WO2021093881A1 - Composition permettant de réguler la réponse immunitaire dans un environnement acide, son procédé de préparation et son utilisation - Google Patents

Composition permettant de réguler la réponse immunitaire dans un environnement acide, son procédé de préparation et son utilisation 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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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
    • 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
    • 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

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.

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Abstract

L'invention concerne une composition permettant de réguler une réponse immunitaire dans un environnement acide, son procédé de préparation et son utilisation. La composition se compose d'une molécule de ciblage d'environnement acide et d'une cellule immunitaire, la molécule de ciblage d'environnement acide étant une molécule de couplage ou une protéine de fusion d'un peptide pHLIP (peptide d'insertion à faible pH) ou d'un variant associé, et la cellule immunitaire étant une cellule immunitaire endogène ou exogène. La composition peut être utilisée pour traiter des tumeurs solides par régulation d'une réponse immunitaire dans un environnement acide, en particulier une réponse immunitaire dans un environnement acide tumoral.
PCT/CN2020/128957 2019-11-14 2020-11-16 Composition permettant de réguler la réponse immunitaire dans un environnement acide, son procédé de préparation et son utilisation WO2021093881A1 (fr)

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CN114149510B (zh) * 2021-10-29 2024-01-30 上海鑫湾生物科技有限公司 一种条件控制的可剪接嵌合抗原受体分子及其应用
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CN114181319A (zh) * 2021-11-26 2022-03-15 北京双赢科创生物科技有限公司 用于靶向肿瘤细胞的多肽偶联物及其制备方法与应用
CN114716566A (zh) * 2022-02-22 2022-07-08 广东粤港澳大湾区国家纳米科技创新研究院 一种融合蛋白及其在制备肿瘤药物中的应用
CN116640229B (zh) * 2023-04-10 2024-01-30 中国人民解放军总医院第五医学中心 一种低pH靶向性CAR-T细胞的构建及应用

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