CD19 CAR T cell product and disease attributes predict leukemia remission durability

Abstract

Background: Chimeric antigen receptor (CAR) T cells can induce remission in highly refractory leukemia and lymphoma subjects, yet the parameters for achieving sustained relapse-free survival are not fully delineated.

Methods: We analyzed 43 pediatric and young adult subjects participating in a Phase I trial of defined composition CD19CAR T cells (NCT02028455). CAR T cell phenotype, function and expansion, as well as starting material T cell repertoire, were analyzed in relation to therapeutic outcome (defined as achieving complete remission within 63 days) and duration of leukemia free survival and B cell aplasia.

Results: These analyses reveal that initial therapeutic failures (n = 5) were associated with attenuated CAR T cell expansion and/or rapid attrition of functional CAR effector cells following adoptive transfer. The CAR T products were similar in phenotype and function when compared to products resulting in sustained remissions. However, the initial apheresed peripheral blood T cells could be distinguished by an increased frequency of LAG-3+/TNF-αlow CD8 T cells and, following adoptive transfer, the rapid expression of exhaustion markers. For the 38 subjects who achieved an initial sustained MRD-neg remission, remission durability correlated with therapeutic products having increased frequencies of TNF-α-secreting CAR CD8+ T cells, and was dependent on a sufficiently high CD19+ antigen load at time of infusion to trigger CAR T cell proliferation.

Conclusion: These parameters have the potential to prospectively identify patients at risk for therapeutic failure and support the development of approaches to boost CAR T cell activation and proliferation in patients with low levels of CD19 antigen.

Trial registration: ClinicalTrials.gov NCT02028455.

Funding: Partial funding for this study was provided by Stand Up to Cancer & St. Baldrick's Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113), RO1 CA136551-05, Alex Lemonade Stand Phase I/II Infrastructure Grant, Conquer Cancer Foundation Career Development Award, Washington State Life Sciences Discovery Fund, Ben Towne Foundation, William Lawrence & Blanche Hughes Foundation, and Juno Therapeutics, Inc., a Celgene Company.

Keywords: Cancer immunotherapy; Immunology; Leukemias; Oncology.

Figure 1. Expansion in dysfunctional response group is less robust than in functional response group.

(A) AUC of percentage of EGFRt⁺CD3⁺ cells in the peripheral blood between D0 and D63. Percentage of CD8⁺EGFRt⁺CD3⁺ cells (B) and CD4⁺EGFRt⁺CD3⁺ cells (C) in the peripheral blood at peak engraftment. (D) AUC of number of EGFRt⁺CD3⁺ cells per microliter in the peripheral blood between D0 and D63. Number of CD8⁺EGFRt⁺CD3⁺ cells (E) and CD4⁺EGFRt⁺CD3⁺ cells (F) per microliter in the peripheral blood at peak engraftment (n = 43). Percentage of CD8⁺EGFRt⁺ cells expressing PD-1 (G), LAG-3 (H), and TIM-3 (I) at peak expansion (n = 26). Percentage of CD4⁺EGFRt⁺ cells expressing PD-1 (J), LAG-3 (K), and TIM-3 (L) at peak expansion (n = 26). Bars represent the median. P values calculated using a Mann-Whitney test. Green circles: functional response; orange circles: dysfunctional response.

Figure 2. Higher frequency of inhibitory receptors in starting material from dysfunctional group.

(A) Representative gating of CD8⁺ SM. Percentage of CD8⁺ SM cells expressing PD-1 (B), LAG-3 (C), and TIM-3 (D) (n = 41). Percentage of CD4⁺ SM cells expressing PD-1 (E), LAG-3 (F), and TIM-3 (G) (n = 41). Bars represent the median. P values calculated using a Mann-Whitney test. Green circles: functional response; orange circles: dysfunctional response.

Figure 3. Phenotypic and functional characteristics of CD8⁺ SM associated with dysfunctional response.

(A) Clustering and regression tree analysis of CD8⁺ SM attributes. (B) Scatter plot representing the percentage of CD8⁺ SM cells expressing TNF-α and LAG-3 in the high antigen burden subjects (n = 40). Lines represent cutoff values determined by the tree analysis.

Figure 4. The combination of high leukemia burden and shortBCA is related to high risk of relapse.

(A) Kaplan-Meier of LFS of all patients in the functional response group (n = 38). Median follow-up was 26.2 months. Dotted line represents 95% confidence. (B) Effect of BCA duration on LFS (n = 33). (C) Effect of BCA duration on CD19⁺ relapse. P values calculated using the log-rank test (n = 30). (D) Percentage of CD19⁺ cells in the bone marrow before infusion in different BCA groups (n = 30). (E) Correlation between frequency of CD19⁺ cells in bone marrow and duration of BCA (n = 43). Eight patients were excluded from BCA group analysis due to being censored prior to 6 months. Data was censored on February 15, 2018. Green: longBCA; purple: mediumBCA; blue: shortBCA.

Figure 5. Expansion in shortBCA group is less robust than in longBCA group.

(A) AUC of number of EGFRt⁺CD3⁺ cells in the peripheral blood between D0 and D63. Number of CD8⁺EGFRt⁺CD3⁺ cells (B) and CD4⁺EGFRt⁺CD3⁺ cells (C) in the peripheral blood at peak engraftment. (D) AUC of percentage of EGFRt⁺CD3⁺ cells in the peripheral blood between D0 and D63. Percentage of CD8⁺EGFRt⁺CD3⁺ cells (E) and CD4⁺EGFRt⁺CD3⁺ cells (F) in the peripheral blood at peak engraftment. (G) Correlation between BCA and AUC of absolute engraftment. Bars represent the median. P values calculated using a Mann-Whitney test. Correlation statistics based on Spearman correlation. Green circles: longBCA; purple circles: mediumBCA; blue circles: shortBCA.

Figure 6. Higher frequency of functional cells in the final product from longBCA subjects.

Percentage of CD8⁺EGFRt⁺ FP cells secreting IFN-γ (A), TNF-α (B), and IL-2 (C) following antigen-specific stimulation (n = 26). Percentage of CD8⁺EGFRt⁺ FP cells expressing PD-1 (D), LAG-3 (E), and TIM-3 (F) (n = 29). Bars represent the median. P values calculated using a Mann-Whitney test. Green circles: longBCA; purple circles: mediumBCA; blue circles: shortBCA.