Next-generation genetic engineering of the pan-leukocyte antigen CD45 to facilitate CAR-T cell therapy against hematologic malignancies

下一代全白细胞抗原 CD45 基因工程促进 CAR-T 细胞治疗血液恶性肿瘤

• 批准号: 10713201
• 负责人: Saar Gill
• 金额: $ 47.26万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713201
• 关键词: Acceleration; Address; Adoptive Immunotherapy; Alanine; Allogenic; Antibody-drug conjugates; Antigens; B lymphoid malignancy; Biotechnology; Bone Marrow Purging; CAR T cell therapy; CD19 gene; CD34 gene; CD45 Antigens; CRISPR/Cas technology; Cell Therapy; Cells; Cessation of life; Clinical Research; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Credentialing; Disease; Engineering; Engraftment; Epitopes; Extracellular Domain; Functional disorder; Genes; Genetic Engineering; Goals; HLA Antigens; Hematologic Neoplasms; Hematology; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Human; Immunodeficient Mouse; Immunotherapy; In Vitro; Individual; Infusion procedures; Knock-out; Lead; Malignant Neoplasms; Medical; Mission; Multiple Myeloma; Mutagenesis; Mutation; PTPRC gene; Patients; Pharmaceutical Preparations; Physicians; Protein Tyrosine Phosphatase; Public Health; Reporting; Research; Resistance; Scientist; Site; Stem cell transplant; Surface; T cell therapy; T-Lymphocyte; Technology; Testing; Translating; Work; base editing; chemotherapy; chimeric antigen receptor; chimeric antigen receptor T cells; clinical development; conditioning; design; donor stem cell; drug development; engineered T cells; immunological synapse; in vivo; leukemia; next generation; novel therapeutics; prevent; receptor; screening; self-renewal; stem cells; stemness; tool

SUMMARY/ABSTRACT (PROJECT 2): Next-generation genetic engineering of the pan-leukocyte antigen CD45 to facilitate CAR-T cell therapy against hematologic malignancies Currently, chimeric antigen receptor (CAR)-T cells must be individually designed for each disease, targeting lineage-associated antigens such as CD19 for B-cell malignancies or BCMA for myeloma. While this approach has led to the successful treatment of hundreds of patients on clinical trials or with commercial CAR-T cell therapies, from a drug development standpoint this approach is inefficient. By targeting a pan-hematologic antigen, a single “drug” could be used for all indications, thereby accelerating clinical research. CD45 is a receptor tyrosine phosphatase that is expressed on the surface of most hematopoietic cells, from immature stem cells to differentiated progeny. As expected, antibody-drug conjugates targeting CD45 cause profound myeloablation and are in clinical development as conditioning agents prior to stem cell transplantation. A unique hurdle limiting the use of anti-CD45 CAR T cells (CART-45) is that T cells themselves express CD45, and CART- 45 are therefore vulnerable to fratricide. Furthermore, CD45 is crucial for proper function of the T cell immune synapse. The overarching goal of this project is to render CD45 targetable using genetic engineering of T cells and of hematopoietic stem cells (HSC). To achieve this goal, we will test the central hypothesis that site-specific mutation of CD45 in hematopoietic cells (including T cells) can abrogate recognition by CART-45 while retaining all other domains required for the function of this molecule. We expect that this approach will prevent T cell dysfunction and myeloablation. This will be accomplished in three specific aims. In Aim 1, we will generate fratricide-resistant CD45-targeting CAR-T cells. We have identified a candidate CAR construct based on unbiased functional screening and shown that CART-45 are indeed subject to fratricide. While this fratricide could be rescued by CRISPR-based knock out of CD45, CD45-deficient CART cells were dysfunctional. Using alanine mutagenesis, we then identified the target epitope on CD45 that is recognized by our lead CAR candidate and we have now successfully edited this epitope to abrogate fratricide, resulting in CAR-T cells that recognize native CD45 in trans without themselves being the target of anti-CD45 CAR. In this aim we will test the hypothesis that the function of CD45-edited T cells is equivalent to unmodified control T cells, and superior to that of CD45- deleted T cells. In Aim 2, we will genetically engineer hematopoietic stem cells to generate a CART-resistant hematopoietic system. To demonstrate that engineered CD34+ HSC’s are resistant to CD45 targeted CAR-T cell therapy, engineered HSPC’s will be engrafted into immunodeficient mice followed by treatment with CD45 CAR-T cells. Finally, to target CD45 in human AML we will disrupt CD45 expression in primary human AML and test leukemogenicity in vitro and in vivo. In Aim 3 up to 6 patients will undergo allogeneic stem cell transplantation using donor stem cells where CD45 expression has been edited using CRISPR/Cas9, followed by infusion of donor-derived CART-45. Successful completion of this Aim will provide proof-of-concept for our overall strategy to create a leukemia-specific antigen using gene editing tools.

摘要/摘要(项目2)：泛白细胞的下一代基因工程 抗原CD45促进CAR-T细胞治疗血液系统恶性肿瘤 目前，嵌合抗原受体(CAR)-T细胞必须针对每种疾病单独设计，靶向 血统相关抗原，如B细胞恶性肿瘤的CD19或骨髓瘤的BCMA。虽然这种方法 已经成功地治疗了数百名正在进行临床试验或使用商业CAR-T细胞的患者 从药物开发的角度来看，这种方法是低效的。通过瞄准泛血液学 抗原，一种“药物”可以用于所有适应症，从而加快临床研究。CD45是一种 受体酪氨酸磷酸酶，表达在大多数造血细胞表面，来自未成熟的干细胞 细胞传给分化的后代。正如预期的那样，针对CD45的抗体-药物结合物导致深远的 作为干细胞移植前的调理剂，目前正处于临床开发阶段。独一无二的 限制抗CD45 CAR T细胞(CART-45)使用的障碍是T细胞本身表达CD45，而CART-45 因此，45人容易受到自相残杀的伤害。此外，CD45对于T细胞免疫的正常功能至关重要 Synapse。该项目的首要目标是利用基因工程使CD45具有靶向性 T细胞和造血干细胞(HSC)。为了实现这一目标，我们将检验以下核心假设 造血细胞(包括T细胞)中CD45的定点突变可使CART-45识别功能丧失 同时保留该分子功能所需的所有其他结构域。我们预计，这种做法将 预防T细胞功能障碍和骨髓清除术。这将通过三个具体目标来实现。在目标1中，我们将 产生抗杀菌剂CD45靶向CAR-T细胞。我们已经确定了一个候选的汽车构造，基于 关于无偏见的功能筛查，并表明CART-45确实受到自相残杀的影响。虽然这起自相残杀 可以通过基于CRISPR的CD45基因敲除来拯救，CD45缺陷的CART细胞功能障碍。vbl.使用 丙氨酸突变，然后我们确定了CD45上的目标表位，它是我们的主要候选CAR识别的 我们现在已经成功地编辑了这个表位，以消除兄弟会，导致CAR-T细胞识别 天然CD45反式表达，自身不是抗CD45 Car的靶标。在这个目标中，我们将检验这一假设 CD45编辑的T细胞的功能与未修饰的对照T细胞相当，而优于CD45- 删除了T细胞。在目标2中，我们将对造血干细胞进行基因改造，以产生一种抗CART的 造血系统。证明工程CD34+造血干细胞对CD45靶向CAR-T具有抵抗力 细胞疗法，工程HSPC将被植入免疫缺陷小鼠，然后用CD45治疗 CAR-T细胞。最后，针对人AML中的CD45，我们将干扰原发人AML中CD45的表达，并 检测体外和体内的致白血病作用。在AIM，3至6名患者将接受异基因干细胞移植。 使用已使用CRISPR/Cas9编辑了CD45表达的供体干细胞，然后输注 供体来源的CART-45。成功实现这一目标将为我们的整体战略提供概念验证 使用基因编辑工具创建白血病特异性抗原。