Preclinical Development of a CAR-T Cell Approach Targeting the CD84 Antigen Associated to Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) treatment outcomes significantly improved over the past 3 decades, but the development of new therapies still remains paramount for ameliorating the prognosis of patients with relapsed, refractory life-threatening forms. Indeed, refractory disease and relapse still remain the main causes of mortality in pediatric AML despite the use of intensive chemotherapy protocols and allogeneic hematopoietic stem cell transplantation. Numerous clinical trials and pre-clinical studies are nowadays testing new immunotherapy-based drugs in patients with hematological malignancies, the most promising results having been obtained employing the CAR-T (Chimeric Antigen Receptor T) cell technology. This approach is based on ex vivo engineered T cells expressing a membrane receptor combining the specificity of the variable component of immunoglobulins with molecules playing a major role in the cytotoxic machinery of T cells.

With the goal of developing a novel and highly specific CAR-T cell approach for pediatric AML, we performed a large analysis of a gene expression dataset from AML patients and identified for the first time the CD84 as an AML specific antigen. CD84 was highly expressed on AML blasts of 99% of the tested validation cohort composed of newly diagnosed and relapsed AML patients. This antigen is also characterized by a very low/null expression on healthy regenerating bone marrow cells and hematopoietic stem cells. Two novel single-chain variable fragment (ScFv) recognizing CD84 were thus generated by phage display. The new CAR cassettes containing these ScFv chains were cloned into a plasmid transfer to produce third-generation lentiviral vectors (LVs). We then applied a standard protocol for producing second-generation 4-1BB CAR-T cells by LV transduction of IL-7 and IL-15 activated T cells. After 14 days of expansion, the CAR-T cells generated at a 2-20 fold expansion rate were mostly CD4⁺ (>70%), with central memory (Tcm, 65% of CD4⁺ cells), naive (Tn) and stem cell memory (Tscm) differentiation (cumulatively > 20% of CD4⁺ cells), and no effect of the ScFv chains on cell activation or exhaustion. The CAR-T cells were co-cultured with several target cell lines expressing CD84 and non-target CD84^neg cell lines, to assess specificity, at 1:1 effector to target (E:T) ratio. After 48 hours of co-culture, CAR-T lytic activity was analyzed by flow-cytometry with Annexin V and 7-AAD, and by luciferase viability assay on luciferase-transduced AML cell lines. We documented a significant in vitro antitumor activity with a lysis potency ranging between 10 to 50% on target AML cell lines (SHI-1 and HL-60), with specificity towards CD84 (K562 and KASUMI-1). Importantly, the CAR-T cells did not exert toxicity on hematopoietic stem and progenitor cells, as they induced neither a significant reduction in the number of colony-forming units (CFUs) generated by CD34⁺ cells nor a reduction in cell viability. Interestingly, single in vivo infusion of the anti-CD84 CAR-T cells in NOD/SCID gamma mice engrafted with target SHI-1 AML cell line (3:1 E:T ratio) showed a 75% reduction of leukemia burden to almost undetectable levels (8x10¹⁰ of total flux of control mice vs 2x10¹⁰ of total flux of anti-CD84 CAR-T cell injected mice) from day 14 post AML cell injection up 80 days of observation.

Overall, these results support further in vivo testing of our two novel ScFv(s) recognizing CD84 in AML-PDXs to assess and develop their potential as novel immunotherapy approach to treat pediatric AML.

Pigazzi:Altheia Science: Consultancy. Rizzardi:Altheia Science: Current Employment. Locatelli:SOBI: Speakers Bureau; JAZZ PHARMACEUTICALS: Speakers Bureau; MILTENYI: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BLUEBIRD BIO: Speakers Bureau; AMGEN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TAKEDA: Speakers Bureau; MEDAC: Speakers Bureau; GILEAD: Speakers Bureau; SANOFI: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NEOVII: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Membership on an entity's Board of Directors or advisory committees. Biffi:Altheia Science: Consultancy.