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

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 CD19 CAR T cells (ClinicalTrials.gov, NCT02028455). CAR T cell phenotype, function, and expansion, as well as starting material T cell repertoire, were analyzed in relationship 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 with products resulting in sustained remissions. However, the initial apheresed peripheral blood T cells could be distinguished by an increased frequency of LAG-3⁺/TNF-α^lo CD8 T cells and, following adoptive transfer, the rapid expression of exhaustion markers. For the 38 subjects who achieved an initial sustained minimal residual disease–negative remission, 15 are still in remission, 10 of whom underwent allogenic hematopoietic stem cell transplantation (alloHSCT) following CAR T treatment. Subsequent remission durability correlated with therapeutic products having increased frequencies of TNF-α–secreting CAR CD8⁺ T cells, but 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 a Stand Up to Cancer and St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113), R01 CA136551-05, an Alex Lemonade Stand Phase I/II Infrastructure Grant, a Conquer Cancer Foundation Career Development Award, the Washington State Life Sciences Discovery Fund, the Ben Towne Foundation, the William Lawrence & Blanche Hughes Foundation, and Juno Therapeutics Inc., a Celgene Company.

Remission induction failures associated with attenuated CAR T cell expansion and acquisition of LAG3, TIM3, and PD-1 expression in vivo. We have previously reported the results from a phase I safety study of the CD19 CAR T cell product to treat pediatric ALL (ClinicalTrials.gov, NCT02028455) (9). Of 43 subjects infused, 3 did not achieve an MRD-negative CR, for an overall induction failure rate of 7% (9). Two of these 3 induction failures were associated with blast reduction in marrow or peripheral blood. An additional 2 subjects attained an MRD-negative CR, and were notable for having subsequent rapid CD19⁺ disease progression within 63 days of the CAR T cell infusion while still having detectable CAR T cells in the bone marrow. These 2 subjects were therefore grouped with those who did not achieve CR, as there was an early failure of the CAR T cells to effectively eradicate the disease. In order to assess whether T cell intrinsic factors contributed to therapeutic failure in these subjects, we compared T cell repertoire status in the apheresis products and final expanded CAR T cell products of these subjects (dysfunctional responders) with the cohort of subjects who experienced MRD-negative remission and, subsequently, more than 63 days of leukemia-free survival (LFS) (functional responders).

CD19 CAR T cell engraftment was quantitatively tracked by determining the frequency of CD3⁺EGFRt⁺ T cells in blood and marrow specimens (13, 19). Following adoptive transfer, there was no significant difference between functional and dysfunctional responders when assessing the overall percentage of engraftment area under the curve (AUC) of peripheral blood CD3⁺EGFRt⁺ cells (Figure 1A), or peak percentage of EGFRt⁺ CD4⁺ (Figure 1B) and CD8⁺ (Figure 1C) subsets (13, 19). However, the magnitude of absolute EGFRt⁺ cell engraftment AUC was attenuated in the dysfunctional response group (AUC 150.3, range 0.54–752.8, Mann-Whitney P = 0.0033, Figure 1D) as compared with the functional response group (median AUC 1309, range 5.23–9537). Furthermore, the absolute number of CD8⁺EGFRt⁺ cells (Figure 1E) and CD4⁺EGFRt⁺ cells (Figure 1F) at peak engraftment was significantly higher in the functional response subjects compared with the dysfunctional response. The phenotype of the EGFRt⁺ cells was analyzed at peak engraftment by multiparameter flow cytometry (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI125423DS1). EGFRt⁺ cells from both groups had similar frequencies of PD-1⁺ cells in both CD4⁺ and CD8⁺ EGFRt⁺ cells (Figure 1, G and J), whereas the dysfunctional response group showed a significantly higher frequency of LAG-3⁺ T cells, both in the EGFRt⁺CD8⁺ cells and the EGFRt⁺CD4⁺ cells (Figure 1, H and K). A similar trend was seen with the expression of TIM-3 (Figure 1, I and L). These data indicate that deficiencies in CAR T cell–intrinsic capacity for replicative expansion and/or survival is operative in poor initial antileukemic responses and is associated with increased frequencies of CAR T cells that acquire expression of LAG3, TIM3, and PD-1 during the initial phase of leukemia clearance (13, 19).

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.

Phenotypic and functional attributes of CAR T cell products do not distinguish initial dysfunctional responders from subjects that achieve MRD-negative CR and prolonged leukemia-free survival. In order to determine whether the quality of CAR T cell FPs could predict their subsequent therapeutic potency, we performed detailed phenotypic and in vitro functional profiling of CD8 and CD4 products. The manufacturing of the FPs has been previously described (9). Briefly, immunomagnetically purified CD8⁺ and CD4⁺ T cells were separately transduced and grown for 10–22 days with IL-2/IL-15 or IL-7/IL-15, respectively, with a mid-process selection for EGFRt. At the end of culture, both CD8⁺EGFRt⁺ and CD4⁺EGFRt⁺ cryopreserved products were extensively polyclonal, as demonstrated by TCR Vβ repertoire analysis (Supplemental Figure 2). Frequencies of CD8⁺EGFRt⁺ and CD4⁺EGFRt⁺ FP cells expressing surface phenotypic markers associated with engraftment fitness, namely CD127, CCR7, and CD45RA, were not significantly different between functional and dysfunctional responders (Supplemental Figure 2) (16–18). Likewise, FP T cell phenotypic markers associated with activation and/or attenuated effector function, namely LAG-3, PD-1, and TIM-3, were present at similar frequencies (Supplemental Figure 2). Subject product analysis for additional phenotypic and functional attributes are presented in Supplemental Figure 2 and Supplemental Figure 3. No readily identifiable defects in CD19⁺ target cell cytolysis or CAR-mediated activation for cytokine production between the 2 groups were observed (Supplemental Figure 3). These data prompted the analysis of the T cell repertoire in subject apheresis units used to generate these CD19 CAR T cell products to ascertain if remission induction/engraftment durability could be ascribed to differences observable in apheresis-derived SM.

Higher frequencies of LAG3- and PD-1–expressing CD8⁺ T cells distinguish apheresis products from dysfunctional responders. Several reports have shown that using SM rich in terminally differentiated cells resulted in CD19 CAR T cell products having limited replicative capacity and attenuated ability to transition to long-lived memory cells (12, 14, 16). As has been previously shown in adults with B cell malignancies (14), we observed that pediatric R/R ALL subject’s T cell repertoires were skewed toward more differentiated effector cells as compared with healthy donors, likely as a consequence of chronic chemotherapy-induced lymphopenia and recursive infection (Supplemental Figure 4). Flow cytometric analysis comparing apheresis-derived SM from the functional and dysfunctional response subject groups for markers associated with functional exhaustion (LAG-3, TIM-3, PD-1; Figure 2A), showed a significantly higher percentage of CD8⁺ T cells expressing PD-1 (Figure 2B) and LAG-3 (Figure 2C) in the dysfunctional response group compared with the functional response subjects (P = 0.0266 and P = 0.0052, respectively). We also observed a higher frequency of CD4⁺ cells expressing PD-1 in the dysfunctional group (Figure 2E). We did not observe any differences in the percentage of CD8⁺ T cells expressing TIM-3 (Figure 2D) or the percentage of CD4⁺ T cells expressing LAG-3 or TIM-3 (Figure 2, F and G). 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 (Supplemental Figure 5) between the groups.

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.

Frequencies of LAG3⁺ and TNF-α⁺ CD8⁺ T cells in apheresis SM predict subjects who will experience therapeutic failure due to remission induction failure or very early relapse. In an attempt to define analytic signals that prospectively predict CD19 CAR T cell performance in our patient population, we performed a classification and regression tree (CART) analysis of our SM data set to determine what phenotypic or functional variables of the SM T cells could accurately distinguish between the dysfunctional and functional response subjects. Using CART 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, Figure 3). Subjects with fewer than 0.745% SM CD8⁺ T cells expressing LAG-3 were all in the functional response group (n = 26/43, Figure 3A). Subjects with at least 0.745% SM CD8⁺ T cells expressing LAG-3 (n = 16) could be further subdivided into 2 groups: subjects with at least 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). This is further illustrated in a scatter plot displaying the frequency of CD8⁺ T cells secreting TNF-α and expressing LAG-3, where all dysfunctional patients cluster in the bottom right quadrant (Figure 3B). Of the 3 subjects from the functional response group with high frequencies of LAG3⁺ cells and low frequencies of TNF-α–secreting cells, all 3 had short duration BCA and relapsed within 6 months. Thus, the combination of an elevated frequency of CD8⁺ T cells expressing LAG-3 and a reduced capacity to secrete TNF-α may serve to prospectively identify patients at high risk for early therapeutic failure and who may benefit from alternative therapies. Further prospective analysis in larger patient cohorts will be required to fully validate this signature.

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.

Prolonged functional persistence of CD19 CAR T cell grafts reduces the incidence of postremission CD19⁺ leukemic relapse in subjects that do not undergo consolidative allogeneic HSCT. In our previous study, we observed a relationship between event-free survival (EFS) and the duration of BCA (used as a measure for in vivo CD19 CAR T cell functional persistence) and antigen burden, defined as the percentage of CD19⁺ cells in the marrow (leukemia and nonmalignant B cells) at the time of adoptive therapy, with a median follow-up period of 9.6 months (9). We have now followed these subjects for a median follow-up period of 26.4 months (range 1–47.4). The median LFS of the 38 patients in the functional response group, censoring patients that received post–CAR consolidative alloHSCT, was 13.87 months (Figure 4A). The duration of BCA varied greatly among this cohort of subjects, with a median BCA duration of 3.2 months (range 0.7–38.2 months, Supplemental Figure 6). At the time of manuscript preparation, 13 of the 38 subjects in the functional responders cohort underwent alloHSCT after CAR T therapy while still in remission. Of these 13 patients, 10 remained in remission at the time of manuscript preparation, while 2 relapsed with CD19⁺ disease and one of CD19^– disease. Of the 25 subjects that did not undergo alloHSCT, 20 subjects have relapsed, 10 with CD19^– disease, 10 with CD19⁺ disease. Five subjects remain in remission without subsequent treatment or therapy.

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.

We report here an updated analysis of LFS in relation to BCA duration. We grouped subjects from the functional response cohort into 3 groups based on the duration of BCA: subjects who lost BCA in the first 63 days (shortBCA), subjects who lost BCA between 63 days and 6 months (mediumBCA), and subjects who retained BCA for at least 6 months (longBCA). Eight patients were censored from BCA analysis within the first 6 months due to transplant or CD19^– recurrence, so these patients were excluded from the analysis. We observed a significant positive correlation of LFS and BCA duration across the 3 groups (log-rank test for trend, P = 0.0131, Figure 4B). This effect was further accentuated when CD19^– relapses are excluded (P = 0.0033, Figure 4C). In the shortBCA group, all subjects that did not receive post–CAR consolidative alloHSCT (n = 8) recurred, 75% with CD19⁺ disease. In contrast, of the 9 longBCA subjects that did not receive post–CAR alloHSCT, 4 of 6 recurrences were CD19^– (Table 1).

Table 1

Relapse rates in subjects who did not receive HSCT post–CAR T treatment

A multivariate cox regression analysis was performed to identify independent predicators of BCA durability after adjusting for other factors. Variables included: age, sex, prior HSCT, absolute lymphocyte count at apheresis, CD19 antigen burden, dose level, lymphodepletion regimen, and relapse/refractory status (1 vs 2 vs 3–4 vs refractory). CD19 antigen burden (χ² P = 0.0197) was the only independent variable that affected BCA durability: antigen burden of more than 15% was associated with longer BCA. These data verify our prior reported results, now with a significantly longer duration of follow-up (9). In fact, of the 9 patients in the longBCA group, 8 of them (88.9%) are in the high antigen group, while 10 of 15 shortBCA subjects had less than 15% CD19⁺ cell antigen burden at time of infusion (Table 2). Furthermore, there were significantly higher frequencies of CD19⁺ cells of total bone marrow cells in the longBCA group compared with the shortBCA group (P = 0.0409, Figure 4D). Finally, the percent of CD19⁺ cells in the BM was positively correlated to the duration of BCA (Figure 4E). These results demonstrate that the capacity of functional CD19 CAR T cells to persist more than 6 months is a critical determinant of remission durability when CD19^– relapse is excluded in subjects not undergoing consolidative alloHSCT, and is positively associated with CD19 antigen burden at the time of infusion.

Table 2

Antigen burden in BCA groups

Prolonged functional persistence of CD19 CAR T cell grafts associated with robust engraftment. We have previously shown that the magnitude (AUC) and peak engraftment in peripheral blood positively correlates with the total level of CD19⁺ antigen load in the marrow (9). We show here that the peak magnitude of CD3⁺EGFRt⁺ CAR T cell engraftment is significantly more robust in the longBCA group (median AUC 2430, range 241.2–9088) compared with the shortBCA group (AUC 932.9, range 5.23–5131; Mann-Whitney P = 0.0177; Figure 5A). A similar trend was seen for the peak magnitude of the frequency of CD3⁺EGFRt⁺ CAR T cells (Figure 5D). Furthermore, the absolute number of CD8⁺EGFRt⁺ cells at peak engraftment was significantly higher in longBCA subjects compared with the short BCA (P = 0.0349, Figure 5B). A similar trend was seen for CD4⁺EGFRt⁺ cells (Figure 5C). The frequency of CD8⁺EGFRt⁺ and CD4⁺EGFRt⁺ cells at peak engraftment was not significantly different (Figure 5, E and F). Both the magnitude of the EGFRt expansion (Figure 5G) and the absolute number of CD8⁺EGFRt⁺ cells at peak engraftment (Spearman correlation, r = 0.4231, P = 0.0313, data not shown) was positively correlated to the duration of BCA. There was no difference in the frequency of CD45RO, CD45RA, CCR7, CD27, PD-1, LAG-3, and TIM-3 CD8⁺EGFRt⁺ and CD4⁺EGFRt⁺ at peak engraftment between the different groups (Supplemental Figure 7). These data suggest that duration of functional CAR T cell engraftment is associated with a robust expansion and set by a high antigen load.

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.

Functional engraftment duration is associated with phenotypic and functional attributes of CAR CD8 T cell FP. We analyzed the phenotype and function of FP in relation to the duration of BCA. The percentage of FP CD8⁺EGFRt⁺ T cells secreting TNF-α in response to CD19 antigen was significantly higher in the longBCA group compared with the shortBCA group (Figure 6B). In contrast, the percent of FP CD8⁺EGFRt⁺ T cells expressing TIM-3 was significantly lower in the longBCA group compared with both the mediumBCA and the shortBCA group (Figure 6F). There was no significant difference in the frequency of CD8⁺EGFRt⁺ cells producing IFN-γ or IL-2 (Figure 6, A and C), the frequency of CD8⁺EGFRt⁺ cells expressing PD-1 or LAG-3 (Figure 6, D and E), or in the functional profile of CD4⁺EGFRt⁺ FP cells (Supplemental Figure 8). However, there was a significant difference in the percentage of CD4⁺ cells expressing CD45RO in the longBCA group compared with the shortBCA group. These data indicate that subjects whose CD19 CAR T cell products have a low frequency of TNF-α⁺/TIM-3^– CD8⁺EGFRt⁺ T cells may be at higher risk for shortBCA. If confirmed in larger cohorts with highly predictive indices, this could prospectively identify patients requiring early postremission consolidative measures.

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.

OCF, RG, and MCJ designed, conducted, and analyzed the study and wrote the paper. HB and SRR conducted and analyzed the study and reviewed the paper. RH, DD, ML, BF, and DL conducted the study. RJO wrote the paper. DL analyzed the data and reviewed the paper.

View Supplemental data

View ICMJE disclosure forms

Partial funding for this study was provided by a Stand Up to Cancer and St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113), R01 CA136551-05, an Alex Lemonade Stand Phase I/II Infrastructure Grant, a Conquer Cancer Foundation Career Development Award, the Washington State Life Sciences Discovery Fund, the Ben Towne Foundation, the William Lawrence and Blanche Hughes Foundation, and Juno Therapeutics Inc., a Celgene Company.

Address correspondence to: Michael C. Jensen, Ben Towne Center for Childhood Cancer Research Seattle Children’s Research Institute, 1100 Olive Way, Suite 100, Seattle, Washington 98101, USA. Phone: 206.884.2129; Email: michael.jensen@seattlechildrens.org.

Conflict of interest: DL is an employee of, and has an equity interest in, Juno Therapeutics Inc. MCJ has received consulting fees and grants from and is an inventor of patents licensed to Juno Therapeutics Inc., in which he has an equity interest. Seattle Children’s Hospital received funds from Juno Therapeutics Inc.

Copyright: © 2019, American Society for Clinical Investigation.

Reference information: J Clin Invest. 2019;129(5):2123–2132.https://doi.org/10.1172/JCI125423.

See the related Commentary at Improving CAR T cell immunotherapy–mediated remissions for pediatric leukemia.

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