Session: 703. Cellular Immunotherapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Acute Myeloid Malignancies, Biological therapies, Clinical Practice (Health Services and Quality), AML, APL, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Diversity, Equity, Inclusion, and Accessibility (DEI/DEIA) , Diseases, Therapies, Myeloid Malignancies
Methods: To generate CAR T cells, negatively selected CD8+ T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG4 hinge only), intermediate (hinge plus IgG4 CH3 domain), or long (hinge plus IgG4 CH3 domain plus IgG4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants.
Results: CD33V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33V-set CAR T cells, optimized CD33PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33PAN CAR T cells extended survival in immunodeficient NOD.SCID.IL2rg-/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion.
Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33PAN CAR T cells toward clinical testing.
Disclosures: Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.
See more of: Oral and Poster Abstracts