Refractory Chronic Lymphocytic Leukemia Research Articles

Chimeric antigen receptor (CAR) T cell therapies have transformed the treatment of hematologic malignancies, leading to FDA approval in B-cell acute lymphoblastic leukemia, non-Hodgkin lymphoma, and multiple myeloma. While many patients with previously untreatable disease have achieved excellent responses and long-term remissions, a substantial number do not respond or relapse after months to years. Most prior studies of CAR T cell-intrinsic determinants of response and resistance have focused on infusion products. Functional expansion and persistence of CAR T cells in patients constitute an important feature of treatment success, yet the trajectories of CAR T cells and their molecular underpinnings remain poorly understood due to technical challenges. Here we performed single-cell multiomics with T-cell receptor (TCR) lineage tracing to study the molecular attributes of CAR T cell fitness, which we define as the ability to expand and/or persist to at least 12 months after infusion. We recently published long-term follow-up from a prospective single-center phase 2 clinical trial in which CD19-directed CAR T cells with 4-1BB co-stimulation were administered to patients with relapsed or refractory chronic lymphocytic leukemia (CLL) not in complete remission despite at least 6 months of ibrutinib. In this trial, we found high rates of deep and durable remissions with prolonged persistence of functional CAR T cells determined by flow cytometric detection of CAR T cells with concomitant B-cell aplasia. We obtained CAR T cell infusion products and peripheral blood at day 10 and month 12 from five patients with durable remissions. We then performed single-cell multiomics sequencing to measure gene expression, TCRs, surface proteins, and chromatin accessibility to define the multidimensional phenotypes and clonal trajectories of CAR T cells over time ( Figure). We first evaluated CAR T cell dynamics from infusion product to day 10 and identified clones that expanded using TCR as a molecular barcode. While ~90% of CAR T cells in the infusion product are CD4 +, peak expansion at day 10 is driven by effector CD8 + CAR T cells. Clonally expanded CAR T cells at day 10, compared to non-expanded CAR T cells, showed higher expression of multiple cytotoxicity genes ( GZMH, GZMB, GNLY, PRF1) and co-inhibitory receptors ( LAG3, PDCD1, and KLRB1) and lower expression of memory genes ( SELL, LTB, TCF7). Using TCR lineage tracing and a machine learning classifier, we identified similar markers predictive of clonal expansion from the infusion product, e.g. CD8A, GNLY, KLRG1, KLRK1 among the top positive markers and CD4 and SELL among the top negative markers. Along with clonal expansion, we found that CD8 + CAR T cells undergo significant evolution in their chromatin accessibility landscape from infusion product to day 10. Genomic regions that are more open at infusion are enriched for Fos/Jun (AP-1) and Batf family of transcription factor (TF) motifs, consistent with their function in promoting effector T cell differentiation and proliferation. Genomic regions that are more open at day 10 are enriched for Eomes, T-bet, and other T-box TF motifs, consistent with their roles in regulating T cell cytolytic function, memory formation, and exhaustion. These data provide insights into the regulatory mechanisms of rapid CAR T cell expansion post-infusion in patients which have not yet been reported. We found that most clonally expanded CAR T cells at day 10 contracted or disappeared by month 12. However, a subset of CD8 + CAR T cells in 2 patients that did not undergo early expansion surprisingly demonstrated late clonal expansion and persistence at month 12, coinciding with hyper-expansion (&gt;200 cells per clone) of their TCR clonotypes with or without CAR expression. We then investigated the specificity of TCRs using publicly available databases and found late expansions of clones predicted to bind viral peptides. This observation suggests that the TCR antigen specificity may contribute to the long-term persistence or fitness of CAR T cell clones, in contrast to recent studies of long-lived CAR T cells in a limited number of patients which argue that persistence is driven by specific phenotypes rather than clonotype-specific features. Further validation of this finding is ongoing through flow enrichment of rare persistent CAR T cells for deeper single-cell sequencing and in vitro cellular immunology assays.

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is absent or expressed at low levels in normal adult tissues but overexpressed in a range of malignancies including CLL and MCL. NVG-111 is a humanized first in class, tandem scFv, ROR1xCD3 bispecific T cell engager. NVG-111 mediates potent killing of ROR1 + tumors by engaging a unique epitope on the Frizzled domain of ROR1 and redirecting T cell activity via the CD3 binder engineered for attenuated cytokine release. Methods: In this phase 1, first-in-human, dose-escalation study (ClinicalTrials.gov identifier: NCT04763083), NVG-111 was evaluated in relapsed/refractory CLL and MCL subjects with an ECOG PS of 0-2 who received ≥2 prior lines of treatment including a Bruton tyrosine kinase inhibitor (BTKi). Bayesian continual reassessment method with overdose control was implemented to guide escalation over a dose range of 0.3-45ug/day. Each subject received at least one dose of NVG-111, administered as a continuous intravenous infusion (cIV) over 21 days followed by 7 days off drug (=1 cycle). NVG-111 was administered in combination (N=8) with ibrutinib to subjects who had achieved a partial response to &gt;1 year of ibrutinib therapy, or as monotherapy in subjects that progressed after covalent BTKi/B-cell lymphoma 2 inhibitor (BCL2i) (N=4). Primary objective was safety, with the secondary objectives being efficacy (overall response rates [ORR], minimal residual disease (MRD) measured by ERIC-compliant flow cytometry and duration of response [DoR]). Serum cytokine levels were determined using a human high sensitivity cytokine premixed magnetic Luminex assay and T cell function was longitudinally evaluated using cytometry by time of flight (CyTOF) analysis. Results: Between May 2021 and July 2023 12 subjects (10 males and 2 females, median age 60 years) completed a maximum of 6 cycles of treatment with NVG-111 (median=3 cycles [range 1-6 cycles]). Pharmacokinetic and pharmacodynamic data demonstrated systemic NVG-111 exposure and exposure-dependent NVG-111 targeting of ROR1 on circulating tumor cells. Adverse events (AEs) related to NVG-111 occurred in 10 subjects (83%), with the most common being nausea (58%), headaches (67%), and fatigue (33%), majority of which were grade 1 or 2. Grade 1(71%) or 2(29%) cytokine release syndrome (CRS) occurred in 58% of subjects during week 1 of cycle 1 except in one subject who had a second grade 1 CRS in cycle 2. Grade 3 dose limiting toxicities (DLTs) occurred in three subjects (25%) consisting of transient elevation of liver enzymes (ALT and AST) in one subject dosed at 3ug/day, immune effector cell-associated neurotoxicity syndrome-like symptoms (ICANS) in one subject dosed at 30ug/day and headache in one subject dosed at 45ug/day. All AEs were fully reversible with no late emergent grade 3 or greater toxicities. Evidence of T cell activation was observed in all 12 subjects with peak cytokine levels at the 30µg/day dose levels (mean±SEM): TNFα, 43±17pg/ml; IL6, 566±541pg/ml; IFNɣ, 18±12pg/ml; and IL10, 103±56pg/ml. Furthermore, T cell profiling showed an increase in cytotoxic CD8 + T cell activation marker expression during each NVG-111 treatment cycle without evidence of T cell exhaustion. Efficacy was evaluable in 11 subjects with objective clinical responses observed in 55% (6/11), including two subjects with clear evidence of single agent activity. Amongst these, three CLL subjects were rendered MRD negative in peripheral blood and one MCL subject achieved complete metabolic response by the Lugano criteria. Two subjects had stable disease but in one of these subjects there was a &gt;50% reduction in peripheral blood lymphocytosis and a 40% reduction in lymphadenopathy. Despite time limited exposure to NVG-111, the restricted mean survival time for DoR is currently 13.6 months (SEM 3.07), the median is not yet calculable with response ongoing in four subjects. The median progression-free survival currently is 18.7 months (95% CI 2.6 - not calculable). Conclusion: These data provide clinical proof of concept for selective targeting of ROR1 with a TCE leading to objective evidence of antitumor activity with encouraging response durability even in CLL patients known to have defective T cell function. The safety profile was consistent with the mechanism of action. Further evaluation of this promising molecule is ongoing.

Refractory Chronic Lymphocytic Leukemia Research Articles

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