PROJECT SUMMARY/ABSTRACT:
The goal of this study is to develop a novel therapeutic strategy for AML, a malignancy of myeloid lineage, that
represents a disease with enormous unmet therapeutic need. Hematopoietic stem cell transplant (HSCT) is the
only curative treatment option for patients with relapsed/refractory AML but up to 40% patients further relapse
after HSCT. Targeting of AML cells using agents directed against a lineage specific antigen such as CD33 using
the antibody drug conjugate Gemtuzimab Ozagamycin (GO; Mylotarg) has improved outcomes, but the use of
Mylotarg is associated with severe myelosuppression. This is due to targeting of both the leukemia cells and
normal myeloid cells (including stem, progenitor and myeloid cells in the donor graft) that also express CD33
(the normal myeloid cells are concomitantly killed by Mylotarg, which also has other toxicities, including veno-
occlusive disease at high doses). We reasoned that by ablating CD33 expression using genomic engineering
methods in donor stem/progenitor cells, we could generate stem/progenitor cells for transplant that are resistant
to Mylotarg treatment, while rendering the AML cells uniquely sensitive to anti-CD33 therapy such as low-
dose Mylotarg, anti-CD33 CAR-T or CD33 bi-specific T cell engagers (BiTEs). Indeed, in a recently published
“proof of concept study”, we demonstrated that this strategy (either Mylotarg alone, CART-33 or both) enabled
the complete killing of an engrafted human CD33+ AML cell line, while allowing fully functional hematopoietic
repopulation by CD33- HSPCs.
In preliminary experiments with primary human AML cells, however, we found that despite AML clearance with
Mylotarg, a small fraction of AML persisted in the bone marrow after 16 weeks of treatment. These AML cells
are CD33+ but also express a second lineage antigen, CLL-1+ (Clec12a). We reasoned that targeting multiple
antigens will result reduce the chances of relapse. In this proposal, we plan to test the hypothesis that long-term
leukemia remission is achieved when two antigens on AML cells are targeted, either serially or simultaneously.
To test this hypothesis, we will measure the engraftment, differentiation, and functional potential of HSPCs that
are gene-edited to ablate expression of CLL-1 antigen alone or in combination with CD33 (Aim 1). We will test
this two-hit treatment approach using patient derived xenograft (PDX) models of primary human AML and also
test the engraftment, differentiation, and functional potential of single (CLL-1) and double (CLL-1 and CD33)
deletion HSPC in this context (Aim 2). The long-term objective of this study is to develop novel cell-based gene
therapeutic strategies in combination with immunotherapeutic approaches for the treatment of AML and related
cancers such myelodysplastic syndromes.
