PR1-specific CB T cells for Patients with Myeloid Malignancies

脐带血移植（CBT）是一种越来越有效的治疗血液恶性肿瘤患者的合适的hla匹配供体是不可用的。如果能够将介导移植物病毒白血病（GVL）的T细胞与介导移植物抗宿主病（GVHD）的T细胞区分开来，就可以开发出更有效的免疫治疗策略。我们的长期目标是通过改造供体T细胞来增加GVL而不增加GVHD，从而改善AML患者CBT治疗的结果。我们假设，靶向白血病相关抗原（LAA）的供体T细胞，如AML上hla - a2限制性PR1肽，优先介导GVL活性，而不是GVHD，脐带血（CB）供体来源的PR1特异性细胞毒性T淋巴细胞（PR1- ctl）可以在体外被诱导和扩增，用于临床，选择性地诱导CBT受体的GVL。因此，我们从PR1疫苗临床应答者中克隆了高亲和力和低亲和力的PR1特异性T细胞受体- α β (TCR)异源二聚体，它们可以转导到多克隆T细胞中，以重定向抗原特异性并介导抗白血病作用。我们还生产了一种对PR1/HLA-A2 （8F4）的特定构象表位具有高亲和力的单克隆抗体，该单克隆抗体介导对急性髓性白血病（AML）的有效和特异性细胞毒性，并且8F4的单链Fv与CD3zeta + CD28融合作为嵌合抗原受体（CAR）将用于基因修饰t细胞以研究GVL效应。此外，与成人外周血相比，CB中前体PR1-CTL的数量增加了约1000倍，CB pr - ctl在体外可被激活和扩增5倍以上。基于这些进展，我们建议(1)通过比较(a)单个CB单位的细胞扩增，(b)来自多个供体的细胞纯化，(c) pr1 - tcr - α β基因修饰和(d) 8F4-CAR基因修饰，确定一种最佳方法来诱导和扩增有效的CB衍生的PR1-CTL体外；(2)利用异种小鼠模型验证PR1-CTL在体内抗人AML的效力，研究AML诱导PR1-CTL的持久性和可能的耐受性，并利用生物发光和PET/CT成像技术确定cb源性PR1-CTL的时空GVL效应、持久性和可能的耐受性。最后，根据确定的最佳获得PR1-CTL的方法，我们将(3)测试cb来源的PR1-CTL作为CBT后AML患者过继细胞治疗的临床可行性和安全性。

Cord blood transplantation (CBT) is an increasingly effective treatment for patients with hematological malignancies for whom suitable HLA-matched donors are not available. If T cells that mediate graft verus leukemia (GVL) could be distinguished from those that mediate graft versus host disease (GVHD), more effective immunotherapy strategies could be developed. Our long-term goal is to improve the outcome of CBT for patients with AML by engineering donor T cells to increase GVL without increasing GVHD. We hypothesize that donor T-cells targeting leukemia-associated antigens (LAA), such as the HLA-A2-restricted PR1 peptide on AML, preferentially mediate GVL activity over GVHD and that cord blood (CB) donor-derived PR1-specific cytotoxic T lymphocytes (PR1-CTL) can be elicited and expanded ex vivo for clinical use to selectively induce GVL in CBT recipients. Thus, we have cloned high- and low-affinity PR1-specific T cell receptor-alphaBeta (TCR) heterodimers from PR1 vaccine clinical responders, which can be transduced into polyclonal T-cells to redirect antigen specificity and mediate antileukemic effects. We have also produced a monoclonal antibody with high affinity for a specific conformational epitope of PR1/HLA-A2 (8F4) that mediates potent and specific cytotoxicity against acute myeloid leukemia (AML), and a single chain Fv of 8F4 fused with CD3zeta + CD28 as a chimeric antigen receptor (CAR) will be used to gene modify T-cells to study GVL effects. In addition, the number of precursor PR1-CTL is ~1000-fold higher in CB compared to adult peripheral blood and CB PRI-CTL can be activated and expanded more than 5-fold in vitro. On the strength of these advances, we propose to (1) identify an optimal method to elicit and expand potent CB-derived PR1-CTL ex vivo by comparing (a) cell expansion from single CB units, (b) cell purification from multiple donors, (c) PR1-TCR-alphaBeta gene modification, and (d) 8F4-CAR gene modification; (2) use a xenogeneic mouse model to validate the potency of PR1-CTL against human AML in vivo to study the persistence and possible tolerance induction of PR1-CTL by AML, and to determine the spatial and temporal GVL effects, persistence, and possible tolerance of CB-derived PR1-CTL using bioluminescence and PET/CT imaging. Finally, based on the method identified to optimally obtain PR1-CTL, we will (3) test the clinical feasibility and safety of CB-derived PR1-CTL as adoptive cell therapy for AML patients after CBT.