Developing novel targeted immunotherapy for infant and childhood MLL-rearranged acute lymphoblastic leukaemia (co-funded by Children with Cancer UK)

Background: MLL-rearranged infant acute lymphoblastic leukaemia (MLLr-iALL) has a very poor prognosis (40% cure rates), highlighting the need for novel treatment strategies.

Chimaeric antigen receptor (CAR)-based immunotherapy in the form of CAR-T-cells directed against antigens that are expressed on the surface of leukaemic cells (CD19/CD22) is the most recent novel therapy against childhood ALL.

A limitation to CAR-T therapy for iALL is the need for autologous T-cells, often difficult to obtain reliably from such young children after intensive chemotherapy.

CAR-invariant natural killer T (CAR-iNKT) cell immunotherapy is an attractive alternative approach because of their potential for ‘off-the-shelf’ availability and superior performance compared to CAR-T therapy in preclinical CD19+ lymphoma.

In this project, we aim to design and validate bispecific iNKT-cell immunotherapy against CD19 and CD133 antigens expressed on MLLr leukaemic cells. Aims and methods: 1.

Define the pattern of co-expression of CD19, CD133 and CD1d surface antigens on normal fetal and paediatric haematopoietic stem and progenitor cells (HSPC) and MLLr-ALL blasts using multiparameter flow cytometry. 2.

Develop and validate anti-CD133 CAR iNKT-cell immunotherapy by developing first a lentiviral CAR133 followed by generation of CAR-iNKT-cells using optimised protocols.

Efficacy of CAR133-iNKT-cells will be validated using in vitro and in vivo assays against MLLr cell lines, MLLr ALL patient samples and a novel MLL-AF4 leukaemia in vivo model and compared to CAR133-T cells.

The functional profile of anti-leukaemic CAR133-iNKT-cells will be studied using the Chromium 10x platform to simultaneously analyse transcriptome and TCR repertoire in single CAR-iNKT-cells; and imaging mass cytometry to investigate the spatial relationship between tissue resident CAR-iNKT-cells and leukaemic cells. 3.

Develop and validate optimal bi-specific CAR19/133-iNKT immunotherapy by combining the optimal CAR133 developed in (2) with CAR19 to generate bispecific CAR19/133-iNKT-cells.

The efficacy of these cells will be validated as in (2) and compared to monospecific CAR19-iNKT and CAR133-iNKT-cells. 4.

Determine whether CAR CD19/133 -iNKT cells cause major haematological toxicity (given CD133 expression on a wide range of normal HSPC) using in vivo models of humanised haematopoiesis.

Expected impact: Bispecific CAR19/133-iNKT cell immunotherapy will be an effective ‘off-the-shelf’ treatment for MLLr-ALL. We envisage that upon completion of this project, we will be able to swiftly proceed to clinical development.

This novel immunotherapy will allow us to deliver clinically meaningful improvements in outcome for infant ALL, which so far has been impossible to achieve.