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

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.