CN112426438B - Composition for regulating immune response in acidic environment, preparation method and application thereof - Google Patents

Composition for regulating immune response in acidic environment, preparation method and application thereof Download PDF

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CN112426438B
CN112426438B CN202011276641.1A CN202011276641A CN112426438B CN 112426438 B CN112426438 B CN 112426438B CN 202011276641 A CN202011276641 A CN 202011276641A CN 112426438 B CN112426438 B CN 112426438B
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CN112426438A (en
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徐建青
张晓燕
廖启彬
丁相卿
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Shanghai Sinobay Bio Tech Co ltd
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung

Abstract

The present invention provides a composition for modulating an acidic environment immune response, the composition consisting of an acidic environment targeting molecule which is a coupling molecule or fusion protein of a low pH intercalating peptide (low pH insertion peptide, pHLIP) or variant thereof, and an immune cell which is an endogenous or exogenous immune cell. The invention also provides a preparation method and application of the composition. The composition of the invention can be used for treating solid tumors by regulating the immune response in the acidic environment, especially the tumor acidic environment.

Description

Composition for regulating immune response in acidic environment, preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, relates to a composition for regulating and controlling immune response in an acidic environment, and also relates to a preparation method and application of the composition.
Background
Immunotherapies for tumors involve monoclonal antibodies, vaccines, gene therapy, immune cell therapy, etc., and among them, monoclonal antibodies targeting tumor antigens, immune checkpoint inhibitors, T cell receptor-modified T cells (TCR-T), chimeric antigen receptor T cells (CAR-T), etc., and have been shown to have remarkable antitumor effects in clinical studies. However, the off-target effect, particularly the tumor heterogeneity of solid tumors, the negative regulation of immune cell infiltration and function by tumor-inhibiting microenvironment, and the like, caused by the lack of tumor-specific antigens severely restrict the effect of immunotherapy and cause the safety concern of the novel tumor intervention technology in the technical field of biological medicine.
Due to abnormal vascular structure of tumor microenvironment and excessive proliferation of tumor cells, metabolism of tumor tissue is more dependent on anaerobic glycolysis, and metabolic product lactic acid and CO generated by respiration 2 Together with the acidification of the tumor microenvironment, which is a common feature of many solid tumors, the extracellular pH of tumors (6.5-6.8) is significantly lower than the pH of peripheral blood or normal tissue (7.4). Research shows that the acidic environment of tumor can regulate various biological processes including cell proliferation, angiogenesis, immunosuppression, invasion, drug resistance, etc., even promote the formation of tumor with high invasion phenotype, and the malignancy of tumor is higher. Therefore, the acidic environment of the targeted tumor is a brand-new diagnosis and treatment method, which is not dependent on specific tumor antigens and is not limited by tumor heterogeneity, and can be used for predicting the malignancy of the tumor and delivering therapeutic drugs to various tumor tissues so as to achieve the aim of treatment.
The low pH intercalating peptide (pH low insertion peptide, pHLIP) is a water-soluble polypeptide derived from bacterial rhodopsin transmembrane helix protein C, capable of intercalating into the bilayer lipid membrane of cells and forming a stable transmembrane alpha helix. pHLIP has three main forms, either neutral or alkaline pH (pH > 7) to form water-soluble unstructured form I, or unstructured form II which adheres to the cell membrane surface, intercalates into the cell membrane at acidic pH (pH < 7) and forms form III of transmembrane alpha helices. Conventional cell penetrating peptides penetrate the cell membrane and then directly into the cytoplasm. In contrast, there is no specific interaction between pHLIP and lipid bilayer, and it is only affected by the pH of the environment in which it is located, i.e. it is inserted into the cell membrane in an acidic environment, with the N-terminal outside the cell membrane and the C-terminal into the cytoplasm. Thus, pHLIP has a strong transfer ability: the cargo molecules are connected with the N end of the molecular sieve and tied on the surface of a cell membrane, and then cargo molecules without penetrating the membrane are targeted into cytoplasm through the C end of the molecular sieve. Because of this particular physicochemical property of pHLIP, it is widely used in the following fields: (1) pHLIP is labeled by fluorescent dye, imaging probe, etc. and delivered to acidic tissue for disease diagnosis or fluorescent image navigation surgery; (2) Nuclear imaging (PET/SPECT) by radionuclide labeled pHLIP delivery to acidic tissues; (3) Coupled to toxins, drugs, therapeutic genes to treat diseases; (4) specific delivery of nanomaterials to acidified tissue.
However, there is no report in the prior art that pHLIP coupled fluorescent molecules or linked bioactive cytokines or chemokines or various antigens have been associated with immune cell therapies for diseases characterized by acidity. In particular, no report has been made regarding the potential use of pHLIP in: (1) pHLIP linked bioactive cytokines or chemokines or conjugated immunomodulatory drugs in combination with endogenous or exogenous immune cells specifically targeted to the acidic environment to modulate immune responses; (2) pHLIP linked to polypeptide antigens such as tumor-associated antigens, bacterial antigens, viral antigens, etc., in combination with immunopotentiating therapies of genetically engineered immune cells (T cells, NK cells, NKT cells, macrophages, etc.); (3) The magnetic nanomaterial labeled pHLIP is delivered to acidic tissue for Magnetic Resonance Imaging (MRI).
That is, there is currently a need for more advanced compositions of pHLIP.
Disclosure of Invention
Based on this, it is an object of the present invention to provide a composition for modulating an acidic environment immune response comprising an acidic environment targeting molecule and an immune cell. The invention also provides a preparation method and application of the composition.
In one aspect, the invention provides a composition consisting of an acidic environment targeting molecule which is a coupling molecule or fusion protein of a low pH insertion peptide (low pH insertion peptide, pHLIP) or variant thereof, and an immune cell which is an endogenous or exogenous immune cell;
wherein the coupling molecule or fusion protein of the pHLIP or the variant thereof is obtained by connecting the N-terminal of the pHLIP or the variant thereof with cytokines, fluorescent molecules, immunoregulatory drugs, magnetic resonance imaging contrast agents, magnetic nanomaterials, tumor-associated antigens, bacterial antigens, viral antigens, fungal antigens, plant antigens, polypeptide antigens or antibody Fc fragments.
The composition of the invention, wherein the amino acid sequence of the low pH insertion peptide is shown as SEQ ID NO. 1 or SEQ ID NO. 2;
preferably, the amino acid sequence of the variant of the low pH insertion peptide is as shown in any one of SEQ ID NO. 3-SEQ ID NO. 18;
preferably, the fusion protein of pHLIP or a variant thereof is as shown in any one of SEQ ID NO. 19-SEQ ID NO. 25.
In particular, the low pHLIP and variants thereof include the following polypeptides:
WT-1(SEQ ID NO:1)；WT-2(SEQ ID NO:2)；Var1(SEQ ID NO:3)；Var2(SEQ ID NO:4)；Var3(SEQ ID NO:5)；Var4(SEQ ID NO:6)；Var5(SEQ ID NO:7)；Var6(SEQ ID NO:8)；Var7(SEQ ID NO:9)；Var8(SEQ ID NO:10)；Var9(SEQ ID NO:11)；Var10(SEQ ID NO:12)；Var11(SEQ ID NO:13)；Var12(SEQ ID NO:14)；Var13(SEQ ID NO:15)；Var14(SEQ ID NO:16)；Var15(SEQ ID NO:17)；Var16(SEQ ID NO:18)。
the composition of the invention, wherein the immune cells are endogenous or exogenous immune cells, including but not limited to autoimmune cells, allogenic immune cells, autologous genetically engineered immune cells, allogenic genetically engineered immune cells.
Preferably, the immune cells include, but are not limited to, NK cells, NKT cells, CD16/64chimeric receptor (CD 16/64chimeric receptor,CD16/64 CR) modified NK/NKT/T cells, chimeric antigen receptor (chimeric antigen receptor, CAR) modified NK/NKT/T cells;
more preferably, the immune cells are CD16/64CR-T cells and CAR-T cells.
The composition according to the present invention, wherein the cytokines include, but are not limited to, interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemokines and growth factors. Wherein the interleukins include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21; such interferons include, but are not limited to, IFN- α, IFN- β, IFN- γ, IFN- κ, and IFN- λ; wherein the tumor necrosis factor includes, but is not limited to, TNF- α and TNF- β; wherein the colony stimulating factor includes, but is not limited to, SCF, flt3L, G-CSF, M-CSF and GM-CSF; wherein the chemokines include, but are not limited to, CCL2, CCL5, CCL19, CCL21, CXCL8, CXCL9, CXCL10, CXCL11, XCL1, and CX3CL1; wherein the growth factors include, but are not limited to, TGF-beta, EGF, FGF, NGF, PDGF, and VEGF.
The composition of the present invention, wherein the immunomodulating drug comprises an immunopotentiator and an immunosuppressant. Wherein the immunosuppressive drugs include, but are not limited to, cyclosporine, tacrolimus, and azathioprine; such immunopotentiators include, but are not limited to, thymosins, transfer factors and recombinant human interferons.
The composition of the invention, wherein the tumor-associated antigen includes, but is not limited to, CD19, CD20, CD22, CD30, CD33, ROR1, ROR2, CD38, CD123, CD133, NKG2D ligand, ERBB2, MUC1, CD44v6, CD44v7, CD44v8, CEA, epCAM, TAG, KIT, IL-13Rα2, EGFR, EGFRvIII, GD2, GD3, HMW-MAA, MAGE1, MAGEA3, CD171, NCAM, IL-11Rα, FR α, PSCA, PSMA, TARP, CAIX, VEGFR, BCMA, CTLA-4, PD-L1, PD-L12, GPC3, CD47, AXL, FAP, α5β1, αvβ3, TGFβ R, ER, PR, P53, IGFR, CD25, CD117, CD34, CD138, BCMA, mesothelin, S100, CD70, ALK, RANK and HER3.
The composition of the invention, wherein the bacterial antigen includes, but is not limited to, a staphylococcal antigen, a streptococcal antigen, a streptococcus pneumoniae antigen, a neisseria meningitidis antigen, and a neisseria gonorrhoeae antigen.
The composition of the invention, wherein the viral antigen includes, but is not limited to, a cytomegalovirus antigen, a balvirus antigen, a hepatitis virus antigen, a herpes simplex virus antigen, an HIV antigen, a human T-lymphocyte virus antigen, a rubella virus antigen, a SARS coronavirus antigen, a varicella-zoster virus, a rabies virus antigen, an influenza virus antigen, a rotavirus antigen, and a human papilloma virus antigen.
The composition of the invention, wherein the fungal antigens include, but are not limited to, dermatophyte fungal antigens, cryptococcus neoformans antigens, and candida albicans antigens.
The composition according to the invention, wherein the polypeptide antigen includes, but is not limited to, aviTag (SEQ ID NO: 26), calmulin Tag (SEQ ID NO: 27), polyglutamate tag (SEQ ID NO: 28), E-Tag (SEQ ID NO: 29), FLAG Tag (SEQ ID NO: 30), HA-Tag (SEQ ID NO: 31), his-Tag (SEQ ID NO: 32), myc-Tag (SEQ ID NO: 33), S-Tag (SEQ ID NO: 34), SBP-Tag (SEQ ID NO: 35), sof-Tag 1 (SEQ ID NO: 36), softag3 (SEQ ID NO: 37), strep-Tag (SEQ ID NO: 38), TC-Tag (SEQ ID NO: 39), V5 Tag (SEQ ID NO: 40), T7 Tag (SEQ ID NO: 41), V-Tag (SEQ ID NO: 42), xpress Tag (SEQ ID NO: 43), 3X FLAG (SEQ ID NO: 44), snoop ID NO:45, and Snoop ID NO: 46.
The composition of the invention, wherein the fluorescent molecules include, but are not limited to FITC, FAM, PE, APC, PB, cy, cy5, texas Red, TRITC, GFP, RFP, CFP and BFP.
The composition of the invention, wherein the antibody Fc fragment includes, but is not limited to, an Fc fragment of IgG1, an Fc fragment of IgG2, an Fc fragment of IgG3, and an Fc fragment of IgG 4.
More preferably, the magnetic nanomaterial includes, but is not limited to, gadopentetate meglumine (Gd-DTPA), dysprosium acid meglumine (Dy-DTPA), paramagnetic metal ion chelate represented by manganese fosfodrin trisodium (Mn-DPDP), and Fe 2 O 3 、Fe 3 O 4 Superparamagnetic metal oxide particles as cores.
The composition of the invention, wherein the acidic environment targeting molecule is selected from FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP, fc-pHLIP or CCL19-pHLIP; the immune cells are FITC CAR-T cells, CD16/64CR-T cells and T cells.
According to one embodiment of the invention, the composition for modulating an acidic environment immune response is an acidic environment targeting molecule pHLIP fusion protein CCL19-pHLIP and T cells, the amino acid sequence of CCL19-pHLIP is shown as SEQ ID NO. 49. Specifically, chemokine CCL19 is linked to the N-terminus of wild-type pHLIP by a GS flexible linker amino acid sequence (GSSGGSGGSGGSG), interspersed with protease cleavage sequences (LSGRSDNH) for cleavage and release within the tumor microenvironment. In addition, the histidine tag sequence (HHHHH) was used to detect and purify CCL19-pHLIP in vitro.
According to one embodiment of the invention, the composition for modulating an acidic environment immune response is an acidic environment targeting molecule, pHLIP coupling molecule, FITC-pHLIP and FITC CAR-T cells. Specifically, the pHLIP is wild pHLIP, and the amino acid sequence is shown as SEQ ID NO. 1. The coupling molecule is fluorescein isothiocyanate FITC which is a fluorescent molecule. Wherein FITC is coupled with pHLIP through aminocaproic Acid (ACP) to obtain FITC-pHLIP, and the structural formula is as follows: FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT.
The immune cells of the composition are FITC CAR-T cells targeting FITC, and the sequence of the FITC CAR is shown as SEQ ID NO. 50.
According to one embodiment of the invention, the composition for modulating an acidic environment immune response is an acidic environment targeting molecule, pHLIP fusion protein FLAG-pHLIP and CAR-T cells.
Specifically, the amino acid sequence of pHLIP is shown as SEQ ID NO. 1, the fusion protein is a polypeptide antigen FLAG tag, and the FLAG tag is connected to the N-terminal of pHLIP, and the sequence is shown as SEQ ID NO. 51. Alternatively, the polypeptide antigen is PNE tag, the PNE tag is connected to the N end of pHLIP to obtain PNE-pHLIP, and the sequence is shown as SEQ ID NO. 52. The immune cells are FLAG CAR-T cells or PNE CAR-T cells targeting FLAG tag or PNE tag respectively, wherein the amino acid sequence of the PNE CAR is shown as SEQ ID NO. 53.
According to one embodiment of the invention, the composition for modulating an acidic environment immune response is an acidic environment targeting molecule pHLIP fusion protein and CD16/64CR-T cells. The fusion proteins are pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc, and the sequences are shown as SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21. The CD16/64CR-T cell is a CD16 CR-T cell targeting the Fc fragment of IgG1, and the sequence of the CD16 CR is shown as SEQ ID NO. 54.
In a further aspect, the invention also provides the use of a composition according to the invention for the manufacture of a medicament for the diagnosis and/or treatment of a tumour, an autoimmune disease, a disease associated with ischemia and hypoxia and/or an inflammatory disease.
In yet another aspect, the present invention also provides a method for diagnosing and/or treating various diseases comprising administering to a patient in need thereof a therapeutically effective amount of the composition.
Wherein the disease is selected from tumor, autoimmune disease, diseases accompanied by ischemia and anoxia and/or inflammatory disease.
Although the present invention has been described with reference to pHLIP shown in SEQ ID NO. 1, due to the similarity of physicochemical properties of pHLIP and its variants, those skilled in the art can certainly confirm that all pHLIP and its variants can be applied to the technical solution of the present invention and the same effects can be obtained.
In particular, the inventors have found that the composition of the invention has the following uses: (1) When the fusion protein in the composition is a cytokine with biological activity, the fusion protein is combined with endogenous or exogenous immune cells to specifically target the cell to an acidic environment, so that immune response is enhanced or inhibited; (2) When the fusion protein in the composition is a chemotactic factor with biological activity, the fusion protein is combined with endogenous or exogenous immune cells, so that the fusion protein is specifically targeted to an acidic environment, and the immune cells are recruited to strengthen or inhibit immune response; (3) When the coupling molecules in the composition are immunoregulation drugs, endogenous or exogenous immune cells are combined, and the coupling molecules are specifically targeted to an acidic environment to play a role in local immunity enhancement or immunosuppression; (4) The fusion protein in the composition is antigens such as tumor-related antigens, bacterial antigens, viral antigens, fluorescent molecules and the like, and is combined with genetically engineered immune cells (T cells, NK cells, NKT cells, macrophages and the like) to target the fusion protein specifically to the tumor acidic environment, so that the purpose of broad-spectrum anti-tumor is achieved; (5) When the coupling molecules in the composition are magnetic nanomaterials, they are delivered to acidic tissues for Magnetic Resonance Imaging (MRI).
In yet another aspect, the present invention provides a method of preparing the composition for modulating an immune response in an acidic environment, the method comprising the steps of:
synthesizing the composition by a solid phase method by using an automatic polypeptide synthesizer; or (b)
The above composition is expressed and purified by engineering bacteria and cells by biosynthesis.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the composition of the invention can be coupled or connected with antigens such as tumor-associated antigens, antigens derived from pathogens, fluorescent molecules, polypeptide antigens and the like, and can be used for developing general innovative cell immunotherapy by combining endogenous or exogenous immune cells to regulate and control immune response in an acidic environment, thereby being expected to treat almost all solid tumors. In addition, in addition to solid tumors, extracellular acidification is associated with a number of pathological states including ischemic stroke, nerve injury, infection, tissue tear damage, and the like. The abnormal metabolic activity of cells in damaged or diseased tissues often causes acidification of the extracellular environment, so that the extracellular acidification is expected to become a universal marker for diagnosis and treatment of various diseases, and the pHLIP can be used for targeting the characteristic of the acidic environment and can also be used for diagnosis and treatment of the pathological states.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the detection of the expression of the acidic environment targeting molecule of the invention on tumor cells (A549) using a flow cytometer under microenvironment at different pH conditions, wherein the coupling molecules or fusion proteins are FITC (fluorescein isothiocyanate), FLAG tag polypeptide, and PNE polypeptide (14 amino acid polypeptides derived from the yeast transcription factor GCN 4), respectively;
FIG. 2 shows the detection of the expression of the acidic environment targeting molecule of the invention on tumor cells (A549) using a flow cytometer under microenvironment at different pH conditions, wherein the fusion proteins are immune cell recruitment molecules, respectively an antibody crystallizable fragment (Fc), an antibody crystallizable fragment carrying trimerization foldon sequence (foldon-Fc), and a crystallizable fragment carrying trimerization p24 sequence (p 24-Fc);
FIG. 3 shows the effect of the acidic environment targeting molecule pHLIP fusion protein CCL19-pHLIP chemotactic T cells of the present invention;
FIG. 4 shows the localization of the acidic environment targeting molecule FITC-pHLIP on tumor tissue and other organs using the immunohistochemical/fluorescence analysis system TissueFAXS, with the white triangle indicating FITC-pHLIP anchored on tumor cells;
FIG. 5 shows that FITC-pHLIP in combination with FITC CAR-T cells in the composition of the present invention specifically kills tumor cells in an acidic environment, wherein A549 is lung adenocarcinoma cells (alveolar basal epithelium) and NCI-H292 is lung cancer cells (lymph node metastasis);
FIG. 6 shows the tumor growth curve of FITC-pHLIP in combination with FITC CAR-T cells in tumor-bearing mice of the compositions of the present invention.
Detailed Description
Example 1 acidic Environment targeting molecules of the compositions of the invention in tumor cells under microenvironment at different pH conditions Expression on cells (A549)
Cell culture: a549 cells (american type culture collection) were cultured using RPMI1640 complete medium (abbreviated as R10), R10 RPMI1640 medium (Corning) complete medium containing 10% (v/v) fetal bovine serum (Biological Industries), 1X streptomycin/penicillin (100X streptomycin/penicillin, gibco) at 37 ℃,5% co 2 Is cultured in a cell culture incubator. The cells were passaged at 1:5 after confluence.
pHLIP expression analysis: a549 cells pass through pancreatin cellsThe digestion solution is digested for 1 to 2 minutes, neutralized with 10 times volume of R10 medium, 500Xg and centrifuged for 5 minutes. Discarding supernatant, and re-suspending with R10 culture medium to obtain A549 single cell suspension, each 1×10 5 Cells were placed in 3 1.5mL EP tubes, 800Xg, centrifuged for 3 minutes, the medium was discarded, 1xPBS at pH6.0, pH6.5 and pH7.4 was added, 5. Mu.g/mL of the acidic environment targeting molecule of the present invention, FITC-pHLIP (structural formula:
FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT), FLAG-pHLIP (SEQ ID NO: 51) and PNE-pHLIP (SEQ ID NO: 52) (a polypeptide synthesized by Shanghai, shanghai Biotechnology Co., ltd.). After incubation at room temperature for 1 hour, the supernatant was discarded, the cells were resuspended with 1xPBS at pH7.4 by washing 3 times with 1xPBS at the corresponding pH, and the adsorption levels of FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP at A549 cells were detected by flow cytometry. The cell samples tested were divided into 6 groups: (1) pH6.0 untreated group; (2) pH6.0+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP treated group; (3) pH6.5 untreated group; (4) pH6.5+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP treated group; (5) pH7.4 untreated group: (6) pH7.4+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP.
Results: FIG. 1A, B, C shows that in a neutral solution environment (pH 7.4), FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP are less efficient at inserting into the cell membrane of A549 at 30%, 30% and 15%, respectively. In an acidic solution environment, FITC-pHLIP is inserted into A549 cell membranes with high efficiency, the insertion efficiency of pH6.5 and pH6.0 are respectively 85% and 99%, and the insertion efficiency is obviously higher than pH7.4; the FLAG-pHLIP polypeptide can be inserted into A549 cell membranes with high efficiency, the insertion efficiency of pH6.5 and pH6.0 is 73% and 91%, which is obviously higher than pH7.4; the PNE-pHLIP polypeptide of the invention can also be effectively inserted into A549 cell membranes, and the insertion efficiencies of pH6.5 and pH6.0 are 57% and 53%, respectively, which are significantly higher than pH7.4.
EXAMPLE 2 acidic Environment targeting molecules of the invention in tumor cells under microenvironment at different pH conditions (A549) Expression on
2.1 in vitro expression and purification of pHLIP fusion proteins of pHLIP-Fc, pHLIP-foldon-Fc and pHLIP-p24-Fc
2.1.1 construction of eukaryotic expression vectors: the eukaryotic expression vector was prepared by total gene synthesis of the designed DNA sequences of the fusion proteins according to the invention (SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO: 21) delivered to Shanghai Jieli bioengineering Co., ltd.
2.1.2 in vitro expression of pHLIP fusion proteins of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p 24-Fc: HEK293T cells were plated on 15cm dishes at intervals of 1.5X10 each 7 Cells, at 37 ℃,5% co 2 Is cultured overnight in a cell incubator. The next day, eukaryotic expression vectors carrying fusion protein DNA sequences were transfected with the total amount of DNA at 30. Mu.g/dish using the transfection reagent PEIpro, and replaced with serum-free medium for 6-8 hours at 37℃with 5% CO 2 The cell culture is continued for 5 to 7 days.
Purification of pHLIP fusion proteins of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p 24-Fc: cell culture supernatants were collected, centrifuged at 2000rpm at 4℃for 10 minutes, and cell pellets were discarded. The culture supernatant collected by centrifugation was purified by using Protein G agarose column (GE company, cat. No. 28-9031-34), and the purified antibody was dissolved in PBS buffer.
2.2pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc pHLIP fusion proteins were expressed on A549 cells
Cell culture: a549 cells were cultured in a cell incubator at 37 ℃ with 5% co2 using R10 medium. The cells were passaged at 1:5 after confluence.
Flow cytometry observation expression: a549 cells are digested by pancreatin cell digestive juice for 1-2 minutes, 10 times of volume of R10 culture medium is neutralized, 500xg is centrifuged for 5 minutes, and the R10 culture medium is resuspended, so that the A549 single cell suspension is finally obtained. Take 1x10 5 Cells were placed in 3 1.5mL EP tubes, centrifuged at 800Xg for 3 min, 1xPBS at pH6.0 and pH7.4 was added to the discarded medium, pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc fusion proteins (20. Mu.g/mL) prepared as described above were added, incubated at room temperature for 1 hour, the supernatant was discarded, 1xPBS at the corresponding pH was washed 3 times, cells were resuspended at the corresponding pH 1xPBS, and the expression levels of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc fusion proteins on A549 cells were detected using a flow cytometer. Grouping: (1) pH6.0 untreated group; (2) pH6.0+pHLIP-Fc group; (3) pH6.0+pHLIP-delA den-Fc group; (4) pH6.0+pHLIP-p24-Fc group; (5) pH7.4 untreated group: (6) pH7.4+ pHLIP-Fc group;
(7) pH7.4+pHLIP-folder-Fc group; (8) pH7.4+pHLIP-p24-Fc group.
Results: FIG. 2 shows that in a neutral solution environment (pH 7.4), the pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc fusion proteins of the present invention have very low efficiency of insertion into the cell membrane of A549, below 4%. In the acidic solution environment, the pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc fusion proteins can be inserted into A549 cell membranes, and the insertion efficiencies of pH6.0 are respectively 21%, 19.1% and 21.4%, and are higher than pH7.4.
Example 3 acidic Environment targeting molecule CCL19-pHLIP highly potent chemotactic T cells
3.1 In vitro expression and identification of CCL19-pHLIP fusion proteins
3.1.1 construction of eukaryotic expression vectors
The designed DNA sequence of the fusion protein amino acid sequence (SEQ ID NO: 49) according to the present invention was delivered to Shanghai Jieli bioengineering Co.Ltd for total gene synthesis to prepare a eukaryotic expression vector.
3.1.2 In vitro expression of CCL19-pHLIP fusion proteins
HEK293T cells (american type culture collection) were cultured with DMEM complete medium (abbreviated as D10), D10 complete medium containing 10% (v/v) fetal bovine serum (Biological Industries), 1X streptomycin/penicillin (100X streptomycin/penicillin, gibco) in DMEM medium (Corning) at 37 ℃,5% co 2 Is cultured in a cell culture incubator. The cells were passaged at 1:5 after confluence.
HEK293T cells are digested by pancreatin cell digestive juice for 1-2 minutes, neutralized by D10 culture medium with 10 times volume, centrifuged for 5 minutes at 500Xg, and resuspended by DMEM complete culture medium, thus finally obtaining HEK293T single cell suspension. HEK293T cells were plated in six well plates, 1X10 per well 6 Cells, at 37 ℃,5% co 2 Is cultured overnight in a cell incubator. The next day, eukaryotic expression vectors carrying the fusion protein DNA sequences were transfected with 4. Mu.g/well of DNA using the transfection reagent Turbofect at 37℃with 5% CO 2 In a cell incubator of (C)Culturing for 2 days, collecting culture supernatant, centrifuging at 2000rpm and 4 ℃ for 10 minutes, collecting cell sediment for WB detection, collecting the centrifuged culture supernatant, and freezing for later use.
3.2T cell chemotaxis assay
A5 μm-pore-diameter Transwell plate was used, 600. Mu.L of the culture supernatant collected by the centrifugation was added to the lower chamber, 100. Mu.L of T cell suspension was added to the upper chamber, and the cell density was 1X10 6 /mL, at 37 ℃,5% CO 2 The number of cells in the lower chamber was counted after culturing for 2, 4 or 6 hours, respectively.
Results: FIG. 3A shows that eukaryotic expression vectors carrying the DNA sequence of the CCL19-pHLIP fusion protein are efficiently expressed in HEK293T cells. FIG. 3B shows that the acidic environment targeting molecule CCL19-pHLIP fusion protein of the invention can effectively chemotactic T cells, and the chemotactic efficiency is equivalent to that of a positive control CCL 19.
EXAMPLE 4 distribution of FITC-pHLIP of acidic Environment targeting molecule in tumor tissue and organs in vivo
5x10 5 The individual melanocyte B16 cells were inoculated subcutaneously on the right side of C57BL/6 mice (purchased from Shanghai Schleman laboratory animal Limited), when the tumor diameter was as large as 0.5-1cm, 1mg/kg of FITC-pHLIP was intravenously injected, the mice were sacrificed at 4, 12, 24, 48, 72 and 96 hours after injection, frozen sections were made from tumor tissue, heart, liver, spleen, lung and kidney, and 4% paraformaldehyde was fixed and stained with PE-anti-FITC (Abcam, ab 25539) and DAPI, and analyzed by the immunohistochemical/fluorescence analysis system TissueFAXS.
Results: FIG. 4 shows that FITC-pHLIP is enriched in tumor tissue at least 6 hours after reinfusion, and that significant FITC-pHLIP localization to tumor cells is visible until 24 hours after reinfusion. However, none of the 4, 12, 24, 48 hours after FITC-pHLIP reinfusion was enriched in heart, liver, spleen, lung and kidney.
Example 5 in vitro anti-tumor effect of the acidic Environment targeting molecule FITC-pHLIP in combination with anti-FITC CAR-T cells
5.1 cell culture and plating
Lung cancer cell A549-luciferases/NCI-H292-luciferases (Shanghai Xinwan Biotechnology Co., ltd.) stably expressing luciferase was cultured using R10 medium containing 10% fetal bovine serum, 1x streptomycin/penicillin at 37℃and 5% CO 2 Is cultured in a cell culture incubator. The cells were passaged at 1:5 after confluence.
A549-luciferase/NCI-H292-luciferase cells are digested by pancreatin cell digestive juice for 1-2 minutes, R10 culture medium with 10 times volume is neutralized, 500xg is centrifugated for 5 minutes, and R10 culture medium is resuspended, so that single cell suspension of A549-luciferase/NCI-H292-luciferase is finally obtained. A549-luciferase/NCI-H292-luciferase cells were plated in 96-well black matrix plates at 1X10 per well 4 Individual cells, total 100. Mu.L, at 37℃and 5% CO 2 Is cultured overnight in a cell incubator.
5.2 FITC-pHLIP treatment
A549-luciferase/NCI-H292-luciferase cells were cultured overnight on a culture plate, R10 medium at pH6.5 and pH6.0 was added to the culture supernatant, FITC-pHLIP (5. Mu.g/mL) of example 1 was added, incubation was performed at 37℃for 1 hour, the supernatant was discarded, washing 3 times with 1xPBS at the corresponding pH was performed, and 100. Mu.L of R10 medium at pH6.5 and pH6.0 was added. Grouping: ph6.5 untreated group; (2) pH6.5+ FITC-pHLIP treated group; (3) pH6.0 untreated group; (4) pH6.0+FITC-pHLIP treatment group.
5.3 anti-FITC CAR-T cell treatment
Resting anti-FITC CAR-T cells were collected and resuspended to the corresponding cell concentration with R10 medium. anti-FITC CAR-T cells were added to the 4 treatment groups at a ratio of 5:1 for effector cells to target cells, with 100. Mu.L of anti-FITC CAR-T cell suspension per well.
Results: FIG. 5A shows that FITC-pHLIP in combination with anti-FITC CAR-T cells specifically kills lung adenocarcinoma cells A549-luciferases in an acidic solution environment. FIG. 5B shows that FITC-pHLIP in combination with anti-FITC CAR-T cells specifically kills highly metastatic lung cancer cells NCI-H292-luciferases in an acidic solution environment.
Example 6 in vivo anti-tumor efficacy of the acidic Environment targeting molecule FITC-pHLIP in combination with anti-FITC CAR-T cells Fruit evaluation
NCI-H292 lung cancer cells (Shanghai Xinwan Biotechnology Co., ltd.) were inoculated subcutaneously in the right flank of B-NDG immunodeficient mice (purchased from Beijing Bai Osai Gene Biotechnology Co., ltd.) at 2X10 6 Individual cells/individual, mice with substantially uniform tumor size are randomly grouped when tumors grow to 0.5-1cm in diameter, and oversized and undersized tumors are knocked out. The total groups were 3: PBS alone in the injection group, 5; anti-FITC CAR-T cells were injected alone into the group, 5; FITC-pHLIP in combination with anti-FITC CAR-T cell injection groups, 5. Tumor size was measured every 3 days.
The administration method comprises the following steps: FITC-pHLIP dosing: each mouse was injected intravenously 20 μg (125 μl) and 10 consecutive doses were given starting from the day after the completion of the grouping, once a day, once every other day. anti-FITC CAR-T cell dosing: anti-FITC CAR-T cells were infused back intravenously 10 per mouse 7 mu.L/125. Mu.L, 2 times in total. Reinfusion was initiated the first time from the day after the completion of the grouping, with the injection time 12 hours after the first FITC-pHLIP administration, and a second cell reinfusion was performed nine days later.
Results: figure 6 shows that FITC-pHLIP in combination with anti-FITC CAR-T cells significantly inhibited tumor growth in lung cancer tumor-bearing mice, neither PBS nor anti-FITC CAR-T cells alone controlled tumor growth in tumor-bearing mice.
While only examples of the embodiments of the present invention have been described above, it will be understood by those skilled in the art that the foregoing is illustrative only and the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the principles and spirit of the invention, but such changes and modifications should not be construed as falling within the scope of the invention.
Sequence listing
<110> Shanghai Xinwan biotechnology Co., ltd
<120> composition for modulating immune response in acidic environment, method for preparing the same and use thereof
<130> DIC19110078R
<150> 2019111120438
<151> 2019-11-14
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Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe
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Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp
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Glu Gly Thr
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Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu
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Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala
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Asp Glu Gly Thr
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Ala Cys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu
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Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly
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Ala Cys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe
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Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly
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Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe
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Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp
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Ala Cys Glu Glu Gln Asn Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe
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Ala Cys Asp Asp Gln Asn Pro Trp Ala Arg Tyr Leu Asp Trp Leu Phe
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Cys Asp Asn Asn Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro
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Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe
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Cys Glu Glu Gln Gln Pro Trp Ala Gln Tyr Leu Glu Leu Leu Phe Pro
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Cys Glu Glu Gln Gln Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe Pro
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Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe
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Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Trp Leu Phe
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Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe
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Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Leu Leu Phe
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Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro
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Cys Ala
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Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Asn
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Glu Ala Glu Glu Thr
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Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Pro
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Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Pro Gly Ala Leu Leu Val
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Glu Ala Glu Glu Thr
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Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
50 55 60
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
65 70 75 80
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
85 90 95
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
100 105 110
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
115 120 125
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
130 135 140
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
145 150 155 160
Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly
165 170 175
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
180 185 190
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
195 200 205
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
210 215 220
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
225 230 235 240
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
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Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
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Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
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Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
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Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
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Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val
50 55 60
Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Gly
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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser
85 90 95
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
100 105 110
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
115 120 125
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
130 135 140
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
145 150 155 160
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
165 170 175
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro
195 200 205
Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
210 215 220
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
225 230 235 240
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
245 250 255
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
260 265 270
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
275 280 285
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
290 295 300
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
305 310 315 320
Ser Pro Gly Lys
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Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Pro
50 55 60
Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys
65 70 75 80
Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly
85 90 95
Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His
100 105 110
Gln Ala Ala Met Gln Ile Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala
115 120 125
Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly
130 135 140
Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Asn
145 150 155 160
Leu Gln Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Val Pro Val
165 170 175
Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
180 185 190
Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Lys Gln Gly Pro Lys
195 200 205
Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala
210 215 220
Glu Gln Ala Thr Gln Gly Val Lys Asn Trp Met Thr Asp Thr Leu Leu
225 230 235 240
Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly
245 250 255
Pro Gly Ala Ser Ile Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly
260 265 270
Gly Pro Ser His Lys Ala Lys Val Leu Gly Gly Gly Gly Ser Gly Gly
275 280 285
Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr
290 295 300
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
305 310 315 320
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
325 330 335
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
340 345 350
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
355 360 365
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
370 375 380
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
385 390 395 400
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr
405 410 415
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
420 425 430
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
435 440 445
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
450 455 460
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
465 470 475 480
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
485 490 495
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
500 505 510
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
515 520 525
<210> 22
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Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu
50 55 60
Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile
65 70 75 80
Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr
145 150 155 160
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
165 170 175
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
180 185 190
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
195 200 205
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
210 215 220
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
225 230 235 240
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
245 250 255
Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser
260 265 270
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
275 280 285
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
290 295 300
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
305 310 315 320
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
325 330 335
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
340 345 350
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
355 360 365
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
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<213> Artificial sequence (Artificial Sequence)
<400> 23
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu
50 55 60
Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile
65 70 75 80
Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala
145 150 155 160
Leu Asp Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg
165 170 175
Ala Trp Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly
180 185 190
Gly Gly Ser Gly Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu
195 200 205
Leu Ala Leu Asp Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala
210 215 220
Tyr Arg Ala Trp Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp
245 250 255
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
260 265 270
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
275 280 285
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
290 295 300
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
305 310 315 320
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
325 330 335
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
340 345 350
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu
355 360 365
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
370 375 380
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
385 390 395 400
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
405 410 415
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
420 425 430
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
435 440 445
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
450 455 460
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
465 470 475 480
Gly Lys
<210> 24
<211> 382
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp
50 55 60
Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp
65 70 75 80
Ala Asp Glu Gly Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr
145 150 155 160
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
165 170 175
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
180 185 190
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
195 200 205
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
210 215 220
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
225 230 235 240
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
245 250 255
Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser
260 265 270
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
275 280 285
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
290 295 300
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
305 310 315 320
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
325 330 335
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
340 345 350
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
355 360 365
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
<210> 25
<211> 482
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp
50 55 60
Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp
65 70 75 80
Ala Asp Glu Gly Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg
145 150 155 160
Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala
165 170 175
Leu Leu Val Asp Ala Asp Glu Gly Thr Gly Gly Gly Gly Ser Gly Gly
180 185 190
Gly Gly Ser Gly Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu
195 200 205
Leu Ala Leu Asp Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala
210 215 220
Tyr Arg Ala Trp Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp
245 250 255
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
260 265 270
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
275 280 285
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
290 295 300
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
305 310 315 320
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
325 330 335
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
340 345 350
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu
355 360 365
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
370 375 380
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
385 390 395 400
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
405 410 415
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
420 425 430
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
435 440 445
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
450 455 460
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
465 470 475 480
Gly Lys
<210> 26
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15
<210> 27
<211> 26
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg
1 5 10 15
Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu
20 25
<210> 28
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Glu Glu Glu Glu Glu Glu
1 5
<210> 29
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg
1 5 10
<210> 30
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 31
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 32
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
His His His His His His
1 5
<210> 33
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 34
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
1 5 10 15
<210> 35
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 35
Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly
1 5 10 15
Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro
20 25 30
Gln Gly Gln Arg Glu Pro
35
<210> 36
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser
1 5 10
<210> 37
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 37
Thr Gln Asp Pro Ser Arg Val Gly
1 5
<210> 38
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 38
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 39
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Cys Cys Pro Gly Cys Cys
1 5
<210> 40
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 40
Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr
1 5 10
<210> 41
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly
1 5 10
<210> 42
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys
1 5 10
<210> 43
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Asp Leu Tyr Asp Asp Asp Asp Lys
1 5
<210> 44
<211> 23
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
1 5 10 15
Tyr Lys Asp Asp Asp Asp Lys
20
<210> 45
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Thr Asp Lys Asp Met Thr Ile Thr Phe Thr Asn Lys Lys Asp Ala Glu
1 5 10 15
<210> 46
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys
1 5 10
<210> 47
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys
1 5 10
<210> 48
<211> 14
<212> PRT
<213> Artificial work sequence (Artificial Sequence)
<400> 48
Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu
1 5 10
<210> 49
<211> 139
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
His His His His His His Glu Asp Cys Cys Leu Ser Val Thr Gln Lys
1 5 10 15
Pro Ile Pro Gly Tyr Ile Val Arg Asn Phe His Tyr Leu Leu Ile Lys
20 25 30
Asp Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr Leu Arg Gly Arg
35 40 45
Gln Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu Arg Ile Ile Gln
50 55 60
Arg Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg Ser Ser Gly Ser
65 70 75 80
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser
85 90 95
Asp Asn His Gly Ser Ser Gly Thr Ala Glu Gln Asn Pro Ile Tyr Trp
100 105 110
Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp
115 120 125
Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr
130 135
<210> 50
<211> 806
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Asn Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Gly Ser Phe Ser Met Ser Trp Val Arg Gln Ala Pro Gly Gly
50 55 60
Gly Leu Glu Trp Val Ala Gly Leu Ser Ala Arg Ser Ser Leu Thr His
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Arg Ser Tyr Asp Ser Ser Gly Tyr Trp
115 120 125
Gly His Phe Tyr Ser Tyr Met Asp Val Trp Gly Gln Gly Thr Leu Val
130 135 140
Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Ser Val Leu Thr Gln Pro Ser Ser Val Ser Ala Ala Pro Gly
165 170 175
Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Asn
180 185 190
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
195 200 205
Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Lys Ser Gly Asn Ser Ala Ser Leu Asp Ile Ser Gly Leu
225 230 235 240
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser
245 250 255
Leu Ser Glu Phe Leu Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly
260 265 270
Ala Ser Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys
275 280 285
Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser
290 295 300
Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val
305 310 315 320
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
325 330 335
Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr
340 345 350
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
355 360 365
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser
370 375 380
Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
385 390 395 400
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
405 410 415
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
420 425 430
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
435 440 445
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
450 455 460
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg
465 470 475 480
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
485 490 495
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
500 505 510
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
515 520 525
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly
530 535 540
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
545 550 555 560
Glu Asn Pro Gly Pro His Met Met Val Ser Lys Gly Glu Glu Leu Phe
565 570 575
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
580 585 590
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
595 600 605
Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
610 615 620
Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser
625 630 635 640
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
645 650 655
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
660 665 670
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
675 680 685
Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile
690 695 700
Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
705 710 715 720
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg
725 730 735
His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
740 745 750
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
755 760 765
Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
770 775 780
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
785 790 795 800
Met Asp Glu Leu Tyr Lys
805
<210> 51
<211> 43
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Glu Gln Asn Pro Ile Tyr Trp
1 5 10 15
Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp
20 25 30
Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr
35 40
<210> 52
<211> 59
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Gly Gly Gly Gly Ser Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu
1 5 10 15
Lys Lys Leu Gly Gly Gly Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp
20 25 30
Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp
35 40 45
Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr
50 55
<210> 53
<211> 497
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 53
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ala Val
20 25 30
Val Thr Gln Glu Ser Ala Leu Thr Ser Ser Pro Gly Glu Thr Val Thr
35 40 45
Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala
50 55 60
Ser Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly
65 70 75 80
Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser
85 90 95
Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu
100 105 110
Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp Tyr Ser Asp His Trp Val
115 120 125
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
145 150 155 160
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
165 170 175
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Leu Leu Thr Asp Tyr Gly
180 185 190
Val Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly
195 200 205
Val Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys Ser
210 215 220
Arg Leu Ser Val Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
225 230 235 240
Met Asn Ser Leu Gln Ser Gly Asp Ser Ala Arg Tyr Tyr Cys Val Thr
245 250 255
Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
260 265 270
Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
275 280 285
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
290 295 300
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
305 310 315 320
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
325 330 335
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
340 345 350
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
355 360 365
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
370 375 380
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
385 390 395 400
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
405 410 415
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
420 425 430
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
435 440 445
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
450 455 460
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
465 470 475 480
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
485 490 495
Arg
<210> 54
<211> 433
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 54
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val
165 170 175
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190
Gly Leu Ala Val Pro Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205
Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
210 215 220
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
225 230 235 240
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
245 250 255
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
260 265 270
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
275 280 285
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
290 295 300
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
305 310 315 320
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
325 330 335
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
340 345 350
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
355 360 365
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
370 375 380
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
385 390 395 400
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
405 410 415
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
420 425 430
Arg

Claims (3)

1. A composition for modulating an acidic environment immune response, the composition consisting of an acidic environment targeting molecule and immune cells;
wherein the acidic environment targeting molecule has the structural formula: FITC-pHLIP of FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT; the immune cells are FITC CAR-T cells targeting FITC.
2. Use of a composition according to claim 1 for the preparation of a medicament for the treatment of tumors.
3. A process for preparing the composition of claim 1, said process comprising the steps of:
synthesizing by a solid phase method by using an automatic polypeptide synthesizer; or (b)
And expressing and purifying by engineering bacteria and cells by a biosynthesis method.
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CN113896803B (en) * 2021-10-20 2023-09-22 郑州大学第一附属医院 Tumor slightly acid responsive fusion protein and application thereof
CN114149510B (en) * 2021-10-29 2024-01-30 上海鑫湾生物科技有限公司 Condition-controlled splicing chimeric antigen receptor molecule and application thereof
CN116102658A (en) * 2021-11-09 2023-05-12 四川大学华西医院 Preparation and application of chimeric antigen receptor immune cells constructed based on GAS6
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