WO2021093881A1 - Composition for regulating immune response in acidic environment, and preparation method therefor and use thereof - Google Patents

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

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WO2021093881A1
WO2021093881A1 PCT/CN2020/128957 CN2020128957W WO2021093881A1 WO 2021093881 A1 WO2021093881 A1 WO 2021093881A1 CN 2020128957 W CN2020128957 W CN 2020128957W WO 2021093881 A1 WO2021093881 A1 WO 2021093881A1
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phlip
cells
antigen
tag
seq
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PCT/CN2020/128957
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French (fr)
Chinese (zh)
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徐建青
张晓燕
廖启彬
丁相卿
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上海鑫湾生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/62—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 the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung

Definitions

  • the invention belongs to the technical field of biomedicine, and relates to a composition for regulating the immune response of an acidic environment.
  • the invention also relates to a preparation method and application of the composition.
  • Tumor immunotherapy involves monoclonal antibodies, vaccines, gene therapy, immune cell therapy, etc., among which monoclonal antibodies targeting tumor antigens, immune checkpoint inhibitors, T cell receptor modified T cells (TCR-T), chimeric Immunotherapy methods such as antigen receptor T cells (CAR-T) have shown remarkable anti-tumor effects in clinical studies.
  • TCR-T T cell receptor modified T cells
  • CAR-T antigen receptor T cells
  • targeting the acidic environment of tumors is a new diagnosis and treatment method that does not depend on specific tumor antigens and is not limited to tumor heterogeneity. It can be used to predict the malignancy of tumors and deliver treatments. Sex drugs to a variety of tumor tissues to achieve the purpose of treatment.
  • pH low insertion peptide is a water-soluble peptide derived from bacterial rhodopsin transmembrane helix protein C, which can be inserted into the double lipid membrane of cells and form a stable transmembrane alpha helix.
  • pHLIP has three main forms: water-soluble unstructured form I at neutral or alkaline pH (pH>7), or unstructured form II adhered to the cell membrane surface at acidic pH (pH ⁇ 7) Insert into the cell membrane and form a transmembrane alpha helix III.
  • the traditional cell penetrating peptides directly enter the cytoplasm after passing through the cell membrane.
  • pHLIP has a powerful delivery ability: the cargo molecules are connected to the cell membrane surface through its N-terminus, and then cargo molecules that do not have the ability to penetrate the membrane are targeted to the cytoplasm through its C-terminus. Due to the special physical and chemical properties of pHLIP, it is widely used in the following fields: (1) pHLIP is labeled with fluorescent dyes, imaging probes, etc.
  • pHLIP coupling fluorescent molecules or linking biologically active cytokines or chemokines or various antigens combined with immune cells to treat diseases with acidic characteristics.
  • pHLIP is linked to biologically active cytokines or chemokines or coupled with immunomodulatory drugs combined with endogenous or exogenous immune cells to specifically target Acidic environment to regulate immune response;
  • pHLIP connects tumor-associated antigens, bacterial antigens, viral antigens and other polypeptide antigens, combined with genetically engineered immune cells (T cells, NK cells, NKT cells, macrophages, etc.) immune-enhancing therapy ;
  • Magnetic nanomaterials labeled pHLIP delivered to acidic tissues for magnetic resonance imaging (MRI).
  • the purpose of the present invention is to provide a composition for regulating an acidic environment immune response, which includes an acidic environment targeting molecule and immune cells.
  • the invention also provides a preparation method and application of the composition.
  • the present invention provides a composition composed of an acidic environment targeting molecule and immune cells, the acidic environment targeting molecule is a low pH insertion peptide (low pH insertion peptide, pHLIP) or a variant thereof.
  • the acidic environment targeting molecule is a low pH insertion peptide (low pH insertion peptide, pHLIP) or a variant thereof.
  • Body coupling molecules or fusion proteins, the immune cells are endogenous or exogenous immune cells;
  • the coupling molecule or fusion protein of pHLIP or its variants is connected to cytokines, fluorescent molecules, immunomodulatory drugs, magnetic resonance imaging contrast agents, magnetic nanomaterials, tumor-associated antigens, through the N-terminus of pHLIP or its variants.
  • Bacterial antigens, viral antigens, fungal antigens, plant antigens, polypeptide antigens or antibody Fc fragments are connected to obtain.
  • composition according to the present invention wherein the amino acid sequence of the low pH insert peptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2;
  • amino acid sequence of the variant of the low pH insert peptide is shown in any one of SEQ ID NO: 3-SEQ ID NO: 18.
  • the fusion protein of pHLIP or a variant thereof is shown in any one of SEQ ID NO: 19-SEQ ID NO: 25.
  • the low pHLIP and its variants 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 immune cells are endogenous or exogenous immune cells, including but not limited to autoimmune cells, allogeneic immune cells, autologous genetically engineered immune cells, and allogeneic genetically engineered immune cells.
  • the immune cells include but are not limited to NK cells, NKT cells, CD16/64 chimeric receptor (CD16/64 chimeric receptor, CD16/64 CR) modified NK/NKT/T cells, chimeric antigen receptors (chimeric antigen receptor, CAR) modified NK/NKT/T cells;
  • the immune cells are CD16/64 CR-T cells and CAR-T cells.
  • the cytokines include, but are not limited to, interleukin, interferon, tumor necrosis factor, colony stimulating factor, chemokine and growth factor.
  • the interleukin includes but not limited to IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21;
  • the interferon includes but not limited to IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ ;
  • the tumor necrosis factor includes but not limited to TNF- ⁇ and TNF- ⁇ ;
  • the colony stimulating factor includes but is not limited to SCF, Flt3L, G-CSF , M-CSF and GM-CSF;
  • the chemokines include but are not limited to CCL2, CCL5, CCL19, CCL21, CXCL8, CXCL9, CXCL10, CXCL11, XCL1 and CX3CL1;
  • the growth factors include but are not limited to T
  • the immunomodulatory drug includes an immune enhancing drug and an immunosuppressive drug.
  • the immunosuppressive drugs include but are not limited to cyclosporine, tacrolimus and azathioprine;
  • the immune enhancing drugs include but are not limited to thymosin, transfer factor and recombinant human interferon.
  • composition according to the present invention wherein the tumor-associated antigens include but are not limited to CD19, CD20, CD22, CD30, CD33, ROR1, ROR2, CD38, CD123, CD133, NKG2D ligand, ERBB2, MUC1, CD44v6, CD44v7, CD44v8, CEA, EpCAM, TAG72, KIT, IL-13R ⁇ 2, EGFR, EGFRvIII, GD2, GD3, HMW-MAA, MAGE1, MAGEA3, CD171, NCAM, IL-11R ⁇ , FR ⁇ , PSCA, PSMA, TARP, CAIX, VEGFR, BCMA, CTLA-4, PD-L1, PD-L12, GPC3, CD47, AXL, FAP, ⁇ 5 ⁇ 1, ⁇ v ⁇ 3, TGF ⁇ R, ER, PR, P53, IGFR, CD25, CD117, CD34, CD138, BCMA, Mesothelin, S100, CD70, ALK, RANK and
  • composition according to the present invention wherein the bacterial antigens include, but are not limited to, Staphylococcus antigen, Streptococcus antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis antigen, and Neisseria gonorrhoeae antigen.
  • composition according to the present invention wherein the viral antigens include but are not limited to cytomegalovirus antigen, Barr virus antigen, hepatitis virus antigen, herpes simplex virus antigen, HIV antigen, human T-lymphocyte virus antigen, rubella virus Antigen, SARS coronavirus antigen, varicella-zoster virus, rabies virus antigen, influenza virus antigen, rotavirus antigen and human papilloma virus antigen.
  • the viral antigens include but are not limited to cytomegalovirus antigen, Barr virus antigen, hepatitis virus antigen, herpes simplex virus antigen, HIV antigen, human T-lymphocyte virus antigen, rubella virus Antigen, SARS coronavirus antigen, varicella-zoster virus, rabies virus antigen, influenza virus antigen, rotavirus antigen and human papilloma virus antigen.
  • composition according to the present invention wherein the fungal antigens include but are not limited to dermatophyte fungal antigens, Cryptococcus neoformans antigens and Candida albicans antigens.
  • composition according to the present invention wherein the polypeptide antigen includes but not limited to AviTag (SEQ ID NO: 26), Calmodulin 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-tag1 (SEQ ID NO: 36), Softag3 (SEQ ID NO: 37), Strep-tag (SEQ ID NO: 38), TC tag (SEQ ID NO: 39), V5tag (SEQ ID NO: 40), T7tag (SEQ ID NO: 41), VSV tag (SEQ ID NO: 42), Xpress tag (SEQ ID NO: 43), 3X FLAG tag (SEQ ID NO: 44), Isopeptag (SEQ ID NO:
  • composition according to the present invention wherein the fluorescent molecules include but are not limited to FITC, FAM, PE, APC, PB, Cy3, Cy5, Texas Red, TRITC, GFP, RFP, CFP and BFP.
  • composition according to the present invention wherein the antibody Fc fragment includes, but is not limited to, the Fc fragment of IgG1, the Fc fragment of IgG2, the Fc fragment of IgG3, and the Fc fragment of IgG4.
  • the magnetic nanomaterials include, but are not limited to, paramagnetism represented by gadopentetate meglumine (Gd-DTPA), dysprosium pentetate meglumine (Dy-DTPA), and Mn-DPDP.
  • Gd-DTPA gadopentetate meglumine
  • Dy-DTPA dysprosium pentetate meglumine
  • Mn-DPDP Mn-DPDP.
  • composition according to the present 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 , CAR-T cells, CD16/64CR-T cells, T cells.
  • the composition for regulating an acidic environment immune response is an acidic environment targeting molecule pHLIP fusion protein CCL19-pHLIP and T cells, and the amino acid sequence of CCL19-pHLIP is shown in SEQ ID NO: 49.
  • the chemokine CCL19 is connected to the N-terminus of wild-type pHLIP through the GS flexible linker amino acid sequence (GSSGGSGGSGGSG), separated by a protease cleavage sequence (LSGRSDNH) in order to be cleaved and released in the tumor microenvironment.
  • the histidine tag sequence HHHHHH is used for in vitro detection and purification of CCL19-pHLIP.
  • the composition for regulating the immune response of an acidic environment is an acidic environment targeting molecule pHLIP coupling molecule FITC-pHLIP and FITC CAR-T cells.
  • pHLIP is a wild-type pHLIP, and the amino acid sequence is shown in SEQ ID NO:1.
  • the coupling molecule is a fluorescent molecule fluorescein isothiocyanate FITC.
  • FITC is obtained by coupling aminocaproic acid (ACP) and pHLIP to obtain FITC-pHLIP, the structural formula is: FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT.
  • the immune cells of the composition are FITC CAR-T cells targeting FITC, and the FITC CAR sequence is shown in SEQ ID NO: 50.
  • the composition for regulating an acidic environment immune response is the acidic environment targeting molecule pHLIP fusion protein FLAG-pHLIP and CAR-T cells.
  • the amino acid sequence of pHLIP is shown in SEQ ID NO: 1
  • the fusion protein is the polypeptide antigen FLAG tag, which is obtained by connecting the FLAG tag to the N-terminus of pHLIP, and the sequence is shown in SEQ ID NO: 51.
  • the polypeptide antigen is a PNE tag, and the PNE tag is connected to the N-terminus of pHLIP to obtain PNE-pHLIP.
  • the sequence is shown in 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 as SEQ ID NO:53.
  • the composition for regulating 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 their sequences are shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • the CD16/64CR-T cells are CD16CR-T cells targeting the Fc fragment of IgG1, and the sequence of CD16CR is shown in SEQ ID NO: 54.
  • the present invention also provides the use of the composition of the present invention to prepare a medicine for diagnosing and/or treating tumors, autoimmune diseases, diseases accompanied by ischemia and hypoxia, and/or inflammatory diseases.
  • the present invention also provides a method for diagnosing and/or treating various diseases, the method comprising administering a therapeutically effective amount of the composition to a patient in need.
  • the disease is selected from tumors, autoimmune diseases, diseases accompanied by ischemia and hypoxia, and/or inflammatory diseases.
  • the composition of the present invention has the following uses: (1) When the fusion protein in the composition is a cytokine with biological activity, it is combined with endogenous or exogenous immune cells to combine it with Specific targeting to acidic environment to strengthen or suppress immune response; (2) When the fusion protein in the composition is a chemokine with biological activity, it is specifically targeted in combination with endogenous or exogenous immune cells In an acidic environment, immune cells are recruited to enhance or suppress the immune response; (3) When the coupled molecule in the composition is an immunomodulatory drug, it is combined with endogenous or exogenous immune cells to specifically target it to the acidic environment.
  • the environment plays the role of local immune enhancement or immunosuppression;
  • the fusion protein in the composition is tumor-associated antigen, bacterial antigen, viral antigen, fluorescent molecule and other antigens, combined with genetically engineered immune cells (T cells, NK cells, etc.) , NKT cells, macrophages, etc.), to specifically target the tumor acidic environment to achieve the purpose of broad-spectrum anti-tumor;
  • T cells, NK cells, etc. genetically engineered immune cells
  • NKT cells nuclear-specific kinases, etc.
  • the present invention also provides a method for preparing the composition for regulating and controlling an immune response in an acidic environment, the method comprising the following steps:
  • the above composition is expressed and purified by engineering bacteria and cells.
  • the present invention has the following advantages and beneficial effects:
  • composition of the present invention can be coupled or connected to tumor-associated antigens, pathogen-derived antigens, fluorescent molecules, polypeptide antigens and other antigens.
  • endogenous or exogenous immune cells to regulate the immune response in an acidic environment
  • a universal type can be developed
  • innovative cellular immunotherapy is expected to treat almost all solid tumors.
  • extracellular acidification is also associated with many pathological conditions, including ischemic stroke, nerve damage, infection, tissue tearing and other pathological conditions. Abnormal metabolic activities of cells in damaged or diseased tissues often cause acidification of the extracellular environment. Therefore, extracellular acidification is expected to become a universal marker for diagnosis and treatment of many diseases.
  • pHLIP can be used to target the characteristics of acidic environment and can also be used for the above Diagnosis and treatment of pathological conditions.
  • Figure 1 shows the use of flow cytometry to detect the expression of the acidic environment targeting molecules of the present invention on tumor cells (A549) in microenvironments with different pH conditions, wherein the coupling molecules or fusion proteins are FITC respectively (Fluorescein isothiocyanate), FLAG tag polypeptide and PNE polypeptide (14 amino acid polypeptide derived from yeast transcription factor GCN4);
  • FITC Fluorescein isothiocyanate
  • FLAG tag polypeptide FLAG tag polypeptide
  • PNE polypeptide 14 amino acid polypeptide derived from yeast transcription factor GCN4
  • Figure 2 shows the use of flow cytometry to detect the expression of the acidic environment targeting molecules of the present invention on tumor cells (A549) in microenvironments with different pH conditions, where the fusion protein is an immune cell recruitment molecule, respectively It is an antibody crystallizable fragment (Fc), an antibody crystallizable fragment carrying a trimerized foldon sequence (foldon-Fc), and a crystallizable fragment carrying a trimerized p24 sequence (p24-Fc);
  • Figure 3 shows the chemotaxis effect of the acidic environment targeting molecule pHLIP fusion protein CCL19-pHLIP of the present invention on T cells;
  • Figure 4 shows the use of immunohistochemistry/fluorescence analysis system TissueFAXS to observe the location of acidic environment targeting molecule FITC-pHLIP on tumor tissues and other organs.
  • the white triangle indicates FITC-pHLIP anchored on tumor cells;
  • Figure 5 shows that the composition of the present invention FITC-pHLIP combined with FITC CAR-T cells specifically kills tumor cells in an acidic environment, where A549 is lung adenocarcinoma (alveolar basal epithelial cells) and NCI-H292 is lung cancer cell (lymph node metastasis);
  • Figure 6 shows the composition of the present invention FITC-pHLIP combined with FITC CAR-T cells to control the tumor growth curve of tumor-bearing mice.
  • Example 1 The acidic environment targeting molecule of the composition of the present invention is in the microenvironment of different pH conditions Down, the expression on tumor cells (A549)
  • R10 RPMI1640 complete medium (abbreviated as R10).
  • R10 is RPMI1640 medium (Corning) containing 10% (volume ratio) fetal bovine serum (Biological Industries), The complete medium of 1X streptomycin/penicillin (100X streptomycin/penicillin, Gibco) was cultured in a cell incubator at 37°C and 5% CO 2. After the cells are full, pass at 1:5.
  • pHLIP expression analysis A549 cells were digested with trypsin cell digestion solution for 1 to 2 minutes, neutralized with 10 times the volume of R10 medium, 500xg, and centrifuged for 5 minutes. The supernatant was discarded, and the A549 single cell suspension was resuspended in R10 medium to prepare A549 single cell suspension. Each 1x10 5 cells were placed in three 1.5mL EP tubes and centrifuged at 800xg for 3 minutes. The medium was discarded and added to pH 6.0 and pH 6. 5 and 1xPBS with pH 7.4, adding 5 ⁇ g/mL of the acidic environment targeting molecule of the present invention, respectively, are FITC-pHLIP (the structural formula is:
  • FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT FLAG-pHLIP
  • PNE-pHLIp SEQ ID NO: 52
  • the tested cell samples were divided into 6 groups: (1) pH6.0 untreated group; (2) pH6.0+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP treatment group; (3) pH6.5 untreated group ; (4) pH6.5+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP treatment group; (5) pH7.4 untreated group: (6) pH7.4+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP .
  • Figure 1A, B, and C show that in a neutral solution environment (pH 7.4), FITC-pHLIP, FLAG-pHLIP, and PNE-pHLIP are inserted into the cell membrane of A549 with low efficiency, respectively 30%, 30% and 15%. In an acidic solution environment, FITC-pHLIP can be efficiently inserted into the A549 cell membrane.
  • the insertion efficiencies of pH6.5 and pH6.0 are 85% and 99%, respectively, which are significantly higher than pH7.4; the FLAG-pHLIP polypeptide of the present invention can also be inserted efficiently
  • the insertion efficiency of A549 cell membrane at pH 6.5 and pH 6.0 is 73% and 91%, respectively, which is significantly higher than pH 7.4; the PNE-pHLIP polypeptide of the present invention can also be effectively inserted into A549 cell membrane at pH 6.5 and pH 6.0.
  • the insertion efficiency was 57% and 53%, respectively, which was significantly higher than pH 7.4.
  • Example 2 The acidic environment targeting molecule of the present invention is in microenvironment with different pH conditions, Expression on tumor cells (A549)
  • eukaryotic expression vector The DNA sequence designed according to the amino acid sequence of the fusion protein of the present invention (SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21) is delivered to Shanghai Jerui Bioengineering Co., Ltd. for complete Gene synthesis to prepare eukaryotic expression vectors.
  • HEK293T cells were plated the next day in 15cm culture dishes, each dish 1.5x10 7 cells were plated at 37 °C, Cultivate overnight in a 5% CO 2 cell incubator.
  • the transfection reagent PEIpro was used to transfect the eukaryotic expression vector carrying the DNA sequence of the fusion protein at a total amount of DNA of 30 ⁇ g/dish, and the medium was replaced with a serum-free medium at 37°C and 5% CO 2 for 6 to 8 hours.
  • the cell incubator for 5-7 days.
  • pHLIP fusion proteins of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc Collect the cell culture supernatant, centrifuge at 2000 rpm, 4°C for 10 minutes, and discard the cell pellet. The culture supernatant collected by centrifugation was purified on a Protein G Sepharose column (GE Company, Catalog No. 28-9031-34), and the purified antibody was dissolved in PBS buffer.
  • pHLIP fusion proteins of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc are expressed on A549 cells
  • Cell culture A549 cells are cultured in a cell incubator at 37°C and 5% CO2 using R10 medium. After the cells are full, pass at 1:5.
  • A549 cells were digested with trypsin cell digestion solution for 1 to 2 minutes, 10 times volume of R10 medium was neutralized, 500xg, centrifuged for 5 minutes, R10 medium was resuspended, and A549 single cell suspension was finally obtained .
  • pHLIP-p24-Fc fusion protein (20 ⁇ g/mL), incubate for 1 hour at room temperature, discard the supernatant and wash 3 times with 1xPBS of corresponding pH, add 1xPBS of corresponding pH to resuspend cells, and detect pHLIP-Fc, pHLIP by flow cytometer -The expression levels of folden-Fc and pHLIP-p24-Fc fusion proteins on A549 cells.
  • pH6.0 untreated group (2) pH6.0+pHLIP-Fc group; (3) pH6.0+pHLIP-folden-Fc group; (4) pH6.0+pHLIP-p24-Fc Group; (5) pH7.4 untreated group: (6) pH7.4+pHLIP-Fc group; (7) pH7.4+pHLIP-folden-Fc group; (8) pH7.4+pHLIP-p24-Fc group.
  • Figure 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 insertion efficiency into the cell membrane of A549, which is lower than 4 %.
  • the pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc fusion proteins of the present invention can all be inserted into the A549 cell membrane, and the insertion efficiency of pH6.0 is 21%, 19.1% and 21.4%, respectively. All are higher than pH7.4.
  • the DNA sequence designed according to the amino acid sequence of the fusion protein (SEQ ID NO: 49) of the present invention is delivered to Shanghai Jierui Bioengineering Co., Ltd. for full gene synthesis to prepare eukaryotic expression vectors.
  • HEK293T cells (American Model Culture Collection) are cultured in DMEM complete medium (abbreviated as D10), D10 is DMEM medium (Corning) containing 10% (volume ratio) fetal bovine serum (Biological Industries), 1X streptomycin / Penicillin (100X Streptomycin / Penicillin, Gibco) complete medium, cultured in a cell culture incubator at 37°C and 5% CO 2. After the cells are full, pass at 1:5.
  • D10 DMEM complete medium (abbreviated as D10)
  • D10 is DMEM medium (Corning) containing 10% (volume ratio) fetal bovine serum (Biological Industries), 1X streptomycin / Penicillin (100X Streptomycin / Penicillin, Gibco) complete medium, cultured in a cell culture incubator at 37°C and 5% CO 2. After the cells are full, pass at 1:5.
  • HEK293T cells were digested with trypsin cell digestion solution for 1-2 minutes, 10 times the volume of D10 medium was neutralized, 500xg, centrifuged for 5 minutes, and resuspended in DMEM complete medium to finally obtain HEK293T single cell suspension. Put HEK293T cells in a six-well plate, spread 1 ⁇ 10 6 cells per well, and culture them overnight in a 37°C, 5% CO 2 cell incubator.
  • transfection reagent Turbofect to transfect the eukaryotic expression vector carrying the DNA sequence of the fusion protein at a total amount of 4 ⁇ g/well of DNA, and continue to culture for 2 days in a cell incubator at 37°C and 5% CO 2 to collect and culture The supernatant was centrifuged at 2000 rpm, 4°C for 10 minutes, the cell pellet was collected for WB detection, and the culture supernatant after centrifugation was collected and frozen for later use.
  • Figure 3A shows that the eukaryotic expression vector carrying the DNA sequence of the CCL19-pHLIP fusion protein was highly expressed in HEK293T cells.
  • Figure 3B shows that the acidic environment targeting molecule CCL19-pHLIP fusion protein of the present invention can effectively chemoattract T cells, and the chemotaxis efficiency is equivalent to the positive control CCL19.
  • 5x10 5 melanin B16 cells were inoculated subcutaneously into the right abdomen of C57BL/6 mice (purchased from Shanghai Shrek Laboratory Animal Co., Ltd.). When the tumor diameter grows to 0.5-1 cm, 1 mg/kg FITC-pHLIP is injected intravenously. Mice were sacrificed at 4, 12, 24, 48, 72, and 96 hours after injection. The tumor tissues, heart, liver, spleen, lung and kidney were taken to make frozen sections, and fixed with 4% paraformaldehyde using PE-anti-FITC (Abcam, ab25539) and DAPI staining, using immunohistochemistry/fluorescence analysis system TissueFAXS for analysis.
  • PE-anti-FITC Abcam, ab25539
  • DAPI staining using immunohistochemistry/fluorescence analysis system TissueFAXS for analysis.
  • Figure 4 shows that FITC-pHLIP was enriched in tumor tissue at least 6 hours after reinfusion, and it was observed that FITC-pHLIP was clearly localized on tumor cells until 24 hours after reinfusion. However, FITC-pHLIP was not enriched in the heart, liver, spleen, lung and kidney at 4, 12, 24, and 48 hours after reinfusion.
  • Lung cancer cells A549-luciferase/NCI-H292-luciferase (Shanghai Xinwan Biotechnology Co., Ltd.) that stably express luciferase use R10 medium containing 10% fetal bovine serum, 1x streptomycin/penicillin, at 37°C, 5 %CO 2 cell culture incubator. After the cells are full, pass at 1:5.
  • A549-luciferase/NCI-H292-luciferase cells were digested with trypsin cell digestion solution for 1 to 2 minutes, and 10 times volume of R10 medium was neutralized, 500xg, centrifuged for 5 minutes, and R10 medium was resuspended to obtain A549-luciferase/ NCI-H292-luciferase single cell suspension.
  • the A549-luciferase/NCI-H292-luciferase cells were plated in a 96-well black bottom plate, 1 ⁇ 10 4 cells per well, with a total volume of 100 ⁇ L, and cultured overnight in a cell incubator at 37°C and 5% CO 2.
  • the A549-luciferase/NCI-H292-luciferase cells were cultured overnight on the culture plate, the culture supernatant was discarded and the R10 medium with pH 6.5 and pH 6.0 was added, and the FITC-pHLIP (5 ⁇ g/mL) of Example 1 was added. Incubate at 37°C for 1 hour, discard the supernatant and wash 3 times with 1xPBS of corresponding pH, and add 100 ⁇ L of R10 medium with pH 6.5 and pH 6.0. Grouping: pH6.5 untreated group; (2) pH6.5+FITC-pHLIP treatment group; (3) pH6.0 untreated group; (4) pH6.0+FITC-pHLIP treatment group.
  • Figure 5A shows that in an acidic environment, FITC-pHLIP combined with anti-FITC CAR-T cells can specifically kill lung adenocarcinoma cells A549-luciferase.
  • Figure 5B shows that in an acidic solution environment, FITC-pHLIP combined with anti-FITC CAR-T cells can specifically kill the highly metastatic lung cancer cell NCI-H292-luciferase.
  • Example 6 Combination of FITC-pHLIP, an acidic environment targeting molecule, combined with anti-FITC CAR-T cells Evaluation of anti-tumor effect in vivo
  • NCI-H292 lung cancer cells (Shanghai Xinwan Biotechnology Co., Ltd.) were inoculated subcutaneously in the right abdomen of B-NDG immunodeficiency mice (purchased from Beijing Biocytogene Biotechnology Co., Ltd.), 2x10 6 cells/mouse, When the diameter of the tumor grows to 0.5-1 cm, the tumors that are too large and too small are eliminated, and mice with the same tumor size are randomly grouped. Divided into 3 groups: PBS alone injection group, 5 animals; anti-FITC CAR-T cell injection group, 5 animals; FITC-pHLIP combined with anti-FITC CAR-T cell injection group, 5 animals. The tumor size was measured every 3 days.
  • FITC-pHLIP administration each mouse is intravenously injected 20 ⁇ g (125 ⁇ L) each time, starting from the day after the grouping is completed, once a day, once every other day, and 10 times in a row.
  • Anti-FITC CAR-T cells administration intravenous reinfusion each time per mouse anti-FITC CAR-T 10 7 cells / 125 ⁇ L, a total of 2 times. The first reinfusion starts on the day after the grouping is completed. The injection time is 12 hours after the first injection of FITC-pHLIP, and the second cell reinfusion is performed nine days later.
  • Figure 6 shows that FITC-pHLIP combined 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 could control tumor growth in tumor-bearing mice.

Abstract

Provided are a composition for regulating an immune response in an acidic environment, and a preparation method therefor and a use thereof. The composition is composed of an acidic-environment-targeting molecule and an immune cell, wherein the acidic-environment-targeting molecule is a coupling molecule or a fusion protein of a pH low insertion peptide (pHLIP) or a variant thereof, and the immune cell is an endogenous or exogenous immune cell. The composition can be used to treat solid tumors by means of regulating an immune response in an acidic environment, especially an immune response in a tumor acidic environment.

Description

用于调控酸性环境免疫应答的组合物、其制备方法和用途Composition for regulating and controlling acidic environment immune response, preparation method and application thereof 技术领域Technical field
本发明属于生物医药技术领域，涉及一种用于调控酸性环境免疫应答的组合物，本发明还涉及所述组合物的制备方法和用途。The invention belongs to the technical field of biomedicine, and relates to a composition for regulating the immune response of an acidic environment. The invention also relates to a preparation method and application of the composition.
背景技术Background technique
肿瘤的免疫治疗涉及单克隆抗体、疫苗、基因治疗、免疫细胞治疗等，其中靶向肿瘤抗原的单克隆抗体、免疫检查点抑制剂、T细胞受体修饰T细胞(TCR-T)、嵌合抗原受体T细胞(CAR-T)等免疫治疗方法，在临床研究中显示出令人瞩目的抗肿瘤效果。然而，由于肿瘤特异性抗原缺乏而带来的脱靶效应、特别是实体瘤的肿瘤异质性以及肿瘤抑制性微环境对免疫细胞浸润与功能的负向调控等，严重制约了免疫治疗的效果，并引起生物医药技术领域对该种新型肿瘤干预技术的安全性的担忧。Tumor immunotherapy involves monoclonal antibodies, vaccines, gene therapy, immune cell therapy, etc., among which monoclonal antibodies targeting tumor antigens, immune checkpoint inhibitors, T cell receptor modified T cells (TCR-T), chimeric Immunotherapy methods such as antigen receptor T cells (CAR-T) have shown remarkable anti-tumor effects in clinical studies. However, the off-target effects caused by the lack of tumor-specific antigens, especially the tumor heterogeneity of solid tumors and the negative regulation of immune cell infiltration and function by the tumor suppressive microenvironment, have severely restricted the effectiveness of immunotherapy. And it has caused concerns about the safety of this new tumor intervention technology in the field of biomedical technology.
由于肿瘤微环境异常的血管结构以及肿瘤细胞的过度增殖，使得肿瘤组织的代谢更多地依赖于无氧糖酵解，其代谢产物乳酸与呼吸作用产生的CO 2共同造成了肿瘤微环境的酸化，肿瘤细胞外pH(6.5-6.8)显著低于外周血或正常组织的pH(7.4)，酸性环境是多种实体肿瘤的一个共有特征。研究表明，肿瘤的酸性环境可以调控多种生物过程，包括细胞增殖、血管新生、免疫抑制、侵袭和耐药等，甚至促使高侵袭表型的肿瘤形成，肿瘤恶性程度更高。因此，靶向肿瘤的酸性环境是一种全新的诊断和治疗方法，其不依赖于特定的肿瘤抗原，不受限于肿瘤异质性，既可以用于预测肿瘤的恶性程度，又可以递送治疗性药物至多种肿瘤组织从而达到治疗的目的。 Due to the abnormal vascular structure of the tumor microenvironment and the excessive proliferation of tumor cells, the metabolism of tumor tissues is more dependent on anaerobic glycolysis. The metabolite lactic acid and CO 2 produced by respiration together cause the acidification of the tumor microenvironment The extracellular pH (6.5-6.8) of tumor cells is significantly lower than the pH (7.4) of peripheral blood or normal tissues. Acidic environment is a common feature of many solid tumors. Studies have shown that the acidic environment of tumors can regulate a variety of biological processes, including cell proliferation, angiogenesis, immunosuppression, invasion and drug resistance, etc., and even promote the formation of highly invasive phenotype tumors, and tumors are more malignant. Therefore, targeting the acidic environment of tumors is a new diagnosis and treatment method that does not depend on specific tumor antigens and is not limited to tumor heterogeneity. It can be used to predict the malignancy of tumors and deliver treatments. Sex drugs to a variety of tumor tissues to achieve the purpose of treatment.
低pH插入肽(pH low insertion peptide，pHLIP)是来源于细菌的视紫红质跨膜螺旋蛋白C的一种水溶性多肽，能够插入细胞双层脂膜并形成稳定的跨膜α螺旋。pHLIP具有三种主要形态，中性或碱性pH(pH＞7)下形成溶于水的无结构形态I，或粘附到细胞膜表面的无结构形态II，于酸性pH(pH＜7)下插入细胞膜并形成跨膜α螺旋的形态III。传统的细胞穿透肽穿过细胞膜后则直接进入细胞质中。与此截然不同，pHLIP和脂质双分子层之间不存在特异性的相互作用，仅仅受到所处环境下的pH影响即在酸性环境下插入细胞膜，其N端位于细胞膜外而C端则进入细胞质中。因此，pHLIP具有强大的传递能力：通过其N端连接货物分子并系于细胞膜表面，进而通过其C端将不具穿膜能力的货物分子靶向到细胞质中。由于pHLIP所具有的这种特殊的理化性质，它被广泛应用于以下领域：(1)通过荧光染料、成 像探针等标记pHLIP并递送至酸性组织，用于疾病诊断或荧光图像导航手术；(2)通过放射性核素标记pHLIP递送至酸性组织，进行核成像(PET/SPECT)；(3)与毒素、药物、治疗性基因偶联以治疗疾病；(4)特异递送纳米材料至酸化组织。Low pH insertion peptide (pH low insertion peptide, pHLIP) is a water-soluble peptide derived from bacterial rhodopsin transmembrane helix protein C, which can be inserted into the double lipid membrane of cells and form a stable transmembrane alpha helix. pHLIP has three main forms: water-soluble unstructured form I at neutral or alkaline pH (pH>7), or unstructured form II adhered to the cell membrane surface at acidic pH (pH<7) Insert into the cell membrane and form a transmembrane alpha helix III. The traditional cell penetrating peptides directly enter the cytoplasm after passing through the cell membrane. In contrast to this, there is no specific interaction between pHLIP and lipid bilayers. It is only affected by the pH of the environment that it is inserted into the cell membrane in an acidic environment. Its N-terminus is outside the cell membrane and the C-terminus is entering the cell membrane. In the cytoplasm. Therefore, pHLIP has a powerful delivery ability: the cargo molecules are connected to the cell membrane surface through its N-terminus, and then cargo molecules that do not have the ability to penetrate the membrane are targeted to the cytoplasm through its C-terminus. Due to the special physical and chemical properties of pHLIP, it is widely used in the following fields: (1) pHLIP is labeled with fluorescent dyes, imaging probes, etc. and delivered to acidic tissues for disease diagnosis or fluorescent image navigation surgery; ( 2) Use radionuclide labeled pHLIP to deliver to acidic tissues for nuclear imaging (PET/SPECT); (3) Coupling with toxins, drugs, and therapeutic genes to treat diseases; (4) Specific delivery of nanomaterials to acidified tissues.
但是，现有技术中并没有pHLIP偶联荧光分子或连接生物活性的细胞因子或趋化因子或各种抗原联合免疫细胞治疗具有酸性特征疾病的相关报道。具体地，关于pHLIP在以下方面的潜在用途尚未见报道：(1)pHLIP连接生物活性的细胞因子或趋化因子或偶联免疫调节药物联合内源性或外源性免疫细胞，特异靶向至酸性环境以调节免疫应答；(2)pHLIP连接肿瘤相关抗原、细菌抗原、病毒抗原等多肽抗原，联合基因工程免疫细胞(T细胞、NK细胞、NKT细胞、巨噬细胞等)的免疫增强型疗法；(3)磁性纳米材料标记pHLIP，递送至酸性组织进行磁共振成像(MRI)。However, in the prior art, there is no report about pHLIP coupling fluorescent molecules or linking biologically active cytokines or chemokines or various antigens combined with immune cells to treat diseases with acidic characteristics. Specifically, there are no reports on the potential use of pHLIP in the following aspects: (1) pHLIP is linked to biologically active cytokines or chemokines or coupled with immunomodulatory drugs combined with endogenous or exogenous immune cells to specifically target Acidic environment to regulate immune response; (2) pHLIP connects tumor-associated antigens, bacterial antigens, viral antigens and other polypeptide antigens, combined with genetically engineered immune cells (T cells, NK cells, NKT cells, macrophages, etc.) immune-enhancing therapy ; (3) Magnetic nanomaterials labeled pHLIP, delivered to acidic tissues for magnetic resonance imaging (MRI).
也就是说，当前对更先进的pHLIP的组合物存在需求。In other words, there is currently a demand for more advanced pHLIP compositions.
发明内容Summary of the invention
基于此，本发明的目的是提供一种用于调控酸性环境免疫应答的组合物，其包括酸性环境靶向分子和免疫细胞。本发明还提供了所述组合物的制备方法及其用途。Based on this, the purpose of the present invention is to provide a composition for regulating an acidic environment immune response, which includes an acidic environment targeting molecule and immune cells. The invention also provides a preparation method and application of the composition.
一方面，本发明提供了一种组合物，所述组合物由酸性环境靶向分子和免疫细胞组成，所述酸性环境靶向分子为低pH插入肽(low pH insertion peptide，pHLIP)或其变体的偶联分子或融合蛋白，所述免疫细胞为内源性或外源性免疫细胞；In one aspect, the present invention provides a composition composed of an acidic environment targeting molecule and immune cells, the acidic environment targeting molecule is a low pH insertion peptide (low pH insertion peptide, pHLIP) or a variant thereof. Body coupling molecules or fusion proteins, the immune cells are endogenous or exogenous immune cells;
其中，所述pHLIP或其变体的偶联分子或融合蛋白通过pHLIP或其变体的N端与细胞因子、荧光分子、免疫调节药物、磁共振成像造影剂、磁性纳米材料、肿瘤相关抗原、细菌抗原、病毒抗原、真菌抗原、植物抗原、多肽抗原或抗体Fc片段连接获得。Wherein, the coupling molecule or fusion protein of pHLIP or its variants is connected to cytokines, fluorescent molecules, immunomodulatory drugs, magnetic resonance imaging contrast agents, magnetic nanomaterials, tumor-associated antigens, through the N-terminus of pHLIP or its variants. Bacterial antigens, viral antigens, fungal antigens, plant antigens, polypeptide antigens or antibody Fc fragments are connected to obtain.
根据本发明所述的组合物，其中，所述低pH插入肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；The composition according to the present invention, wherein the amino acid sequence of the low pH insert peptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2;
优选地，所述低pH插入肽的变体的氨基酸序列如SEQ ID NO:3-SEQ ID NO:18中任一项所示。Preferably, the amino acid sequence of the variant of the low pH insert peptide is shown in any one of SEQ ID NO: 3-SEQ ID NO: 18.
优选地，所述pHLIP或其变体的融合蛋白如SEQ ID NO:19-SEQ ID NO:25中任一项所示。Preferably, the fusion protein of pHLIP or a variant thereof is shown in any one of SEQ ID NO: 19-SEQ ID NO: 25.
具体地，所述低pHLIP及其变体包括以下多肽：Specifically, the low pHLIP and its variants 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)。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).
根据本发明所述的组合物，其中，所述免疫细胞为内源性或外源性免疫细胞，包括但不限于自体免疫细胞、异体免疫细胞、自体基因工程免疫细胞、异体基因工程免疫细胞。According to the composition of the present invention, the immune cells are endogenous or exogenous immune cells, including but not limited to autoimmune cells, allogeneic immune cells, autologous genetically engineered immune cells, and allogeneic genetically engineered immune cells.
优选地，所述免疫细胞包括但不限于NK细胞、NKT细胞、CD16/64嵌合受体(CD16/64 chimeric receptor，CD16/64 CR)修饰的NK/NKT/T细胞、嵌合抗原受体(chimeric antigen receptor，CAR)修饰的NK/NKT/T细胞；Preferably, the immune cells include but are not limited to NK cells, NKT cells, CD16/64 chimeric receptor (CD16/64 chimeric receptor, CD16/64 CR) modified NK/NKT/T cells, chimeric antigen receptors (chimeric antigen receptor, CAR) modified NK/NKT/T cells;
更优选地，所述免疫细胞为CD16/64 CR-T细胞和CAR-T细胞。More preferably, the immune cells are CD16/64 CR-T cells and CAR-T cells.
根据本发明所述的组合物，其中所述的细胞因子包括但不限于白细胞介素、干扰素、肿瘤坏死因子、集落刺激因子、趋化因子和生长因子。其中，所述白细胞介素包括但不限于IL-2、IL-7、IL-10、IL-12、IL-15和IL-21；所述干扰素包括但不限于IFN-α、IFN-β、IFN-γ、IFN-κ和IFN-λ；其中，所述肿瘤坏死因子包括但不限于TNF-α和TNF-β；其中，所述集落刺激因子包括但不限于SCF、Flt3L、G-CSF、M-CSF和GM-CSF；其中，所述趋化因子包括但不限于CCL2、CCL5、CCL19、CCL21、CXCL8、CXCL9、CXCL10、CXCL11、XCL1和CX3CL1；其中，所述生长因子包括但不限于TGF-β、EGF、FGF、NGF、PDGF和VEGF。According to the composition of the present invention, the cytokines include, but are not limited to, interleukin, interferon, tumor necrosis factor, colony stimulating factor, chemokine and growth factor. Wherein, the interleukin includes but not limited to IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21; the interferon includes but not limited to IFN-α, IFN-β , IFN-γ, IFN-κ and IFN-λ; wherein, the tumor necrosis factor includes but 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-β, EGF, FGF, NGF, PDGF and VEGF.
根据本发明所述的组合物，其中，所述免疫调节药物包括免疫增强药物和免疫抑制药物。其中，所述免疫抑制药物包括但不限于环孢素、他克莫司和硫唑嘌呤；所述免疫增强药物包括但不限于胸腺素、转移因子和重组人干扰素。The composition according to the present invention, wherein the immunomodulatory drug includes an immune enhancing drug and an immunosuppressive drug. Wherein, the immunosuppressive drugs include but are not limited to cyclosporine, tacrolimus and azathioprine; the immune enhancing drugs include but are not limited to thymosin, transfer factor and recombinant human interferon.
根据本发明所述的组合物，其中，所述肿瘤相关抗原包括但不限于CD19、CD20、CD22、CD30、CD33、ROR1、ROR2、CD38、CD123、CD133、NKG2D配体、ERBB2、MUC1、CD44v6、CD44v7、CD44v8、CEA、EpCAM、TAG72、KIT、IL-13Rα2、EGFR、EGFRvIII、GD2、GD3、HMW-MAA、MAGE1、MAGEA3、CD171、NCAM、IL-11Rα、FRα、PSCA、PSMA、TARP、CAIX、VEGFR、BCMA、CTLA-4、PD-L1、PD-L12、GPC3、CD47、AXL、FAP、α5β1、αvβ3、TGFβR、ER、PR、P53、IGFR、CD25、CD117、CD34、CD138、BCMA、Mesothelin、S100、CD70、ALK、RANK和HER3。The composition according to the present invention, wherein the tumor-associated antigens include but are not limited to CD19, CD20, CD22, CD30, CD33, ROR1, ROR2, CD38, CD123, CD133, NKG2D ligand, ERBB2, MUC1, CD44v6, CD44v7, CD44v8, CEA, EpCAM, TAG72, KIT, IL-13Rα2, EGFR, EGFRvIII, GD2, GD3, HMW-MAA, MAGE1, MAGEA3, CD171, NCAM, IL-11Rα, FRα, PSCA, PSMA, TARP, CAIX, VEGFR, BCMA, CTLA-4, PD-L1, PD-L12, GPC3, CD47, AXL, FAP, α5β1, αvβ3, TGFβR, ER, PR, P53, IGFR, CD25, CD117, CD34, CD138, BCMA, Mesothelin, S100, CD70, ALK, RANK and HER3.
根据本发明所述的组合物，其中，所述细菌抗原包括但不限于葡萄球菌抗原、链球菌抗原、肺炎链球菌抗原、脑膜炎奈瑟菌抗原和淋病奈瑟菌抗原。The composition according to the present invention, wherein the bacterial antigens include, but are not limited to, Staphylococcus antigen, Streptococcus antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis antigen, and Neisseria gonorrhoeae antigen.
根据本发明所述的组合物，其中，所述病毒抗原包括但不限于巨细胞病毒抗原、巴尔病毒抗原、肝炎病毒抗原、单纯疱疹病毒抗原、HIV抗原、人T-淋巴细胞病毒抗原、风疹病毒抗原、SARS冠状病毒抗原、水痘-带状疱疹病毒、狂犬病毒抗原、流感病毒抗原、轮状病毒抗原和人乳头瘤病毒抗原。The composition according to the present invention, wherein the viral antigens include but are not limited to cytomegalovirus antigen, Barr virus antigen, hepatitis virus antigen, herpes simplex virus antigen, HIV antigen, human T-lymphocyte virus antigen, rubella virus Antigen, SARS coronavirus antigen, varicella-zoster virus, rabies virus antigen, influenza virus antigen, rotavirus antigen and human papilloma virus antigen.
根据本发明所述的组合物，其中，所述真菌抗原包括但不限于皮肤癣真菌抗原、新生隐球菌抗原和白假丝念珠菌抗原。The composition according to the present invention, wherein the fungal antigens include but are not limited to dermatophyte fungal antigens, Cryptococcus neoformans antigens and Candida albicans antigens.
根据本发明所述的组合物，其中，所述多肽抗原包括但不限于AviTag(SEQ ID NO:26)、Calmodulin 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-tag1(SEQ ID NO:36)、Softag3(SEQ ID NO:37)、Strep-tag(SEQ ID NO:38)、TC tag(SEQ ID NO:39)、V5tag(SEQ ID NO:40)、T7tag(SEQ ID NO:41)、VSV tag(SEQ ID NO:42)、Xpress tag(SEQ ID NO:43)、3X FLAG tag(SEQ ID NO:44)、Isopeptag(SEQ ID NO:45)、Spytag(SEQ ID NO:46)、Snooptag(SEQ ID NO:47)和PNE tag(SEQ ID NO:48)。The composition according to the present invention, wherein the polypeptide antigen includes but not limited to AviTag (SEQ ID NO: 26), Calmodulin 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-tag1 (SEQ ID NO: 36), Softag3 (SEQ ID NO: 37), Strep-tag (SEQ ID NO: 38), TC tag (SEQ ID NO: 39), V5tag (SEQ ID NO: 40), T7tag (SEQ ID NO: 41), VSV tag (SEQ ID NO: 42), Xpress tag (SEQ ID NO: 43), 3X FLAG tag (SEQ ID NO: 44), Isopeptag (SEQ ID NO: 45), Spytag (SEQ ID NO: 46), Snooptag (SEQ ID NO: 47) and PNE tag (SEQ ID NO: 48) .
根据本发明所述的组合物，其中，所述荧光分子包括但不限于FITC、FAM、PE、APC、PB、Cy3、Cy5、Texas Red、TRITC、GFP、RFP、CFP和BFP。The composition according to the present invention, wherein the fluorescent molecules include but are not limited to FITC, FAM, PE, APC, PB, Cy3, Cy5, Texas Red, TRITC, GFP, RFP, CFP and BFP.
根据本发明所述的组合物，其中，所述抗体Fc片段包括但不限于IgG1的Fc片段、IgG2的Fc片段、IgG3的Fc片段和IgG4的Fc片段。The composition according to the present invention, wherein the antibody Fc fragment includes, but is not limited to, the Fc fragment of IgG1, the Fc fragment of IgG2, the Fc fragment of IgG3, and the Fc fragment of IgG4.
更优选地，所述磁性纳米材料包括但不限于钆喷酸葡胺(Gd-DTPA)、镝喷酸葡胺(Dy-DTPA)、锰福地吡三钠(Mn-DPDP)为代表的顺磁性金属离子螯合物和Fe 2O 3、Fe 3O 4为核心的超顺磁性金属氧化物粒子。 More preferably, the magnetic nanomaterials include, but are not limited to, paramagnetism represented by gadopentetate meglumine (Gd-DTPA), dysprosium pentetate meglumine (Dy-DTPA), and Mn-DPDP. Superparamagnetic metal oxide particles with metal ion chelate and Fe 2 O 3 and Fe 3 O 4 as the core.
根据本发明所述的组合物，其中，所述酸性环境靶向分子选自FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP、Fc-pHLIP或CCL19-pHLIP；所述免疫细胞为FITC CAR-T细胞、CAR-T细胞、CD16/64CR-T细胞、T细胞。The composition according to the present 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 , CAR-T cells, CD16/64CR-T cells, T cells.
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP融合蛋白CCL19-pHLIP和T细胞，CCL19-pHLIP的氨基酸序列如SEQ ID NO:49所示。具体地，趋化因子CCL19通过GS柔性连接子氨基酸序列(GSSGGSGGSGGSG)连接至野生型pHLIP的N端，中间以蛋白酶切割序列(LSGRSDNH)间隔，以便在肿瘤微环境内切 割并释放。另外，组氨酸标签序列(HHHHHH)则用于体外检测和纯化CCL19-pHLIP。According to an embodiment of the present invention, the composition for regulating an acidic environment immune response is an acidic environment targeting molecule pHLIP fusion protein CCL19-pHLIP and T cells, and the amino acid sequence of CCL19-pHLIP is shown in SEQ ID NO: 49. Specifically, the chemokine CCL19 is connected to the N-terminus of wild-type pHLIP through the GS flexible linker amino acid sequence (GSSGGSGGSGGSG), separated by a protease cleavage sequence (LSGRSDNH) in order to be cleaved and released in the tumor microenvironment. In addition, the histidine tag sequence (HHHHHH) is used for in vitro detection and purification of CCL19-pHLIP.
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP偶联分子FITC-pHLIP和FITC CAR-T细胞。具体地，所述pHLIP为野生型pHLIP，氨基酸序列如SEQ ID NO:1所示。所述偶联分子为荧光分子异硫氰酸荧光素FITC。其中，FITC通过氨基己酸(ACP)与pHLIP偶联获得FITC-pHLIP，结构式为：FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT。According to an embodiment of the present invention, the composition for regulating the immune response of an acidic environment is an acidic environment targeting molecule pHLIP coupling molecule FITC-pHLIP and FITC CAR-T cells. Specifically, the pHLIP is a wild-type pHLIP, and the amino acid sequence is shown in SEQ ID NO:1. The coupling molecule is a fluorescent molecule fluorescein isothiocyanate FITC. Among them, FITC is obtained by coupling aminocaproic acid (ACP) and pHLIP to obtain FITC-pHLIP, the structural formula is: FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT.
其中，所述组合物的免疫细胞为靶向FITC的FITC CAR-T细胞，FITC CAR序列如SEQ ID NO:50所示。Wherein, the immune cells of the composition are FITC CAR-T cells targeting FITC, and the FITC CAR sequence is shown in SEQ ID NO: 50.
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP融合蛋白FLAG-pHLIP和CAR-T细胞。According to one embodiment of the present invention, the composition for regulating an acidic environment immune response is the acidic environment targeting molecule pHLIP fusion protein FLAG-pHLIP and CAR-T cells.
具体地，pHLIP的氨基酸序列如SEQ ID NO:1所示，所述融合蛋白为多肽抗原FLAG tag，FLAG tag连接至pHLIP的N端获得，序列如SEQ ID NO:51所示。或者，多肽抗原为PNE tag，PNE tag连接至pHLIP的N端获得PNE-pHLIP，序列如SEQ ID NO:52所示。所述免疫细胞分别为靶向FLAG tag或PNE tag的FLAG CAR-T细胞或PNE CAR-T细胞，其中，所述PNE CAR的氨基酸序列如SEQ ID NO:53。Specifically, the amino acid sequence of pHLIP is shown in SEQ ID NO: 1, and the fusion protein is the polypeptide antigen FLAG tag, which is obtained by connecting the FLAG tag to the N-terminus of pHLIP, and the sequence is shown in SEQ ID NO: 51. Alternatively, the polypeptide antigen is a PNE tag, and the PNE tag is connected to the N-terminus of pHLIP to obtain PNE-pHLIP. The sequence is shown in 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 as SEQ ID NO:53.
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP融合蛋白和CD16/64CR-T细胞。所述融合蛋白为pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc，其序列如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示。所述CD16/64CR-T细胞为靶向IgG1的Fc片段的CD16CR-T细胞，CD16CR的序列如SEQ ID NO:54所示。According to one embodiment of the present invention, the composition for regulating 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 their sequences are shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. The CD16/64CR-T cells are CD16CR-T cells targeting the Fc fragment of IgG1, and the sequence of CD16CR is shown in SEQ ID NO: 54.
另一方面，本发明还提供了本发明的组合物制备用于诊断和/或治疗肿瘤、自身免疫性疾病、伴有缺血缺氧的疾病和/或炎性疾病的药物的用途。On the other hand, the present invention also provides the use of the composition of the present invention to prepare a medicine for diagnosing and/or treating tumors, autoimmune diseases, diseases accompanied by ischemia and hypoxia, and/or inflammatory diseases.
再一方面，本发明还提供了用于诊断和/或治疗各种疾病的方法，所述方法包括给予有需要的患者治疗有效量的组合物。In another aspect, the present invention also provides a method for diagnosing and/or treating various diseases, the method comprising administering a therapeutically effective amount of the composition to a patient in need.
其中，所述疾病选自肿瘤、自身免疫性疾病、伴有缺血缺氧的疾病和/或炎性疾病等。Wherein, the disease is selected from tumors, autoimmune diseases, diseases accompanied by ischemia and hypoxia, and/or inflammatory diseases.
虽然本发明仅仅以SEQ ID NO:1所示的pHLIP进行了说明，但由于pHLIP及其变体理化性质的相似性，所以本领域技术人员可以毫无疑义地地确认，所有的pHLIP及其变体均可以应用于本发明的技术方案并获得相同的 效果。Although the present invention is only described with the pHLIP shown in SEQ ID NO:1, due to the similarity of the physicochemical properties of pHLIP and its variants, those skilled in the art can undoubtedly confirm that all pHLIPs and their variants Any body can be applied to the technical solution of the present invention and obtain the same effect.
具体地，发明人发现，本发明的组合物具有以下的用途：(1)所述组合物中的融合蛋白为具有生物活性的细胞因子时，联合内源性或外源性免疫细胞，将其特异靶向至酸性环境，加强或抑制免疫应答；(2)所述组合物中的融合蛋白为具有生物活性的趋化因子时，联合内源性或外源性免疫细胞，将其特异靶向至酸性环境，招募免疫细胞，加强或抑制免疫应答；(3)所述组合物中的偶联分子为免疫调节药物时，联合内源性或外源性免疫细胞，将其特异靶向至酸性环境，发挥局部免疫增强或免疫抑制的作用；(4)所述组合物中的融合蛋白为肿瘤相关抗原、细菌抗原、病毒抗原、荧光分子等抗原，联合基因工程免疫细胞(T细胞、NK细胞、NKT细胞、巨噬细胞等)，将其特异靶向至肿瘤酸性环境，达到广谱抗肿瘤的目的；(5)所述组合物中的偶联分子为磁性纳米材料时，递送至酸性组织进行磁共振成像(MRI)。Specifically, the inventors found that the composition of the present invention has the following uses: (1) When the fusion protein in the composition is a cytokine with biological activity, it is combined with endogenous or exogenous immune cells to combine it with Specific targeting to acidic environment to strengthen or suppress immune response; (2) When the fusion protein in the composition is a chemokine with biological activity, it is specifically targeted in combination with endogenous or exogenous immune cells In an acidic environment, immune cells are recruited to enhance or suppress the immune response; (3) When the coupled molecule in the composition is an immunomodulatory drug, it is combined with endogenous or exogenous immune cells to specifically target it to the acidic environment. The environment plays the role of local immune enhancement or immunosuppression; (4) The fusion protein in the composition is tumor-associated antigen, bacterial antigen, viral antigen, fluorescent molecule and other antigens, combined with genetically engineered immune cells (T cells, NK cells, etc.) , NKT cells, macrophages, etc.), to specifically target the tumor acidic environment to achieve the purpose of broad-spectrum anti-tumor; (5) When the coupling molecule in the composition is a magnetic nanomaterial, it is delivered to the acidic tissue Perform magnetic resonance imaging (MRI).
再另一方面，本发明还提供了所述的调控酸性环境免疫应答的组合物的制备方法，所述方法包括以下步骤：In yet another aspect, the present invention also provides a method for preparing the composition for regulating and controlling an immune response in an acidic environment, the method comprising the following steps:
利用多肽自动合成仪，通过固相法合成上述组合物；或Use an automatic peptide synthesizer to synthesize the above-mentioned composition by solid-phase method; or
用生物合成法，通过工程菌和细胞表达并纯化上述组合物。Using biosynthesis, the above composition is expressed and purified by engineering bacteria and cells.
与现有技术相比，本发明具有以下优点和有益效果:Compared with the prior art, the present invention has the following advantages and beneficial effects:
本发明的组合物可以偶联或连接肿瘤相关抗原、病原体来源的抗原、荧光分子、多肽抗原等抗原，通过联合内源性或外源性免疫细胞调控酸性环境的免疫应答，开发出通用型的创新细胞免疫疗法，有望治疗几乎所有的实体肿瘤。此外，除了实体肿瘤，细胞外酸化还与许多病理状态相关，包括缺血性中风、神经损伤、感染、组织撕裂受损等多种病理状态。细胞在损伤或病变组织中的异常代谢活动常常造成细胞外环境的酸化，所以胞外酸化有望成为多种疾病的诊断和治疗通用标志物，利用pHLIP可靶向酸性环境的特征，亦可用于上述病理状态的诊断和治疗。The composition of the present invention can be coupled or connected to tumor-associated antigens, pathogen-derived antigens, fluorescent molecules, polypeptide antigens and other antigens. By combining endogenous or exogenous immune cells to regulate the immune response in an acidic environment, a universal type can be developed Innovative cellular immunotherapy is expected to treat almost all solid tumors. In addition, in addition to solid tumors, extracellular acidification is also associated with many pathological conditions, including ischemic stroke, nerve damage, infection, tissue tearing and other pathological conditions. Abnormal metabolic activities of cells in damaged or diseased tissues often cause acidification of the extracellular environment. Therefore, extracellular acidification is expected to become a universal marker for diagnosis and treatment of many diseases. pHLIP can be used to target the characteristics of acidic environment and can also be used for the above Diagnosis and treatment of pathological conditions.
附图的简要说明Brief description of the drawings
以下，结合附图来详细说明本发明的实施方案，其中：Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, in which:
图1显示利用流式细胞仪检测本发明的酸性环境靶向分子在不同pH条件的微环境下，在肿瘤细胞(A549)上的表达情况，其中，所述偶联分子或融合蛋白分别为FITC(异硫氰酸荧光素)、FLAG标签多肽和PNE多肽(来源于酵母转录因子GCN4的14个氨基酸多肽)；Figure 1 shows the use of flow cytometry to detect the expression of the acidic environment targeting molecules of the present invention on tumor cells (A549) in microenvironments with different pH conditions, wherein the coupling molecules or fusion proteins are FITC respectively (Fluorescein isothiocyanate), FLAG tag polypeptide and PNE polypeptide (14 amino acid polypeptide derived from yeast transcription factor GCN4);
图2显示利用流式细胞仪检测本发明的酸性环境靶向分子在不同pH条 件的微环境下，在肿瘤细胞(A549)上的表达情况，其中，所述融合蛋白为免疫细胞募集分子，分别为抗体可结晶片段(Fc)、携带三聚化foldon序列的抗体可结晶片段(foldon-Fc)和携带三聚化p24序列的可结晶片段(p24-Fc)；Figure 2 shows the use of flow cytometry to detect the expression of the acidic environment targeting molecules of the present invention on tumor cells (A549) in microenvironments with different pH conditions, where the fusion protein is an immune cell recruitment molecule, respectively It is an antibody crystallizable fragment (Fc), an antibody crystallizable fragment carrying a trimerized foldon sequence (foldon-Fc), and a crystallizable fragment carrying a trimerized p24 sequence (p24-Fc);
图3显示本发明的酸性环境靶向分子pHLIP融合蛋白CCL19-pHLIP趋化T细胞的效果；Figure 3 shows the chemotaxis effect of the acidic environment targeting molecule pHLIP fusion protein CCL19-pHLIP of the present invention on T cells;
图4显示利用免疫组化/荧光分析系统TissueFAXS观察酸性环境靶向分子FITC-pHLIP在肿瘤组织和其它脏器上的定位，白色三角指示处为锚定在肿瘤细胞上的FITC-pHLIP；Figure 4 shows the use of immunohistochemistry/fluorescence analysis system TissueFAXS to observe the location of acidic environment targeting molecule FITC-pHLIP on tumor tissues and other organs. The white triangle indicates FITC-pHLIP anchored on tumor cells;
图5显示酸性环境下本发明的组合物FITC-pHLIP联合FITC CAR-T细胞特异杀伤肿瘤细胞，其中A549为肺腺癌细胞(肺泡基底上皮细胞)，NCI-H292为肺癌细胞(淋巴结转移)；Figure 5 shows that the composition of the present invention FITC-pHLIP combined with FITC CAR-T cells specifically kills tumor cells in an acidic environment, where A549 is lung adenocarcinoma (alveolar basal epithelial cells) and NCI-H292 is lung cancer cell (lymph node metastasis);
图6显示本发明的组合物FITC-pHLIP联合FITC CAR-T细胞控制荷瘤小鼠的肿瘤生长曲线。Figure 6 shows the composition of the present invention FITC-pHLIP combined with FITC CAR-T cells to control the tumor growth curve of tumor-bearing mice.
实施发明的最佳方式The best way to implement the invention
实施例1 本发明组合物的酸性环境靶向分子在不同pH条件的微环境Example 1 The acidic environment targeting molecule of the composition of the present invention is in the microenvironment of different pH conditions 下，在肿瘤细胞(A549)上的表达Down, the expression on tumor cells (A549)
细胞培养：A549细胞(美国模式培养物集存库)使用RPMI1640完全培养基培养(简写为R10)培养，R10为RPMI1640培养基(Corning)含有10％(体积比)胎牛血清(Biological Industries)、1X链霉素/青霉素(100X链霉素/青霉素，Gibco)的完全培养基，在37℃，5％CO 2的细胞培养箱中培养。细胞长满后按1:5传代。 Cell culture: A549 cells (American Model Culture Collection) are cultured in RPMI1640 complete medium (abbreviated as R10). R10 is RPMI1640 medium (Corning) containing 10% (volume ratio) fetal bovine serum (Biological Industries), The complete medium of 1X streptomycin/penicillin (100X streptomycin/penicillin, Gibco) was cultured in a cell incubator at 37°C and 5% CO 2. After the cells are full, pass at 1:5.
pHLIP表达分析：A549细胞经过胰酶细胞消化液消化1～2分钟，采用10倍体积的R10培养基中和，500xg，离心5分钟。弃上清，采用R10培养基重悬制备A549单细胞悬液，各取1x10 5细胞置于3个1.5mL EP管中，800xg，离心3分钟，弃去培养基分别加入pH6.0、pH6.5和pH7.4的1xPBS，加入本发明的酸性环境靶向分子5μg/mL，分别为FITC-pHLIP(结构式为： pHLIP expression analysis: A549 cells were digested with trypsin cell digestion solution for 1 to 2 minutes, neutralized with 10 times the volume of R10 medium, 500xg, and centrifuged for 5 minutes. The supernatant was discarded, and the A549 single cell suspension was resuspended in R10 medium to prepare A549 single cell suspension. Each 1x10 5 cells were placed in three 1.5mL EP tubes and centrifuged at 800xg for 3 minutes. The medium was discarded and added to pH 6.0 and pH 6. 5 and 1xPBS with pH 7.4, adding 5μg/mL of the acidic environment targeting molecule of the present invention, respectively, are FITC-pHLIP (the structural formula is:
FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT)，FLAG-pHLIP(SEQ ID NO:51)和PNE-pHLIp(SEQ ID NO:52)(上海昕浩生物科技有限公司化学合成多肽)。室温孵育1小时，弃上清用相应pH的1xPBS洗涤3次，加入pH7.4的1xPBS重悬细胞，采用流式细胞仪检测FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP在A549细胞吸附水平。所 检测细胞样本共分为6组：(1)pH6.0未处理组；(2)pH6.0+FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP处理组；(3)pH6.5未处理组；(4)pH6.5+FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP处理组；(5)pH7.4未处理组：(6)pH7.4+FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP。FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT), FLAG-pHLIP (SEQ ID NO: 51) and PNE-pHLIp (SEQ ID NO: 52) (Shanghai Xinhao Biotechnology Co., Ltd. chemically synthesized peptides). Incubate for 1 hour at room temperature, discard the supernatant and wash 3 times with 1xPBS with corresponding pH, add 1xPBS with pH7.4 to resuspend the cells, and use flow cytometry to detect the adsorption level of FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP on A549 cells. The tested cell samples were divided into 6 groups: (1) pH6.0 untreated group; (2) pH6.0+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP treatment group; (3) pH6.5 untreated group ; (4) pH6.5+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP treatment group; (5) pH7.4 untreated group: (6) pH7.4+FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP .
结果：图1A、B、C显示，在中性溶液环境中(pH7.4)，FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP插入A549的细胞膜的效率较低，分别为30％、30％和15％。在酸性溶液环境中，FITC-pHLIP高效插入A549细胞膜，pH6.5和pH6.0的插入效率分别为85％和99％，显著高于pH7.4；本发明的FLAG-pHLIP多肽亦可高效插入A549细胞膜，pH6.5和pH6.0的插入效率分别为73％和91％，显著高于pH7.4；本发明的PNE-pHLIP多肽亦能有效插入A549细胞膜，pH6.5和pH6.0的插入效率分别为57％和53％，显著高于pH7.4。Results: Figure 1A, B, and C show that in a neutral solution environment (pH 7.4), FITC-pHLIP, FLAG-pHLIP, and PNE-pHLIP are inserted into the cell membrane of A549 with low efficiency, respectively 30%, 30% and 15%. In an acidic solution environment, FITC-pHLIP can be efficiently inserted into the A549 cell membrane. The insertion efficiencies of pH6.5 and pH6.0 are 85% and 99%, respectively, which are significantly higher than pH7.4; the FLAG-pHLIP polypeptide of the present invention can also be inserted efficiently The insertion efficiency of A549 cell membrane at pH 6.5 and pH 6.0 is 73% and 91%, respectively, which is significantly higher than pH 7.4; the PNE-pHLIP polypeptide of the present invention can also be effectively inserted into A549 cell membrane at pH 6.5 and pH 6.0. The insertion efficiency was 57% and 53%, respectively, which was significantly higher than pH 7.4.
实施例2 本发明的酸性环境靶向分子在不同pH条件的微环境下，在肿Example 2 The acidic environment targeting molecule of the present invention is in microenvironment with different pH conditions, 瘤细胞(A549)上的表达Expression on tumor cells (A549)
2.1 pHLIP-Fc、pHLIP-foldon-Fc和pHLIP-p24-Fc的pHLIP融合蛋白的体外表达和纯化2.1 In vitro expression and purification of pHLIP fusion proteins of pHLIP-Fc, pHLIP-foldon-Fc and pHLIP-p24-Fc
2.1.1真核表达载体构建：将根据本发明的融合蛋白氨基酸序列(SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21)设计DNA序列交付上海捷瑞生物工程有限公司进行全基因合成制备真核表达载体。2.1.1 Construction of eukaryotic expression vector: The DNA sequence designed according to the amino acid sequence of the fusion protein of the present invention (SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21) is delivered to Shanghai Jerui Bioengineering Co., Ltd. for complete Gene synthesis to prepare eukaryotic expression vectors.
2.1.2 pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc的pHLIP融合蛋白的体外表达：隔天铺HEK293T细胞于15cm培养皿，每个培养皿铺1.5x10 7细胞，在37℃，5％CO 2的细胞培养箱中培养过夜。第二天，采用转染试剂PEIpro以30μg/皿的DNA总量转染携带融合蛋白DNA序列的真核表达载体，6～8小时更换为无血清培养基，在37℃，5％CO 2的细胞培养箱中继续培养5～7天。 2.1.2 pHLIP-Fc, pHLIP-folden -Fc pHLIP-p24-Fc and the protein expressed in vitro fusion PHLIP: HEK293T cells were plated the next day in 15cm culture dishes, each dish 1.5x10 7 cells were plated at 37 ℃, Cultivate overnight in a 5% CO 2 cell incubator. On the second day, the transfection reagent PEIpro was used to transfect the eukaryotic expression vector carrying the DNA sequence of the fusion protein at a total amount of DNA of 30 μg/dish, and the medium was replaced with a serum-free medium at 37°C and 5% CO 2 for 6 to 8 hours. Continue to culture in the cell incubator for 5-7 days.
2.1.3 pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc的pHLIP融合蛋白的纯化：收集细胞培养上清，2000rpm，4℃，离心10分钟，弃去细胞沉淀。离心收集的培养上清采用Protein G琼脂糖柱子(GE公司，货号28-9031-34)进行纯化，纯化后的抗体用PBS缓冲液溶解。2.1.3 Purification of pHLIP fusion proteins of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc: Collect the cell culture supernatant, centrifuge at 2000 rpm, 4°C for 10 minutes, and discard the cell pellet. The culture supernatant collected by centrifugation was purified on a Protein G Sepharose column (GE Company, Catalog No. 28-9031-34), and the purified antibody was dissolved in PBS buffer.
2.2 pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc的pHLIP融合蛋白在A549细胞上表达2.2 The pHLIP fusion proteins of pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc are expressed on A549 cells
细胞培养：A549细胞使用R10培养基，在37℃，5％CO2的细胞培养箱中培养。细胞长满后按1:5传代。Cell culture: A549 cells are cultured in a cell incubator at 37°C and 5% CO2 using R10 medium. After the cells are full, pass at 1:5.
流式细胞仪观察表达：A549细胞经过胰酶细胞消化液消化1～2分钟， 10倍体积的R10培养基中和，500xg，离心5分钟，R10培养基重悬，最终获得A549单细胞悬液。取1x10 5细胞置于3个1.5mL EP管中，800xg，离心3分钟，弃去培养基分别加入pH6.0和pH7.4的1xPBS，加入上述制备的pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白(20μg/mL)，室温孵育1小时，弃上清用相应pH的1xPBS洗涤3次，加入相应pH的1xPBS重悬细胞，采用流式细胞仪检测pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白在A549细胞上的表达水平。分组：(1)pH6.0未处理组；(2)pH6.0+pHLIP-Fc组；(3)pH6.0+pHLIP-folden-Fc组；(4)pH6.0+pHLIP-p24-Fc组；(5)pH7.4未处理组：(6)pH7.4+pHLIP-Fc组；(7)pH7.4+pHLIP-folden-Fc组；(8)pH7.4+pHLIP-p24-Fc组。 Flow cytometry observation and expression: A549 cells were digested with trypsin cell digestion solution for 1 to 2 minutes, 10 times volume of R10 medium was neutralized, 500xg, centrifuged for 5 minutes, R10 medium was resuspended, and A549 single cell suspension was finally obtained . Place 1x10 5 cells in three 1.5mL EP tubes, centrifuge at 800xg for 3 minutes, discard the medium, add 1xPBS with pH 6.0 and pH 7.4, and add the pHLIP-Fc, pHLIP-folden-Fc and pHLIP-folden-Fc prepared above. pHLIP-p24-Fc fusion protein (20μg/mL), incubate for 1 hour at room temperature, discard the supernatant and wash 3 times with 1xPBS of corresponding pH, add 1xPBS of corresponding pH to resuspend cells, and detect pHLIP-Fc, pHLIP by flow cytometer -The expression levels of folden-Fc and pHLIP-p24-Fc fusion proteins on A549 cells. Grouping: (1) pH6.0 untreated group; (2) pH6.0+pHLIP-Fc group; (3) pH6.0+pHLIP-folden-Fc group; (4) pH6.0+pHLIP-p24-Fc Group; (5) pH7.4 untreated group: (6) pH7.4+pHLIP-Fc group; (7) pH7.4+pHLIP-folden-Fc group; (8) pH7.4+pHLIP-p24-Fc group.
结果：图2显示，在中性溶液环境中(pH7.4)，本发明的pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白插入A549的细胞膜的效率极低，低于4％。而在酸性溶液环境中，本发明的pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白均能插入A549细胞膜，pH6.0的插入效率分别为21％、19.1％和21.4％，均高于pH7.4。Results: Figure 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 insertion efficiency into the cell membrane of A549, which is lower than 4 %. In an acidic solution environment, the pHLIP-Fc, pHLIP-folden-Fc and pHLIP-p24-Fc fusion proteins of the present invention can all be inserted into the A549 cell membrane, and the insertion efficiency of pH6.0 is 21%, 19.1% and 21.4%, respectively. All are higher than pH7.4.
实施例3 酸性环境靶向分子CCL19-pHLIP高效趋化T细胞Example 3 Acidic environment targeting molecule CCL19-pHLIP highly efficient chemotaxis of T cells
3.1 CCL19-pHLIP融合蛋白的体外表达和鉴定3.1 In vitro expression and identification of CCL19-pHLIP fusion protein
3.1.1真核表达载体构建3.1.1 Construction of eukaryotic expression vector
将根据本发明的融合蛋白氨基酸序列(SEQ ID NO:49)设计DNA序列交付上海捷瑞生物工程有限公司进行全基因合成制备真核表达载体。The DNA sequence designed according to the amino acid sequence of the fusion protein (SEQ ID NO: 49) of the present invention is delivered to Shanghai Jierui Bioengineering Co., Ltd. for full gene synthesis to prepare eukaryotic expression vectors.
3.1.2 CCL19-pHLIP融合蛋白的体外表达3.1.2 In vitro expression of CCL19-pHLIP fusion protein
HEK293T细胞(美国模式培养物集存库)使用DMEM完全培养基培养(简写为D10)，D10为DMEM培养基(Corning)含有10％(体积比)胎牛血清(Biological Industries)、1X链霉素/青霉素(100X链霉素/青霉素，Gibco)的完全培养基，在37℃，5％CO 2的细胞培养箱中培养。细胞长满后按1:5传代。 HEK293T cells (American Model Culture Collection) are cultured in DMEM complete medium (abbreviated as D10), D10 is DMEM medium (Corning) containing 10% (volume ratio) fetal bovine serum (Biological Industries), 1X streptomycin / Penicillin (100X Streptomycin / Penicillin, Gibco) complete medium, cultured in a cell culture incubator at 37°C and 5% CO 2. After the cells are full, pass at 1:5.
HEK293T细胞经过胰酶细胞消化液消化1-2分钟，10倍体积的D10培养基中和，500xg，离心5分钟，DMEM完全培养基重悬，最终获得HEK293T单细胞悬液。将HEK293T细胞于六孔板中，每孔铺1x10 6细胞，在37℃，5％CO 2的细胞培养箱中培养过夜。第二天，采用转染试剂Turbofect以4μg/孔的DNA总量转染携带融合蛋白DNA序列的真核表达载体，在37℃，5％CO 2的细胞培养箱中继续培养2天，收集培养上清，2000rpm，4℃，离心10分钟，收集细胞沉淀用于WB检测，收集离心后的培养上清，冻存备用。 HEK293T cells were digested with trypsin cell digestion solution for 1-2 minutes, 10 times the volume of D10 medium was neutralized, 500xg, centrifuged for 5 minutes, and resuspended in DMEM complete medium to finally obtain HEK293T single cell suspension. Put HEK293T cells in a six-well plate, spread 1×10 6 cells per well, and culture them overnight in a 37°C, 5% CO 2 cell incubator. On the second day, use the transfection reagent Turbofect to transfect the eukaryotic expression vector carrying the DNA sequence of the fusion protein at a total amount of 4μg/well of DNA, and continue to culture for 2 days in a cell incubator at 37°C and 5% CO 2 to collect and culture The supernatant was centrifuged at 2000 rpm, 4°C for 10 minutes, the cell pellet was collected for WB detection, and the culture supernatant after centrifugation was collected and frozen for later use.
3.2 T细胞趋化试验3.2 T cell chemotaxis test
取一块5μm孔径的Transwell培养板，下室加入上述离心收集的培养上清600μL，上室加入100μL的T细胞悬液，细胞密度为1x10 6/mL，在37℃，5％CO 2的细胞培养箱中分别培养2、4或6小时取出计数下室细胞数目。 Take a 5μm Transwell culture plate, add 600μL of the culture supernatant collected by centrifugation to the lower chamber, add 100μL of T cell suspension to the upper chamber, cell density 1x10 6 /mL, at 37 ℃, 5% CO 2 cell culture Incubate in the box for 2, 4, or 6 hours, take out and count the cells in the lower chamber.
结果：图3A显示，携带CCL19-pHLIP融合蛋白DNA序列的真核表达载体在HEK293T细胞中高效表达。图3B显示，本发明的酸性环境靶向分子CCL19-pHLIP融合蛋白可以有效地趋化T细胞，趋化效率与阳性对照CCL19相当。Results: Figure 3A shows that the eukaryotic expression vector carrying the DNA sequence of the CCL19-pHLIP fusion protein was highly expressed in HEK293T cells. Figure 3B shows that the acidic environment targeting molecule CCL19-pHLIP fusion protein of the present invention can effectively chemoattract T cells, and the chemotaxis efficiency is equivalent to the positive control CCL19.
实施例4 酸性环境靶向分子的FITC-pHLIP在体内肿瘤组织和脏器的分Example 4 Analysis of FITC-pHLIP of acidic environment targeting molecules in tumor tissues and organs in vivo 布cloth
5x10 5个黑色素B16细胞接种于C57BL/6小鼠(购自上海史莱克实验动物有限责任公司)右侧腹部皮下，待肿瘤直径长至0.5-1cm时，静脉注射1mg/kg的FITC-pHLIP，分别于注射后4、12、24、48、72和96小时处死小鼠，取肿瘤组织、心脏、肝脏、脾脏、肺脏和肾脏制作冰冻切片，4％多聚甲醛固定后采用PE-anti-FITC(Abcam，ab25539)和DAPI染色，采用免疫组化/荧光分析系统TissueFAXS进行分析。 5x10 5 melanin B16 cells were inoculated subcutaneously into the right abdomen of C57BL/6 mice (purchased from Shanghai Shrek Laboratory Animal Co., Ltd.). When the tumor diameter grows to 0.5-1 cm, 1 mg/kg FITC-pHLIP is injected intravenously. Mice were sacrificed at 4, 12, 24, 48, 72, and 96 hours after injection. The tumor tissues, heart, liver, spleen, lung and kidney were taken to make frozen sections, and fixed with 4% paraformaldehyde using PE-anti-FITC (Abcam, ab25539) and DAPI staining, using immunohistochemistry/fluorescence analysis system TissueFAXS for analysis.
结果：图4显示，FITC-pHLIP于回输后至少6小时富集在肿瘤组织，一直持续到回输后24小时均可见明显的FITC-pHLIP定位在肿瘤细胞上。然而，FITC-pHLIP回输后4、12、24、48小时均不在心脏、肝脏、脾脏、肺脏和肾脏富集。Results: Figure 4 shows that FITC-pHLIP was enriched in tumor tissue at least 6 hours after reinfusion, and it was observed that FITC-pHLIP was clearly localized on tumor cells until 24 hours after reinfusion. However, FITC-pHLIP was not enriched in the heart, liver, spleen, lung and kidney at 4, 12, 24, and 48 hours after reinfusion.
实施例5 酸性环境靶向分子FITC-pHLIP联合抗FITC CAR-T细胞的体外Example 5 In vitro of acidic environment targeting molecule FITC-pHLIP combined with anti-FITC CAR-T cells 抗肿瘤作用Anti-tumor effect
5.1细胞培养和铺板5.1 Cell culture and plating
稳定表达荧光素酶的肺癌细胞A549-luciferase/NCI-H292-luciferase(上海鑫湾生物科技有限公司)使用含有10％胎牛血清、1x链霉素/青霉素的R10培养基，在37℃，5％CO 2的细胞培养箱中培养。细胞长满后按1:5传代。 Lung cancer cells A549-luciferase/NCI-H292-luciferase (Shanghai Xinwan Biotechnology Co., Ltd.) that stably express luciferase use R10 medium containing 10% fetal bovine serum, 1x streptomycin/penicillin, at 37℃, 5 %CO 2 cell culture incubator. After the cells are full, pass at 1:5.
A549-luciferase/NCI-H292-luciferase细胞经过胰酶细胞消化液消化1～2分钟，10倍体积的R10培养基中和，500xg，离心5分钟，R10培养基重悬，最终获得A549-luciferase/NCI-H292-luciferase单细胞悬液。将A549-luciferase/NCI-H292-luciferase细胞铺在96孔黑底平板中，每孔1x10 4个细胞，总体积为100μL，在37℃，5％CO 2的细胞培养箱中培养过夜。 A549-luciferase/NCI-H292-luciferase cells were digested with trypsin cell digestion solution for 1 to 2 minutes, and 10 times volume of R10 medium was neutralized, 500xg, centrifuged for 5 minutes, and R10 medium was resuspended to obtain A549-luciferase/ NCI-H292-luciferase single cell suspension. The A549-luciferase/NCI-H292-luciferase cells were plated in a 96-well black bottom plate, 1 ×10 4 cells per well, with a total volume of 100 μL, and cultured overnight in a cell incubator at 37°C and 5% CO 2.
5.2 FITC-pHLIP处理5.2 FITC-pHLIP treatment
A549-luciferase/NCI-H292-luciferase细胞在培养板上培养过夜，弃去培养上清分别加入pH6.5和pH6.0的R10培养基，加入实施例1的FITC-pHLIP (5μg/mL)，37℃孵育1小时，弃去上清用相应pH的1xPBS洗涤3次，加入pH6.5和pH6.0的R10培养基100μL。分组：pH6.5未处理组；(2)pH6.5+FITC-pHLIP处理组；(3)pH6.0未处理组；(4)pH6.0+FITC-pHLIP处理组。The A549-luciferase/NCI-H292-luciferase cells were cultured overnight on the culture plate, the culture supernatant was discarded and the R10 medium with pH 6.5 and pH 6.0 was added, and the FITC-pHLIP (5 μg/mL) of Example 1 was added. Incubate at 37°C for 1 hour, discard the supernatant and wash 3 times with 1xPBS of corresponding pH, and add 100 μL of R10 medium with pH 6.5 and pH 6.0. Grouping: pH6.5 untreated group; (2) pH6.5+FITC-pHLIP treatment group; (3) pH6.0 untreated group; (4) pH6.0+FITC-pHLIP treatment group.
5.3抗FITC CAR-T细胞处理5.3 Anti-FITC CAR-T cell treatment
收集静息期的抗FITC CAR-T细胞，用R10培养基重悬至相应细胞浓度。以效应细胞:靶细胞为5:1的比例加入抗FITC CAR-T细胞至上述4个处理组，每孔加入100μL的抗FITC CAR-T细胞悬液。Collect the anti-FITC CAR-T cells in the resting phase and resuspend them to the corresponding cell concentration in R10 medium. Add anti-FITC CAR-T cells to the above 4 treatment groups at a ratio of 5:1 effector cells: target cells, and add 100 μL of anti-FITC CAR-T cell suspension to each well.
结果：图5A显示，在酸性溶液环境中，FITC-pHLIP联合抗FITC CAR-T细胞可以特异杀伤肺腺癌细胞A549-luciferase。图5B显示，在酸性溶液环境中，FITC-pHLIP联合抗FITC CAR-T细胞可以特异杀伤高转移肺癌细胞NCI-H292-luciferase。Results: Figure 5A shows that in an acidic environment, FITC-pHLIP combined with anti-FITC CAR-T cells can specifically kill lung adenocarcinoma cells A549-luciferase. Figure 5B shows that in an acidic solution environment, FITC-pHLIP combined with anti-FITC CAR-T cells can specifically kill the highly metastatic lung cancer cell NCI-H292-luciferase.
实施例6 酸性环境靶向分子FITC-pHLIP联合抗FITC CAR-T细胞的Example 6 Combination of FITC-pHLIP, an acidic environment targeting molecule, combined with anti-FITC CAR-T cells 体内抗肿瘤效果评价Evaluation of anti-tumor effect in vivo
将NCI-H292肺癌细胞(上海鑫湾生物科技有限公司)接种于B-NDG免疫缺陷小鼠(购自北京百奥赛图基因生物技术有限公司)的右侧腹部皮下，2x10 6个细胞/只，待肿瘤直径长至0.5-1cm，剔除过大和过小的肿瘤，将肿瘤大小基本一致的小鼠随机分组。共分为3组：PBS单独注射组，5只；抗FITC CAR-T细胞单独注射组，5只；FITC-pHLIP联合抗FITC CAR-T细胞注射组，5只。每隔3天测量肿瘤大小。 NCI-H292 lung cancer cells (Shanghai Xinwan Biotechnology Co., Ltd.) were inoculated subcutaneously in the right abdomen of B-NDG immunodeficiency mice (purchased from Beijing Biocytogene Biotechnology Co., Ltd.), 2x10 6 cells/mouse, When the diameter of the tumor grows to 0.5-1 cm, the tumors that are too large and too small are eliminated, and mice with the same tumor size are randomly grouped. Divided into 3 groups: PBS alone injection group, 5 animals; anti-FITC CAR-T cell injection group, 5 animals; FITC-pHLIP combined with anti-FITC CAR-T cell injection group, 5 animals. The tumor size was measured every 3 days.
给药方法：FITC-pHLIP给药：每次每只小鼠静脉注射20μg(125μL)，从分组完成后当天开始注射，一天注射一次，每隔一天注射一次，连续给药10次。抗FITC CAR-T细胞给药：每次每只小鼠静脉回输抗FITC CAR-T细胞10 7个/125μL，总共2次。从分组完成后当天开始回输第一次，注射时间为第一次给FITC-pHLIP后12小时，九天后进行第二次细胞回输。 Administration method: FITC-pHLIP administration: each mouse is intravenously injected 20μg (125μL) each time, starting from the day after the grouping is completed, once a day, once every other day, and 10 times in a row. Anti-FITC CAR-T cells administration: intravenous reinfusion each time per mouse anti-FITC CAR-T 10 7 cells / 125μL, a total of 2 times. The first reinfusion starts on the day after the grouping is completed. The injection time is 12 hours after the first injection of FITC-pHLIP, and the second cell reinfusion is performed nine days later.
结果：图6显示，FITC-pHLIP联合抗FITC CAR-T细胞显著抑制肺癌荷瘤小鼠的肿瘤生长，PBS和单独抗FITC CAR-T细胞均不能控制荷瘤小鼠的肿瘤生长。Results: Figure 6 shows that FITC-pHLIP combined 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 could control tumor growth in tumor-bearing mice.
虽然以上仅描述了本发明的具体实施方式的范例，但本领域的技术人员应当理解，以上这些仅为举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均应落入本发明的保护范围。Although only examples of specific implementations of the present invention are described above, those skilled in the art should understand that the above are only examples, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principle and essence of the present invention, but these changes or modifications should fall within the protection scope of the present invention.

Claims (11)

  1. 一种用于调控酸性环境免疫应答的组合物，所述组合物由酸性环境靶向分子和免疫细胞组成，所述酸性环境靶向分子为pHLIP或其变体的偶联分子或融合蛋白，所述免疫细胞为内源性或外源性免疫细胞；A composition for regulating an acidic environment immune response, the composition is composed of an acidic environment targeting molecule and immune cells, the acidic environment targeting molecule is a coupling molecule or fusion protein of pHLIP or a variant thereof, and The immune cells are endogenous or exogenous immune cells;
    其中，所述pHLIP或其变体的偶联分子或融合蛋白通过pHLIP或其变体的N端与细胞因子、荧光分子、免疫调节药物、磁共振成像造影剂、磁性纳米材料、肿瘤相关抗原、细菌抗原、病毒抗原、真菌抗原、植物抗原、多肽抗原或抗体Fc片段连接获得。Wherein, the coupling molecule or fusion protein of pHLIP or its variants is connected to cytokines, fluorescent molecules, immunomodulatory drugs, magnetic resonance imaging contrast agents, magnetic nanomaterials, tumor-associated antigens, through the N-terminus of pHLIP or its variants. Bacterial antigens, viral antigens, fungal antigens, plant antigens, polypeptide antigens or antibody Fc fragments are connected to obtain.
  2. 根据权利要求1所述的组合物，其中，所述连接为偶联或接头连接。The composition according to claim 1, wherein the connection is coupling or linker connection.
  3. 根据权利要求1或2所述的组合物，其中，所述低pH插入肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；The composition according to claim 1 or 2, wherein the amino acid sequence of the low pH insert peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2;
    优选地，所述低pH插入肽的变体的氨基酸序列如SEQ ID NO:3-SEQ ID NO:18中任一项所示；Preferably, the amino acid sequence of the variant of the low pH insert peptide is shown in any one of SEQ ID NO: 3-SEQ ID NO: 18;
    优选地，所述pHLIP或其变体的融合蛋白如SEQ ID NO:19-SEQ ID NO:25中任一项所示。Preferably, the fusion protein of pHLIP or a variant thereof is shown in any one of SEQ ID NO: 19-SEQ ID NO: 25.
  4. 根据权利要求1所述的组合物，其中，所述免疫细胞选自自体免疫细胞、异体免疫细胞、自体基因工程免疫细胞或异体基因工程免疫细胞；The composition according to claim 1, wherein the immune cells are selected from autoimmune cells, allogeneic immune cells, autologous genetically engineered immune cells or allogeneic genetically engineered immune cells;
    优选地，所述免疫细胞选自NK细胞、NKT细胞、CD16/64嵌合受体修饰的NK/NKT/T细胞、嵌合抗原受体修饰的NK/NKT/T细胞；Preferably, the immune cells are selected from NK cells, NKT cells, CD16/64 chimeric receptor modified NK/NKT/T cells, and chimeric antigen receptor modified NK/NKT/T cells;
    更优选地，所述免疫细胞为CD16/64 CR-T细胞和CAR-T细胞。More preferably, the immune cells are CD16/64 CR-T cells and CAR-T cells.
  5. 根据权利要求1至4中任一项所述的组合物，其中，所述细胞因子选自白细胞介素、干扰素、肿瘤坏死因子、集落刺激因子、趋化因子和生长因子；The composition according to any one of claims 1 to 4, wherein the cytokine is selected from the group consisting of interleukin, interferon, tumor necrosis factor, colony stimulating factor, chemokine and growth factor;
    其中，所述白细胞介素选自IL-2、IL-7、IL-10、IL-12、IL-15和IL-21；所述干扰素选自IFN-α、IFN-β、IFN-γ、IFN-κ和IFN-λ；Wherein, the interleukin is selected from IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21; the interferon is selected from IFN-α, IFN-β, IFN-γ , IFN-κ and IFN-λ;
    其中，所述肿瘤坏死因子选自TNF-α和TNF-β；Wherein, the tumor necrosis factor is selected from TNF-α and TNF-β;
    其中，所述集落刺激因子选自SCF、Flt3L、G-CSF、M-CSF和GM-CSF；Wherein, the colony stimulating factor is selected from SCF, Flt3L, G-CSF, M-CSF and GM-CSF;
    其中，所述趋化因子选自CCL2、CCL5、CCL19、CCL21、CXCL8、CXCL9、CXCL10、CXCL11、XCL1和CX3CL1；Wherein, the chemokine is selected from CCL2, CCL5, CCL19, CCL21, CXCL8, CXCL9, CXCL10, CXCL11, XCL1 and CX3CL1;
    其中，所述生长因子选自TGF-β、EGF、FGF、NGF、PDGF和VEGF。Wherein, the growth factor is selected from TGF-β, EGF, FGF, NGF, PDGF and VEGF.
  6. 根据权利要求1至5中任一项所述的组合物，其中，所述免疫调节药物包括免疫抑制药物和免疫增强药物；The composition according to any one of claims 1 to 5, wherein the immunomodulatory drug comprises an immunosuppressive drug and an immune enhancing drug;
    优选地，所述免疫抑制药物选自环孢素、他克莫司和硫唑嘌呤；所述免疫增强药物选自胸腺素、转移因子和重组人干扰素。Preferably, the immunosuppressive drug is selected from cyclosporine, tacrolimus, and azathioprine; the immune enhancing drug is selected from thymosin, transfer factor and recombinant human interferon.
  7. 根据权利要求1至6中任一项所述的组合物，其中，所述肿瘤相关抗原选自CD19、CD20、CD22、CD30、CD33、ROR1、ROR2、CD38、CD123、CD133、NKG2D配体、ERBB2、MUC1、CD44v6、CD44v7、CD44v8、CEA、EpCAM、TAG72、KIT、IL-13Rα2、EGFR、EGFRvIII、GD2、GD3、HMW-MAA、MAGE1、MAGEA3、CD171、NCAM、IL-11Rα、FRα、PSCA、PSMA、TARP、CAIX、VEGFR、BCMA、CTLA-4、PD-L1、PD-L12、GPC3、CD47、AXL、FAP、α5β1、αvβ3、TGFβR、ER、PR、P53、IGFR、CD25、CD117、CD34、CD138、BCMA、Mesothelin、S100、CD70、ALK、RANK和HER3；The composition according to any one of claims 1 to 6, wherein the tumor-associated antigen is selected from CD19, CD20, CD22, CD30, CD33, ROR1, ROR2, CD38, CD123, CD133, NKG2D ligand, ERBB2 , MUC1, CD44v6, CD44v7, CD44v8, CEA, EpCAM, TAG72, KIT, IL-13Rα2, EGFR, EGFRvIII, GD2, GD3, HMW-MAA, MAGE1, MAGEA3, CD171, NCAM, IL-11Rα, FRα, PSCA, PSMA , TARP, CAIX, VEGFR, BCMA, CTLA-4, PD-L1, PD-L12, GPC3, CD47, AXL, FAP, α5β1, αvβ3, TGFβR, ER, PR, P53, IGFR, CD25, CD117, CD34, CD138 , BCMA, Mesothelin, S100, CD70, ALK, RANK and HER3;
    优选地，所述细菌抗原选自葡萄球菌抗原、链球菌抗原、肺炎链球菌抗原、脑膜炎奈瑟菌抗原和淋病奈瑟菌抗原；Preferably, the bacterial antigen is selected from the group consisting of Staphylococcus antigen, Streptococcus antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis antigen and Neisseria gonorrhoeae antigen;
    优选地，所述病毒抗原选自巨细胞病毒抗原、巴尔病毒抗原、肝炎病毒抗原、单纯疱疹病毒抗原、HIV抗原、人T-淋巴细胞病毒抗原、风疹病毒抗原、SARS冠状病毒抗原、水痘-带状疱疹病毒、狂犬病毒抗原、流感病毒抗原、轮状病毒抗原和人乳头瘤病毒抗原；Preferably, the viral antigen is selected from the group consisting of cytomegalovirus antigen, Barr virus antigen, hepatitis virus antigen, herpes simplex virus antigen, HIV antigen, human T-lymphocyte virus antigen, rubella virus antigen, SARS coronavirus antigen, and varicella-belt Herpes virus, rabies virus antigen, influenza virus antigen, rotavirus antigen and human papilloma virus antigen;
    优选地，所述真菌抗原选自皮肤癣真菌抗原、新生隐球菌抗原和白假丝念珠菌抗原；Preferably, the fungal antigen is selected from the group consisting of dermatophyte fungal antigen, Cryptococcus neoformans antigen and Candida albicans antigen;
    优选地，所述多肽抗原选自AviTag、Calmodulin tag、polyglutamate tag、E-tag、FLAG tag、HA-tag、His-tag、Myc-tag、S-tag、SBP-tag、Sof-tag 1、Sof-tag3、Strep-tag、TC tag、V5 tag、T7 tag、VSV tag、Xpress tag、3X FLAG tag、Isopep tag、Spytag、Snoop tag和PNE tag；Preferably, the polypeptide antigen is selected from AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, His-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof -tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag;
    优选地，所述荧光分子选自FITC、FAM、PE、APC、PB、Cy3、Cy5、Texas Red、TRITC、GFP、RFP、CFP和BFP；Preferably, the fluorescent molecule is selected from FITC, FAM, PE, APC, PB, Cy3, Cy5, Texas Red, TRITC, GFP, RFP, CFP and BFP;
    优选地，所述抗体Fc片段选自IgG1的Fc片段、IgG2的Fc片段、IgG3的Fc片段和IgG4的Fc片段。Preferably, the antibody Fc fragment is selected from the Fc fragment of IgG1, the Fc fragment of IgG2, the Fc fragment of IgG3 and the Fc fragment of IgG4.
    优选地，所述磁性纳米材料选自顺磁性金属离子螯合物或超顺磁性金属氧化物粒子；更优选地，所述顺磁性金属离子螯合物为钆喷酸葡胺、镝喷酸葡胺或锰福地吡三钠；所述超顺磁性金属氧化物粒子以Fe 2O 3、Fe 3O 4为核心。 Preferably, the magnetic nanomaterial is selected from paramagnetic metal ion chelates or superparamagnetic metal oxide particles; more preferably, the paramagnetic metal ion chelate is gadopentetate meglumine, dysprosium pentetate meglumine Amine or manganese fodipir trisodium; the superparamagnetic metal oxide particles have Fe 2 O 3 and Fe 3 O 4 as the core.
  8. 根据权利要求1至7中任一项所述的组合物，其中，所述酸性环境 靶向分子选自FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP、Fc-pHLIP或CCL19-pHLIP；所述免疫细胞为FITC CAR-T细胞、CAR-T细胞、CD16/64CR-T细胞、T细胞；The composition according to any one of claims 1 to 7, wherein the acidic environment targeting molecule is selected from FITC-pHLIP, FLAG-pHLIP, PNE-pHLIP, Fc-pHLIP or CCL19-pHLIP; the immune The cells are FITC CAR-T cells, CAR-T cells, CD16/64CR-T cells, T cells;
    优选地，所述组合物为CCL19-pHLIP和T细胞；更优选地，所述CCL19-pHLIP的氨基酸序列如SEQ ID NO:49所示；Preferably, the composition is CCL19-pHLIP and T cells; more preferably, the amino acid sequence of CCL19-pHLIP is shown in SEQ ID NO: 49;
    优选地，所述组合物为FITC-pHLIP和FITC CAR-T细胞；更优选地，所述FITC-pHLIP的结构式为：Preferably, the composition is FITC-pHLIP and FITC CAR-T cells; more preferably, the structural formula of the FITC-pHLIP is:
    FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTFITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT
    优选地，所述组合物为FLAG-pHLIP和CAR-T细胞；Preferably, the composition is FLAG-pHLIP and CAR-T cells;
    优选地，所述组合物为PNE-pHLIP和CAR-T细胞；Preferably, the composition is PNE-pHLIP and CAR-T cells;
    优选地，所述组合物为PNE-pHLIP和CAR-T细胞；Preferably, the composition is PNE-pHLIP and CAR-T cells;
    优选地，所述组合物为pHLIP-Fc、pHLIP-folden-Fc或pHLIP-p24-Fc和CD16/64 CR-T细胞。Preferably, the composition is pHLIP-Fc, pHLIP-folden-Fc or pHLIP-p24-Fc and CD16/64 CR-T cells.
  9. 如权利要求1-8中任一项所述的组合物在制备用于诊断和/或治疗肿瘤、自身免疫性疾病、伴有缺血缺氧的疾病和/或炎性疾病的药物中的用途。Use of the composition according to any one of claims 1-8 in the preparation of a medicament for the diagnosis and/or treatment of tumors, autoimmune diseases, diseases accompanied by ischemia and hypoxia and/or inflammatory diseases .
  10. 一种用于诊断和/或治疗疾病的方法，所述方法包括给予有需要的患者治疗有效量的如权利要求1-8中任一项所述的组合物；A method for diagnosing and/or treating diseases, the method comprising administering to a patient in need a therapeutically effective amount of the composition according to any one of claims 1-8;
    优选地，所述疾病选自肿瘤、自身免疫性疾病、伴有缺血缺氧的疾病和/或炎性疾病等。Preferably, the disease is selected from tumors, autoimmune diseases, diseases accompanied by ischemia and hypoxia, and/or inflammatory diseases.
  11. 如权利要求1-8中任一项所述的组合物的制备方法，所述方法包括以下步骤：The preparation method of the composition according to any one of claims 1-8, the method comprising the following steps:
    利用多肽自动合成仪，通过固相法合成；或Use an automatic peptide synthesizer to synthesize by solid-phase method; or
    用生物合成法，通过工程菌和细胞表达并纯化。Using biosynthesis, express and purify by engineering bacteria and cells.
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