Phase 1 study of CD19 CAR T-cell therapy harboring a fully human scFv in CAR-naïve adult patients with B-ALL

Treatment with T cells engineered to express CD19-directed chimeric antigen receptors (CD19 CAR T-cell therapy) induces measurable residual disease (MRD)–negative complete responses (CRs) in ∼70% to 95% of children^1-4 and adults^5-10 with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). These high response rates led to the US Food and Drug Administration (FDA) approval of 3 CD19 CAR T-cell products for R/R B-ALL,^1,5,10 adding invaluable treatment options for this high-risk patient population. Yet high rates of CD19⁺ relapses are still observed after CD19 CAR T-cell therapy, typically following loss of in vivo CAR T-cell persistence. This highlights a critical need to identify strategies enhancing in vivo CAR T-cell function and persistence. We have previously shown an association between impaired CAR T-cell pharmacokinetics (PK) and anti-CAR immune responses in patients undergoing CD19 CAR T-cell therapy on a phase 1/2 clinical trial (ClinicalTrials.gov identifier: NCT01865617).^6,11,12 Immune rejection of CAR T-cells was mediated by endogenous CD8⁺ T-cell recognizingt peptides that could be mapped to the murine single-chain variable fragment (scFv) of the CAR. In an effort to mitigate the impact of T-cell–mediated immune responses, improve in vivo CAR T-cell persistence, and enhance antitumor effects, we constructed a novel CAR including a fully human CD19-directed scFv¹³ instead of a murine scFv. We report here results and extended follow-up of a first-in-human phase 1 clinical trial of treatment with fully human scFv-bearing CD19 CAR T cells (JCAR021, previously named huJCAR014) in adult patients with R/R B-ALL.

We conducted at our institution a phase 1 clinical trial investigating treatment with JCAR021 in patients with R/R B-cell malignancies (ClinicalTrials.gov identifier: NCT03103971). Patients received lymphodepletion (LD) chemotherapy with cyclophosphamide (Cy) 300 mg/m² per day and fludarabine (Flu) 30 mg/m² per day for 3 days and planned to be completed 36 to 96 hours before JCAR021. Patients with B-ALL were enrolled between May 2018 and June 2021 in 1 of 2 cohorts: (1) high marrow burden (HMB; >5% blasts in bone marrow [BM] before LD by morphology or multiparameter flow cytometry [MFC]), (2) low marrow burden (LMB; ≤5% blasts in BM before LD by morphology or MFC). Bridging therapy after leukapheresis was allowed. The starting JCAR021 dose was 7 × 10⁴ or 7 × 10⁵ CAR T-cells per kg for the HMB and LMB cohorts, respectively. The study also allowed enrollment of CAR-naïve and CAR-exposed patients with large B-cell lymphoma whose outcomes will be reported in a separate manuscript. Dose escalation and de-escalation followed a modified toxicity probability interval-2 algorithm¹⁴ with a target dose-limiting toxicity rate of 30% and an equivalence interval of 25% to 35%. The primary objective of the study was to assess the safety of JCAR021. Secondary objectives included (1) to characterize the PK profile of JCAR021, (2) to assess the antitumor activity of JCAR021, and (3) to estimate the relapse-free survival (RFS) and overall survival (OS) in patients with ALL treated with JCAR021. Study objectives and end points are detailed in supplemental Table 1. Informed consent was obtained from each participant, and this study was approved by the Fred Hutchinson Cancer Center Institutional Review Board.

BM aspirates and biopsies were obtained before LD chemotherapy and ∼4 weeks after CAR T-cell infusion. Responses were assessed using the Acute Lymphoblastic Leukemia National Comprehensive Cancer Network (NCCN) Guidelines criteria.¹⁵ Extramedullary disease (EMD) was assessed before LD in patients with history of EMD or with clinical signs of EMD, and defined as extramedullary lesions measurable by computed tomography (CT; longest transverse diameter ≥15 mm) and/or fluorodeoxyglucose (FDG)-avid lesions by positron emission tomography (PET)–CT (FDG uptake above the liver uptake) before LD, excluding leptomeningeal involvement. EMD responses were evaluated by PET-CT and/or high-resolution CT ∼28 days after JCAR021 infusion in patients with EMD before LD, as defined above, and thereafter as clinically indicated. MRD-negative BM response was defined as the absence of immunophenotypically abnormal blasts in the BM by morphology and MFC (limit of detection 1:10,000). When available from patients with MRD-negative BM response, an aliquot of each BM aspirate was submitted for IGH deep sequencing (ClonoSEQ; Adaptive Biotechnologies). Cytokine release syndrome (CRS) severity was graded according to the Lee 2014 consensus criteria.¹⁶ The severity of neurotoxicity events was graded according to the Common Terminology Criteria for Adverse Events version 4.03. Immune effector cell (IEC)–associated hematotoxicity severity was graded per the European Hematology Association and European Bone Marrow Transplantation Society consensus criteria¹⁷ and computed as previously described.¹⁸ IEC-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) was defined according to the American Society of Transplantation and Cellular Therapy consensus criteria.¹⁹ 

For the detailed protocol regarding JCAR021 manufacturing, CAR T-cell PK and B-cell recovery monitoring, cellular immunogenicity assays, and statistical analyses, refer to supplemental Methods.

Of 25 patients who underwent screening, 23 (92%) proceeded to leukapheresis, LD chemotherapy, and JCAR021 infusion (Figure 1). Baseline characteristics are shown in Table 1. Five patients with LMB had no detectable BM disease by MFC before LD; however, all had measurable FDG-avid EMD. A high proportion (14/23 [61%]) of the treated population had EMD before LD. In the LMB cohort, 10 patients (91%) had EMD, including 6 (55%) with bulky disease (≥5 cm; excluding the spleen size). In the HMB cohort, there were 4 patients (33%) with EMD, and only 1 (8%) with bulky tumor. Postleukapheresis bridging therapies are listed in supplemental Table 2.

All patients received the target total CAR T-cell dose level. Of 23 patients who received JCAR021 infusion, 22 (96%) received a 1:1 ratio of CD8⁺:CD4⁺ CAR T cells as planned. One patient treated in the LMB cohort received a 0.34:0.66 ratio of CD8⁺:CD4⁺ CAR T cells due to insufficient growth of the CD8⁺ fraction during manufacturing. JCAR021 was administered at one of the following dose levels: 7 × 10⁴ (n = 3), 2 × 10⁵ (n = 4), 7 × 10⁵ (n = 8), 2 × 10⁶ (n = 7), or 7 × 10⁶ (n = 1) CAR T cells per kg. The median vein-to-vein time was 20 days (interquartile range [IQR], 19-27).

Nineteen patients developed CRS of any grade (83%; grade 2, 61%). No patient developed grade ≥3 CRS. CRS occurred more frequently and with higher severity in the HMB cohort (any grade, 92%; grade 2, 67%) compared to the LMB cohort (any grade, 73%; grade 2, 55%). The median time to first CRS symptom after JCAR021 infusion with patients who developed CRS was 1.5 days (IQR, 1-6). Neurotoxicity events occurred in 12 patients (52%; grade ≥3, 35%). The most common neurotoxicity events included headaches (30%), encephalopathy (26%), and delirium (9%). The median time to the first neurotoxicity event after JCAR021 infusion in patients who experienced neurotoxicity was 11.5 days (IQR, 7-18.5). We observed higher rates of neurotoxicity in the HMB cohort (67%; grade ≥3, 42%) compared to the LMB cohort (36%; grade ≥3, 27%). Eleven (48%) and 13 patients (57%) required tocilizumab and dexamethasone, respectively, for CRS and/or neurotoxicity management. Seven patients (30%) required intensive care unit admission (HMB, n = 6; LMB, n = 1). Exploratory univariate regression analyses of factors associated with grade ≥2 CRS and grade ≥3 neurotoxicity are shown in supplemental Tables 3 and 4, respectively. We observed grade ≥3 (Common Terminology Criteria for Adverse Events, 4.03) neutropenia, anemia, and thrombocytopenia in 100%, 74%, and 70% of patients, respectively, and grade ≥3 early IEC-associated hematotoxicity in 35% of patients (ANC ≤0.5 × 10³/μL for ≥14 days or ≤0.1 × 10³/μL for ≥7 days). No patients developed IEC-HS or a secondary malignancy.

Dose-limiting toxicities occurred in 2 patients treated with 7 × 10⁵ CAR T cells per kg in the HMB cohort, resulting in no further dose escalation in that cohort: 1 patient developed prolonged lower extremity muscle weakness and 1 patient died on day +10 after JCAR021 infusion from septic shock due to Clostridium septicum bacteremia. No dose-limiting toxicities occurred in the LMB cohort, allowing escalation to a higher CAR T-cell dose than that of the HMB cohort (Table 1).

The data demonstrate more severe CRS and neurotoxicity in the HMB cohort compared to the LMB cohort, despite higher EMD burden and higher cell doses in the LMB cohort. This suggests that severe toxicity may be associated more with BM leukemia burden than with EM disease. Grade ≥3 adverse events are listed in Table 2.

Of 23 patients who received JCAR021 infusion, 22 were evaluable for disease response assessment (HMB, n = 11; LTB, n = 11; Figure 1). The overall response and CR/CR with incomplete hematologic recovery (CRi) rates were 82% and 64%, respectively. In patients with measurable BM disease by MFC before LD (n = 17), we observed MFC MRD-negative BM responses in 14 patients (82%). We confirmed absence of MRD by ClonoSEQ in 5 of 7 patients (71%) with available data and a trackable clone. We observed MFC MRD–negative BM responses in 6 of 6 LMB patients with measurable BM disease by MFC before LD. Two of these 6 LMB patients had BCR::ABL1 fusion B-ALL and quantitative polymerase chain reaction testing of the BM for BCR::ABL1 was also negative. In patients with measurable EMD before LD (n = 14), we observed overall responses by PET-CT in 11 patients (79%) and CRs in 7 patients (50%). Higher BM burden, higher EMD burden, and factors reflecting lower levels of in vivo CAR T-cell expansion were associated with lower odds of CR/CRi (supplemental Table 5). Despite the high fraction of patients with EMD in the LMB cohort, we observed superior responses in that cohort (overall response, 91%; MRD-negative BM response, 100%) compared to the HMB cohort (overall response, 73%; MRD-negative BM response, 73%; Figure 2).

Kaplan-Meier estimates of long-term outcomes after JCAR021 treatment are shown in Figure 3. The median RFS and OS in all infused patients were 5.5 (95% confidence interval [CI], 3-19.5) and 23 months (95% CI, 11 to not estimable [NE]), respectively. After a median follow-up of 50 months, the 4-year RFS and OS were 17% (95% CI, 7-42) and 29% (95% CI, 15-56), respectively. The median duration of response (DOR) was 10 months (95% CI, 5-NE; censoring for allogeneic hematopoietic cell transplantation [allo-HCT], median 8 months [95% CI, 4 to NE]) with a 4-year DOR probability of 29% (95% CI, 12-65; censoring for allo-HCT, 20% [95% CI, 6-70]). Four patients underwent allo-HCT while in CR/CRi after JCAR021 in the absence of subsequent therapy. The 4-year cumulative incidence of relapse after JCAR021 was 57% (95% CI, 25-80). Two patients received a tyrosine kinase inhibitor after treatment with JCAR021 (ponatinib for relapse post-JCAR021, n = 1; maintenance therapy after allo-HCT with imatinib followed by dasatinib, n = 1). No patient subsequently received the FDA-approved CD19 CAR T-cell therapy brexucabtagene autoleucel (brexu-cel). Exploratory univariate analyses of factors associated with DOR are shown in supplemental Table 6.

The 4-year RFS in the LMB and HMB cohorts were 36% (median, 11 months) and 0% (median, 4 months), respectively (P = .036). The 4-year DOR in the LMB and HMB cohorts were 57% (median, not reached) and 0% (median, 4.5 months), respectively (P = .007); the 4-year cumulative incidence of relapse in the LMB and HMB cohorts was 29% (95% CI, 2-66) and 86% (95% CI, 5-99), respectively (P = .017). Kaplan-Meier and cumulative incidence curves of outcomes stratified by study cohort and CAR T-cell dose levels are shown in supplemental Figures 1 and 2, respectively. Outcomes of LMB patients with nonbulky (<5 cm) EMD are detailed in supplemental Table 7.

Despite high rates of initial response, durable remission was not achieved for patients in the HMB cohort, primarily due to a high incidence of relapse. In conjunction with the association of inferior outcomes with low infused cell dose (supplemental Figure 2), the dose-limitation imposed by toxicities in the HMB cohort likely contributed to the poor outcomes in those with high BM leukemia burden.

All relapses occurred after loss of CAR transgene persistence in the blood (Figure 4). Median time from confirmed loss of CAR transgene persistence to relapse was 137 days (IQR, 11-279). We confirmed B-cell recovery in the blood before relapse in 6 of 9 patients with available B-cell reconstitution data (missing data for patient ALL-LMB-1, n = 1) with a median time to B-cell recovery of 140 days (IQR, 93-228). Median time from B-cell recovery to relapse was 153 days (IQR, 68-274).

Of 10 relapses occurring in patients who achieved MRD-negative BM or extramedullary CR after JCAR021 and with available CD19 expression data on leukemic blasts, CD19 expression on lymphoblasts was confirmed in 8 cases by MFC and/or immunohistochemistry (80%; diminished CD19 expression by MFC, n = 1; missing data, n = 1). A CD19^– relapse associated with a concurrent CD19⁺ subset was observed by flow cytometry in 1 patient with persistent B-cell aplasia at the time of relapse (patient ALL-HMB-7). The data suggest the dominant mechanism of relapse after JCAR021 was loss of CD19-targeted immune pressure.

We measured in vivo expansion of CAR T cells by MFC or quantitative polymerase chain reaction in the blood in all but 1 patient who received high-dose chemotherapy at day +4 after CAR T-cell infusion due to rapid disease progression. In patients with available data and excluding patients who subsequently underwent allo-HCT without confirmed loss of CAR persistence at HCT (n = 15), the median time to confirmed loss of CAR persistence was 95 days (IQR, 74-174; HMB: median, 95 days; LMB: median, 93.5 days). In 10 patients with confirmed B-cell recovery after JCAR021, excluding patients who subsequently underwent allo-HCT without B-cell recovery at HCT, the median time to B-cell recovery was 97 days (IQR, 91-133). The observations of median duration of CAR T-cell persistence and B-cell aplasia of ∼3 months after infusion are consistent with moderate duration of JCAR021-associated CD19–targeted immune pressure. Summary statistics and longitudinal CAR T-cell kinetics categorized by dose level and by cohort are shown in supplemental Table 8 and supplemental Figure 3, respectively. Because the CAR T-cell dose was determined based on BM burden, we assessed the impact of the CAR T-cell dose on CAR T-cell PK measurements using linear regression models that also included the BM burden to reduce confounding. In these models (supplemental Table 9), higher CAR T-cell doses remained independently associated with higher CD4⁺ CAR T-cell maximum concentration (Cmax), higher CD3⁺ CAR T-cell area under the curve (AUC)_{day 0-28} and higher CD3⁺ CAR T-cell AUC_{day 0-90}.

To test the hypothesis that CAR T cells engineered with a fully human scFv might be associated with superior antileukemic activity, we compared clinical outcomes of CAR-naïve patients with B-ALL treated with JCAR021 to CAR-naïve patients with B-ALL who were treated with CD19 CAR T cells expressing a murine scFv-containing CAR (JCAR014; n = 51) on a previous clinical trial at our center (NCT01865617).^6,21 Both JCAR021 and JCAR014 were similarly manufactured using the same lentivirus backbone, CAR design (with the exception of the human and murine scFvs), and processing and formulation in a 1:1 CD8⁺:CD4⁺ CAR T-cell ratio; both were infused after Cy/Flu LD. Key baseline characteristics for each cohort are shown in supplemental Table 10.

We observed comparable rates of CR/CRi (JCAR021, 64%; JCAR014, 60%) and MRD-negative BM response rates in patients with measurable BM disease by MFC (JCAR021, 82%; JCAR014, 82%), any grade CRS (JCAR021, 83%; JCAR014, 76%), grade ≥2 CRS (JCAR021, 61%; JCAR014, 57%), and any grade neurotoxicity (JCAR021, 52%; JCAR014, 47%). We observed higher rates of grade ≥3 neurotoxicity after JCAR021 (JCAR021, 35%; JCAR014, 22%). The 4-year DOR after JCAR021 and JCAR014 were 29% (95% CI, 12-65) and 36% (95% CI, 23-59; P = .5), respectively. The 4-year DOR after JCAR021 and JCAR014 censoring for allo-HCT were 20% (95% CI, 6-70) and 13% (95% CI, 3-45; P = .8), respectively. The 4-year cumulative incidence of relapse after JCAR021 and JCAR014 was 57% (95% CI, 25-80) and 47% (95% CI, 27-64), respectively (P = .6).

Next, we estimated the effect of the CAR T-cell product type using cause-specific Cox models censoring for death in CR and allo-HCT. In a univariate cause-specific Cox model, we could not confirm an impact of the CAR T-cell product type on DOR (hazard ratio, 0.95 for JCAR021 vs JCAR014; 95% CI, 0.40-2.27; P = .91). Similarly, we could not confirm an independent impact of the CAR T-cell product type on DOR (adjusted hazard ratio, 1.10 for JCAR021 vs JCAR014; 95% CI, 0.36-3.41; P = .86) in a multivariable model including the following covariates: CAR T-cell dose, pre-LD BM blasts percentage, and presence or absence of EMD.

Because CAR T-cell PK are affected by the CAR T-cell dose level and LD intensity, we performed exploratory comparisons in a subset of patients who received the same low-intensity Cy (300 mg/m² per day for 3 days) and Flu (30 mg/m² per day for 3 days) LD matched by CAR T-cell dose (2 × 10⁵/kg, n = 18; 2 × 10⁶/kg, n = 11). We measured numerically lower CD8⁺ and CD4⁺ JCAR021 Cmax compared to JCAR014 at either dose level, although this did not reach statistical significance (supplemental Table 11), and comparable transgene persistence (supplemental Figure 4).

The data do not show an appreciable difference between outcomes of CAR-naïve patients with B-ALL after fully human scFv-containing compared to murine scFv-containing CD19 CAR T cells.

CD19 CAR T-cell therapy achieves MRD-negative responses in a high proportion of patients with R/R B-ALL, yet there remains a critical need to improve response durability, particularly in adults. We previously demonstrated the immunogenic potential of the broadly used murine CD19-targeted FMC63 scFv, which was associated with impaired in vivo CAR T-cell expansion and antitumor effects after second CAR T-cell infusions in our phase 1/2 clinical trial.^6,11,12 The identification of anti-CAR immune responses suggested reducing the immunogenicity of CD19 CAR T-cells may improve outcomes. This led us to conduct a first-in-human phase 1 clinical trial evaluating the safety and efficacy of treatment with CD19 CAR T-cells engineered with a fully human scFv (JCAR021) in patients with R/R B-ALL.

We successfully manufactured a product for all patients; all but one product was formulated in the planned 1:1 ratio of CD8⁺:CD4⁺ CAR T cells. We safely dose-escalated to 7 × 10⁵ CAR T cells per kg and 7 × 10⁶ CAR T cells per kg in the HMB and LMB cohorts, respectively. CRS and neurotoxicity events were common but manageable in most patients, and consistent with rates observed in other trials of CD19 CAR T cells in B-ALL.^3,5 There were no deaths attributed to JCAR021 toxicity. Although we observed high response rates after JCAR021 (CR/CRi rate, 64%; MRD-negative BM responses, 82%), remissions were not durable in most patients (median DOR in CR/CRi, 10 months). Cellular immunogenicity assays, reported in the supplemental Material (“Cellular Immunogenicity”; supplemental Figure 5), did not suggest patients developed CD8⁺ T-cell–mediated anti-CAR immune responses after treatment with JCAR021.

Comparing our findings to other single-arm clinical trials and external cohorts is challenging given differences in patient and disease characteristics, in addition to variations in follow-up duration and end point definitions. Acknowledging these limitations, our observed response rates after JCAR021 compare favorably with published studies to date utilizing other CD19 CAR T-cell immunotherapies in adults with R/R B-ALL. In the ZUMA-3 clinical trial, investigating the FDA-approved murine CD19 scFv-bearing CAR T-cell product brexu-cel, investigators reported CR/CRi in 71%, and MRD negativity in 76%.²² In a recent report in the nontrial setting, brexu-cel was associated with MRD-negative BM responses in 71% of patients.²³ Although response rates after JCAR021 compared favorably with other CD19 CAR T-cell immunotherapies in adult patients with R/R B-ALL, we observed shorter median DOR after JCAR021 (10 months) compared with brexu-cel (median DOR, 14.6 months²²). The higher CAR T-cell dose associated with brexu-cel (1 × 10⁶/kg) may contribute to the longer DOR observed after this product compared with JCAR021. A recent report from the FELIX study investigators demonstrated durable responses (median event-free survival in all patients who received infusion, 11.9 months) after treatment with the now FDA approved, fast-off murine scFv-bearing CD19 CAR T-cell product obecabtagene autoleucel.¹⁰ These data compare with a median RFS of 5.5 months after JCAR021. Compared with adults with R/R B-ALL previously treated at our center with Cy/Flu LD and murine scFv-bearing CD19 CAR T-cell therapy (JCAR014) of similar 1:1 CD8⁺:CD4⁺ composition, JCAR021 achieved comparable CR/CRi rates (JCAR021, 64%; JCAR014, 60%) and 4-year DOR (JCAR021, 29%; JCAR014, 36%). We acknowledge the limitations of retrospective comparisons across noncontemporaneous single-arm trials, as reflected by the higher proportion of JCAR021 patients exposed to blinatumomab and inotuzumab ozogamicin compared with those previously treated with JCAR014. Yet, we believe our findings suggest that the incorporation of a fully human scFv alone may not be sufficient to improve outcomes of CD19 CAR T-cell therapy in adult CAR-naïve patients with B-ALL.

To our knowledge, we are the first to report the use of CAR T-cell immunotherapy engineered with a fully human CD19 scFv in a clinical trial exclusively enrolling adult patients with B-ALL. This is noteworthy because worse outcomes are generally reported in adults compared to children with R/R B-ALL.^24,25 More favorable outcomes have been reported in a pediatric clinical trial of CD19 CAR T-cell therapy using a humanized scFv conducted at Children’s Hospital of Pennsylvania.²⁶ In this study the CR/CRi rate in the CAR-naïve cohort was 98% and the 24-month RFS in CR/CRi patients was 74%. Although our study specifically investigated a CAR-naïve population, an important question is whether fully human or humanized scFv-bearing CD19 CAR T-cell products can be efficacious after failure of a murine CAR. In the study from the Children’s Hospital of Pennsylvania, the authors also reported high rates of CR/CRi (79%) and a 58% RFS probability at 2 years in CAR-exposed patients with only 1 patient receiving allo-HCT consolidation. An et al²⁷ from Beijing Boren Hospital also reported high CR rates (68%) after humanized scFv-bearing CD19 CAR T-cell therapy in a study including 19 pediatric and adult patients with B-ALL after failure of a previous murine scFv-bearing CD19 CAR T-cell therapy. Most responders (84%) subsequently received consolidative allo-HCT. The event-free survival probability at 12 to 18 months was 91% in the 11 patients who received subsequent allo-HCT and 69% in all CR patients. Taken together, these 2 studies suggest CD19 CAR T-cell therapy with a humanized scFv can induce high response rates in patients with B-ALL who were previously treated with a murine scFv-containing CAR. More data are needed to evaluate the DOR and the need for subsequent allo-HCT consolidation. The apparent superiority of a second infusion with a human compared to murine scFv-containing CAR T-cell product in murine scFv CAR T-cell–exposed patients may be due to less rejection mediated by endogenous memory T cells with capacity for an anti-CAR recall response to neoantigens in the murine scFv. In CAR-naïve patients, there are typically no preexisting endogenous anti-CAR T cells; therefore, human and murine scFv-containing CAR T-cell products are similarly effective.

As previously shown by our group²¹ and others,^28-30 BM disease burden remains a key determinant of outcomes in patients with R/R B-ALL who are receive CD19 CAR T-cell therapy. We observed favorable outcomes in the LMB cohort after JCAR021 treatment with a 100% MRD-negative BM response rate and a 4-year DOR estimate of 57% (median, not reached). In this cohort, relatively low rates of CRS (73%) and neurotoxicity (36%) occurred after JCAR021 compared with brexu-cel (CRS, 89%; ICANS, 60%). Notably, the lower toxicity was observed despite a very high proportion of bulky EMD (55%) among the LMB JCAR021 patients. Importantly, 2 patients with LMB remained alive and in remission in the absence of consolidative allo-HCT with an extended follow-up of over 4 years, suggesting JCAR021 treatment alone may be curative in a subset of patients with LMB. In contrast, despite a relatively high MRD-negative BM response rate of 73% after JCAR021, the 4-year DOR in patients with HMB was 0% (median, 4.5 months). This highlights a critical need to develop novel strategies for patients with B-ALL with HMB disease (eg, improvement of debulking treatments pre-CAR T-cell therapy, development of CAR T-cell products with enhanced antitumor effects, novel consolidative strategies, and better toxicity management). Besides the higher BM disease burden, the administration of lower CAR T-cell doses in patients with HMB likely contributed to poor outcomes in this group. Lower JCAR021 doses were overall associated with worse outcomes, lower CD4⁺ CAR T-cell Cmax, and CD3⁺ CAR T-cell AUC. Taken together, our findings suggest higher CAR T-cell doses may elicit superior outcomes in patients with higher BM disease burden, but with an increased risk of toxicities. New studies will be needed to determine whether recently introduced strategies to manage toxicities will allow JCAR021 escalation to higher dose levels and improve efficacy in those with high BM disease burden.

We observed limited CAR T-cell persistence and B-cell aplasia in treated patients, consistent with moderate duration of in vivo CAR T-cell function. CD19 was expressed by all but 1 evaluated case of relapsed leukemia. The high rate of CD19⁺ relapse may reflect insufficiently sustained CAR T-cell–mediated persistence and systemic immune pressure on CD19⁺ lymphoblasts or the presence of leukemia sanctuary sites that are protected from CAR T-cell–mediated immune pressure. In support of the latter, we noted lower response rates in EM disease (CR, 50%) compared to BM disease (MRD negativity, 82%), suggesting that differences between the EM and BM tumor microenvironments may affect local CAR T-cell efficacy. In concert with our observation that bulky EMD was associated with less CAR T-cell–mediated toxicity, this is consistent with the effects of a more suppressive tumor microenvironment in EM compared with BM tumor.

In summary, treatment with the human scFv-bearing CD19 CAR T-cell therapy JCAR021 achieved high rates of MRD-negative BM and EM responses in CAR-naïve adults with R/R B-ALL, even in those with high disease burden. While those with LMB had durable remissions, the DOR was limited in patients with HMB, highlighting a remaining critical need to identify new strategies to prolong remissions.

The authors thank the Cell Processing Facility and Cell Therapy Laboratory, and the staff of the Integrated Immunotherapy Research Center and the Bezos Family Immunotherapy Clinic at the Fred Hutchinson Cancer Center.

This work was supported by Juno Therapeutics, a Bristol Myers Squibb company, and grants from the Fred Hutch Cancer Center Immunotherapy Integrated Research Center Paul Estate, Swim Across America Foundation, ClearBridge Foundation, Australian National Health and Medical Research Council Investigator Grant, the National Institutes of Health (NIH) National Cancer Institute (grants P30 CA015704-45, P01CA18029, 5T32CA951539, and 5K12CA076930-24), NIH National Institute of Diabetes and Digestive and Kidney Diseases (grant P30 DK56465), NIH National Heart, Lung, and Blood Institute (grant 5T32HL007093), the Life Sciences Discovery Fund, the Bezos Family Foundation, the National Gene Vector Biorepository at Indiana University funded by NIH National Heart, Lung, and Blood Institute (grant 75N92019D00018).

Contribution: J.G., E.C.L., D.G.M., and C.J.T. conceived and designed the study; J.G., E.C.L., J.J.H., C.J.T., and J.M.V collected and assembled the data; J.G., E.C.L., and C.J.T. analyzed and interpreted the data; and all authors wrote and approved the manuscript and are accountable for all aspects of the work.

Conflict-of-interest disclosure: J.G. reports consultancy with Kite, a Gilead company, MorphoSys, Legend Biotech, Janssen, and Sobi; received honoraria from Kite, a Gilead company, Legend Biotech, Janssen, and Sobi; received research funding from MorphoSys, Angiocrine Bioscience, CARGO Therapeutics, Celgene, a BMS company, CytoAgents, Faron Pharmaceuticals, Juno Therapeutics, a BMS company, and Sobi; and serves on an independent data review committee at Century Therapeutics. E.C.L. reports consultancy with Glass Health. E.L.K. received research funding from Juno Therapeutics, a BMS company. A.V.H. received honoraria from Novartis, BMS, and Nektar Therapeutics; and research funding from BMS, Nektar Therapeutics, and Juno Therapeutics, a BMS company. S.F. filed and received license fees on patents for optimizing CAR T-cell function; received research funding from BMS; and reports consultancy with Prescient Therapeutics. M.S. reports consultancy with Fate Therapeutics, Genmab, MorphoSys/Incyte, Eli Lilly, BMS, Genentech, Kite, a Gilead company, AbbVie, Mustand Bio, ADC Therapeutics, BeiGene, AstraZeneca, Pharmacyclics, Janssen, MEI Pharma, and Regeneron; and received research funding from Genmab, Vincerx, MorphoSys/Incyte, BMS, Genentech, AbbVie, Mustang Bio, BeiGene, AstraZeneca, Pharmacyclics, and TG Therapeutics. R.D.C. reports consultancy with Amgen, Pfizer, Jazz, and Kite, a Gilead company/Gilead; received honoraria from Amgen, Autolus, Pfizer, Jazz, and Kite, a Gilead company/Gilead; received research funding from Amgen, Merck, Incyte, Pfizer, Servier, Kite, a Gilead company/Gilead, and Vanda Pharmaceuticals; has membership on the board of directors or advisory committees at Autolus and PeproMene Bio; and has a spouse who was employed by and owned stock in Seagen within the last 24 months. S.R.R. is the cofounder of Lyell Immunopharma and Juno Therapeutics, a BMS company; received research funding from Lyell Immunopharma and BMS; has an intellectual property license agreement with Lyell Immunopharma and BMS; serves on advisory boards at Juno Therapeutics, a BMS company and Adaptive Biotechnologies; and is a member of board of directors at Ozette Technologies. D.G.M. reports consultancy with A2 Biotherapeutics, Juno Therapeutics, a BMS company, Janssen, Legend Biotech, Mustang Bio, Novartis, Incyte, Gilead Sciences, Kite, a Gilead company, a Gilead Sciences, Pharmacyclics, Umoja, Celgene, a BMS company, Genentech, MorphoSys, BMS, Amgen, Navan Technologies, and Bioline Rx; is a current holder of stock options in privately held companies A2 Biotherapeutics and Navan Technologies; received honoraria from A2 Biotherapeutics, Juno Therapeutics, a BMS company, Janssen, Legend Biotech, Mustang Bio, Novartis, Incyte, Gilead Sciences, Kite, a Gilead company, a Gilead Sciences, Pharmacyclics, Umoja, Celgene, a BMS company, Genentech, MorphoSys, BMS, Amgen, and Navan Technologies; serves as a member of the scientific advisory Boards at A2 Biotherapeutics, Navan Technologies, and Chimeric Therapeutics; has rights to royalties from Fred Hutch for patents licensed to Juno Therapeutics, a BMS company; received research funding from Juno Therapeutics, a BMS company, Legend Biotech, Kite, a Gilead company, a Gilead Sciences, Celgene, a BMS company, and BMS; is a member of the CAR T steering committee at Lyell Immunopharma; is a member of the scientific review committee of Gilead Sciences; is a part of the Research Scholars Program in Hematologic Malignancies at Gilead Sciences; reports membership on board of directors or advisory committees and participation on data safety monitory boards at Celgene, a BMS company and Bioline Rx; is chair and member of the lymphoma steering committee at Genentech; is a member of the JCAR017 EAP-001 safety review committee, member of the Chronic Lymphocytic Leukemia Strategic Council, and member of the JCAR017-BCM-03 scientific steering committee under BMS; is a member of the clinical advisory board, CD19/CD20 bispecific CAR T-Cell Therapy Program at ImmPACT Bio; and a member of the clinical advisory board at Interius. C.J.T. received research funding from Juno Therapeutics, a BMS company, Nektar Therapeutics, and 10x Genomics; serves on scientific advisory boards at Caribou Biosciences, T-CURX, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics, Celgene, a BMS company Cell Therapy, Differentia Bio, eGlint, and Advesya; is a data safety monitory board member at Kyverna; reports ad hoc advisory roles/consulting (last 12 months with Prescient Therapeutics, Century Therapeutics, IGM Biosciences, AbbVie, Boxer Capital, Novartis, and Merck Sharp & Dohme; has stock options in Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics, and eGlint; reports speaker engagement (last 12 months) with Pfizer and Novartis; and is an inventor on patents related to CAR T-cell therapy. The remaining authors declare no competing financial interests.

Correspondence: Jordan Gauthier, Fred Hutchinson Cancer Center, 1100 Fairview Ave N, Mail Stop D3-100, Seattle, WA 98109; email: jgauthier@fredhutch.org.