BACKGROUND:
Discovery of chimeric antigen receptors (CARs) specific for tumour-associated antigens are emerging to be an effective form of immunotherapy for cancer treatment in recent years. However, the lack of a compelling acute myeloid leukemia (AML)-specific cell surface antigen and the safety concerns for myeloid-directed CAR T therapies causing prolonged myeloablation/aplasia which necessitate bone marrow transplantation make it challenging to develop CARTs for AML. In spite of these challenges, the high relapse rate of the disease i.e. 43% and 18% patients never attaining clinical remission (CR) with front-line induction treatments highlight an unmet need for developing improved CAR T vectors with enhanced specificity towards leukemic blasts in refractory/resistant cases with poor cytogenetics. Therefore, we aim to develop improved CAR T vectors for AML that produce safe and consistent responses in patients with high-risk disease. Herein, we demonstrate the in-vitro data for (a) efficacy of a second-generation CAR expressing six single-chain variable fragments (scFv) with different affinities for CD123 [interleukin three receptor alpha (IL3RA; CD123), a molecule over expressed on AML blasts and leukemic stem cells (LSC)] and (b) evaluate the cytotoxic effects of a dual targeting CARCD123/CD33 (against CD123 and CD33; an important myeloid marker specifically expressed on bulk AML disease) to enhance specificity towards leukemic cells therefore reducing "on-target off-organ effects".
METHOD:
Six lentiviral vectors with CAR against CD123 were constructed i.e. two high affinity (4nM kD & 4nM kD with a point mutation resulting in amino acid change K136Q), two moderate (56nM kD & 56nM kD with mutation at A105G) and two low affinity vectors (101nM kD & 101nM kD with mutation at V24G). To improve the specificity of the single targeting CARCD123, the high affinity (4nM kD, K136Q) vector was utilized to generate two dual targeting (CARCD123/CD33) constructs containing the activation domain (CD3ζ) directed against CD33 and the costimulatory domain (either CD28 or 41BB) directed against CD123. All constructs were transduced (MOI 1:5) into peripheral blood mononuclear cells (PBMCs) from healthy donors or AML patients and their cytotoxicity was examined by flowcytometry on leukemic cell lines; Kg1, U937, K562 [Fig:1a], Ramos wild type (CD19+, CD123-), artificially engineered Ramos cells (transduced by lentiviral vectors with CD123 and/or CD33 cDNA) i.e. Ramos 123 (CD123+), Ramos 33 (CD33+), Ramos 123/33 (CD123+ and CD33+) and AML mononucelar cells (MNCs).
RESULTS:
Flowcytometric analysis confirmed the expansion of T cells from PBMCs and cytotoxicity of the eight CAR constructs against target cells in in-vitro co-culture assay. High affinity CARCD123 (4nM kD & 4nM kD K136Q) T cells demonstrated enhanced cytotoxicity [Fig 1a] compared to moderate (56nM kD, 56nM kD A105G) CARCD123 in all leukemic cell lines while the low affinity (101nM kD, 101nM kD V24G) vectors had no effect. Efficacy of the high affinity CARCD123 constructs was validated on Kg1 [Fig 1b] and Ramos 123+ target cells by the increasing effector: target ratios (1:2, 1:4 & 1:10). Similar cytotoxic effects were also consistently observed against autologous AML MNCs (target cells) [n=4] and allogenic (effector cells are PBMCs from healthy donors) AML MNCs [Fig 1c] [n=3]. T cell activation was confirmed by ELISA and showed increased IFN-γ (500-2000 fold) and TNF-α (150-200 fold) levels after 24hr co-culture.
Furthermore, we also elucidated the exclusive cytotoxicity of the two dual targeting CARCD123/CD33 in Ramos 123+/33+ cells [Fig 1d] with no effect being observed on Ramos 123+, Ramos 33+ and Ramos wild type (123-/33-) cell lines. This confirmed the absence of non-specific targeting, validated the improved specificity of the CARs towards leukemic cells and demonstrated a potential to reduce deleterious "on-target but off organ effects".
CONCLUSION:
In summary, we illustrate in-vitro data establishing the importance of scFv on CAR T cell cytotoxicity and exemplify for the first time an improved specificity of CARTs by targeting two antigens simultaneously in AML. Future work will involve examining the in-vivo dynamics of CAR CD123 and CAR CD123 CD33 on the hematopoietic system and on disease pathogenesis with an aim to proceed to phase I clinical trial.
Mufti:BMS, Novartis: Research Funding; Abbvie, Novartis: Consultancy.
