Development of CD371 shielded hematopoietic stem cell grafts for protection against AML-directed immunotherapies

Abstract

Abstract CAR T cells are a promising immunotherapy for AML. However, a major obstacle in the success of CAR T cells for AML has been that the most commonly evaluated AML surface antigens are expressed on normal hematopoietic stem and progenitor cells (HSPCs) and/or essential mature myeloid cells. An emerging strategy to overcome this hurdle is genetic editing of immunotherapy targets in normal hematopoietic stem cells (HSCs) thereby shielding them from T cell-mediated elimination. Recently, robust clinical efficacy of an IL-18 armored CD371-directed CAR T cell platform has been demonstrated in patients with refractory AML (NCT06017258). Although several patients achieved MRD-negative remissions, clinical courses were complicated by severe myelosuppression and the need for allogeneic stem cell rescue. We therefore set out to investigate the requirement of CD371 for normal human hematopoiesis with the goal of developing CD371-deficient HSC grafts to allow for effective anti-leukemia T cell therapy with preserved hematopoietic reconstitution. We performed CRISPR-mediated deletion of CD371 using two distinct sgRNAs in human cord blood CD34+ cells and achieved >95% CD371 surface protein depletion on HSCs and mature myeloid cells. Deletion of CD371 in HSCs did not alter the number or proliferation of mature myeloid (CD11b, CD33, or CD14) cells generated in liquid culture containing myeloid inducing cytokines (SCF, TPO, G-CSF, and GM-CSF). Moreover, stimulation of CD371 null mature myeloid cells with LPS did not impact production of inflammatory cytokines (IL-8, TNF-a, and MIP-1b). Similarly, CD371 deletion did not influence the number of mature myeloid or erythroid colonies yielded from CD34+ cells seeded in methylcellulose semi-solid media. In each of these experiments, CD371 protein-level deletion was verified at the end of each culture period. Following the above in vitro studies, we evaluated the impact of CD371 deletion on human hematopoiesis in vivo. CD371 deletion on human CD34+ cells allowed for effective human hematopoiesis after four months of primary and secondary transplantation in NSGS mice with successful qualitative and quantitative recovery of human mature B-, T-, or myeloid cells in vivo. At each time point of in vivo evaluation, CD371 deletion at the DNA and protein level was maintained. To further interrogate the impact of CD371 deletion on human hematopoiesis, we performed CITE-seq on human CD371 wild-type (WT) versus null human CD45+ cells sorted from mouse bone marrow four months post-transplantation. These studies revealed that CD371 was indeed most abundantly expressed (at RNA and protein level) in antigen presenting cells (monocytes, macrophages, and dendritic cells) as well as a subset of HSCs. Nonetheless, we achieved stable CD371 deletion on all human cell populations from mice engrafted with CD371-null cells without any alterations in frequency of HSPCs or mature myeloid or lymphoid cells. Moreover, CD371 KO mature myeloid and dendritic cells retained comparable gene expression profiles relative to CD371-expressing counterparts. Having confirmed minimal effects of CD371 loss on human hematopoiesis, we next evaluated the impact of CD371 deletion in response to CD371-directed immunotherapies. We isolated CD34+ and CD3+ cells from the same donors to simultaneously create CD371-null CD34+ HSPCs and autologous anti-CD371 CAR T cells. CD371 CAR T cells effectively killed autologous normal human HSCs in vitro and in vivo, as well as human AML cell lines (with IL-18 armored CARs yielding more potent killing), while CD371-null human HSCs were spared from CAR T killing. Similarly, CD371 HSC modification also protected HSCs from the anti-leukemic activity of a newly generated CD371xCD3 bispecific antibody. Here we identified that CD371 is dispensable for human hematopoiesis in vitro and in vivo using xenotransplantation studies in mice as well as functional studies of B-, T-, and myeloid cells derived from long-term engrafted human CD371-null HSCs. Genetic depletion of CD371 in human CD34+ cells yields functional long-term HSCs and blood progeny which are shielded from a clinically validated CAR approach for patients with AML, as well as an investigational off-the-shelf bispecific product. This approach thereby creates CD371-shielded CD34+ grafts to mitigate on-target/off-tumor myelotoxicity for emerging T cell immunotherapies for AML without compromising post-transplant immune reconstitution.