Evaluation of the Efficacy and Safety of LILRB3-Targeted Immunotherapies in Preclinical Models of Human AML

急性髓性白血病（AML）是最常见的急性白血病，超过80%的病例发生在18岁以上的患者中。临床上的主流治疗通常以化疗为中心，其次是异基因造血干细胞移植或低甲基化剂。不幸的是，目前的疗法是不够的，因为患者仍然死于这种疾病或经历与这种治疗方案相关的相当多的副作用。尽管吉妥珠单抗（CD 33抗体药物偶联物）已被批准用于AML免疫治疗，但它仅适用于FLT 3突变患者。鉴于嵌合抗原受体（CAR）疗法在治疗患有B细胞淋巴瘤和白血病的患者亚组中的成功，几种AML靶向CAR疗法（例如，CD 33、CD 123）正在接受（临床前）评价。以前，我们的小组产生并进行了LILRB 3特异性单克隆抗体（mAb）的广泛表征。他们进一步证明，LILRB 3是一种有效的骨髓检查点，可以增强免疫调节作用，并且可能是AML免疫治疗的可行靶点。在这方面，来自其他小组的补充数据也证实LILRB 3可能提供合适的靶点，并且其在AML细胞上的表达可能通过抑制T细胞活性提供额外的免疫抑制。此外，高LILRB 3表达已被证明与AML预后和生存率较差相关。为了预测这些LILRB 3 mAb及其CAR衍生物的作用机制、有效性和安全性，在此我们着手建立表达LILRB 3的AML异种移植小鼠模型，并研究使用结构域4（克隆A1）或结构域2（克隆A20）LILRB 3 mAb靶向肿瘤细胞的治疗潜力。初步数据表明，与A1和同种型匹配的对照相比，用A20处理显著增加了NSG小鼠的存活10天。平行地，我们成功地产生了CAR构建体并转导了原代人T细胞以表达A1或A20 scFv。我们还成功地将原代人造血干细胞（HSC）移植到新生的NSG小鼠中，并对重建的“人源化小鼠”（hu-NSG）进行了广泛的表征。迄今为止，所有移植的小鼠均显示出成功的重建，具有可接受百分比的人CD 45+白细胞（30- 55%），主要由B和T淋巴细胞组成。我们目前正在开发用于产生自体CAR T细胞并将其再输注回携带AML的hu-NSG受体的稳健方案。总的来说，我们的目标是进一步优化和利用这些先进的小鼠模型（即，人AML异种移植物和人源化），以测试我们的LILRB 3-tageted免疫疗法对人AML的有效性和安全性，并以长远的眼光来转化我们的研究。

Acute myeloid leukaemia (AML) is the most common acute leukaemia with over 80% of cases present in patients over 18 years of age. The mainstream treatments in the clinic typically centre around chemotherapy, followed by allogenic haematopoietic stem cell transplantation or hypomethylating agents. Unfortunately, the current therapies are insufficient as patients still die from the disease or experience a considerable number of side effects associated with such therapeutic regimens. Although gemtuzumab ozogamicin (CD33 antibody drug conjugate) has been approved for AML immunotherapy, it is only suitable for patients with the FLT3-mutation. Given the success of chimeric antigen receptor (CAR) therapies in treating a subset of patients with B-cell lymphoma and leukaemia, several AML-targeted CAR therapies (e.g., CD33, CD123) are undergoing (pre)-clinical evaluation. Previously, our group generated and performed an extensive characterisation of LILRB3 specific monoclonal antibodies (mAb). They further demonstrated that LILRB3 is a potent myeloid checkpoint that elicits profound immunomodulation and that it may additionally be a viable target for AML immunotherapy. In this regard, complementary data from other groups have also confirmed that LILRB3 may provide a suitable target and its expression on AML cells may provide additional immunosuppression via suppressing T cell activity. Moreover, high LILRB3 expression has been shown to associate with worse AML prognosis and survival. In order to predict the mechanism of action, efficacy and safety of these LILRB3 mAbs and their CAR derivatives, here we set out to establish a LILRB3-expressing AML xenograft mouse model and investigated the therapeutic potential of targeting the tumour cells using a domain 4 (clone A1) or a domain 2 (clone A20) LILRB3 mAb. Preliminary data indicate that treatment with A20 significantly increases the survival of NSG mice by 10 days, compared to A1 and an isotypematched control In parallel, we have successfully generated CAR constructs and transduced primary human T cells to express either the A1 or A20 scFv. We have also successfully engrafted new-born NSG mice with primary human haematopoietic stem cells (HSC) and performed extensive characterisation of the reconstituted 'humanised mice' (hu-NSG). To date, all engrafted mice showed successful reconstitution with an acceptable percentage of human CD45+ leukocytes (30- 55%), mostly composed of B and T lymphocytes We are currently in the process of developing a robust protocol for generating autologous CAR T cells and reinfusing them back into AML-bearing hu-NSG recipients. Collectively, we aim to further optimise and utilise these advanced mouse models (i.e., human AML xenografts and humanised) to test the efficacy and safety of our LILRB3-tageted immunotherapies against human AML with a long view to translate our research.