Effector Differentiation and Exhaustion Attenuate on-Target, Off-Tumor Toxicity of CD24 CAR T Cells
Authors: Boardman Alexander P. , Rajagopalan Adhithi , Chen Sophia , Ng Brandon , Fischman Jacob , Massa Alyssa , Elias Harold K , Manuele Dylan , Atmane Mohamed I. , Miranda Ileana , James Scott E. , van den Brink Marcel R.M. 
Published: December 2024

Introduction
CD24 is a “don't eat me” signal that inhibits macrophage phagocytosis and has emerged as a potential immunotherapy target due to expression on solid tumors and hematologic malignancies and its proposed role in promoting immune evasion. CD24-CAR T cells have been described, however widespread expression of CD24 on healthy tissues introduces therapeutic challenges such as on-target, off-tumor toxicity. Little is known about the molecular drivers of CAR T cell off-tumor toxicity. In this study, we evaluated determinants of efficacy and toxicity of CD24-CAR T cells by modulating signal transduction via CD3ζ and costimulatory signals.
Methods
To assess toxicity in a syngeneic mouse model, we generated mouse-specific CD24-CAR T cells incorporating a CD28 costimulatory domain and a CD3ζ activation domain. To compare efficacy and toxicity of CAR designs with varying strengths of signal 1 and signal 2, we additionally constructed and evaluated CD24-CARs with inactivation of immunoreceptor tyrosine activation motifs (ITAMs) 2 and 3 (1XX), CD24-CARs with CD28 costimulatory domain mutations, and those lacking costimulation (first generation). Quantitative live cell microscopy was performed to measure T cell proliferation, NFkB activation, and target lysis. We evaluated CD24-CAR T cells in syngeneic mouse models of pre-B cell acute lymphoblastic leukemia (BM185), B cell lymphoma, and acute myeloid leukemia (AML) following sublethal irradiation as pre-conditioning. We evaluated mice for tumor clearance by bioluminescence imaging, and toxicity via weight assessments, serum cytokine quantification, and multiparameter flow cytometry of bone marrow.
Results
CD28-costimulated CD24-CAR T cells (CD24.28z) showed modest anti-tumor activity against BM185 in vivo, without evidence of toxicity. To boost T cell activity and restrict differentiation and exhaustion, we modulated signal 1 by mutating ITAMs. Treatment with CD24.28.1XX T cells improved anti-tumor activity, but mice required euthanasia due to moribund state and weight loss, associated with elevation in serum cytokines IL-6, IFNg, and MCP-1 suggesting cytokine release syndrome (CRS), and pulmonary parenchymal CAR T cell infiltration causing pneumocyte apoptosis and vasculitis. CD24.28.1XX T cells were similarly active and toxic in lymphoma and AML models. CRS and toxicity were fully abrogated in CD24-deficient recipient mice, which cleared leukemia and demonstrated long-term survival after CD24.28.1XX T cell infusion. In vitro, CD24.28.1XX T cells exhibited increased antigen-dependent proliferation and IFNg production relative to CD24.28z. First generation 1XX CD24-CAR T cells did not cause early lethal toxicity, but enabled late leukemia outgrowth, suggesting the requirement of CD28 costimulation for efficacy and toxicity. Mutation of CD28 YMNM and PRRP, but not PYAP, motifs attenuated in vivo toxicity of CD24.28.1XX T cells, which correlated with decreased in vitro NFkB activation. Overexpression of a degradation-resistant IkBalpha canonical NFkB pathway inhibitor (NFkBIAm) attenuated cytokine production in vitro and in vivo, inhibited pulmonary toxicity, and eliminated early toxicity, but also impeded tumor clearance. Transcriptomic analysis identified that compared with CD24.28.1XX T cells, both CD24.28z and CD24.28.1XX.NFKBIAm T cells exhibited reduced type I/II interferon responses, enhanced CD8 cytotoxic differentiation, and increased exhaustion scores. NFkBIAm additionally downregulated pathways related to metabolism and cell cycle. Co-expression of PD-1-OX40 and Fas-4-1BB switch receptors with CD24.28.1XX.NFkBIAm transiently increased NFkB activation, enhancing anti-leukemia activity and prolonging survival of tumor-bearing mice.
Conclusions
Our findings demonstrate robust anti-tumor activity of CD24-CAR T cells against multiple hematologic malignancies, but also identified non-relapse mortality from on-target, off-tumor toxicity with prominent CRS and pulmonary pathology, which required CD24 expression on normal tissues. Promoting T cell differentiation and exhaustion via full-strength CD3 zeta signaling and NFkB attenuation mitigated toxicity. Addition of OX40 and 4-1BB costimulation to NFkB-inhibited CD24 CAR T cells enhanced CAR T cell expansion and anti-tumor activity without early lethal toxicity, enabling prolonged survival in a syngeneic mouse model.