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Efficacy of second CAR-T (CART2) infusion limited by poor CART expansion and antigen modulation

Chimeric antigen receptor T-cells (CART) are active in relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but relapse remains a substantial challenge. Reinfusion with the same CART product (CART2) in patients with suboptimal response or antigen positive relapse following first infusion (CART1) represents a potential treatment strategy, though early experiences suggest limited efficacy of CART2 with CD19 targeting. We report on our experience with CART2 across a host of novel CAR T-cell trials. This was a retrospective review of children and young adults with B-ALL who received reinfusion with an anti-CD19, anti-CD22, or anti-CD19/22 CART construct on one of 3 CAR T-cells trials at the National Cancer Institute (NCT01593696, NCT02315612, NCT0344839) between July 2012 and January 2021. All patients received lymphodepletion (LD) pre-CART (standard LD: 75 mg/m2 fludarabine, 900 mg/m2 cyclophosphamide; or intensified LD: 120 mg/m2 fludarabine, 1200 mg/m2 cyclophosphamide). Primary objectives were to describe response to and toxicity of CART2. Indication for CART2, impact of LD intensity, and CAR T-cell expansion and leukemia antigen expression between CART infusions was additionally evaluated. Eighteen patients proceeded to CART2 due to persistent (n=7) or relapsed antigen positive disease (n=11) following CART1. Seven of 18 (38.9%) demonstrated objective response (responders) to CART2: 5 achieved a minimal residual disease (MRD) negative CR, 1 had persistent MRD level disease, and 1 showed a partial remission, the latter with eradication of antigen positive disease and emergence of antigen negative B-ALL. Responders included four patients who had not achieved a CR with CART1. Limited cytokine release syndrome was seen following CART2. Peripheral blood CART1 expansion was higher than CART2 expansion (p=0.03). Emergence of antigen negative/dim B-ALL in 6 (33.3%) patients following CART2 contributed to lack of CR. Five of seven (71.4%) responders received intensified LD pre-CART2, which corresponded with higher CART2 expansion than in those receiving standard LD (p=0.029). Diminished CAR T-cell expansion and antigen downregulation/loss impeded robust responses to CART2. A subset of patients, however, may derive benefit from CART2 despite suboptimal response to CART1. Intensified LD may be one strategy to augment CART2 responses, though further study of factors associated with CART2 response, including serial monitoring of antigen expression, is warranted.

ABSTRACT Chimeric antigen receptor T-cells (CART) are active in relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but relapse remains a substantial challenge. Reinfusion with the same CART product (CART2) in patients with suboptimal response or antigen positive relapse following first infusion (CART1) represents a potential treatment strategy, though early experiences suggest limited efficacy of CART2 with CD19 targeting. We report on our experience with CART2 across a host of novel CAR T-cell trials. This was a retrospective review of children and young adults with B-ALL who received reinfusion with an anti-CD19, anti-CD22, or anti-CD19/22 CART construct on one of 3 CAR T-cells trials at the National Cancer Institute (NCT01593696, NCT02315612, NCT0344839) between July 2012 and January 2021. All patients received lymphodepletion (LD) pre-CART (standard LD: 75 mg/m 2 fludarabine, 900 mg/m 2 cyclophosphamide; or intensified LD: 120 mg/m 2 fludarabine, 1200 mg/m 2 cyclophosphamide). Primary objectives were to describe response to and toxicity of CART2. Indication for CART2, impact of LD intensity, and CAR T-cell expansion and leukemia antigen expression between CART infusions was additionally evaluated. Eighteen patients proceeded to CART2 due to persistent (n=7) or relapsed antigen positive disease (n=11) following CART1. Seven of 18 (38.9%) demonstrated objective response (responders) to CART2: 5 achieved a minimal residual disease (MRD) negative CR, 1 had persistent MRD level disease, and 1 showed a partial remission, the latter with eradication of antigen positive disease and emergence of antigen negative B-ALL. Responders included four patients who had not achieved a CR with CART1. Limited cytokine release syndrome was seen following CART2. Peripheral blood CART1 expansion was higher than CART2 expansion (p=0.03). Emergence of antigen negative/dim B-ALL in 6 (33.3%) patients following CART2 contributed to lack of CR. Five of seven (71.4%) responders received intensified LD pre-CART2, which corresponded with higher CART2 expansion than in those receiving standard LD (p=0.029). Diminished CAR T-cell expansion and antigen downregulation/loss impeded robust responses to CART2. A subset of patients, however, may derive benefit from CART2 despite suboptimal response to CART1. Intensified LD may be one strategy to augment CART2 responses, though further study of

Chimeric antigen receptor T-cells (CART)
have induced remarkable remission rates of 70%-90% in children and young adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). 1-6 Many patients, however, will not achieve a durable remission with CART therapy alone. Relapse following CART therapy remains a significant challenge: approximately 50% will relapse in the first year, 2 7 8 with many retaining expression of the CART target antigen. In patients with residual disease or antigen-positive relapse following CART1, reinfusion of the same CAR T-cell product (CART2) represents a potential treatment strategy; which is to Key message ⇒ Suboptimal response and relapse following chimeric antigen receptor T-cell (CART) infusion remain significant challenges in children and young adults receiving CART therapy for relapsed/refractory Bcell acute lymphoblastic leukemia. Reinfusion of the same CART construct (CART2) represents a potential treatment strategy. Evaluation of CART2 outcomes across a host of novel CART constructs and targets revealed minimal CART2 toxicity, with a subset of patients deriving benefit from CART2 despite suboptimal CART1 response. Notable biological limitations, including diminished CART expansion and target antigen downregulation/loss, however, impeded both robust CART2 responses and duration of remission. Given the potential need for and benefit of CART2, strategies to augment CART2 responses, including increased cell dose, routine use of intensified lymphodepletion and postremission consolidative approaches warrant further study.
Open access be distinguished from infusion of a unique CAR T-cell construct (eg, targeting an alternate antigen). Early experiences with CD19 CART reinfusion in B-ALL suggest limited efficacy of CART2. 9 10 Diminished CART expansion, persistence, and development of anti-CAR immune response inhibiting CAR T-cell activity are potential mechanisms of CART2 failure. Further characterization of factors associated with CART2 response is needed. We evaluated complete remission (CR) rates following CART2 in patients who received reinfusion across several of our phase I CAR T-cell trials using a host of CART constructs and targets. To elucidate factors associated with CART2 response, we additionally evaluated indications for CART2, CART expansion and antigen expression following CART2, and the impact of pre-CART2 lymphodepletion (LD) intensity.

Study population
This was a retrospective review (NCT03827343) of children and young adults with r/r B-ALL receiving reinfusion of the same CART product (CART2) on one of three institutional review board approved phase I CAR T-cell trials at the National Cancer Institute (NCI) between 7/2012 and 1/2021. Patients received anti-CD19-28ζ (NCT01593696), anti-CD22-.BBζ (NCT02315612), or anti-CD19/CD22.BBζ (NCT0344839) CAR T-cells. CART2 was offered only to patients who had (1) relapse following a CR with CART1 or (2) suboptimal CART1 response with evidence for CART1 expansion in whom strategies to augment CART2 response were feasible (eg, intensified LD, increased cell dose or reduced disease burden pre-CART2). Patients with disease progression without CART1 expansion were not offered CART2. Four (22.2%) patients received CART2 by individualized compassionate use following CART1 infusion on-study (online supplemental file).

Objectives
The primary objective was to evaluate CART2 CR rate and toxicity incidence. Secondary objectives included identifying indication for CART2, characterizing CART1 versus CART2 expansion, describing pre-CART1 and pre-CART2 antigen expression, and evaluating the impact of LD intensity on CART2 outcomes.

Disease assessments
Bone marrow disease was assessed by standard bone marrow morphology and classified as M1 (<5%), M2 (5%-25%), or M3 (>25%). Flow cytometry (FC) was used to evaluate disease at baseline, day 28 (±4 days) post-CART infusion and at best response. The NCI Flow Cytometry Laboratory performed minimal residual disease (MRD) assessment with validated limit of detection of ALL blasts at 0.002% of total cells. 11 12 Disease in the cerebrospinal fluid (CSF) was analyzed by routine cytopathology and FC. Non-central nervous system (CNS) extramedullary disease (EMD) was assessed with 18-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography/CT.
Objective marrow response was defined as achieving a bone marrow CR (with or without detectable MRD) or partial remission (PR) following CART infusion (online supplemental file). The lower limit of detection for CARpositive (CAR + ) T-cells in the bone marrow by FC was 0.1% for CD19% and 1% for CD22 CAR.

Toxicity evaluations
Cytokine release syndrome (CRS) and neurological toxicity were prospectively evaluated on all studies. For the purposes of consistency, all CRS grading has been reconciled with the American Society for Transplantation and Cellular Therapy CRS consensus criteria. 13 As ICANS grading was not established prior to 2018 and cannot be retrospectively applied to patients and trials conducted prior to this time, neurologic toxicity was assessed by CTCAE and considered severe if the AE was >grade 3.

Statistical analyses
Standard descriptive statistics were used to characterize demographics of the cohort. Given the small sample size, non-parametric tests were used for all analyses. Wilcoxon signed-rank tests were used for paired analyses and Mann-Whitney U tests used for all unpaired analyses and performed with Prism GraphPad using threshold of significance p<0.05.

Patient characteristics
Eighteen of 136 (13.2%) patients with B-ALL receiving CART1 proceeded to CART2 with the same CART product. Median age at CART1 and CART2 was 18.5 years (range, 7-30 years) and 19 years (range, 8-31 years), respectively. Prior to CART1, patients were heavily pretreated and received a median of 6 lines of therapy (range, 2-13), with 77.8% (n=14) having undergone prior hematopoietic stem cell transplantation (HSCT). A substantial portion had previous immunotherapy exposures: 8 (44.4%) received blinatumomab or inotuzumab ozogamicin and 8 (44.4%) received an alternate anti-CD19-and/or CD22 CART therapy prior to CART1 at our institution (table 1). No patient had interim HSCT between CART1 and CART2. Response to CART1 and indications for CART2 CART2 was indicated for suboptimal response (PR or stable disease (SD)) to CART1 in 7 (38.9%) patients or for antigen-positive relapse in 11 (61.1%) following CART1 (figure 1A, online supplemental table 1). Two patients proceeded to CART2 for persistent or relapsed   Open access clearance of antigen positive disease but emerged from CART2 with antigen loss. Among four patients with CNS involvement, two (50.0%) had full eradication of CNS disease with CART2, including the patient with CNS3 disease who had simultaneous eradication of non-CNS EMD. Among seven CART2 responders, four had not achieved a CR with CART1 (PR, n=1; SD, n=3). Of these four, three (75%) received intensified LD, including one patient who also had improved disease control pre-CART2 and one who received a higher CART2 dose. One CART1 non-responder who required early steroids due to rapid disease progression abrogating CART1 expansion achieved a CR with CART2 following standard LD with lower disease burden ( figure 1B,C).
Few experienced symptoms of neurological toxicity with CART1 or CART2, all of which were mild (grade 1 or grade 2). Four of 18 (22.2%) had neurotoxicity following CART1, while 2 (11.1%) had neurotoxicity with CART2 ( figure 1E). Of four patients with CART1-associated neurotoxicity, only one subsequently experienced neurotoxicity with CART2.
Long-term survival across six patients achieving a CR with CART2 was limited. No patient was eligible for consolidative HSCT and all patients experienced relapse, either with persistent CAR T-cells and antigen escape (n=3); or with loss of CAR T-cells and preserved antigen expression (n=3). Median duration of remission was 77 days (range, 54-292 days) (figure 1F). Relapse included two patients with CNS disease; one with combined EMD and medullary relapse and three with medullary relapse.

CART2 expansion
Peripheral blood absolute CAR T-cell expansion was substantially higher with CART1 (median, 24.05 cells/ mL; range, 0.35-13 653.0 c/mL) than with CART2 (median, 1.69 c/mL; range, 0-2886.0 c/mL) (p=0.03). Specifically, 5 (27.8%) of 18 failed to demonstrate any CART2 expansion which correlated with non-response (figure 2A). CD22 CART expansion was generally higher with both CART1 and CART2, as previously reported (online supplemental file). 15 CAR T-cell trafficking to the CSF was seen in 5 (35.7%) of 14 patients where lumbar punctures were performed and largely correlated with those who also had more robust PB expansion (online supplemental file).

Antigen density and modulation
Paired analysis of antigen binding capacity (ABC) pre-CART1 and pre-CART2 revealed a trend toward decreased antigen expression pre-CART2 in a subset of patients (n=10) with evaluable serial data (figure 2B,C). In two (11.1%) with increased antigen expression pre-CART2, higher ABC did not correlate with CART2 response. Emergence of antigen negative/dim B-ALL in six (33.3%) patients following CART2 also contributed to suboptimal responses (figure 2D-F). Antigen modulation was evident post-CART1/pre-CART2 with partial expression (figure 2D) and diminution (figure 2E), highlighting the importance of serial monitoring of antigen expression.

DISCUSSION
CAR T-cells induce remissions in children and young adults with r/r B-ALL, 1-6 but relapse after and suboptimal response to first infusion (CART1) remain significant challenges. Optimization strategies for CART2 are needed, particularly for those with antigen positive relapse or poor CART1 response. Prospective study of reinfusion has been limited, and early data from retrospective reviews suggests that CART2 responses with CD19 targeting are suboptimal. 9 10 Our data describe outcomes of reinfusion across a host of CAR T-cell targets and constructs, providing biological insights into limitations of CART2 response and a strategic framework for considering CART2 optimization strategies.
Responses to CART2 were limited in our heavily pretreated B-ALL cohort. Objective response rate with CART2 was 38.9% in contrast to 72.2% with CART1. Poor CART2 response correlated with a significant lack of CART2 expansion, though intensified LD to enhance expansion represents a potential strategy to overcome rejection mechanisms. Decreased antigen expression pre-CART2 and antigen escape following CART2 was also observed. Our results suggest that diminished CAR T-cell expansion alongside antigen downregulation and loss impeded robust responses to CART2. Further exploration of the mechanisms underlying CART2 response is needed.

Open access
We also investigated indication for reinfusion as a potential prognostic factor affecting CART2 outcomes. In our small cohort, four of seven (57.1%) patients with suboptimal CART1 response had objective response to CART2. Of 11 who underwent CART2 for antigen positive relapse, only 3 (27.3%) had objective response (online supplemental table 1). Our numbers were small but suggest that even those without an initial response to CART therapy have potential to derive benefit from reinfusion. Furthermore, we demonstrate that CART2 has the potential to traffic to and eradicate CNS disease. Importantly, CART2 was strategically offered only to those who had evidence of CART1 expansion and for whom some aspect of CART2 infusion could be modified. This included: (1) change in CART dose; (2) change in LD or (3) change in pre-CART2 disease burden to collectively augment CART expansion and potential for response. We postulate that these factors collectively may explain the differences between our results and those of Myers et al, who found reinfusion ineffective in patients with CART1 non-response. 16 Consideration of individual patient parameters and Open access CART1 response will be critical to identifying who may benefit from CART2. Still, likelihood of response with CART2 remains low, and high relapse risk following CART2 limited long-term cure (figure 1F). 2 7 8 Therefore, in eligible patients achieving a CR with CART2, consolidation measures such as HSCT should be considered to extend durable remission.
Interestingly, nearly half (n=8, 44.4%) of patients in this analysis received prior CART therapy elsewhere before treatment on our studies. Of six with a CART2 CR, three (50%) had received an alternate CART construct before CART1 infusion. While we only report on CART1 and CART2 outcomes with our constructs here, we expect to see an increasing frequency of patients receiving multiple CART products as post-CART relapse occurs. Our findings suggest that receipt of an alternate CART construct does not preclude potential benefit from reinfusion, though the impact of multiple prior CART therapies on subsequent responses warrants investigation.
Limitations of our study included the heterogeneity of CART constructs analyzed and retrospective nature of our analysis. However, prospective reinfusion studies can be challenging to enroll on, as seen with the recently completed tisagenlecleucel reinfusion trial (NCT04225676). Furthermore, given challenges in testing and assessing the impact of immunogenicity on CART outcomes, 17 18 our study does not include these analyses. CART constructs incorporating fully humanized single chain variable fragments (scFvs) have been hypothesized to be less immunogenic than those with murinederived components. 16 19 This may potentially explain the more robust CART2 expansion seen with our fully humanized CD22.BBζ construct (figure 2A) compared with the more immunogenic murine-based CD19 CART constructs (online supplemental file). Further study is needed to clarify how reinfusion strategies may differ between murine versus humanized CART constructs. Additionally, while our studies required active disease at CART2, given improved CART responses in low-disease burden, efforts to use reinfusion for relapse prevention are underway. 10 Such strategies have since been incorporated into our protocols.
In conclusion, our study provides important insights into CART2 outcomes, including with antigen targets beyond CD19. Future strategies to augment response to CART reinfusion may include strategic planning for modifications to CART2 such as routine use of intensified LD for patients with prior CART exposure. Consideration of the most appropriate antigen target for additional CART infusions will also be critical, particularly since antigen downregulation/loss impeded CART2 response. Given the limitations of CART reinfusion in an era with emerging novel CART constructs, our future efforts will further explore optimal timing of reinfusion strategies and when treatment with an alternate CART construct should be prioritized, especially in those receiving sequential CART infusions.
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Data availability statement Data are available on reasonable request.
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