Poster Presentation NSW State Cancer Conference 2023

TIM3-targeting chimeric antigen receptor T-cells for treating acute myeloid leukaemia (#244)

Daochen Tong 1 , Koon Lee 1 2 3 4 , Ken Micklethwaite 1 2 3 4
  1. Cell Therapies Group, Centre for Cancer Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
  2. Western Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
  3. Department of Haematology, Westmead Hospital, Westmead, NSW, Australia
  4. Blood Transplant and Cell Therapies Program, Westmead Hospital, Westmead, NSW, Australia

Background

Chimeric antigen receptor (CAR) T-cell is a rapidly emerging novel treatment modality for cancer, which has been especially successful against B-cell leukaemia and lymphoma. However, application to other haematological malignancies such as acute myeloid leukemia (AML) remains challenging due to the lack of suitable cancer-associated antigens. T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is an attractive target as it is overexpressed in AML bulk disease and leukemic stem cells, but they are also expressed on activated T-cells. Here, we report in vitro efficacy of a novel locally developed TIM3-specific CAR T-cell, following knock-out (KO) of TIM3 in modified T-cells to prevent fratricide.

Aim

To generate TIM3- targeting CAR (CARTIM3) T-cells and evaluate in vitro activity.

Methods

CAR short-chain variable fragment (scFv) was based upon an antibody patent. The scFv was cloned into an in-house CAR construct and electroporated into primary T-cells using the PiggyBat transposon/transposase system. TIM3 is a T-cell exhaustion marker upregulated upon T-cell activation, therefore two TIM3 guide RNAs (gRNAs) and Cas9 were co-electroporated with the CAR construct to prevent possible fratricide (n=3), then expanded in vitro using irradiated feeder cells and interleukin 15, for 2 weeks. At the end of culture period, CAR T-cells were assayed for CAR expression, exhaustion and memory phenotypes by flow cytometry. CAR T-cell function was determined by intracellular cytokine flow cytometry release and calcein-AM cytotoxicity assays upon co-culture with TIM3-overexpressing HEK293 and K562 cell lines. CAR without TIM3-targeting domain and CAR T-cells without TIM3 KO served as negative controls.

Results

The TIM3KO CARTIM3 T-cells expanded 3.8-fold with CAR expression of 75%±14%. There was no difference in phenotypes between cultures, besides lower TIM3 expression in the TIM3KO cultures as expected (7.5±7.9% vs 51.2±21%; p=0.0413).

Cytokines TNF-α and IFN-γ produced in response to TIM3 overexpressing cell lines were not significantly different to no-target controls. However, a strong cytotoxic response was observed exclusively for TIM3KO CARTIM3 T-cells, but none of the other control CARs at 20:1 effector:target ratio against TIM3 overexpressing HEK293 (60.1±12.9% vs 0.1±5.6%, p=0.0214) and K562 (90.5± 23.7% vs 13.2± 15.6%, p= 0.0128).

Discussion

We have demonstrated the successful generation of an anti-TIM3 CAR construct able to produce significant specific cytotoxicity against TIM3 overexpressing cell lines when expressed in T-cells with TIM3 KO. TIM3 is an extremely attractive AML target due to presence on leukaemic stem cells and this work could inform further translational studies.