Pharmacodynamic Profile and Clinical Response in Patients with B-Cell Malignancies of Anti-CD19 CAR T-Cell Therapy

This study is supported in part by funding from the Cooperative Research and Development Agreement (CRADA) between the National Cancer Institute and Kite Pharma

Introduction: Chimeric antigen receptor (CAR) engineered autologous T-cell therapy has shown promising efficacy in B‑cell malignancies in an ongoing phase 1 study (Kochenderfer et al. J Clin Oncol 2014). Anti-CD19 CAR T‑cell product characteristics and potential pharmacodynamic markers from patients in this study were evaluated together with updated clinical responses.  

Methods: In this National Cancer Institute (NCI) clinical trial (NCT00924326), patients with relapsed/refractory B‑cell malignancies received conditioning with cyclophosphamide and fludarabine daily for 3 days starting on day -5; followed by 1-2 x 10⁶/kg anti-CD19 CAR T cells engineered with a CAR expressing CD28 and CD3‑zeta signaling domains. Forty one cytokines, chemokines and immune response related markers were measured in the serum of patients prior to conditioning and CAR T-cell infusion, and during an interval of 4 weeks post-CAR T-cell infusion. EMD Millipore Luminex® xMAP® multiplex assays were used to measure all analytes. A Luminex 200™ instrument and xPONENT® 3.1 software were used for data acquisition and analysis. Major T-cell phenotypic markers including CD4, CD8, CD45RA and CCR7 were evaluated by multicolor flow cytometry on CAR-expressing T cells prior to and post-infusion, using a BD FACSCanto II. FlowJo software was used for data analysis.  T-cell marker expression, as well as cytokine and chemokine levels were analyzed together with the clinical response to anti-CD19 CAR T cells. Maximum fold increase (MFI) was defined as the maximum fold change of measured analytes above baseline (pre-conditioning, day -5) across sampling timepoints.

Results: Anti-CD19 CAR T-cell products, PBMCs from 12 patients, and serum samples from 15 patients have been evaluated. In 12 patient lots evaluated to date, the median CD4⁺/CD8⁺ CAR T‑cell ratio was 0.48 (range 0.02-6.12).  In addition, the median ratio between naïve (T_N) plus central memory T cells (T_CM), and more differentiated effector memory (T_EM) plus effector cells (T_E), was 0.48 (range 0.1-16.8).  Post-hoc analyses adjusted for multiple comparisons showed that the frequency of CD4⁺ T_N and T_CM cells in the 6-8 day T‑cell lots was significantly greater than that of CD4⁺ T_N and T_CM cells in the 10 day T-cell lots. The corresponding frequencies of CD8⁺ T_N and T_CM cells in the 6-8 day T‑cell lots compared to 10 day T-cell lots approached significance, but did not meet the threshold after multiplicity adjustment. Clinical responses were seen across broad ranges of CD4⁺/CD8⁺ and (T_N+T_CM)/(T_EM+T_E) ratios in the CAR T-cell product.

CAR T cells upregulated T-cell activation and immune modulating markers, as well as released measurable levels of cytokines and chemokines in response to CAR engagement of CD19 in vitro, or post-infusion. Cytokine and chemokine levels achieved their peak 3-10 days post T-cell infusion and returned to baseline generally within 3 weeks. Key pro-inflammatory cytokines and markers were upregulated: IL-6 median fold increase (MFI) at peak of 66 (interquartile range 5-152), IFN-g MFI 57 (13-126), C-reactive protein MFI 6 (4-42); immune homeostatic cytokines IL-15 MFI 19 (7-54), IL-2 MFI 20 (4-22), IL-10 MFI 10 (4-15); chemokines monocyte chemotactic protein (MCP)-1 MFI 7 (5-9), MCP-4 MFI 4 (2-5); and the immune effector molecules granzyme A MFI 7 (6-17) and granzyme B MFI 5 (3-9).  Further analyses are ongoing.

Conclusion:  Clinical responses were observed irrespective of the CD4⁺/CD8⁺ ratio in the CAR T cell product.  Cytokines and immune effector mediators peaked and cleared within 3 weeks. This pharmacodynamic profile reveals a rapid and coordinated sequence of T cell activation underlying durable responses in patients with B-cell malignancies.