WO2021233411A1 - 逆转肿瘤微环境的融合蛋白及其应用 - Google Patents

逆转肿瘤微环境的融合蛋白及其应用 Download PDF

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WO2021233411A1
WO2021233411A1 PCT/CN2021/095107 CN2021095107W WO2021233411A1 WO 2021233411 A1 WO2021233411 A1 WO 2021233411A1 CN 2021095107 W CN2021095107 W CN 2021095107W WO 2021233411 A1 WO2021233411 A1 WO 2021233411A1
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sirpγ
car
fusion protein
seq
tumor
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PCT/CN2021/095107
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English (en)
French (fr)
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徐艳敏
梅恩典
赵永春
单娟娟
齐亚男
赵文旭
陈军
黄霞
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重庆精准生物技术有限公司
重庆精准生物产业技术研究院有限公司
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Priority to EP21809833.3A priority Critical patent/EP4151660A4/en
Priority to BR112022023371A priority patent/BR112022023371A2/pt
Priority to KR1020227044207A priority patent/KR20230013257A/ko
Priority to US17/926,790 priority patent/US20230203125A1/en
Priority to JP2022572364A priority patent/JP2023527194A/ja
Priority to IL298346A priority patent/IL298346A/en
Publication of WO2021233411A1 publication Critical patent/WO2021233411A1/zh

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Definitions

  • the invention belongs to the technical field of immunotherapy, and specifically relates to a fusion protein that reverses the tumor microenvironment, a new type of tumor immunosuppressive resistance CAR, expression vectors, immune cells and applications.
  • CAR-T is chimeric antigen receptor T cells, that is, T lymphocytes expressing chimeric antigen receptor (CAR).
  • CAR-T therapy has made breakthrough progress in hematological tumors.
  • the effect in solid tumors is not as good as in hematological tumors.
  • CAR-T is difficult to enter the solid tumor.
  • CAR-T cells even if CAR-T cells enter the solid tumor, they cannot function normally because of the tumor microenvironment.
  • CAR-T cell treatment of solid tumors is indispensable to solve tumor microenvironment inhibition, that is, to implement tumor microenvironment resistance.
  • CD47 acts as a new immune checkpoint after PD-1/PD-L1 and CTLA-4, and suppresses the progress of innate immunity by transmitting a "don't eat me” signal to macrophages.
  • CD47ScFv can contribute to the immune microenvironment for tumor escape.
  • the efficacy of targeting CD47 alone is limited, and exogenous ScFv may cause excessive activation of CAR-T cells due to its strong affinity. Poor survival and non-specific causes Security risks. Therefore, it is very important to adopt a safer and more effective solution to identify the escape signal of CD47 to destroy the tumor microenvironment.
  • SIRP ⁇ Signal regulatory protein ⁇
  • SIRP ⁇ is one of the ligands of CD47, which can bind to CD47 and inhibit the phagocytosis of macrophages.
  • SIRP ⁇ can also bind to CD47. It exists on the surface of T cells. The extracellular region is composed of a V domain and two C1 domains. There is no intracellular signal and only one-way signal is transmitted through CD47.
  • SIRP ⁇ is modified, and we think it is a more suitable ligand for CD47 to destroy the tumor microenvironment. Therefore, we selected the extracellular segment of SIRP ⁇ protein for modification and design, and further designed a tumor immunosuppressive resistant CAR.
  • the purpose of the present invention is to provide a fusion protein that reverses the tumor microenvironment and an expression vector and immune cells containing the protein.
  • the fusion protein can reverse the tumor microenvironment and target tumor cells to kill.
  • the extracellular segment of SIRP ⁇ protein is modified and designed to obtain a fusion protein that can break the inhibitory signal in tumor tissue and reverse the tumor microenvironment.
  • the fusion protein is a SIRP ⁇ fusion protein, and the structure of the SIRP ⁇ fusion protein includes an extracellular segment, a transmembrane region and an intracellular signal region.
  • transmembrane structure is derived from the transmembrane region of human CD28 or the transmembrane region derived from human CD8.
  • amino acid sequence of the transmembrane structure is shown in SEQ ID NO: 7 or SEQ ID NO: 8.
  • the intracellular signal region is derived from CD28, and the sequence is shown in SEQ ID NO: 9 or SEQ ID NO: 38.
  • SIRP ⁇ fusion protein is SIRP ⁇ -CD28TM-CD28 and SIRP ⁇ -CD8TM-4-1BB.
  • amino acid sequence of the extracellular segment of SIRP ⁇ is shown in SEQ ID NO: 1 or a functional variant thereof.
  • amino acid sequence of the SIRP ⁇ fusion protein SIRP ⁇ -CD28TM-CD28 is shown in SEQ ID NO: 2 or a functional variant thereof.
  • amino acid sequence of the SIRP ⁇ fusion protein SIRP ⁇ -CD8TM-4-1BB is shown in SEQ ID NO: 3 or a functional variant thereof.
  • nucleotide sequence of the SIRP ⁇ fusion protein being SIRP ⁇ -CD28TM-CD28 is shown in SEQ ID NO: 13.
  • nucleotide sequence of the SIRP ⁇ fusion protein SIRP ⁇ -CD8TM-4-1BB is shown in SEQ ID NO: 14.
  • an immune cell containing the expression vector is an immune cell containing the expression vector.
  • the immune cells are T cells, T cell precursors or NK cells.
  • the T cells can be ⁇ T cells or ⁇ T cells; ⁇ T cells are a group of unique T cell subgroups containing receptor ⁇ and ⁇ chains on the surface, accounting for 0.5-5% of all T lymphocytes.
  • the cell population was first discovered in 1987. Although it has ⁇ TCR, its recognition of antigen or ligand is not MHC restricted, which is different from traditional ⁇ T cells.
  • many patents such as CN107810267A and CN107771215A have disclosed the application of ⁇ T cells in cell therapy and CAR-T cell therapy.
  • the PD-1 fusion protein PD-1-28TM-28 and PD-1-8TM-BB were also designed.
  • the designed SIRP ⁇ fusion protein when used in combination with CAR1, it can be expressed alone or co-expressed with CAR1 to play a role. Moreover, the SIRP ⁇ fusion protein can also be used alone in immunotherapy.
  • the inventor believes that the SIRP ⁇ protein is more suitable for CD47 as a ligand for tumor microenvironment destruction. Therefore, the extracellular segment of SIRP ⁇ protein was selected for modification and design, and the tumor immunosuppressive resistant CAR was further designed; therefore, the present invention creatively combines the designed and constructed hypoxia adjustable promoter with CAR-T cell technology and hypoxia adjustable Promoter, SIRP ⁇ protein combined with CAR-T cell technology are applied to tumor immunotherapy, that is, the traditional CAR structure is further modified to improve the therapeutic efficacy of CAR-T on solid tumors and the safety of CAR-T.
  • the immune cell also includes a chimeric antigen receptor structure that recognizes tumor antigens, and the chimeric antigen receptor includes an extracellular segment, hinge region, transmembrane region, and intracellular signal region that recognize tumor antigens.
  • Antigens include but are not limited to PSCA, PSMA, CD19, BCMA, CD123, CD20, CD22, CEA, EGFR, EGFRVIII, GPC3, 5T4, CD33, Her2, GD2, CD70, CLL-1, Trop2, CD47, GPC3, CLND18.2 , CD133, CS1, CD155, CD30, ROR1, MUC1, IL13RA ⁇ 2, or mesothelin can be used as antigen molecules for tumor targeting recognition.
  • the purpose of the present invention is to also provide a novel tumor immunosuppressive resistant CAR, and an expression vector and immune cells containing the CAR.
  • the novel tumor immunosuppressive resistant CAR and immune cells eliminate the influence of inhibitory signals in tumor tissues on CAR-T functions, realize the effectiveness of CAR-T therapy, and at the same time ensure certain safety.
  • the novel tumor immunosuppressive resistant CAR includes the fusion protein for reversing the tumor microenvironment described in the purpose one and CAR1, and the CAR1 includes an extracellular segment that recognizes tumor antigens, a hinge area, a transmembrane area, and an intracellular signal area.
  • the fusion protein is connected to CAR1 through a polycistronic structure, the polycistronic structure is a self-cleaving polypeptide or an internal ribosome entry site IRES, and the self-cleaving polypeptide is T2A, P2A, E2A or F2A.
  • the structure of the CAR is ScFv-hinge-TM-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ fusion protein or ScFv-hinge-TM-4-1BB-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ fusion protein.
  • the structure of the CAR is ScFv-hinge-TM-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ -CD28TM-CD28 or ScFv-hinge-TM-4-1BB-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ -CD28TM-CD28.
  • the CAR structure a can be a conventional first-generation, second-generation, and third-generation CAR structure, or a new type of CAR structure such as an improved dual CAR, a controllable CAR structure (such as FRB/FKBP12 regulation).
  • the hinge region sequence in CAR1 can be derived from: IgG, CD8, CD7, CD4; the transmembrane region in the CAR structure can be derived from: CD8, CD28, CD3 ⁇ , CD4, CD16, CD137, CD80, and CD86; the cell in the CAR structure
  • the internal signal area can be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function related antigen-1 (LFA-1), ICAM- 1. CD7, NKG2C, CD83, CD86 and CD127.
  • the ScFv can recognize CD19, CD123, MOv- ⁇ , PSMA, IL13R ⁇ 2, EGFRvIII, EGFR, EPCAM, GD2, MUC1, HER2, GPC3, CEA, Meso, CD133, NKG2D, CD138, LeY, k-Light, CD33 , ROR1, BCMA, CD30, CD20, CD22, PSCA, CLL-1, CD70, CD47.
  • the ScFv can recognize CD47 or CEA or PSCA or CD19 or BCMA.
  • the CAR structure has a truncated EGFRt regulatory tag; in certain embodiments, the PSCA-targeted CAR structure is a universal CAR structure; in certain embodiments, the The CAR structure targeting PSCA carries suicide genes such as iCasp9.
  • the CAR structure includes one or more components of the natural killer cell receptor (NKR), thereby forming an NKR-CAR.
  • the NKR component can be a transmembrane domain, hinge domain or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptor (KIR), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A , KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural cytotoxic receptors (NCR), for example, NKp30, NKp44, NKp46; signal transduction of immune cell receptors Cell activation molecule (SLAM) family, for example, CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME and CD2F-10; Fc receptor
  • the amino acid sequence of the transmembrane region derived from human CD28 is shown in SEQ ID NO: 7; the amino acid sequence of the intracellular signal region derived from human CD28 is shown in SEQ ID NO: 9 Shown.
  • amino acid sequence of the ScFv is shown in SEQ ID NO: 25 or a functional variant thereof.
  • this preferred plan designed to express a new type of immunosuppressive resistant CAR ScFv-hinge-TM-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ -28TM-28, ScFv-hinge-TM-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ - 8TM-BB, ScFv-hinge-TM-4-1BB-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ -28TM-28, ScFv-hinge-TM-4-1BB-CD3 ⁇ -self-cleaving peptide-SIRP ⁇ -8TM-BB and ScFv-hinge-TM-CD3 ⁇ -self-cleaving peptide-PD-1-28TM-28 and ScFv-hinge-TM-CD3 ⁇ -self-cleaving peptide-PD-1-8TM-BB genetically modified T lymphocytes to verify the new type The role of immunosuppressive resistance CAR.
  • the method for preparing the structure of the CAR is to co-express the CAR structure that does not contain the SIRP ⁇ fusion protein and the SIRP ⁇ fusion protein in a vector to transfect immune cells; or to transfect immune cells without the SIRP ⁇ fusion protein.
  • the CAR structure and the SIRP ⁇ fusion protein are respectively expressed in two vectors to transfect immune cells.
  • the CAR structure composed of the aforementioned SIRP ⁇ fusion protein and CAR1, wherein the CAR1 comprises an anti-CEA single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, CD137 and CD3 ⁇ dual stimulation signals.
  • the CAR structure a is a CEA ScFv-CD8 hinge region-CD8 transmembrane region-CD137-CD3 ⁇ structure, and its amino acid sequence includes the sequence shown in SEQ ID NO:26.
  • the nucleic acid sequence encoding the CEA single-chain antibody is shown in SEQ ID NO: 36; the nucleic acid sequence encoding the CD8 hinge region-CD8 transmembrane region-CD137-CD3 ⁇ structure is shown in SEQ ID NO: 37.
  • nucleic acid sequence encoding the CAR structure including the hypoxia-regulatable promoter includes the sequence shown in SEQ ID NO: 31.
  • the CAR structure composed of the aforementioned SIRP ⁇ fusion protein and CAR1, wherein the CAR1 includes CD19 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD137 and CD3 ⁇ dual stimulation signals;
  • amino acid sequence of the CAR structure a is shown in SEQ ID NO: 27 or a functional variant thereof.
  • nucleic acid sequence encoding the CAR structure includes the sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33.
  • the method for preparing the structure of the CAR is to co-express the CAR structure that does not contain the SIRP ⁇ fusion protein and the SIRP ⁇ fusion protein in a vector to transfect immune cells; or to transfect immune cells without the SIRP ⁇ fusion protein.
  • the CAR structure and the SIRP ⁇ fusion protein are respectively expressed in two vectors to transfect immune cells.
  • the CAR structure composed of the aforementioned SIRP ⁇ fusion protein and CAR1, wherein the CAR structure a includes PSCA single-chain antibody, hinge region, CD28 transmembrane region, CD28, CD137 and CD3 ⁇ tristimulus signals;
  • the hinge region is G4H or 7H.
  • the amino acid sequence of the CAR structure a is shown in SEQ ID NO: 28 or a functional variant thereof; or shown in SEQ ID NO: 29 or a functional variant thereof.
  • the amino acid sequence is shown in SEQ ID NO: 28 or its functional variant is the CAR structure a with the hinge region of G4H; the amino acid sequence is shown in SEQ ID NO: 29 or its functional variant is the CAR structure with the hinge region of 7H a.
  • nucleic acid sequence encoding the CAR structure includes the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 35; wherein SEQ ID NO: 35 includes a hypoxia-regulated promoter.
  • the method for preparing the structure of the CAR is to co-express the CAR structure that does not contain the SIRP ⁇ fusion protein and the SIRP ⁇ fusion protein in a vector to transfect immune cells; or to transfect immune cells without the SIRP ⁇ fusion protein.
  • the CAR structure and the SIRP ⁇ fusion protein are respectively expressed in two vectors to transfect immune cells.
  • the CAR structure also includes a hypoxia promoter, and the hypoxia can be regulated
  • the nucleic acid sequence of the promoter includes the sequence shown in SEQ ID NO: 30.
  • hypoxia adjustable promoter is composed of Hifla regulatory elements and a mini promoter; the mini promoter is selected from the group consisting of cell virus promoters, HSV thymidine kinase promoters, simian virus 40 promoters, and adenosine Any one of viral late promoters and synthetic promoters.
  • the method for preparing the structure of the CAR is: co-expressing the CAR structure that does not contain the SIRP ⁇ fusion protein and the SIRP ⁇ fusion protein in a vector to transfect immune cells; or transfecting immune cells without the SIRP ⁇ fusion protein
  • the CAR structure and the SIRP ⁇ fusion protein are respectively expressed in two vectors to transfect immune cells.
  • the purpose of the present invention is to also provide an expression vector comprising any one of the aforementioned novel tumor immunosuppressive resistance CAR (including the schemes in the preferred scheme 1 to the preferred scheme 5 and the non-preferred scheme), and immune cells containing the expression vector .
  • the expression vector is any one of a lentiviral expression vector, a retroviral expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector, and a plasmid.
  • the immune cells are T cells, T cell precursors or NK cells.
  • the method for preparing the immune cell is: co-expressing the CAR structure that does not contain the SIRP ⁇ fusion protein and the SIRP ⁇ fusion protein in a vector to transfect immune cells; or the CAR structure that does not contain the SIRP ⁇ fusion protein The structure and the SIRP ⁇ fusion protein are respectively expressed in two vectors to transfect immune cells.
  • the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Wisner- May Virus (visna-maedi virus, VMV), goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunity Defective virus (SIV).
  • HAV-1 human immunodeficiency virus 1
  • HMV-2 human immunodeficiency virus 2
  • Wisner- May Virus visna-maedi virus, VMV
  • CAEV goat arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • SIV simian immunity Defective virus
  • the vector comprises a left (5') retroviral LTR, a Psi( ⁇ ) packaging signal, a central polypurine segment/DNA flap (cPPT/FLAP), a retroviral export element, and is operably linked to The promoter of the polynucleotide encoding the CAR covered herein and the right (3') retroviral LTR.
  • the CAR comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).
  • HPRE hepatitis B virus post-transcriptional regulatory element
  • WPRE woodchuck post-transcriptional regulatory element
  • oPRE optimized woodchuck post-transcriptional regulatory element
  • the promoter of the 5'LTR is replaced with a heterologous promoter.
  • the heterologous promoter is a cytomegalovirus (CMV) promoter, Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • SV40 Simian Virus 40
  • the 5'LTR or 3'LTR is a lentiviral LTR.
  • the 3'LTR is a self-inactivating (SIN) LTR.
  • the nucleic acid sequence of the CAR structure comprises an optimized Kozark sequence.
  • the promoter is operably linked to the polynucleotide encoding the CAR covered herein and the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 ⁇ promoter (EF1- ⁇ ), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer/chicken ⁇ -actin promoter (CAG), multiple Oncovirus enhancer/herpes simplex thymidine kinase promoter (MC1), ⁇ -actin promoter ( ⁇ -ACT), simian virus 40 promoter (SV40) and myelodysplastic sarcoma virus enhancer, the negative control area is missing, dl587rev primer binding site substitution (MND) promoter.
  • CMV cytomegalovirus immediate early gene promoter
  • EF1- ⁇ elongation factor 1 ⁇ promoter
  • PGK phosphoglycerate kina
  • the CAR-containing vector may contain secreted anti-PD-1 ScFv; in certain embodiments, the CAR-containing vector contains PD-1 conjugated transduction peptide (such as PD-1-CD28-CD137- CD3 signal structure); In some embodiments, a carrier containing CAR is a combination of multiple CARs, such as two CAR combinations that target different antigens or different recognition sites of the same antigen.
  • PD-1 conjugated transduction peptide such as PD-1-CD28-CD137- CD3 signal structure
  • a carrier containing CAR is a combination of multiple CARs, such as two CAR combinations that target different antigens or different recognition sites of the same antigen.
  • the purpose of the present invention is to also provide a pharmaceutical composition and its application.
  • the pharmaceutical composition comprises the fusion protein or an expression vector comprising the fusion protein and immune cells or the novel tumor immunosuppressive resistant CAR or the immune cells comprising the novel tumor immunosuppressive resistant CAR .
  • the fusion protein can be selected from SIRP ⁇ or SIRP ⁇ partial domains, and the structure of the fusion protein can be SIRP ⁇ -28TM-28, SIRP ⁇ -8TM-137, SIRP ⁇ -8TM-OX40, SIRP ⁇ -8TM-ICOS, etc.
  • kind of fusion protein can be used alone, or the above SIRP ⁇ fusion protein and CAR molecule may be co-expressed in the same immune cell; or immune cells expressing SIRP ⁇ fusion protein and CAR molecule may be mixed in a certain ratio .
  • the active agent and/or treatment may be surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate , Mycophenolate and FK506, antibodies or other immune scavengers (immunoablative agents) such as CAMPATH, anti-CD3 antibodies or other antibody treatments, cyclophosphamide (cytoxan), fludarabine (fludarabine), cyclosporine ( cyclosporin), FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate , Mycophenolate and FK506, antibodies or other immune scavengers (immunoablative agents) such as CAMPATH, anti-CD3 antibodies or other antibody treatments, cyclophosphamide (cytoxan), fludarabine (fludarabine), cyclosporine (
  • the cells may express other active agents, for example, agents that enhance the activity of CAR expressing cells.
  • the active agent may be an active agent that blocks inhibitory molecules.
  • Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to launch immune effector responses in some embodiments.
  • Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG , LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC Class I, MHC Class II, GAL9, Adenosine, TGFR (TGFR ⁇ ) and TGFR ⁇ .
  • the extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as PD1CAR.
  • any one of the aforementioned fusion protein or any one of the aforementioned CAR structure or any one of the aforementioned nucleic acid sequences, any one of the aforementioned expression vectors, or any one of the aforementioned immune cells in the preparation of tumor drugs.
  • the tumor is a malignant tumor, including acute lymphoid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, prostate cancer, colorectal cancer, breast cancer, and ovarian cancer , Cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer and skin cancer.
  • Z refers to the human CD3 intracellular signal CD3 ⁇
  • BB refers to the human 4-1BB intracellular signal
  • BBZ refers to the intracellular domain of 4-1BB ICD-CD3 ⁇
  • 28Z refers to CD28ICD -The intracellular domain of CD3 ⁇ .
  • the "functional variant” generally refers to having substantially the same function (for example, may possess the properties of the chimeric antigen receptor) and having at least 85% (for example, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity
  • the amino acid sequence In some embodiments, the amino acid sequence variant has substantially the same function as it.
  • CEAZ refers to the CEA-targeting CAR structure with only CD3 ⁇ in the cell
  • CEA-28Z refers to the intracellular costimulatory signal derived from the CD28 intracellular domain and the CD3 ⁇ -targeting CEA CAR structure
  • CEA-BBZ refers to the intracellular costimulatory signal derived from the intracellular domain of 4-1BB and CD3 ⁇ targeting CEA CAR structure
  • BCMA-BBZ refers to the intracellular costimulatory signal from the intracellular domain derived from 4-1BB Signal and CD3 ⁇ targeting the BCMA CAR structure
  • SIRP ⁇ -28TM-28 refers to the SIRP ⁇ fusion peptide with only the intracellular signal derived from CD28, using the abbreviation SIRP ⁇ -28
  • SIRP ⁇ -8TM-BB refers to the intracellular only The fusion peptide derived from the intracellular domain signal of 4-1BB uses the abbreviation SIRP ⁇ -BB.
  • the fusion protein for reversing the tumor microenvironment provided by the present invention can reverse the tumor microenvironment and target and kill CD47-positive tumor cells; the novel tumor immunosuppressive resistant CAR and immune cells containing the fusion protein can destroy the inhibitory signal in tumor tissues
  • the effect on CAR-T function realizes the effectiveness of CAR-T treatment while ensuring a certain degree of safety.
  • Figure 1 is a structural diagram of immunosuppressive destructive fusion protein.
  • Figure 2 is a schematic diagram of the immunosuppressive CAR structure.
  • Figure 3 is a verification diagram of CAR construction plasmid.
  • Figure 4 shows the results of target cell construction.
  • Figure 5a shows the CAR-positive rate of the new immunosuppressive resistant CAR-T.
  • Figure 5b shows the expression intensity of the new immunosuppressive resistant CAR-T.
  • Figure 6a shows the in vitro function verification of a new type of immunosuppressive resistant CAR-T.
  • Figure 6b shows the in vitro killing rate of the new immunosuppressive resistant CAR-T.
  • Figure 7 shows the verification of a new type of immunosuppressive resistant CAR-T on the destruction of the tumor suppressive environment.
  • Figure 8a shows the three positive expressions of CEA, PD-L1 and CD47 in NCG mice.
  • Figure 8b is the tumor volume measurement data of NCG mice to plot the tumor volume growth curve.
  • Figure 9 shows the in vivo effectiveness of the second-generation immunosuppressive CAR.
  • Figure 10 shows the average fluorescence intensity of each experimental group of CEA targets.
  • Figure 11 shows the positive rates of CEA targets in each experimental group.
  • Figure 12 shows the in vitro expansion curve of CAR-T for each experimental group of CEA targets.
  • Figure 13 shows the killing efficiency of CAR-T in each experimental group of CEA targets on target cells DLDL1-CEA.
  • Figure 14 shows the secretion of IFN- ⁇ in DLD1-CEA and DLD1-CEA (CD47-) cells in each experimental group of CEA targets.
  • Figure 15 shows the secretion of IL-2 in DLD1-CEA and DLD1-CEA (CD47-) cells in each experimental group of CEA targets.
  • Figure 16 shows the secretion of TNF- ⁇ in DLD1-CEA and DLD1-CEA (CD47-) cells in each experimental group of CEA targets.
  • Figure 17 is a physical map of the tumor growth in mice verified by the in vivo hypoxia model of CEA target screening and comparison group a.
  • Figure 18 is the CEA target screening comparison group a in vivo hypoxia model to verify the amount of bioluminescence in tumor mice.
  • Figure 19 shows the positive rate of CAR-T in each experimental group targeting CD19 by flow cytometry.
  • Figure 20 shows the killing of cells in each experimental group targeting CD19.
  • Figure 21 shows the secretion of IFN- ⁇ factor in each experimental group targeting CD19.
  • Figure 22 shows the bioluminescence in mice of each experimental group targeting CD19.
  • Figure 23 shows the tumor growth in mice of each experimental group targeting CD19.
  • Figure 24 shows the secretion of IFN- ⁇ factor by PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28.
  • Figure 25 shows the secretion of IFN- ⁇ factor by PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28.
  • Figure 26 shows the secretion of IFN- ⁇ factor by PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28.
  • Figure 27 shows the secretion of IFN- ⁇ factor by PSCA-28BBZ-7H-28TM+SIRP ⁇ -28.
  • Figure 28 shows the secretion of IFN- ⁇ factor by PSCA-28BBZ-G4H-28TM and PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28.
  • Figure 29 shows the secretion of IFN- ⁇ factor by PSCA-28BBZ-7H-28TM and PSCA-28BBZ-7H-28TM+SIRP ⁇ -28.
  • Figure 30 shows the cell killing status of PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28 in positive/negative cells, respectively.
  • Figure 31 shows the cell killing status of PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28 in positive/negative cells, respectively.
  • Figure 32 shows the cell killing status of PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28 in positive/negative cells, respectively.
  • Figure 33 shows the cell killing status of PSCA-28BBZ-7H-28TM+SIRP ⁇ -28 in positive/negative cells, respectively.
  • Figure 34 shows the killing of cells in PSCA-28BBZ-G4H-28TM and PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28.
  • Figure 35 shows the killing of cells in PSCA-28BBZ-7H-28TM and PSCA-28BBZ-7H-28TM+SIRP ⁇ -28.
  • 5HCEA-BBZ refers to the CAR structure that contains hypoxia and has costimulatory signals derived from the intracellular domain of 4-1BB and CD3 ⁇ targeting CEA
  • CEA-BBZ refers to intracellular It has a costimulatory signal derived from the intracellular domain of 4-1BB and a CEA-targeted CAR structure of CD3 ⁇
  • CEA-28Z refers to the intracellular costimulatory signal derived from the intracellular domain of CD28 and a CEA-targeted CAR structure of CD3 ⁇
  • SIRP ⁇ -28TM-28 refers to the fusion peptide that only originates from the intracellular domain signal of CD28 in the cell and subsequently uses the abbreviation SIRP ⁇ -28.
  • 5HCD19-BBZ refers to the intracellular hypoxic promoter, the costimulatory signal derived from the intracellular domain of 4-1BB (abbreviated as BB) and the CD19-targeting CAR structure of CD3 ⁇ ;
  • SIRP ⁇ -28TM- 28 refers to the fusion peptide with only the intracellular domain signal derived from CD28;
  • 5HCD19-BBZ-SIRP ⁇ -28 refers to the CAR with the following structure: 5HCD19-8H-8TM-CD137-CD3 ⁇ ;
  • SIRP ⁇ -28-5HCD19-BBZ refers to CAR-T obtained by transfecting immune cells by expressing two vectors of CAR fusion protein containing hypoxia promoter.
  • RT4-Luc-GFP is a positive cell
  • PC-3-Luc-GFP is a negative cell
  • SIRP ⁇ -28TM-28 refers to the intracellular only source of CD28 intracellular domain signal Fusion peptide
  • PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM+SIRP ⁇ -28 refers to the combination of PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and SIRP ⁇ -28
  • the fusion protein SIRP ⁇ -28 is expressed in the products obtained by transfecting immune cells with two vectors respectively.
  • PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28 refers to the combination of PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and SIRP ⁇ -28 It is a product obtained by co-expressing with the fusion protein SIRP ⁇ -28 in a vector transfected with immune cells.
  • CEA ScFv To SEQ ID NO: 25 CEAZ SEQ ID NO: 15 SEQ ID NO: 4 CEA-BB-CD3 ⁇ To SEQ ID NO: 23 SIRP ⁇ -28TM-28 SEQ ID NO: 13 SEQ ID NO: 2 SIRP ⁇ -8TM-BB SEQ ID NO: 14 SEQ ID NO: 3 CEAZ-P2A-SIRP ⁇ -28 SEQ ID NO: 16 SEQ ID NO: 5 CEAZ-P2A-SIRP ⁇ -BB SEQ ID NO: 17 SEQ ID NO: 6
  • Z refers to CD3 ⁇
  • CEAZ refers to the CAR structure (ScFv(CEA)-hinge-TM-CD3 ⁇ ) with only CD3 ⁇ in the cell
  • CEA-28Z refers to the intracellular source
  • CEA-BBZ refers to the costimulation of intracellular 4-1BB intracellular domain Signal and CAR structure of CD3 ⁇ targeting CEA (ScFv(CEA)-hinge-TM-4-1BB-CD3 ⁇ )
  • BCMA-BBZ refers to the costimulation of the intracellular domain of 4-1BB (abbreviated as BB) Signal and CD3 ⁇ target the CAR structure of BCMA
  • SIRP ⁇ -28TM-28 refers to the fusion peptide with only the source of CD28 intracellular domain signal in the cell.
  • 5HCD19-BBZ refers to a CAR structure that has a hypoxic promoter, a costimulatory signal derived from the intracellular domain of 4-1BB (abbreviated as BB), and CD3 ⁇ targeting CD19;
  • SIRP ⁇ -28TM-28 (Abbreviation SIRP ⁇ -28) refers to the fusion peptide with only source CD28 intracellular domain signal in the cell;
  • SIRP ⁇ -28+5HCD19-BBZ refers to the CAR fusion protein containing hypoxia promoter expressing two vectors respectively to transfect immune cells CAR-T obtained.
  • 5HCD19-BBZ-P2A-SIRP ⁇ -28 refers to the CAR-T obtained by transfecting immune cells with a CAR fusion protein containing a hypoxia promoter and co-expressing a vector.
  • PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM+SIRP ⁇ -28 refers to the combination of PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and SIRP ⁇ -28 and the fusion protein SIRP ⁇ -28 are respectively expressed in the products obtained by transfecting immune cells with two vectors.
  • PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28 refers to the combination of PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and SIRP ⁇ -28 It is a product obtained by co-expressing with the fusion protein SIRP ⁇ -28 in a vector transfected with immune cells.
  • the method of packaging lentivirus by the calcium phosphate method is specifically: culturing 293T cells to a better state with DMEM medium containing 10% FBS (w/v), packaging plasmid (RRE:REV:2G) and The expression plasmid was added to a 1.5 centrifuge tube according to a certain ratio, and CaCl2 and 2 ⁇ HBS were added. After mixing, the mixture was allowed to stand at room temperature and then added to the processed 293T cell culture medium. After 3-5 hours, the medium was changed again to 10 mL containing 10 %FBS in DMEM medium, after 48h or 72h, the cell supernatant was collected and the virus was purified.
  • the antibody is: Protein-L-PE
  • Protein-L can recognize the light chain of the antibody
  • the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L
  • Protein-L can be used to detect CAR positive Rate and CAR expression intensity.
  • the method for detecting the killing ability of different CAR-Ts on target cells is: using ACEA xCELLigence RTCA MP instrument, and the experimental steps are carried out according to the instrument manual.
  • the principle of ACEA xCELLigence RTCA MP is to record the resistance index of tumor cells attached to the bottom of the well every 15 minutes, and use the resistance index to judge the proliferation or death of the adherent target cells.
  • CAR-T cell killing rate baseline resistance index-real-time resistance index.
  • IFN- ⁇ detection uses BD IFN- ⁇ kit detection, and the experimental steps are carried out according to product instructions;
  • IL-2 detection uses inritrogen IL-2 kit detection, and the experimental steps are carried out according to product instructions;
  • TNF- ⁇ detection The Biolegend kit was used for detection, and the experimental procedures were carried out according to the product instructions.
  • the method for verifying whether the hypoxia model is completed is: construct an in vitro hypoxic cell model using recombinant plasmid virus-infected activated PBMC, culture for 12-18h and then change the medium; then use CoCl 2 to induce a hypoxic environment and culture By the Nth day, the expression of CAR was detected by detecting the light chain antibody on the CAR structure.
  • the mouse used for in vivo verification is NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl, referred to as NOG mouse, which was cultivated by Mamoru Ito of the Japan Institute of Experimental Animals (CIEA) and is an international CAR-T in vivo related product. The most common strains for tumor experiments.
  • the method for verifying the in vivo hypoxia model is: select 6-8 week old female NOG mice, mark the ear tags, and inject the target cells subcutaneously on the back of the mice at a rate of 1 ⁇ 10 ⁇ 6/cell , The tumor volume of mice was measured on the 12th day of tumor formation.
  • the restriction enzyme digestion identification of the recombinant plasmid 1-3: pL-CAG-CEAZ-PD1-28 plasmid, followed by: original plasmid, CEAZ (1371bp), PD1-28 (783bp); 4-6: pL-CAG-CEAZ-PD1-BB plasmid, in order: original plasmid, CEAZ (1371bp), PD1-BB (798bp); 7-9: pL-CAG-CEAZ- SIRP ⁇ -28 plasmid, in order: original plasmid, CEAZ (1371bp), SIRP ⁇ -28 (1374bp); 10-12: pL-CAG-CEAZ-SIRP ⁇ -BB plasmid, in order: original plasmid, CEAZ (1371bp), SIRP ⁇ -BB (1368bp); M1: DL5000 DNA molecular weight standard; M2: DL15000 DNA molecular weight standard; 13-15: pL-CAG-S
  • the virus containing CEA, PD-L1, and CD47 antigens was prepared by calcium phosphate method, and CHO cells were respectively infected to construct CHO-CEA cells, CHO-CEA-PD-L1 cell lines and CHO-CEA-CD47 cell lines.
  • Signal regulatory protein ⁇ SIRP ⁇
  • CD47-positive target cells can be used to evaluate CEAZ-SIRP ⁇ -BB, CEAZ-SIRP ⁇ -28, SIRP ⁇ , SIRP ⁇ -28, and SIRP ⁇ -BB .
  • the positive rates of the three cell lines were tested after ten subcultures. The results are shown in Figure 4.
  • the positive rate of CHO-CEA is 97.1%
  • the double positive rate of CHO-CEA-CD47 is 97.6%
  • CHO-CEA-PD-L1 The double positive rate of 87% is in line with experimental requirements, indicating that the cell line has been successfully constructed and can be used as target cells for subsequent CAR-T drug efficacy evaluation.
  • the calcium phosphate method was used to package the lentivirus, and the 5 single-expressed CARs (CEAZ, PD1-28, PD1-BB, SIRP ⁇ -28, SIRP ⁇ -BB) and 6 new immunosuppressive-resistant CARs (CEAZ-BB) in Example 1 were obtained.
  • Lymphocytes were separated by gradient centrifugation; after centrifugation, the second layer of white lymphocytes was taken, washed with physiological saline, and cultured with RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After the obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected according to a certain multiplicity of infection (MOI). The positive rate of CAR-T was detected on the 12th day of virus infection. The detection method was flow cytometry.
  • MOI multiplicity of infection
  • the antibody is: Protein-L-PE
  • Protein-L can recognize the light chain of the antibody
  • the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L, so Protein-L can be used to detect the CAR positive rate and CAR expression strength.
  • Control T cells that did not express the immunosuppressive destructive fusion protein were used as controls to verify the function of immunosuppressive destructive fusion proteins SIRP ⁇ -28TM-28 and SIRP ⁇ -8TM-BB.
  • the target cell is a CHO cell line expressing CD47. The results are shown in Figure 6a. Both SIRP ⁇ -28TM-28 and SIRP ⁇ -8TM-BB fusion proteins have killing effects on CD47-positive target cells.
  • CEAZ group Take CEAZ group as positive control, Control-T group as negative control, set CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRP ⁇ -28, CEAZ-SIRP ⁇ -BB group as experimental group, set CHO-CEA-CD47 And CHO-CEA-PD-L1 as target cells to verify the in vitro effectiveness of the new immunosuppressive resistant CAR-T.
  • the results are shown in Figure 6b and Table 2 below.
  • the cell killing rate of the CEAZ-PD1-28 and CEAZ-SIRP ⁇ -28 groups was significantly higher than that of the CEAZ group, while the CEAZ-PD1-BB and CEAZ-SIRP ⁇ -BB groups had a greater effect on target cells. There was no significant increase in killing compared to the CEAZ group.
  • the new immunosuppressive resistant CAR-T group has killing specificity
  • CEAZ-PD1-28 and CEAZ-SIRP ⁇ -28 have significant killing effects on target cells.
  • CAR-T cells After CAR-T cells infiltrate tumor tissues, they are often affected by tumor immunosuppression, and high expression of PD1, LAG-3, and Tim-3 attenuating molecules, which in turn weakens the effective function of killing tumor cells, and increases the apoptosis of CART cells.
  • CAR-T cells in SIRP ⁇ -BB and Control-T groups were cultured to Day 7
  • CAR-T cells and DLD-1-CEA-Luc-GFP cells were co-cultured in a 12-well cell culture plate, and CAR-T cells were collected after 48 hours .
  • CEAZ-PD1-28 and CEAZ-PD1-BB groups express PD1 exogenously, the PD1 positive rate of these two groups is not used to evaluate the degree of CAR-T cell failure.
  • the detection results of failure molecules are shown in Figure 7 and Table 3 below: Under CD3+PL+, the expression levels of PD1, LAG-3, and Tim-3 of CEAZ-SIRP ⁇ -28 were lower than those of CEAZ group, indicating that CEAZ-SIRP ⁇ -28 The degree of exhaustion after antigen stimulation is lower.
  • BCMA-BBZ-P2A-SIRP ⁇ -28 also showed lower expression of exhaustion molecules after stimulation with BCMA antigen.
  • mice used for in vivo verification are NCG mice. Thirty NCG mice were selected and injected with DLD-1-CEA-Luc-GFP (CEA, PD-L1, and CD47 positive expression, as shown in Figure 8a) for tumor-bearing. When the tumor grows to a measurable size of mung bean, the tumor size is measured. During the experiment, mice in the experimental group appeared malaise and dying, half-length or whole body paralysis, weight loss of 20% (compared with the beginning of the experiment), and tumor volume ⁇ 1500mm3. The experiment will be terminated in some cases.
  • DLD-1-CEA-Luc-GFP CEA, PD-L1, and CD47 positive expression, as shown in Figure 8a
  • mice in the experimental group appeared malaise and dying, half-length or whole body paralysis, weight loss of 20% (compared with the beginning of the experiment), and tumor volume ⁇ 1500mm3. The experiment will be terminated in some cases.
  • the fluorescence values of the tumors of the mice were measured on Day 5, and the fluorescence values of intravital imaging were used for random grouping to ensure that there was no significant difference in the body weight and fluorescence value of the mice in each group, and the average body weight was calculated.
  • CAR-T cells were reinfused with a volume of 100 ⁇ L (containing 3 ⁇ 10 6 effective CAR-T cells), and untransfected T cells with the same total number of cells were given as a control group.
  • the tumor volume measurement data of NCG mice draws a tumor volume growth curve, and it is found that on Day 27-30, CEAZ-SIRP ⁇ -28 has a more obvious inhibitory effect on tumors, and the results are shown in Figure 8b.
  • the in vivo effectiveness of the immunosuppressive CAR designed by the second-generation CAR and the fusion protein, namely the CEABBZ-P2A-SIRP ⁇ -28 structure was verified.
  • NCG mice were also injected with DLD-1-CEA-Luc-GFP cells to bear tumors.
  • CAR-T cell reinfusion was carried out 13 days after tumor-bearing, and the total cell number was 8 ⁇ 10 6 .
  • the results are shown in Fig. 9 that the immunosuppressive CAR-T (CEABBZ-P2A-SIRP ⁇ -28) of the present invention can function well in the body, and the effect is significantly better than the CEABBZ of the second-generation CAR structure of the control group.
  • the second part of the experimental part of the CAR structure targeting CEA (where the amino acid sequence of CAR1 is 27)
  • the hypoxia promoter sequence 5HRE-CMVmini promoter is synthesized from the mini promoter miniCMV, and its nucleotide sequence is shown in SEQ ID NO:1. Then the CAR structure of 5HRE-CMVmini promoter, lentiviral expression vector, CEAScFv-CD8 hinge region-CD8 transmembrane region-CD137-CD3 ⁇ -P2A-SIRP ⁇ -CD28 (5HCEA-BBZ-P2A-SIRP ⁇ -28) was digested with double enzymes The fragments were cut and recovered respectively, the gene fragments were ligated, transformed, and single clones were selected to construct the recombinant plasmid PBKL1-5H1P-CEA-OPRE (SIRP ⁇ fusion protein) containing SIRP ⁇ fusion protein for CAR-T cell preparations targeting CEA.
  • SIRP ⁇ fusion protein SIRP ⁇ fusion protein
  • CAR and fusion protein are co-expressed in a vector to transfect immune cells or CAR and fusion protein are co-expressed in a vector to transfect immune cells to obtain the structure of our CAR containing fusion protein.
  • CAR and fusion protein are co-expressed in a vector to transfect immune cells to obtain the structure of our CAR containing fusion protein.
  • CAR and fusion protein are co-expressed in a vector to transfect immune cells to obtain the structure of our CAR containing fusion protein.
  • CAR and The products obtained by expressing the fusion proteins together in a vector and transfecting immune cells are represented by the abbreviation "5HCEA-BBZ+SIRP ⁇ -28".
  • the CEA-positive DLD1-CEA and DLD1-CEA (CD47-) cells were used as target cells, respectively. After hypoxia treatment of effector cells (conventional CAR-T cells and CAR-T cells with hypoxic promoter CAR expression), they are plated in target cells according to the ratio of 1:1 to detect different CAR-T against target cells. Lethality.
  • Table 4 The killing efficiency of each group of CAR-T targeting CEA on target cells DLDL1-CEA
  • the cell supernatant was collected 24 hours after killing, and the IFN- ⁇ , IL-2, and TNF- ⁇ secretion ability of CAR-T cells was tested after stimulation by target cells.
  • the collected supernatant was used to detect the secretion of IFN- ⁇ and IL-2 using the ELISA method.
  • DLD1-CEA-Luc-GFP cells were selected as tumor-forming target cells used in the in vivo verification to construct a human CEA+ solid tumor tumor-bearing model.
  • Control T mice were randomly divided into Control T (CT) group, 5HCEA-BBZ-8H-8, 5HCEA-BBZ-P2A-SIRP ⁇ -28, and the control group was Control T group.
  • CT Control T
  • 5HCEA-BBZ-8H-8 5HCEA-BBZ-P2A-SIRP ⁇ -28
  • the control group was Control T group.
  • 1*10 ⁇ 7Copies of CAR-T cells were injected into the tail vein of mice in different groups; the Control T group was infused with the same total number of T lymphocytes on the 812th day.
  • the tumor volume of each group of mice was measured every three days.
  • the experimental results are shown in Figure 17 and Figure 18. It can be seen that the 5HCEA-BBZ-8H-8 is more effective than the 5HCEA-BBZ-P2A-SIRP ⁇ -28 group of mice in vivo. Significantly improved, the 5HCEA-BBZ-8H-8 group also has a significant effect on tumor elimination.
  • the lentivirus was packaged by the calcium phosphate method to obtain 3 virus particles (SIRP ⁇ -28, 5HCD19-BBZ, 5HCD19-BBZ-P2A-SIRP ⁇ -28) in Example 1.
  • Lymphocytes were separated by gradient centrifugation. After centrifugation, the second layer of white lymphocytes was taken, washed with physiological saline, and cultured with RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After the obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected according to a certain multiplicity of infection (MOI), and the positive rate of CAR-T was detected by flow cytometry on the 8th day of virus infection. The results are as follows Figure 19 and Table 5 below.
  • MOI multiplicity of infection
  • Control T As the control group, the experimental group was set to SIRP ⁇ -28 group, 5HCD19-BBZ group, 5HCD19-BBZ-P2A-SIRP ⁇ -28 group, and Nam6-Luc-GFP (CD19 positive), K562-Luc-GFP (CD19 negative) ) Is the target cell, and its effectiveness in vitro is verified by in vitro killing and in vitro factor secretion. The results are shown in Figure 20 and Table 6 below.
  • the product obtained by transfecting immune cells with one co-expressed vector (5HCD19-BBZ-P2A-SIRP ⁇ -28) and the product obtained by co-transfecting immune cells with two separately expressed vectors
  • the in vitro killing of the product (SIRP ⁇ -28+5HCD19-BBZ) was significantly higher than that of the SIRP ⁇ -28 group and 5HCD19-BBZ group, and it did not kill negative cells.
  • NCG mice female, 6 weeks old, at 1 ⁇ 10 6 Cells / only subcutaneous injections Nalm6-Luc-GFP cells in vivo established tumor-bearing model (SC), the tumor-bearing 8d to 1 ⁇ 10 7 CAR-T Cells/only dose tail vein injection (iv) was given to different groups (Control T, 5HCD19-BBZ, 5HCD19-BBZ-P2A-SIRP ⁇ -28) CAR-T. In vivo imaging is used to observe the growth of tumors in vivo, and to evaluate the therapeutic effects of different CAR-Ts on lymphoma in vivo. The results are shown in Figure 22 and Figure 23. Compared with the Control T group and the 5HCD19-BBZ group, 5HCD19-BBZ-P2A-SIRP ⁇ -28 has a significant anti-tumor effect in vivo and can clear tumors significantly.
  • PSCA ScFv-G4H hinge region-CD28 transmembrane region-CD28-CD137-CD3 ⁇ -P2A-SIRP ⁇ -28 PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28 uses double enzyme digestion The fragments were cut and recovered separately, and the gene fragments were ligated, transformed, and single clones were picked.
  • the CAR structure of the lentiviral expression vector, PSCA ScFv-7H hinge region-CD28 transmembrane region-CD28-CD137-CD3 ⁇ -P2A-SIRP ⁇ -28 (PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28) is digested with double enzymes The fragments were cut and recovered separately, and the gene fragments were ligated, transformed, and single clones were picked.
  • the product PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28 obtained by transfecting immune cells with a co-expression vector is co-transformed with two separately expressed vectors
  • the IFN- ⁇ factor secretion of the product PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM+SIRP ⁇ -28 obtained by infecting immune cells was significantly higher than that of the control group and PSCA-28BBZ-G4H-28TM group or PSCA-28BBZ-7H-28TM group, no killing of negative cells
  • the experimental group was set with PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28, PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28, PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28, PSCA-28BBZ-7H- 28TM+SIRP ⁇ -28, PSCA-28BBZ-G4H-28TM, PSCA-28BBZ-7H-28TM, using RT4-Luc-GFP (PSCA positive) and PC-3-Luc-GFP (PSCA negative) as target cells, respectively.
  • Table 14-Table 19 and Figure 30- Figure 30- Figure 35 The results as shown in Table 14-Table 19 and Figure 30- Figure 30- Figure 35.
  • the product PSCA-28BBZ-G4H-28TM-P2A-SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM-P2A-SIRP ⁇ -28 obtained by transfecting immune cells with a co-expression vector is co-transformed with two separately expressed vectors
  • the in vitro killing of the product PSCA-28BBZ-G4H-28TM+SIRP ⁇ -28 or PSCA-28BBZ-7H-28TM+SIRP ⁇ -28 obtained by infecting immune cells was significantly higher than that of the control group and PSCA-28BBZ-G4H-28TM group or PSCA-28BBZ The -7H-28TM group did not kill the negative cells.

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Abstract

本发明属于免疫治疗技术领域，具体涉及一种逆转肿瘤微环境的融合蛋白、一种肿瘤免疫抑制抵抗型CAR以及表达载体、免疫细胞和应用。该融合蛋白与CEA、CD19、PSCA、BCMA等不同靶点的不同CAR结构结合成肿瘤免疫抑制抵抗型CAR或联合使用，靶向杀伤CD47阳性的的肿瘤细胞；所述肿瘤免疫抑制抵抗型CAR和免疫细胞破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性。

Description

逆转肿瘤微环境的融合蛋白及其应用 技术领域
本发明属于免疫治疗技术领域，具体涉及一种逆转肿瘤微环境的融合蛋白、一种新型肿瘤免疫抑制抵抗型CAR以及表达载体、免疫细胞和应用。
背景技术
CAR全称是嵌合抗原受体，CAR-T是嵌合抗原受体T细胞即表达嵌合抗原受体(CAR)的T淋巴细胞，目前CAR-T治疗在血液系统肿瘤中取得了突破性进展，但在实体瘤中的效果不如血液系统瘤，其原因一方面因为CAR-T较难进入实体瘤内部，一方面即便CAR-T细胞进入实体瘤内部也因为肿瘤微环境而不能正常发挥功能，这些都影响CAR-T细胞在实体瘤治疗的疗效；肿瘤微环境与肿瘤的发生、生长和转移有着密切关系，近年来的临床研究表明，肿瘤微环境尤其是实体瘤的免疫微环境对免疫治疗(包含免疫细胞治疗)的疗效和预后都有着显著的影响。因此，CAR-T细胞治疗实体肿瘤，解决肿瘤微环境抑制即实施肿瘤微环境抵抗必不可少。
CD47作为继PD-1/PD-L1,CTLA-4之后新的免疫检查点，通过向巨噬细胞传递“不要吃我”信号，抑制固有免疫的进行。目前有文献报道采用靶向CD47ScFv制备CAR-T进行肿瘤治疗。CD47可以促成肿瘤逃逸的免疫微环境，不过由于肿瘤的异质性，单独靶向CD47疗效有限，并且外源的ScFv由于其强的亲和力可能会导致CAR-T细胞过度活化存续性差和非特异性引起安全隐患。因此，采用更安全有效的方案去识别CD47破除肿瘤微环境的逃逸信号十分重要。
信号调节蛋白α(SIRPα)是CD47的配体之一，可与CD47结合，抑制巨噬细胞的吞噬作用，目前绝大多数关于CD47的研究均集中于SIRPα。SIRPγ也可与CD47结合，其存在于T细胞表面，胞外区由一个V结构域和两个C1结构域组成，无胞内信号，仅通过CD47传递单向信号，目前很少有研究者针对SIRPγ进行改造，而我们认为其是更适合进行针对CD47进行肿瘤微环境破除的配体。因此，我们选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR。
发明内容
本发明的目的在于提供一种逆转肿瘤微环境的融合蛋白和包含该蛋白的表达载体和免疫细胞，所述融合蛋白可以逆转肿瘤微环境，靶向杀伤肿瘤细胞。
为了实现上述目的，本发明采用以下方案：
对SIRPγ蛋白的胞外段进行改造设计，得到可以破除肿瘤组织中抑制性信号的逆转肿瘤微环境的融合蛋白。
所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段，跨膜区和胞内信号区。
进一步，跨膜结构来源于人CD28跨膜区或人CD8来源跨膜区。
进一步，所述跨膜结构氨基酸序列如SEQ ID NO:7或SEQ ID NO:8所示。
优选地，所述胞内信号区来源于CD28，序列如SEQ ID NO:9或SEQ ID NO:38所示。
进一步，所述SIRPγ融合蛋白的结构为SIRPγ-CD28TM-CD28、SIRPγ-CD8TM-4-1BB。
进一步，所述SIRPγ胞外段氨基酸序列如SEQ ID NO:1或其功能性变体所示。
进一步，所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的氨基酸序列为SEQ ID NO:2或其功能性变体 所示。
进一步，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的氨基酸序列为SEQ ID NO:3或其功能性变体所示。
进一步，所述SIRPγ融合蛋白为SIRPγ-CD28TM-CD28的核苷酸序列如SEQ ID NO:13所示。
进一步，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的核苷酸序列如SEQ ID NO:14所示。
进一步，包含所述表达载体的免疫细胞。
进一步，所述免疫细胞为T细胞、T细胞前体或NK细胞。
具体的所述的T细胞可以是αβT细胞，也可以是γδT细胞；γδT细胞是一群表面含有受体γ和δ链的独特的T细胞亚群，占所有t淋巴细胞的0.5-5％，该细胞群于1987年首次发现。其虽然具有γδTCR，但是其识别抗原或配体为非MHC限制的，与传统的αβT细胞不同。目前已经有CN107810267A和CN107771215A等多篇专利公开将γδT细胞应用于细胞治疗和CAR-T细胞治疗中。
具体的，在设计SIRPγ融合蛋白的同时还设计了PD-1的融合蛋白PD-1-28TM-28、PD-1-8TM-BB。
具体地，所述设计的SIRPγ融合蛋白在与CAR1联合应用时，既可单独表达，也可以与CAR1共同表达发挥作用。并且所述SIRPγ融合蛋白也可单独应用到免疫治疗中。
具体地，本发明中，发明人认为SIRPγ蛋白更适合进行针对CD47进行肿瘤微环境破除的配体。因此，选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR；因此本发明创造性的将设计构建的缺氧可调控启动子联合CAR-T细胞技术和缺氧可调控启动子、SIRPγ蛋白联合CAR-T细胞技术应用到肿瘤的免疫治疗当中，即进一步对传统的CAR结构进行改造，提高CAR-T在实体瘤上的治疗药效及CAR-T的安全性。
进一步，所述免疫细胞还包含具有识别肿瘤抗原的嵌合抗原受体结构，所述嵌合抗原受体包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区，其中肿瘤抗原包含但不限于PSCA、PSMA、CD19、BCMA、CD123、CD20、CD22、CEA、EGFR、EGFRVIII、GPC3、5T4、CD33、Her2、GD2、CD70、CLL-1、Trop2、CD47、GPC3、CLND18.2、CD133、CS1、CD155、CD30、ROR1、MUC1、IL13RAα2或mesothelin等可以作为肿瘤靶向性识别的抗原分子。
本发明的目的在于还提供一种新型肿瘤免疫抑制抵抗型CAR，以及包含所述CAR的表达载体和免疫细胞。所述新型肿瘤免疫抑制抵抗型CAR和免疫细胞破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性。
为实现上述目的，本发明采用以下方案：
所述新型肿瘤免疫抑制抵抗型CAR包含目的一所述的逆转肿瘤微环境的融合蛋白和CAR1，所述CAR1包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区。
进一步，所述融合蛋白通过多顺反子结构与CAR1连接，所述多顺反子结构为自剪切多肽或内部核糖体进入位点IRES，所述自剪切多肽为T2A、P2A、E2A或F2A。
进一步，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ融合蛋白或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ融合蛋白。
进一步，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28或ScFv -hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28。
进一步，所述CAR结构a可以是常规的第一代、第二代、第三代CAR结构，也可以是改进的双CAR、可调控CAR结构(如FRB/FKBP12调控)等新型CAR结构。
进一步，CAR1中的铰链区序列可以来源于：IgG、CD8、CD7、CD4；CAR结构中跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；CAR结构中胞内信号区可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127。
进一步，所述ScFv可以识别CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70、CD47中的任一种。
进一步，所述ScFv可以识别CD47或CEA或PSCA或CD19或BCMA。
在某些实施例中，所述CAR结构带有截短的EGFRt调控标签；在某些实施例中，所述靶向PSCA的CAR结构为通用型CAR结构；在某些实施例中，所述靶向PSCA的CAR结构带有自杀基因如iCasp9。
在某些实施例中，所述CAR结构包含天然杀伤细胞受体(NKR)的一种或多种组分，因而形成NKR-CAR。NKR组分可以是来自以下任何天然杀伤细胞受体的跨膜结构域、铰链结构域或胞质结构域：杀伤细胞免疫球蛋白样受体(KIR)，例如KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、DIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3、KIR2DP1和KIR3DP1；天然细胞毒性受体(NCR)，例如，NKp30、NKp44、NKp46；免疫细胞受体的信号传导淋巴细胞活化分子(SLAM)家族，例如，CD48、CD229、2B4、CD84、NTB-A、CRA、BLAME和CD2F-10；Fc受体(FcR)，例如，CD16、和CD64；和Ly49受体，例如，LY49A、LY49C。所述的NKR-CAR分子可以与衔接分子或胞内信号结构域(例如，DAP12)相互作用。
作为一种优选方案1，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR1包含：hinge的氨基酸序列如SEQ ID NO:24或其功能性变体所示，TM的氨基酸序列如SEQ ID NO:7或SEQ ID NO:8，CD3ζ的氨基酸序列如SEQ ID NO:11或其功能性变体所示所示；融合蛋白结构：融合蛋白结构：SIRPγ的胞外段的氨基酸序列如SEQ ID NO:1或功能性变体所示；来源于人CD28的跨膜区氨基酸序列如SEQ ID NO:7所示；来源于人CD28的胞内信号区氨基酸序列如SEQ ID NO:9所示。
进一步，所述ScFv的氨基酸序列如SEQ ID NO:25所示或其功能性变体。
具体的，本优选方案设计了表达新型免疫抑制抵抗型CAR：ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-28TM-28，ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-8TM-BB，ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-28TM-28，ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-8TM-BB以及ScFv-hinge-TM-CD3ζ-自剪切肽-PD-1-28TM-28和ScFv-hinge-TM-CD3ζ-自剪切肽-PD-1-8TM-BB的基因修饰的T淋巴细胞验证新型免疫抑制抵抗型CAR的作用。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
作为一种优选方案2，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR1包含抗CEA单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号。
优选地，所述CAR结构a为CEA ScFv-CD8铰链区-CD8跨膜区-CD137-CD3ξ结构，其氨基酸序列包含如SEQ ID NO：26所示的序列。其中，编码CEA单链抗体的核酸序列如SEQ ID NO：36所示；编码CD8铰链区-CD8跨膜区-CD137-CD3ξ结构的核酸序列如SEQ ID NO：37所示。
进一步，编码包含缺氧可调控的启动子的所述CAR结构的核酸序列包含如SEQ ID NO：31所示的序列。
作为一种优选方案3，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR1包括CD19单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；
优选地，所述CAR结构a氨基酸序列如SEQ ID NO：27所示或其功能性变体。
进一步，编码所述CAR结构的核酸序列包含如SEQ ID NO：32或SEQ ID NO：33所示的序列。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
作为一种优选方案4，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR结构a包括PSCA单链抗体、铰链区、CD28跨膜区、CD28、CD137和CD3ξ三刺激信号；所述铰链区为G4H或7H。
优选地，所述CAR结构a氨基酸序列如SEQ ID NO：28所示或其功能性变体；或如SEQ ID NO：29所示或其功能性变体。氨基酸序列如SEQ ID NO：28所示或其功能性变体为铰链区为G4H的CAR结构a；氨基酸序列如SEQ ID NO：29所示或其功能性变体为铰链区为7H的CAR结构a。
进一步，编码所述CAR结构的核酸序列包含如SEQ ID NO：34或SEQ ID NO：35所示的序列；其中SEQ ID NO：35包含了缺氧可调控的启动子。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
作为一种优选方案5，包含前述的SIRPγ融合蛋白，和优选方案1-优选方案4中的任一一种CAR1组成CAR结构，所述CAR结构还包括缺氧启动子，所述缺氧可调控启动子的核酸序列包含如SEQ ID NO：30所示的序列。包含缺氧启动子的靶向CD19的CAR结构能够在缺氧微环境中有效的清除体内肿瘤作用,并且利用缺氧微环境诱导的启动子能够在缺氧环境强化目的基因、蛋白等因子的表达，提高肿瘤的药物疗效和CAR-T治疗的有效性和安全性，而且不仅可以有效的表达于T淋巴细胞，而且在缺氧环境中能够加强CAR分子表达，并且使CAR-T细胞有高的IFN-γ分泌能力，提高CAR-T细胞针对肿瘤靶细胞的杀伤，能够用于肿瘤的靶向治疗。
进一步，所述缺氧可调控启动子，由Hifla调节元件和迷你启动子连接构成；所述迷你启动子选自细胞病毒启动子、HSV胸苷激酶的启动子、猿猴病毒40的启动子、腺病毒晚期启动子和合成启动子中的任一项。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ 融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
本发明目的在于还提供一种包含前任一所述的新型肿瘤免疫抑制抵抗型CAR(包括优选方案1-优选方案5中的方案和非优选地方案)的表达载体、包含该表达载体的免疫细胞。
进一步，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
进一步，所述免疫细胞为T细胞、T细胞前体或NK细胞。
进一步，制备所述的免疫细胞的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
在某些实施例中，所述慢病毒载体选自基本上由以下组成的群组：人免疫缺陷病毒1(HIV-1)、人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(visna-maedi virus，VMV)病毒、山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿猴免疫缺陷病毒(SIV)。
在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。
在某些实施例中，CAR包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)以及优化的土拔鼠转录后调节元件(oPRE)。
在某些实施例中，所述5'LTR的启动子经异源启动子置换。
在某些实施例中，所述异源启动子是巨细胞病毒(CMV)启动子、劳斯肉瘤病毒(Rous Sarcoma Virus，RSV)启动子或猿猴病毒40(SV40)启动子。
在某些实施例中，所述5'LTR或3'LTR是慢病毒LTR。
在某些实施例中，所述3'LTR是自我失活(SIN)LTR。
在某些实施例中，所述CAR结构的核酸序列包含优化的Kozark序列。
在某些实施例中，可操作地连接到编码本文所涵盖的CAR的多核苷酸的所述启动子以及以下组成的群组：巨细胞病毒立即早期基因启动子(CMV)、延伸因子1α启动子(EF1-α)、磷酸甘油酸激酶-1启动子(PGK)、泛素-C启动子(UBQ-C)、巨细胞病毒增强子/鸡β-肌动蛋白启动子(CAG)、多瘤病毒增强子/单纯疱疹胸苷激酶启动子(MC1)、β肌动蛋白启动子(β-ACT)、猿猴病毒40启动子(SV40)和骨髓增生肉瘤病毒增强子，阴性对照区缺失的、dl587rev引物结合位点取代的(MND)启动子。
在某些实施例中，包含CAR的载体可以包含分泌型抗PD-1ScFv；在某些实施例中，包含CAR的载体包含PD-1共轭转导肽(如PD-1-CD28-CD137-CD3信号结构)；在某些实施例中，包含CAR的载体多个CAR组合，如2个靶向不同抗原或同一抗原的不同识别位点的CAR组合。
本发明的目的在于还提供了一种药物组合物及其应用。
为实现上述目的，本发明采用以下方案：
所述药物组合物包含所述的融合蛋白或包含所述融合蛋白的表达载体和免疫细胞或所述的新型肿 瘤免疫抑制抵抗型CAR或包含所述新型肿瘤免疫抑制抵抗型CAR所述的免疫细胞。
在某些实施例中，融合蛋白可以选择SIRPα或者SIRPα的部分结构域，融合蛋白的结构可以是SIRPα-28TM-28，SIRPα-8TM-137，SIRPα-8TM-OX40、SIRPα-8TM-ICOS等多种融合蛋白。在有些实施例中可以单独使用表达上述SIRPα融合蛋白的免疫细胞，也可以将上述SIRPα融合蛋白和CAR分子共表达于同一免疫细胞；或分别表达SIRPα融合蛋白、CAR分子的免疫细胞以一定比例混合。
在某些具体实施例中，所述活性剂和/或治疗可以是手术、化疗、放射、免疫抑制剂，例如环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(methotrexate)、霉酚酸酯(mycophenolate)和FK506、抗体或其它免疫清除剂(immunoablativeagents)例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、环孢素(cyclosporin)、FK506、雷帕霉素(rapamycin)、霉酚酸(mycophenolicacid)、类固醇(steroids)、FR901228、细胞因子和辐射。
在某些实施例中，所述细胞可以表达其它活性剂，例如，增强CAR表达细胞活性的活性剂。活性剂可以是阻断抑制性分子的活性剂。抑制性分子如PD1可以在一些实施方案中降低CAR表达细胞发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT,LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1CAR。
进一步，种前任一所述的融合蛋白或前任一所述的CAR结构或前任一所述的核酸序列或前任一所述的表达载体或前任一所述的免疫细胞在制备肿瘤药物中的应用。
进一步，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、前列腺癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌。
在本发明中，在CAR结构中，Z是指人CD3胞内信号CD3ζ，BB是指人4-1BB胞内信号，BBZ是指4-1BB ICD-CD3ζ的胞内结构域，28Z是指CD28ICD-CD3ζ的胞内结构域。
本发明中，所述“功能性变体”通常是指包括与其具有基本上相同的功能(例如，可以具备所述嵌合抗原受体的性质)，且与其具有至少85％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的氨基酸序列。在某些实施方式中，所述氨基酸序列的变体为与其具有基本上相同的功能。
本发明中，在CAR结构中，CEAZ是指胞内结构仅有CD3ζ的靶向CEA的CAR结构，CEA-28Z是指胞内具有来源CD28胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构，CEA-BBZ是指胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构，BCMA-BBZ是指胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向BCMA的CAR结构；SIRPγ-28TM-28是指胞内仅有来源CD28胞内域信号的SIRPγ融合肽，使用SIRPγ-28简写；SIRPγ-8TM-BB是指胞内仅有来源4-1BB胞内域信号的融合肽，使用SIRPγ-BB简写。
本发明的有益效果在于：
本发明提供的逆转肿瘤微环境的融合蛋白可以逆转肿瘤微环境，靶向杀伤CD47阳性的的肿瘤细胞；包含所述融合蛋白的新型肿瘤免疫抑制抵抗型CAR和免疫细胞破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性。
附图说明
图1为免疫抑制破除融合蛋白结构图。
图2为免疫抑制型CAR结构示意图。
图3为CAR构建质粒验证图。
图4为靶细胞构建结果。
图5a为新型免疫抑制抵抗型CAR-T的CAR阳性率。
图5b为新型免疫抑制抵抗型CAR-T的表达强度。
图6a为新型免疫抑制抵抗型CAR-T体外功能验证。
图6b为新型免疫抑制抵抗型CAR-T体外杀伤率。
图7为新型免疫抑制抵抗型CAR-T对肿瘤抑制环境破除验证。
图8a为NCG小鼠CEA，PD-L1，CD47三阳性表达。
图8b为NCG小鼠的肿瘤体积测量数据绘制肿瘤体积增长曲线。
图9为二代免疫抑制型CAR的体内有效性。
图10为CEA靶点各实验组平均荧光强度情况。
图11为CEA靶点各实验组阳性率情况。
图12为CEA靶点各实验组CAR-T的体外扩增倍数曲线。
图13为CEA靶点各实验组CAR-T对靶细胞DLDL1-CEA的杀伤效率。
图14为CEA靶点各实验组分别在DLD1-CEA和DLD1-CEA(CD47-)细胞中IFN-γ分泌情况。
图15为CEA靶点各实验组分别在DLD1-CEA和DLD1-CEA(CD47-)细胞中IL-2分泌情况。
图16为CEA靶点各实验组分别在DLD1-CEA和DLD1-CEA(CD47-)细胞中TNF-α分泌情况。
图17为CEA靶点筛选对比组a体内缺氧模型验证小鼠肿瘤生长情况实体图。
图18为CEA靶点筛选对比组a体内缺氧模型验证肿瘤小鼠体内生物发光量情况。
图19为流式检测靶向CD19的各实验组CAR-T的阳性率情况。
图20为靶向CD19的各实验组细胞杀伤情况。
图21为靶向CD19的各实验组IFN-γ因子分泌情况。
图22为靶向CD19的各实验组小鼠体内生物发光情况。
图23为靶向CD19的各实验组小鼠体内肿瘤生长情况。
图24为PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况。
图25为PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况。
图26为PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图27为PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图28为PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图29为PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图30为PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图31为PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图32为PSCA-28BBZ-G4H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图33为PSCA-28BBZ-7H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图34为PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28中细胞杀伤情况。
图35为PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28中细胞杀伤情况。
图10-图18中，5HCEA-BBZ指的是含缺氧的并且胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构；CEA-BBZ指的是胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构；CEA-28Z指的是胞内具有来源CD28胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构，SIRPγ-28TM-28是指胞内仅有来源CD28胞内域信号的融合肽后续使用SIRPγ-28简写。
图19-图23中，5HCD19-BBZ是指胞内具有缺氧启动子、来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向CD19的CAR结构；SIRPγ-28TM-28(SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；5HCD19-BBZ-SIRPγ-28指的是指的是如下结构的CAR：5HCD19-8H-8TM-CD137-CD3ζ；SIRPγ-28-5HCD19-BBZ是指是包含缺氧启动子的CAR融合蛋白分别表达两个载体转染免疫细胞获得的CAR-T。
图24-图35中，RT4-Luc-GFP为阳性细胞，PC-3-Luc-GFP为阴性细胞；SIRPγ-28TM-28(SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28分别表达于两个载体转染免疫细胞获得的产品。PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28共同表达于一个载体转染免疫细胞获得的产品。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或按照制造厂商所建议的条件。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
本发明实施例中，涉及到的载体结构原件的序列如下表1所示。
表1 载体结构原件的序列
CAR结构原件 核苷酸序列 氨基酸序列
SIRPγ SEQ ID NO:12 SEQ ID NO:1
hinge   SEQ ID NO:24
CD28TM SEQ ID NO:18 SEQ ID NO:7
CD8TM SEQ ID NO:19 SEQ ID NO:8
CD28 ICD SEQ ID NO:20 SEQ ID NO:9
4-1BB ICD SEQ ID NO:21 SEQ ID NO:10
CD3ζ SEQ ID NO:22 SEQ ID NO:11
CEA ScFv   SEQ ID NO:25
CEAZ SEQ ID NO:15 SEQ ID NO:4
CEA-BB-CD3ζ   SEQ ID NO:23
SIRPγ-28TM-28 SEQ ID NO:13 SEQ ID NO:2
SIRPγ-8TM-BB SEQ ID NO:14 SEQ ID NO:3
CEAZ-P2A-SIRPγ-28 SEQ ID NO:16 SEQ ID NO:5
CEAZ-P2A-SIRPγ-BB SEQ ID NO:17 SEQ ID NO:6
本发明实施例中(表1中)：Z是指CD3ζ，CEAZ是指胞内结构仅有CD3ζ的CAR结构(ScFv(CEA)-hinge-TM-CD3ζ)，CEA-28Z是指胞内具有来源CD28胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构(ScFv(CEA)-hinge-TM-CD28-CD3ζ)，CEA-BBZ是指胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构(ScFv(CEA)-hinge-TM-4-1BB-CD3ζ)，BCMA-BBZ是指胞内具有来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向BCMA的CAR结构；SIRPγ-28TM-28是指胞内仅有来源CD28胞内域信号的融合肽后续使用SIRPγ-28简写，SIRPγ-8TM-BB是指胞内仅有来源4-1BB胞内域信号的融合肽，后续使用SIRPγ-BB简写。
本发明实施例中，5HCD19-BBZ是指胞内具有缺氧启动子、来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向CD19的CAR结构；SIRPγ-28TM-28(简写SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；SIRPγ-28+5HCD19-BBZ是指是包含缺氧启动子的CAR融合蛋白分别表达两个载体转染免疫细胞获得的CAR-T。5HCD19-BBZ-P2A-SIRPγ-28是指是包含缺氧启动子的CAR融合蛋白共同表达一个载体转染免疫细胞获得的CAR-T。
本发明实施例中，PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28分别表达于两个载体转染免疫细胞获得的产品。PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28共同表达于一个载体转染免疫细胞获得的产品。
本发明实施例中采用磷酸钙法包装慢病毒的方法，具体为：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态，包装质粒(RRE:REV:2G)和表达质粒按一定比列加入到1.5的离心管中，加入CaCl2和2×HBS，混匀后室温静置后加入到处理好的293T细胞培养液中，3-5h后再次换液至10mL含10％FBS的DMEM培养基，48h或72h后收集细胞上清，纯化病毒。
本发明实施例中，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率和CAR表达强度。SIRPγ-28后有GFP标签，以GFP阳性率确定其表达情况。
本发明实施例中，检测不同CAR-T对靶细胞杀伤能力的方法为：利用ACEA xCELLigence RTCA MP仪器进行，实验步骤依据仪器说明书进行。ACEA xCELLigence RTCA MP原理为对附着于孔底的肿瘤细胞以电阻指数为数据每15分钟记录一次，通过电阻指数判断贴壁的靶细胞的增殖或者死亡情况。利用电阻指数分析结果公式为：CAR-T细胞杀伤率＝基线电阻指数-实时电阻指数。
本发明实施例中，IFN-γ检测采用BD IFN-γ试剂盒检测，实验步骤依据产品说明书进行；IL-2检测采用inritrogen IL-2试剂盒检测，实验步骤依据产品说明书进行；TNF-α检测采用Biolegend试剂 盒检测，实验步骤依据产品说明书进行。
本发明实施例中，验证缺氧模型是否构建完成的方法为：利用重组质粒病毒感染活化的PBMC构建体外缺氧细胞模型，培养12-18h后换液；然后利用CoCl 2诱导缺氧环境，培养至第N天通过检测CAR结构上轻链抗体检测CAR表达情况。
本发明实施例中，体内验证使用小鼠为NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl，简称NOG小鼠，由日本实验动物研究所(CIEA)的Mamoru Ito培育而成，为国际上CAR-T体内相关成瘤实验最常见品系。
本发明实施例中，体内缺氧模型验证的方法为：选择6-8周鼠龄雌性NOG小鼠，标记耳号后，在小鼠背部以1×10^6/只细胞量皮下注射靶细胞，成瘤第12天测量小鼠肿瘤体积。
第一部分
实施例1 质粒构建
基于如图1所示的CAR模式图，以SIRPγ及CD47全长的质粒、pL-CAG-2AGFP、pL-CAG-PD1-CD28-2ACherry、pL-CAG-PD1-BB-2Acherry为模板，构建如图2所示的CAR结构和对应的单靶点CAR结构。构建获得载体：CEAZ-PD1-28、CEAZ-PD1-BB、CEAZ-SIRPγ-BB、CEAZ-SIRPγ-28、CEABBZ-P2A-SIRPγ-28，CEABBZ-P2A-SIRPγ-BB，BCMA-BBZ-P2A-SIRPγ-28和单靶点CAR：CEAZ，PD1-28，PD1-BB，SIRPγ-28TM-28，SIRPγ-8TM-BB。通过酶切及测序比对验证后，结果如图3所示，重组质粒的酶切鉴定:1-3:pL-CAG-CEAZ-PD1-28质粒，依次为:原质粒，CEAZ(1371bp)，PD1-28(783bp)；4-6:pL-CAG-CEAZ-PD1-BB质粒，依次为:原质粒，CEAZ(1371bp)，PD1-BB(798bp)；7-9:pL-CAG-CEAZ-SIRPγ-28质粒，依次为:原质粒，CEAZ(1371bp)，SIRPγ-28(1374bp)；10-12:pL-CAG-CEAZ-SIRPγ-BB质粒，依次为:原质粒，CEAZ(1371bp)，SIRPγ-BB(1368bp)；M1:DL5000DNA分子量标准；M2:DL15000DNA分子量标准；13-15:pL-CAG-SIRPγ-28-2AGFP质粒，依次为:原质粒，SIRPγ-28(1292bp)，2AGFP(785bp)；16-18:pL-CAG-SIRPγ-BB-2AGFP质粒，依次为:原质粒，SIRPγ-BB(1286bp)，2AGFP(785bp)19-20:pL-CAG-CD47质粒，依次为:CD47(974bp)，原质粒；M3：DL5000DNA分子量标准；构建成功。
实施例2 靶细胞构建
利用磷酸钙法制备获得CEA，PD-L1，CD47抗原的病毒，分别感染CHO细胞构建CHO-CEA细胞、CHO-CEA-PD-L1细胞系和CHO-CEA-CD47细胞系。信号调节蛋白α(SIRPγ)是CD47的配体之一，可与CD47结合，因此可用CD47阳性靶细胞对CEAZ-SIRPγ-BB、CEAZ-SIRPγ-28、SIRPγ、SIRPγ-28，SIRPγ-BB进行评估。
三个细胞系经十次传代培养后检测其阳性率，结果如图4显示，CHO-CEA的阳性率为97.1％，CHO-CEA-CD47的双阳率为97.6％，CHO-CEA-PD-L1的双阳率为87％，符合实验需求，表明细胞系已构建成功，可作为靶细胞用于后续的CAR-T药效评价。
实施例3 制备慢病毒及感染T淋巴细胞
采用磷酸钙法包装慢病毒，获得实施例1中5个单表达的CAR(CEAZ，PD1-28，PD1-BB，SIRPγ-28，SIRPγ-BB)及6个新型免疫抑制抵抗型CAR(CEAZ-PD1-28，CEAZ-PD1-BB，CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB，CEABBZ-SIRPγ-28，CEABBZ-SIRPγ-BB)病毒颗粒。
利用梯度离心法进行淋巴细胞分离；离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有 10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC，在病毒感染的第12天检测CAR-T的阳性率，检测方法为流式检测，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率和CAR表达强度。
结果如图5a所示：免疫抑制破除融合蛋白可在T细胞表面表达成功，如图5b所示新型免疫抑制抵抗型CAR表达成功。
实施例4 新型免疫抑制抵抗型CAR-T体外功能验证
以未表达免疫抑制破除融合蛋白的Control T细胞作为对照，验证免疫抑制破除融合蛋白SIRPγ-28TM-28，SIRPγ-8TM-BB的功能。靶细胞为表达CD47的CHO细胞系。结果如图6a所示，SIRPγ-28TM-28和SIRPγ-8TM-BB融合蛋白在针对CD47阳性的靶细胞均有杀伤作用。
以CEAZ组作为阳性对照，Control-T组作为阴性对照，设置CEAZ-PD1-28，CEAZ-PD1-BB，CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB组为实验组，将CHO-CEA-CD47和CHO-CEA-PD-L1作为靶细胞，验证新型免疫抑制抵抗型CAR-T体外有效性。结果如图6b及下表2所示，CEAZ-PD1-28和CEAZ-SIRPγ-28组的细胞杀伤率显著高于CEAZ组，而CEAZ-PD1-BB和CEAZ-SIRPγ-BB组对靶细胞的杀伤对比CEAZ组无明显增强。此外，CEAZ-PD1-28，CEAZ-PD1-BB组在与CHO-CEA-CD47共培养情况下以及CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB组在与CHO-CEA-PD-L1共培养情况下杀伤效率较CEAZ组均无明显的差异，表明新型免疫抑制抵抗型CAR-T组具有杀伤特异性，且CEAZ-PD1-28和CEAZ-SIRPγ-28对靶细胞有较显著的杀伤效果。
表2 免疫抑制抵抗型CAR-T体外杀伤率
Figure PCTCN2021095107-appb-000001
以K562-BCMA为靶细胞，Control-T组作为阴性对照，验证BCMA-BBZ-P2A-SIRPγ-28的功能，结果显示BCMA-BBZ-P2A-SIRPγ-28可以显示很好的杀伤功能。
实施例5 新型免疫抑制抵抗型CAR-T对肿瘤抑制环境破除验证
CAR-T细胞在浸润肿瘤组织后往往受到肿瘤免疫抑制为环境影响，高表达PD1，LAG-3，Tim-3衰减分子，进而杀伤肿瘤细胞有效功能减弱，CART细胞自身凋亡增加。为了验证新型免疫抑制抵抗型CAR-T是否能够破除肿瘤抑制微环境信号，对CEAZ，CEA-28Z，CEA-BBZ，CEAZ-PD1-28，CEAZ-PD1-BB，CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB及Control-T组CAR-T细胞培养至Day 7时将CAR-T细胞与DLD-1-CEA-Luc-GFP细胞12孔细胞培养板中进行共培养，48h后收集CAR-T细胞，标记抗人CD3，PL，PD1，LAG-3，Tim-3流式抗体后进行流式检测，分析检测结果以评价其效应能力。由于CEAZ-PD1-28和CEAZ-PD1-BB组中外源表达PD1，因此这两组的PD1阳性率不用于评价CAR-T细胞的衰竭程度。衰竭分子检测结果如图7以及下 表3所示：在CD3+PL+下，CEAZ-SIRPγ-28的PD1，LAG-3，Tim-3的表达程度低于CEAZ组，表明CEAZ-SIRPγ-28经抗原刺激后衰竭程度较低。
表3 免疫抑制抵抗型CAR-T对肿瘤抑制环境破除验证情况
Figure PCTCN2021095107-appb-000002
同样的，BCMA-BBZ-P2A-SIRPγ-28经BCMA抗原刺激后也显示较低的衰竭分子表达。
实施例6 新型免疫抑制抵抗型CAR-T体内功能验证
体内验证使用小鼠为NCG小鼠。选取30只NCG小鼠，并注DLD-1-CEA-Luc-GFP(CEA，PD-L1，CD47三阳性表达，如图8a所示)进行荷瘤。肿瘤生长到如绿豆大小可测量时，测量肿瘤大小，在实验过程中，实验组小鼠出现萎靡濒死、半身或全身瘫痪、体重丢失20％(与实验开始前比较)、肿瘤体积≥1500mm3任一情况时终止实验。
荷瘤后Day 5测量小鼠肿瘤荧光值，以活体成像荧光值进行随机分组，保证各组小鼠体重和荧光度值无显著差异，计算体重平均值。Day 6回输CAR-T细胞，体积为100μL(含有效CAR-T细胞数量3×10 6)，给予相同总细胞数的未转染的T细胞作为对照组。NCG小鼠的肿瘤体积测量数据绘制肿瘤体积增长曲线，发现Day 27-30，CEAZ-SIRPγ-28对肿瘤有较为明显的抑制效果，结果如图8b所示。
进一步，验证了二代CAR与融合蛋白联合设计的免疫抑制型CAR即CEABBZ-P2A-SIRPγ-28结构的体内有效性，同样采用NCG小鼠注射DLD-1-CEA-Luc-GFP细胞荷瘤，荷瘤13天后进行CAR-T细胞回输，总细胞数8×10 6。结果如图9所示，本发明的免疫抑制型CAR-T(CEABBZ-P2A-SIRPγ-28)能够很好的在体内发挥功能，效果明显优于对照组二代CAR结构的CEABBZ。
第二部分 靶向CEA的CAR结构(其中CAR1氨基酸序列为27)的实验部分
实施例7 靶向CEA的质粒构建
(1)实验组质粒构建
由迷你启动子miniCMV合成缺氧启动序列5HRE-CMVmini promoter，其核苷酸序列如SEQ ID NO：1所示。然后由5HRE-CMVmini promoter、慢病毒表达载体、CEAScFv-CD8铰链区-CD8跨膜区-CD137-CD3ξ-P2A-SIRPγ-CD28(5HCEA-BBZ-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆，构建靶向CEA的CAR-T细胞制剂的含有SIRPγ融合蛋白的重组质粒PBKL1-5H1P-CEA-OPRE(SIRPγ融合蛋白)，该步骤中，使用了CAR和融合蛋白共同表达于一个载体转染免疫细胞或者CAR和融合蛋白共同分别表达于一个载体转染免疫细胞两种方法来获得我们的含有融合蛋白的CAR的结构，其中CAR和融合蛋白共同分别表达于一个载体转染免疫细胞获得的产品用缩写 “5HCEA-BBZ+SIRPγ-28”表示。
(2)对照组质粒构建
按照实施例1中(1)的方法构建5HCEA-BBZ-8H-8。
实施例8 靶向CEA的质粒体外功能模型验证
分别设置5HCEA-BBZ-8H-8、5HCEA-BBZ-P2A-SIRPγ-28、SIRPγ-28加CoCl 2为实验组验证缺氧模型，结果如图10和图11所示，平均荧光强度和阳性率5HCEA-BBZ-8H-8、5HCEA-BBZ-P2A-SIRPγ-28、SIRPγ-28相近，如图12所示，5HCEA-BBZ-P2A-SIRPγ-28扩增倍数更具有优势。
实施例9 靶向CEA的CAR-T有效性验证
(1)靶细胞DLDL1-CEA的杀伤效率
分别以CEA阳性的DLD1-CEA和DLD1-CEA(CD47-)细胞为靶细胞。将效应细胞(常规CAR-T细胞和含缺氧启动子CAR表达的CAR-T细胞)缺氧处理后，按照1：1的效靶比铺于靶细胞中，检测不同CAR-T对靶细胞杀伤能力。
加入CAR-T的24h后，各组CAR-T对靶细胞DLDL1-CEA的杀伤效率结果如图13以及下表4所示，5HCEA-BBZ-8H-8、5HCEA-BBZ+SIRPγ-28和5HCEA-BBZ-P2A-SIRPγ-28具有较强杀伤功能、SIRPγ-28组也具有杀伤功能。
表4 靶向CEA的各组CAR-T对靶细胞DLDL1-CEA的杀伤效率
结构 Specific Lysis(％)
5HCEA-BBZ+SIRPγ-28 84.4752
5HCEA-BBZ-P2A-SIRPγ-28 99.4934
5HCEA-BBZ-8H-8 99.7738
SIRPγ 45.2293
Control T 29.721
Medium 0
(2)IFN-γ、IL-2、TNF-α分泌检测
接着(1)，在杀伤24小时后收集细胞上清，进行CAR-T细胞受到靶细胞刺激后IFN-γ、IL-2、TNF-α分泌能力的检测。收集的上清，使用试剂盒利用ELISA方法检测IFN-γ和IL-2的分泌情况。
结果如图14、图15、图16所示，靶细胞为DLD1-CEA时，5HCEA-BBZ-8H-8分泌IFN-γ、IL-2、TNF-α较少，5HCEA-BBZ-P2A-SIRPγ-28分泌IFN-γ、IL-2、TNF-α远高于5HCEA-BBZ-8H-8组，靶细胞为DLD1-CEA(CD47-)细胞，IFN-γ、IL-2、TNF-α较低或达不到检测线。本发明5HCEA-BBZ-P2A-SIRPγ-28更有利于CAR-T的增值和杀伤肿瘤相关的因子分泌，说明其确实能够提高CAR-T的有效性。
实施例8 含有SIRPγ融合蛋白的质粒体内功能验证
体内验证使用的成瘤靶细胞选择DLD1-CEA-Luc-GFP细胞，构建人CEA+实体瘤荷瘤模型。
根据肿瘤体积随机分为Control T(CT)组、5HCEA-BBZ-8H-8、5HCEA-BBZ-P2A-SIRPγ-28,对照组为Control T组。在成瘤第12天向不同分组小鼠尾静脉注射对应的CAR-T细胞1*10^7Copies/只；Control T组于第812天回输总数相同的T淋巴细胞。每三天测量一次各组小鼠肿瘤体积，实验结果如图17和图18所示，可见进行5HCEA-BBZ-8H-8相较于5HCEA-BBZ-P2A-SIRPγ-28组小鼠体内有效性显著提高，5HCEA-BBZ-8H-8组对肿瘤也具有明显的消除作用。
第三部分 CD19靶点的CAR结构的实验部分
实施例9 靶向CD19质粒构建
以SIRPγ及CD47全长的质粒、pL-CAG-2AGFP、pL-CAG-PD1-CD28-2ACherry、pL-CAG-PD1-BB-2Acherry为模板，CD19靶点CAR结构。构建获得载体：SIRPγ-28、5HCD19-BBZ、5HCD19-BBZ-SIRPγ-28。通过测序比对验证后，结构建成功。
实施例10 制备慢病毒及感染T淋巴细胞
采用磷酸钙法包装慢病毒，获得实施例1中3个病毒颗粒(SIRPγ-28、5HCD19-BBZ、5HCD19-BBZ-P2A-SIRPγ-28)。
利用梯度离心法进行淋巴细胞分离，离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC，在病毒感染的第8天利用流式检测CAR-T的阳性率，结果如图19以及下表5所示。
表5 流式检测靶向CD19的各实验组CAR-T的阳性率
结构 ％of CD3+T Cells
Control T 0.26
SIRPγ-28 51.25
5HCD19-BBZ 53.15
5HCD19-BBZ-P2A-SIRPγ-28 54.89
SIRPγ-28+5HCD19-BBZ 18.04
实施例11 靶向CD19的体外药效学评价
以Control T为对照组，实验组设置SIRPγ-28组、5HCD19-BBZ组、5HCD19-BBZ-P2A-SIRPγ-28组，以Nam6-Luc-GFP(CD19阳性)、K562-Luc-GFP(CD19阴性)为靶细胞，通过体外杀伤和体外因子分泌验证体外有效性。结果如图20及下表6所示，采用一个共表达的载体转染免疫细胞获得的产品(5HCD19-BBZ-P2A-SIRPγ-28)与采用两个分别表达的载体共转染免疫细胞获得的产品(SIRPγ-28+5HCD19-BBZ)的体外杀伤显著高于SIRPγ-28组和5HCD19-BBZ组，对阴性细胞无杀伤。
和图21及表7所示,采用一个共表达的载体转染免疫细胞获得的产品(5HCD19-BBZ-P2A-SIRPγ-28)与采用两个分别表达的载体共转染免疫细胞获得的产品(SIRPγ-28+5HCD19-BBZ)的因子分泌显著高于高于SIRPγ-28组和5HCD19-BBZ组。
表6 靶向CEA的各实验组细胞杀伤情况
结构 Specific Lysis(％)
Control T 37.2867
SIRPγ-28 28.6995
5HCD19-BBZ 84.3876
5HCD19-BBZ-P2A-SIRPγ-28 97.5187
SIRPγ-28+5HCD19-BBZ 97.4608
表7 靶向CEA的各实验组IFN-γ因子分泌情况
结构 IFN-γ(pg/ml)
Control T 135
SIRPγ-28 453.97
5HCD19-BBZ 17296.67
5HCD19-BBZ-P2A-SIRPγ-28 21820
SIRPγ-28+5HCD19-BBZ 14363.33
实施例12 靶向CD19的体内药效学评价
选用NCG小鼠(雌性，6周龄)，以1×10 6Cells/只剂量皮下注射(s.c.)Nalm6-Luc-GFP细胞建立体内荷瘤模型，荷瘤后8d以1×10 7CAR-T Cells/只剂量尾静脉注射(i.v.)给予不同组别(Control T、5HCD19-BBZ、5HCD19-BBZ-P2A-SIRPγ-28)CAR-T。通过活体成像观察肿瘤体内生长情况，体内评价不同CAR-T对淋巴瘤治疗效果。结果如图22和图23所示，相比Control T组和5HCD19-BBZ组，5HCD19-BBZ-P2A-SIRPγ-28体内抗肿瘤效果明显，能明显清除肿瘤。
第四部分 PSCA靶点的CAR结构的实验部分
实施例13 靶向PSCA的质粒构建及感染T细胞
(1)质粒构建
慢病毒表达载体、PSCA ScFv-G4H铰链区-CD28跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-28(PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆。
慢病毒表达载体、PSCA ScFv-7H铰链区-CD28跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-28(PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆。
(2)感染T细胞
将获得的质粒感染T细胞，得CAR-T细胞。
实施例14 靶向PSCA的IFN-γ因子分泌情况
以Control T为对照组，实验组分别设置PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-G4H-28TM+SIRPγ-28、PSCA-28BBZ-7H-28TM+SIRPγ-28、PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-7H-28TM，以RT4-Luc-GFP(PSCA阳性)为靶细胞，通过体外因子分泌验证体外有效性。结果如表8-表13及图24-图29所示。采用一个共表达的载体转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28与采用两个分别表达的载体共转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28的IFN-γ因子分泌显著高于对照组和PSCA-28BBZ-G4H-28TM组或PSCA-28BBZ-7H-28TM组，对阴性细胞无杀伤
表8 PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 4060.33
Control T 234.23
表9 PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 4024.67
Control T 234.23
表10 PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33
Control T 234.23
表11 PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67
Control T 234.23
表12 PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM 6998.67
PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33
Control T 234.23
表13 PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM 6660.33
PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67
Control T 234.23
实施例14 靶向PSCA的细胞杀伤情况
实验组分别设置PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-G4H-28TM+SIRPγ-28、PSCA-28BBZ-7H-28TM+SIRPγ-28、PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-7H-28TM，分别以RT4-Luc-GFP(PSCA阳性)、PC-3-Luc-GFP(PSCA阴性)为靶细胞，结果如表14-表19及图30-图35所示。采用一个共表达的载体转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28与采用两个分别表达的载体共转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28的体外杀伤显著高于对照组和PSCA-28BBZ-G4H-28TM组或PSCA-28BBZ-7H-28TM组，对阴性细胞无杀伤。
表14 PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure PCTCN2021095107-appb-000003
表15 PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure PCTCN2021095107-appb-000004
表16 PSCA-28BBZ-G4H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure PCTCN2021095107-appb-000005
表17 PSCA-28BBZ-7H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure PCTCN2021095107-appb-000006
表18 PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28中细胞杀伤情况
Figure PCTCN2021095107-appb-000007
表19 PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28中细胞杀伤情况
Figure PCTCN2021095107-appb-000008
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (32)

  1. 一种逆转肿瘤微环境的融合蛋白，其特征在于，所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段，跨膜结构和胞内信号区。
  2. 根据权利要求1的融合蛋白，其特征在于，跨膜结构来源于人CD28跨膜区或人CD8来源跨膜区，优选地，所述胞内信号区来源于CD28或4-1BB。
  3. 根据权利要求2所述的融合蛋白，其特征在于，所述跨膜结构氨基酸序列如SEQ ID NO:7或SEQ ID NO:8所示；优选地，所述胞内信号区来源于CD28，序列如SEQ ID NO:9或SEQ ID NO:38所示。
  4. 根据权利要求1所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白的结构为SIRPγ-CD28TM-CD28或SIRPγ-CD8TM-4-1BB。
  5. 根据权利要求1所述的融合蛋白，其特征在于，所述SIRPγ胞外段氨基酸序列如SEQ ID NO:1或其功能性变体所示。
  6. 根据权利要求4所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的氨基酸序列为SEQ ID NO:2或其功能性变体所示。
  7. 根据权利要求4所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的氨基酸序列为SEQ ID NO:3或其功能性变体所示。
  8. 根据权利要求6所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白为SIRPγ-CD28TM-CD28的核苷酸序列如SEQ ID NO:13所示。
  9. 根据权利要求7所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的核苷酸序列如SEQ ID NO:14所示。
  10. 包含权利要求1-9任一项所述的融合蛋白的表达载体。
  11. 根据权利要求10所述的表达载体，其特征在于，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
  12. 包含权利要求10-11任一项所述的表达载体的免疫细胞。
  13. 根据权利要求12所述的免疫细胞，其特征在于，所述免疫细胞为T细胞、T细胞前体或NK细胞。
  14. 根据权利要求13所述的免疫细胞，其特征在于，包含具有识别肿瘤抗原的嵌合抗原受体结构，所述嵌合抗原受体包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区，所述肿瘤抗原包含但不限于PSCA、PSMA、CD19、BCMA、CD123、CD20、CD22、CEA、EGFR、EGFRVIII、GPC3、5T4、CD33、Her2、GD2、CD70、CLL-1、Trop2、CD47、GPC3、CLND18.2、CD133、CS1、CD155、CD30、ROR1、MUC1、IL13RAα2或mesothelin等可以作为肿瘤靶向性识别的抗原分子。
  15. 一种肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR包含权利要求1-9任一项所述的融合蛋白和CAR1，所述CAR1包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区。
  16. 根据权利要求15所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述融合蛋白通过多顺反子结构与CAR1连接，所述多顺反子结构为自剪切多肽或内部核糖体进入位点IRES，所述自剪切多肽为T2A、P2A、E2A或F2A。
  17. 根据权利要求15所述新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ融合蛋白或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ融 合蛋白。
  18. 根据权利要求15所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28。
  19. 根据权利要求15所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，CAR1结构：hinge的氨基酸序列如SEQ ID NO:24或其功能性变体所示，TM的氨基酸序列如SEQ ID NO:7或SEQ ID NO:8，CD3ζ的氨基酸序列如SEQ ID NO:11或其功能性变体所示；融合蛋白结构：SIRPγSIRPγ胞外段氨基酸序列如SEQ ID NO:1所示或功能性变体；来源于人CD28的跨膜区氨基酸序列如SEQ ID NO:7所示；来源于人CD28的胞内信号区氨基酸序列如SEQ ID NO:9所示。
  20. 根据权利要求17或18或19所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述ScFv可以识别CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70、CD47中的任一种或多种。
  21. 根据权利要求20所述的CAR，其特征在于，所述ScFv的氨基酸序列如SEQ ID NO:25所示或其功能性变体。
  22. 根据权利要求15或16所述的肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR1包含以下几种的一种，
    a)CAR1包含包括CEA单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：26所示或其功能性变体；
    或b)所述CAR1包含CD19单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：27所示或其功能性变体；
    或c)所述CAR1包含PSCA单链抗体、铰链区、CD28跨膜区、CD28、CD137和CD3ξ三刺激信号；所述铰链区为G4H或7H；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：28所示或其功能性变体；
    或如SEQ ID NO：29所示或其功能性变体。
  23. 根据权利要求22所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述a)和b)的CAR1结构中还包括缺氧启动子，所述缺氧可调控启动子的核酸序列包含如SEQ ID NO：30所示的序列。
  24. 核酸序列，其特征在于，编码权利要求21或22所述的肿瘤免疫抑制抵抗型CAR，所述核酸序列包含如SEQ ID NO：31或SEQ ID NO：32或SEQ ID NO：33或SEQ ID NO：34或SEQ ID NO：35所示的序列。
  25. 包含权利要求15-23任一项所述的肿瘤免疫抑制抵抗型CAR的表达载体,其特征在于，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
  26. 包含权利要求25所述的表达载体的免疫细胞。
  27. 根据权利要求26所述的免疫细胞，其特征在于，所述免疫细胞为T细胞、T细胞前体或NK细胞。
  28. 一种制备如权利要求14所述的免疫细胞的方法，其特征在于,将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的 CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
  29. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-9任一项所述的融合蛋白或权利要求15-23任一所述的肿瘤免疫抑制抵抗型CAR或权利要求12-14任一项所述的免疫细胞或26-27任一所述的免疫细胞或权利要求10-11任一项所述的表达载体或权利要求25所述的表达载体或权利要求24所述的核酸序列。
  30. 根据权利要求19所述的药物组合物，其特征在于，所述药物组合物还包括可增强CAR表达活性的活性剂和/或治疗剂。
  31. 权利要求29或30所述的药物组合物在制备肿瘤药物中的应用。
  32. 根据权利要求31所述的应用，其特征在于，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、前列腺癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌，所述肿瘤高表达CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70、CD47中的任一种或多种。
PCT/CN2021/095107 2020-05-22 2021-05-21 逆转肿瘤微环境的融合蛋白及其应用 WO2021233411A1 (zh)

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