TW202233662A - Membrane fusion protein and use thereof in immune cells - Google Patents

Membrane fusion protein and use thereof in immune cells Download PDF

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TW202233662A
TW202233662A TW110149647A TW110149647A TW202233662A TW 202233662 A TW202233662 A TW 202233662A TW 110149647 A TW110149647 A TW 110149647A TW 110149647 A TW110149647 A TW 110149647A TW 202233662 A TW202233662 A TW 202233662A
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董琦
江鵬斐
連軍 沈
曹衛
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大陸商亘喜生物科技（上海）有限公司
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Abstract

Provided are a chimeric antigen receptor immune cell expressing interleukin-15, and the use thereof. Specifically, provided is a genetically recombinant chimeric antigen receptor immune cell, which expresses a fusion protein of an interleukin-15 mutant and IL-15Ra on the cell surface of the immune cell. Experiments show that under continuous antigen stimulation, the mutant immune cell significantly improves the survival of immune cells, promotes the expansion of the immune cells, reduces the inhibition of TME on the immune cells, and enhances the persistence and anti-tumor activity of CAR-T cells.

Description

膜融合蛋白及其在免疫細胞中的應用Membrane fusion protein and its application in immune cells

本發明涉及免疫細胞治療領域，更具體地涉及共表現介白素-15突變體和IL-15Rα融合蛋白且標靶固態腫瘤和血液瘤的基因工程化免疫細胞及其應用。The present invention relates to the field of immune cell therapy, and more particularly to genetically engineered immune cells that co-express interleukin-15 mutants and IL-15Rα fusion proteins and target solid tumors and hematological tumors, and applications thereof.

免疫細胞療法是一種全新的藥物開發模式。利用基因工程化改造人類免疫細胞使其成為活性藥物，在治療B細胞惡性腫瘤方面已經取得了顯著的進展。Immune cell therapy is a new mode of drug development. Significant progress has been made in the treatment of B-cell malignancies by genetically engineering human immune cells into active drugs.

目前已開發了多種免疫療法，包括LAK、DC、CIK、DC-CIK、TCR-T、CAR-T、NK、CAR-NK等等。常用的CAR-T細胞免疫療法包括自體和通用型CAR-T療法，其中自體CAR-T療法採用病人的免疫細胞，而異體免疫療法採用同種異體對象的CAR-T細胞，將所需的免疫細胞進行體外培養、基因編輯和擴增後回輸病人體內，這些細胞以非MHC限制方式辨識腫瘤細胞，不需要抗原呈現的機制辨識腫瘤抗原，減少腫瘤細胞透過向下調控MHC以及抗原呈現介導的免疫逃逸。針對腫瘤相關抗原標的(TAA)，設計細胞表面嵌合抗原受體（CAR）分子特異辨識腫瘤細胞表現的抗原標的，透過分泌干擾素、穿孔素等細胞因子直接攻擊並毒殺腫瘤細胞，從而達到治療或緩解疾病的目的。A variety of immunotherapies have been developed, including LAK, DC, CIK, DC-CIK, TCR-T, CAR-T, NK, CAR-NK, and more. Commonly used CAR-T cell immunotherapy includes autologous and universal CAR-T therapy, in which autologous CAR-T therapy uses the patient's immune cells, while allogeneic immunotherapy uses CAR-T cells from allogeneic subjects, and the required Immune cells are cultured in vitro, gene-edited and expanded and then infused back into the patient. These cells recognize tumor cells in a non-MHC-restricted manner, and do not require an antigen-presenting mechanism to recognize tumor antigens. induced immune escape. For tumor-associated antigen targets (TAA), the chimeric antigen receptor (CAR) molecules on the cell surface are designed to specifically recognize the antigen targets expressed by tumor cells, and directly attack and kill tumor cells by secreting cytokines such as interferon and perforin, so as to achieve treatment. or the purpose of alleviating the disease.

目前，免疫治療血液瘤已有突出的療效，而在固態腫瘤中療效並不理想。主要原因包括三方面，一是安全的腫瘤特異性抗原標的，二是實體腫瘤的異質性，三是存在複雜的腫瘤細胞微環境等因素，包括免疫抑制細胞Tregs和MDSCs等以及免疫抑制因子的存在，不利於免疫細胞存活，從而抑制免疫治療效果。間皮素是一種存在於正常間皮細胞上的分化抗原，在間皮瘤、肺癌、胰腺癌、乳腺癌、卵巢癌等腫瘤中均有高度表現，正常組織如正常胸膜、心包和腹膜的間皮細胞中有限表現，在氣管，卵巢，睾丸，扁桃腺和輸卵管的上皮細胞表面上表現最低。因此間皮素可作為有效的免疫治療標的，目前針對MSLN設計免疫治療策略，主要包括抗體療法、免疫毒素和嵌合抗原受體T細胞療法。抗體類藥物包括Amatuximab（MORAb-009），Anetumab Ravtansine (BAY94-9343)，DMOT4039A，MDX-1204等，但其治療固態腫瘤療效並不理想，尚需完善現有的治療技術，增強療效，降低治療過程中的風險。At present, immunotherapy for hematological tumors has outstanding curative effect, but the curative effect in solid tumors is not ideal. The main reasons include three aspects, one is safe tumor-specific antigen targets, the other is the heterogeneity of solid tumors, and the third is the existence of complex tumor cell microenvironment and other factors, including immunosuppressive cells Tregs and MDSCs, and the existence of immunosuppressive factors. , is not conducive to the survival of immune cells, thereby inhibiting the effect of immunotherapy. Mesothelin is a differentiation antigen that exists on normal mesothelial cells. It is highly expressed in mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer and other tumors, and normal tissues such as normal pleura, pericardium and peritoneum. Limited expression in the epithelium, with minimal expression on the epithelial surface of the trachea, ovary, testis, tonsils, and fallopian tubes. Therefore, mesothelin can be used as an effective immunotherapy target. Currently, immunotherapy strategies are designed for MSLN, mainly including antibody therapy, immunotoxin and chimeric antigen receptor T cell therapy. Antibody drugs include Amatuximab (MORAb-009), Anetumab Ravtansine (BAY94-9343), DMOT4039A, MDX-1204, etc., but their efficacy in the treatment of solid tumors is not satisfactory, and it is necessary to improve the existing treatment technology to enhance the efficacy and reduce the treatment process risk in.

綜上所述，本領域尚需進一步開發更有效、持久、安全標靶固態腫瘤的免疫療法。In conclusion, there is still a need for further development of more effective, durable and safe immunotherapy targeting solid tumors in the art.

本發明的目的在於提供一種表現介白素-15的嵌合抗原受體免疫細胞及其應用。The purpose of the present invention is to provide a chimeric antigen receptor immune cell expressing interleukin-15 and its application.

在本發明的第一方面，提供了一種嵌合抗原受體(CAR)建構物，所述CAR建構物的結構如下式I或II所示， X-A-E    (I) E-A-X    (II) 式中， 各“-”獨立地為連接肽或肽鍵； X為標靶腫瘤抗原的CAR； A為自剪切元件； E為IL-15/IL-15Rα複合體。 In a first aspect of the present invention, a chimeric antigen receptor (CAR) construct is provided, and the structure of the CAR construct is shown in the following formula I or II, X-A-E (I) E-A-X (II) In the formula, each "-" is independently a linking peptide or peptide bond; X is a CAR targeting tumor antigen; A is the self-shearing element; E is the IL-15/IL-15Rα complex.

在另一優選例中，所述的CAR建構物的結構如式I所示。In another preferred embodiment, the structure of the CAR construct is shown in formula I.

在另一優選例中，所述的IL-15/IL-15Rα複合體包含IL-15和IL-15Rα。In another preferred embodiment, the IL-15/IL-15Rα complex comprises IL-15 and IL-15Rα.

在另一優選例中，所述的IL-15和IL-15Rα來源於人。In another preferred embodiment, the IL-15 and IL-15Rα are derived from human.

在另一優選例中，所述的IL-15和IL-15Rα透過連接肽連接。In another preferred embodiment, the IL-15 and IL-15Rα are linked through a linking peptide.

在另一優選例中，所述的IL-15/IL-15Rα複合體還包含信號肽元件。In another preferred embodiment, the IL-15/IL-15Rα complex further comprises a signal peptide element.

另一優選例中，所述的IL-15/IL-15Rα複合體的結構如下式III所示， L’-M-I-R    (III) 式中， 各“-”獨立地為連接肽或肽鍵； L’為無或信號肽； M為IL-15或其突變體； I為柔性連接子； R為IL-15Rα。 In another preferred example, the structure of the IL-15/IL-15Rα complex is shown in the following formula III, L’-M-I-R (III) In the formula, each "-" is independently a linking peptide or peptide bond; L' is none or signal peptide; M is IL-15 or a mutant thereof; I is a flexible linker; R is IL-15Rα.

在另一優選例中，所述的IL-15突變體具有IL-15的生物活性。In another preferred embodiment, the IL-15 mutant has the biological activity of IL-15.

在另一優選例中，所述的IL-15的胺基酸序列如SEQ ID NO.: 1所示。In another preferred example, the amino acid sequence of the IL-15 is shown in SEQ ID NO.: 1.

在另一優選例中，所述的IL-15突變體(IL-15N72D)的胺基酸序列如SEQ ID NO.: 2所示。In another preferred example, the amino acid sequence of the IL-15 mutant (IL-15N72D) is shown in SEQ ID NO.: 2.

在另一優選例中，所述的IL-15Rα為完整的IL-15Rα元件。In another preferred embodiment, the IL-15Rα is a complete IL-15Rα element.

在另一優選例中，所述的IL-15Rα包含跨膜區和胞內區。In another preferred embodiment, the IL-15Rα comprises a transmembrane domain and an intracellular domain.

在另一優選例中，所述的IL-15Rα從N端到C端依次包括sushi結構域(與IL-15結合的活性片段)胞外區、跨膜區和胞內區。In another preferred example, the IL-15Rα includes an extracellular region, a transmembrane region and an intracellular region of the sushi domain (active fragment that binds to IL-15) in sequence from the N-terminus to the C-terminus.

在另一優選例中，所述的IL-15Rα的胺基酸序列如SEQ ID NO.: 3所示。In another preferred example, the amino acid sequence of the IL-15Rα is shown in SEQ ID NO.: 3.

在另一優選例中，所述的柔性連接子為連接肽，較佳地，所述連接肽的胺基酸序列如SEQ ID NO.: 4所示(SGGGSGGGGSGGGGSGGGGSGGGSLQ)。In another preferred embodiment, the flexible linker is a connecting peptide, preferably, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.: 4 (SGGGGSGGGGSGGGGSGGGGSGGGSLQ).

在另一優選例中，所述的L’為來源IgE, IL-2的信號肽。In another preferred embodiment, the L' is a signal peptide derived from IgE, IL-2.

在另一優選例中，所述的IL-15 /IL-15Rα複合體的胺基酸序列如SEQ ID NO.: 5所示。In another preferred example, the amino acid sequence of the IL-15/IL-15Rα complex is shown in SEQ ID NO.: 5.

在另一優選例中，所述的IL-15突變體/IL-15Rα複合體的胺基酸序列如SEQ ID NO.: 6所示。In another preferred example, the amino acid sequence of the IL-15 mutant/IL-15Rα complex is shown in SEQ ID NO.: 6.

在另一優選例中，所述的自剪切元件包括T2A、P2A。In another preferred embodiment, the self-shearing element includes T2A and P2A.

在另一優選例中，所述的X(CAR)的結構如下式IV所示， L-scFv-H-TM-C-CD3ζ    (IV) 各“-”獨立地為連接肽或肽鍵； L為無或信號肽； scFv為標靶腫瘤抗原的抗體單鏈可變區； H為無樞紐區； TM為跨膜結構域； C為共刺激信號分子； CD3ζ為源於CD3ζ的細胞質液信號傳導序列； In another preferred embodiment, the structure of described X (CAR) is shown in the following formula IV, L-scFv-H-TM-C-CD3ζ (IV) each "-" is independently a linking peptide or peptide bond; L is none or signal peptide; scFv is an antibody single-chain variable region targeting a tumor antigen; H is no hub area; TM is the transmembrane domain; C is a costimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

在另一優選例中，所述的腫瘤抗原選自下組：間皮素、Claudin18.2、MUC1、GPC3、PSCA、Her2、CD19、或其組合。In another preferred embodiment, the tumor antigen is selected from the group consisting of mesothelin, Claudin18.2, MUCl, GPC3, PSCA, Her2, CD19, or a combination thereof.

在另一優選例中，所述的腫瘤抗原為間皮素。In another preferred embodiment, the tumor antigen is mesothelin.

在另一優選例中，所述的L為選自下組的蛋白的信號肽：CD8、CD28、GM-CSF、CD4、CD137、或其組合。In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

在另一優選例中，所述的L為CD8來源的信號肽。In another preferred embodiment, the L is a signal peptide derived from CD8.

在另一優選例中，所述的H為選自下組的蛋白的樞紐區：CD8、CD28、CD137、或其組合。In another preferred embodiment, the H is a hub region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

在另一優選例中，所述的H為CD8來源的樞紐區。In another preferred embodiment, the H is a CD8-derived hub region.

在另一優選例中，所述的TM為選自下組的蛋白的跨膜區：ICOS、CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、MUC1-Tn、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其組合。In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of ICOS, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, MUCl-Tn, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

在另一優選例中，所述的TM為CD8或CD28來源的跨膜區。In another preferred embodiment, the TM is a transmembrane region derived from CD8 or CD28.

在另一優選例中，所述的C為選自下組的蛋白的共刺激信號分子：ICOS、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1 (CD11a/CD18)、ICOS (CD278)、NKG2D、GITR、TLR2、或其組合。In another preferred embodiment, the C is a costimulatory signal molecule selected from the following group of proteins: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

在另一優選例中，所述的C為CD28來源的共刺激信號分子。In another preferred embodiment, the C is a costimulatory signal molecule derived from CD28.

在另一優選例中，所述的X的胺基酸序列如SEQ ID NO.: 8所示。In another preferred embodiment, the amino acid sequence of the X is shown in SEQ ID NO.: 8.

在另一優選例中，所述的CAR建構物的胺基酸序列如SEQ ID NO.: 7所示。In another preferred embodiment, the amino acid sequence of the CAR construct is shown in SEQ ID NO.: 7.

在本發明的第二方面，提供了一種核酸分子，所述的核酸分子編碼本發明第一方面所述的CAR建構物，或者，In the second aspect of the present invention, there is provided a nucleic acid molecule encoding the CAR construct described in the first aspect of the present invention, or,

所述的核酸分子包含編碼標靶腫瘤抗原的CAR的第一核酸分子和編碼IL-15/IL-15Rα複合體的第二核酸分子，其中所述的標靶腫瘤抗原的CAR和IL-15/IL-15Rα複合體的定義如上所述。The nucleic acid molecule comprises the first nucleic acid molecule encoding the CAR of the target tumor antigen and the second nucleic acid molecule encoding the IL-15/IL-15Rα complex, wherein the CAR and the IL-15/IL-15Rα complex of the target tumor antigen are included. The IL-15Rα complex is defined as described above.

在另一優選例中，所述的第一核酸分子和第二核酸分子可以是串聯的，也可以是獨立存在的。In another preferred embodiment, the first nucleic acid molecule and the second nucleic acid molecule may be connected in series, or may exist independently.

在本發明的第三方面，提供了一種載體，所述的載體含有本發明第二方面所述的核酸分子。In the third aspect of the present invention, a vector is provided, and the vector contains the nucleic acid molecule described in the second aspect of the present invention.

在另一優選例中，所述的載體選自下組：DNA、RNA、質體、慢病毒載體、腺病毒載體、腺相關病毒載體(AAV)、反轉錄病毒載體、轉座子、或其組合。In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plastid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or its combination.

在另一優選例中，所述的載體選自下組：質體、病毒載體。In another preferred embodiment, the vector is selected from the group consisting of plastid and viral vector.

在另一優選例中，所述載體為病毒顆粒的形式。In another preferred embodiment, the vector is in the form of virus particles.

在另一優選例中，所述載體為慢病毒載體。In another preferred embodiment, the vector is a lentiviral vector.

在本發明的第四方面，提供了一種宿主細胞，所述的宿主細胞中含有本發明第三方面所述的載體或染色體中整合有外源的本發明第二方面所述的核酸分子或表現本發明第一方面所述的CAR建構物。In the fourth aspect of the present invention, a host cell is provided, the host cell contains the vector described in the third aspect of the present invention or the exogenous nucleic acid molecule or expression of the second aspect of the present invention is integrated into the chromosome. The CAR construct of the first aspect of the present invention.

在另一優選例中，所述的宿主細胞包括真核細胞和原核細胞。In another preferred embodiment, the host cells include eukaryotic cells and prokaryotic cells.

在另一優選例中，所述的宿主細胞包括大腸桿菌。In another preferred embodiment, the host cell includes Escherichia coli.

在本發明的第五方面，提供了一種基因工程化的免疫細胞，所述的免疫細胞表現有本發明第一方面所述的CAR建構物，或者所述的免疫細胞表現有標靶腫瘤抗原的CAR和IL-15/IL-15Rα複合體，其中所述的標靶腫瘤抗原的CAR和IL-15/IL-15Rα複合體的定義如上所述。In a fifth aspect of the present invention, a genetically engineered immune cell is provided, the immune cell expressing the CAR construct described in the first aspect of the present invention, or the immune cell expressing a target tumor antigen CAR and IL-15/IL-15Rα complex, wherein the CAR and IL-15/IL-15Rα complex that target tumor antigen are defined as above.

在另一優選例中，標靶腫瘤抗原的CAR和IL-15/IL-15Rα複合體獨立地表現於所述免疫細胞的細胞膜上。In another preferred embodiment, the CAR targeting tumor antigen and the IL-15/IL-15Rα complex are independently expressed on the cell membrane of the immune cells.

在另一優選例中，所述細胞為分離的細胞，和/或所述細胞為基因工程化的細胞。In another preferred embodiment, the cells are isolated cells, and/or the cells are genetically engineered cells.

在另一優選例中，所述的免疫細胞來自人或非人哺乳動物(如鼠)。In another preferred embodiment, the immune cells are derived from human or non-human mammals (eg, mice).

在另一優選例中，所述細胞包括T細胞、NK細胞。In another preferred example, the cells include T cells and NK cells.

在另一優選例中，所述的基因工程化的免疫細胞可以是嵌合抗原受體T細胞（CAR-T 細胞）或嵌合抗原受體NK細胞（CAR-NK 細胞）。In another preferred example, the genetically engineered immune cells may be chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

在本發明的第六方面，提供了一種製劑，所述製劑含有本發明第一方面所述的CAR建構物、本發明第二方面所述的核酸分子、本發明第三方面所述的載體、或本發明第五方面所述的免疫細胞，以及藥學上可接受的載劑。In the sixth aspect of the present invention, there is provided a preparation comprising the CAR construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, Or the immune cells described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.

在另一優選例中，所述製劑為液態製劑。In another preferred embodiment, the preparation is a liquid preparation.

在另一優選例中，所述製劑的劑型為注射劑。In another preferred embodiment, the dosage form of the preparation is an injection.

在另一優選例中，所述製劑中所述CAR-T細胞的濃度為1×10 3-1×10 8個細胞/ml，較佳地1×10 4-1×10 7個細胞/ml。 In another preferred embodiment, 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 .

在另一優選例中，所述的製劑還包含抗腫瘤的第二活性成分，較佳地包括第二抗體、或化療劑。In another preferred embodiment, the preparation further comprises a second anti-tumor active ingredient, preferably a second antibody, or a chemotherapeutic agent.

在另一優選例中，所述的化療劑選自下組：多西他賽、卡鉑、或其組合。In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.

在本發明的第七方面，提供了一種本發明第一方面所述的CAR建構物、本發明第二方面所述的核酸分子、本發明第三方面所述的載體、或本發明第五方面所述的免疫細胞、或本發明第六方面所述的製劑的用途，用於製備預防和/或治療癌症或腫瘤的藥物或製劑。In the seventh aspect of the present invention, there is provided a CAR construct according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, or the fifth aspect of the present invention The use of the immune cells or the preparation described in the sixth aspect of the present invention is for preparing a medicine or preparation for preventing and/or treating cancer or tumor.

在另一優選例中，所述腫瘤選自下組：血液腫瘤、固態腫瘤、或其組合。In another preferred embodiment, the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.

在另一優選例中，所述血液腫瘤選自下組：急性骨髓性白血病(AML)、多發性骨髓瘤(MM)、慢性淋巴細胞白血病(CLL)、急性淋巴白血病(ALL)、彌漫性大B細胞淋巴瘤(DLBCL)、或其組合。In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse B-cell lymphoma (DLBCL), or a combination thereof.

在另一優選例中，所述固態腫瘤選自下組：胃癌、胃癌腹膜轉移、肝癌、白血病、腎臟腫瘤、肺癌、小腸癌、骨癌、***癌、結直腸癌、乳腺癌、大腸癌、宮頸癌、卵巢癌、淋巴癌、鼻咽癌、腎上腺腫瘤、膀胱腫瘤、非小細胞肺癌(NSCLC)、腦膠質瘤、子宮內膜癌、或其組合。In another preferred embodiment, the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.

在另一優選例中，所述的腫瘤為間皮素陽性腫瘤，較佳地為間皮素高度表現的腫瘤。In another preferred embodiment, the tumor is a mesothelin-positive tumor, preferably a tumor with a high expression of mesothelin.

在本發明的第八方面，提供了一種用於製備本發明第四方面所述的宿主細胞的套組，所述套組含有容器，以及位於容器內的本發明第二方面所述的核酸分子、或本發明第三方面所述的載體。In an eighth aspect of the present invention, there is provided a kit for preparing the host cell according to the fourth aspect of the present invention, the kit comprising a container and the nucleic acid molecule according to the second aspect of the present invention located in the container , or the carrier of the third aspect of the present invention.

在本發明的第九方面，提供了一種製備本發明第五方面所述的基因工程化的免疫細胞的方法，所述方法包括以下步驟： (a)提供待改造的免疫細胞；和 (b)將本發明第二方面所述的核酸分子或本發明第三方面所述的載體轉導入所述免疫細胞內，從而獲得所述基因工程化的免疫細胞。 In the ninth aspect of the present invention, a method for preparing the genetically engineered immune cells described in the fifth aspect of the present invention is provided, the method comprising the following steps: (a) providing immune cells to be engineered; and (b) transfecting the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the immune cells, thereby obtaining the genetically engineered immune cells.

在另一優選例中，所述基因工程化的免疫細胞為CAR-T細胞或CAR-NK細胞。In another preferred embodiment, the genetically engineered immune cells are CAR-T cells or CAR-NK cells.

在另一優選例中，所述的方法還包括對獲得的基因工程化免疫細胞進行功能和有效性檢測的步驟。In another preferred embodiment, the method further includes the step of testing the function and effectiveness of the obtained genetically engineered immune cells.

在本發明的第十方面，提供了一種治療疾病的方法，包括給需要治療的對象施用適量的本發明第三方面所述的載體、本發明第五方面所述的免疫細胞、或本發明第六方面所述的製劑。In the tenth aspect of the present invention, there is provided a method for treating a disease, comprising administering an appropriate amount of the carrier of the third aspect of the present invention, the immune cell of the fifth aspect of the present invention, or the first aspect of the present invention to a subject in need of treatment. The preparation described in the sixth aspect.

在另一優選例中，所述疾病為癌症或腫瘤。In another preferred embodiment, the disease is cancer or tumor.

在本發明的第十一方面，提供了一種IL-15/IL-15Rα複合體，所述的IL-15/IL-15Rα複合體的結構如下式III所示， L’-M-I-R    (III) 式中， 各“-”獨立地為連接肽或肽鍵； L’為無或信號肽； M為IL-15或其突變體； I為柔性連接子； R為IL-15Rα， 其中，所述的IL-15Rα包含跨膜區和胞內區。 In the eleventh aspect of the present invention, an IL-15/IL-15Rα complex is provided, and the structure of the IL-15/IL-15Rα complex is shown in the following formula III, L’-M-I-R (III) In the formula, each "-" is independently a linking peptide or peptide bond; L' is none or signal peptide; M is IL-15 or a mutant thereof; I is a flexible linker; R is IL-15Rα, Wherein, the IL-15Rα comprises a transmembrane region and an intracellular region.

在本發明的第十二方面，提供了一種本發明的第十一方面所述的IL-15/IL-15Rα複合體的用途，用於製備一製劑，所述製劑用於增強CAR-T細胞的持久性和/或增強CAR-T細胞的細胞毒性。In the twelfth aspect of the present invention, there is provided a use of the IL-15/IL-15Rα complex according to the eleventh aspect of the present invention, for preparing a preparation for enhancing CAR-T cells persistence and/or enhanced cytotoxicity of CAR-T cells.

在另一優選例中，所述製劑用於基於CAR-T細胞的過繼免疫治療。In another preferred embodiment, the preparation is used for adoptive immunotherapy based on CAR-T cells.

在另一優選例中，所述的增強CAR-T細胞的持久性是指增強CAR-T細胞對腫瘤細胞的持續毒殺能力。In another preferred example, the enhancing the persistence of CAR-T cells refers to enhancing the ability of CAR-T cells to continuously kill tumor cells.

應理解，在本發明範圍內中，本發明的上述各技術特徵和在下文(如實施例)中具體描述的各技術特徵之間都可以互相組合，從而構成新的或優選的技術方案。限於篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

本發明人經過廣泛而深入地研究，首次意外地發現一種基因重組的嵌合抗原受體免疫細胞及其應用，所述嵌合抗原受體免疫細胞表面表現超級介白素-15，即為介白素-15突變體與IL-15Ra融合蛋白，體外實驗顯示該突變體能顯著增強免疫細胞的增殖能力、存活能力，持續毒殺力和促進NK細胞的擴增；體內實驗顯示該突變體持久抗腫瘤活性。在此基礎上完成了本發明。 術語 After extensive and in-depth research, the present inventor unexpectedly discovered for the first time a genetically recombined chimeric antigen receptor immune cell and its application. The surface of the chimeric antigen receptor immune cell expresses super interleukin-15, which is an The fusion protein of leukin-15 mutant and IL-15Ra, in vitro experiments show that the mutant can significantly enhance the proliferation and survival ability of immune cells, sustained lethality and promote the expansion of NK cells; in vivo experiments show that the mutant has durable anti-tumor active. The present invention has been completed on this basis. the term

為了可以更容易地理解本公開，首先定義某些術語。如本申請中所使用的，除非本文另有明確規定，否則以下術語中的每一個應具有下面給出的含義。在整個申請中闡述了其它定義。In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

術語“約”可以是指在本領域普通技術人員確定的特定值或組成的可接受誤差範圍內的值或組成，其將部分地取決於如何測量或測定值或組成。The term "about" may refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

術語“給予”是指使用本領域技術人員已知的各種方法和遞送系統中的任一種將本發明的產品物理引入受試者，包括靜脈內，肌內，皮下，腹膜內，脊髓或其它腸胃外給藥途徑，例如透過注射或輸液。The term "administration" refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other gastrointestinal External routes of administration, such as by injection or infusion.

術語“抗體”(Ab)應包括但不限於免疫球蛋白，其特異性結合抗原並包含透過二硫鍵互連的至少兩條重(H)鏈和兩條輕(L)鏈，或其抗原結合部分。每條H鏈包含重鏈可變區 (本文縮寫為VH)和重鏈恆定區。重鏈恆定區包含三個恆定結構域CH1、CH2和CH3。每條輕鏈包含輕鏈可變區(本文縮寫為VL)和輕鏈恆定區。輕鏈恆定區包含一個恆定結構域CL。VH和VL區可以進一步細分為稱為互補決定區(CDR)的高度變異，其散佈有更守恆的稱為框架區(FR)的區域。每個VH和VL包含三個CDR和四個FR，從胺基末端到羧基末端按照以下順序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重鏈和輕鏈的可變區含有與抗原相互作用的結合結構域。The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigens thereof combined part. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one constant domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

應理解，本文中胺基酸名稱採用國際通用的單英文字母標識，與其相對應的胺基酸名稱三英文字母簡寫分別是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、I1e(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。It should be understood that the names of amino acids in this article are identified by the internationally accepted single English letters, and the three English letter abbreviations of the corresponding amino acid names are: Ala(A), Arg(R), Asn(N), Asp( D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), I1e(I), Leu(L), Lys(K), Met(M), Phe( F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).

術語“嵌合抗原受體”、“嵌合膜抗原受體”、“膜融合蛋白”、和“重組的膜融合蛋白”可互換使用，均代表本發明所建構的IL-15/IL-15Rα複合體。 IL-15 The terms "chimeric antigen receptor", "chimeric membrane antigen receptor", "membrane fusion protein", and "recombinant membrane fusion protein" are used interchangeably, and all represent the IL-15/IL-15Rα constructed by the present invention Complex. IL-15

IL-15是一種可以刺激免疫細胞生長的細胞因子。基於IL-15的藥物開發長期以來集中在可溶型的IL-15改造上。已有文獻和專利報導，集中在開發可溶型IL-15藥物，主要有RL1，ALT803，HRP008，P22339四種結構。IL-15 is a cytokine that can stimulate the growth of immune cells. IL-15-based drug development has long focused on soluble IL-15 engineering. There have been literature and patent reports, focusing on the development of soluble IL-15 drugs, mainly including RL1, ALT803, HRP008, and P22339 four structures.

RL1是IL-15與IL-15受體α的 Sushi domain的複合體，兩者之間透過一個Linker連接。ALT803是兩個IL-15N72D突變體與二聚體IL-15受體α的 Sushi domain /Fc融合蛋白的複合體，即IL-15（N72D）: IL-15Rα Su / Fc的複合體。HRP00018是IL-15 /Fc融合蛋白與IL-15受體α的 Sushi domain /Fc融合蛋白透過Knob-into-Hole形式相連形成複合體。P22339是IL-15(L52C)突變體與IL-15Rα Sushi domain（S40C）/Fc 複合體在突變位點形成二硫鍵，透過Knob-into-Hole形式連接，進而穩定複合物的結構。RL1 is a complex of IL-15 and the Sushi domain of IL-15 receptor α, which is connected by a Linker. ALT803 is a complex of two IL-15N72D mutants with a Sushi domain/Fc fusion protein of dimeric IL-15 receptor α, namely IL-15(N72D): IL-15Rα Su/Fc complex. HRP00018 is a complex of IL-15/Fc fusion protein and Sushi domain/Fc fusion protein of IL-15 receptor α linked through Knob-into-Hole form. P22339 is the IL-15(L52C) mutant and the IL-15Rα Sushi domain (S40C)/Fc complex forms a disulfide bond at the mutation site, which is connected through the Knob-into-Hole form, thereby stabilizing the structure of the complex.

綜上所述的四種結構，克服了重組的IL-15半衰期短的缺點，都明顯延長體內的半衰期，並提高其生物學活性，如促進了CD8+記憶 T細胞、NKT和NK細胞增殖。但其仍舊存在毒副作用和半衰期短的問題，在臨床使用中需多次給藥，限制其臨床實用性。 IL-15/IL-15Rα 複合體 In summary, the four structures above overcome the short half-life of recombinant IL-15, significantly prolong the half-life in vivo, and improve its biological activity, such as promoting the proliferation of CD8+ memory T cells, NKT and NK cells. However, it still has the problems of toxic side effects and short half-life, and needs to be administered multiple times in clinical use, which limits its clinical practicability. IL-15/IL-15Rα complex

本發明提供了一種“IL-15/IL-15Rα複合體”，其包含IL-15或其突變體，以及完整的包含跨膜區和胞內區的IL-15Rα，可以用於增強CAR-T細胞的持久性和/或增強CAR-T細胞的細胞毒性。The present invention provides an "IL-15/IL-15Rα complex", which comprises IL-15 or a mutant thereof, and a complete IL-15Rα comprising a transmembrane region and an intracellular region, which can be used to enhance CAR-T Persistence of cells and/or enhanced cytotoxicity of CAR-T cells.

在優選的實施方式中，本發明的IL-15/IL-15Rα複合體可以包含IL-15N72D/IL-15Rα複合體，可以包含信號肽(IgE, IL-2等)，可以與CAR基本結構共表現。In a preferred embodiment, the IL-15/IL-15Rα complex of the present invention may comprise an IL-15N72D/IL-15Rα complex, may comprise a signal peptide (IgE, IL-2, etc.), and may share the basic structure of the CAR. Performance.

本發明的IL-15(N72D)-IL-15Rα結構是表現在免疫治療細胞表面的複合物。IL-15和IL-15Rα透過linker相連，其中IL-15Rα結構是指完整的IL-15Rα分子包括胞外區（含sushi domain）、跨膜區和胞內區。IL-15Rα可透過召集免疫治療細胞表面自身表現的IL-2Rβ和IL-2Rγc鏈，活化下游信號途徑信號比如JAK1/JAK3，Stat3/Stat5達到促進免疫治療細胞增殖，抑制免疫治療細胞凋亡的目的，從而獲得更好的治療效果。同時將該複合物限制在免疫治療細胞表面，避免活化免疫抑制型T細胞、NK和NKT細胞的增殖（比如Treg）可能帶來的免疫抑制作用，也能夠避免過度活化T細胞、NK和NKT細胞帶來的系統性或者全身性的毒副作用。 嵌合抗原受體（ CAR ） The IL-15(N72D)-IL-15Rα structure of the present invention is a complex expressed on the surface of immunotherapy cells. IL-15 and IL-15Rα are connected through linker, and the structure of IL-15Rα refers to the complete IL-15Rα molecule including extracellular domain (including sushi domain), transmembrane domain and intracellular domain. IL-15Rα can promote the proliferation of immunotherapy cells and inhibit the apoptosis of immunotherapy cells by recruiting the self-expressed IL-2Rβ and IL-2Rγc chains on the surface of immunotherapy cells and activating downstream signaling signals such as JAK1/JAK3, Stat3/Stat5 , so as to obtain a better therapeutic effect. At the same time, the complex is restricted to the surface of immunotherapy cells to avoid the possible immunosuppressive effects caused by the proliferation of activated immunosuppressive T cells, NK and NKT cells (such as Treg), and to avoid excessive activation of T cells, NK and NKT cells. systemic or systemic side effects. Chimeric Antigen Receptor ( CAR )

本發明例示性的採用Meso-CAR基本結構，即標靶間皮素的CAR結構進行表現IL-15/IL-15Rα複合體的CAR-T細胞的建構。The present invention exemplifies the construction of CAR-T cells expressing the IL-15/IL-15Rα complex using the basic structure of Meso-CAR, that is, the CAR structure targeting mesothelin.

在優選的實施方式中，所述IL-15/IL-15Rα複合體透過自剪切元件連接至基本CAR結構的C端，並在剪切後在CAR-T細胞的膜上表現。In a preferred embodiment, the IL-15/IL-15Rα complex is attached to the C-terminus of the basic CAR structure via a self-cleaving element, and is expressed on the membrane of the CAR-T cell after cleavage.

在優選的實施方式中，所述Meso-CAR由信號肽、Mesothelin單鏈可變區、CD28/4-1bb/ICOS、CD3ζ串聯而成。In a preferred embodiment, the Meso-CAR is composed of a signal peptide, a single-chain variable region of Mesothelin, CD28/4-1bb/ICOS, and CD3ζ in series.

具體地，本發明的嵌合抗原受體(CAR)包括細胞外結構域、跨膜結構域、和細胞內結構域。胞外結構域包括標的-特異性結合元件(也稱為抗原結合結構域)。細胞內結構域包括共刺激信號傳導區和ζ鏈部分。共刺激信號傳導區指包括共刺激分子的細胞內結構域的一部分。共刺激分子為淋巴細胞對抗原的有效反應所需要的細胞表面分子，而不是抗原受體或它們的配體。在優選的實施方式中，本發明的CAR包含來源CD28的共刺激信號分子。Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes the costimulatory signaling region and the zeta chain portion. A costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for effective lymphocyte responses to antigens. In a preferred embodiment, the CAR of the present invention comprises a costimulatory signaling molecule derived from CD28.

在CAR的胞外結構域和跨膜結構域之間，或在CAR的細胞質液結構域和跨膜結構域之間，可併入連接子。如本文所用的，術語“連接子”通常指起到將跨膜結構域連接至多肽鏈的胞外結構域或細胞質液結構域作用的任何寡肽或多肽。連接子可包括0-300個胺基酸，優選地2至100個胺基酸和最優選地3至50個胺基酸。A linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

在本發明的一個較佳的實施方式中，本發明提供的CAR的胞外結構域包括標靶Meso的抗原結合結構域。本發明的CAR當在T細胞中表現時，能夠基於抗原結合特異性進行抗原辨識。當其結合其關聯抗原時，影響腫瘤細胞，導致腫瘤細胞不生長、被促使死亡或以其他方式被影響，並導致患者的腫瘤負荷縮小或消除。抗原結合結構域優選與來自共刺激分子和ζ鏈中的一個或多個的細胞內結構域融合。In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes the antigen-binding domain of the target Meso. The CAR of the present invention is capable of antigen recognition based on antigen binding specificity when expressed in T cells. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to not grow, being driven to die, or otherwise being affected, and resulting in a reduction or elimination of the patient's tumor burden. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain.

如本文所用，“抗原結合結構域”“單鏈抗體片段”均指具有抗原結合活性的Fab片段，Fab’片段，F(ab’) 2片段，或單一Fv片段。Fv抗體含有抗體重鏈可變區、輕鏈可變區，但沒有恆定區，並具有全部抗原結合位點的最小抗體片段。一般的，Fv抗體還包含VH和VL結構域之間的多肽連接子，且能夠形成抗原結合所需的結構。抗原結合結構域通常是scFv(single-chain variable fragment)。scFv的大小一般是一個完整抗體的1/6。單鏈抗體優選是由一條核苷酸鏈編碼的一條胺基酸鏈序列。作為本發明的優選方式，所述scFv包含特異性辨識Meso的抗體，較佳地為人源化的單鏈抗體。 As used herein, "antigen-binding domain" and "single-chain antibody fragment" each refer to a Fab fragment, Fab' fragment, F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). The size of scFv is generally 1/6 of that of a complete antibody. A single chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain. As a preferred mode of the present invention, the scFv comprises an antibody that specifically recognizes Meso, preferably a humanized single-chain antibody.

對於絞鏈區和跨膜區(跨膜結構域)，CAR可被設計以包括融合至CAR的胞外結構域的跨膜結構域。在一個實施方式中，使用天然與CAR中的結構域之一相關聯的跨膜結構域。在一些例子中，可選擇跨膜結構域，或透過胺基酸置換進行修飾，以避免將這樣的結構域結合至相同或不同的表面膜蛋白的跨膜結構域，從而最小化與受體複合物的其他成員的相互作用。 序列 For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain naturally associated with one of the domains in the CAR is used. In some instances, transmembrane domains may be selected, or modified by amino acid replacement, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing complexation with receptors interactions with other members of the species. sequence

本申請序列表中涉及的各序列如下： SEQ ID NO 序列名稱 序列 1 IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 2 IL-15突變體 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 3 IL-15Rα ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 4 柔性連接子 SGGGSGGGGSGGGGSGGGGSGGGSLQ 5 IL-15 /IL-15α複合物 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 6 IL-15突變體/IL-15α複合物 MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 7 CAR建構物 MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 8 CAR MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSG 載體 The sequences involved in the sequence listing of the present application are as follows: SEQ ID NO sequence name sequence 1 IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 2 IL-15 mutant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 3 IL-15Rα ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 4 flexible linker SGGGSGGGGSGGGGSGGGGSGGGSLQ 5 IL-15/IL-15α complex MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 6 IL-15 mutant/IL-15α complex MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 7 CAR construct MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 8 CAR MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSG carrier

編碼期望分子的核酸序列可利用在本領域中已知的重組方法獲得，諸如例如透過從表現基因的細胞中篩選文庫，透過從已知包括該基因的載體中得到該基因，或透過利用標準的技術，從包含該基因的細胞和組織中直接分離。可選地，感興趣的基因可被合成生產。Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology to isolate directly from cells and tissues that contain the gene. Alternatively, the gene of interest can be produced synthetically.

本發明也提供了其中***本發明的表現匣的載體。源於反轉錄病毒諸如慢病毒的載體是實現長期基因轉移的合適工具，因為它們允許轉基因長期、穩定的整合並且其在子細胞中增殖。慢病毒載體具有超過源自致癌反轉錄病毒諸如鼠科白血病病毒的載體的優點，因為它們可轉導非增殖的細胞，諸如肝細胞。它們也具有低免疫原性的優點。The present invention also provides vectors into which the expression cassettes of the present invention are inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

簡單概括，通常可操作地連接本發明的表現匣或核酸序列至啟動子，並將其併入表現載體。該載體適合於複製和整合真核細胞。典型的選殖載體包含可用於調節期望核酸序列表現的轉錄和翻譯終止子、初始序列和啟動子。Briefly summarized, an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

本發明的表現建構體也可利用標準的基因傳遞方案，用於核酸免疫和基因療法。基因傳遞的方法在本領域中是已知的。見例如美國專利號5,399,346、5,580,859、5,589,466，在此透過引用全文併入。在另一個實施方式中，本發明提供了基因療法載體。The expression constructs of the present invention may also utilize standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides gene therapy vectors.

該核酸可被選殖入許多類型的載體。例如，該核酸可被選殖入如此載體，其包括但不限於質體、噬質體、噬菌體衍生物、動物病毒和黏接質體。特定的感興趣載體包括表現載體、複製載體、探針產生載體和定序載體。The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plastids, phages, phage derivatives, animal viruses, and cohesoplasts. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

進一步地，表現載體可以以病毒載體形式提供給細胞。病毒載體技術在本領域中是公知的並在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒學和分子生物學手冊中進行了描述。可用作載體的病毒包括但不限於反轉錄病毒、腺病毒、腺相關病毒、皰疹病毒和慢病毒。通常，合適的載體包含在至少一種有機體中起作用的複製起點、啟動子序列、方便的限制酶位點和一個或多個可選擇的標記(例如，WO01/96584；WO01/29058；和美國專利號6,326,193)。Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and US Pat. No. 6,326,193).

已經開發許多基於病毒的系統，用於將基因轉移入哺乳動物細胞。例如，反轉錄病毒提供了用於基因傳遞系統的方便的平台。可利用在本領域中已知的技術將選擇的基因***載體並包封入反轉錄病毒顆粒。該重組病毒可隨後被分離和傳遞至體內或離體的對象細胞。許多反轉錄病毒系統在本領域中是已知的。在一些實施方式中，使用腺病毒載體。許多腺病毒載體在本領域中是已知的。在一個實施方式中，使用慢病毒載體。A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and encapsulated into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

額外的啟動子元件，例如增強子，可以調節轉錄開始的頻率。通常地，這些位於起始位點上游的30-110bp區域中，儘管最近已經顯示許多啟動子也包含起始位點下游的功能元件。啟動子元件之間的間隔經常是柔性的，以便當元件相對於另一個被倒置或移動時，保持啟動子功能。在胸苷激酶(tk)啟動子中，啟動子元件之間的間隔可被增加隔開50bp，活性才開始下降。取決於啟動子，表現出單個元件可合作或獨立地起作用，以起動轉錄。Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to act cooperatively or independently to initiate transcription.

合適的啟動子的一個例子為即時早期巨細胞病毒(CMV)啟動子序列。該啟動子序列為能夠驅動可操作地連接至其上的任何多核苷酸序列高位準表現的強組成型啟動子序列。合適的啟動子的另一個例子為延伸生長因子-1α(EF-1α)。然而，也可使用其他組成型啟動子序列，包括但不限於類人猿病毒40(SV40)早期啟動子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)長末端重複(LTR)啟動子、MoMuLV啟動子、鳥類白血病病毒啟動子、艾伯斯坦-巴爾(Epstein-Barr)病毒即時早期啟動子、魯斯氏肉瘤病毒啟動子、以及人基因啟動子，諸如但不限於肌動蛋白啟動子、肌球蛋白啟動子、血紅素啟動子和肌酸激酶啟動子。進一步地，本發明不應被限於組成型啟動子的應用。誘導型啟動子也被考慮為本發明的一部分。誘導型啟動子的使用提供了分子開關，其能夠當這樣的表現是期望的時，打開可操作地連接誘導型啟動子的多核苷酸序列的表現，或當表現是不期望的時關閉表現。誘導型啟動子的例子包括但不限於金屬硫蛋白啟動子、糖皮質激素啟動子、孕酮啟動子和四環素啟動子。An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse breast cancer virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russell sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, Myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

為了評估CAR多肽或其部分的表現，被引入細胞的表現載體也可包含可選擇的標記基因或報導基因中的任一個或兩者，以便於從透過病毒載體尋求被轉染或感染的細胞群中鑑定和選擇表現細胞。在其他方面，可選擇的標記可被攜帶在單獨一段DNA上並用於共轉染程序。可選擇的標記和報導基因兩者的側翼都可具有適當的調節序列，以便能夠在宿主細胞中表現。有用的可選擇標記包括例如抗生素抗性基因，諸如neo等等。To assess the performance of a CAR polypeptide or portion thereof, the expression vector introduced into a cell may also contain either or both a selectable marker gene or a reporter gene to facilitate the search for transfected or infected cell populations through the viral vector Identify and select expressing cells. In other aspects, the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

報導基因用於鑑定潛在轉染的細胞並用於評價調節序列的功能性。通常地，報導基因為以下基因：其不存在於受體有機體或組織或由受體有機體或組織進行表現，並且其編碼多肽，該多肽的表現由一些可容易檢測的性質例如酶活性清楚表示。在DNA已經被引入受體細胞後，報導基因的表現在合適的時間下進行測定。合適的報導基因可包括編碼螢光素酶、β-半乳糖苷酶、氯黴素乙醯轉移酶、分泌型鹼性磷酸酶或綠色螢光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合適的表現系統是公知的並可利用已知技術製備或從商業上獲得。通常，顯示最高位準的報導基因表現的具有最少5個側翼區的建構體被鑑定為啟動子。這樣的啟動子區可被連接至報導基因並用於評價試劑調節啟動子-驅動轉錄的能力。Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property such as enzymatic activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is determined at an appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (eg, Ui-Tei et al., 2000 FEBS Letters 479 :79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs with a minimum of 5 flanking regions showing the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

將基因引入細胞和將基因表現入細胞的方法在本領域中是已知的。在表現載體的內容中，載體可透過在本領域中的任何方法容易地引入宿主細胞，例如，哺乳動物、細菌、酵母或昆蟲細胞。例如，表現載體可透過物理、化學或生物學手段轉移入宿主細胞。Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, an expression vector can be transferred into a host cell by physical, chemical or biological means.

將多核苷酸引入宿主細胞的物理方法包括磷酸鈣沉澱、脂質轉染法、基因槍、微注射、電穿孔等等。生產包括載體和/或外源核酸的細胞的方法在本領域中是公知的。見例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。將多核苷酸引入宿主細胞的優選方法為磷酸鈣轉染。Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, gene gun, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

將感興趣的多核苷酸引入宿主細胞的生物學方法包括使用DNA和RNA載體。病毒載體，特別是反轉錄病毒載體，已經成為最廣泛使用的將基因***哺乳動物例如人細胞的方法。其他病毒載體可源自慢病毒、痘病毒、單純皰疹病毒I、腺病毒和腺相關病毒等等。見例如美國專利號5,350,674和5,585,362。Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.

將多核苷酸引入宿主細胞的化學手段包括膠體分散系統，諸如大分子複合物、奈米膠囊、微球、珠；和基於脂質的系統，包括水包油乳劑、微胞、混合微胞和脂質體。用作體外和體內傳遞工具(delivery vehicle)的例示性膠體系統為脂質體(例如，人造膜囊)。Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids body. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).

在使用非病毒傳遞系統的情況下，例示性傳遞工具為脂質體。考慮使用脂質製劑，以將核酸引入宿主細胞(體外、離體(ex vivo)或體內)。在另一方面，該核酸可與脂質相關聯。與脂質相關聯的核酸可被封裝入脂質體的水性內部中，散佈在脂質體的脂雙層內，經與脂質體和寡核苷酸兩者都相關聯的連接分子附接至脂質體，陷入脂質體，與脂質體複合，分散在包含脂質的溶液中，與脂質混合，與脂質聯合，作為懸浮液包含在脂質中，包含在微胞中或與微胞複合，或以其他方式與脂質相關聯。與組合物相關聯的脂質、脂質/DNA或脂質/表現載體不限於溶液中的任何具體結構。例如，它們可存在於雙分子層結構中，作為微胞或具有“塌縮的(collapsed)”結構。它們也可簡單地被散佈在溶液中，可能形成大小或形狀不均一的聚集體。脂質為脂肪物質，其可為天然發生或合成的脂質。例如，脂質包括脂肪小滴，其天然發生在細胞質以及包含長鏈脂肪族烴和它們的衍生物諸如脂肪酸、醇類、胺類、胺基醇類和醛類的該類化合物中。Where non-viral delivery systems are used, exemplary delivery vehicles are liposomes. The use of lipid formulations is contemplated to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid can be encapsulated into the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via linker molecules associated with both the liposome and the oligonucleotide, entrapped in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise with lipids Associated. The lipid, lipid/DNA or lipid/expression vehicle associated with the composition is not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include lipid droplets, which occur naturally in the cytoplasm and in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

在本發明的一個優選地實施方式中，所述載體為慢病毒載體。 製劑 In a preferred embodiment of the present invention, the vector is a lentiviral vector. preparation

本發明提供了一種含有本發明的CAR-T細胞，以及藥學上可接受的載劑、稀釋劑或賦形劑。在一個實施方式中，所述製劑為液態製劑。優選地，所述製劑為注射劑。優選地，所述製劑中所述CAR-T細胞的濃度為1×10 3-1×10 8個細胞/ml，更優地1×10 4-1×10 7個細胞/ml。 The present invention provides a CAR-T cell containing the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells/ml, more preferably 1×10 4 -1×10 7 cells/ml.

在一個實施方式中，所述製劑可包括緩衝液諸如中性緩衝鹽水、硫酸鹽緩衝鹽水等等；碳水化合物諸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白質；多肽或胺基酸諸如甘胺酸；抗氧化劑；螯合劑諸如EDTA或谷胱甘肽；佐劑(例如，氫氧化鋁)；和防腐劑。本發明的製劑優選配製用於靜脈內施用。 治療性應用 In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration. therapeutic application

本發明包括用編碼本發明表現匣的慢病毒載體(LV)轉導的細胞(例如，T細胞)進行的治療性應用。轉導的T細胞可標標的腫瘤細胞的標誌物Meso，協同活化T細胞，引起T細胞免疫反應，從而顯著提高其對腫瘤細胞的毒殺效率。The present invention includes therapeutic applications of cells (eg, T cells) transduced with lentiviral vectors (LVs) encoding the expression cassettes of the present invention. The transduced T cells can be labeled with Meso, a marker of tumor cells, to synergistically activate T cells and cause T cell immune responses, thereby significantly improving their killing efficiency against tumor cells.

因此，本發明也提供了刺激對哺乳動物的標的細胞群或組織的T細胞-介導的免疫反應的方法，其包括以下步驟：給哺乳動物施用本發明的CAR-T細胞。Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the steps of: administering to the mammal a CAR-T cell of the present invention.

在一個實施方式中，本發明包括一類細胞療法，分離病人自體T細胞（或者異源供體），活化並進行基因改造產生CAR-T細胞，隨後注入同一病人體內。這種方式患移植物抗宿主病概率極低，抗原被T細胞以無MHC限制方式辨識。此外，一種CAR-T就可以治療表現該抗原的所有癌症。不像抗體療法，CAR-T細胞能夠體內複製，產生可導致持續腫瘤控制的長期持久性。In one embodiment, the present invention includes a type of cell therapy in which autologous T cells (or allogeneic donors) are isolated from a patient, activated and genetically engineered to produce CAR-T cells, and then infused into the same patient. In this way, the probability of graft-versus-host disease is extremely low, and the antigen is recognized by T cells in an MHC-free manner. In addition, a single CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

在一個實施方式中，本發明的CAR-T細胞可經歷穩固的體內T細胞擴展並可持續延長的時間量。另外，CAR介導的免疫反應可為過繼免疫療法步驟的一部分，其中CAR-修飾T細胞誘導對CAR中的抗原結合結構域特異性的免疫反應。例如，抗Meso的CAR-T細胞引起抗Meso陽性的細胞的特異性免疫反應。In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time. Alternatively, a CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-Meso CAR-T cells elicit a specific immune response against Meso-positive cells.

儘管本文公開的數據具體公開了包括抗-Meso scFv、樞紐和CD28跨膜區和胞內區和CD3ζ信號傳導結構域的慢病毒載體，但本發明應被解釋為包括對建構體組成部分中的每一個的任何數量的變化。Although the data disclosed herein specifically disclose lentiviral vectors comprising anti-Meso scFvs, hub and CD28 transmembrane and intracellular domains and CD3ζ signaling domains, the present invention should be construed to include reference to the construct components Any number of variations of each.

可治療的癌症包括沒有被血管化或基本上還沒有被血管化的腫瘤，以及血管化的腫瘤。癌症可包括非固態腫瘤(諸如血液學腫瘤，例如白血病和淋巴瘤)或可包括固態腫瘤。用本發明的CAR治療的癌症類型包括但不限於癌、胚細胞瘤和肉瘤，和某些白血病或淋巴惡性腫瘤、良性和惡性腫瘤、和惡性瘤，例如肉瘤、癌和黑色素瘤。也包括成人腫瘤/癌症和兒童腫瘤/癌症。Cancers that can be treated include tumors that are not vascularized or substantially not vascularized, as well as tumors that are vascularized. Cancers may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemic or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and pediatric tumors/cancers.

血液學癌症為血液或骨髓的癌症。血液學(或血原性)癌症的例子包括白血病，包括急性白血病(諸如急性淋巴細胞白血病、急性骨髓性白血病、急性骨髓性白血病和成髓細胞性、前髓細胞性、顆粒-單核細胞型、單核細胞性和紅白血病)、慢性白血病(諸如慢性髓細胞(顆粒細胞性)白血病、慢性骨髓性白血病和慢性淋巴細胞白血病)、真性紅血球增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(無痛和高等級形式)、多發性骨髓瘤、瓦爾登斯特倫氏巨球蛋白血症、重鏈疾病、骨髓增生異常綜合症、多毛細胞白血病和脊髓發育不良。Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, granulosa-monocyte type) , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

固態腫瘤為通常不包含囊腫或液體區的組織的異常腫塊。固態腫瘤可為良性或惡性的。不同類型的固態腫瘤以形成它們的細胞類型命名(諸如肉瘤、癌和淋巴瘤)。固態腫瘤諸如肉瘤和癌的例子包括纖維肉瘤、黏液肉瘤、脂肪肉瘤間皮瘤、淋巴惡性腫瘤、胰腺癌卵巢癌。Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that forms them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

在優選的實施方式中，可治療的癌症為Meso陽性腫瘤。In a preferred embodiment, the treatable cancer is a Meso positive tumor.

本發明的CAR-修飾T細胞也可用作對哺乳動物離體免疫和/或體內療法的疫苗類型。優選地，哺乳動物為人。The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

對於離體免疫，以下中的至少一項在將細胞施用進入哺乳動物前在體外發生：i)擴增細胞，ii)將編碼CAR的核酸引入細胞，和/或iii)冷凍保存細胞。For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into the mammal: i) expanding the cells, ii) introducing the CAR-encoding nucleic acid into the cells, and/or iii) cryopreserving the cells.

離體程序在本領域中是公知的，並在以下更完全地進行討論。簡單地說，細胞從哺乳動物(優選人)中分離並用表現本文公開的CAR的載體進行基因修飾(即，體外轉導或轉染)。CAR-修飾的細胞可被施用給哺乳動物接受者，以提供治療益處。哺乳動物接受者可為人，和CAR-修飾的細胞可相對於接受者為自體的。可選地，細胞可相對於接受者為同種異基因的、同基因的(syngeneic)或異種的。Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.

除了就離體免疫而言使用基於細胞的疫苗之外，本發明也提供了體內免疫以引起針對患者中抗原的免疫反應的組合物和方法。In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

本發明提供了治療腫瘤的方法，其包括施用給需要其的對象治療有效量的本發明的CAR-修飾的T細胞。The present invention provides methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the present invention.

本發明的CAR-修飾的T細胞可被單獨施用或作為藥物組合物與稀釋劑和/或與其他組分諸如IL-2、IL-17或其他細胞因子或細胞群結合施用。簡單地說，本發明的藥物組合物可包括如本文所述的標的細胞群，與一種或多種藥學或生理學上可接受載體、稀釋劑或賦形劑結合。這樣的組合物可包括緩衝液諸如中性緩衝鹽水、硫酸鹽緩衝鹽水等等；碳水化合物諸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白質；多肽或胺基酸諸如甘胺酸；抗氧化劑；螯合劑諸如EDTA或谷胱甘肽；佐劑(例如，氫氧化鋁)；和防腐劑。本發明的組合物優選配製用於靜脈內施用。The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may include a target cell population as described herein in association with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; Antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

本發明的藥物組合物可以以適於待治療(或預防)的疾病的方式施用。施用的數量和頻率將由這樣的因素確定，如患者的病症、和患者疾病的類型和嚴重度——儘管適當的劑量可由臨床試驗確定。The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate doses may be determined by clinical trials.

當指出“免疫學上有效量”、“抗腫瘤有效量”、“腫瘤-抑制有效量”或“治療量”時，待施用的本發明組合物的精確量可由醫師確定，其考慮患者(對象)的年齡、重量、腫瘤大小、感染或轉移程度和病症的個體差異。可通常指出：包括本文描述的T細胞的藥物組合物可以以10 4至10 9個細胞/kg體重的劑量，優選10 5至10 6個細胞/kg體重的劑量(包括那些範圍內的所有整數值)施用。T細胞組合物也可以以這些劑量多次施用。細胞可透過使用免疫療法中公知的注入技術(見例如Rosenberg等，NewEng.J. of Med.319:1676，1988)施用。對於具體患者的最佳劑量和治療方案可透過監測患者的疾病跡象並因此調節治療由醫學領域技術人員容易地確定。 When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) individual differences in age, weight, tumor size, degree of infection or metastasis, and condition. It may generally be noted that the pharmaceutical compositions comprising the T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight (including all integers within those ranges). value) application. The T cell composition can also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

對象組合物的施用可以以任何方便的方式進行，包括透過噴霧法、注射、吞咽、輸液、植入或移植。本文描述的組合物可被皮下、皮內、瘤內、結內、脊髓內、肌肉內、透過靜脈內(i.v.)注射或腹膜內施用給患者。在一個實施方式中，本發明的T細胞組合物透過皮內或皮下注射被施用給患者。在另一個實施方式中，本發明的T細胞組合物優選透過i.v.注射施用。T細胞的組合物可被直接注入腫瘤，淋巴結或感染位置。Administration of the composition to a subject can be carried out in any convenient manner, including by nebulization, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into tumors, lymph nodes or the site of infection.

在本發明的某些實施方式中，利用本文描述的方法或本領域已知的其他將T細胞擴展至治療性層級的方法活化和擴展的細胞，與任何數量的有關治療形式結合(例如，之前、同時或之後)施用給患者，所述治療形式包括但不限於用以下試劑進行治療：所述試劑諸如抗病毒療法、西多福韋和白細胞介素-2、阿糖胞苷(也已知為ARA-C)或對MS患者的那他珠單抗治療或對牛皮癬患者的厄法珠單抗治療或對PML患者的其他治療。在進一步的實施方式中，本發明的T細胞可與以下結合使用：化療、輻射、免疫抑制劑，諸如，環孢菌素、硫唑嘌呤、甲氨喋呤、麥考酚酯和FK506，抗體或其他免疫治療劑。在進一步的實施方式中，本發明的細胞組合物與骨髓移植、利用化療劑諸如氟達拉濱、外部光束放射療法(XRT)、環磷醯胺結合(例如，之前、同時或之後)而施用給患者。例如，在一個實施方式中，對象可經歷高劑量化療的標準治療，之後進行周邊血液幹細胞移植。在一些實施方式中，在移植後，對象接受本發明的擴展的免疫細胞的注入。在一個額外的實施方式中，擴展的細胞在外科手術前或外科手術後施用。In certain embodiments of the invention, cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to the therapeutic level, are combined with any number of relevant therapeutic modalities (eg, previously , concurrently or subsequently) to a patient in a form of treatment including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients. In a further embodiment, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutics. In a further embodiment, the cellular compositions of the invention are administered in combination with (eg, before, concurrently or after) bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide to the patient. For example, in one embodiment, a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

施用給患者的以上治療的劑量將隨著治療病症的精確屬性和治療的接受者而變化。人施用的劑量比例可根據本領域接受的實踐實施。通常，每次治療或每個療程，可將1×10 6個至1×10 10個本發明經修飾的T細胞(如，CAR-T細胞)，透過例如靜脈回輸的方式，施用於患者。 The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be carried out in accordance with art-accepted practice. Typically, 1 x 106 to 1 x 1010 modified T cells (eg, CAR-T cells) of the invention can be administered to a patient per treatment or per course of treatment, eg, by intravenous infusion .

本發明的主要優點包括： (a) 本發明CAR-T細胞的CAR結構中同時包含CAR的基本結構和IL-15/IL-15Rα複合體或IL-15突變體/IL-15Rα複合體，各自發揮功能，互不干擾。 (b) 體內實驗結果顯示本發明免疫細胞Meso-E1m1能明顯增強CAR-T細胞的細胞毒性和持久性，明顯優於Meso-E1。 (c) 體外實驗結果顯示，與Meso-1 CAR-T 比較，本發明免疫細胞Meso-E1m1在標的細胞和TGF-β1持續共培養情況下，明顯提高免疫細胞存活率，促進免疫細胞的擴增，減少TGF-β1對免疫細胞的抑制，同時增強CAR-T 細胞的細胞毒性和持久性。 (d) 與Meso-1 CAR-T 比較，本發明免疫細胞Meso-E1m1在體外暫態毒殺實驗中，明顯減少發炎因子TNF-α分泌。 (e) 本發明的免疫細胞可明顯增強NK細胞擴增。 The main advantages of the present invention include: (a) The CAR structure of the CAR-T cell of the present invention includes both the basic structure of the CAR and the IL-15/IL-15Rα complex or the IL-15 mutant/IL-15Rα complex, each of which functions without interfering with each other. (b) The results of in vivo experiments show that the immune cell Meso-E1m1 of the present invention can significantly enhance the cytotoxicity and persistence of CAR-T cells, which is significantly better than that of Meso-E1. (c) The results of in vitro experiments show that, compared with Meso-1 CAR-T, the immune cell Meso-E1m1 of the present invention significantly improves the survival rate of immune cells and promotes the expansion of immune cells when the target cells and TGF-β1 are continuously co-cultured. , reducing the suppression of immune cells by TGF-β1, while enhancing the cytotoxicity and persistence of CAR-T cells. (d) Compared with Meso-1 CAR-T, the immune cell Meso-E1m1 of the present invention significantly reduced the secretion of inflammatory factor TNF-α in the in vitro transient poisoning experiment. (e) The immune cells of the present invention can significantly enhance NK cell expansion.

下面結合具體實施例，進一步闡述本發明。應理解，這些實施例僅用於說明本發明而不用於限制本發明的範圍。下列實施例中未註明具體條件的實驗方法，通常按照常規條件，例如Sambrook等人，分子選殖：實驗室手冊(New York: Cold Spring Harbor Laboratory Press, 1989)中所述的條件，或按照製造廠商所建議的條件。除非另外說明，否則百分比和份數是重量百分比和重量份數。 實施例 1 從供體血液中分離 PBMC 和擴增 T 細胞 The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions, people such as Sambrook, molecular colonization: conditions described in laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacture conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise specified. Example 1 Isolation of PBMC and expansion of T cells from donor blood

從周邊血液中分離單核細胞，使用Ficoll進行密度梯度離心，並富集T細胞（EasySep human T cell enrichment kit，Stemcell Technologies），使用偶聯anti-CD3/CD28磁珠活化培養和擴增T細胞，細胞培養系統採用x-vivo 15(5% FBS，300IU/mL rhIL-2)，細胞培養在37℃，5% CO 2培養箱連續培養。 實施例 2 細胞培養及建構 Mononuclear cells were isolated from peripheral blood, subjected to density gradient centrifugation using Ficoll, and enriched for T cells (EasySep human T cell enrichment kit, Stemcell Technologies), and activated to culture and expand T cells using coupled anti-CD3/CD28 magnetic beads , the cell culture system used x-vivo 15 (5% FBS, 300IU/mL rhIL-2), and the cells were cultured at 37°C in a 5% CO 2 incubator for continuous culture. Example 2 Cell Culture and Construction

從ATCC獲得表現MSLN的細胞株： OVCAR3(人卵巢癌細胞株，ATCC HTB-161)，HCC70(人乳腺癌細胞株)，MDA-MB-231(人乳腺癌細胞株)，中MDA-MB-231MESO在MDA-MB231細胞基礎上透過慢病毒將MSLN抗原轉入，再經過單株選殖篩選後獲得高度表現MSLN的穩定細胞株。以上細胞按照ATCC指引配製培養基並培養細胞。 實施例 3 CAR 結構設計與轉導 Cell lines expressing MSLN were obtained from ATCC: OVCAR3 (human ovarian cancer cell line, ATCC HTB-161), HCC70 (human breast cancer cell line), MDA-MB-231 (human breast cancer cell line), MDA-MB- On the basis of MDA-MB231 cells, 231MESO transferred MSLN antigen into MDA-MB231 cells through lentivirus, and then obtained a stable cell line with high expression of MSLN after single clone selection. The above cells were prepared according to ATCC guidelines and the cells were cultured. Example 3 CAR structure design and transduction

本實施例例示性的採用Meso-CAR基本結構，即標靶間皮素的CAR結構進行表現IL-15/IL-15Rα複合物的CAR-T細胞建構。This example exemplifies the construction of CAR-T cells expressing the IL-15/IL-15Rα complex using the basic structure of Meso-CAR, that is, the CAR structure targeting mesothelin.

本實施例建構了二代和四代CAR，結構如表1所示。In this example, second- and fourth-generation CARs were constructed, and the structures are shown in Table 1.

CAR的核心結構包括CD8胞外信號肽，P4 scFv(特異性標靶間皮素的scFv)，CD8來源的樞紐區和CD8/CD28跨膜區，並採用CD28胞內段共刺激信號，建構了3種Meso-CAR。 表1 命名 共刺激信號 胞內活化區 增強子 Meso-1 CD28 3ζ N/A Meso-E1 CD28 3ζ IL-15/IL-15Rα Meso-E1m1 CD28 3ζ IL-15突變體/IL-15Rα The core structure of CAR includes CD8 extracellular signal peptide, P4 scFv (scFv that specifically targets mesothelin), CD8-derived hub region and CD8/CD28 transmembrane region, and uses CD28 intracellular segment costimulatory signal to construct a 3 Meso-CARs. Table 1 name costimulatory signal intracellular activation domain enhancer Meso-1 CD28 3ζ N/A Meso-E1 CD28 3ζ IL-15/IL-15Rα Meso-E1m1 CD28 3ζ IL-15 mutant/IL-15Rα

將3種Meso-CAR基因分別選殖至FUW慢病毒載體中，與慢病毒包封質體pMD2.G（Addgene,Plasmid#12259）和psPAX2（Addgene, Plasmid#12260）使用PEI轉染試劑轉入293T細胞中，表現載體，分別收集48小時和72小時病毒，超高速離心濃縮後，感染活化T細胞。The three Meso-CAR genes were cloned into the FUW lentiviral vector, and transfected with lentiviral encapsulated plasmids pMD2.G (Addgene, Plasmid#12259) and psPAX2 (Addgene, Plasmid#12260) using PEI transfection reagent. In 293T cells, the expression vector was collected for 48 hours and 72 hours, respectively, and after concentration by ultracentrifugation, activated T cells were infected.

結果顯示，利用三種Meso-CAR基因，成功建構了慢病毒載體。 實施例 4 CAR-T 細胞製備 - 慢病毒感染、 CAR 陽性率檢測和體外增殖 The results showed that the lentiviral vector was successfully constructed using three Meso-CAR genes. Example 4 CAR-T cell preparation - lentivirus infection, CAR positive rate detection and in vitro proliferation

分離純化的原代T細胞在活化48小時後，分別加入實施例3中濃縮的慢病毒，細胞感染72小時後檢測CAR陽性率，分別使用生物素標記MSLN 抗原作為一抗，APC-Streptavidin(BD)作為二抗，以及anti-IL-15Rα抗體檢測scFv和IL-15Rα表現。After 48 hours of activation, the isolated and purified primary T cells were added with the concentrated lentivirus in Example 3, and the CAR positive rate was detected 72 hours after cell infection. Biotin-labeled MSLN antigen was used as primary antibody, APC-Streptavidin (BD) ) as secondary antibody, and anti-IL-15Rα antibody to detect scFv and IL-15Rα expression.

流式檢測結果如圖4所示。The flow detection results are shown in Figure 4.

轉染後T細胞置於37℃，5%CO 2培養箱中連續培養，隔天補液，在第10天收取細胞，凍存、計數，計算體外增殖率，E1 CAR-T細胞與E1m1 CAR-T細胞的增殖無明顯差異。 實施例 5 體外毒殺實驗和細胞因子檢測 After transfection, T cells were continuously cultured in a 37°C, 5% CO 2 incubator, supplemented every other day, harvested on the 10th day, cryopreserved, counted, and calculated in vitro proliferation rate. E1 CAR-T cells were compared with E1m1 CAR-T cells. There was no significant difference in the proliferation of T cells. Example 5 In vitro poisoning experiment and cytokine detection

對實施例4中收取的T細胞進行體外毒殺實驗。透過RTCA方法，以1x10 4/孔將標的細胞平盤培養於96孔RTCA平盤，培養18小時後，CAR T與標的細胞OVCAR3按比例1:1,1:3,1:6共培養，連續培養1-2天，即時記錄標的細胞的生長狀況，檢測標的細胞的存活率，計算CAR-T細胞的毒殺效率。連續培養1-2天後，將RTCA平盤取出，離心取共培養上清液，凍存-20℃。 The T cells collected in Example 4 were subjected to in vitro poisoning experiments. Through the RTCA method, the target cells were cultured in a 96-well RTCA plate at 1×10 4 /well. After 18 hours of culture, CAR T and the target cells OVCAR3 were co-cultured at a ratio of 1:1, 1:3, and 1:6, continuously. After culturing for 1-2 days, the growth status of the target cells was recorded immediately, the survival rate of the target cells was detected, and the killing efficiency of the CAR-T cells was calculated. After 1-2 days of continuous culture, the RTCA plate was taken out, the co-culture supernatant was collected by centrifugation, and frozen at -20°C.

結果如圖5A所示，Meso-1，Meso-E1與Meso-E1m1 CAR-T細胞與OVCAR3標的細胞共培養下，有明顯毒殺，且三者之間毒殺無明顯差異，NT （Non-transduced T cell）分別與腫瘤細胞進行共培養時(E:T=1:1,1:3,1:6)對腫瘤細胞沒有明顯毒殺。The results are shown in Figure 5A, Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells co-cultured with OVCAR3-targeted cells had obvious poisoning, and there was no significant difference in poisoning between the three, NT (Non-transduced T cell) were co-cultured with tumor cells (E:T=1:1, 1:3, 1:6) without obvious toxicity to tumor cells.

按說明書推薦方法，使用HumanTh1/Th2 Cytokine kit Ⅱ（BD，Cat.551809）檢測共培養上清液細胞因子，先將共培養上清液解凍，配置mixed capture beads和Human Th1/Th2-Ⅱ PE Detection, 與樣品或標準品避光培育3小時，培育後，300g離心5min，甩掉上清液。加入100µl washing buffer 重新懸浮，震盪5min，流式細胞儀上機檢測。用FCAP Array v.3軟體數據分析。結果見圖5B所示，Meso-1，Meso-E1與Meso-E1m1 CAR-T細胞與OVCAR3標的細胞共培養的上清液中，IFN-γ含量無明顯差異；Meso-E1與Meso-E1m1 CAR-T細胞與OVCAR3標的細胞共培養上清液，TNF-α含量明顯低於Meso-1。HumanTh1/Th2 Cytokine kit Ⅱ (BD, Cat. 551809) was used to detect cytokines in the co-culture supernatant according to the method recommended in the instructions. First, the co-culture supernatant was thawed, and mixed capture beads and Human Th1/Th2-Ⅱ PE Detection were arranged. , Incubate with samples or standards in the dark for 3 hours, after incubation, centrifuge at 300g for 5min, and discard the supernatant. Add 100µl of washing buffer to resuspend, shake for 5min, and detect by flow cytometer. Data analysis was performed with FCAP Array v.3 software. The results are shown in Figure 5B, there is no significant difference in IFN-γ content in the supernatant of Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells co-cultured with OVCAR3 target cells; Meso-E1 and Meso-E1m1 CAR - The supernatant of T cells co-cultured with OVCAR3-targeted cells has significantly lower TNF-α content than Meso-1.

結果如圖6A所示，Meso-1，Meso-E1與Meso-E1m1CAR-T細胞與MDA-MB-231-MESO標的細胞共培養下，有明顯毒殺，且三者之間毒殺無明顯差異， NT （Non-transduced T cell）分別與腫瘤細胞進行共培養時(E:T=3:1,1:1,1:3)對腫瘤細胞沒有明顯毒殺。用HumanTh1/Th2 Cytokine kit Ⅱ（BD）檢測共培養上清液細胞因子，結果如圖6B， Meso-E1與Meso-E1m1 CAR-T細胞與MDA-MB-231-MESO標的細胞共培養上清液，TNF-α含量明顯低於Meso-1。 實施例 6 體內藥效研究 -1 The results are shown in Figure 6A, Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells co-cultured with MDA-MB-231-MESO target cells had obvious poisoning, and there was no significant difference in poisoning among the three, NT (Non-transduced T cell) did not significantly kill tumor cells when co-cultured with tumor cells (E:T=3:1, 1:1, 1:3). HumanTh1/Th2 Cytokine kit Ⅱ (BD) was used to detect cytokines in the co-culture supernatant, the results are shown in Figure 6B, Meso-E1 and Meso-E1m1 CAR-T cells and MDA-MB-231-MESO target cells co-culture supernatant , the content of TNF-α was significantly lower than that of Meso-1. Example 6 In vivo efficacy study -1

選取NOD小鼠，皮下注射5E6 HCC70細胞，連續檢測腫瘤負荷，待腫瘤處於高速生長時，分組，每組2-3隻小鼠，分組後一天尾靜脈注射200uL DPBS/鼠， 5E6 E1/E1m1-CAR-T/鼠，CAR-T細胞注射後第1天，取少量小鼠血檢測CAR-T細胞體內存活數量，其後每週取一次血樣，檢測CAR-T細胞各種表型，每週兩次檢測皮下瘤大小。NOD mice were selected, subcutaneously injected with 5E6 HCC70 cells, and the tumor burden was continuously detected. When the tumors were growing rapidly, they were divided into groups, 2-3 mice in each group, and 200uL DPBS/mouse was injected into the tail vein one day after grouping, 5E6 E1/E1m1- CAR-T/mouse, on the first day after CAR-T cell injection, a small amount of mouse blood was taken to detect the number of CAR-T cells in vivo, and then blood samples were taken once a week to detect various phenotypes of CAR-T cells. Subcutaneous tumor size was measured.

結果如圖7A所示，注射Meso-E1 CAR-T細胞與Meso-E1m1 CAR-T細胞的小鼠在D10出現較為明顯的腫瘤體積下降；在D17、D21、D24繼續觀察動物腫瘤體積，E1組動物腫瘤體積回升，E1m1組繼續下降。The results are shown in Figure 7A, the mice injected with Meso-E1 CAR-T cells and Meso-E1m1 CAR-T cells showed a significant decrease in tumor volume on D10; the tumor volume of the animals was continued to be observed on D17, D21, and D24, and the E1 group The tumor volume of animals recovered, and continued to decrease in the E1m1 group.

小鼠體重如圖7B所示，注射CAR-T細胞後，Meso-E1m1組小鼠的體重基本沒有變化。 實施例 7 體內藥效研究 -2 The body weight of the mice is shown in Figure 7B. After the injection of CAR-T cells, the body weight of the mice in the Meso-E1m1 group basically did not change. Example 7 In vivo efficacy study -2

選取NOD小鼠，皮下注射5E6 HCC70細胞，連續檢測腫瘤負荷，待腫瘤處於高速生長時，分組，每組2-3隻小鼠，分組後一天尾靜脈注射200uL DPBS/鼠，高（表示HD）低劑量（表示LD）兩組CAR-T，每隻小鼠注射劑量分別為5E6或2E6，CAR-T分別為 Meso-1/Meso-E1m1，CAR-T細胞注射後第1天，取少量小鼠血檢測CAR-T細胞體內存活數量，其後每週取一次血樣，檢測CAR-T細胞各種表型，每週兩次檢測皮下瘤大小。NOD mice were selected, subcutaneously injected with 5E6 HCC70 cells, and the tumor burden was continuously detected. When the tumors were growing rapidly, they were divided into groups, with 2-3 mice in each group. One day after grouping, 200uL DPBS/mouse was injected into the tail vein, high (indicates HD) Low-dose (indicated LD) two groups of CAR-T, each mouse was injected with a dose of 5E6 or 2E6, respectively, CAR-T was Meso-1/Meso-E1m1, the first day after CAR-T cell injection, a small amount of small Mouse blood was used to detect the number of viable CAR-T cells in vivo, then blood samples were taken once a week to detect various phenotypes of CAR-T cells, and the size of subcutaneous tumors was measured twice a week.

結果如圖8A所示，注射Meso-1 CAR-T細胞與Meso-E1m1 CAR-T細胞的小鼠在D10開始出現較為明顯的腫瘤體積下降；在D17、D21、D24繼續觀察動物腫瘤體積，高、低劑量Meso-1組動物腫瘤體積回升，高劑量E1m1組動物腫瘤體積沒有上升，低劑量E1m1組動物在D40開始腫瘤體積有復發，與Meso-1組比較，腫瘤體積明顯小。The results are shown in Figure 8A, the mice injected with Meso-1 CAR-T cells and Meso-E1m1 CAR-T cells started to have a more obvious decrease in tumor volume at D10; at D17, D21, and D24, the tumor volume of the animals continued to be observed, and the tumor volume was higher. . The tumor volume of the low-dose Meso-1 group recovered, but the tumor volume of the high-dose E1m1 group did not increase. The tumor volume of the low-dose E1m1 group started to recur at D40. Compared with the Meso-1 group, the tumor volume was significantly smaller.

如圖8B所示，各組小鼠體重無明顯變化。As shown in Figure 8B, there was no significant change in the body weight of the mice in each group.

如圖8C所示，注射CAR-T細胞後，各組中單隻小鼠的腫瘤體積變化，與Meso-1組比較，高劑量Meso-E1m1組動物腫瘤體積無復發，腫瘤體積均一併明顯小於Meso-1組。 實施例 8 體外藥效研究 — 多輪 毒殺 As shown in Figure 8C, after the injection of CAR-T cells, the tumor volume of a single mouse in each group changed. Compared with the Meso-1 group, the tumor volume of the high-dose Meso-E1m1 group had no recurrence, and the tumor volume was uniformly smaller than that of the Meso-1 group. Meso-1 group. Example 8 In vitro drug efficacy study — multiple rounds of poisoning

對實施例4中收取的T細胞進行體外多輪毒殺實驗。實驗方法見示意圖(圖9A)，將CAR-T細胞與標的細胞按1：3共培育，連續培養2-3天，在顯微鏡下觀察標的細胞的生長狀況，當其中一種CAR-T細胞將標的細胞全部裂解時，取一半T細胞檢測T細胞的表型及細胞數，並用胰蛋白酶消化標的細胞，將其計數作為標的細胞殘餘值，CAR-T細胞毒殺效率的計算公式： CAR-T細胞的毒殺效率=（腫瘤only組-實驗組)/腫瘤only組*100%，餘下一半細胞繼續與新的標的細胞共培養，按上述方法繼續連續毒殺。The T cells collected in Example 4 were subjected to multiple rounds of in vitro poisoning experiments. The experimental method is shown in the schematic diagram (Figure 9A). The CAR-T cells and the target cells were co-incubated at a ratio of 1:3 for 2-3 days. The growth of the target cells was observed under a microscope. When all the cells are lysed, take half of the T cells to detect the phenotype and cell number of the T cells, digest the target cells with trypsin, and count them as the residual value of the target cells. The calculation formula of the killing efficiency of CAR-T cells is: Poisoning efficiency=(tumor-only group-experimental group)/tumor-only group*100%, and the remaining half of the cells continue to co-culture with new target cells, and continue to kill continuously according to the above method.

結果如圖9B所示，各組CAR-T與OVCAR3共培養下(E:T=1:3)，Meso-E1/E1m1 在第三輪連續培養後顯示明顯毒殺優勢，如圖9C，顯示Meso-E1/E1m1在每一輪連續毒殺後，根據CAR陽性率和T細胞的計數，計算CAR-T細胞值，結果顯示Meso-E1/E1m1連續擴增明顯多於Meso-1 CAR-T, Meso-E1/E1m1之間無顯著差異。The results are shown in Figure 9B. Under the co-culture of CAR-T and OVCAR3 in each group (E:T=1:3), Meso-E1/E1m1 showed obvious poisoning advantage after the third round of continuous culture, as shown in Figure 9C, showing that Meso-E1/E1m1 -After each round of continuous poisoning of E1/E1m1, the CAR-T cell value was calculated according to the CAR positive rate and the count of T cells. The results showed that the continuous expansion of Meso-E1/E1m1 was significantly more than There was no significant difference between E1/E1m1.

結果如圖9D所示，各組CAR-T與MDA-MB231-Meso共培養下(E:T=1:3)，Meso-E1/E1m1 在第四輪連續培養後顯示明顯毒殺優勢。如圖9E，顯示Meso-E1/E1m1在每一輪連續毒殺後，根據CAR陽性率和T細胞的計數，計算CAR-T細胞值，結果顯示Meso-E1/E1m1連續擴增明顯多於Meso-1 CAR-T， Meso-E1/E1m1之間無顯著差異。 實施例 9 體外藥效研究 — TGF-β 存在下多輪毒殺 The results are shown in Figure 9D. Under the co-culture of CAR-T and MDA-MB231-Meso in each group (E:T=1:3), Meso-E1/E1m1 showed obvious poisoning advantage after the fourth round of continuous culture. Figure 9E shows that after each round of continuous poisoning of Meso-E1/E1m1, the CAR-T cell value was calculated according to the CAR positive rate and the count of T cells. The results showed that the continuous expansion of Meso-E1/E1m1 was significantly more than that of Meso-1. There was no significant difference between CAR-T, Meso-E1/E1m1. Example 9 In vitro pharmacodynamic study — multiple rounds of poisoning in the presence of TGF-β

對實施例4中收取的T細胞進行體外多輪毒殺實驗。實驗方案如圖9A所示。將CAR-T細胞與標的細胞按1：5共培育，培養過程中加入不同濃度重組TGF-β1，每兩天補加一次，連續培養2-3天，即時記錄標的細胞生長狀況，當其中一種CAR-T細胞將標的細胞全部裂解時，取一半T細胞檢測T細胞的表型，細胞數，胰蛋白酶消化標的細胞後，計數即得到CAR-T細胞的毒殺效率=(對照組-實驗組)/對照組*100%，對照組為腫瘤only組，餘下一半細胞繼續與標的細胞共培養，按上述方法繼續連續毒殺。The T cells collected in Example 4 were subjected to multiple rounds of in vitro poisoning experiments. The experimental protocol is shown in Figure 9A. The CAR-T cells were co-cultured with the target cells at a ratio of 1:5. During the culture, different concentrations of recombinant TGF-β1 were added, supplemented every two days, and cultured for 2-3 days. The growth status of the target cells was recorded immediately. When the CAR-T cells lyse all the target cells, take half of the T cells to detect the phenotype and cell number of the T cells. After the target cells are digested with trypsin, the killing efficiency of the CAR-T cells is obtained by counting = (control group - experimental group) /Control group*100%, the control group is the tumor-only group, and the remaining half of the cells continue to co-culture with the target cells, and continue to kill continuously according to the above method.

結果如圖10A所示，各組CAR-T與OVCAR3共培養下(E:T=1:5)，Meso-E1m1在第二(R2)、四輪(R4)連續培養後顯示明顯毒殺優勢。表1顯示每輪毒殺後根據細胞陽性率，T細胞計數值和稀釋係數，計算出Meso-1、Meso-E1m1 CAR-T細胞數。圖10B為根據表2和表3所繪製CAR-T細胞擴增曲線，結果顯示Meso-E1m1在連續毒殺第四輪，CAR-T細胞擴增明顯比Meso-1多；Meso-E1m1在添加1.25、2.5、5ng/mL TGFβ1培養條件下，在連續毒殺第二輪，CAR-T細胞擴增明顯比Meso-1多17倍、19倍、15倍；Meso-E1m1在添加1.25 ng/mL TGFβ1培養條件下，連續毒殺第四輪有明顯毒殺標的細胞能力明顯優於Meso-1，CAR-T細胞的擴增也明顯多於相同培養條件的Meso-1 CAR-T，實驗結果顯示Meso-E1m1 CAR-T能有效減少TGFβ抑制T細胞增殖、活化能力，從而減少TGFβ的免疫抑制作用。 表2 Meso-1   未處理組 +1.25ng/mL TGF-β1 +2.5ng/mL TGF-β1 +5ng/mL TGF-β1 R0 1.20E+05 1.20E+05 1.20E+05 1.20E+05 R1 2.57E+06 1.76E+06 1.51E+06 1.45E+06 R2 1.31E+07 6.66E+05 3.46E+05 5.36E+05 R4 8.48E+06 1.93E+05 9.22E+04 7.55E+04 表3 Meso-E1m1   未處理組 +1.25ng/mL TGF-β1 +2.5ng/mL TGF-β1 +5ng/mL TGF-β1 R0 1.20E+05 1.20E+05 1.20E+05 1.20E+05 R1 2.84E+06 1.59E+06 1.39E+06 1.37E+06 R2 2.16E+07 1.12E+07 6.76E+06 7.92E+06 R4 2.61E+07 1.27E+07 1.38E+06 7.71E+05 實施例 10 Annexin V 染色檢測細胞凋亡 The results are shown in Figure 10A, under the co-culture of CAR-T and OVCAR3 in each group (E:T=1:5), Meso-E1m1 showed obvious poison-killing advantage after the second (R2) and four rounds (R4) of continuous culture. Table 1 shows the number of Meso-1 and Meso-E1m1 CAR-T cells calculated according to the positive rate of cells, T cell count value and dilution factor after each round of poisoning. Figure 10B is the CAR-T cell expansion curve drawn according to Table 2 and Table 3. The results show that Meso-E1m1 in the fourth round of continuous poisoning, the CAR-T cell expansion is significantly more than that of Meso-1; Meso-E1m1 is added 1.25 , 2.5, 5ng/mL TGFβ1 culture conditions, in the second round of continuous poisoning, CAR-T cells expanded significantly more than Meso-1 17 times, 19 times, 15 times; Meso-E1m1 was cultured with 1.25 ng/mL TGFβ1 Under these conditions, the ability to kill cells with obvious poisoning targets in the fourth round of continuous poisoning was significantly better than that of Meso-1, and the expansion of CAR-T cells was also significantly more than that of Meso-1 CAR-T under the same culture conditions. The experimental results showed that Meso-E1m1 CAR -T can effectively reduce the ability of TGFβ to inhibit the proliferation and activation of T cells, thereby reducing the immunosuppressive effect of TGFβ. Table 2 Meso-1 untreated group +1.25ng/mL TGF-β1 +2.5ng/mL TGF-β1 +5ng/mL TGF-β1 R0 1.20E+05 1.20E+05 1.20E+05 1.20E+05 R1 2.57E+06 1.76E+06 1.51E+06 1.45E+06 R2 1.31E+07 6.66E+05 3.46E+05 5.36E+05 R4 8.48E+06 1.93E+05 9.22E+04 7.55E+04 Table 3 Meso-E1m1 untreated group +1.25ng/mL TGF-β1 +2.5ng/mL TGF-β1 +5ng/mL TGF-β1 R0 1.20E+05 1.20E+05 1.20E+05 1.20E+05 R1 2.84E+06 1.59E+06 1.39E+06 1.37E+06 R2 2.16E+07 1.12E+07 6.76E+06 7.92E+06 R4 2.61E+07 1.27E+07 1.38E+06 7.71E+05 Example 10 Annexin V staining to detect apoptosis

對實施例4中收取的T細胞進行體外毒殺實驗，CAR-T細胞與標的細胞(MDA-MB231-Meso)按1：2共培育，連續培養3天後，將CAR-T細胞移到新鮮的標的細胞中，繼續培養3天後，檢測CAR-T細胞的凋亡情況，培養過程中分兩組分別為不添加/添加圖示濃度的TGF-β1組。The T cells collected in Example 4 were subjected to in vitro poisoning experiments. The CAR-T cells were co-cultured with the target cells (MDA-MB231-Meso) at a ratio of 1:2. After continuous culture for 3 days, the CAR-T cells were moved to fresh In the target cells, after culturing for 3 days, the apoptosis of CAR-T cells was detected. During the culturing process, two groups were divided into groups without addition/addition of TGF-β1 at the concentration shown in the figure.

結果如圖10C所示，Annexin V和7-AAD雙陰細胞，Meso-E1m1 CAR-T活細胞比例最高為60%，加入不同濃度TGF-β，其存活率也沒有明顯變化，而Meso-1 CAR組在TGF-β1培養條件下，凋亡細胞隨著TGF-β1濃度而升高，早期凋亡和晚期細胞明顯增多，實驗結果顯示Meso-E1m1 CAR-T能有效減少TGFβ促進T細胞凋亡能力，從而減少TGFβ的免疫抑制作用。 實施例 11 CAR-T 細胞與 PBNK 細胞共培育 The results are shown in Figure 10C, Annexin V and 7-AAD double negative cells, Meso-E1m1 CAR-T cells have the highest proportion of viable cells of 60%, adding different concentrations of TGF-β, the survival rate did not change significantly, while Meso-1 Under the TGF-β1 culture condition in the CAR group, the apoptotic cells increased with the concentration of TGF-β1, and the early apoptotic cells and the late-stage cells increased significantly. The experimental results showed that Meso-E1m1 CAR-T can effectively reduce TGFβ and promote T cell apoptosis. ability to reduce the immunosuppressive effects of TGFβ. Example 11 Co-culture of CAR-T cells and PBNK cells

對實施例4中收取的T細胞與體外培養的自體PBNK細胞按1：1比例共培養，PBNK細胞先用CFSE標記，分別檢測48小時，96小時的PBNK細胞數量，採用流式檢測方法，檢測培養系統中FITC+ 細胞。The T cells collected in Example 4 were co-cultured with the autologous PBNK cells cultured in vitro at a ratio of 1:1. The PBNK cells were first labeled with CFSE, and the number of PBNK cells at 48 hours and 96 hours were detected respectively. The flow detection method was used. Detection of FITC+ cells in the culture system.

CFSE標記方法： PBNK細胞離心後用PBS重新懸浮至1E6/mL，加入終濃度4uM CFSE，室溫培育2分鐘，再加入培養基終止反應。CFSE labeling method: PBNK cells were resuspended to 1E6/mL with PBS after centrifugation, added with a final concentration of 4uM CFSE, incubated at room temperature for 2 minutes, and then added medium to stop the reaction.

自體PBNK來源於T細胞同一Donor的單核細胞，使用anti-CD56 microbeads 分離、純化，具體方法見美天旎的protocol。Autologous PBNK is derived from monocytes of the same Donor as T cells, and is isolated and purified using anti-CD56 microbeads. For specific methods, see Miltenyi's protocol.

實驗結果如圖11所示，E1 CAR-T 細胞與E1m1 CAR-T 細胞與PBNK細胞共培養48-72小時，與NT 細胞相比，E1和E1-m1 CAR-T細胞能明顯促進PBNK細胞的擴增。The experimental results are shown in Figure 11. E1 CAR-T cells were co-cultured with E1m1 CAR-T cells and PBNK cells for 48-72 hours. Compared with NT cells, E1 and E1-m1 CAR-T cells could significantly promote the growth of PBNK cells. Amplification.

在本發明提及的所有文獻都在本申請中引用作為參考，就如同每一篇文獻被單獨引用作為參考那樣。此外應理解，在閱讀了本發明的上述講授內容之後，本領域技術人員可以對本發明作各種改動或修改，這些等價形式同樣落於本申請所附申請專利範圍所限定的範圍。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended patent scope of the present application.

圖1顯示了本發明所述膜融合蛋白的作用機理示意圖。Figure 1 shows a schematic diagram of the mechanism of action of the membrane fusion protein of the present invention.

圖2A顯示本發明採用的CAR結構的示意圖。Figure 2A shows a schematic diagram of the CAR structure employed in the present invention.

圖2B顯示huIL-15/IL-15突變體複合物示意圖。Figure 2B shows a schematic diagram of the huIL-15/IL-15 mutant complex.

圖2C顯示huIL-15/IL-15突變體複合物的huIL-15Rα的結構。Figure 2C shows the structure of huIL-15Rα of the huIL-15/IL-15 mutant complex.

圖3A顯示MESO-E1m1基因的胺基酸序列。Figure 3A shows the amino acid sequence of the MESO-E1m1 gene.

圖3B顯示E1m1基因的胺基酸序列。Figure 3B shows the amino acid sequence of the E1m1 gene.

圖4顯示CAR-T細胞的陽性率檢測圖。Figure 4 shows the detection chart of the positive rate of CAR-T cells.

圖5A顯示Meso-1，Meso-E1和Meso-E1m1 CAR-T細胞與卵巢癌細胞OVCAR3體外共培養系統中，CAR-T細胞對標的細胞裂解率。Figure 5A shows the cell lysis rate of CAR-T cells against the target in the in vitro co-culture system of Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells and ovarian cancer cell OVCAR3.

圖5B顯示CAR-T細胞與卵巢癌OVCAR3體外共培養後， CAR-T細胞的細胞因子分泌。Figure 5B shows the cytokine secretion of CAR-T cells after co-culture of CAR-T cells with ovarian cancer OVCAR3 in vitro.

圖6A顯示Meso-1，Meso-E1和Meso-E1m1 CAR-T細胞與三陰性乳腺癌MDA-MB-231-MESO細胞體外共培養系統中，CAR-T細胞對標的細胞裂解率。Figure 6A shows the lysis rate of CAR-T cells against the target in the in vitro co-culture system of Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells and triple-negative breast cancer MDA-MB-231-MESO cells.

圖6B顯示CAR-T細胞與三陰性乳腺癌MDA-MB-231-MESO細胞體外共培養後，CAR-T細胞的細胞因子分泌。Figure 6B shows the cytokine secretion of CAR-T cells after in vitro co-culture of CAR-T cells with triple-negative breast cancer MDA-MB-231-MESO cells.

圖7A顯示Meso-E1和Meso-E1m1 CAR-T 體內藥效實驗-1， HCC70（乳腺癌細胞）腫瘤體積變化曲線。Figure 7A shows the in vivo efficacy experiment of Meso-E1 and Meso-E1m1 CAR-T-1, HCC70 (breast cancer cells) tumor volume change curve.

圖7B顯示Meso-E1和Meso-E1m1 CAR-T細胞體內藥效實驗-1，動物體重變化曲線。Figure 7B shows the in vivo efficacy test-1 of Meso-E1 and Meso-E1m1 CAR-T cells, and the change curve of animal body weight.

圖8A顯示Meso-1和Meso-E1m1 CAR-T體內藥效實驗-2，HCC70(乳腺癌細胞)腫瘤體積變化曲線。Figure 8A shows Meso-1 and Meso-E1m1 CAR-T in vivo efficacy experiment-2, HCC70 (breast cancer cells) tumor volume change curve.

圖8B顯示Meso-1和Meso-E1m1 CAR-T體內藥效實驗-2，動物體重變化。Figure 8B shows the in vivo efficacy test of Meso-1 and Meso-E1m1 CAR-T-2, and changes in animal body weight.

圖8C顯示Meso-1和Meso-E1m1 CAR-T體內藥效實驗-2，每組單隻小鼠的腫瘤體積變化。Figure 8C shows the in vivo efficacy of Meso-1 and Meso-E1m1 CAR-T experiments-2, the tumor volume changes of a single mouse in each group.

圖9A顯示多輪毒殺實驗設計方案。Figure 9A shows the multi-round poisoning experimental design.

圖9B、圖9C顯示體外共培養系統Meso-1，Meso-E1和Meso-E1m1 CAR-T細胞分別與卵巢癌細胞OVCAR3多輪毒殺後，標的細胞裂解率和CAR-T細胞擴增比較。Figure 9B and Figure 9C show the comparison of target cell lysis rate and CAR-T cell expansion after multiple rounds of poisoning between Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells in the in vitro co-culture system and ovarian cancer cells OVCAR3, respectively.

圖9D、圖9E顯示體外共培養系統Meso-1，Meso-E1和Meso-E1m1 CAR-T細胞分別與三陰性乳腺癌MDA-MB-231-MESO細胞多輪刺激後，標的細胞裂解率和CAR-T細胞擴增比較。Figure 9D and Figure 9E show the target cell lysis rate and CAR-T cells after multiple rounds of stimulation of the in vitro co-culture system Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells with triple-negative breast cancer MDA-MB-231-MESO cells, respectively - Comparison of T cell expansion.

圖10A顯示添加不同濃度TGF-β1體外共培養系統，Meso-1和Meso-E1m1 CAR-T細胞分別與卵巢癌細胞OVCAR3多輪刺激後，標的細胞的裂解率。Figure 10A shows the lysis rate of target cells after multiple rounds of stimulation of Meso-1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 after adding different concentrations of TGF-β1 in vitro co-culture system.

圖10B顯示添加不同濃度TGF-β1體外共培養系統，Meso-1和Meso-E1m1 CAR-T細胞分別與卵巢癌細胞OVCAR3多輪刺激後， CAR-T細胞擴增曲線。Figure 10B shows the expansion curve of CAR-T cells after multiple rounds of stimulation of Meso-1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 after adding different concentrations of TGF-β1 in vitro co-culture system, respectively.

圖10C顯示添加不同濃度TGF-β1體外共培養系統，Meso-1和Meso-E1m1 CAR-T細胞分別與卵巢癌細胞OVCAR3第二輪多輪毒殺後， CAR-T細胞凋亡的檢測。Figure 10C shows the detection of apoptosis of CAR-T cells after the second round of multi-round poisoning of Meso-1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 by adding different concentrations of TGF-β1 in vitro co-culture system.

圖11顯示不同CAR-T與PBNK共培養後，PBNK細胞在48和72小時擴增值。Figure 11 shows the expansion values of PBNK cells at 48 and 72 hours after co-culture of different CAR-Ts with PBNK.

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0015

Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0016

Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0017

Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0018

Claims (15)

一種嵌合抗原受體(CAR)建構物，其特徵在於，所述CAR建構物的結構如下式I或II所示， X-A-E    (I) E-A-X    (II) 式中， 各“-”獨立地為連接肽或肽鍵； X為標靶腫瘤抗原的CAR； A為自剪切元件； E為IL-15/IL-15Rα複合體。 A chimeric antigen receptor (CAR) construct, characterized in that the structure of the CAR construct is shown in the following formula I or II, X-A-E (I) E-A-X (II) In the formula, each "-" is independently a linking peptide or peptide bond; X is a CAR targeting tumor antigen; A is the self-shearing element; E is the IL-15/IL-15Rα complex. 如請求項1所述的CAR建構物，其特徵在於，所述的IL-15/IL-15Rα複合體的結構如下式III所示， L’-M-I-R    (III) 式中， 各“-”獨立地為連接肽或肽鍵； L’為無或信號肽； M為IL-15或其突變體； I為柔性連接子； R為IL-15Rα。 The CAR construct according to claim 1, wherein the structure of the IL-15/IL-15Rα complex is shown in the following formula III, L’-M-I-R (III) In the formula, each "-" is independently a linking peptide or peptide bond; L' is none or signal peptide; M is IL-15 or a mutant thereof; I is a flexible linker; R is IL-15Rα. 如請求項2所述的CAR建構物，其特徵在於，所述的IL-15突變體為IL-15N72D，其胺基酸序列如SEQ ID NO.: 2所示。The CAR construct according to claim 2, wherein the IL-15 mutant is IL-15N72D, and its amino acid sequence is shown in SEQ ID NO.: 2. 如請求項2所述的CAR建構物，其特徵在於，所述的IL-15Rα為完整的IL-15Rα元件，其胺基酸序列如SEQ ID NO.: 3所示。The CAR construct according to claim 2, wherein the IL-15Rα is a complete IL-15Rα element, and its amino acid sequence is shown in SEQ ID NO.: 3. 如請求項2所述的CAR建構物，其特徵在於，所述的IL-15/IL-15Rα複合體的胺基酸序列如SEQ ID NO.: 5或6所示。The CAR construct according to claim 2, wherein the amino acid sequence of the IL-15/IL-15Rα complex is shown in SEQ ID NO.: 5 or 6. 如請求項1所述CAR建構物，其特徵在於，所述的X(CAR)的結構如下式IV所示， L-scFv-H-TM-C-CD3ζ    (IV) 各“-”獨立地為連接肽或肽鍵； L為無或信號肽； scFv為標靶腫瘤抗原的抗體單鏈可變區； H為無樞紐區； TM為跨膜結構域； C為共刺激信號分子； CD3ζ為源於CD3ζ的細胞質液信號傳導序列。 The CAR construct according to claim 1, wherein the structure of the X (CAR) is shown in the following formula IV, L-scFv-H-TM-C-CD3ζ (IV) each "-" is independently a linking peptide or peptide bond; L is none or signal peptide; scFv is an antibody single-chain variable region targeting a tumor antigen; H is no hub area; TM is the transmembrane domain; C is a costimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ. 一種核酸分子，其特徵在於，所述核酸分子編碼請求項1所述的嵌合抗原受體(CAR)建構物，或者， 所述的核酸分子包含編碼標靶腫瘤抗原的CAR的第一核酸分子和編碼IL-15/IL-15Rα複合體的第二核酸分子。 A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the chimeric antigen receptor (CAR) construct described in claim 1, or, The nucleic acid molecule comprises the first nucleic acid molecule encoding the CAR of the target tumor antigen and the second nucleic acid molecule encoding the IL-15/IL-15Rα complex. 一種載體，其特徵在於，所述的載體含有請求項7所述的核酸分子。A vector, characterized in that the vector contains the nucleic acid molecule of claim 7. 一種基因工程化的免疫細胞，其特徵在於，所述的免疫細胞表現有請求項1所述的CAR建構物，或者 所述的免疫細胞表現有標靶腫瘤抗原的CAR和IL-15/IL-15Rα複合體。 A genetically engineered immune cell, characterized in that the immune cell expresses the CAR construct described in claim 1, or The immune cells express CAR and IL-15/IL-15Rα complexes targeting tumor antigens. 如請求項9所述的免疫細胞，其特徵在於，所述標靶腫瘤抗原的CAR和IL-15/IL-15Rα複合體獨立地表現於所述免疫細胞的細胞膜上。The immune cell according to claim 9, wherein the CAR and the IL-15/IL-15Rα complex targeting the tumor antigen are independently expressed on the cell membrane of the immune cell. 一種製劑，其特徵在於，所述製劑含有請求項1所述的嵌合抗原受體建構物、請求項3所述的核酸分子、請求項4所述的載體、或請求項5所述的免疫細胞，以及藥學上可接受的載劑。A preparation, characterized in that the preparation contains the chimeric antigen receptor construct described in claim 1, the nucleic acid molecule described in claim 3, the vector described in claim 4, or the immunization described in claim 5 cells, and a pharmaceutically acceptable carrier. 一種請求項1所述的嵌合抗原受體建構物、請求項7所述的核酸分子、請求項8所述的載體、或請求項9所述的免疫細胞、或請求項11所述的製劑的用途，用於製備預防和/或治療癌症或腫瘤的藥物或製劑。A chimeric antigen receptor construct according to claim 1, a nucleic acid molecule according to claim 7, a vector according to claim 8, or the immune cell according to claim 9, or the preparation according to claim 11 for the preparation of medicaments or preparations for the prevention and/or treatment of cancer or tumors. 一種製備請求項9所述的基因工程化的免疫細胞的方法，所述方法包括以下步驟： (a)     提供待改造的免疫細胞；和 (b)     將請求項7所述的核酸分子或請求項8所述的載體轉導入所述免疫細胞內，從而獲得所述基因工程化的免疫細胞。 A method for preparing the genetically engineered immune cell of claim 9, the method comprising the following steps: (a) providing immune cells to be engineered; and (b) Transduce the nucleic acid molecule described in claim 7 or the vector described in claim 8 into the immune cell, thereby obtaining the genetically engineered immune cell. 一種IL-15/IL-15Rα複合體的用途，用於製備一製劑，所述製劑用於增強CAR-T細胞的持久性和/或增強CAR-T細胞的細胞毒性， 其中，所述的IL-15/IL-15Rα複合體的結構如下式III所示， L’-M-I-R    (III) 式中， 各“-”獨立地為連接肽或肽鍵； L’為無或信號肽； M為IL-15或其突變體； I為柔性連接子； R為IL-15Rα， 其中，所述的IL-15Rα包含跨膜區和胞內區。 Use of an IL-15/IL-15Rα complex for the preparation of a preparation for enhancing the persistence of CAR-T cells and/or enhancing the cytotoxicity of CAR-T cells, Wherein, the structure of the IL-15/IL-15Rα complex is shown in the following formula III, L’-M-I-R (III) In the formula, each "-" is independently a linking peptide or peptide bond; L' is none or signal peptide; M is IL-15 or a mutant thereof; I is a flexible linker; R is IL-15Rα, Wherein, the IL-15Rα comprises a transmembrane region and an intracellular region. 一種治療疾病的方法，其特徵在於，所述方法包括給需要治療的對象施用適量的如請求項8所述的載體、如請求項9所述的免疫細胞、或如請求項11所述的製劑，其中所述疾病為癌症或腫瘤。A method for treating a disease, characterized in that the method comprises administering an appropriate amount of the carrier according to claim 8, the immune cell according to claim 9, or the preparation according to claim 11 to an object in need of treatment , wherein the disease is cancer or tumor.
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