CN108285493A - It is a kind of restore debilitating immune cell function fusion protein and its application - Google Patents
It is a kind of restore debilitating immune cell function fusion protein and its application Download PDFInfo
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Abstract
The present invention relates to a kind of fusion proteins restoring failure immune cell function, and it includes the functional areas of the functional areas of identification failure immunocyte and activation amplification failure immunocyte, two functional areas are connected by the non-functional amino acid fragment of certain length；Wherein, the amino acid sequence of the fusion protein is SEQ ID NO：Sequence shown in 5 or at least 90% with similar functions similar sequences；The invention further relates to the applications of the fusion protein.Artificial fusion protein of the present invention can identify debilitating immunocyte, the immunocyte that amplification can be activated to identify again, restore the function of immunocyte killing antigen-positive cell, fusion protein prepared by the present invention is compared with prior art, which further improves the proliferation effectiveness to CD8 effector cell, also increase the activation of effector cell and the expression of costimulatory molecules simultaneously, the function of inhibiting tumour growth and control virus infection can be further enhanced, there are good clinical landscapes and be widely applied range.
Description
Technical field
The present invention relates to fusion protein technology field more particularly to a kind of fusion eggs restoring debilitating immune cell function
It is white and its preparation method and application.
Background technology
Tumour is due to generating gene mutation in body cell fission process, and the growth of mutant cell loses adjusting
The result of control.If tumour cell cannot be eliminated in a short time, lasting antigenic stimulus can gradually cause tumour antigen special
Function sexual exhaustion (the Nature Medicine 1999,5 of specific T cell:677-685), the tolerance to tumour, siberian crabapple are formed
System is no longer sensitive to tumour cell, and tumour is caused further to grow and spread, and formation cancer (PNAS.2002,99:12293-
7).The generation of chronic disease is caused to be also the result (Trends of antigen-specific cellular failure by virus infection
Immunol.2014,35:51-60；Blood 2007,109:4671-4678；Cell Death and Disease 2015,
6:e1694).Studies have shown that all high expression of most of tumor-infiltrating lymphocyte (TIL) and antiviral specific T-cells is exempted from
Epidemic disease Inhibitory receptor (PNAS 2010,107:7875-7880；JI 2007,178:2714-2720), it is in depletion, is lost
The function of specific recognition and killing antigen positive target cell.Debilitating T cell loses the energy of secreting function cell factor
Power, such as gamma interferon (IFN γ) (Blood 2013,121:1367-1376；J.Biomed.Biotechnol. 2011,
451694).Even if there are a large amount of specific T-cells in body, it is thin that the immunocyte of failure can not remove antigen positive target
Born of the same parents (J Immunol 2005；175:6169-6176).In phenotype, the immunosupress inspection of the universal high expression of debilitating T cells
It includes programmed death receptor (PD-1), programmed death ligand (PD-L1/L2), T cell immunoglobulin 3 to make an inventory of receptor
(Tim-3), (Nat such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphocyte activation gene 3 (Lag-3)
Immunol.2011；12:492-9).The activation of these signal paths can be slackened by inducing phosphatase to inhibit phosphorylation reaction
Activated immune cell signal inhibits the amplification of T cell, reduces the function of effector T cell, promotes the apoptosis of T cell, causes to be immunized
Tolerance (J Clin Invest.2011,121:2350-2360).So activating the immunocyte group of this kind of antigentic specificity
Body allows them to restore to kill the function of antigen positive target cell again, could remove tumour cell or virus infected cell
(Trends Immunol.2015；36:265-276), it is finally reached the purpose of healing.
Antibody can specifically identify that the proteantigen of target cell surface, checkpoint inhibiting antibody can suppress failure
The inhibitive ability of immunity signal of property cell surface, such as pass through PD-1 antibody (Pembrolizumab and Nivolumab) and PD-1
Combination prevent the conduction and activation of inhibition signal, restore the function of debilitating immunocyte, clinically display is controlled
The clinical effectiveness for treating cancer, has especially obtained cases of complete remission and longer clinical effectiveness duration with some patientss
(N Engl J Med 2012,366:2455-2465；N Engl J Med 2012,366:2443-2454).But to lacking
Also undesirable (the NATURE 2014,515 of patient clinical effect of effector cell:568-571), and the recovery of this T cell is held
The continuous time short (Science 2016,354:1160-1165), the specific immune cell of patient's body can return again quickly again
To depletion (Science 2016,354:1165-1169).Meanwhile only blocking t cell inhibits signal not cause to be immunized
The increase of cell quantity, and the patient for lacking T effector cell not will produce clinical effectiveness (Nature 2014,515 substantially:
568-571), so further limiting the clinical therapeutic efficacy of checkpoint antibody.
Has the effect of certain and different degrees of side effect (Semin using white blood cell growth factors treatment tumour
Oncol.2015,42:539–548).The clinical application of interleukin-22 (IL-2) is the immunotherapy for the treatment of of cancer first, can be thorough
The tumour cell of patient's body is removed at bottom, and in part, cancer patient achievees the effect that cure and long term survival (Cancer completely
2008,113:293–301；JCO 2005,23:133-141).But cell factor all can to all cells for expressing its receptor
Activation is generated, leads to undershooting-effect so as to activate non-mesh cell, clinical application will produce a series of secondary work of poison
With limiting its clinical application (JI 2014,192:5451-5458), so the clinical application range and curative effect of current IL-2
There is very big limitation (Immunity 2013,38:13-25).
Up to now, the tumor specific T cells of failure can pointedly be identified and restore its work(there are no a kind of
It can and expand the protein drug of its quantity.Therefore it is badly in need of a kind of immune substance that can identify simultaneously specific amplification immunocyte of exploitation
Object, to overcome the shortcomings of existing clinical medicine.
Invention content
It is an object of the invention to overcome the shortcomings of existing clinical medicine, a kind of recovery debilitating immune cell function is provided
Fusion protein, not only can recognize that debilitating immunocyte, but can specific amplification immunocyte quantity, restore immunocyte
Function.
To achieve the above object, the present invention adopts the following technical scheme that：
The first purpose of the invention is to provide a kind of fusion proteins restoring debilitating immune cell function, including identification
The functional areas of the functional areas and activation amplification debilitating immunocyte of debilitating immunocyte, two functional areas pass through certain length
Non-functional amino acid fragment connection, to avoid the protein structure of two functional areas from interfering with each other, ensure fusion protein
Difunctional feature, while having the function of new immune function.
In order to advanced optimize above-mentioned technical proposal, the technical measures that the present invention is taken further include：
Further, the functional areas of identification debilitating immunocyte are immune thin using PD-1 single-chain antibodies identification debilitating
Debilitating immunocyte is activated in the functional areas of the receptor PD-1 of born of the same parents' phenotype, activation amplification debilitating immunocyte using IL-2,
Fusion protein amino acid sequence after being connected by nonfunctional area is SEQ ID NO：Shown in 5.
Further, it is described identification debilitating immunocyte functional areas be identify debilitating immunocyte phenotype by
Body, the phenotype receptor of the debilitating immunocyte are the immunologic test point PD-1 for having co-suppression sexual function.This receptoroid base
Because that can be over-expressed on immunocyte surface, the activation of this signal path can cause the failure of immunocyte and function to be lost.
Further, the amino acid sequence of the PD-1 is SEQ ID NO：Sequence (GenBank shown in 1:
L27440.1)。
Further, identify that the antibody of the PD-1 is PD-1 single-chain antibodies, the amino acid sequence of the PD-1 single-chain antibodies
It is classified as SEQ ID NO：Sequence shown in 2 or at least 90% similar sequences.
Further, the functional areas of the activation amplification debilitating immunocyte are similar prominent using cell factor or function
Variant activity antibody activates debilitating immunocyte, and the functional areas of the activation amplification debilitating immunocyte use cell
The amino acid series of the factor IL-2, the IL-2 are SEQ ID NO：Sequence (GenBank shown in 3:S77834.1) or or at least
90% similar sequences, receptor are mainly expressed in T cell and NK cell surfaces.Cell factor or function are similar to mutant
Refer under the premise of not changing its basic function, by Amino acid sequence mutants or just with functional areas polypeptide, it is right to increase its
The activating reaction of aim cell reduces its effect to non-aim cell.For example, by the change of IL-2 upper amino acid sequences,
Mutant may be decreased or increase and the activation of T regulatory cells, effect of the reduction to non-target cell and histoorgan, to subtract
The side effect of few clinical application.
Further, the amino acid sequence of the nonfunctional area amino acid fragment is SEQ ID NO：Sequence shown in 4, or extremely
Few 90% similar sequences.
Further, the debilitating immunocyte be failure specific T-cells or failure NK cells at least
It is a kind of.
Above-mentioned each amino acid sequence is as shown in the table：
Further, the preparation method of the fusion protein includes the following steps：
Step 1) passes through non-functional amino acid by the C-terminal of people's PD-1 single chain antibodies and the N-terminal amino acid sequence of interleukin-22
Segment connects, and forms fusion protein structural gene；
Fusion protein structural gene described in step 1) is transferred to eukaryotic expression vector by step 2), and is transfected to hamster
Gonad cell (CHO)；
Hamster ovary cell described in step 2) is placed in incubator and cultivates a period of time by step 3), takes supernatant, passes through
Recombination fusion protein is made after affinity purification.
Further, in above-mentioned preparation method, the amino acid sequence such as SEQ ID NO of the PD-1 single chain antibodies：2 institutes
Show, the amino acid sequence such as SEQ ID NO of the interleukin-22：Shown in 3, the amino acid sequence of the non-functional amino acid fragment
Row such as SEQ ID NO：Shown in 4, the amino acid sequence such as SEQ ID NO of the recombination fusion protein：Shown in 5.
Further, the eukaryotic expression vector is pcDNA3.1.
Second purpose of volume of the present invention is to provide a kind of fusion protein application restoring debilitating immune cell function, this melts
The medical usage of hop protein is used for non-diagnostic or non-treatment purpose.
Further, the application is being used alone or being fusion protein and chemotherapy, targeted drug, antibody for fusion protein
The use in conjunction of drug, cell therapy composition and radiotherapy causes disease for preparing treatment because of immunocyte failure
Drug.
Further, the disease includes cancer and chronic viral infection disease.
Further, the cancer includes clear-cell carcinoma, melanoma, lymthoma, colorectal cancer, liver cancer, soft nest cancer, head
Neck squamous cell carcinoma, carcinoma of urinary bladder, lung cancer；Virus in the chronic viral infection disease includes HIV, HBV, HCV, EBV, HPV,
CMV。
Compared with prior art, fusion protein of the present invention has unique inside and outside immunocompetence, and has
Following advantageous effect：
Fusion protein of the present invention disclosure satisfy that the needs for restoring patient immune function, which can
It identifies the immunocyte of failure, and its quantity can be expanded, restore its function；And its clinical application can enhance inhibition tumour growth
With the function of control virus infection, there are good clinical landscapes and be widely applied range.
Inventor has applied for that this is specially about a kind of patent for the fusion protein restoring debilitating immune cell function before
Sharp Publication No. CN107082812A, the patent Example prepare fusion protein be double-strand, use PD-1 heavy chain of antibody and
It is prepared by PD-1 antibody light chains.The present invention is further innovated on the basis of above-mentioned patent, is prepared using PD-1 single-chain antibodies
Fusion protein improves the proliferation effect to CD8 effector cell compared with fusion protein prepared by patent CN107082812A
With, while the activation of effector cell and the expression of costimulatory molecules are also increased, it can preferably inhibit tumour growth and control
System virus infection.
Description of the drawings
Fig. 1 is the recombination fusion protein gel electrophoresis analysis figure prepared in one embodiment of the invention；A bands are under reducing condition
Albumen, B band be reset condition under albumen, MK be protein standard molecular weight label.
Fig. 2 is the CD137 of the recombination fusion protein human PBMC's in vitro test lymphocyte prepared in one embodiment of the invention
Activation expression schematic diagram.
Fig. 3 is that the recombination fusion protein human PBMC's in vitro test prepared in one embodiment of the invention shields in lymphocyte
The schematic diagram of PD-1 signals.
Fig. 4 is that the recombination fusion protein people's ascites in vitro test prepared in one embodiment of the invention increases in CD8+T cells
The schematic diagram of cytokine-expressing.
Fig. 5 be one embodiment of the invention in recombination fusion protein In-vivo test in mice CD8+T cells and NK cells outside
Ratio schematic diagram in all blood lymphocytes.
Fig. 6 is that the recombination fusion protein in one embodiment of the invention increases the ratio illustration that mouse tumor invades profit CD8+T cells
It is intended to.
Fig. 7 is that the ratio of the recombination fusion protein increase NSG mouse transplanted with human PBMCs cells in one embodiment of the invention illustrates
It is intended to.
Fig. 8 is that the recombination fusion protein in one embodiment of the invention increases mouse transplanting human lymphocyte in NSG tumours
Invade the ratio schematic diagram of profit.
Fig. 9 be not plus protein groups compare (CTRL), the fusion protein (α PD1scFvFcIL2) in one embodiment of the invention,
Recombination fusion protein (α PD1-IL2) induces the ratio schematic diagram of CD8+ cells；
Figure 10 is not plus protein groups compare (CTRL), using the fusion protein (α in one embodiment of the invention
PD1scFvFcIL2 it) handles, illustrated using the ratio of the CD8 cells of CD137+ after recombination fusion protein (α PD1-IL2) processing
Figure.
Specific implementation mode
The present invention provides a kind of fusion proteins restoring debilitating immune cell function, including identification debilitating is immune thin
The functional areas of the functional areas and activation amplification debilitating immunocyte of born of the same parents, the non-functional ammonia that two functional areas pass through certain length
Base acid fragment connects.Draw because of immunocyte failure the present invention also provides the preparation method of above-mentioned fusion protein and its in treatment
Play the application in disease.
With reference to the accompanying drawings and examples, the specific implementation mode of the present invention is further described.Following embodiment is only
For clearly illustrating technical scheme of the present invention, and not intended to limit the protection scope of the present invention.
Embodiment 1
The present embodiment is the gene constructed of recombination fusion protein and production purifying.
According to people PD-1 single-chain antibodies (SEQ ID NO：2) C-terminal and interleukin-22 (SEQ ID NO：3) N-terminal amino acid
Sequence, by gene chemical synthesis, digestion and further clone pass through non-functional artificial constructed amino acid (SEQ ID NO：4) by two
Part connects, composition fusion protein manual construction gene order (SEQ ID NO：5), then it is transferred to eukaryotic expression vector
pcDNA3.1(-).Finally the expression vector of fusion protein is transfected into Chinese hamster ovary cell (CHO).The cell of transfection
It is placed in 37 DEG C, 5%CO2It is cultivated in incubator, supernatant is taken after 72 hours, further by the affinitive layer purification of Protein A,
The albumen of final purification is that artificial protein (α PD1scFvFcIL2) is merged in difunctional recombination.Meanwhile utilizing PD-1 antibody sequences
The Fc fusion proteins prepared with interleukin-22 sequence are as non pregnant women experimental control (α PD1scFvFc, IL2Fc).Such as Fig. 1
It is shown, confirm that the fusion protein molecule amount of purifying is about 67kD by electrophoresis detection, it was demonstrated that the recombination designed according to the present invention
Fusion artificial protein can be produced by Chinese hamster ovary celI.It finally with spectrophotometer test proteins concentration and is diluted in PBS, uses
Make active testing and the functional study of further inside and outside.
Embodiment 2
The present embodiment is activity and functional examination of the difunctional recombination fusion protein to human PBMC's cell injuring model.
Human peripheral uses X- through being isolated and purified using lymphocyte density-gradient centrifugation method (Ficoll) in 24 orifice plates
It is 5 × 10 that Vivo15 culture mediums, which are diluted to cell density,6/ ml, it is 200ng/ml that experiment albumen to ultimate density, which is added,.Then
It is placed in 37 DEG C, 5%CO2Cultivated 72 hours in incubator, cell collect after with streaming antibody dye, after washing with flow cytometer into
Row phenotype test and data analysis.It is shown in Fig. 2, with not plus compared with protein groups control, PD-1 antibody (α PD1scFvFc) cannot
The expression (0.06%vs 0.09%) of CD137 is induced, and fusion protein (α PD1scFvFcIL2) processing can dramatically increase CD8-
With the expression (0.96%) of CD137 in CD8+ cells.Meanwhile as Fig. 3 is shown, culture rear fusion protein (α PD1scFvFcIL2)
(0.037%, 0.01%) is reduced with PD-1+ fluorescence intensities in PD-1 antibody (α PD1scFvFc) processing group, and interleukin-22
(IL2Fc) fluorescence intensity of processing group PD-1+ increases (0.81%).Experiment proves that egg is merged in the recombination designed according to the present invention
It is white to be not only able to activation human PBMC's cell, while reducing or shielding the signal of cell surface PD-1+.
Embodiment 3
The present embodiment is measurement of the difunctional recombination fusion protein to people's ascites immunologic cellular activity and function.
Human lung cancer patient's ascites is taken, it is 200ng/ml that experiment albumen to ultimate density is added in 24 orifice plates.It is subsequently placed in
37 DEG C, 5%CO2It is taken out after being cultivated 72 hours in incubator, cell is dyed after collecting with CD8 and PD-1 streaming antibody, then with wearing
Dye in TNF α and the IFN γ streaming antibody in CD8+ cells is carried out after the element broken cell film of hole, is carried out with flow cytometer after washing
Phenotype test and data analysis.As shown in Fig. 4, compared with the PD-1 antibody (α PD1scFvFc) not merged, fusion protein (α
PD1scFvFcIL2) processing can dramatically increase the expression of the TNF α (13.0%vs 5.29%) of PBMC in ascites.
Embodiment 4
The present embodiment is influence of the difunctional recombination fusion protein to vivo immunization effector cell.
Inject within every 2 days fusion proteins (α PD1scFvFcIL2) (10 μ g/ are only) or interleukin-22 respectively by abdominal cavity (IP)
(IL2Fc) (10 μ g/ only) or anti-PD1 albumen (α PD1scFvFc) (10 μ g/ are only) or the PBS (control group) of respective volume in
In C57B6 test mices, peripheral blood is taken from tail portion within the 7th day, dyed with the streaming antibody of anti-mouse CD8, CD4, CD25 and NK1.1
After detect, data analysis is as shown in Figure 5.With control group (PBS), interleukin-22 (IL2Fc) and anti-PD1 albumen (α PD1scFvFc)
Group is compared, and recombination fusion protein (antiPD1scFvFcIL2) can significantly increase CD8 and NK cells in lymphocyte
Ratio.In vivo studies proves that the fusion protein immunization effect that the present invention synthesizes is more than the immunization of non pregnant women.
Embodiment 5
The present embodiment is that fusion protein increases the ratio that profit lymphocyte is invaded in tumour.
In the subcutaneous vaccination 1 × 10 of every C57B6 mouse6Mouse lung carcinoma cell (LLC), waits gross tumor volumes to rise to 100mm3
When pass through every 2 days of abdominal cavity injection fusion proteins (α PD1scFvFcIL2) (10 μ g/ are only) or interleukin-22 (IL2Fc) (10 μ respectively
G/ is only) or anti-PD1 albumen (α PD1scFvFc) (10 μ g/ only) or respective volume PBS (control group), total co-injection 5 times.20th
It when take tumor tissues grinding and be filtered into unicellular, the ratio of staining analysis tumour medium size lymphocyte is carried out with streaming antibody,
Data analysis is as shown in Figure 6.Compared with anti-PD1 albumen (α PD1scFvFc) (0.22%) or PBS groups (0.32%), recombination fusion
Albumen (α PD1scFvIL2) (0.95%) significantly increases the ratio of CD8+ lymphocytes in tumor tissues, it was demonstrated that fusion protein
Tumor lympha cell can be increased invades profit.
Embodiment 6
The present embodiment is the amplification that fusion protein increases that NSG mouse transplant human lymphocyte.
From the tail vein of NSG mouse inoculation 4 × 106Human PBMC's cell starts to inject within every 2 days by abdominal cavity respectively on the 8th day
Fusion protein (α PD1scFvFcIL2) (10 μ g/ are only) or interleukin-22 (IL2Fc) (10 μ g/ are only) or anti-PD1 albumen (α
PD1scFvFc) the PBS (control group) of (10 μ g/ are only) or respective volume, total co-injection 3 times.At the 12nd day 50 are taken by tail vein
μ l peripheral bloods, be added streaming antibody dyed, break it is red after with stream type cell analyzer analyze people CD45+ cells in periphery blood strangury
Ratio in bar cell, data analysis are as shown in Figure 7.Before no progress protein injection (the 7th day), people CD45+ in each group
Cell proportion is essentially identical.And at the 12nd day, interleukin-22 (IL2Fc) or anti-PD1 albumen (α PD1scFvFc) or PBS group bases
Originally people's CD45 positive cells are not detected, and recombination fusion protein (α PD1scFvIL2) group people's CD45+ cells are more than 27%.
Originally experiments have shown that, fusion protein has the function of that non pregnant women is not had, and only fusion protein of the invention could be significantly
The ratio for increasing people CD45+ in NSG mouse, causes immune cell expansion.
Embodiment 7
The present embodiment is that fusion protein increases mouse in NSG tumours and transplants human lymphocyte and invades profit.
In the subcutaneous vaccination 1 × 10 of NSG mouse6Human lung cancer cell A549, etc. tumour growths to area 100mm2, pass through tail
Intravenous inoculation 2 × 106Human PBMC's cell, while passing through every 2 days injection fusion protein (α PD1scFvFcIL2) (the 10 μ g/ in abdominal cavity
Only) or interleukin-22 (IL2Fc) (10 μ g/ only) or anti-PD1 albumen (α PD1scFvFc) (10 μ g/ are only), total co-injection 2 times.The
Tumor tissues are taken at 21 days, the unicellular rear streaming antibody that is added is ground into and is dyed, people is analyzed with stream type cell analyzer
Ratio of the CD45+ cells in tumour, data analysis are as shown in Figure 8.At the 21st day, interleukin-22 (IL2Fc) or anti-PD1 albumen
(α PD1scFvFc) group people's CD45 positive cell ratios are respectively 1.18% and 0.11%, and recombination fusion protein (α
PD1scFvIL2) group people's CD45+ cell proportions reach 10.4%.Originally experiments have shown that, fusion protein can significantly increase NSG mouse
The ratio of people CD45+ in human tumour, induces the neoplasm invasiveness of immunocyte.
Comparative example
The recombination fusion protein (α PD1-IL2) prepared using embodiment 1 in Chinese patent CN107082812A, with this hair
Recombination fusion protein (α PD1scFvFcIL2) prepared by bright embodiment 1 is compared, external activity, immune effector cell ratio
Example and active comparison are as follows：
Human peripheral uses X- through being isolated and purified using lymphocyte density-gradient centrifugation method (Ficoll) in 24 orifice plates
It is 5 × 10 that Vivo15 culture mediums, which are diluted to cell density,6/ ml, it is 200ng/ml that experiment albumen to ultimate density, which is added,.Then
It is placed in 37 DEG C, 5%CO2Cultivated 72 hours in incubator, cell collect after with streaming antibody dye, after washing with flow cytometer into
Row phenotype test and data analysis.It shows in Fig. 9, with not plus compared with protein groups control (CTRL), implements in CN107082812A
Recombination fusion protein (α PD1-IL2) induction cd8 cell ratio prepared by example 1 is (6.29%vs 5.03%), and in the present invention
The CD8+ cells of fusion protein (α PD1scFvFcIL2) processing are 7.94%.Meanwhile as Figure 10 is shown, the present invention is melted after culture
The cd8 cell ratio of CD137+ is 21.4% after hop protein processing, and the CD137+ of control group (CTRL) and α PD1-IL2 processing
Cd8 cell ratio be 2.06% and 4.07%.Experiment proves that the recombination fusion protein designed according to the present invention not only increases
CD8+ effector cell's ratio, while promoting the expression of effector cell's activation signals (CD137).
By above-described embodiment it is found that the fusion protein of the present invention for restoring debilitating immune cell function, Ji Nengshi
Other debilitating immunocyte, and amplification immunocyte can be activated, restore the function of immunocyte killing antigen-positive cell.Fusion
Albumen has the function of that non pregnant women has, and immune cell activated and can shield the signal of PD1, while only merging
The quantity of people's CD45+ cells in albumen ability directed expansion NSG mouse, parent's profit of induction immunocyte tumour.Due to tumour
Generation and diffusion and chronic viral infection are that immunocyte failure is resistant to immune system as a result, and restoring debilitating and being immunized carefully
The function of born of the same parents and the quantity of amplification immunocyte can enhance the ability of the antitumor and anti-chronic viral infection of body, and the present invention
Compared with prior art, which further improves the proliferation effectiveness to CD8 effector cell for the fusion protein prepared, while
The activation of effector cell and the expression of costimulatory molecules are increased, by parity of reasoning, and the clinical application of above-mentioned fusion protein can be more
Inhibit tumour growth and control virus infection well, there are good clinical landscapes and be widely applied range；And
Above-mentioned difunctional recombination fusion protein can be used alone or and chemotherapy, targeted drug, antibody drug, cell therapy and
Radiotherapy forms use in conjunction, is used to prepare the drug for the treatment of disease because of caused by immunocyte failure, such as treats cancer
The drug etc. of disease drug and treatment chronic viral infection.
Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the practicality carry out equivalent modifications and replace
In generation, is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and repair
Change, all should be contained within the scope of the invention.
Sequence table
<110>Shanghai Bioisystech Co., Ltd of Ke Yi Linkages
<120>It is a kind of restore debilitating immune cell function fusion protein and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 268
<212> PRT
<213> PD-1(Artificial Sequence)
<400> 1
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr
1 5 10 15
Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe
20 25 30
Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr
35 40 45
Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu
50 55 60
Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu
65 70 75 80
Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn
85 90 95
Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala
100 105 110
Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg
115 120 125
Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Ser Ala Gly
130 135 140
Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly Leu Leu Gly Ser
145 150 155 160
Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala
165 170 175
Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp
180 185 190
Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe
195 200 205
Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu
210 215 220
Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser
225 230 235 240
Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro
245 250 255
Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
260 265
<210> 2
<211> 243
<212> PRT
<213>PD-1 single-chain antibodies (Artificial Sequence)
<400> 2
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
130 135 140
Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser
145 150 155 160
Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
165 170 175
Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp
180 185 190
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
195 200 205
Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
210 215 220
Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
<210> 3
<211> 133
<212> PRT
<213> IL-2（Proleulzin）(Artificial Sequence)
<400> 3
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 4
<211> 231
<212> PRT
<213>Non-functional amino acid fragment (Artificial Sequence)
<400> 4
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro
1 5 10 15
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
20 25 30
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
35 40 45
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
50 55 60
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
65 70 75 80
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
85 90 95
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
100 105 110
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
115 120 125
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
130 135 140
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
145 150 155 160
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
165 170 175
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
180 185 190
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
195 200 205
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
210 215 220
Gly Gly Ser Gly Gly Gly Ser
225 230
<210> 5
<211> 607
<212> PRT
<213>Recombination fusion protein (Artificial Sequence)
<400> 5
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
130 135 140
Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser
145 150 155 160
Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
165 170 175
Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp
180 185 190
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
195 200 205
Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
210 215 220
Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Leu Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser
465 470 475 480
Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln
485 490 495
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg
500 505 510
Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys
515 520 525
His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu
530 535 540
Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile
545 550 555 560
Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
565 570 575
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu
580 585 590
Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
595 600 605
Claims (10)
1. a kind of fusion protein restoring debilitating immune cell function, including identifying the functional areas of debilitating immunocyte and swashing
The functional areas of amplification debilitating immunocyte living, two functional areas are connected by the non-functional amino acid fragment of certain length,
The functional areas of the identification debilitating immunocyte are the phenotype receptors for identifying debilitating immunocyte, and the activation expands failure
Property immunocyte functional areas be to activate debilitating immune thin similar to mutant activity antibody using cell factor or function
Born of the same parents, which is characterized in that the amino acid sequence of the fusion protein is SEQ ID NO：Sequence shown in 5 or with similar functions extremely
Few 90% similar sequences.
2. a kind of fusion protein restoring debilitating immune cell function according to claim 1, which is characterized in that described
The phenotype receptor of debilitating immunocyte is PD-1, the amino acid sequence such as SEQ ID NO of the PD-1：Shown in 1.
3. a kind of fusion protein restoring debilitating immune cell function according to claim 2, which is characterized in that be used for
Identify that the antibody of the PD-1 is PD-1 single-chain antibodies, the amino acid sequence such as SEQ ID NO of the PD-1 single-chain antibodies：2 institutes
Show or at least 90% similar sequences.
4. a kind of fusion protein restoring debilitating immune cell function according to claim 1, which is characterized in that described
Cell factor or function are IL-2, the amino acid sequence such as SEQ ID NO of the IL-2 similar to mutant：Shown in 3 or at least
90% similar sequences.
5. a kind of fusion protein restoring debilitating immune cell function according to claim 1, which is characterized in that described
The amino acid sequence of non-functional amino acid fragment such as SEQ ID NO：Shown in 4 or at least 90% similar sequences.
6. a kind of fusion protein restoring debilitating immune cell function according to claim 1, which is characterized in that described
Debilitating immunocyte be the specific T-cells of failure, failure at least one of NK cells.
7. a kind of fusion protein restoring debilitating immune cell function according to claim 1, which is characterized in that described
The preparation method of fusion protein includes the following steps：
Step 1) passes through non-functional amino acid fragment by the C-terminal of people's PD-1 single chain antibodies and the N-terminal amino acid sequence of interleukin-22
Connection forms fusion protein structural gene；
Fusion protein structural gene described in step 1) is transferred to eukaryotic expression vector by step 2), and is transfected to Hamster Qvary
Cell；
Hamster ovary cell described in step 2) is placed in incubator and cultivates a period of time by step 3), supernatant is taken, through affine
Recombination fusion protein is made after purification；
Wherein, the amino acid sequence of the PD-1 single chain antibodies such as SEQ ID NO：Shown in 2, the amino acid sequence of the interleukin-22
Row such as SEQ ID NO：Shown in 3, the amino acid sequence such as SEQ ID NO of the non-functional amino acid fragment：It is described shown in 4
The amino acid sequence of recombination fusion protein such as SEQ ID NO：Shown in 5.
8. a kind of fusion protein restoring debilitating immune cell function according to claim 7, which is characterized in that described
Eukaryotic expression vector is pcDNA3.1.
9. a kind of application of the fusion protein such as according to any one of claims 1 to 8 restoring debilitating immune cell function.
10. application according to claim 9, which is characterized in that the application is that fusion protein is preparing treatment because immune
Cell failure and the application in causing the drug of disease.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734498A (en) * | 2019-10-15 | 2020-01-31 | 上海科棋药业科技有限公司 | fusion protein for relieving immunosuppression and application thereof |
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|---|---|---|---|---|
| CN104334573A (en) * | 2012-04-30 | 2015-02-04 | 比奥孔有限公司 | Targeted/immunomodulatory fusion proteins and methods for making same |
| WO2017058859A1 (en) * | 2015-09-29 | 2017-04-06 | Celgene Corporation | Pd-1 binding proteins and methods of use thereof |
| CN107082812A (en) * | 2017-03-29 | 2017-08-22 | 上海科医联创生物科技有限公司 | A kind of fusion protein for recovering debilitating immune cell function and its application |
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2018
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|---|---|---|---|---|
| CN104334573A (en) * | 2012-04-30 | 2015-02-04 | 比奥孔有限公司 | Targeted/immunomodulatory fusion proteins and methods for making same |
| WO2017058859A1 (en) * | 2015-09-29 | 2017-04-06 | Celgene Corporation | Pd-1 binding proteins and methods of use thereof |
| CN107082812A (en) * | 2017-03-29 | 2017-08-22 | 上海科医联创生物科技有限公司 | A kind of fusion protein for recovering debilitating immune cell function and its application |
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| CN110734498A (en) * | 2019-10-15 | 2020-01-31 | 上海科棋药业科技有限公司 | fusion protein for relieving immunosuppression and application thereof |
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Address after: Room 302, Building 1, Zhebei Life and Health Small and Micro Park, Yuedu Middle Road, Dipu Street, Anji County, Huzhou City, Zhejiang Province, 313399 (self declared) Patentee after: Keyi (Zhejiang) Pharmaceutical Technology Co.,Ltd. Address before: 200127 floor 5, building 6, No. 1, Lane 1800, Xinyang Road, Lingang New Area, China (Shanghai) pilot Free Trade Zone, Pudong New Area, Shanghai Patentee before: SHANGHAI KEYI PHARMACEUTICAL TECHNOLOGY Co.,Ltd. |