Starting T Cell and Cell Product Phenotype Are Associated with Durable Remission of Leukemia Following CD19 CAR-T Cell Immunotherapy
Authors: Gardner Rebecca , Finney Olivia , Brakke Hannah , Rhea Stephanie , Hicks Roxana , Doolittle Danielle , Lopez Marisa , Orentas Rimas J , Li Daniel , Jensen Michael C. 
Published: October 2019

Abstract
Chimeric antigen receptor (CAR) redirected T cells can induce remission in highly refractory leukemia and lymphoma subjects. Despite the potential of this emerging therapeutic modality, treatment with CD19 CAR T cells is not uniformly effective for remission induction, and remissions can be short-lived in a significant proportion of treated subjects.
Here, we analyzed 43 pediatric and young adult subjects participating in the phase 1 trial (NCT02028455) and correlated their outcomes with in vivo performances of SCRI-CAR19v1(a CD19 specific CAR T cell product), as well as starting material (SM) T cell repertoire- and final cell product (FP) -intrinsic attributes. Subjects were allocated to the dysfunctional response group defined as early treatment failure (no remission or early disease progression after remission while still having CAR engraftment) (n=5) and the functional response group defined as those who obtained an MRD-negative remission that was sustained beyond 63 days (n=37).
We found the magnitude of absolute CAR T cell engraftment area under the curve (AUC) was attenuated in the dysfunctional response group (AUC 150.3, range 0.54-752.8, Mann-Whitney p=0.0033) as compared to the functional response group (median AUC 1309, range 5.23-9537). The absolute number of CD8+CAR+ cells and CD4+CAR+ cells at peak engraftment was also significantly higher in the functional response versus the dysfunctional response. The phenotype of the CAR+ cells was analyzed at peak engraftment by multiparameter flow cytometry. CAR+ CD8+ cells from both groups had similar frequencies of PD-1+ cells, whereas the dysfunctional response group showed a significantly higher frequency of LAG-3+ T cells, both in the CAR+CD8+ cells and the CAR+CD4+ cells. A similar trend was seen with the expression of TIM-3.
In order to assess whether T cell intrinsic factors contributed to therapeutic failure in these subjects, we studied T cell repertoire status in apheresis products and final expanded CAR T cell products. Several reports have shown that using SM rich in terminally differentiated cells result in CD19 CAR-T cell products having limited replicative capacity and attenuated ability to transition to long lived memory cells . When comparing apheresis derived SM from the functional and dysfunctional response subject groups by flow cytometry for markers associated with functional exhaustion (LAG-3, TIM-3, PD-1), we observed a significantly higher percentage of CD8+ T cells expressing PD-1 and LAG-3 in the dysfunctional response group compared to the functional response subjects (p=0.0266 and p=0.0052). We also observed a higher frequency of CD4+ cells expressing PD-1 in the dysfunctional group. There was no difference in the frequency of cells expressing CD45RA, CD45RO, CCR7, CD27, or in the frequency of cells expressing TNF-α, IFN-γ or IL-2 in response to CD3/CD28 stimulation in both CD4 and CD8 SM T cells between the groups.
Lastly, using classification and regression tree analysis, subjects could be classified by the frequency of SM CD8+ T cells expressing LAG-3 and the frequency of SM CD8+ T cells capable of secreting TNF-α upon CD3/CD28 bead activation (r2=0.636). Subjects with fewer than 0.745% SM CD8+ T cells expressing LAG-3 were all in the functional response group (n=26/43). Subjects with equal or more than 0.745% SM CD8+ T cells expressing LAG-3 (n=16) could be further sub-divided into two groups: subjects with equal or more than 25.283% of CD8+ T cells expressing TNF-α were also functional responders (n=8/8), while subjects with fewer than 25.283% of CD8+ T cells expressing TNF-α were in majority in the dysfunctional response group (n=5/8). Of the three subjects from the functional response group with high frequencies of LAG3+ cells and low frequencies of TNF- α -secreting cells, all three had short duration BCA and relapsed within 6 months. The combination of elevated frequency of cells expressing LAG-3 and a reduced capacity to secrete cytokines in response to stimulation potential serve as biomarkers for patients with perturbated T cell repertoires that generate CAR T cell products with attenuated anti-leukemic potency.
The parameters identified herein, should they be validated in larger trial cohorts, have the potential to prospectively identify patients at risk for initial therapeutic failure thus requiring alternative therapies from those who have an excellent prognosis.

Disclosures
Li: Juno Therapeutics: Employment, Equity Ownership. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.