Tumor-Infiltrating Donor Derived Lymphocyte Therapy After Allogeneic Transplant

同种异体移植后肿瘤浸润供体来源的淋巴细胞治疗

• 批准号: 8158352
• 负责人: Frances Hakim
• 金额: $ 20.95万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158352
• 关键词: Allogenic; Lymphocyte; Transplantation; tumor

We supported the preclinical development of the TDL clinical protocol (P.I. Michael Bishop; 07-C-0064) by establishing the feasibility of expanding TDL in cultures through the use of anti CD3/anti CD8 beads, resulting in a product with high viability, donor origin and T cell content, very low residual B cell numbers, free of endotoxin or contamination; In this process, we developed methods for viably dissociating cells and culturing them under good manufacturing process (GMP) standards. Furthermore we supported the clinical approval process by developing the necessary documentation of standard operating procedures and certificates of analysis for product release, assembling GMP reagents lists, and providing links to established investigational new drug (IND) protocols for manufacture of an clinical product suitable for infusion into patients. Following the approval of this protocol, we have continued to assess T cell expansion and clearance of B cell lymphoma and Hodgkins disease populations in the TDL expansion cultures in the first nine patients. We have characterized the TDL product from both preclinical test cultures and from the first clinical trial cultures, using multiparameter flow cytometry and cytokine production assays. We have demonstrated that the TDL expansion cultures result in the disappearance of lymphoma cells and a marked decline in the frequency of regulatory T cells found in the original tumor population. The final product contains more than 90% T cells, primarily T-Bet+ Th1/Tc1 cells, that have elevated expression of effector molecules including CD40L, NKG2D, and perforin, and produce primarily IFN-gamma on stimulation. These assays will form the basis for tests of efficacy and specificity of anti-tumor activity that will be used to optimize the TDL product. Furthermore we have supported a clinical initiative to make this therapy available to a broader patient population by demonstrating the feasibility of using patient bone marrow (rather than surgically excised lymphoma nodules) as a basis for generating TDL in patients with marrow-resident tumor populations. We have successfully expanded replicate cultures from patient marrow collected following allogeneic transplant, demonstrating significant expansion of donor-derived T cells that meet criteria for T cell numbers and viability, donor chimerism, removal of tumor cells and microbial standards. These tests have supported submission of a protocol amendment providing for use of marrow. Furthermore preclinical studies on marrow form patients with relapsed acute myeloid leukemia are continuing in order to support expansion of this therapy to these patients. Finally we have initiated development of alternative culture conditions to optimize not only the numerical expansion of donor-derived T cells from the lymphoma tissue, but also anti-tumor activity and persistence in vivo after re-infusion. We have shortened the culture and are examining alterations in the cytokine milieu of the culture to enhance retention of activated CD8 effectors. As part of this trial, we are evaluating changes in the peripheral blood following infusion of the TDL product and have been preserving aspirates of tumor sites after TDL therapy to assess molecular changes indicative of donor anti lymphoma activity. These flow cytometric and molecular monitoring studies have been extended to the first of additional planned trials of immune therapies for relapse. In this trial (09-C-0224, P.I.: Nancy Hardy) donor lymphocyte infusions (a standard method of immune therapy) are infused following irradiation of selected tumor sites. Monitoring focuses on whether the irradiation process has produced activation and trafficking of antigen presenting cells into the tumor and subsequent in anti-tumor immune activation. Finally, ongoing clinical trials (04-C-0055, 08-C-0088: P.I. Daniel Fowler) have utilized expanded donor-derived CD4 Type 2 T helper cells grown in rapamycin (Th2rapa). Our support for these efforts has included scaling up research laboratory products into clinical size expansion cultures using GMP materials and practices, preparing Standard Operating Protocols for generation of cultures, developing release criteria for products and providing documentation of these procedure for clinical IND. These efforts have supported the continued development of Th2rapa therapy for B cell malignancies and renal cell carcinoma.

我们通过使用抗CD3/抗CD8珠子来建立扩展TDL在培养物中扩展TDL的可行性，从而支持TDL临床方案的临床前开发（P.I. Michael Bishop; 07-C-0064），从而导致具有高活力，供体的生存力和T细胞含量，非常低的B细胞数量，非常剩余的B细胞数量，自由氧氧蛋白或contantination，Ondiantination，Ondiantination of Endoxin of Endoxin of Endoxin或Contam of form of Endoxin;在此过程中，我们开发了在良好的制造过程（GMP）标准下逐步解离细胞和培养它们的方法。此外，我们通过制定标准操作程序的必要文档和产品释放，组装GMP试剂列表的必要文档，并提供与已建立的研究新药（IND）制造适合于输注患者的临床产品的链接的必要文档。在获得该方案的批准后，我们​​继续评估前九名患者TDL扩张培养物中B细胞淋巴瘤和Hodgkins病种群的T细胞扩张和清除率。我们使用多参数流式细胞术和细胞因子生产测定法表征了临床前测试培养物和第一个临床试验培养物的TDL产物。我们已经证明，TDL膨胀培养物导致淋巴瘤细胞消失，并且在原始肿瘤种群中发现的调节性T细胞的频率显着下降。最终产物含有90％以上的T细胞，主要是T-Bet+ Th1/Tc1细胞，它们的效应分子表达升高，包括CD40L，NKG2D和Perforin，并且主要在刺激上产生IFN-Gamma。这些测定将构成用于优化TDL产品的抗肿瘤活性功效和抗肿瘤活性特异性的基础。 此外，我们还支持一项临床倡议，以证明使用患者骨髓（而不是手术切除的淋巴瘤结节）作为在骨髓抑制肿瘤群体中产生TDL的基础，从而使更广泛的患者人群可以使用这种疗法。我们已经成功地扩展了从同种异体移植后收集的患者骨髓中的重复培养物，表明符合T细胞数量和生存能力标准的供体衍生的T细胞显着扩展，供体嵌合，去除肿瘤细胞和微生物标准。这些测试支持提交规程修正案，以使用骨髓。此外，对骨髓的临床前研究形成了复发性急性髓样白血病的患者，以支持将这种疗法扩展向这些患者。最后，我们启动了替代培养条件的发展，不仅优化了淋巴瘤组织中供体衍生的T细胞的数值扩张，而且还优化了抗肿瘤活性和抗肿瘤活性和重新输注后体内的持久性。我们已经缩短了培养物，并正在研究培养物细胞因子环境中的改变，以增强活化的CD8效应子的保留。作为这项试验的一部分，我们正在评估输注TDL产物后外周血的变化，并在TDL治疗后一直在保留肿瘤部位的抽吸物，以评估指示供体抗淋巴瘤活性的分子变化。这些流式细胞仪和分子监测研究已扩展到免疫疗法的其他计划试验中的第一个复发试验。在这项试验中（09-C-0224，P.I。：南希·哈迪）供体淋巴细胞输注（一种标准的免疫治疗方法）在辐照选定的肿瘤部位后被注入。监测的重点是辐照过程是否已产生抗原呈递细胞的激活和运输到肿瘤中，然后在抗肿瘤的免疫激活中产生激活和运输。最后，正在进行的临床试验（04-C-0055，08-C-0088：P.I。Daniel Fowler）利用了在雷帕霉素（TH2RAPA）生长的供体衍生的2型T型助手细胞。我们对这些努力的支持包括使用GMP材料和实践将研究实验室产品扩展到临床规模的扩展培养物中，为生成培养物的标准操作方案，为产品制定释放标准以及为临床IND提供这些程序的文档。这些努力支持B细胞恶性肿瘤和肾细胞癌的Th2RAPA治疗的持续发展。