WO2021136538A1 - Cellule cart améliorée favorisant l'infiltration dans des tumeurs solides, procédé de préparation associé et médicament cellulaire correspondant - Google Patents

Cellule cart améliorée favorisant l'infiltration dans des tumeurs solides, procédé de préparation associé et médicament cellulaire correspondant Download PDF

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WO2021136538A1
WO2021136538A1 PCT/CN2020/142571 CN2020142571W WO2021136538A1 WO 2021136538 A1 WO2021136538 A1 WO 2021136538A1 CN 2020142571 W CN2020142571 W CN 2020142571W WO 2021136538 A1 WO2021136538 A1 WO 2021136538A1
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light
cells
cancer
nucleic acid
cart
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张娜
杜冰
刘小红
吴诗佳
席在喜
刘明耀
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华东师范大学
上海邦耀生物科技有限公司
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Definitions

  • the present disclosure relates to the technical field of CART cells, in particular, to an enhanced CART cell that promotes solid tumor infiltration, a preparation method thereof, and a cell medicine.
  • CD-19-specific CAR-T cells can have complete remission in the treatment of relapsed and refractory acute lymphoblastic leukemia.
  • the main treatment process is: first isolate the patient’s own T cells (or T cells from allogeneic donors), and then activate and genetically modify them to obtain chimeric antigen receptor T cells. CAR-T), and finally returned to the patient's body.
  • the chimeric antigen receptor consists of an extracellular antigen recognition domain (usually an antibody single-chain variable fragment scFv) and an intracellular signal transduction domain (the CD3 ⁇ chain of the T cell receptor, or the simultaneous introduction of one or more co-stimulatory signals such as CD28 and 4-1BB) are connected together, and its extracellular part can make T cells have the ability to recognize specific antigens. Able to overcome the MHC restriction and directly bind to the antigen recognized by it, it will stimulate the proliferation of T cells through the signal transduction domain, activate the cytotoxicity of T cells and promote the secretion of cytokines, and finally eliminate the cells with the antigen. Better specificity and persistence.
  • an extracellular antigen recognition domain usually an antibody single-chain variable fragment scFv
  • an intracellular signal transduction domain the CD3 ⁇ chain of the T cell receptor, or the simultaneous introduction of one or more co-stimulatory signals such as CD28 and 4-1BB
  • CAR-T cells are effective in the treatment of hematomas, in the application of solid tumors, there are challenges such as less infiltration of CART cells, low persistence, and the weakening of CART function by the tumor immunosuppressive microenvironment, which makes the traditional second-generation CAR- The therapeutic effect of T in clinical trials of solid tumors is not ideal.
  • the ratio of survival and proliferation and memory T cells can also significantly enhance the infiltration of CART cells into the tumor microenvironment, and can also activate the functions of other non-CART cells, such as promoting the migration of DC and NK cells to the tumor site, and promoting NK Amplification, promoting the maturation of DC, enhancing the secretion of tumor-related fibroblast chemokines (such as CCL19, CCL21), etc., to fully mobilize the anti-tumor immune response of non-CART cells, so as to achieve the functional amplification of enhanced CART.
  • it has multiple functions of promoting infiltration and mobilizing the patient's own immune cells to respond to tumors, and has more practical value in clinical applications.
  • the purpose of the present disclosure includes, but is not limited to, providing an enhanced CART cell that promotes infiltration of solid tumors, and a preparation method and cytomedicine thereof.
  • the CART cells provided in the present disclosure have higher infiltration capacity for tumor tissues of solid tumors, and improve With its ability to kill tumor cells, it has broad clinical application prospects, and provides a new treatment idea and strategy for using CART cells to treat solid tumors.
  • embodiments of the present disclosure provide an enhanced CART that promotes the infiltration of CART cells into solid tumors, the CART cells containing a first nucleic acid sequence and a second nucleic acid sequence;
  • the first nucleic acid sequence contains a first coding sequence encoding a chimeric antigen receptor, and the antigen binding domain of the chimeric antigen receptor can target the solid tumor;
  • the second nucleic acid sequence contains a second coding sequence encoding a LIGHT protein and a signal peptide coding sequence located upstream of the second coding sequence;
  • the expression of the second coding sequence is driven by a constitutive promoter.
  • HEV high endothelial venules
  • Carcinoma-associated fibroblasts can also secrete TGF- Cytokines such as ⁇ inhibit T cell function.
  • LIGHT co-expressed with CAR can improve and activate the tumor microenvironment and promote the formation of HEV, thereby promoting the intratumor infiltration of CART cells and other immune cells such as DC and NK, and can also activate CAF and other cells to release more chemotaxis Factors such as CCL19, CCL21, etc., under the action of these chemokines, more CCR7-positive cell populations (such as DC, NK and naive T cells, etc.) can be recruited to infiltrate the tumor microenvironment, thereby increasing the killing of tumors , Amplify the role of anti-tumor immunity.
  • DC cells discover new antigens and present them to the cell surface through MHC. By forming immune protrusions with T cells, they complete the activation signal transmission of T cells, thereby activating adaptive immunity, which is an immune response. Starter.
  • the presentation event mainly occurred in secondary lymphatic organs, including lymph nodes (LN), spleen and Peyer's lymph nodes.
  • the antigen presentation process is summarized as: the establishment of cellular immune synapses between T cells and DCs occurs through the interaction of T cell receptors (TCR) with peptide-major histocompatibility (MHC) complexes present on the surface of DCs ( signal 1).
  • TCR T cell receptors
  • MHC peptide-major histocompatibility
  • T cell-DC contact also involves the binding of different receptors and ligands, including the binding of costimulatory molecules and adhesion molecules (signal 2) and the secretion of cytokines (signal 3). These three integrated signals jointly drive the full activation and differentiation of T cells, so DC is very important in the immune regulatory network.
  • Keishi Adachi et al. Co-expression of IL7 and CCL19 through CART enhanced the infiltration of DC and T cells in tumors, allowing more DCs and T cells to form immune synapses and signal transmission in the tumor microenvironment, thereby enhancing CART cells and DCs Cells infiltrate in solid tumors and at the same time mobilize their own anti-tumor immunity (Adachi, K., et al.
  • Tertiary lymphatic structure is an ectopic lymphoid organ formed in non-lymphoid tissues such as chronic inflammation and tumors. It is composed of T cells, B cells, dendritic cells, and other cells. Studies have reported that tertiary lymphatic structure and B Cells are positively correlated with patient prognosis and response to immunotherapy. The tertiary lymphatic structure in the tumor microenvironment can also interact with DC-T cells and maintain the memory of T cells. Inducing the formation of more tertiary lymphatic structures in the tumor can improve the tumor's response to immunotherapy.
  • LIGHT protein (homologous to lymphotoxins, inducible, competes With HSV glycoprotein D for HVEM, expressed by T Lymphocyte, TNFSF14) is a member of the TNF superfamily of cytokines, and is an inducible inflammatory cytokine.
  • the CART cells provided in the embodiments of the present disclosure use a constitutive promoter to drive the continuous secretion and expression of LIGHT protein in large quantities, that is, overexpress the LIGHT protein.
  • the CART cells provided in the embodiments of the present disclosure are used to contact tumor cells, these secreted and expressed LIGHT proteins, On the one hand, it can be used as a costimulatory molecule of T cells to enhance the immune function of T cells.
  • CART cells can normalize the abnormal blood vessels of the tumor and activate the tumor microenvironment (expressing favorable chemokines and adhesion molecules), so that the infiltration ability of CART cells, DC, NK and other immune cells to solid tumors can be effectively improved, and TLS in tumors can be induced
  • the formation of CART cells further improves the ability of CART cells to kill tumor cells.
  • the CART cells have broad clinical application prospects.
  • the present disclosure provides a new treatment idea and strategy for using CART cells to treat solid tumors.
  • the LIGHT protein is a secreted LIGHT protein.
  • the second nucleic acid sequence is located downstream of the first nucleic acid sequence
  • the constitutive promoter is located upstream of the first nucleic acid sequence
  • the first coding sequence and the second coding sequence are The sequences are all driven and expressed by the constitutive promoter.
  • Placing the first coding sequence and the second coding sequence under the same constitutive promoter to be driven by the expression, and placing the first nucleic acid sequence and the second nucleic acid sequence together, that is, on the same DNA strand, can reduce the overall nucleic acid sequence Length reduces the genome load of CART cells and facilitates the transformation of CART cells.
  • the first coding sequence and the second coding sequence can be expressed by two independent promoters respectively; or even the first nucleic acid sequence and the second nucleic acid sequence are placed on different DNA strands.
  • CART cells that are on or placed on the same DNA strand but have other nucleic acid sequences between the first nucleic acid sequence and the second nucleic acid sequence are also within the protection scope of the present disclosure.
  • the constitutive promoter is selected from the group consisting of elongation growth factor-1 ⁇ (EF-1 ⁇ ), early cytomegalovirus (CMV) promoter sequence, simian virus 40 (SV40) early promoter, mouse Breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Lu Any one of the sarcoma virus promoter, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.
  • EF-1 ⁇ elongation growth factor-1 ⁇
  • CMV early cytomegalovirus
  • SV40 simian virus 40
  • MMTV mouse Breast cancer virus
  • HSV human immunodeficiency virus
  • LTR human immunodeficiency virus
  • MoMuLV promoter MoMuLV promoter
  • avian leukemia virus promoter Epstein-Barr virus immediate
  • constitutive promoter of the present disclosure is not limited to the above-mentioned promoters, and the use of other types of promoters to drive the secretory expression of the secreted LIGHT protein also falls within the protection scope of the present disclosure.
  • the constitutive promoter is EF-1 ⁇ .
  • the constitutive promoter of the present disclosure can be selected according to actual needs, and it includes but is not limited to EF-1 ⁇ , as long as it can drive the secretory expression of the secreted LIGHT protein in large quantities.
  • mass expression here means that under the drive of the constitutive promoter, the amount of secreted LIGHT protein secreted and expressed by CART cells is higher than the amount of expression without the drive of the constitutive promoter.
  • the secreted LIGHT protein is an extracellular segment of the full-length LIGHT protein or a functional fragment thereof. This extracellular segment is an active segment with secretory characteristics.
  • amino acid sequence of the secreted LIGHT protein is shown in SEQ ID NO.15.
  • the secretory LIGHT (SEQ ID NO.15) lacking intracellular and transmembrane regions still has normal biological functions.
  • the secreted LIGHT protein is fused with a VTP polypeptide or a tumor targeting polypeptide. That is, the second coding sequence encodes LIGHT protein and VTP polypeptide or tumor targeting polypeptide.
  • amino acid sequence of the VTP polypeptide is shown in SEQ ID NO.17.
  • VTP polypeptides can target abnormal blood vessels in tumors. Through the fusion of VTP polypeptides, they can play a role in targeted transport, bringing the LIGHT protein to abnormal blood vessels in tumors, thereby promoting the normalization of abnormal blood vessels in tumors and promoting the formation of lymphoid structures in tumor sites. Guide CART cells to gather and activate here, mobilize autoimmune function, and reshape the inhibitory effect of tumor microenvironment, thereby changing the effect limitations of CART cells due to the inability to penetrate tumor tissues and immunosuppression, and enhance CAR-T cells against solid tumors The activity of CART cells greatly improves the effect of CART cells in the treatment of solid tumors.
  • Tumor-targeting polypeptides are a type of polypeptide that specifically binds to tumor surface markers. They can regulate the tumor microenvironment and transport tumor drugs. Through fusion with secretory LIGHT, LIGHT can be specifically delivered to the tumor microenvironment.
  • the fusion protein of LIGHT protein and VTP polypeptide has a secretory signal peptide.
  • the signal peptide encoded by the signal peptide coding sequence is selected from a membrane-integrated signal peptide or a secreted signal peptide.
  • the membrane-integrated signal peptide is selected from any one of CD8 signal peptide, CD28 signal peptide, GM-CSF signal peptide, CD4 signal peptide and CD137 signal peptide.
  • the secretory signal peptide is selected from any one of IgG, IgK signal peptide and cytokine signal peptide.
  • the base sequence of the signal peptide coding sequence is shown in SEQ ID NO.13.
  • the chimeric antigen receptor also has a transmembrane domain and a costimulatory signal transduction region.
  • the transmembrane domain is selected from the transmembrane domain of at least one of the following protein molecules: CD5, CD28, CD137, CD3 ⁇ , CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134 and CD8 ⁇ .
  • the transmembrane domain is a CD8 ⁇ transmembrane domain.
  • the costimulatory signal transduction region comprises an intracellular domain of at least one of the following costimulatory molecules: OX40, CD3 ⁇ , CD3 ⁇ , CD134, CD5, CD79a, CD137, ICD3 ⁇ , CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, COS, 4-1BB and CD3 ⁇ .
  • the costimulatory signal transduction area includes the intracellular costimulatory element of 4-1BB and the intracellular domain of CD3 ⁇ .
  • the solid tumor is selected from the group consisting of liver cancer, head and neck cancer, melanoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, and osteosarcoma Tumor, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma and Any kind of breast cancer.
  • the solid tumor is prostate cancer
  • the solid tumors of the present disclosure include, but are not limited to, liver cancer, head and neck cancer, melanoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma Tumor, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma and Breast cancer, for other types of solid tumors, also belongs to the protection scope of the present disclosure.
  • the antigen-binding domain can target specific membrane antigens of prostate cancer, and the antigen-binding domain is selected from any one of Fab, Fab', F(ab')2 and scFv Kind.
  • the antigen binding domain is scFv.
  • antigen-binding domains in the examples of the present disclosure can be selected according to the type of solid tumor to be treated. No matter which scFv is used as the antigen-binding domain, it belongs to the protection scope of the present disclosure.
  • amino acid sequence of the light chain variable region of the antigen-binding domain is shown in SEQ ID NO. 3
  • amino acid sequence of the heavy chain variable region of the antigen-binding domain is shown in SEQ ID.
  • amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is shown in SEQ ID NO. 5.
  • the antigen binding domain composed of SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7 has a prostate-specific membrane antigen and can target prostate cancer.
  • first nucleic acid sequence and second nucleic acid sequence only need to encode the corresponding protein, and there is no particular limitation. On the basis of a clear protein sequence, those skilled in the art can easily obtain the protein encoding the protein. Therefore, no matter how the specific nucleotide sequences of the first nucleic acid sequence and the second nucleic acid sequence change, as long as they can encode the respective corresponding proteins mentioned above, they belong to the protection scope of the present disclosure.
  • the embodiments of the present disclosure provide a method for preparing the enhanced CART cell that promotes solid tumor infiltration as described in any one of the foregoing embodiments, which comprises: making a target T cell contain the first nucleic acid sequence and the first nucleic acid sequence. Two nucleic acid sequence.
  • embodiments of the present disclosure provide a cellular medicine for treating solid tumors, which contains the enhanced CART cells that promote solid tumor infiltration as described in any one of the foregoing embodiments as an active ingredient.
  • the CART cells provided in the present disclosure can not only be used alone as active drugs to treat solid tumors, but also can be combined with other active drugs to treat solid tumors. Therefore, the CART cells provided in the present disclosure can be combined with other anti-tumor drugs. The combined use also belongs to the protection scope of the present disclosure.
  • embodiments of the present disclosure provide a treatment method for treating tumors, which includes: administering to a subject in need of treatment a therapeutically effective amount of a cellular drug as described above.
  • the tumor suffered by the subject is a solid tumor.
  • the solid tumor is selected from the group consisting of liver cancer, head and neck cancer, melanoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, and osteosarcoma Tumor, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma and Any kind of breast cancer.
  • the solid tumor is prostate cancer.
  • the subject is a human.
  • a chimeric antigen receptor (CAR) construct is provided, the structure of the CAR construct is shown in the following formula I or II,
  • Each "-" is independently a connecting peptide or a peptide bond
  • X is a CAR that targets tumor antigens
  • A is a self-shearing element
  • E is LIGHT fusion protein.
  • the structure of the CAR construct is as shown in Formula I.
  • the LIGHT fusion protein comprises a secreted signal peptide and a LIGHT protein.
  • the LIGHT protein includes LIGHT full-length protein or functional fragments thereof;
  • the functional fragment includes the extracellular segment of LIGHT protein
  • the extracellular segment is an active fragment with secretory characteristics
  • amino acid sequence of the LIGHT protein is shown in SEQ ID NO.15.
  • the LIGHT fusion protein further comprises a targeting polypeptide, and the targeting polypeptide targets the tumor microenvironment, tumor blood vessels or tumor cells.
  • the structure of the LIGHT fusion protein is shown in the following formula III or IV,
  • Each "-" is independently a connecting peptide or a peptide bond
  • M is LIGHT protein
  • I is a flexible joint
  • V is a tumor-targeting polypeptide.
  • the LIGHT fusion protein is represented by formula III.
  • the targeting polypeptide includes a VTP polypeptide.
  • amino acid sequence of the VTP polypeptide is shown in SEQ ID NO.17.
  • the flexible linker is a connecting peptide.
  • the LIGHT protein and the targeting polypeptide are connected by a connecting peptide, and the amino acid sequence of the connecting peptide is GGG.
  • the secreted signal peptide is selected from any one of IgG, IgK signal peptide and cytokine signal peptide.
  • amino acid sequence of the LIGHT fusion protein is shown in SEQ ID NO.20.
  • the self-shearing element includes T2A and P2A.
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is no or signal peptide
  • scFv is an antibody single-chain variable region targeting tumor antigens
  • H is no hinge area
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ ;
  • the tumor antigen is selected from the following group: PSMA, mesothelin, Her2, CD19, GPC3, GD2, CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand , Or a combination thereof.
  • the scFv is a single-chain variable region of an antibody targeting PSMA.
  • the structure of the scFv is shown in the following formula A or B:
  • V H antibody heavy chain variable region V L is an antibody light chain variable region; and "-" connecting peptide or a peptide bond.
  • the structure of the antigen binding domain is shown in Formula I.
  • amino acid sequence of X (CAR) is shown in SEQ ID NO.22.
  • nucleic acid molecule encoding the CAR construct according to the fifth aspect of the present invention, or,
  • the nucleic acid molecule includes a first nucleic acid molecule encoding a tumor antigen-targeting CAR and a second nucleic acid molecule encoding a LIGHT fusion protein, wherein the definitions of the tumor antigen-targeting CAR and LIGHT fusion protein are as described above.
  • first nucleic acid molecule and the second nucleic acid molecule may be connected in series, or may exist independently.
  • a vector is provided, and the vector contains the nucleic acid molecule according to the sixth aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, adeno-associated virus vector (AAV), retroviral vector, transposon, or a combination thereof .
  • the vector is selected from the group consisting of plasmids and viral vectors.
  • the vector is in the form of virus particles.
  • the vector is a lentiviral vector.
  • the vector contains a constitutive promoter, and the first nucleic acid molecule and the second nucleic acid molecule are expressed by the constitutive promoter.
  • a host cell containing the vector according to the seventh aspect of the present invention or the nucleic acid molecule according to the sixth aspect of the present invention integrated into the chromosome or Express the CAR construct described in the fifth aspect of the present invention.
  • the host cell includes eukaryotic cells and prokaryotic cells.
  • the host cell includes Escherichia coli.
  • an engineered immune cell is provided, the immune cell expressing the CAR construct according to the fifth aspect of the present invention, or
  • the immune cells express a fusion protein of CAR and LIGHT targeting tumor antigens, and the definition of the fusion protein of CAR and LIGHT targeting tumor antigens is as described above.
  • the tumor antigen-targeting CAR is expressed on the cell membrane of the immune cell.
  • the LIGHT fusion protein is secreted and expressed.
  • the cell is an isolated cell, and/or the cell is a genetically engineered cell.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • the cells include T cells and NK cells.
  • the engineered immune cells may be chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).
  • CAR-T cells chimeric antigen receptor T cells
  • CAR-NK cells chimeric antigen receptor NK cells
  • a preparation which contains the CAR construct according to the fifth aspect of the present invention, the nucleic acid molecule according to the sixth aspect of the present invention, and the vector according to the seventh aspect of the present invention. , Or the immune cell of the ninth aspect of the present invention, and a pharmaceutically acceptable carrier.
  • the formulation is a liquid formulation.
  • the dosage form of the preparation is injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml .
  • the preparation also contains a second active ingredient for anti-tumor, preferably a second antibody or a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.
  • a CAR construct according to the fifth aspect of the present invention, a nucleic acid molecule according to the sixth aspect of the present invention, a vector according to the seventh aspect of the present invention, and a ninth aspect of the present invention.
  • the use of the immune cells described in this aspect or the preparation described in the tenth aspect of the present invention is used to prepare drugs or preparations for preventing and/or treating cancer or tumors.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B cell lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
  • NSCLC non
  • the tumor is prostate cancer.
  • kits for preparing the host cell according to the fourth aspect of the present invention contains a container and the sixth aspect of the present invention in the container. Nucleic acid molecule, or the vector according to the seventh aspect of the present invention.
  • the engineered immune cells are CAR-T cells or CAR-NK cells.
  • the method further includes the step of performing function and effectiveness testing on the obtained engineered immune cells.
  • a LIGHT fusion protein is provided.
  • the structure of the LIGHT fusion protein is shown in the following formula III or IV,
  • Each "-" is independently a connecting peptide or a peptide bond
  • M is LIGHT protein
  • I is a flexible joint
  • V is a tumor-targeting polypeptide.
  • the fifteenth aspect of the present invention there is provided a use of the LIGHT fusion protein according to the fourteenth aspect of the present invention for the preparation of a preparation for enhancing the tumor infiltration of CAR-T cells and/ Or recruit immune cells.
  • the preparation is used for CAR-T cell-based adoptive immunotherapy.
  • A is a schematic diagram of the structure of the first nucleic acid sequence
  • B is a schematic diagram of the structure of the connection of the first nucleic acid and the second nucleic acid.
  • Figure 2 is a schematic diagram of the structure of the CJ-PSMA-LIGHT-VTP plasmid vector backbone.
  • Figure 3 is a schematic diagram of the structure of the lentivirus packaging helper plasmid pMD2.G.
  • Figure 4 is a schematic diagram of the structure of the lentivirus packaging helper plasmid psPAX2.
  • Figure 5 shows the detection results of the positive rate of different viruses infecting 293T cells in Example 1.
  • Figure 6 shows the detection results of the CAR positive rate of different CARTs in Example 1.
  • Figure 7 shows the LIGHT expression levels of different CARTs in Example 1.
  • Figure 8 shows the results of the killing efficiency of different CARTs and the original data table under different target ratios.
  • Figure 9 shows the detection results of the secreted LIGHT content of different CARTs under the condition of E:T of 1:1.
  • Figure 10 is a table of the proliferation and original data of different CARTs in the same culture system.
  • Figure 11 shows the results of in vivo imaging of tumor size in mice after injection of PSMA-CART or PSMA-CART+hLIGHT.
  • Figure 12 shows the visual size of tumors in mice after injection of PSMA-CART or PSMA-CART+hLIGHT.
  • Figure 13 shows the results of in vivo imaging of tumor size in mice after injection of PBS or hLIGHT.
  • Figure 14 shows the comparison of the secretion volume of full-length transmembrane LIGHT and free secreted LIGHT.
  • Figure 14A shows the corresponding viral stock solutions of full-length LIGHT (Full length LIGHT) and free secreted LIGHT (Secreted LIGHT) under different gradients. CAR positive rate.
  • Figure 14B shows the secretion of LIGHT under different gradients.
  • Figure 15 shows the detection results of the CAR positive rate of different CARTs in Example 5.
  • Figure 16 shows the secretion of LIGHT, IFN ⁇ , and TNF ⁇ after co-incubation of different CARTs with tumor cells in Example 6
  • Figure 17 is a table of the proliferation and original data of different CARTs in the same culture system within two weeks.
  • Figure 18 shows the memory typing of different CARTs in the same culture system.
  • Figure 19 shows the results of the LIGHT-VTP-CART activating tumor microenvironment experiment.
  • A is the qPCR that LIGHT-VTP-CART and PC3-PSMA cells were incubated with 1:1 E:T and the supernatant activated CAF cells up-regulated CCL19 The result graph.
  • B is the result of qPCR of LIGHT-VTP-CART and PC3-PSMA cells co-incubated with E:T as 1:1 to activate CAF cells to up-regulate CCL21.
  • C is the result of qPCR of LIGHT-VTP-CART supernatant activated PC3 tumor cells up-regulating CCL19.
  • D is the qPCR result graph of LIGHT-VTP-CART supernatant activated PC3 tumor cells up-regulating CCL21.
  • E is the result of qPCR of LIGHT-VTP-CART supernatant activating C57 mouse bone marrow cells up-regulating CCL19.
  • F is the qPCR result of C57 mouse bone marrow cells upregulated by LIGHT-VTP-CART supernatant.
  • G is the qPCR result of LIGHT protein activating HUVEC cells to up-regulate CCL19.
  • H is the qPCR result of LIGHT protein activating HUVEC cells to up-regulate CCL21.
  • I is the qPCR result of HUVEC cells activated by LIGHT protein to up-regulate the adhesion molecule VCAM-1.
  • J is the multiple relationship of the number of T cells adhered to the surface of HUVEC cells after stimulating the supernatant of LIGHT-VTP-CART for 24 hours.
  • FIG. 20 is a migration experiment.
  • A is a schematic diagram of a migration experiment
  • B is the number of migration cells of PC3 cells to T cells after LIGHT protein stimulation
  • C is the number of cells that CAF cells migrate to T cells after LIGHT protein stimulation
  • D is the number of migrating cells of LIGHT protein to T cells (F12 is CAF cell blank medium)
  • E is the migration and killing of PC3-PSMA after LIGHT stimulates CAF+PC3-PSMA mixed cells
  • F is mouse bone marrow cells on different CARTs The number of migrating cells on T cells after clearing stimulation.
  • Figure 21 shows the enrichment analysis results of different CART RNA-seq differential genes.
  • Figure 22 shows the results of the NSG animal experiment.
  • A is a schematic diagram of the construction process of the PC3-PSMA subcutaneous tumor model, and B is the in vivo imaging result of the tumor size after different CART treatment in mice.
  • Figure 23 shows the results of the NSG animal experiment.
  • A is the expression of CCR7 in the body of the NSG mouse model after CART treatment
  • B is the immunofluorescence image of CART infiltration in the tumor tissue of the NSG mouse model after CART treatment
  • C is LIGHT-VTP-CART and LIGHT CART treatment result comparison chart
  • D is the treatment effect chart of LIGHT-VTP-CART in NSG bone metastasis model.
  • Figure 24 is a graph showing changes in tumor volume after LIGHT-OT-1 treatment of B16F10-OVA mouse model.
  • Figure 25 is an immunofluorescence staining image of tumor tissue after LIGHT-OT-1 treatment of B16F10-OVA mouse model.
  • Figure 26 shows the results of NK expansion (A), CD3 infiltration (B), and NK infiltration (C) after LIGHT-OT-1 treatment of the B16F10-OVA mouse model.
  • prostate cancer is taken as an example to construct CART cells that can specifically target prostate cancer and secrete and express LIGHT-VTP protein.
  • the first nucleic acid sequence expressing the chimeric antigen receptor targeting prostate specific membrane antigen was synthesized by GenScript.
  • the first nucleic acid sequence includes: CD8 ⁇ signal peptide, PSMA single-chain antibody heavy chain variable region , Linker1, PSMA single-chain antibody light chain, CD8 hinge region, CD8 ⁇ transmembrane domain, 4-1BB intracellular costimulatory element and CD3 ⁇ intracellular domain ( Figure 1 A), connect the above sequences in sequence, The Kozac sequence and the corresponding restriction site are introduced at the forefront.
  • the first nucleic acid sequence was transferred to the plasmid pELPS transfervector by double enzyme digestion with XbaI and SalI, and the chimeric antigen receptor expression vector was named CJ-PSMA-CAR after enzyme ligation, which was used as a control plasmid.
  • CJ-PSMA-CAR plasmid as the initial plasmid, a second nucleic acid sequence expressing LIGHT-VTP is added, the first nucleic acid sequence and the second nucleic acid sequence are connected by P2A, and the second nucleic acid sequence (LIGHT-VTP) is in turn Including: secretion signal peptide (SP), LIGHT-VTP ( Figure 1 B).
  • the resulting plasmid was named CJ-PSMA-LIGHT-VTP plasmid (see Figure 2 for the structure).
  • sequence of each element of the expression cassette expressing the chimeric antigen receptor targeting prostate specific membrane antigen is as follows:
  • CD8 ⁇ Leader The base sequence of CD8 ⁇ signal peptide (CD8 ⁇ Leader) is shown in SEQ ID NO.1:
  • PSMA-ScFv VL The base sequence of the PSMA single-chain antibody light chain variable region (PSMA-ScFv VL) is shown in SEQ ID NO. 2:
  • amino acid sequence of the light chain variable region of the PSMA single-chain antibody is shown in SEQ ID NO. 3:
  • PSMA-ScFv VH The base sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO.6:
  • PSMA-ScFv VH The amino acid sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO.7:
  • CD8 hinge region (CD8hinge) is shown in SEQ ID NO.8:
  • CD8 ⁇ transmembrane domain CD8a-TM
  • SEQ ID NO.9 The base sequence of CD8 ⁇ transmembrane domain (CD8a-TM) is shown in SEQ ID NO.9:
  • the base sequence of the intracellular costimulatory element of 4-1BB is shown in SEQ ID NO.10:
  • the base sequence of the intracellular domain of CD3 ⁇ is shown in SEQ ID NO.11.
  • the base sequence of P2A is shown in SEQ ID NO.12:
  • SP secretion signal peptide
  • the base sequence of the secreted LIGHT is shown in SEQ ID NO.14:
  • the amino acid sequence of LIGHT in this embodiment is shown in SEQ ID NO. 15.
  • the secreted LIGHT fragment is selected in this embodiment, and the amino acid sequence is as follows:
  • amino acid sequence of the full-length LIGHT is shown in SEQ ID NO.18, and the details are as follows:
  • SEQ ID NO. 19 The base sequence of the above-mentioned full-length LIGHT is shown in SEQ ID NO. 19, which is specifically as follows:
  • VTP The base sequence of VTP is shown in SEQ ID NO. 16 or 23:
  • amino acid sequence of VTP is shown in SEQ ID NO. 17 or 24: GGGCRGRRSTG (SEQ ID NO. 17); CRGRRST (SEQ ID NO. 24).
  • LIGHT and the targeting polypeptide are connected by a connecting peptide, and the base sequence of the connecting peptide is shown in SEQ ID NO. 21: ggcggcggc.
  • LIGHT and the targeting polypeptide are connected by a connecting peptide, and the amino acid sequence of the connecting peptide is GGG.
  • LIGHT-VTP The amino acid sequence of the LIGHT fusion protein (LIGHT-VTP) is shown in SEQ ID NO. 20:
  • PSMA prostate specific membrane antigen
  • the method is as follows: Use E. coli to amplify the above-mentioned CJ-PSMA-CAR and CJ-PSMA-LIGHT-VTP plasmids and lentivirus packaging auxiliary plasmids pMD2.G (see Figure 3) and psPAX2 (see Figure 4), and then extract the plasmids. Agarose gel electrophoresis and sequencing to identify the correctness of the plasmid. The 293T with the highest generation number in good condition was selected as the lentivirus packaging cell, and the above three plasmids were transfected into 293T cells with the transfection reagent PEI. Transfection was completed in a 10cm culture dish with a total system of 10mL.
  • the culture supernatant was harvested at 48h and 72h, respectively, and the virus expressing chimeric antigen receptor and LIGHT-VTP was obtained after ultrafiltration and ultraionization concentration.
  • the resulting virus was named LIGHT-VTP-CAR virus.
  • CJ-PSMA-CAR plasmid compared to CJ-PSMA-LIGHT-VTP plasmid, CJ-PSMA-CAR plasmid lacks the second expression cassette that expresses LIGHT-VTP) as a control, and refer to the above methods for transfection and treatment. Named PSMA-CAR virus.
  • the titer detection results are shown in Figure 5.
  • the titer detection is based on the above-mentioned titer detection method.
  • the control viruses PSMA-CAR and LIGHT-VTP-CAR viruses are set with two volume gradients of 2 ⁇ l and 5 ⁇ l respectively.
  • CTRL To avoid false positives caused by non-specific staining, it is necessary to set CTRL to carry out the CAR positive circle gate.
  • the CAR positive cells fall into the APC positive gate, and the ratio value shown is the CAR positive rate.
  • PBMCs are separated from human blood using lymphatic separation fluid, and then T cells are separated using CD4 and CD8 magnetic bead sorting. After 48 hours of activation by the CD3/CD28 complex, the packaged PSMA-CAR and LIGHT-VTP- can be used.
  • the two CART cells were named PSMA-CART cells and LIGHT-VTP-CART cells, respectively.
  • the CAR expression levels of the above two CARTs were detected according to the same method of titer detection 48h after the infection was changed.
  • LIGHT-VTP-CAR For LIGHT-VTP-CAR, it is not only necessary to detect the expression of its CAR, but also to verify whether it has the ability to express LIGHT-VTP. Therefore, the above two CART cells under the same culture system conditions (the positive rate is adjusted to be consistent) are collected for 48h. Clear, verify the expression of LIGHT by ELISA.
  • the average killing efficiency of LIGHT-VTP-CART is 43.12%, and the killing efficiency of PSMA-CART is 27.19%, indicating that LIGHT- is overexpressed.
  • the CART cells of VTP can enhance its ability to kill tumor cells.
  • the above 12h co-incubation supernatant with E:T of 1:1 was selected to detect the expression of LIGHT by ELISA.
  • the result is shown in Figure 9.
  • the LIGHT-VTP-CART stimulated by the antigen secreted more LIGHT (at the same culture concentration). (1 ⁇ 10 6 cells/mL), LIGHT-VTP-CART secreted LIGHT concentration of 423.75pg/mL, PSMA-CART secreted LIGHT concentration of 223.75pg/mL, CTRL-T was 68.75pg/mL), further confirmation Therefore, the function of LIGHT-VTP enhances the function of LIGHT-VTP-CART.
  • the combination of the two methods was used for experiments in the prostate cancer NSG mouse model.
  • the PC3-PSMA cell line (labeled with Luciferase) was injected subcutaneously into the mouse, and the volume of the tumor was tracked using vernier calipers and mouse in vivo imaging technology. When it reached a certain size, the following four groups of experiments were set up: PBS, hLIGHT, PSMA-CART, PSMA -CART+hLIGHT.
  • PSMA-CART is only infused once through the tail vein (3.5 ⁇ 10 6 cells/head)
  • PBS is only infused once through the tail vein (100 ⁇ l PBS/head)
  • hLIGHT recombinant human secreted LIGHT, 100ng/mL
  • Internal injection is injected once a week, 25 ⁇ l each time. After treatment, the patients were followed by intravital imaging twice a week for 40 days.
  • Figure 13 is the control group, which only injected PBS or hLIGHT. It can be seen from the figure that the tumors of the mice did not become smaller during the tracking process and died at 34 days, indicating that hLIGHT alone has no effect on the treatment of prostate cancer NSG mouse model (high immunodeficiency) . It shows that LIGHT needs the help of immune cells to function.
  • a full-length LIGHT CAR plasmid expressing fusion VTP corresponding to the CJ-PSMA-LIGHT-VTP plasmid was constructed, and the lentivirus was packaged using the above method and collected
  • the virus stock solution after 48h. Choose good 293T to inoculate 500 ⁇ l of cells with a density of 4*10 ⁇ 5/mL in a 24-well plate. After the cells adhere to the wall, add different gradient volumes of virus stock solution, respectively 20ul, 50ul, 100ul, 200ul, and infect 24 After hours, the medium was changed. After 48 hours, the cells were collected to test the positive rate and the cell supernatant was collected.
  • LIGHT-VTP-CART After LIGHT-VTP-CART is incubated with tumor cells, more LIGHT, IFN ⁇ , and TNF ⁇ are secreted.
  • the inventors used the above-mentioned CART and target cells to incubate for 24 hours with an E:T ratio of 2:1 (the target cell was 0.5M and the culture system volume was 1.5 mL), and the supernatant was collected.
  • ELISA was used to detect the secretion of LIGHT, IFN ⁇ , and TNF ⁇ in the system before and after the co-incubation. The results are shown in Figure 16, which is consistent with the previous results.
  • PSMA-CART can also increase the release of factors after co-incubation with target cells, LIGHT- VTP-CART secretes more LIGHT, IFN ⁇ , and TNF ⁇ than PSMA-CART.
  • LIGHT-VTP-CART has a high proportion of Tcm (central memory T cells) phenotype
  • LIGHT-VTP-CART supernatant activates tumor microenvironment cells to up-regulate the expression of chemokines and adhesion molecules
  • LIGHT Under the action of LIGHT, not only CAF cells are activated, but also PC3 tumor cells, C57 mouse bone marrow cells and vascular endothelial cells HUVEC can be significantly activated (CH in Figure 19). In addition to upregulating chemokines, LIGHT can activate vascular endothelial cells HUVEC The expression of cell adhesion molecules, thereby increasing the adhesion of CART on its surface (IJ in Figure 19)
  • LIGHT can activate tumor cells, tumor-associated fibroblasts, and mouse bone marrow cells after secreting large amounts of chemokines such as CCL19 and CCL21 (data not shown).
  • LIGHT-VTP-CART has significantly different gene expression profiles than PSMA-CART.
  • These differential genes are mainly enriched (GSEA) in IFNG, TNFA, IL -2, inflammatory response. The result is shown in Figure 21.
  • PC3-PSMA cells a PSMA-positive cell line stably expressing firefly luciferase
  • the specific process of modeling is shown in Figure 22-A.
  • the effector cells injected for treatment are PSMA- with an adjusted positive rate.
  • CART and LIGHT-VTP-CART the control group is the Ctrl T cell group without virus infection.
  • 5 ⁇ 10 6 effector cells/mouse are injected into the tail vein at one time.
  • the IVIS intravital imaging system is used to take pictures and imaging every 2-4 days after the injection.
  • the tumor growth is shown in Figure 22-B.
  • LIGHT-VTP-CART has an enhanced ability to infiltrate the tumor.
  • LIGHT-VTP-CART has stronger anti-tumor ability than PBBZ-L CART.
  • LIGHT-CART and LIGHT-VTP-CART were prepared according to the method of the present invention, and PC3-PSMA cells were transplanted into NSG mice for subcutaneous modeling. After successful tail vein injection of 5 ⁇ 106 effector cells/mouse, the IVIS live imaging system is used to take pictures and image to track tumor changes every 2-4 days. Both LIGHT-VTP-CART and LIGHT-CART show anti-tumor effects, but they are obvious LIGHT-VTP-CART has faster and stronger anti-tumor ability.
  • LIGHT-VTP-CART has a faster and stronger ability to regress tumors than PSMA-CART in the treatment of bone metastasis prostate cancer models constructed by injecting PC3-PSMA cells into the tibia.
  • LIGHT-VTP enhances the treatment of solid tumors in mice.
  • LIGHT-VTP promotes the formation of HEV and promotes the infiltration of T, DC and NK cells.
  • B16F10-OVA cells are used to construct a subcutaneous transplantation model.
  • NK cells in the blood of mice treated with LIGHT-OT-1 cells have more expansion than mice treated with ordinary OT-1 cells, and tumor tissues are also found There was also more NK infiltration in the medium ( Figure 26-C).
  • LIGHT-VTP can significantly enhance the infiltration of T cells and other immune cells into different solid tumor models, thereby further enhancing the "mutual dialogue" between immune cells and enhancing anti-tumor immunity.
  • TIL tumor infiltrating lymphocytes
  • the infiltration of T cells into tumor tissues and contact with tumor cells is the first condition for CART cells to fully function.
  • the embodiments of the present disclosure are based on the typical "cold” tumor prostate cancer among solid tumors, and the second-generation CAR-T technology that targets PSMA as a basis to design and construct a new "infiltrating type” targeting PSMA that can secrete LIGHT.
  • "CART cells, and the LIGHT is fused with the tumor abnormal blood vessel targeting polypeptide VTP, namely LIGHT-VTP.
  • LIGHT-VTP secreted by CAR-T cells can specifically target the abnormal blood vessels of the tumor, thereby promoting the normalization of abnormal blood vessels of the tumor, and can promote the infiltration of T, DC, and NK cells into the tumor tissue, and promote the lymphoid pattern of the tumor site.
  • the structure (TLS) is formed to guide the aggregation and activation of CAR-T cells, mobilize autoimmune function, and reshape the inhibitory effect of the tumor microenvironment, thereby changing the limitation of the effect of CART cells due to the inability to penetrate tumor tissues and immunosuppression. Enhance the activity of CART cells against solid tumors. Under the synergistic effect of LIGHT-VTP, it greatly enhances the effect of CAR-T in the treatment of solid tumors.
  • the enhanced CART cells that promote the infiltration of solid tumors provided by the present disclosure effectively improve the infiltration ability of CART cells to tumor tissues of solid tumors by secreting and expressing LIGHT protein, and enhance their killing ability to tumor cells, and they have broad clinical applications.
  • the use of CART cells to treat solid tumors provides a new drug choice and treatment strategy.

Abstract

La présente invention se rapporte au champ technique des cellules CART. L'invention concerne une cellule CART améliorée favorisant l'infiltration dans des tumeurs solides, un procédé de préparation associé et un médicament cellulaire correspondant. La cellule CART comprend une première séquence d'acide nucléique et une deuxième séquence d'acide nucléique et présente une capacité élevée d'infiltration dans un tissu tumoral solide et une forte capacité de tuer une cellule tumorale.
PCT/CN2020/142571 2019-12-31 2020-12-31 Cellule cart améliorée favorisant l'infiltration dans des tumeurs solides, procédé de préparation associé et médicament cellulaire correspondant WO2021136538A1 (fr)

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