CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

Abstract

Background: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells.

Methods: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy.

Results: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival.

Conclusion: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival.

Trial registration: ClinicalTrials.gov NCT01865617.

Funding: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Figures

Figure 1. Heterogeneity in distribution of TN, TCM, and TEM/EMRA cells within CD4+ and CD8+ T cell subsets in normal donors and patients with B-ALL.

(A) Study participant flow chart. (B) Representative flow cytometry plots showing the immunophenotype of T cell subsets in blood from a B-ALL patient are shown. TN (CD45RA+CD62L+), TCM (CD45RA–CD62L+), and TEM/EMRA (CD62L–) cells can be identified in the CD3+CD4+ and CD3+CD8+ T cell populations. (C) The absolute CD4+ and CD8+ T cell counts in blood from healthy individuals (n = 14) and B-ALL patients (n = 30) are shown. Mann-Whitney U test was used for statistical analysis. (D) The percentages of TN, TCM, and TEM/EMRA cells in the CD3+CD4+ T cell population are shown. (E) The percentages of TN, TCM, and TEM/EMRA cells in the CD3+CD8+ T cell population are shown.

Figure 2. Serious toxicity due to CRS is mainly seen in B-ALL patients with high BM-tumor burden.

(A) The peak IFN-γ and IL-6 concentrations in serum in the first 28 days after CAR–T cell infusion are shown in patients who had high (> 20% blasts; n = 15), intermediate (5%–20% blasts; n = 5), or low (≤ 5% blasts; n = 10) tumor burden before lymphodepletion chemotherapy and CAR–T cell infusion. Each point represents data from 1 patient. (B) The peak IFN-γ and IL-6 concentrations in serum in the first 28 days after CAR–T cell infusion are shown in patients who did (n = 7) or did not (n = 23) require ICU care. (C and D) The percentages of BM blasts before lymphodepletion chemotherapy and the peak absolute CD3+CD4+ (C) and CD3+CD8+ (D) EGFRt+ CAR–T cell counts in the first 28 days after CAR–T cell infusion are shown in patients who did (n = 7) or did not (n = 23) require ICU care after CAR–T cell infusion. (E) The peak ferritin and CRP concentrations in serum in the first 28 days after CAR–T cell infusion are shown in patients who had high (> 20% blasts; n = 15), intermediate (5%–20% blasts; n = 5), or low (≤ 5% blasts; n = 10) tumor burden before lymphodepletion chemotherapy and CAR–T cell infusion. (F) The peak ferritin and CRP concentrations in serum in the first 28 days after CAR–T cell infusion are shown in patients who did (n = 7) or did not (n = 23) require ICU care.

Figure 3. Relationship between serum cytokine, ferritin, and CRP levels and severe neurotoxicity.

(A) The peak IL-6 and IFN-γ concentrations in serum in the first 28 days after CAR–T cell infusion in patients who developed NCI CTCAE grade 3 or higher neurotoxicity (n = 15) compared with those without neurotoxicity (n = 15). (B) The peak ferritin and CRP concentrations in serum in the first 28 days after CAR–T cell infusion in patients who developed NCI CTCAE grade 3 or higher neurotoxicity (n = 15) compared with those without neurotoxicity (n = 15). (C) Serum IL-6, IFN-γ, and TNF-α concentrations on day 1 after CAR–T cell infusion in patients who subsequently developed grade 3 to 5 neurotoxicity compared with those without neurotoxicity. Data represent the mean ± SEM. The Mann-Whitney U test was used for statistical analysis. (D) Serum IL-6, IFN-γ, and TNF-α concentrations on day 1 after CAR–T cell infusion in patients who subsequently required ICU care compared with those who did not require ICU care. Data represent the mean ± SEM. The Mann-Whitney U test was used for statistical analysis. (E) Serum IL-6 and IFN-γ concentrations on day 1 after CAR–T cell infusion in patients who subsequently developed the indicated grades of neurotoxicity. Data represent the mean ± SEM. (F) Predicted probability curve with bounding 95% CI limits showing the relationship between log2-transformed serum IL-6 concentration on day 1 after CAR–T cell infusion and the occurrence of grade 3 or higher neurotoxicity. Circles, observed; line, predicted.

Figure 4. Kinetics of CAR–T cell expansion, migration, and peak blood levels in relation to cell dose.

(A) Flow cytometry plots showing EGFRt+ CAR–T cells detected in the CD3+CD4+ and CD3+CD8+ T cell subsets in blood of a representative B-ALL patient at the indicated times after CAR–T cell infusion. (B) EGFRt+ CD4+ and CD8+ CAR–T cells infiltrate the BM and CSF. A representative flow cytometry analysis of a BM and CSF sample from 1 patient is shown. (C) Graphs show the absolute count (top) and percentage (bottom) of EGFRt+ CAR–T cells in the CD3+CD4+ (left) and CD3+CD8+ (right) T cell subsets in blood at intervals after CAR–T cell infusion in patients treated at DL1 (n = 6) or DL2 (n = 12) prior to incorporation of risk-adjusted CAR–T cell dosing. Data represent the mean ± SEM.

Figure 5. CAR–T cells are detected at higher levels in blood from patients with high tumor burden.

(A) The graphs show the absolute count (top) and percentage (bottom) of EGFRt+ CAR–T cells in the CD3+CD4+ (left) and CD3+CD8+ (right) T cell subsets in blood at intervals after CAR–T cell infusion in patients with high (≥ 5% BM blasts by flow cytometry; n = 15) or low (< 5% BM blasts by flow cytometry; n = 5) BM disease burden prior to incorporation of risk-adjusted CAR–T cell dosing. Data represent the mean ± SEM. The Mann-Whitney U test was used for statistical analysis. *P < 0.05; **P < 0.01. (B) ImmunoSEQ analysis of the TCRB genes of CAR–T cells sorted from blood of treated patients demonstrates polyclonality of CD4+ (left) and CD8+ (right) CAR–T cells in the recipient after adoptive transfer and sharing of sequences between the infusion product and the recipient after adoptive transfer. Each point represents 1 detected TCRB gene sequence. The axes indicate the percentage of TCRB reads. TCRB sequences identified by points in red were detected only in the infused CAR–T cell product (x axes). TCRB sequences identified by points in green are detected only in the recipient at the indicated day after CAR–T cell infusion (y axes). TCRB sequences identified by points in blue are detected both in the infused CAR–T cell product and in the recipient at the indicated day after infusion.

Figure 6. Failure to achieve engraftment of CAR–T cells after second infusions.

Patients who had persistent MRD after the first CAR–T cell infusion or subsequently relapsed after attaining a CR (n = 5) received a second infusion of CAR–T cells at an equivalent (n = 1) or 10-fold higher EGFRt+ dose (n = 4) compared with their first infusion. Engraftment after each infusion was analyzed by QPCR to detect a transgene vector sequence. Each graph shows the number of copies of integrated transgene (WPRE copies/μg DNA, n = 1; FlapEF1α copies/μg DNA, n = 4) detected in PBMCs collected at the indicated times after the first and second CAR–T cell infusions. The times and doses of the first (blue) and second (red) CAR–T cell infusions are noted on the graphs.

Figure 7. Incorporation of Flu into Cy-based lymphodepletion increases the expansion and persistence of CAR–T cells.

(A) Clinical outcomes of patients treated with CD19 CAR–T cells. (B) Graphs show the absolute count (top) and percentage (bottom) of EGFRt+ CAR–T cells in the CD3+CD4+ (left) and CD3+CD8+ (right) T cell subsets at intervals after CAR–T cell infusion in patients who received Cy and Flu lymphodepletion chemotherapy (DL2: Flu, n = 10) compared with those who received Cy alone or Cy/etoposide lymphodepletion (DL2: No Flu, n = 5). Day 0 represents a preinfusion sample and the background staining of the monoclonal antibody used to detect EGFRt+ T cells. All patients received EGFRt+ CAR–T cells at DL2. Data represent the mean ± SEM. The Mann-Whitney U test was used for statistical analysis. *P < 0.05; **P < 0.01. (C) The number of copies of integrated transgene (FlapEF1α copies/μg DNA) detected in PBMCs collected at the indicated times after the first CAR–T cell infusion in patients who received lymphodepletion with Cy/Flu (Flu, n = 17) compared with those who received Cy alone or Cy/etoposide (no Flu, n = 12). Time points after a second CAR–T cell infusion or allogeneic HCT are excluded. (D) DFS from the day of CAR–T cell infusion is shown for patients who received lymphodepletion with Cy/Flu (Flu, n = 17) compared with those who received Cy alone or Cy/etoposide (No Flu, n = 13). The median follow-up for Cy/Flu patients who were alive and in CR was 300 days. We compared the group who received Cy/Flu lymphodepletion and the group who received Cy-based lymphodepletion without Flu using the log-rank test, where P = 0.001.