Preclinical development and clinical monitoring of adoptive immune therapy

过继免疫疗法的临床前开发和临床监测

• 批准号: 8763747
• 负责人: Frances Hakim
• 金额: $ 20.42万
• 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/8763747
• 关键词: Acute Graft Versus Host Disease; Adoptive Transfer; Allogenic; Amendment; Antibodies; Antigen Receptors; Antigen-Presenting Cells; Antigens; Archives; Aspirate substance; Autologous; B lymphoid malignancy; B-Cell Lymphomas; B-Lymphocytes; Biological Assay; Blood; Blood Circulation; Bone Marrow; CD19 gene; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Count; Cell Culture Techniques; Cell Survival; Cell Therapy; Cell physiology; Cells; Cessation of life; Chimerism; Clinic; Clinical; Clinical Protocols; Clinical Trials; Development; Documentation; Donor Lymphocyte Infusion; Effector Cell; Endotoxins; Engraftment; Excision; Feasibility Studies; Flow Cytometry; Frequencies; Generations; Goals; Helper-Inducer T-Lymphocyte; Hematologic Neoplasms; Hodgkin Disease; Homing; Homologous Transplantation; Immune; Immunologic Monitoring; Immunotherapy; Infusion procedures; Interferon Type II; Interleukin-4; Investigational Drugs; Laboratories; Laboratory Research; Link; Lymphocyte; Lymphoma; Malignant Neoplasms; Marrow; Medicine; Methods; Molecular; Monitor; Multiple Myeloma; Nodule; Patients; Phenotype; Play; Population; Preclinical Testing; Procedures; Process; Production; Protocols documentation; Reagent; Regulatory T-Lymphocyte; Relapse; Reporting; Residual state; Role; Schedule; Services; Sirolimus; Site; Specificity; Stem cells; T-Cell Receptor; T-Lymphocyte; TNFSF5 gene; Therapeutic; Tissues; Transfusion; Translations; Transplant Recipients; Transplantation; Transplantation Immunology; Tumor-Derived; Tumor-Infiltrating Lymphocytes; United States National Institutes of Health; Work; anergy; arm; cytokine; flexibility; graft vs host disease; immune activation; immune function; in vivo; irradiation; lymph nodes; lymphocyte product; manufacturing process; meetings; member; microbial; neoplastic cell; novel; patient population; perforin; peripheral blood; pre-clinical; prevent; reconstitution; scale up; trafficking; tumor; validation studies

The Preclinical Development and Clinical Monitoring Facility of the Experimental Transplantation and Immunology Branch supports the development and implementation of new protocols involving ex vivo expanded adoptive cell therapies through a core staff member working with Cell Processing in the Department of Transfusion Medicine and through support of archiving and analysis of patient blood and tissues during clinical trials. Our support for new adoptive cell therapies 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 procedures for clinical IND. Following initation of each trial, we have characterized the transfered cell product and the subsequent changes in circulating patient lymphocytes. Three novel protocols involving adoptive transfer of expanded donor-derived T cells have been implemented in recent years as a result of this process. In 04-C-0055 Arm 4A, Daniel Fowler has previously utilized expanded donor-derived CD4 helper cells grown for 12 days in IL-4 and rapamycin (T.Rapa.12) to enhance donor engraftment and reduce GVHD. The Preclinical Service has supported implementation of clinical trial Arm 4B utilizing a shorter expansion period to generate cells after only 6 days of expansion (T.Rapa.6), to produce a population with increased anti-tumor potency. This product has been used this past year in an additional trial arm (Arm 4C) that combined the the T.Rapa.6 product with an altered schedule of post transplant rapamycin. Following completion of each trial arm, we have characterized the early immune reconstitution following infusion of the T.Rapa product and have assessed the T cell receptor repertoire diversity of the infused T.Rapa cells and the tranplant recipients at day 60 through spectratyping. These studies have been reported in 2013(Fowler et al, Blood, 2013). The second novel product involves expansion of autologous T cells (both CD4 and CD8) in the presence of IFNalpha and rapamycin (T1.rapa), to generate a cell product that potentially has activity against residual myeloma plasma cells. In protocol 11-C-0016 (P.I. Claude Sportes), patients with multiple myeloma undergo an autologous transplant followed by infusion of T1.Rapa cells. Clinical production of these cells was developed through our staff in Cell Processing. Following initiation of this trial, we have monitored serial changes in lymphocyte populations in blood and in bone marrow. Finally, we have supported the implementation of the first trial of the use of donor-derived anti-CD19 Chimeric Antigen Receptor (CAR)T cells in patients with relapsed or persistent lymphoma following allogenic transplant (Protocol 10-C-0052, P.I. James Kochenderfer). We have tracked the presence of CAR T cells following adoptive transfer and have characterized expression of markers of activation and anergy. These studies have demonstrated the expansion of anti-CD19 CAR T cells in the blood concurrent with the period of anti-tumor activity approximately one week after adoptive transfer (J. Kochenderfer, submitted). We have also supported the preclinical development of the TDL (tumor infiltrating donor derived lymphocyte) clinical protocol (P.I. Michael Bishop; 07-C-0064), a protocol that has now closed to further accrual with Dr. Bishop's departure from NIH.(Results reported in Hardy et al, Blood, 2012). Dr. Bishop hypothesized that the donor-derived T cells found in tumor sites after alloHSCT would be enriched for effector cells that were tumor-specific in their homing and antigen specificity. He proposed that activation and ex vivo expansion of these cells through CD3/CD28 costimulation might yield a more effective form of cell therapy after alloHSCT, with enhanced anti-tumor effects and less GVHD. The PCS staff supported this project by assessing the feasibility of expanding TDL in cultures through the use of anti CD3/CD28 beads, generating a donor T cell product with high viability, very low residual B cell content, free of endotoxin or contamination; In this process, we developed methods for viably dissociating cells and culturing them under good manufacturing process (GMP) standards. We further 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. We supported a clinical initiative to make this therapy available to a broader patient population. 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; this work supported the amendment permitting expansion of the protocol to generation of TDL from bone marrow. Two patients with hematologic malignancies that are mainly localized in bone marrow were able to be treated with TDL grown from patient marrow collected under this protocol. We have also supported the clinical implementation and assessment of this protocol. We have assessed T cell expansion and clearance of B cell lymphoma and Hodgkins disease populations in the lymph node derived TDL expansion cultures in eight patients and in the bone marrow derived TDL from an additional two patients. As part of this trial, we evaluated 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. 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 in the clinical trial resulted in a marked decline in the frequency of regulatory T cells found in the original tumor population. The final product contained more than 90% T cells, primarily T-Bet+ Th1/Tc1 cells, that had elevated expression of effector molecules including CD40L, NKG2D, and perforin, and produced primarily IFN-gamma on stimulation. These assays have been used to optimize the TDL product in terms of numerical, anti-tumor activity and persistence in vivo after re-infusion. We have shortened the culture period and investigated alterations in the cytokine milieu of the culture to enhance retention of activated CD8 effectors. 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 and subsequent localized deaths of tumor cells has produced activation and trafficking of antigen presenting cells into the tumor and increased anti-tumor immune activation.

实验移植和免疫学分支机构的临床前开发和临床监测机构支持涉及离体的新方案的开发和实施，通过在临床试验期间通过与输血医学系的细胞处理以及对患者血液和组织分析的核心人员处理，通过与细胞处理的核心人员处理，扩大了涉及的涉及的新方案。我们对新的收养细胞疗法的支持包括使用GMP材料和实践将研究实验室产品扩展到临床规模扩展培养物中，为生成培养物的标准操作方案准备，为产品制定释放标准以及为临床IND提供这些程序的文档。在每次试验中进行了沉迷之后，我们表征了转移的细胞产物以及随后的循环患者淋巴细胞的变化。近年来，由于此过程，已经实施了三种涉及扩展供体衍生T细胞的收养供体衍生T细胞的新型方案。在04-C-0055 ARM 4A中，丹尼尔·福勒（Daniel Fowler）此前曾在IL-4中使用扩展的供体衍生的CD4辅助细胞，在IL-4和雷帕霉素（T.Rapa.12）中生长了12天，以增强供体插入并减少GVHD。临床前服务已支持实施临床试验臂4B，该临床试验臂4b利用较短的扩展期仅在扩展6天后就会产生细胞（T.Rapa.6），以产生抗肿瘤效力增加的人群。去年，该产品已在另一个试验臂（ARM 4C）中使用，该臂将T.RAPA.6产品与移植后雷帕霉素的时间表更改相结合。每个试验臂完成后，我们表征了在t.Rapa产物输注后的早期免疫重建，并评估了注入的T.RAPA细胞的T细胞受体曲目多样性和在第60天通过频谱型的tranplant受体。这些研究已在2013年报道（Fowler等，《血液》，2013年）。第二种新型产物涉及在IFNALPHA和雷帕霉素（T1.RAPA）存在下自体T细胞（CD4和CD8）的扩展，以产生一种细胞产物，该细胞产物可能具有对残留骨髓瘤浆细胞活性的细胞产物。在方案11-C-0016（P.I. Claude Sportes）中，多发性骨髓瘤的患者进行自体移植，然后输注T1.RAPA细胞。这些细胞的临床生产是通过我们的细胞加工的员工开发的。在开始这项试验之后，我们监测了血液和骨髓中淋巴细胞种群的系列变化。最后，我们支持实施第一次使用供体衍生的抗CD19嵌合抗原受体（CAR）T细胞中同种异体移植后复发或持续性淋巴瘤的患者（方案10-C-0052，P.I. James Kochenderfer）。我们已经跟踪了产物转移后的CAR T细胞的存在，并表征了激活和反感标志物的表达。这些研究表明，抗CD19 CAR T细胞在血液同时发生的抗肿瘤活性周期大约在收养后的一周后扩展（J. Kochenderfer，提交）。我们还支持TDL（肿瘤浸润供体衍生的淋巴细胞）临床方案（P.I. Michael Bishop; 07-C-0064）的临床前开发，该方案现已在Bishop博士离开NIH的情况下与BiShop博士的进一步应计（Hardy等人，Hardy等人，血液，2012年）。 Bishop博士假设，在AllOHSCT之后，在肿瘤部位发现的供体衍生的T细胞将富集在其归因和抗原特异性的肿瘤特异性的效应细胞中。他提出，这些细胞通过CD3/CD28共刺激对这些细胞的激活和离体扩展可能会产生AlloHSCT后更有效的细胞疗法形式，具有增强的抗肿瘤作用，GVHD较少。 PCS的工作人员通过使用抗CD3/CD28珠子来评估扩展TDL在培养物中扩展TDL的可行性，从而支持该项目，从而产生具有高生存能力的供体T细胞产物，不含内毒素或污染的供体T细胞产品；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。我们通过制定必要的标准操作程序和分析证书，以释放产品释放，组装GMP试剂清单，并提供与已建立的研究新药（IND）协议的链接，以制造适合输入患者的临床产品，以进一步支持临床批准过程。我们支持一项临床计划，使该疗法可用于更广泛的患者人群。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。这项工作支持修正案，允许将方案扩展为从骨髓中产生TDL。两名主要位于骨髓中的血液学恶性肿瘤患者可以用根据该方案收集的患者骨髓生长的TDL治疗。我们还支持该协议的临床实施和评估。我们已经评估了八名患者的淋巴结中B细胞淋巴瘤和Hodkins病种群的T细胞膨胀和HODKINS疾病群的清除率，而在骨髓中，TDL膨胀培养物的TDL培养物在另外两名患者中得出了TDL。作为这项试验的一部分，我们评估了输注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。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，临床试验中的TDL膨胀培养物导致原始肿瘤种群中发现的调节性T细胞的频率显着下降。最终产物包含超过90％的T细胞，主要是T-BET+ TH1/TC1细胞，这些细胞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。这些分析已用于通过数值，抗肿瘤活性和重新输注后体内的持久性来优化TDL产品。我们缩短了培养期，并研究了培养物细胞因子环境的改变，以增强活化的CD8效应子的保留。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。监测的重点是肿瘤细胞的辐射和随后的局部死亡是否已激活和运输抗原呈递细胞进入肿瘤并增加抗肿瘤免疫激活。