Background CD19-specific chimeric antigen receptor (CAR) T cell therapy has achieved high efficacy in acute lymphoblastic leukemia patients. However, the treatment of acute myeloid leukemia (AML) has remained a particular challenge due to the heterogeneity of AML bearing cells, which renders single antigen targeting CAR T cell therapy ineffective. CLL1 and CD33 are often used as targets for AML CAR T cell therapy. CLL1 is associated with leukemia stem cells and disease relapse, and CD33 is expressed on the bulk AML disease. Previously, we demonstrated the profound anti-tumor activity of CLL1-CD33 compound CAR (cCAR) T cells. Here we present the efficacy of cCAR in preclinical study and update the success in level 1 dose escalation clinical trial on relapsed/refractory AML patients. Methods We engineered a cCAR comprising of an anti-CLL1 CAR linked to an anti- CD33 CAR via a self-cleaving P2A peptide and expressing both functional CAR molecules on the surface of a T-cell cell. We tested the anti-leukemic activities of CLL1-CD33 cCAR using multiple AML cell lines, primary human AML samples, human leukemia cell line (REH cells) expressing either CLL1 or CD33, and multiple mouse models. An alemtuzumab safety switch has also been established to ensure the elimination of CAR T cells following tumor eradication. Children and adults with relapsed/refractory AML were enrolled in our phase 1 dose escalation trial with primary objective to evaluate the safety of cCAR and secondary objective to assess the efficacy of cCAR anti-tumor activity. Results Co-culture assays results showed that cCAR displayed profound tumor killing effects in AML cell lines, primary patient samples and multiple mouse model systems. Our preclinical findings suggest that cCAR, targeting two discrete AML antigens: CLL1 and CD33, is an effective two-pronged approach in treating bulk AML disease and eradicating leukemia stem cells. Patients enrolled in the phase 1 dose escalation trial have shown remarkable response to cCAR treatment. Noticeably, a 6-yr-old female patient diagnosed with a complex karyotype AML including FLT3-ITD mutation had achieved complete remission. The patient was diagnosed with Fanconi anemia, which had progressed to juvenile myelomonocytic leukemia and eventually transformed into AML. The patient had been resistant to multiple lines of treatments, including 5 cycles of chemotherapy with FLT3 inhibitor prior to receiving cCAR. Before the treatment, patient's leukemia blasts comprised 73% of the peripheral blood mononuclear cells and 81% of the bone marrow. Patient underwent lymphodepletion therapy (Fludarabine and Cyclophosphamide) prior to cCAR infusion. Two split doses, each consisting of 1x106/kg CAR T cells, were infused on day 1 and day 2 respectively. On day 12, while leukemia blast still counting up to 98% of the bone marrow (Fig. 1A), robust CAR T cell expansion was detected in both peripheral blood and bone marrow. On day 19, patient achieved MRD- complete remission with bone marrow aspirates revealing complete ablation of myeloid cells (Fig. 1B). Flow cytometry confirmed the absence of leukemia blasts and showed that CAR T cells comprised 36% of the PBMC and 60% of the bone marrow. The patient later underwent non-myeloablative hematopoietic cell transplantation with less toxicities compared to conventional total body radiation and high dose chemotherapies. Updated results on other patients enrolled in this clinical trial including adverse events will be presented. Conclusion Our first-in-human clinical trial demonstrates promising efficacy of cCAR therapy in treating patients with relapsed/ refractory AML. cCAR is able to eradicate leukemia blasts and leukemia stem cells, exerting a profound tumor killing effect that is superior to single target CAR T cell therapies. cCAR is also shown to induce total myeloid ablation in bone marrow, suggesting that it may act as a safer alternative to avoid the severe toxicities caused by standard bone marrow ablation regimens without sacrificing the anti-tumor efficacy. This strategy will likely benefit patients who are unable to tolerate total body radiation or high dose chemotherapies. In addition to AML, cCAR also has the potential to treat blast crisis developed from myelodysplastic syndrome, chronic myeloid leukemia, and chronic myeloproliferative neoplasm. Disclosures Pinz: iCell Gene Therapeutics LLC: Employment. Ma:iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Chen:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC, iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

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The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult β-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation.

Hematopoietic stem cells (HSCs) are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The expansion of HSCs has remained an important goal to develop advanced cell therapies for bone marrow transplantation and many blood disorders. Over the last several decades, there have been numerous attempts to expand HSCs in vitro using purified growth factors that are known to regulate HSCs. However, these attempts have been met with limited success for clinical applications. New developments in the HSC expansion field coupled with gene therapy and stem cell transplant should encourage progression in attractive treatment options for many disorders including hematologic conditions, immunodeficiencies, and genetic disorders.