Project Summary
CAR T cell therapies have been revolutionary for patients with relapsed/refractory hematologic malignancies;
however, the relapse rates are unacceptably high, with ~60% of patients relapsing with leukemia and lymphoma,
and for multiple myeloma (MM), where more follow up is needed, relapse is likely even higher. Notably, adult
patients rarely develop long-term immunologic memory or functional long-term persistence of CAR T cells, with
relapse predictably often occurring after the loss of functional adoptive anti-tumor immunity. One solution to
short-lived adoptive immunity is to genetically modify hematopoietic stem cells (HSCs) with the transgene for the
CAR. This would result in the CAR being expressed across hematopoiesis long-term. However, as cytokine
release syndrome (CRS) is mediated by T and myeloid cells, this long-term pan-hematopoietic expression would
put patients at great risk for cytokine-mediated or other toxicity. We propose an alternative concept, where
HSCs are stably integrated with DNA encoding the CAR; however, transcription is only initiated once
cells have differentiated into NK cells to generate safe and efficacious “HSC CAR-NK factories”.
We selected NK cells as the risk of CRS is minimal, there is no need for thymic differentiation, and they have a
proven track record of CAR-mediated killing; however, the defining limitation of CAR-NK cell therapies has been
persistence, which this approach is designed to address via long-term continuous renewal. We hypothesize that
including NK-specific regulatory elements controlling CAR transgene expression will generate NK-specific, highly
efficacious long-term anti-cancer immunity.
In preliminary data, we demonstrated that patient HSC-based CAR transduction and transplantation in the setting
of murine models that generate minimal T cells can be highly active in preventing tumor growth; NK cells
generated this way and isolated ex vivo maintain cytolytic anti-tumor function through the CAR. We further
identified, via analysis of comparative ATAC-seq data, a library of NK-specific regulatory elements and found a
lead assembly of these regulatory elements that reproducibly led to CAR expression highly enriched in NK cells;
a finding that we validated in different in vivo HSC transplant/NK differentiation models using patient HSCs.
Here, we will investigate if a naturally occurring NK-specific regulatory element assembly, (Aim 1) can be
leveraged to develop a model HSC-based persistent adoptive cellular therapy platform for anti-tumor immunity,
and (Aim 2) seek to understand the biology and maximize the efficacy of a “HSC CAR-NK cell factory” approach.
Specifically, we will use three separate in vivo models that allow for NK development post-huHSC
transplantation.
