CAR T cells mediate antitumor effects in a small subset of cancer patients 1-3 , but dysfunction due to T cell exhaustion is an important barrier to progress 4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmark features of exhaustion 6. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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are coinventors on a patent application for a CD93-CAR. C.L.M. is a cofounder and consultant for Lyell Immunopharma, and Syncopation Life Sciences, which are developing CAR-based therapies, and serves as an advisor and consultant for Roche, NeoImmune Tech, Immatics, Apricity and Nektar. R.G.M. is a cofounder of Syncopation. E.S. is a consultant for Lyell Immunopharma. R.G.M. is a consultant for Lyell Immunopharma, Gamma Delta Therapeutics, Zai Lab, and Aptorum Group. R.M. is on the Board of Directors of BeyondSpring Inc, on the Scientific Advisory Boards of Kodikaz Therapeutic Solutions Inc., Coherus Biosciences, and Zenshine Pharmaceuticals, and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc. K.R.P., A.T.S., and HYC are cofounders of Cartography Biosciences. A.T.S is a scientific founder of Immunai and receives research funding from Arsenal Biosciences. H.Y.C is a cofounder of Accent Therapeutics and Boundless Bio, and an advisor to 10X Genomics, Arsenal Biosciences, and Spring Discovery. The remaining authors declare no competing financial interests.

Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

CD19 CAR T cells have revolutionized the treatment of relapsed and refractory (R/R) large B cell lymphomas (LBCL), mediating durable complete responses in approximately 40-50% of patients. Besides a loss or decrease in CD19 expression, no studies have identified tumor specific factors driving inherent or acquired resistance to CAR T cells in LBCL. Mutations in and loss of expression of LFA-3 (CD58) have been described in approximately 20% of cases of LBCL. As the ligand for CD2 on T cells, CD58 provides costimulation to T cells and CD58 loss or mutation has been linked to immune resistance in LBCL. We evaluated CD58 status in fifty-one R/R LBCL patients treated at Stanford with commercial axicabtagene ciloleucel (axi-cel) through immunohistochemistry (IHC) on tumor biopsy samples and/or deep sequencing of circulating tumor DNA by CAPP-Seq. We identified 12/51 (24%) patients with a CD58 aberration (lack of expression by IHC or mutation by CAPP-Seq). Progression-free survival (PFS) was significantly decreased in patients with a CD58 aberration (median PFS for CD58 aberration 3 months vs. not reached for CD58 intact, p<0.0001). In fact, only 1/12 patients with a CD58 alteration achieved a durable, complete response to axi-cel, while the remaining 11 patients progressed, most commonly after a period of initial response. Partial responses were more common among patients with CD58 aberrations (58% for CD58 aberration vs 10% for CD58 intact, p<0.001), and complete responses were less common (25% for CD58 aberration vs 82% for CD58 intact, p<0.0001). To probe the biology of CAR T cell responses towards tumors lacking functional CD58, we generated a CD58 knockout Nalm6 model. CD19.CD28.ζ, CD19.4-1BB.ζ, and CD22.4-1BB.ζ CAR T cells demonstrated significantly reduced cytokine production and cytolytic activity in response to CD58 KO vs wildtype (WT) tumor cells. Additionally, while mice inoculated with WT Nalm6 and treated with any of the three CARs demonstrate complete responses and prolonged leukemia-free survival, mice inoculated with CD58KO Nalm6 demonstrated only partial responses, eventual tumor progression, and death from leukemia. CD2, the T cell ligand for CD58, plays both an adhesive role and a costimulatory role in T cells. CD2 knockout resulted in significantly reduced cytokine production after CAR stimulation. Re-expression of only the CD2 extracellular domain did not rescue CAR function, indicating that CD2 signaling is essential for full CAR activation. Additionally, when we stimulated CD19 CAR T cells with anti-idiotype antibody (CAR stimulation), soluble CD58 (CD2 stimulation), or both, we observed significantly enhanced phosphorylation of both CD3ζ and ERK by western blot in CAR T cells stimulated through both the CAR and CD2. Phosphorylation analysis by mass spectrometry revealed that CD2 stimulation enhances phosphorylation of proximal signaling molecules in the TCR pathway (LCK, LAT, CD3ε among others) and also mediators of actin-cytoskeletal rearrangement in CAR T cells, consistent with effects in natural T cell responses. To overcome CD58 loss in LBCL, we generated second- and third-generation CAR T cell constructs integrating CD2 costimulatory domains within the CAR molecule. While these cis constructs demonstrated increased potency against CD58KO cells in vitro, they were unable to ultimately overcome CD58 loss in vivo. However, when CARs were co-expressed with an additional CD2 receptor in trans, they mediated significant anti-tumor activity in vivo, overcoming CD58 knockout in tumor cells. In conclusion, we have identified that CD58 status is an important biomarker for durable response to CAR T cells in LBCL. We modeled the biologic basis for this finding and generated CAR T cells capable of overcoming CD58 loss in B cell malignancies. CD58 mutations have been reported in many cancers, including multiple myeloma and colon cancer, and are likely to play a role in immune evasion for CAR T cells as they are developed for additional histologies. These data provide rationale for investigating CD58 status for patients receiving CAR based therapeutics and devising next generation CARs capable of overcoming this newly discovered mechanism of resistance. Disclosures Majzner: Xyphos Biopharma: Consultancy; Zai Lab: Consultancy; Lyell Immunopharma: Consultancy; GammaDelta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aprotum Group: Consultancy; Illumina Radiopharmaceuticals: Consultancy. Kurtz:Roche: Consultancy; Genentech: Consultancy; Foresight Diagnostics: Other: Ownership. Sotillo:Lyell Immunopharma: Consultancy, Other: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding. Miklos:Miltenyi Biotec: Research Funding; Janssen: Consultancy, Other: Travel support; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company.

T cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR-T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. "rest") on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved antitumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.