Intensification of Lymphodepletion Optimizes CAR Re-Treatment Efficacy

Background: Post chimeric antigen receptor (CAR) therapy relapse has generally occurred via two mechanisms; emergence of antigen-negative leukemia, or via loss of CAR T cells. Anecdotally, CAR re-treatment has had limited success, potentially due to immune-mediated rejection of CAR T cells, or from immunogenicity of epitopes on murine constructs-which may be less applicable to humanized constructs. After several re-infusion attempts on our phase I dose-escalation study of CD22 CAR, in an effort to improve response, we incorporated use of an intensified lymphodepletion regimen prior to CAR re-infusion and report on our findings. Furthermore, preclinical models suggest that modulation of antigen expression, despite persistent positivity, may also impact the CAR response. Therefore, in this re-treatment analysis, we also explore the role of antigen expression on treatment response.

Design: Children and young adults with relapsed/refractory CD22+ acute lymphoblastic leukemia (ALL) enrolled on our phase I dose escalation anti-CD22 CAR therapy protocol (NCT02315612) who met eligibility criteria for a second infusion were evaluated. All patients had bone marrow evaluations by morphology and flow cytometry prior to second infusion to demonstrate persistent CD22+ ALL and loss of CD22 CAR T cells, and subsequently had restaging at day 28 (+/- 4 days) post second CAR infusion. Additionally, lymphodepleting chemotherapy, either with the original dosing schema (Fludarabine 25 mg/m² x 3 days and Cyclophosphamide 900 mg/m² x 1 day) or at intensified dosing (Fludarabine 30 mg/m² x 4 days and Cyclophosphamide 600 mg/m² x 2 days) was utilized at the discretion of the treating team. All patients were re-infused with their original cell dose; 3 x 10⁵ CAR T cells/kg (n=1), 1 X 10⁶ CAR T cells/kg (n=4), or 3 X 10⁶ CAR T cells/kg (n=1). Flow cytometry was utilized to assess CD22 antigen-binding capacity (ABC) of leukemia cells pre and post infusion. Patients were analyzed for response and expansion of CAR T cells.

Results: From June 2015 to July 2017, 6 patients with ALL were eligible for a second infusion of CD22 CAR T cells. This included 4 patients who relapsed with CD22+ disease after attaining a complete remission and 2 patients who had stable disease with limited CAR expansion and evidence of clinical benefit following the first infusion. The median time to relapse after a first infusion was 5 months (range: 3-11 months) with a median time to re-infusion of 4 months (range: 2-11 months) from the first infusion. Prior to re-infusion, all patients had <5% circulating CAR T cells, and all had significant disease burden, > M2 marrow (>5% blasts). Our initial re-infusion experience with 4 subjects, which utilized the identical lymphodepletion regimen as with the first infusion, demonstrated CAR expansion in only 2 patients. Following this, the pre-infusion lymphodepleting chemotherapy was intensified, with 2/2 patients having CAR expansion-including one patient who had a minimal expansion with the initial infusion, but robust expansion following the intensified lymphodepletion prior to second infusion. Both patients tolerated the intensified regimen well without any significant chemotherapy related toxicities. Following re-infusion, 4/6 patients had complete clearance of CD22+ leukemia. Notably, in this cohort, 4/6 patients were found to have a drop in their CD22 ABC, between the time from first to second infusion, with a median drop in site density of 3,141 sites/cell (range: 703-6460) with 2 of the re-infusion non-responders having approximately 50% decrease in the ABC from the CAR pre-treatment value.

Conclusion: Our preliminary experience with CAR re-infusions demonstrates that in the presence of preserved antigen expression, re-infusions can be effective, and that an intensified lymphodepletion regimen may optimize re-treatment response, both in patients with limited CAR expansion following a first infusion or with post-CAR relapse. Despite antigen positivity, decreased antigen density post-relapse may be one potential mechanism contributing to lack of CAR efficacy with second infusions, and higher level of antigen density may be needed to optimize anti-tumor response. Future directions will include incorporating an intensified lymphodepletion regimen for all second infusions and exploring methods to enhance antigen expression at the time of re-infusion.