PR1-specific CB T cells for Patients with Myeloid Malignancies

脐带血移植(CBT)是一种日益有效的治疗恶性血液病患者的方法，这些患者没有合适的人类白细胞抗原相合的供者。如果能将介导移植物抗宿主病(GVHD)的T细胞与介导移植物抗白血病(GVL)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使供者T细胞在不增加GVHD的情况下增加GVL来改善AML患者的CBT结局。我们假设，针对白血病相关抗原(LAA)的供者T细胞，如AML上的人类白细胞抗原A2限制性的PR1肽，优先于GVHD介导GVL活性，并且脐带血(CB)供者来源的PR1特异性细胞毒性T淋巴细胞(PR1-CTL)可以被诱导和体外扩增，用于临床选择性地诱导CBT受者GVL。因此，我们从PR1疫苗的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)异二聚体，可以将其转导到多克隆T细胞中，以重定向抗原特异性并介导抗白血病作用。我们还制备了针对PR1/人类白细胞抗原A2(8F4)构象表位(8F4)的高亲和力的单抗，该单链抗体与CD3zeta+CD28融合作为嵌合抗原受体(CAR)，将用于T细胞的基因修饰，以研究GVL效应。此外，脐带血中PR1-CTL前体细胞的数量是成人外周血的1000倍，在体外可被激活和扩增5倍以上。在这些进展的基础上，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-Alphabeta基因修饰和(D)8F4-CAR基因修饰，寻找一种最佳的方法来体外诱导和扩增CB来源的PR1-CTL；(2)用异种小鼠模型验证PR1-CTL对人AML的体内杀伤效力，研究AML对PR1-CTL的持久性和可能的耐受性，并利用生物发光和PET/CT成像技术检测脐带血来源的PR1-CTL的时空GVL效应、持久性和可能的耐受性。最后，在确定最佳获得PR1-CTL方法的基础上，我们将(3)检测脐带源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.