Background: Chimeric antigen receptor T-cells (CART) induce remarkable responses in B-cell acute lymphoblastic leukemia (B-ALL), but relapse remains a challenge. Effective therapies for post-CART relapse are limited but may include infusion of a unique CART construct, a strategy distinct from reinfusion of the same CART product. Given limited data evaluating outcomes of a unique CART as salvage therapy for post-CART relapse, we report on the impact of prior CART constructs on subsequent CART responses. Methods: This was a retrospective review (NCT03827343) of children and young adults receiving CD19 and/or CD22 CART therapy for B-ALL between 7/1/12-12/30/21 at our center. Patients included received at least 2 unique CART constructs at some point in therapy, excluding interim CART reinfusions. CART-A was the first CART construct ever received and CART-B the second unique CART. The primary objective was to evaluate complete remission (CR) rates following CART-A versus CART-B. Results: Of 135 heavily pretreated patients, 54 (40%) received at least one prior CART. The majority (n=37, 68.5%) were male. Median age at CART-A and CART-B was 12.5 (range, 3.3-30.4) and 13.7 years (range, 4.5-30.7), respectively. In 42 (77.8%) patients, CART-B targeted a different antigen than CART-A, primarily due to loss of antigen target after CART-A. CR rate was substantially lower with CART-B (n=35, 64.8%) than with CART-A (n=48, 88.9%, p=0.006) (Table). Still, two (3.7%) patients with CART-A nonresponse attained CR with CART-B. Most CART-B responders (n=31, 88.6%) had CART-B targeting a different antigen than CART-A, suggesting limitations of same antigen targeting even with a unique CART construct. CART-B responses amongst 10 patients with interim hematopoietic stem cell transplantation (HSCT) after CART-A were similar to CART-B responses in those without interim HSCT (5 of 10, 50% vs. 30 of 44, 68.2%, p=0.3). In those where CRS grade was known for both CART infusions (n=40), CRS severity was milder with CART-B (≥grade 3, n=1, 2.5%) than with CART-A (≥grade 3, n=15, 37.5%, p=0.0001). Conclusions: Using an alternative CART for post-CART relapse is effective in a substantial proportion of patients, particularly when targeting a unique antigen. As post-CART relapse occurs more frequently and patients receive multiple CARTs, identifying optimization strategies for CART-B will be critical. Further investigation of indication for CART-B, role of interim HSCT, and optimal timing for sequential CART infusions is underway.