Patients with Deleterious Germline Variants in STXBP2 Develop Toxicity after CAR-T Cell Therapy with Axicabtagene Ciloleucel

Background Chimeric antigen receptor (CAR) T cell therapies have changed the therapeutic landscape for advanced hematologic malignancies. However, toxicity remains a concern and can vary markedly between patients, often in ways not related to tumor burden or established clinical markers. The clinical and biochemical phenotype of the rare hyperinflammatory condition hemophagocytic lymphohistiocytosis (HLH) has long been recognized to be similar to that of CAR-T associated cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity (ICANS). We hypothesized that germline variants in genes known to be associated with primary HLH might also be associated with the development of these toxicities in patients who received CAR-T cell therapy.

Methods We performed whole genome sequencing on 110 patients treated in the ZUMA-1 pivotal clinical trial of axicabtagene ciloleucel (axi-cel) for refractory large B cell lymphoma. We used a case-control study design in which a combined toxicity endpoint consisting of (1) severe CRS (Grade 3+) or (2) severe ICANS (Grade 3+) or (3) treatment with tocilizumab defined the toxicity cohort (n=65) and the remainder were the control cohort (n=45). We used a deep learning variant characterization framework to identify germline variants in 17 HLH-associated genes in cases and controls after appropriate PCA-based population stratification. A correlative analysis was performed to compare serum cytokine levels, clinical toxicity, and outcome variables between patients harboring HLH-related germline variants and wild-type controls.

Results First, we performed a gene-based burden analysis of common germline variants (minor allele frequency (MAF)>5%), and detected no enrichment of any of the HLH genes in cases versus controls. However, when restricted to rare (MAF <5%) variants, we found an enrichment of STXBP2 variants in toxicity cases relative to control patients. We identified 5 unique deleterious missense variants in STXBP2 across 9 (14.52%, 95% CI:6.86% to 25.78%) patients in the toxicity cohort but found no STXBP2 rare variants in the control cohort (OR>1.321, nominal p value: 0.015, q=0.17 for 17 tests). Interestingly, three of the five variants (R190C, T345M, and A433V) present in the toxicity cohort have previously been identified in patients with primary HLH and demonstrated impaired in vitro degranulation. The remaining 2 germline variants in STXBP2 (R474C, R575C) were predicted by multiple in silico prediction algorithms (SIFT, Polyphen, MCAP) to have deleterious effects on protein function. In addition, patients harboring rare deleterious germline STXBP2 variants had higher baseline IFNγ levels than the rest of the toxicity cohort, or the control cohort, and higher levels of peak inflammatory cytokines (IFNγ, IL2RA, TNFa, IL-6, non-parametric, two-tailed Van Elteren test p=0.0033).

To ascertain the effects of deleterious STXBP2 variants on CAR-T cell function, we generated STXBP2 deficient anti-CD19-28z CAR-T cells from primary healthy human donor T cells using multiple CRISPR STXBP2 guides and demonstrated impaired degranulation and increased IFNγ upon stimulation. Furthermore, when these STXBP2 deficient CAR-T cells were activated through their CAR and co-cultured with inflammatory myeloid cells, the STXBP2-deficient CAR-T cells triggered increased release of inflammatory cytokines compared to STXBP2 proficient control CAR-T cells. These cytokine changes were similar to the induction of proinflammatory cytokines in the peripheral blood of patients who harbored STXBP2 variants relative to the control cohort.

Conclusion All patients with rare missense variants in STXBP2 experienced toxicity after treatment with axi-cel and had higher levels of baseline IFNγ and peak levels of inflammatory cytokines. This finding highlights the impact of germline variants in immune cell therapies. Larger studies are warranted to determine the clinical impact, if any, of other germline variants when cells are used as therapeutics.

Gillani:Google: Current equity holder in publicly-traded company; Microsoft: Current equity holder in publicly-traded company; Amazon: Current equity holder in publicly-traded company; Apple: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company; Pfizer: Current equity holder in publicly-traded company; Vertex: Current equity holder in publicly-traded company; Berkshire Hathaway: Current equity holder in publicly-traded company; Target: Current equity holder in publicly-traded company; JP Morgan: Current equity holder in publicly-traded company; Goldman Sachs: Other: Minority stakes. Reilly:RenBio: Consultancy. Budka:Kite, a Gilead Company: Current Employment. Filosto:Tusk Therapeutics: Patents & Royalties; Kite, a Gilead Company: Current Employment, Current equity holder in publicly-traded company. Shen:Atara: Patents & Royalties; Kite, a Gilead Company: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Maus:Adaptimmune: Consultancy; Agenus: Consultancy; Allogene: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellectis: Consultancy, Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding; In8bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine/BendBio: Consultancy; Neximmune: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy, Research Funding; Oncternal: Consultancy, Current holder of stock options in a privately-held company; Sanofi: Consultancy; TCR2: Consultancy, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees; Tmunity: Consultancy; WindMIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Servier: Research Funding; Promab: Patents & Royalties: held by Massachusetts General Hospital; Novartis: Patents & Royalties: held by University of Pennsylvania; 2SeventyBio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Century Therapeutics: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months.