Project 2: Anti-PR1 Immune Therapy for Myeloid Leukemia

项目摘要 我们的长期目标是开发免疫疗法，针对原始细胞中异常表达的蛋白酶和 白血病干细胞。PR1肽(VLQELNVTV)是一种来源于白血病相关抗原的多肽 蛋白酶3(P3)和中性粒细胞弹性蛋白酶(NE)，它存在于人类白细胞抗原A2到PR1特异性细胞毒T细胞上 淋巴细胞(PR1-CTL)。在上一次资助期间，我们发现Pr1被树突状细胞交叉呈递。 (DCs)和B细胞，我们显示了需要蛋白酶体切割外源P3和NE的机制 由抗原提呈细胞上调。在孢子资助的前几年，我们进行了I-II期PR1疫苗 对66名AML、CML和MDS患者进行了试验，观察到58%的患者对PR1疫苗有免疫应答。然而， 目的仅有11例(16%)患者观察到临床反应，且仅限于低血压病患者。 白血病负担(&lt；10%)。我们发现，尽管表达高亲和力的高度细胞溶解的PR1-CTL 在PR1疫苗接种后，一些患者的T细胞受体(TcR-αβ)增加，他们接受了 高表达PR1/HLA-A2表面抗原的白血病细胞凋亡，通过以下途径导致免疫耐受 高亲和力PR1-CTL缺失。此外，虽然可以从脐带血中分离到高亲和力的PR1-CTL 脐带血(CB)细胞，它们很难在体外扩增出足够数量的细胞作为收养细胞 治疗，从而限制了它们的治疗潜力。因此，在一种新的替代治疗方法中，靶向 PR1，我们制备了一株抗PR1/HLA-A2的TCR样单抗(8F4)。我们创造了一个人性化的 8F4(H8F4)对PR1/HLA-A2具有高亲和力(Kd=7.8 nM)，我们发现h8F4消除了AML和 白血病干细胞，而不是临床前模型中的正常人类造血干细胞。有协议的 从支持制造H8F4的行业来看，我们已经生产了足够的临床等级H8F4，这表明 介导AML和LSC的ADCC和凋亡，并建立了PK的配套检测方法， 抗独特型和抗药物抗体检测。此外，我们还建立了AML的并行PDX模型 支持临床试验的研究。因此，这项提议的目的是(1)翻译这部小说的发现 H8F4单抗在急性髓细胞白血病的首次人类I期试验中的临床应用；(2)为了确定药代动力学， 毒性和作用方式；并表征作用机制、潜在的抗性机制和 用基于h8F4的双特异性抗体和h8F4嵌合抗原受体(CAR)T测试新策略 细胞增加8F4的效力，以克服潜在的治疗耐药性。

Project Summary Our long-term goal is to develop immune therapies that target aberrantly expressed proteases in blasts and leukemia stem cells. PR1 peptide (VLQELNVTV) is a peptide derived from the leukemia-associated antigens proteinase 3 (P3) and neutrophil elastase (NE), which is presented on HLA-A2 to PR1-specific cytotoxic T lymphocytes (PR1-CTL). During the last grant period, we showed that PR1 is cross-presented by dendritic cells (DCs) and B cells, and we showed the mechanism required proteasome cleavage exogenous P3 and NE taken up by antigen-presenting cells. In previous years of the SPORE grant, we conducted a Phase I-II PR1 vaccine trial in 66 patients with AML, CML, and MDS, and observed immune responses to PR1 vaccine in 58%. However, objective clinical responses were observed in only 11 (16%) patients, and these were limited to patients with low leukemia burden (<10% blasts). We showed that, although highly cytolytic PR1-CTL that expressed high avidity T cell receptors (TCR-αβ) for PR1/HLA-A2 increased after PR1 vaccination in some patients, they underwent apoptosis by leukemia that expressed high PR1/HLA-A2 surface antigen, resulting in immune tolerance by deletion of high avidity PR1-CTL. Furthermore, although high avidity PR1-CTL could be isolated from umbilical cord blood (CB) units, they are difficult to expand in sufficient quantity ex vivo to be useful as an adoptive cell therapy, thus limiting their therapeutic potential. Therefore, in a novel alternative treatment approach to target PR1, we produced a TCR-like monoclonal antibody (8F4) against PR1/HLA-A2. We have produced a humanized 8F4 (h8F4) with high affinity (KD=7.8 nM) to PR1/HLA-A2 and we showed that h8F4 eliminated AML and leukemia stem cells but not normal human hematopoietic stem cells in preclinical models. With an agreement from industry that supported manufacturing of h8F4, we have produced sufficient clinical grade h8F4, showed it mediated ADCC and apoptosis of AML and LSC in vitro and in vivo, and developed companion assays for PK, anti-idiotype and anti-drug antibody testing. Moreover, we have established PDX models of AML for parallel studies to support a clinical trial. Thus, the goals of this proposal are to (1) translate the discovery of this novel h8F4 monoclonal antibody to the clinic in a first-in-human phase I trial in AML; (2) to determine pharmacokinetics, toxicity, and mode of action; and to characterize the mechanism of action, potential resistance mechanisms and to test novel strategies with an h8F4-based bispecific antibody and an h8F4 chimeric antigen receptor (CAR) T cells to increase the potency of 8F4 to overcome potential treatment resistance.