Leveraging the TCR Repertoire to identify target neoantigens in FLT3-ITD positive Acute Myeloid Leukemia

Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with high-
risk acute myeloid leukemia (AML). However, HSCT is affected by graft-versus-host disease (GvHD) and graft-
versus-leukemia (GvL) effects, both are mediated by donor T lymphocytes and significantly impact treatment
success and thus overall outcome. AML patients commonly harbor FLT3/internal tandem duplication (FLT3-ITD),
a mutation in the receptor tyrosine kinase FLT3 that is associated with poor prognosis. FLT3 targeted therapies
have proven clinical benefit particularly when used in combinational approaches. Midostaurin (a kinase inhibitor)
was recently approved for pre-transplant patients with FLT3-ITD in combination with standard therapy. In addition
to their direct leukemia suppressive effects, FLT3 inhibitors activate leukemia antigen-specific T-cell responses.
T-cell receptors (TCRs) are proteins expressed on the surface of T cells that recognize antigens presented by
MHC molecules. We recently characterized the TCR repertoire in patients who underwent matched donor or
haplo-cord HSCT. We demonstrated that GvHD and relapse (exclusive of each other) are associated with lower
TCR repertoire diversity and expansion of certain T-cell clones. Our data suggest that individual variations in
the immune repertoire significantly impact the clinical outcome in AML patients and underscore the need
for comprehensive quantitative, functional, and mechanistic analyses of the TCR repertoire in a large
cohort of AML patients. Here, we hypothesize: 1) TCR repertoire (diversity, clonal expansion, and V-segment
utilization) affects clinical outcome (GvHD or relapse) and can therefore be used to identify GvL- and GvHD-
associated clones; 2) Somatic mutations in leukemic cells (e.g., FLT3-ITD) affect the TCR repertoire and
subsequent expansion of specific T-cell clones; and 3) FLT3 inhibitors (e.g., midostaurin) modulate the TCR
repertoire and function and enhance GvL effects in patients undergoing HSCT. We will conduct a prospective
longitudinal cohort study characterizing the TCR repertoire and mutational landscape of leukemia cells in ~250
patients (~ 60–80 with FLT3-ITD). GvHD or relapse will be predicted using a proportional hazards model for
competing risks based on TCR repertoire characteristics. TCR sequences and somatic mutations will be
analyzed using a structure based prediction algorithms we developed to predict candidate leukemia neoantigens
and associated TCR clones. Neoantigens will be validated using in vitro and murine models. Finally, functional
analyses will examine the effect of midostaurin on TCR repertoire and function. Our findings will establish the
TCR repertoire as a useful tool for predicting clinical outcomes of HSCT and identify responsible TCR clones.
The identification of TCR clones associated with the GvL effect against FLT3-ITD+ cells will facilitate the
development of engineered T cells expressing GvL-associated TCR clones. Modifying the TCR repertoire
composition via therapies targeting specific somatic mutations will facilitate development of optimized
combinational therapeutic approaches, such as the addition of targeted therapy to post-transplant regimens.

Grant Number: 5R01CA248381-05
NIH Institute/Center: NIH
Principal Investigator: Houda Alachkar