Chimeric antigen receptor-modified iNKT cell therapy for CD7+ malignancies

嵌合抗原受体修饰的 iNKT 细胞治疗 CD7 恶性肿瘤

• 批准号: 10603279
• 负责人: Xianzheng Zhou
• 金额: $ 39.8万
• 依托单位: AKESO THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10603279
• 关键词: Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Acute T Cell Leukemia; Adoptive Transfer; Allogenic; Animals; Antigen Presentation; Antigens; Apoptotic; Autologous; B-Cell Neoplasm; BCL2 gene; BCL2L1 gene; Biological Assay; CAR T cell therapy; CD19 gene; CD28 gene; CD3 Antigens; CD7 gene; CTLA4 gene; Cell Line; Cell Survival; Cell Therapy; Cell physiology; Cells; Cellular biology; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cytotoxic T-Lymphocytes; Data; Development; Failure; Feasibility Studies; Flow Cytometry; Frequencies; Galactosylceramides; Genes; Genomics; Goals; Good Manufacturing Process; Homeostasis; Human; IL7 gene; Immunotherapy; Infusion procedures; Interferons; Interleukin-15; Knowledge; Lentivirus; Lipids; Malignant - descriptor; Malignant Neoplasms; Mediating; Microspheres; Mus; Patients; Peripheral; Peripheral Blood Mononuclear Cell; Phase; Phenotype; Phosphorylation; Physiologic pulse; Population; Positioning Attribute; Production; Proliferating; Reagent; Refractory; Relapse; Research; Safety; Signal Transduction; Small Business Innovation Research Grant; Stat5 protein; T cell therapy; T memory cell; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Therapeutic; Toxic effect; Transact; Translating; Tumor Burden; Xenograft Model; acute myeloid leukemia cell; bioluminescence imaging; cancer therapy; cell killing; checkpoint receptors; chimeric antigen receptor; chimeric antigen receptor T cells; commercialization; cost; cytokine; cytotoxicity; genome editing; leukemia; manufacture; neoplastic cell; novel; nuclease; programmed cell death protein 1; receptor expression; response; tumor

PROJECT SUMMARY/ABSTRACT The major hurdle in developing chimeric antigen receptor (CAR)-T therapy for T-cell malignancies is CAR-T cell fratricide by self-killing, which leads to insufficient numbers of CAR-T cells for infusion. CRISPR- mediated genomic editing provides an alternative approach to enabling expansion of CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based gene editing remain lingering. The objective of this SBIR Phase I project is to develop a non-edited, CD7 CAR-modified allogeneic invariant Natural Killer T (iNKT) cell product for the treatment of relapsed and refractory patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The rationale for this study is based on our strong preliminary data. We propose two Specific Aims. Aim 1: To optimize the iNKT cell primary stimulation for maximal proliferation and CAR transduction. We plan to test major commercially available T-cell activation reagents, i.e., T-Cell Activation/Expansion kit (CD2/CD3/CD28 microbeads), T-Cell TransAct (CD3/CD28 microbeads), and CD3/CD28 Dynabeads in primary stimulation and transduce them with lentivirus CD7 CAR. CD7 CAR-modified iNKT, control CD19 CAR-modified iNKT or mock cells will be evaluated for cytotoxicity and IFN- production against CD7+ T-ALL and AML, CD19+ B-cell tumor cells, and GalCer pulsed CD1d+ target cells. In addition, expansion rate, CAR expression, and expression of CD3 and invariant T-cell receptor (iTCR) will also be assessed. CD7 CAR-modified iNKT cells with a highest expansion and CAR transduction while maintaining iNKT cell function and phenotype in primary culture will be examined for expansion in secondary stimulation. Expanded final cell products will be characterized for their expansion rate, cytotoxicity, cytokine profiling, phenotype, and expression of checkpoint receptors. Aim 2: To determine the effect of IL-7 co-expression on enhancing CD7 CAR-modified iNKT cell survival and efficacy. To test our hypothesis that IL-7 co-expression in CD7 CAR-modified iNKT cells can enhance their survival and anti-tumor efficacy, we will initially examine IL-7 and/or IL-15 co-expression in CD7 CAR-modified iNKT cells in response to CD7+ or CD7- tumor cells and characterize 1) secreted IL-7 and/or IL-15 levels, 2) STAT5 phosphorylation to reflect IL-7 and IL-15 signaling, 3) cytotoxicity and IFN- release, 4) fold expansion, 5) quantification of antiapoptotic factors (Bcl-2, Bcl-xL, MCI-1), 6) serial tumor challenge assay to assess enhanced survival, 7) frequency of naïve, central and effector memory T cell subsets, and 8) expression of checkpoint receptors (PD-1, CTLA4, TIM3, LAG3). We will then evaluate their anti-tumor efficacy in our established aggressive T-ALL xenograft model as assessed by reduced tumor mass via bioluminescence imaging and prolonged survival. iNKT cell survival will also be assessed by flow cytometry.

项目总结/摘要 开发用于T细胞恶性肿瘤的嵌合抗原受体（CAR）-T疗法的主要障碍是 CAR-T细胞通过自我杀伤而自相残杀，这导致用于输注的CAR-T细胞数量不足。CRISPR- 介导的基因组编辑提供了能够扩增CAR转导的T细胞的替代方法 来自同种异体健康捐赠者然而，基于CRISPR的基因编辑的安全性和临床意义 仍然徘徊。该SBIR I期项目的目标是开发一种未经编辑的CD 7 CAR修饰的 用于治疗复发性和难治性患者的同种异体不变自然杀伤T（iNKT）细胞产物 T细胞急性淋巴细胞白血病（T-ALL）和急性髓细胞白血病（AML）。这样做的理由 这项研究是基于我们的初步数据。我们提出两个具体目标。目标1：优化iNKT 用于最大增殖和CAR转导的细胞初级刺激。我们计划测试少校 市售的T细胞活化试剂，即，T细胞活化/扩增试剂盒（CD 2/CD 3/CD 28 微珠）、T细胞transAct（CD 3/CD 28微珠）和CD 3/CD 28 Dynabeads，以及 用慢病毒CD 7 CAR感染它们。CD 7 CAR修饰的iNKT、对照CD 19 CAR修饰的iNKT或模拟物 将评价细胞对CD 7 + T-ALL和AML、CD 19 + B细胞肿瘤的细胞毒性和IFN-γ产生 细胞，和CD 3GalCer脉冲的CD 1d+靶细胞。此外，扩增速率、CAR表达和 还将评估CD 3和恒定T细胞受体（iTCR）。CD 7 CAR修饰的iNKT细胞具有最高的 在原代培养物中维持iNKT细胞功能和表型的同时扩增和CAR转导将是有效的。 在二次刺激中检查膨胀。扩增的最终细胞产物将表征其 扩增率、细胞毒性、细胞因子谱、表型和检查点受体的表达。目标2： 确定IL-7共表达对增强CD 7 CAR修饰的iNKT细胞存活的作用， 功效为了验证我们的假设，即IL-7在CD 7 CAR修饰的iNKT细胞中的共表达可以增强其表达。 为了评估存活率和抗肿瘤功效，我们将首先检查IL-7和/或IL-15在CD 7 CAR修饰的细胞中的共表达。 iNKT细胞响应于CD 7+或CD 7-肿瘤细胞，并表征1）分泌的IL-7和/或IL-15水平，2） STAT 5磷酸化以反映IL-7和IL-15信号传导，3）细胞毒性和IFN-γ释放，4）倍数扩增， 5)抗凋亡因子（Bcl-2、Bcl-xL、MCI-1）的定量，6）连续肿瘤攻击测定以评估 增强的存活率，7）初始、中枢和效应记忆T细胞亚群的频率，和8） 检查点受体（PD-1、CTLA 4、TIM 3、LAG 3）。然后，我们将在我们的研究中评估它们的抗肿瘤功效。 建立侵袭性T-ALL异种移植模型，通过生物发光评估肿瘤质量降低 成像和延长生存期。还将通过流式细胞术评估iNKT细胞存活。