc groups | PD1/FGFR1/Fc groups |
| Mouse 1 | 2.75 | 2.86 | 1.06 | 1.16 | 0.86 |
| Mouse 2 | 2.89 | 2.79 | 1.05 | 1.24 | 0.93 |
| Mouse 3 | 3.16 | 3.04 | 1.37 | 1.18 | 0.81 |
| Mouse 4 | 3.06 | 2.69 | 1.41 | 0.91 | 0.99 |
| Mouse 5 | 3.2 | 2.81 | 1.18 | 1.35 | 0.87 |
| Average knurl weight | 3.01 | 2.84 | 1.21 | 1.17 | 0.89 |
| P values | - | 0.13 | 2.86×e-7 | 2.30×e-7 | 2.30×e-6 |
| Tumour inhibiting rate | - | 5.8% | 59.7% | 61.2% | 70.4% |
Knurl weight after each group mouse successive administration 30d is as shown in fig. 7, specific knurl weight and tumour inhibiting rate are as shown in table 2.
As shown in Fig. 7 and table 2, PD1/FGFR1/Fc groups, FGFR1/Fc groups, PD1/Fc groups and physiological saline group or adjuvant life
Reason brine group is compared, tumor weight there are significant difference, show Fc fusion protein PD1/FGFR1, FGFR1/Fc fusion proteins with
And PD1/Fc fusion proteins all have the significant effect for inhibiting tumour growth, killing tumour.
PD1/FGFR1/Fc groups be can also be seen that for FGFR1/Fc groups, PD1/Fc groups, after 30d is administered
Knurl weight is lower, inhibitory rate to 70.4% shows Fc fusion proteins PD1/FGFR1 and FGFR1/Fc fusion proteins, PD1/Fc
Fusion protein, which is compared, has significantly more tumor inhibition effect, significant in efficacy.
In conclusion this experiment establish 4T1 breast cancer tumour models Fc fusion proteins PD1/FGFR1 is carried out it is antitumor
Functional observation, as a result, it has been found that Fc fusion proteins PD1/FGFR1 is as antitumor drug (independent FGFR1/Fc fusion proteins, PD1/
Fc fusion proteins, physiological saline adjuvant and physiological saline group are as a contrast) it can effectively treat tumour, hence it is evident that inhibit tumour life
Long, gross tumor volume is obviously reduced, and tumour knurl weight is substantially reduced；Independent FGFR1/Fc fusion proteins, PD1/Fc fusion protein immunizations
Treat the work that there is certain antitumor action but its antitumous effect to be not so good as Fc fusion proteins PD1/FGFR1 provided by the invention
With notable.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Hainan Medical College
<120>Fc fusion proteins PD1/FGFR1 and its application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2242
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
aagcttcgag gtccccaatg ggccctggag gtccctcacc ttctacccag cctggctcac 60
agtgtcagag ggagcaaatg ccaccttcac ctgcagcttg tccaactggt cggaggatct 120
tatgctgaac tggaaccgcc tgagtcccag caaccagact gaaaaacagg ccgccttctg 180
taatggtttg agccaacccg tccaggatgc ccgcttccag atcatacagc tgcccaacag 240
gcatgacttc cacatgaaca tccttgacac acggcgcaat gacagtggca tctacctctg 300
tggggccatc tccctgcacc ccaaggcaaa aatcgaggag agccctggag cagagctcgt 360
ggtaacagag agaatcctgg agacctcaac aagatatccc agcccctcgc ccaaaccaga 420
aggccggttt caaggcatgg gtggaggttc gggtggaggt tcaggtggag gttctaggcc 480
agccccaacc ttgcctgaac aagctcagcc ctggggagtc cctgtggaag tggagtctct 540
cctggtccac cctggcgacc tgctacagct tcgctgtcgg cttcgcgatg atgtgcagag 600
catcaactgg ctgcgggatg gggtgcagct ggtggagagc aaccgtaccc gcatcacagg 660
ggaggaggtg gaggtgcggg actccatccc cgctgactct ggcctctacg cttgcgtgac 720
cagcagcccc tctggcagcg ataccaccta cttctccgtc aatgtctcag atgcactccc 780
atcctcggaa gatgatgacg acgacgatga ctcctcctcg gaggagaaag agacggacaa 840
caccaaacca aaccgtaggc ctgtagctcc ctactggaca tccccagaga aaatggagaa 900
gaaactgcat gcggtgcccg ctgccaagac ggtgaagttc aagtgcccgt cgagtgggac 960
acccaacccc actctgcgct ggttgaaaaa tggcaaagag tttaagcctg accaccgaat 1020
tggaggctac aaggttcgct atgccacctg gagcatcata atggattctg tggtgccttc 1080
tgacaagggc aactacacct gcatcgtgga gaatgagtat gggagcatca accacaccta 1140
ccagcttgac gtcgtggaac gatctccgca ccgacccatc cttcaggcag ggctgcctgc 1200
caacaagaca gtggccctgg gcagcaatgt ggagttcatg tgtaaggtgt acagcgatcc 1260
gcagcctcac attcagtggc tgaagcacat cgaggtgaac gggagtaaga tcgggccaga 1320
caacttgccg tatgtccaga tcctgaagac tgctggagtt aataccaccg acaaggaaat 1380
ggaggtgctt catctacgga atgtctcctt tgaggatgcg ggggagtata cgtgcttggc 1440
gggtaactct atcggactct cccatcactc tgcatggttg accgttctgg aagccctgga 1500
agagagacca gctgtgatga cctcaccgct ctacctggag gatcccagag ggcccacaat 1560
caagccctgt cctccatgca aatgcccagc acctaacctc ttgggtggac catccgtctt 1620
catcttccct ccaaagatca aggatgtact catgatctcc ctgagcccca tagtcacatg 1680
tgtggtggtg gatgtgagcg aggatgaccc agatgtccag atcagctggt ttgtgaacaa 1740
cgtggaagta cacacagctc agacacaaac ccatagagag gattacaaca gtactctccg 1800
ggtggtcagt gccctcccca tccagcacca ggactggatg agtggcaagg agttcaaatg 1860
caaggtcaac aacaaagacc tcccagcgcc catcgagaga accatctcaa aacccaaagg 1920
gtcagtaaga gctccacagg tatatgtctt gcctccacca gaagaagaga tgactaagaa 1980
acaggtcact ctgacctgca tggtcacaga cttcatgcct gaagacattt acgtggagtg 2040
gaccaacaac gggaaaacag agctaaacta caagaacact gaaccagtcc tggactctga 2100
tggttcttac ttcatgtaca gcaagctgag agtggaaaag aagaactggg tggaaagaaa 2160
tagctactcc tgttcagtgg tccacgaggg tctgcacaat caccacacga ctaagagctt 2220
ctcccggact ccgggtaaat ga 2242
<210> 2
<211> 432
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gaggtcccca atgggccctg gaggtccctc accttctacc cagcctggct cacagtgtca 60
gagggagcaa atgccacctt cacctgcagc ttgtccaact ggtcggagga tcttatgctg 120
aactggaacc gcctgagtcc cagcaaccag actgaaaaac aggccgcctt ctgtaatggt 180
ttgagccaac ccgtccagga tgcccgcttc cagatcatac agctgcccaa caggcatgac 240
ttccacatga acatccttga cacacggcgc aatgacagtg gcatctacct ctgtggggcc 300
atctccctgc accccaaggc aaaaatcgag gagagccctg gagcagagct cgtggtaaca 360
gagagaatcc tggagacctc aacaagatat cccagcccct cgcccaaacc agaaggccgg 420
tttcaaggca tg 432
<210> 3
<211> 1100
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ggtggaggtt cgggtggagg ttcaggtgga ggttctaggc cagccccaac cttgcctgaa 60
caagctcagc cctggggagt ccctgtggaa gtggagtctc tcctggtcca ccctggcgac 120
ctgctacagc ttcgctgtcg gcttcgcgat gatgtgcaga gcatcaactg gctgcgggat 180
ggggtgcagc tggtggagag caaccgtacc cgcatcacag gggaggaggt ggaggtgcgg 240
gactccatcc ccgctgactc tggcctctac gcttgcgtga ccagcagccc ctctggcagc 300
gataccacct acttctccgt caatgtctca gatgcactcc catcctcgga agatgatgac 360
gacgacgatg actcctcctc ggaggagaaa gagacggaca acaccaaacc aaaccgtagg 420
cctgtagctc cctactggac atccccagag aaaatggaga agaaactgca tgcggtgccc 480
gctgccaaga cggtgaagtt caagtgcccg tcgagtggga cacccaaccc cactctgcgc 540
tggttgaaaa atggcaaaga gtttaagcct gaccaccgaa ttggaggcta caaggttcgc 600
tatgccacct ggagcatcat aatggattct gtggtgcctt ctgacaaggg caactacacc 660
tgcatcgtgg agaatgagta tgggagcatc aaccacacct accagcttga cgtcgtggaa 720
cgatctccgc accgacccat ccttcaggca gggctgcctg ccaacaagac agtggccctg 780
ggcagcaatg tggagttcat gtgtaaggtg tacagcgatc cgcagcctca cattcagtgg 840
ctgaagcaca tcgaggtgaa cgggagtaag atcgggccag acaacttgcc gtatgtccag 900
atcctgaaga ctgctggagt taataccacc gacaaggaaa tggaggtgct tcatctacgg 960
aatgtctcct ttgaggatgc gggggagtat acgtgcttgg cgggtaactc tatcggactc 1020
tcccatcact ctgcatggtt gaccgttctg gaagccctgg aagagagacc agctgtgatg 1080
acctcaccgc tctacctgga 1100
<210> 4
<211> 697
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cagagggccc acaatcaagc cctgtcctcc atgcaaatgc ccagcaccta acctcttggg 60
tggaccatcc gtcttcatct tccctccaaa gatcaaggat gtactcatga tctccctgag 120
ccccatagtc acatgtgtgg tggtggatgt gagcgaggat gacccagatg tccagatcag 180
ctggtttgtg aacaacgtgg aagtacacac agctcagaca caaacccata gagaggatta 240
caacagtact ctccgggtgg tcagtgccct ccccatccag caccaggact ggatgagtgg 300
caaggagttc aaatgcaagg tcaacaacaa agacctccca gcgcccatcg agagaaccat 360
ctcaaaaccc aaagggtcag taagagctcc acaggtatat gtcttgcctc caccagaaga 420
agagatgact aagaaacagg tcactctgac ctgcatggtc acagacttca tgcctgaaga 480
catttacgtg gagtggacca acaacgggaa aacagagcta aactacaaga acactgaacc 540
agtcctggac tctgatggtt cttacttcat gtacagcaag ctgagagtgg aaaagaagaa 600
ctgggtggaa agaaatagct actcctgttc agtggtccac gagggtctgc acaatcacca 660
cacgactaag agcttctccc ggactccggg taaatga 697
<210> 5
<211> 58
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
aaggccggtt tcaaggcatg ggtggaggtt cgggtggagg ttcaggtgga ggttctag 58
<210> 6
<211> 46
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcttgattgt gggccctctg ggatcctcca ggtagagcgg tgaggt 46
<210> 7
<211> 743
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 7
Glu Val Pro Asn Gly Pro Trp Arg Ser Leu Thr Phe Tyr Pro Ala Trp
1 5 10 15
Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe Thr Cys Ser Leu Ser
20 25 30
Asn Trp Ser Glu Asp Leu Met Leu Asn Trp Asn Arg Leu Ser Pro Ser
35 40 45
Asn Gln Thr Glu Lys Gln Ala Ala Phe Cys Asn Gly Leu Ser Gln Pro
50 55 60
Val Gln Asp Ala Arg Phe Gln Ile Ile Gln Leu Pro Asn Arg His Asp
65 70 75 80
Phe His Met Asn Ile Leu Asp Thr Arg Arg Asn Asp Ser Gly Ile Tyr
85 90 95
Leu Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu Glu Ser
100 105 110
Pro Gly Ala Glu Leu Val Val Thr Glu Arg Ile Leu Glu Thr Ser Thr
115 120 125
Arg Tyr Pro Ser Pro Ser Pro Lys Pro Glu Gly Arg Phe Gln Gly Met
130 135 140
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Arg Pro Ala Pro
145 150 155 160
Thr Leu Pro Glu Gln Ala Gln Pro Trp Gly Val Pro Val Glu Val Glu
165 170 175
Ser Leu Leu Val His Pro Gly Asp Leu Leu Gln Leu Arg Cys Arg Leu
180 185 190
Arg Asp Asp Val Gln Ser Ile Asn Trp Leu Arg Asp Gly Val Gln Leu
195 200 205
Val Glu Ser Asn Arg Thr Arg Ile Thr Gly Glu Glu Val Glu Val Arg
210 215 220
Asp Ser Ile Pro Ala Asp Ser Gly Leu Tyr Ala Cys Val Thr Ser Ser
225 230 235 240
Pro Ser Gly Ser Asp Thr Thr Tyr Phe Ser Val Asn Val Ser Asp Ala
245 250 255
Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp Ser Ser Ser Glu
260 265 270
Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Arg Arg Pro Val Ala Pro
275 280 285
Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro
290 295 300
Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn
305 310 315 320
Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His
325 330 335
Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile Ile Met
340 345 350
Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile Val Glu
355 360 365
Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val Val Glu
370 375 380
Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Lys
385 390 395 400
Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr Ser
405 410 415
Asp Pro Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val Asn Gly
420 425 430
Ser Lys Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu Lys Thr
435 440 445
Ala Gly Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His Leu Arg
450 455 460
Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn
465 470 475 480
Ser Ile Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu Glu Ala
485 490 495
Leu Glu Glu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Leu Glu Asp
500 505 510
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
515 520 525
Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
530 535 540
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
545 550 555 560
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
565 570 575
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
580 585 590
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
595 600 605
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
610 615 620
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
625 630 635 640
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
645 650 655
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
660 665 670
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
675 680 685
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
690 695 700
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
705 710 715 720
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
725 730 735
Ser Phe Ser Arg Thr Pro Gly
740
Claims (10)
1. a kind of Fc fusion proteins PD1/FGFR1, which is characterized in that compiled by PD1, FGFR1 and the encoding gene of mouse source antibody section
Code obtains.
2. Fc fusion proteins PD1/FGFR1 according to claim 1, which is characterized in that described PD1, FGFR1 and mouse source are anti-
The sequence of Fc sections of encoding genes of body is followed successively by PD1-FGFR1-Fc by 5 ' ends to 3 ' ends.
3. Fc fusion proteins PD1/FGFR1 according to claim 1 or 2, which is characterized in that
The encoding gene of the PD1 is selected from SEQ ID NO:Nucleotide sequence shown in 2 or with SEQ ID NO:Nucleotides sequence shown in 2
Arrange the nucleotide sequence with 99% or more homology；
The encoding gene of the FGFR1 is selected from SEQ ID NO:Nucleotide sequence shown in 3 or with SEQ ID NO:Nucleotide shown in 3
Sequence has the nucleotide sequence of 99% homology；
The encoding gene of shown mouse source antibody Fc section is selected from SEQ ID NO:Nucleotide sequence shown in 4 or with SEQ ID NO:4 institutes
Show that nucleotide sequence has the nucleotide sequence of 99% or more homology.
4. Fc fusion proteins PD1/FGFR1 according to claim 1, which is characterized in that the coding Fc fusion proteins
The nucleotide sequence of PD1/FGFR1 such as SEQ ID NO:Shown in 1.
5. Fc fusion proteins PD1/FGFR1 according to claim 3, which is characterized in that the amino acid of the fusion protein
Sequence is selected from SEQ ID NO:Amino acid sequence shown in 7 or with SEQ ID NO:Amino acid sequence shown in 7 has 99% homology
Amino acid sequence.
6. a kind of nucleic acid molecules, which is characterized in that Fc fusion proteins PD1/FGFR1 described in coding Claims 1 to 5 any one
Nucleotide sequence.
7. a kind of recombinant expression carrier, which is characterized in that including the nucleic acid molecules described in claim 6.
8. a kind of expression cell, which is characterized in that contain the recombinant expression carrier described in claim 7.
9. nucleic acid molecules described in Fc fusion proteins PD1/FGFR1, claim 6 described in Claims 1 to 5 any one,
Expression cell is in preparing specificity antineoplastic drug described in recombinant expression carrier or claim 8 described in claim 7
Using.
10. a kind of specificity antineoplastic drug, which is characterized in that merge egg including the Fc described in Claims 1 to 5 any one
White PD1/FGFR1 and pharmaceutically acceptable auxiliary material.
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| CN110066344A (en) * | 2019-06-04 | 2019-07-30 | 海南医学院第一附属医院 | A kind of Fc fusion protein F GFR1-MIP3a-CTLA4 and its application |
| CN113121699A (en) * | 2019-12-30 | 2021-07-16 | 国典(北京)医药科技有限公司 | PTTRAP1 bispecific fusion protein and application thereof |
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| CN108440673B (en) | 2021-08-17 |
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