CN110590959B - Recombinant canine PD-1 fusion protein and preparation method and application thereof - Google Patents
Recombinant canine PD-1 fusion protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant canine PD-1 fusion protein and a preparation method and application thereof, wherein the fusion protein is formed by directly or indirectly connecting an extracellular region of a canine PD-1 protein or an extracellular region mutant of the canine PD-1 protein with an Fc fragment. Compared with wild PD-1, the recombinant canine PD-1 fusion protein provided by the invention has higher affinity and better drug effect, and provides more choices for immunotherapy of canine tumors.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to two novel recombinant canine PD-1 fusion proteins and a preparation method and application thereof.
Background
PD-1, called programmed cell death receptor 1 (also called PDCD1), is a type I membrane glycoprotein with the size of 50-55 KD, belongs to a CD28 superfamily receptor, and is an important immunosuppressive molecule. PD-1 protein is mainly expressed on the surface of T cells (including antigen-stimulated activated T cells), B lymphocytes and activated macrophages, and PD-1 is also expressed by bone marrow cells. PD-1 has two ligands-PD-L1 and PD-L2. Under normal physiological conditions, PD-1 and PD-L1/PD-L2 combine to inhibit the activation of T cells and the production of cytokines, thereby protecting the body from being attacked by the autoimmune system. However, a large amount of PD-L1 is also expressed on the cell surfaces of various solid tumors and some hematological malignancies, including melanoma, Hodgkin lymphoma cell lines, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, liver cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer and the like. PD-L1 on the tumor cell membrane is combined with PD-1 on T cells to down regulate T cell activation and cytokine secretion, so that the recognition and attack of the immune system of the body are successfully avoided, and the immune escape of the tumor cells is caused. Simultaneous studies have found that expression of PD-L1 on tumor cells correlates with poor prognosis for multiple tumor types.
Blocking the interaction of PD-1 and PD-L1 becomes an effective way for treating tumors. Most of the current antitumor drugs against the interaction of PD-1/PD-L1 are monoclonal anti-PD-1 or PD-L1 drugs, including PD-1 monoclonal drugs, such as Nivolumab (Nivolumab) of Bethes-Messajou-Bao (BMS), Pembrolizumab (Pembrizumab) of Merck, PD-L1 monoclonal drugs, such as Atezolizumab (Atezolizumab) of Tak, Beluzumab (Durvalumab) of Aslicon, Avelumab of Perele and Mersandon. The monoclonal antibody medicines are mainly used for treating human malignant tumors including melanoma, non-small cell lung cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma and the like, and the monoclonal antibody treatment achieves certain treatment effect, but has certain limitations, such as low average treatment effective rate (about 20% of single-medicine effective rate), high immunogenicity, individual difference, limited adaptation symptoms and the like, so that the development of new and efficient candidate tumor medicines is urgently needed.
The PD-1 fusion protein is a novel tumor medicament aiming at a PD-1/PD-L1 signal path, can competitively bind PDL-1/PD-L2 ligand of a tumor cell, and blocks the combination of the tumor cell and a T cell, thereby restoring the activation of the T cell, improving the secretion level of cytokines and enhancing the immune response. The application and research of PD-1 fusion protein related drugs in human are rarely reported, and the research on canine tumors is still in a blank stage.
Canine neoplastic diseases are relatively common diseases, including melanoma, breast cancer, mast cell tumor, lipoma, and the like, with approximately one-fourth of dogs developing neoplastic disease at some stage in life, with older dogs being more common, and with nearly more than 50% of dogs over the age of ten developing neoplastic disease. The treatment method of the canine tumor mainly comprises surgery, chemotherapy and radiotherapy, and the main treatment medicines applied abroad comprise chemotherapy medicines such as lomustine, vincristine and the like and glucocorticoid medicines such as prednisolone and the like, but the medicines have large toxic and side effects and serious damage to the self; at present, no tumor medicine for dogs is on the market in China and is in the blank of the market.
The invention provides a dog PD-1 fusion protein, which is specially used for treating dog tumor diseases, has high effectiveness and small damage to organisms by enhancing the anti-tumor function of T cells and assisting an autoimmune system to remove tumor cells.
Disclosure of Invention
The invention aims to provide two novel recombinant canine PD-1 fusion proteins and a preparation method and application thereof.
In order to achieve the purpose, the invention provides two novel recombinant canine PD-1 fusion proteins in a first aspect, wherein one fusion protein is formed by directly or indirectly connecting an extracellular region of the canine PD-1 protein with an Fc fragment, and the other fusion protein is formed by directly or indirectly connecting an extracellular region mutant of the canine PD-1 protein with the Fc fragment.
The canine PD-1 protein extracellular region is as follows:
(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 5; or
(b) 5, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 5.
The canine PD-1 protein extracellular region mutant is as follows:
(c) a protein consisting of an amino acid sequence shown as SEQ ID NO. 6; or
(d) And (c) protein which is derived from the (c) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 6.
The Fc fragment comprises an immunoglobulin hinge region, CH2, and CH3 regions.
The immunoglobulin is derived from human or mammal, preferably human or mammal such as dog, cat, pig, mouse, horse, cow, etc., more preferably dog IgG1 immunoglobulin.
The fusion protein is formed by connecting a canine PD-1 protein extracellular region or a canine PD-1 protein extracellular region mutant with an Fc fragment directly or indirectly through a Linker. Wherein the Linker is a flexible polypeptide consisting of 2-20 flexible amino acids, and the flexible amino acids are selected from at least one of Gly, Ser, Ala and Thr.
Preferably, the Linker is (Gly-Gly-Gly-Gly-Ser) n, wherein n is an integer between 2 and 5, and more preferably n is 2.
Further, the two novel recombinant canine PD-1 fusion proteins are cPD1-Fc and cPD1mu-Fc respectively;
fusion protein cPD1-Fc is:
(e) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or
(f) And (e) a protein having 90% or more homology with the amino acid sequence shown in SEQ ID NO. 1 and having the same function.
Fusion protein cPD1mu-Fc is:
(g) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3; or
(h) And (g) a protein having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO. 3 and having the same function.
In a second aspect, the invention provides a biological material containing the recombinant canine PD-1 fusion protein encoding gene, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, an engineering bacterium or a transgenic cell line and the like.
In a third aspect, the invention provides a method for preparing the recombinant canine PD-1 fusion protein, the method comprising: artificially synthesizing the encoding gene of the fusion protein, carrying out codon optimization, constructing the optimized gene into a eukaryotic expression vector, transferring the eukaryotic expression vector into a eukaryotic cell, expressing the eukaryotic expression vector in the eukaryotic cell, and separating and purifying the target protein.
Preferably, the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cell is a CHO cell.
The optimized nucleotide sequences of cPD1-Fc and cPD1mu-Fc are shown in SEQ ID NO 2 and 4, respectively.
In a fourth aspect, the invention provides any one of the following applications of the recombinant canine PD-1 fusion protein:
1) the composition is used for treating canine tumors and related diseases, including promoting tumor shrinkage or effectively controlling tumor growth, and improving the quality of life of the affected dogs;
2) can be used for preparing antitumor drugs or compositions for dogs.
In a fifth aspect, the invention provides a canine antitumor drug or composition, and the effective component is the recombinant canine PD-1 fusion protein.
Such tumors include, but are not limited to, melanoma (e.g., malignant melanoma), lymphoma, mast cell tumor, sarcoma, head and neck tumors, non-small cell lung cancer, breast cancer, urothelial cancer, bladder cancer.
Furthermore, the invention also provides the application of the canine PD-1 fusion protein or the preparation thereof alone or in combination with one or more treatment methods such as operation, radiotherapy, chemotherapy, immunotherapy, hormone therapy, gene therapy, angiogenesis inhibition therapy, palliative treatment and the like for treating canine tumor-related diseases.
In a sixth aspect, the present invention provides a mutant of canine PD-1 protein or a mutant of canine PD-1 protein truncation, wherein the mutant contains a mutation site of T → L located at residue 132 of the amino acid sequence of canine PD-1 protein.
It is understood that engineered proteins, including fusion proteins of canine cPD1-Fc and cPD1mu-Fc fusion proteins or canine PD-1 extracellular domain and mutants thereof, fused to canine Fc or to other proteins without altering protein activity, are within the scope of the present invention.
Modified proteins, including two fusion proteins cPD1-Fc, cPD1mu-Fc or canine PD-1 and mutants thereof, are glycosylated, pegylated, acetylated or combined with BSA and the like, and all belong to the protection scope of the invention.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
compared with wild type PD-1, the two novel recombinant fusion proteins cPD1-Fc and cPD1mu-Fc provided by the invention have higher PD-L1 affinity and better drug effect, and provide more choices for immunotherapy of canine tumors.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis chart of cPD1-Fc and cPD1mu-Fc in example 2 of the present invention. Wherein, A: cPD1-Fc denatured electrophoretogram; b: cPD1-Fc, C: cPD1 denatured electrophoretogram of 1 mu-Fc; d: cPD1 mu-Fc. MK: a protein Marker; 1: clarifying the fermentation liquor; 2: collecting a Protein A liquid; 3: capto Q pool.
FIG. 2 shows cPD1-Fc and cPD1mu-Fc in example 4 of the present invention for the treatment of canine melanoma, interstitial sarcoma, and breast cancer. Wherein A, B, C, D, E indicates the change of tumor load of dogs with No. 6 (melanoma), No. 4 (melanoma), No. 13 (breast cancer), No. 25 (interstitial sarcoma) and No. 7 (melanoma), respectively.
Detailed Description
The technical scheme of the invention is as follows: the amino acid sequence of the canine PD-1 extracellular region or the mutant thereof is directly or indirectly fused to an Fc fragment through a connecting element to synthesize the recombinant canine PD-1 fusion protein: cPD1-Fc and cPD1 mu-Fc.
The cPD1-Fc fusion protein is a fusion protein of canine PD-1 extracellular region and is a protein consisting of an amino acid sequence shown in SEQ ID NO. 1; or a protein which has a homology of more than 90% with SEQ ID NO. 1 and has an amino acid sequence with the same function.
The cPD1mu-Fc fusion protein is a fusion protein of canine PD-1 extracellular region mutant and is a protein consisting of an amino acid sequence shown in SEQ ID NO. 3; or a protein consisting of an amino acid sequence which has homology of more than 90 percent with SEQ ID NO. 3 and has the same function. The fusion protein comprises a canine PD-1 protein mutant part, wherein Thr at the 132 th position of canine PD-1 participates in the binding interface of cPD-1 and cPD-L1, Thr is mutated into Leu, and the affinity of the fusion protein with a PD-L1 ligand is enhanced.
The fusion protein includes an Fc fragment comprising an immunoglobulin hinge region and CH2 and CH3 regions; the immunoglobulin is from mammal such as dog, cat, human, pig, etc.
The connection relation between the canine PD-1 extracellular region or the mutant thereof and Fc is direct splicing connection or connection through a Linker, preferably connection through the Linker; wherein the Linker is a flexible polypeptide consisting of 2-20 flexible amino acids selected from at least one of Gly, Ser, Ala and Thr; preferably, the Linker is (Gly-Gly-Gly-Gly-Ser) n, wherein n is an integer between 2 and 5, and more preferably n is 2.
Furthermore, the invention also provides a biological material of the DNA sequence for coding the fusion protein, and the biological material is an expression cassette, an expression vector, a cloning vector, an engineering bacterium or a transgenic cell line.
The invention provides a preparation method of recombinant canine PD-1 fusion protein, wherein cPD1-Fc and cPD1mu-Fc coding genes are artificially synthesized, codon optimization is carried out, and the optimized genes are cloned into a eukaryotic expression vector; respectively transforming the cPD1-Fc and cPD1mu-Fc recombinant vectors into eukaryotic cells, expressing the eukaryotic cells, and separating and purifying the target protein; preferably, the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cell comprises a CHO cell.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 construction of eukaryotic expression vectors for the fusion proteins of Canine cPD1-Fc and cPD1mu-Fc
Searching the amino acid sequences of canine PD-1 (UniProtKB: A0A024FCJ9) and canine Fc (GenBank: AF354264) in the UniProt library and the GenBank library; thr at position 132 of canine PD-1 is mutated to Leu to form cPD1 mu.
cPD1-Fc and cPD1mu-Fc were codon optimized, respectively, with the nucleotide sequence of cPD1-Fc being shown in SEQ ID NO:2 and the nucleotide sequence of cPD1mu-Fc being shown in SEQ ID NO: 4. The nucleotide is artificially synthesized and respectively constructed in pcDNA3.1 vector to obtain recombinant expression vectors pcDNA3.1-cPD1-Fc and pcDNA3.1-cPD1 mu-Fc. After being linearized, pcDNA3.1-cPD1-Fc and pcDNA3.1-cPD1mu-Fc recombinant vectors are respectively electrotransferred into CHO cells to obtain stable transgenic cell lines of cPD1-Fc and cPD1 mu-Fc.
Example 2 purification of Canine cPD1-Fc and cPD1mu-Fc fusion proteins
The stable transfused cells expressing the canine cPD1-Fc and cPD1mu-Fc are subjected to fermentation culture in a fermentation tank, the fermentation liquor is firstly subjected to two-stage deep filtration membrane package to remove cells and cell debris, and then is filtered by a 0.22 mu m filter membrane to obtain clear fermentation liquor. The fermentation broth was first purified by affinity chromatography Protein A (MabSelect SureTM, GE Healthcare): the column was equilibrated to baseline with an equilibration solution (50mM glycine, 0.15M NaCl, pH 7.2), and then eluted with an eluent (50mM glycine, pH 3.0) and collected. The Protein A pool was purified by anion exchange Capto Q (GE Healthcare) column chromatography: the collected solution was adjusted to pH 8.0 with 1M NaOH, equilibrated to baseline with an equilibration solution (50mM glycine pH 8.0), and then eluted with an eluent (50mM glycine, 0.2M KCl, pH 8.0) to collect the purified protein. The buffer was replaced after concentration by ultrafiltration. The purified target protein was subjected to SDS-PAGE gel electrophoresis (FIG. 1).
Example 3 dog cPD1-Fc and cPD1mu-Fc fusion protein affinity constant assays
Affinity analysis was performed using a BIAcore 3000 instrument. Three sample sets (cPD-1, cPD1-Fc, cPD1mu-Fc) were set up, recombinant canine PD-L1 (recombinant canine PD-L1 was prepared as in example 2, with the amino acid sequence shown in SEQ ID NO: 7) was first coupled to channel 2, and channel 1 was blocked as a control channel. Analytes (cPD-1, cPD1-Fc, cPD1mu-Fc) were flow-diluted in a gradient (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), binding curves to chip surface-coupled PD-L1 were determined, and the signal Fc2-1 was recorded using the software Wizard program. The affinity constant KD was determined by analysis in a 1:1 binding model. The measurement results show that: cPD1-Fc binding constant (K) to recombinant canine PD-L1a) Is 1.60X 105Dissociation constant (K)d) Is 5.12X 10-1Affinity constant (KD) of 3.2X 10-6M; cPD 1K of 1mu-Fc pair recombinant canine PD-L1aIs 2.60 multiplied by 105，KdIs 1.33X 10-2KD of 5.1X 10-8M; and cPD-1K against recombinant canine PD-L1aIs 1.05X 105，KdIs 9.45X 10-1KD of 9.0X 10-6And M. Indicating that both canine cPD1-Fc and cPD1mu-Fc can be usedThe affinity to cPD-L1 is improved, and the improvement effect of cPD1mu-Fc is more obvious.
Example 4 therapeutic Effect of the fusion proteins of Canine cPD1-Fc and cPD1mu-Fc on Canine malignancies
30 tumor dogs (including 12 melanoma dogs, 9 breast cancer dogs and 9 interstitial sarcoma dogs, table 1) were selected for clinical diagnosis in a pet hospital in Beijing, and a preliminary clinical effect study was performed. The test dog breeds include: golden retrievers, toy poodle dogs, giddles, SNARI, Harba dogs, dachshunds, Song Shi dogs, Bixiong dogs, labrador dogs and hybrid dogs, all of which have advanced malignant tumors and are 13 years old (11-16 years old).
TABLE 1 test dog Condition
All dogs were divided into three groups: cPD1-Fc, cPD1mu-Fc and a negative control group, each group comprises 4 melanoma dogs, 3 breast cancer dogs and 3 interstitial sarcoma dogs, the test group is intravenously dripped with cPD1-Fc and cPD1mu-Fc (diluted by physiological saline for injection) according to the dose of 2mg/kg body weight, the negative control group is intravenously dripped with physiological saline for administration once every two weeks for 4 times continuously, and the test is finished after the last administration for one week. In the test, the tumor load is evaluated through gross examination and computed tomography, the tumor size is measured once every two weeks to evaluate the curative effect, and the objective remission rate is compared.
Objective remission rate-complete and partial remission suffering from dog/total suffering from dog x 100%
Wherein complete remission refers to disappearance of all detectable tumors and partial remission refers to at least a 30% reduction in the sum of the maximum diameters of the target lesions.
The experimental results show that in the whole experimental process, all experimental animals are in good mental state, no animal death occurs, normal physical examination, whole blood count and serum chemical detection are carried out, no anaphylactic reaction or autoimmune disease is found, the overall state of a negative control group is poor, and most canine tumors are increased or newly increased. cPD1-Fc group had 2 partial remissions (the sum of the maximum diameter of the target focus is reduced by more than or equal to 30% and maintained for at least 4 weeks), and obvious tumor regression can be observed two weeks after treatment, and the tumor inhibition effect is obvious; the reduction of the tumor size of other dogs except the partial remission cases is not obvious or slightly increased, but the overall state (good spirit, normal diet and increased activity) of the dogs is obviously improved compared with that before treatment, and the objective remission rate of the overall treatment is 20 percent (2/10). cPD1 the dogs in the group 1mu-Fc had 3 partial remissions, and the reduction of the tumor size of dogs except the partial remissions was not obvious or slightly increased, but the overall state was obviously improved compared with that before the treatment, the tumor did not continue to deteriorate, the objective remission rate of the overall treatment was 30% (3/10), most of the tumors of the dogs in the negative control group were increased or new tumors appeared, and the state was poor, and the specific results are shown in Table 2 and FIG. 2.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
TABLE 2 effects of 2 cPD1-Fc and cPD1mu-Fc fusion proteins on tumor treatment
Note: partial Remission (PR): the sum of the maximum diameters of the target lesions is reduced by more than or equal to 30 percent and is maintained for at least 4 weeks; disease Stable (SD): the sum of the maximum diameters of the target lesions is reduced to miss PR or enlarged to miss PD; disease Progression (PD): the sum of the maximum diameters of the target lesions is at least increased by more than or equal to 20 percent, or new lesions appear.
Sequence listing
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<120> recombinant dog PD-1 fusion protein and preparation method and application thereof
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Ser Val Leu Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg Ile Thr
180 185 190
Arg Thr Pro Glu Val Thr Cys Val Val Leu Asp Leu Gly Arg Glu Asp
195 200 205
Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Glu Val His Thr
210 215 220
Ala Lys Thr Gln Ser Arg Glu Gln Gln Phe Asn Gly Thr Tyr Arg Val
225 230 235 240
Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu Thr Gly Lys Glu
245 250 255
Phe Lys Cys Arg Val Asn His Ile Asp Leu Pro Ser Pro Ile Glu Arg
260 265 270
Thr Ile Ser Lys Ala Arg Gly Arg Ala His Lys Pro Ser Val Tyr Val
275 280 285
Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr Val Ser Ile
290 295 300
Thr Cys Leu Ile Lys Asp Phe Tyr Pro Pro Asp Ile Asp Val Glu Trp
305 310 315 320
Gln Ser Asn Gly Gln Gln Glu Pro Glu Arg Lys His Arg Met Thr Pro
325 330 335
Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser
340 345 350
Val Asp Lys Ser Arg Trp Gln Gln Gly Asp Pro Phe Thr Cys Ala Val
355 360 365
Met His Glu Thr Leu Gln Asn His Tyr Thr Asp Leu Ser Leu Ser His
370 375 380
Ser Pro Gly Lys
385
<210> 4
<211> 1164
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctcgatagcc ccgacagacc ttggagccct ttaacattct cccccgctca gctgaccgtg 60
caagaaggag agaacgccac cttcacttgt tctttagccg acatccccga tagcttcgtg 120
ctgaattggt atcgtctgag ccccagaaac cagaccgaca agctcgccgc ctttcaagag 180
gatcgtatcg agcccggtcg tgataggaga tttcgtgtga ccagactccc taacggtcgt 240
gacttccaca tgagcatcgt ggccgctcgt ctgaatgaca gcggcattta tttatgcggc 300
gccatctatt taccccctaa cctccagatt aacgagtccc ctagagctga gctgtccgtg 360
acagagagga ctttagaacc ccctactcag agccccagcc ctcctcctag actgtccgga 420
cagctgcaag gtggcggagg cggctccggc ggaggaggaa gcgtgtttaa tgagtgtcgt 480
tgcacagaca cccccccttg tcccgttccc gaacctctgg gcggacctag cgtgctgatt 540
ttccccccca aacccaagga cattttaagg atcactcgta cccccgaggt gacttgtgtg 600
gtgctggatc tgggtcgtga agatcccgag gtgcagattt cttggttcgt ggacggaaag 660
gaggtgcata ccgccaagac ccagtctcgt gagcagcagt tcaacggcac ctacagagtg 720
gtgtccgtgc tgcccatcga gcatcaagat tggctgaccg gcaaagagtt caagtgtcgt 780
gtgaatcaca tcgacctccc cagccccatc gagaggacca tcagcaaggc tagaggtcgt 840
gctcataagc ctagcgtgta cgtgctcccc cctagcccca aggagctgag cagctccgac 900
accgtgtcca tcacttgtct catcaaggac ttctatcccc ccgacatcga cgtggaatgg 960
cagagcaacg gccagcaaga acccgagaga aaacatcgta tgacacctcc ccagctggac 1020
gaggacggct cctacttttt atattccaag ctgagcgtcg acaagtctcg ttggcaacaa 1080
ggtgacccct tcacatgcgc cgtgatgcac gagactttac aaaaccacta taccgatctg 1140
tctttatccc attcccccgg caag 1164
<210> 5
<211> 144
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Leu Asp Ser Pro Asp Arg Pro Trp Ser Pro Leu Thr Phe Ser Pro Ala
1 5 10 15
Gln Leu Thr Val Gln Glu Gly Glu Asn Ala Thr Phe Thr Cys Ser Leu
20 25 30
Ala Asp Ile Pro Asp Ser Phe Val Leu Asn Trp Tyr Arg Leu Ser Pro
35 40 45
Arg Asn Gln Thr Asp Lys Leu Ala Ala Phe Gln Glu Asp Arg Ile Glu
50 55 60
Pro Gly Arg Asp Arg Arg Phe Arg Val Thr Arg Leu Pro Asn Gly Arg
65 70 75 80
Asp Phe His Met Ser Ile Val Ala Ala Arg Leu Asn Asp Ser Gly Ile
85 90 95
Tyr Leu Cys Gly Ala Ile Tyr Leu Pro Pro Asn Thr Gln Ile Asn Glu
100 105 110
Ser Pro Arg Ala Glu Leu Ser Val Thr Glu Arg Thr Leu Glu Pro Pro
115 120 125
Thr Gln Ser Pro Ser Pro Pro Pro Arg Leu Ser Gly Gln Leu Gln Gly
130 135 140
<210> 6
<211> 144
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Leu Asp Ser Pro Asp Arg Pro Trp Ser Pro Leu Thr Phe Ser Pro Ala
1 5 10 15
Gln Leu Thr Val Gln Glu Gly Glu Asn Ala Thr Phe Thr Cys Ser Leu
20 25 30
Ala Asp Ile Pro Asp Ser Phe Val Leu Asn Trp Tyr Arg Leu Ser Pro
35 40 45
Arg Asn Gln Thr Asp Lys Leu Ala Ala Phe Gln Glu Asp Arg Ile Glu
50 55 60
Pro Gly Arg Asp Arg Arg Phe Arg Val Thr Arg Leu Pro Asn Gly Arg
65 70 75 80
Asp Phe His Met Ser Ile Val Ala Ala Arg Leu Asn Asp Ser Gly Ile
85 90 95
Tyr Leu Cys Gly Ala Ile Tyr Leu Pro Pro Asn Leu Gln Ile Asn Glu
100 105 110
Ser Pro Arg Ala Glu Leu Ser Val Thr Glu Arg Thr Leu Glu Pro Pro
115 120 125
Thr Gln Ser Pro Ser Pro Pro Pro Arg Leu Ser Gly Gln Leu Gln Gly
130 135 140
<210> 7
<211> 217
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Phe Thr Ile Thr Val Ser Lys Asp Leu Tyr Val Val Glu Tyr Gly Gly
1 5 10 15
Asn Val Thr Met Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asn Leu
20 25 30
Phe Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Lys Ile Ile Gln
35 40 45
Phe Val Asn Gly Lys Glu Asp Leu Lys Val Gln His Ser Ser Tyr Ser
50 55 60
Gln Arg Ala Gln Leu Leu Lys Asp Gln Leu Phe Leu Gly Lys Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Arg Leu Gln Asp Ala Gly Val Tyr Cys Cys
85 90 95
Leu Ile Gly Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val
100 105 110
His Ala Pro Tyr Arg Asn Ile Ser Gln Arg Ile Ser Val Asp Pro Val
115 120 125
Thr Ser Glu His Glu Leu Met Cys Gln Ala Glu Gly Tyr Pro Glu Ala
130 135 140
Glu Val Ile Trp Thr Ser Ser Asp His Arg Val Leu Ser Gly Lys Thr
145 150 155 160
Thr Ile Thr Asn Ser Asn Arg Glu Glu Lys Leu Phe Asn Val Thr Ser
165 170 175
Thr Leu Asn Ile Asn Ala Thr Ala Asn Glu Ile Phe Tyr Cys Thr Phe
180 185 190
Gln Arg Ser Gly Pro Glu Glu Asn Asn Thr Ala Glu Leu Val Ile Pro
195 200 205
Glu Arg Leu Pro Val Pro Ala Ser Glu
210 215
Claims (12)
1. The recombinant canine PD-1 fusion protein is characterized in that the fusion protein is formed by directly or indirectly connecting an extracellular region of canine PD-1 protein or an extracellular region mutant of canine PD-1 protein with an Fc fragment;
the canine PD-1 protein extracellular region is as follows:
(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 5;
the canine PD-1 protein extracellular region mutant is as follows:
(c) a protein consisting of an amino acid sequence shown as SEQ ID NO. 6;
wherein the Fc fragment is derived from a canine.
2. The fusion protein of claim 1, wherein the Fc fragment comprises immunoglobulin hinge, CH2, and CH3 regions.
3. The fusion protein of claim 1, wherein the fusion protein is formed by connecting the canine PD-1 protein extracellular region or the canine PD-1 protein extracellular region mutant with an Fc fragment directly or indirectly through a Linker; the Linker is a flexible polypeptide consisting of 2-20 flexible amino acids, and the flexible amino acids are selected from at least one of Gly, Ser, Ala and Thr.
4. The fusion protein of claim 3, wherein the Linker is (Gly-Gly-Gly-Gly-Ser) n, wherein n is an integer between 2 and 5.
5. The fusion protein of claim 4, wherein n is 2.
6. The fusion protein of claim 1, wherein the fusion protein is cPD1-Fc or cPD1 mu-Fc;
fusion protein cPD1-Fc is:
(e) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;
fusion protein cPD1mu-Fc is:
(g) a protein consisting of an amino acid sequence shown as SEQ ID NO. 3.
7. A biological material comprising a gene encoding the fusion protein of any one of claims 1 to 6, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or an engineered bacterium.
8. A method for producing a fusion protein according to any one of claims 1 to 6, said method comprising: artificially synthesizing the encoding gene of the fusion protein, carrying out codon optimization, constructing the optimized gene into a eukaryotic expression vector, transferring the eukaryotic expression vector into a eukaryotic cell, expressing the eukaryotic expression vector in the eukaryotic cell, and separating and purifying the target protein.
9. The method according to claim 8, wherein the eukaryotic expression vector is pcDNA3.1 and the eukaryotic cell is a CHO cell.
10. Use of the fusion protein of any one of claims 1-6 for the preparation of a canine anti-tumor drug or composition, wherein the tumor is canine melanoma, canine interstitial sarcoma, canine breast cancer.
11. A canine antitumor drug or composition, characterized in that the effective ingredient is the fusion protein according to any one of claims 1 to 6.
12. The canine PD-1 protein mutant is characterized in that the mutant contains a mutation site of T → L positioned at the 132 th residue of the canine PD-1 protein amino acid sequence, and the mutant consists of the amino acid sequence shown in SEQ ID NO. 6.
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| PCT/CN2019/122524 WO2021051661A1 (en) | 2019-09-19 | 2019-12-03 | Recombinant canine pd-1 fusion protein and preparation method therefor and application thereof |
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| CN110590959A (en) | 2019-12-20 |
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