WO2022199125A1 - 一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞 - Google Patents

一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞 Download PDF

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WO2022199125A1
WO2022199125A1 PCT/CN2021/135964 CN2021135964W WO2022199125A1 WO 2022199125 A1 WO2022199125 A1 WO 2022199125A1 CN 2021135964 W CN2021135964 W CN 2021135964W WO 2022199125 A1 WO2022199125 A1 WO 2022199125A1
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fusion protein
immune cell
cells
chimeric antigen
autocrine
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French (fr)
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谢海涛
马丽雅
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深圳市先康达生命科学有限公司
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the technical field of immune cells, in particular to immune cells expressing chimeric antigen receptors, in particular to an immune cell that autocrines IL-15 and anti-PD1 fusion proteins.
  • Tumor refers to a new organism (neogrowth) formed by the proliferation of local tissue cells under the action of various tumorigenic factors, because this new organism is mostly space-occupying lumpy protrusions, also known as neoplasms. .
  • malignant tumors are easy to metastasize, easy to relapse after treatment, and extremely difficult to cure under certain special microenvironments.
  • IL-15 plays a critical role in T cells, NK cells and their development, homeostasis and function, and also has various functions on B cells, dendritic cells (DC), macrophages and mast cells .
  • IL-15 is a member of the cytokine 4- ⁇ -helical bundle family with a molecular weight of 14-15 kDa and 114 amino acids.
  • IL-15 has two isotypes: 1SSP: a short signal peptide (SSP, short signal peptide) consisting of 21 amino acids.
  • LSP long signal peptide
  • LSP-IL-15 is secreted extracellularly as an immunomodulator.
  • IL-15 and IL-15R ⁇ are co-expressed by antigen-presenting cells (monocytes and dendritic cells).
  • IL-15 is widely expressed in a variety of cells, including monocytes, macrophages, DC cells, fibroblasts, epithelial cells, and skeletal muscle cells, but does not express the IL-15 cytokine in T cells.
  • IL-15 binds to high-affinity ⁇ receptors expressed on antigen-presenting cells to form IL15R ⁇ ; IL15R ⁇ presents IL-15 to IL-2/
  • the 15R ⁇ dimer forms a ternary complex. It can activate JAK and STAT model pathways, and has the functions of promoting the proliferation and activation of target cells, and increasing the secretion level of IFN- ⁇ and TNF- ⁇ .
  • PD-1 programmed death receptor 1
  • CD279 cluster of differentiation 279
  • PD-1 is an important immunosuppressive molecule. Regulates the immune system and promotes self-tolerance by downregulating the immune system's response to human cells, as well as by suppressing T-cell inflammatory activity. This prevents autoimmune diseases, but it also prevents the immune system from killing cancer cells.
  • PD-1 is a 288 amino acid type I membrane protein, which was originally cloned from apoptotic mouse T cell hybridoma 2B4.11, targeting PD-1 for immunomodulatory anti-tumor, anti-infection, and anti-self Immune diseases and organ transplantation survival are of great significance.
  • the ligand PD-L1 of PD-1 can also be used as a target, and the corresponding antibody can also play the same role.
  • the combination of PD-1 and PD-L1 initiates the programmed death of T cells, enabling tumor cells to obtain immune escape.
  • Anti-PD-1 therapy relieves the immunosuppressive effect, activates T cell function, and enhances the immune surveillance and killing ability of T cells against tumors by binding PD-1 and blocking the combination of PD-1 with PD-L1 and PD-L2. Generate a tumor immune response.
  • An immune cell that autocrines IL-15 and anti-PD1 fusion protein, which can autocrine a fusion protein of IL-15 and anti-PD1 after gene editing, and the immune cell expresses chimeric antigen receptor at the same time; the fusion protein It can improve the activity of immune cells and the killing effect on tumors.
  • the fusion protein and the chimeric antigen receptor gene are realized by constructing expression cassettes, and the number of constructed expression cassettes is one or more; further, the vector delivery method when constructing the expression cassettes These include lentiviruses, retroviruses, common plasmids, episomes, nano-delivery systems, electrotransduction or transposons.
  • the fusion protein of autocrine IL-15 and anti-PD1 is expressed in series in the order of anti-PD1, G4S*4 Linker, IL-15N72D, G4S*4 Linker and IL-15RaSu, and the The gene-edited immune cells express the fusion protein and are influenced by the autocrine fusion protein.
  • the gene-edited immune cells combine the hyperagonist protein of IL-15 and IL15RaSu and the fusion protein of anti-PD1 and successfully obtain a chimeric antigen receptor that secretes the fusion protein, such as T cells (Chimeric antigen receptor CAR). -T).
  • the chimeric antigen receptor is expressed as a chimeric antigen receptor targeting a target or targets.
  • the target of the chimeric antigen receptor includes one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUCl, PSMA and EBV .
  • the binding region of the chimeric antigen receptor and the target can be scFv, Fab or a combination of scFv and Fab; wherein, the structure of the scFv region can be any single-chain antibody, single-chain variable fragment ( scFv) and Fab fragments, etc. one or more substitutions.
  • the chimeric antigen receptor comprises a leader sequence, a scFv that recognizes a tumor-associated antigen, a hinge region and a transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3Zeta; wherein the scFv is an anti-specific scFv of type antibody; hinge region and transmembrane domain are CD28, or CD8 hinge region and transmembrane domain; intracellular costimulatory domain is CD28, CD137 (4-1BB) or ICOS intracellular costimulatory domain.
  • the binding region of the chimeric antigen receptor and the target can be a bispecific antibody that binds to one target, or a bispecific antibody that binds to two targets, or two or more chimeric antigens.
  • Receptors are formed across membranes and recognize different targets.
  • the structure of the chimeric antigen receptor includes one or more of signal peptide CD8SP, transmembrane domain CD8Hinger, CD8TM, intracellular activation element 4-1BB and CD3Zeta.
  • the segmentation method between the chimeric antigen receptor and the fusion protein of autocrine IL-15 and anti-PD1 is a protein cleavage functional element, and in multiple expression boxes, they can be expressed independently or separately Delivery without fragmentation; wherein the protein cleavage functional element is T2A, P2A, E2A, F2A or IRES.
  • the vector for gene transfer of immune cells into the chimeric antigen receptor includes lentivirus, retrovirus, common plasmid, episome, nano-delivery system, electrotransduction, transposon or other delivery system.
  • the immune cells include T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, TCR-T cells, or other tumor killer cells.
  • the present invention has the following beneficial effects:
  • the immune cells of autocrine IL-15 and anti-PD1 fusion proteins provided by the present invention express chimeric antigen receptors and can specifically recognize the targeted tumor cell surface antigens; the immune cells combine IL-15 and IL15RaSu
  • the fusion protein of hyperkinetic protein and anti-PD1, and the fusion protein is successfully secreted by immune cells, so as to enhance the proliferation ability, anti-apoptotic ability and tumor killing ability of immune cells; the immune cell killing effect of the present invention is precise, It is safer, less prone to recurrence, and improves the quality of life of patients.
  • Figure 1 is the structure design diagram of fusion protein and amino acid sequence in immune cells; among them, the fusion protein structure in A# ⁇ D#; 1# ⁇ 4# is the structure design diagram of amino acid sequence;
  • FIG. 2 shows the results of the flow detection of target cell phenotypes; among them, HGC-27-CLDN18.2 cells correspond to FTC-CLDN18.2 and APC-PD-L1 flow detection charts, correspondingly, HGC-27 cells also correspond to FTC- CLDN18.2 and APC-PD-L1 flow detection chart;
  • Figures 3a and 3b are histograms of secretory CAR-T fusion protein secretion;
  • Figure 3a is a histogram corresponding to Secretory PD1-scFv;
  • Figure 3b is a histogram corresponding to Secretory hIL15/Ra
  • Figure 4 shows the growth curve of secreted CAR-T expansion
  • Figures 5a, 5b, 5c, 5d, 5e, and 5f are the flow data of CAR-T cell phenotype;
  • Figure 5a is the flow chart of T cell phenotype;
  • Figure 5b is the flow chart of CAR-T CLDN18.2 cell phenotype Figure;
  • Figure 5c is the flow chart of CAR-T CLDN18.2-il-15/Ra cell phenotype;
  • Figure 5d is the flow chart of CAR-T CLDN18.2-antiPD1 cell phenotype;
  • Figure 5e is CAR-T CLDN18.
  • Figure 5f is the NC (blank control) phenotype flow chart;
  • the abscissa APC channel in the figure represents the expression of CD3, the right side is positive relative to the left side;
  • the ordinate PE channel in the figure Indicates the expression of PD-L1, and the upper part is positive relative to the lower part.
  • Figures 6a and 6b are the in vitro tumoricidal function evaluation curves of CAR-T cells; among them, Figure 6a is the in vitro tumoricidal function evaluation curve of the corresponding cells to HGC-27 target cells; Figure 6b is the corresponding cells to HGC-27-CLDN18. 2. The evaluation curve of the tumoricidal function of target cells in vitro;
  • Fig. 7 is the survival curve of CAR-T animal experiment.
  • the present invention provides an immune cell that autocrines IL-15 and anti-PD1 fusion protein, which can autocrine the fusion protein of IL-15 and anti-PD1 after gene editing, and the immune cell expresses chimeric antigen at the same time Receptor; the fusion protein can improve the activity of immune cells and the killing effect on tumors.
  • the above-mentioned fusion protein of autocrine IL-15 and anti-PD1 is expressed in series according to the sequence of anti-PD1, G4S*4 Linker, IL-15N72D, G4S*4 Linker and IL-15RaSu.
  • the fusion protein is expressed and influenced by the autocrine fusion protein.
  • Immune cells themselves do not express the fusion proteins mentioned above, but in order for immune cells to secrete the fusion proteins, corresponding gene editing is required, such as CAR-T, CAR-NK, TCR-T, IPS, etc.
  • the tumor-treated cells have already undergone corresponding gene editing, and then they secrete fusion proteins for corresponding gene editing. Such cells are collectively referred to here as gene-edited immune cells.
  • the immune cells of autocrine IL-15 and anti-PD1 fusion protein provided by the present invention combine IL-15 and IL15RaSu hyperagonist protein and anti-PD1 fusion protein, and successfully obtain a chimeric antigen receptor that secretes the fusion protein. body, such as T cells (Chimeric antigen receptor CAR-T).
  • the anti-PD1 in the above fusion protein is anti-PD1 VL, anti-PD1 VH or anti-PD1 VL combined with anti-PD1 VH.
  • the immune cells express chimeric antigen receptors, eg, CAR cells.
  • Chimeric antigen receptor expression can be a chimeric antigen receptor targeting one target or multiple targets, eg, CAR cells.
  • the target of the chimeric antigen receptor can also be one of idiotype CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUCl, PSMA and EBV or several.
  • the binding region of the chimeric antigen receptor and the target can be scFv, Fab or a combination of scFv and Fab; wherein, the structure of the scFv region can be any single-chain antibody, single-chain variable fragment (scFv) and Fab fragment of any target. one or more substitutions.
  • the above-mentioned chimeric antigen receptor comprises a leader sequence, a scFv that recognizes a tumor-associated antigen, a hinge region and a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular activation signal CD3 ⁇ .
  • scFv is anti-idiotype antibody scFv
  • hinge region and transmembrane domain are CD28 or CD8 hinge region and transmembrane domain
  • intracellular costimulatory domain is CD28 or CD137 (4-1BB) or ICOS intracellular costimulatory domain.
  • the binding region of the chimeric antigen receptor and the target can be a bispecific antibody that binds to one target, or a bispecific antibody that binds two targets, or two or more chimeric antigen receptors are formed across the membrane respectively. and identify different targets.
  • the structure of the chimeric antigen receptor includes one or more of signal peptide CD8SP, transmembrane domain CD8Hinger, CD8TM, intracellular activation element 4-1BB and CD3Zeta.
  • the segmentation method between the chimeric antigen receptor and the fusion protein with autocrine IL-15 and anti-PD1 is a protein cleavage functional element.
  • the protein cleavage functional element can be T2A, P2A, E2A, F2A or IRES.
  • the gene of fusion protein and chimeric antigen receptor is realized by constructing expression cassettes, and the number of constructed expression cassettes is one or more; when constructing expression cassettes, vector delivery methods include lentivirus, retrovirus, common plasmid , episomes, nano-delivery systems, electrotransduction or transposon; that is, vectors for gene transfer of immune cells into chimeric antigen receptors include lentiviruses, retroviruses, common plasmids, episomes, nano-delivery system, electrotransduction, transposon or other delivery system.
  • the immune cells of the present invention include T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, TCR-T cells or other tumor killer cells.
  • the above-mentioned autocrine IL-15 and anti-PD1 fusion protein immune cells can be prepared into biological preparations, and the biological preparations are pharmaceutically acceptable carriers, diluents or excipients.
  • Administration of the biologic can be carried out in any convenient manner, including by nebulization, injection, swallowing, infusion, implantation or transplantation.
  • the biological preparation can be used in drugs for preventing and/or treating solid tumors.
  • the immune cells of the autocrine IL-15 and anti-PD1 fusion proteins provided by the present invention express chimeric antigen receptors and can specifically recognize the idiotype of anti-autoantibodies; the immune cells combine the supernatants of IL-15 and IL15RaSu.
  • the fusion protein of agonist protein and anti-PD1 is successfully secreted by immune cells, so as to enhance the proliferation ability, anti-apoptosis ability and tumor killing ability of immune cells; in addition, the immune cells of the present invention can also be specific
  • the IL-15 and anti-PD1 fusion protein is secreted by the anti-injury agent, and the killing effect is precise, the safety is higher, the recurrence is not easy, and the quality of life of the patients is improved.
  • the fusion protein structure such as A# ⁇ D# was designed and designed into CAR-T-CLDN18.2; among them, 1 # is the control CAR-T, 2# ⁇ 4# are the secreted CAR-T. in:
  • hIL15 The amino acid sequence of hIL15 is: METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.
  • hIL15RaSu The amino acid sequence of hIL15RaSu is: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.
  • Anti-PD1 VH amino acid sequence is: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS.
  • Anti-PD1 VL amino acid sequence is: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK.
  • the amino acid sequence of Linker is: GSSSSGSSSSGSSSSGSSSS.
  • the structure of the chimeric antigen receptor includes one or more of the signal peptide CD8SP, the transmembrane domain CD8Hinger, CD8TM, the intracellular activation element 4-1BB and CD3Zeta, as shown in Figure 1.
  • Construction of secreted CAR-T cells and in vitro functional tests include the following steps:
  • the base sequence expressing CLDN18.2 was cloned into the backbone of PHBLV lentiviral vector and placed under the promoter of EF1 ⁇ (EF-1 ⁇ ) to form PHBLV-EF1 ⁇ -CLDN18.2, PHBLV-EF1 ⁇ -CLDN18.2, slow Viral envelope plasmid pMD2.G (Addgene, Plasmid #12259) and lentiviral packaging plasmid psPAX2 (Addgene Plasmid #12260) and other three plasmids were transferred into the lentiviral complete expression vector prepared in 293T cells using Lipofectamine3000; the virus supernatants were collected at 48h and 72h respectively, and the collected virus supernatants were concentrated by ultracentrifugation (Merck Millipore); the concentrated virus can be used to infect HGC-27, and finally the HGC-27 cell line overexpressing CLDN18.2 is obtained, which is named as HGC-27-CLDN18.2.
  • the detection results of HGC-27-CLDN18.2 cells expressing CLDN18.2 are shown in Figure 2, in which the detection chart corresponding to HGC-27 is a control chart; according to the detection results corresponding to HGC-27-CLDN18.2 and HGC-27 , it can be seen from the two vertical graphs of part a of the dotted box in Figure 2 that the results of detecting the expression level of CLDN18.2 antigen in the FITC channel show that the expression of CLDN18.2 in HGC-27 is negative (the peak graph is on the left side of the vertical line I), HGC- The expression of CLDN18.2 in 27-CLDN18.2 is positive (the peak graph is on the right side of the vertical line I); from the two vertical graphs in the dashed box b in Figure 2, the results of detecting the expression level of PD-L1 in the APC channel show that HGC- 27 and HGC-27-CLDN18.2 were both positive for PD-L1 expression (peak plots are located to the
  • T cells were isolated from donor peripheral blood, subjected to density gradient centrifugation using the ficol method, and T cells were enriched with T cell sorting kits, such as CD3 MicroBeads, human-lyophilized, or 130-097-043, and using conjugated Anti-CD3/anti-CD28 magnetic beads activate the culture and expansion of T cells;
  • T cell sorting kits such as CD3 MicroBeads, human-lyophilized, or 130-097-043, and using conjugated Anti-CD3/anti-CD28 magnetic beads activate the culture and expansion of T cells;
  • T cells When T cells were cultured, use TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) medium containing 10% FBS, 2mM L-glutamine and 100IU/ml rhIL2. Cells were cultured at 37°C, 5 %CO 2 incubator.
  • the fusion protein in the B# ⁇ D# sequence in the structural design of the first fusion protein above was expressed and purified by the CHO fusion protein expression system and used as an ELISA to detect the positive control standard of protein secretion in 1# ⁇ 4#, and collect the CAR -T culture supernatant, and the protein secretion in the cell culture supernatant was detected, and the detection data were shown in Figures 3a and 3b. It can be seen from Figures 3a and 3b that CART-CLDN18.2-IL15/Ra, CART-CLDN18.2-antiPD1, CART-CLDN18.2-15&PD1 can normally secrete proteins, and have basically the same protein secretion efficiency.
  • CAR-T prepared by lentiviral packaging
  • the obtained CAR-T proliferation is shown in Figure 4.
  • CART-CLDN18.2-IL15/Ra CART-CLDN18.2- AntiPD1 and CART-CLDN18.2 have higher cell proliferation multiples, which proves that they have better cell proliferation ability.
  • HGC-27-CLDN18.2 and HGC-27 cells as positive and negative target cells, respectively, the in vitro tumoricidal function of CAR-T was verified by flow assay.
  • the test results are shown in Figures 6a and 6b.
  • the test results show that, in contrast, CART-CLDN18.2-15&PD1 secreting fusion proteins has the strongest killing effect on HGC-27-CLDN18.2 positive target cells.
  • mice Twenty-four 6-8-week-old NSG mice (body weight 18-22 g) were taken, and after being adapted to feeding for one week, the HGC-27-CLDN18.2 positive tumor cell line was subcutaneously inoculated, and each mouse was inoculated with 1*10 7 tumor cells , closely observe the animal status, use vernier calipers to measure the tumor volume of mice every three days, when the tumor volume reaches 100mm 3 , after random grouping according to the weight and tumor size of mice, CAR-T cells or control T cells are infused through the tail vein. The detailed administration method, dosage and route of administration are shown in Table 2.
  • CART-CLDN18.2-15&PD1 secreted CAR-T can significantly prolong the survival of mice.
  • CAR-T autocrine IL-15 and anti-PD1 fusion protein has stronger proliferative ability than CAR-T that does not secrete other cytokines or CAR-T that only secretes one cytokine In vitro and in vivo tumoricidal activity of tumors.

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Abstract

提供了一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞。该免疫细胞可自分泌IL-15与anti-PD1融合蛋白，能够提高免疫细胞的活性、增强免疫细胞的增殖能力、抗凋亡能力和对于肿瘤的杀伤能力。该免疫细胞按照anti-PD1、G4S*4 Linker、IL-15N72D、G4S*4 Linker和IL-15RaSu顺序串联表达融合蛋白。

Description

一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞 技术领域
本发明涉及免疫细胞技术领域，特别涉及表达嵌合抗原受体的免疫细胞，具体涉及一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞。
 背景技术
肿瘤（tumour）是指机体在各种致瘤因子作用下，局部组织细胞增生所形成的新生物（neogrowth），因为这种新生物多呈占位性块状突起，也称赘生物（neoplasm）。其中，恶性肿瘤因易发生转移，治疗后易复发且在某些特殊的微环境下导致极难治愈。
IL-15 在 T细胞、NK细胞及其发育、稳态和功能中起着至关重要的作用，并且还对B细胞、树突状细胞（DC）、巨噬细胞和肥大细胞具有各种功能。IL-15是细胞因子4-α-螺旋束家族的成员，分子量为14-15kDa，含有114个氨基酸。IL-15具有两种同种类型：①SSP：包含21个氨基酸组成的较短的信号肽（SSP，short signal peptide），SSP型IL-15被充分翻译但并不分泌，因此，它的活动范围被限制在细胞质和细胞核，可能在其转录调控中起着重要作用；②LSP：包含48个氨基酸组成的更长的信号肽（LSP，long signal peptide），LSP-IL-15是作为免疫调节剂被分泌至胞外的。IL-15以及IL-15Rα由抗原呈递细胞（单核细胞和树突细胞）协同表达。IL-15广泛表达于多种细胞中，细胞类型包括单核细胞、巨噬细胞、DC细胞、成纤维细胞、上皮细胞和骨骼肌细胞，但在T 细胞中并不表达IL-15细胞因子。
IL-15与抗原受体的结合方式为反式呈递方式：IL-15与表达在抗原提呈细胞上高亲和力的α受体结合，形成IL15Rα；IL15Rα将IL-15递呈给IL-2/15Rβγ二聚体形成三元复合物。可激活JAK和STAT型号通路，并具有促进靶细胞增殖与活化、IFN-γ、TNF-α分泌水平提升等功能。
PD-1(程序性死亡受体1)，也称为 CD279（分化簇 279），是一种重要的免疫抑制分子。通过向下调节免疫系统对人体细胞的反应，以及通过抑制T细胞炎症活动来调节免疫系统并促进自身耐受。这可以预防自身免疫性疾病，但它也可以防止免疫系统杀死癌细胞。PD-1是288个氨基酸的I型膜蛋白，其最初是从凋亡的小鼠T细胞杂交瘤2B4.11克隆出来，以PD-1为靶点的免疫调节对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义。PD-1的配体PD-L1也可作为靶点，相应的抗体也可以起到相同的作用。PD-1和PD-L1结合启动T细胞的程序性死亡，使肿瘤细胞获得免疫逃逸。抗PD-1疗法通过结合PD-1并阻断PD-1与PD-L1和PD-L2的结合，解除免疫抑制效应，激活T细胞功能，增强T细胞对肿瘤的免疫监视能力和杀伤能力，产生肿瘤免疫应答。
 技术问题
基于此，有必要针对上述问题，提供一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其结合了IL-15和IL15RaSu的超激动蛋白再与anti-PD1结合获得融合蛋白，并使得免疫细胞成功分泌该融合蛋白，以达成增强免疫细胞增殖能力、抗凋亡能力和对于肿瘤的杀伤能力。
 技术解决方案
本发明提供的技术方案如下：
一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其经基因编辑后可自分泌IL-15与anti-PD1的融合蛋白，且免疫细胞同时表达嵌合抗原受体；该融合蛋白可以提高免疫细胞的活性以及对肿瘤的杀伤效果。
一实施例中，所述融合蛋白与嵌合抗原受体的基因是通过构建表达框的方式实现的，其所构建的表达框数量为一个或多个；进一步地，构建表达框时载体递送方式包括慢病毒、逆转录病毒、普通质粒、附加体、纳米递送系统、电转导或转座子。
在其中一个实施例中，自分泌IL-15与anti-PD1的融合蛋白是按照anti-PD1、G4S*4 Linker、IL-15N72D、G4S*4 Linker和IL-15RaSu顺序依次串联表达的，所述接受基因编辑的免疫细胞表达该融合蛋白并接受自分泌融合蛋白的影响。
所述接受基因编辑的免疫细胞，结合了IL-15和IL15RaSu的超激动蛋白及anti-PD1的融合蛋白并成功获得分泌该融合蛋白的嵌合抗原受体，如，T细胞（Chimeric antigen receptor CAR-T）。
在其中一个实施例中，嵌合抗原受体的表达为靶向一个靶点或多个靶点的嵌合抗原受体。
在其中一个实施例中，嵌合抗原受体的靶点包括CLDN18.2、GPC3、HER2、TAA、GD2、MSLN、EGFR、NY-ESO-1、MUC1、PSMA和EBV中的一种或几种。
在其中一个实施例中，嵌合抗原受体和靶点的结合区域可以是scFv、Fab或scFv与Fab组合；其中，scFv区结构可由任意靶点的任意单链抗体、单链可变片段（scFv）及Fab片段等中的一种或多种取代。
在其中一个实施例中，嵌合抗原受体包含前导序列、识别肿瘤相关抗原的scFv、铰链区和跨膜结构域、胞内共刺激结构域以及胞内激活信号CD3Zeta；其中，scFv为抗独特型抗体的scFv；铰链区和跨膜结构域为CD28，或者CD8铰链区与跨膜结构域；胞内共刺激结构域为CD28、CD137 (4-1BB)或者ICOS胞内共刺激结构域。
在其中一个实施例中，嵌合抗原受体和靶点的结合区域可以是结合一个靶点，也可以是结合两个靶点的双特异性抗体，还可以是两个或者多个嵌合抗原受体分别跨膜形成的并分别识别不同的靶点。
在其中一个实施例中，所述嵌合抗原受体的结构中包括信号肽CD8SP、跨膜结构域CD8Hinger、CD8TM、胞内激活元件4-1BB及CD3Zeta中的一种或多种。
在其中一个实施例中，嵌合抗原受体与自分泌IL-15及anti-PD1的融合蛋白之间的分割方式为蛋白切割功能元件，在多个表达框中时，可独立进行表达或分别递送，无需分割；其中，蛋白切割功能元件为T2A、P2A、E2A、F2A或IRES。
在其中一个实施例中，免疫细胞的基因转入嵌合抗原受体的载体包括慢病毒、逆转录病毒、普通质粒、附加体、纳米递送系统、电转导、转座子或其它递送系统。
在其中一个实施例中，免疫细胞包括T细胞、NK细胞、NKT细胞、巨噬细胞、γ-δT细胞、TIL细胞、TCR-T细胞或其它肿瘤杀伤细胞。
 有益效果
与现有技术相比，本发明具有如下有益效果：
本发明提供的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，表达嵌合抗原受体，可以特异性识别所靶向的肿瘤细胞表面抗原；该免疫细胞结合了IL-15和IL15RaSu的超激动蛋白及anti-PD1的融合蛋白，并使得免疫细胞成功分泌该融合蛋白，以达成增强免疫细胞的增殖能力、抗凋亡能力和对于肿瘤的杀伤能力；本发明的免疫细胞杀伤效果精准，安全性更高，不易复发，提高患者的生存质量。
 附图说明
图1 为免疫细胞中的融合蛋白和氨基酸序列结构设计图；其中，A#~D#中的融合蛋白结构；1# ~4#为氨基酸序列结构设计图；
图2 为靶细胞表型流式检测结果；其中，HGC-27-CLDN18.2细胞对应FTC-CLDN18.2和APC-PD-L1流式检测图，相应地，HGC-27细胞也对应FTC-CLDN18.2和APC-PD-L1流式检测图；
图3a、3b 为分泌型CAR-T融合蛋白分泌柱状图；其中，图3a为Secretory PD1-scFv对应柱状图；图3b为Secretory hIL15/Ra对应柱状图
图4 为分泌型CAR-T扩增生长曲线图；
图5a、5b、5c、5d、5e、5f为 CAR-T细胞表型流式数据；其中，图5a为 T细胞表型流式图；图5b为 CAR-T CLDN18.2 细胞表型流式图；图5c为 CAR-T CLDN18.2-il-15/Ra细胞表型流式图；图5d为 CAR-T CLDN18.2-antiPD1细胞表型流式图；图5e为 CAR-T CLDN18.2-15&PD1细胞表型流式图；图5f为NC（空白对照样）表型流式图；图中横坐标APC通道表示CD3表达情况，右侧相对左侧为阳性；图中纵坐标PE通道表示PD-L1表达情况，上方相对下方为阳性。
图6a、6b 为CAR-T细胞体外杀瘤功能评价曲线图；其中，图6a为对应细胞对HGC-27靶细胞体外杀瘤功能评价曲线图；图6b为对应细胞对HGC-27-CLDN18.2靶细胞体外杀瘤功能评价曲线图；
图7为 CAR-T动物实验生存曲线。
 本发明的最佳实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。
本发明提供了一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其经基因编辑后可自分泌IL-15与anti-PD1的融合蛋白，且该免疫细胞表同时达嵌合抗原受体；该融合蛋白可以提高免疫细胞的活性以及对肿瘤的杀伤效果 。
上述自分泌IL-15与anti-PD1的融合蛋白是按照anti-PD1、G4S*4 Linker、IL-15N72D、G4S*4 Linker和IL-15RaSu顺序依次串联表达的，所述接受基因编辑的免疫细胞表达该融合蛋白并接受自分泌融合蛋白的影响。
免疫细胞本身并不会表达上述提及的融合蛋白，但为了使免疫细胞分泌该融合蛋白，就需经过相应的基因编辑，如，CAR-T、CAR-NK、TCR-T、IPS等用于肿瘤治疗的细胞，它们本身已是经过相应基因编辑了，再使其分泌融合蛋白也进行相应基因编辑，这类细胞在此统称为经过基因编辑的免疫细胞。
本发明提供的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，结合了IL-15和IL15RaSu的超激动蛋白及anti-PD1的融合蛋白，并成功获得分泌该融合蛋白的嵌合抗原受体，如，T细胞（Chimeric antigen receptor CAR-T）。
上述融合蛋白中的anti-PD1为anti-PD1 VL、anti-PD1 VH或anti-PD1 VL与anti-PD1 VH组合。
一个实施例中，免疫细胞表达嵌合抗原受体，如，CAR细胞。嵌合抗原受体表达可以是靶向一个靶点或者多个靶点的嵌合抗原受体，如，CAR细胞。
一个实施例中，嵌合抗原受体的靶点还可以为独特型CLDN18.2、GPC3、HER2、TAA、GD2、MSLN、EGFR、NY-ESO-1、MUC1、PSMA和EBV中的一种或几种。
嵌合抗原受体和靶点的结合区域可以是scFv、Fab或scFv与Fab组合；其中， scFv区结构可由任意靶点的任意单链抗体、单链可变片段（scFv）及Fab片段等中的一种或多种取代。
上述嵌合抗原受体包含前导序列、识别肿瘤相关抗原的scFv、铰链区和跨膜结构域、胞内共刺激结构域以及胞内激活信号CD3ζ。其中，scFv为抗独特型抗体的scFv；铰链区和跨膜结构域为CD28或CD8铰链区和跨膜结构域；胞内共刺激结构域为CD28或CD137 (4-1BB)或者ICOS胞内共刺激结构域。
嵌合抗原受体和靶点的结合区域可以是结合一个靶点，也可以是结合两个靶点的双特异性抗体，还可以是两个或者多个嵌合抗原受体分别跨膜形成的并分别识别不同的靶点。
在其中一个实施例中，所述嵌合抗原受体的结构中包括信号肽CD8SP、跨膜结构域CD8Hinger、CD8TM、胞内激活元件4-1BB及CD3Zeta中的一种或多种。
嵌合抗原受体同与自分泌IL-15及anti-PD1的融合蛋白之间的分割方式为蛋白切割功能元件，在多个表达框中时，可独立进行表达或分别递送，无需分割；其中，蛋白切割功能元件可以为T2A、P2A、E2A、F2A或IRES。
融合蛋白与嵌合抗原受体的基因是通过构建表达框的方式实现的，其所构建的表达框数量为一个或多个；构建表达框时载体递送方式包括慢病毒、逆转录病毒、普通质粒、附加体、纳米递送系统、电转导或转座子；也即是说，免疫细胞的基因转入嵌合抗原受体的载体包括慢病毒、逆转录病毒、普通质粒、附加体、纳米递送系统、电转导、转座子或其它递送系统。
本发明的免疫细胞包括T细胞、NK细胞、NKT细胞、巨噬细胞、gamma-delta T细胞、TIL细胞、TCR-T细胞或其它肿瘤杀伤细胞。
上述自分泌IL-15与anti-PD1融合蛋白的免疫细胞，可被制成生物制剂，该生物制剂为药学上可接受的载体、稀释剂或赋形剂。
生物制剂的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。该生物制剂可应用于预防和/或治疗实体肿瘤的药物中。
本发明提供的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，表达嵌合抗原受体，可以特异性识别抗自身抗体的独特型；这种免疫细胞结合了IL-15和IL15RaSu的超激动蛋白及anti-PD1的融合蛋白，并使得免疫细胞成功分泌该融合蛋白，以达成增强免疫细胞的增殖能力、抗凋亡能力和对于肿瘤的杀伤能力；另外本发明的免疫细胞还可以特异性的杀伤分泌IL-15与anti-PD1融合蛋白，且杀伤效果精准，安全性更高，不易复发，提高患者的生存质量。
 本发明的实施方式
以下为具体实施例说明。
下列实施例，以外周血中T细胞制备CAR-T并分泌IL-15与anti-PD1融合蛋白为例，对免疫细胞的制备方法及功能验证等进行详细说明；具体包括以内容：
1、融合蛋白的结构设计；
2、构建分泌型CAR-T细胞并进行体外功能试验；
3、分泌融合蛋白型CAR-T细胞体内功能试验。
    1、融合蛋白的结构设计
根据hIL15、hIL15RaSu、anti-PD1的序列，按照图1所示的融合蛋白结构图，设计如A#~D#中的融合蛋白结构，并将其设计到CAR-T-CLDN18.2中；其中，1#为对照CAR-T，2#~4#为分泌型CAR-T。其中：
hIL15氨基酸序列为：METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS。
hIL15RaSu氨基酸序列为：ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR。
Anti-PD1 VH氨基酸序列为：QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS。
Anti-PD1 VL氨基酸序列为：EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK。
Linker氨基酸序列为：GSSSSGSSSSGSSSSGSSSS。
   其中，嵌合抗原受体的结构中包括信号肽CD8SP、跨膜结构域CD8Hinger、CD8TM、胞内激活元件4-1BB及CD3Zeta中的一种或多种，具体如图1所示。
   2、构建分泌型CAR-T细胞并进行体外功能试验
构建分泌型CAR-T细胞并进行体外功能试验，包括以下步骤：
S100：细胞系培养
将表达CLDN18.2的碱基序列克隆至PHBLV慢病毒载体骨架中，置于EF1α(EF-1α)的启动子下，形成PHBLV-EF1α-CLDN18.2，将PHBLV-EF1α-CLDN18.2、慢病毒包膜质粒pMD2 .G (Addgene，Plasmid#12259)和慢病毒包装质粒psPAX2(Addgene Plasmid#12260)等三个质粒使用Lipofectamine3000转入293T细胞中制备的慢病毒完整表达载体上；分别在48h和72h收集病毒上清，并对所收集的病毒上清进行超速离心浓缩(Merck Millipore)；浓缩后的病毒即可用于感染HGC-27，最终得到过表达CLDN18.2的HGC-27细胞系，命名为HGC-27-CLDN18.2。
HGC-27-CLDN18.2细胞表达CLDN18.2的检测结果如图2所示，其中，HGC-27对应的检测图为对照图；根据HGC-27-CLDN18.2与HGC-27对应的检测结果，由图2中虚线框a部分纵向两图可知，在FITC通道检测CLDN18.2抗原表达水平结果显示，HGC-27中CLDN18.2表达为阴性（峰图位于竖线I左侧），HGC-27-CLDN18.2中CLDN18.2表达为阳性（峰图位于竖线I右侧）；由图2中虚线框b部分纵向两图可知，在APC通道检测PD-L1表达水平结果显示，HGC-27和HGC-27-CLDN18.2中PD-L1表达均为阳性（峰图位于竖线II右侧）。
S200：外周血PBMC的分离和T细胞的扩增
从供体外周血中分离单个核细胞，使用ficol法进行密度梯度离心，并用T细胞分选试剂盒富集T细胞，如，CD3 MicroBeads、 human - lyophilized或130-097-043，并使用偶联anti-CD3/anti-CD28的磁珠激活培养和扩增T细胞；
T细胞培养时，使用TexMACS GMP Medium(Miltenyi Biotec，170-076-309）培养基，培养基中含10％FBS、2mM L-glutamine及100IU/ml rhIL2，细胞培养时均置于37℃，5％CO 2恒温培养箱中培养。
将上述第1项融合蛋白的结构设计中的B#~D#序列中融合度蛋白，通过CHO融合蛋白表达系统表达纯化后作为ELISA，检测1#~4#中蛋白分泌的阳性对照标准品，收集CAR-T培养上清，并对细胞培养上清中蛋白分泌进行检测，检测数据如图3a、3b所示。从图3a、3b中可知，CART-CLDN18.2-IL15/Ra、CART-CLDN18.2-antiPD1、CART-CLDN18.2-15&PD1可以正常分泌蛋白，并且具有基本相同的蛋白分泌效率。
通过慢病毒包装制备得到的CAR-T，所获得的CAR-T增殖情况如图4所示，CART-CLDN18.2-15&PD1相较CART-CLDN18.2-IL15/Ra、CART-CLDN18.2-antiPD1和CART-CLDN18.2具有更高的细胞增殖倍数，证明其有更优异的细胞增殖能力了。
慢病毒侵染制备得到的CAR-T，阳性率及表型结果见表1、图5a、5b、5c、5d、5e及5f所示。
表1 CAR-T细胞阳性率及表型流式检测结果
Figure 589561dest_path_image001
表1中结果显示，慢病毒侵染方法，可以有效制备出CAR-T阳性细胞（阳性率大于50%），并且CART-CLDN18.2、CART-CLDN18.2-IL15/Ra、CART-CLDN18.2-antiPD1和CART-CLDN18.2-15&PD1几种细胞的表型之间无明显差异。在图5a~5f中，以NC为对照划分阴性区域（“十”字象限图中左下部分比例），分泌anti-PD1抗体与IL15&PD1融合蛋白的CART-CLDN18.2-antiPD1及CART-CLDN18.2-15&PD1两种细胞的PD1表达水平（“十”字象限图中右上部分比例），明显低于T细胞、CART-CLDN18.2和CART-CLDN18.2-IL15/Ra，证明IL15&PD1融合蛋白可有效抑制CAR-T细胞表面的PD1表达。
S300：细胞体外杀伤实验
使用HGC-27-CLDN18.2和HGC-27细胞分别作为阳性和阴性靶细胞，采用流式检测方法对CAR-T体外杀瘤功能进行验证。检测结果如图6a和6b所示，检测结果显示，相比之下，分泌融合蛋白的CART-CLDN18.2-15&PD1对HGC-27-CLDN18.2阳性靶细胞具有最强的杀伤效果。
    3、CAR-T细胞体内功能评价
取6-8周龄NSG小鼠（体重18-22g）24只，适应饲养一周后，皮下接种HGC-27-CLDN18.2阳性的肿瘤细胞株，每只小鼠接种1*10 7个肿瘤细胞，密切观察动物状态，每三天使用游标卡尺测量小鼠肿瘤体积，当肿瘤体积达到100mm 3，根据小鼠体重和肿瘤大小随机分组后，经尾静脉输注CAR-T细胞或对照T细胞。详细的给药方法、给药剂量和给药途径见表2所示。
表2  动物实验方案
Figure 166036dest_path_image002
如图7结果显示，CART-CLDN18.2-15&PD1分泌型CAR-T可以大幅延长小鼠生存期。
 工业实用性
以上实施例证明：自分泌IL-15与anti-PD1融合蛋白的CAR-T相对于不分泌其他细胞因子的CAR-T或只分泌一种细胞因子的CAR-T具有更强的增殖能力及对肿瘤的体内体外杀瘤活性。
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Claims (10)

  1. 一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述免疫细胞经基因编辑后可自分泌IL-15与anti-PD1的融合蛋白，且该免疫细胞同时表达嵌合抗原受体。
  2. 根据权利要求1所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述融合蛋白与嵌合抗原受体的基因是通过构建表达框的方式实现的，其所构建的表达框数量为一个或多个。
  3. 根据权利要求2所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，构建表达框时载体递送方式包括慢病毒、逆转录病毒、普通质粒、附加体、纳米递送系统、电转导或转座子。
  4. 根据权利要求1所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述融合蛋白是按照anti-PD1、G4S*4 Linker、IL-15N72D、G4S*4 Linker和IL-15RaSu顺序依次串联表达的。
  5. 根据权利要求1所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述嵌合抗原受体表达为靶向一个或两个以上靶点的嵌合抗原受体。
  6. 根据权利要求5所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述嵌合抗原受体的靶点包括CLDN18.2、GPC3、HER2、TAA、GD2、MSLN、EGFR、NY-ESO-1、MUC1、PSMA和EBV中的一种或几种。
  7. 根据权利要求5所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述嵌合抗原受体与靶点的结合区域包括scFv、Fab及任意靶点的任意单链抗体中的一种或多种；或者所述嵌合抗原受体和靶点的结合区域包括结合一个靶点或者两个靶点的双特异性抗体。
  8. 根据权利要求1所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述嵌合抗原受体的结构中包括信号肽CD8SP、跨膜结构域CD8Hinger、CD8TM、胞内激活元件4-1BB及CD3Zeta中的一种或多种8。
  9. 根据权利要求1所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述嵌合抗原受体与融合蛋白之间的分割在同一表达框中时，可使用蛋白切割功能元件。
  10. 根据权利要求9所述的自分泌IL-15与anti-PD1融合蛋白的免疫细胞，其特征在于，所述蛋白切割功能元件包括T2A、P2A、E2A、F2A及IRES中的任一种。
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WO2023102712A1 (zh) * 2021-12-07 2023-06-15 深圳市先康达生命科学有限公司 一种基因生物制剂及其制备方法和应用
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