Long-Term Follow-up and Single-Cell Multiomics Characteristics of Infusion Products in Patients with Chronic Lymphocytic Leukemia Treated with CD19 CAR-T Cells
Authors: Hirayama Alexandre V. , Zheng Ye , Dowling Mark R. , Sheih Alyssa , Phi Tinh-Doan , Kirchmeier Delaney R. , Chucka Abby W. , Gauthier Jordan , Maloney David G. , Gottardo Raphael , Turtle Cameron J. 
Published: November 2021

Abstract
Introduction
Lymphodepletion chemotherapy followed by infusion of autologous T cells engineered to express a CD19-targeted chimeric antigen receptor (CAR) has shown high response rates in relapsed or refractory high-risk chronic lymphocytic leukemia (CLL). Durable remissions were observed, particularly in patients (pts) who achieved minimal residual disease (MRD)-negative (neg) complete response (CR) in the marrow with no detectable malignant clone by IGH sequencing. Here, we present long-term outcomes of CLL pts treated with CD19 CAR-T cell immunotherapy, and single-cell cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analyses of infusion products (IPs) to identify intrinsic T cell characteristics associated with outcomes.
Methods
Pts were treated on a phase 1/2 clinical trial (NCT01865617) of CD19 CAR-T cell immunotherapy in two cohorts: no ibrutinib (ibr) cohort (No-ibr; ibr discontinued prior to leukapheresis or lymphodepletion) or concurrent ibr cohort (Con-ibr; ibr started at least 2 weeks prior to leukapheresis and continued until at least 3 months after CAR-T cell infusion). Responses were evaluated according to the 2018 International Workshop on CLL (iwCLL) criteria (Hallek et al. Blood 2018). MRD in the marrow was assessed 4 weeks after CAR-T cell infusion using flow cytometry (sensitivity, 10 -4) and IGH sequencing (sensitivity, 10 -6). Multiomics profiling was performed on IPs from 25 pts selected based on distribution of ibr washout time before leukapheresis and clinical response (Table 1). All pts received cyclophosphamide and fludarabine lymphodepletion and 2 x 10 6 CAR-T cells/kg. CD3 + CAR-T cells isolated from IPs underwent CITE-seq (10x Genomics), with generation of gene expression, full-length paired V(D)J, and cell surface protein expression libraries. Differential gene expression (DEG) analyses were performed using Model-based Analysis of Single-cell Transcriptomics (MAST). Blood transcriptome modules were used for Gene Set Enrichment Analysis (GSEA).
Results
Forty-seven pts (median age, 61 years) with CLL (excluding Richter's transformation without coexisting CLL) received lymphodepletion and CD19 CAR-T cell infusion. Among 29 pts with a pretreatment trackable malignant IGH clone who achieved MRD-neg CR by flow cytometry, at a median follow-up of 23.5 months (interquartile range, 11.0-33.8), the median progression-free survival was 8.5 months (95% confidence interval [CI], 2.9-not reached) for those with a detected post-treatment malignant IGH clone in marrow (n = 11) and could not be estimated (95% CI, 22.2-not reached) in those without a detected post-treatment malignant IGH clone (n = 18; P = .0006; Figure 1A). In the pts without a detected malignant IGH clone 4 weeks after infusion, only 3 (17%) relapsed; 1 died of a myocardial infarction while in ongoing response.
We performed multiomics studies on IPs from a selected subset (n = 25) of pts (Table 1) to identify gene expression patterns impacting outcomes. GSEA revealed association with T cell activation, oxidative phosphorylation, transcription initiation and cell cycle modules in IPs of pts who did compared to did not achieve MRD-neg CR (Figure 1B, left), consistent with the hypothesis that CAR-T cell quality is a key determinant of depth and duration of remission in CLL. IPs studied in the multiomics subset were manufactured from pts treated in either the No-ibr or the Con-ibr cohort (Table 1) and our prior data showed a trend towards higher rates of IGH-neg CR in the Con-ibr cohort (85 vs 60%, P = .34). Because ibr administration before leukapheresis might improve CAR-T cell quality and function, we compared IPs of patients in the No-Ibr vs. Con-Ibr cohorts. GSEA revealed that the T cell differentiation via ITK and PKC module was less enriched in IPs of pts in the Con-ibr vs. No-ibr cohort (Figure 1B, right), consistent with ibr-induced inhibition of T cell differentiation as a mechanism for enhanced CAR-T cell quality in the IP. Analyses are ongoing and full results will be presented at the meeting.
Conclusion
Long-term follow-up confirmed that achieving a malignant IGH-neg response after CAR-T cell therapy is associated with durable remissions in high-risk CLL. Integrated multiomics profiling suggests that CAR-T cell IP quality is a key determinant of MRD-neg CR. Ibr-related inhibition of ITK signaling in T cells can further contribute to the quality of infused CAR-T cells.
Figure 1 Figure 1.

Disclosures
Hirayama: Novartis: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Sheih: Umoja Biopharma: Current Employment. Gauthier: Janssen: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Multerra Bio: Consultancy; Larvol: Consultancy; JMP: Consultancy; Eusapharma: Consultancy. Maloney: A2 Biotherapeutics: Divested equity in a private or publicly-traded company in the past 24 months; BioLineRx, Juno, Celgene, Kite, a Gilead Company, Gilead, Novartis, and Pharmacyclics: Honoraria; A2 Biotherapeutics: Consultancy; Kite, a Gilead Company, Juno, and Celgene: Research Funding; Juno: Patents & Royalties. Gottardo: Illumina: Consultancy; 10x Genomics: Current holder of stock options in a privately-held company; Ozette Technologies: Current holder of individual stocks in a privately-held company; Modulus Therapeutics: Current holder of individual stocks in a privately-held company. Turtle: PACT Pharma: Consultancy; TCR2 Therapeutics: Research Funding; Nektar Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; AstraZeneca: Consultancy, Research Funding; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Asher Bio: Consultancy; Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Allogene: Consultancy; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; T-CURX: Other: Scientific Advisory Board.