Different T-Cell Activation Approaches Impact the Resulting CART-Cell Products and Possible Clinical Outcomes
Authors: Deng Biping , Liu Zhaoli , Qu Xiaoming , Zhang Yu'e , Zhang Zhongxin , Luan Rong , Zhang Lingli , Ma Ming , Wu Xiai , Liu Rui , Chang Alex H. , Hu Kai , Tong Chunrong 
Published: December 2024

Introduction ：T-cell activation is critical to the success of the cell manufacturing process as it directly impacts the efficiency of CAR transgene integration and T-cell expansion. At present, manufacturing protocols typically utilize magnetic beads or anti-CD3 and anti-CD28 antibodies to activate T cells. Whether these two activation approaches impact the ex vivo CART-cell products or in vivo cell characteristics and the subsequent clinical outcomes need to be studied.
Patients and Methods:
T cells from 6 relapsed or refractory diffuse large B cell lymphoma patients (r/r DLBCL pts) were activated with Ab and beads to analyze the impact of CART-cell products. Each isolated CD3+T cells were divided into 3 equal parts and activated with Ab (Group1), TLHY beads (Group2), and Gibco beads (Group3). Then, T cells were transduced with lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR cultured for another 4-5 days in media containing cytokines. Cell viability and cell expansion, CD3+T% and transduction efficiency, CD4-to-CD8 CART ratio, Treg cells were all examined in the CART products. PD1 and CD57 expression, TsCM, TCM, TEM proportion were all examined on both CD4+ and CD8+ CART.
T cells from 16 r/r DLBCL pts were respectively activated with Ab (9 pts) or Gibco beads (7 pts) to evaluate the impact of in vivo cell characteristics and subsequent clinical responses. Similar baseline characteristics of pts in the two groups. These pts received cyclophosphamide 250 mg/m2/d X 3d and fludarabine 30 mg/m2/d X 3d, followed by CART infusion (similar dosage). In vivo cell dynamics including peak CART proportion, absolute count and occurrence time, CD4-to-CD8 ratio, cell expansion and persistence were evaluated. The clinical efficacies were further observed.
Cell viability and count was determined using trypan blue exclusion and counting instrument, and cell phenotypes were determined using multicolor FCM assays. Infused pts underwent PET-CT on day 30 and 90 to determine the clinical efficacies.
Results
To 6 pts, results showed significantly increased CAR-transduction efficiency in Group1（34.7±12.6）compared to Group 2 (21.8±12.8, p=0.007) and Group 3 (20.1±12.3, p=0.003), but cell-expanding fold significantly decreased in Group1（12.7±14.2-fold）compared to Group2（22.6±9.8-fold, p=0.01）and Group 3 (24.5±7.1-fold, p=0.043). In addition, CD4-to-CD8 ratio of CART decreased in Group1（1.8±2.0）compared to Group 2 (3.0±3.0, p=0.05) and Group 3 (2.9±2.8, p=0.04) . 3/6 pts had CD4-to-CD8 ratio of CART less than 1:1 in Group 1, but 6/6 pts had more than 1:1 in Group 2 and 3. Similar results were observed between Group 2 and 3 including transduction efficiency, cell-expanding fold and CD4-to-CD8 ratio. There were no significant differences including cell viability, CD3+T %, PD1 and CD57 expression, TsCM、TCM、TEM proportion on CD4+ and CD8+CART cells among three groups. No Treg cells were detected in these culture systems.
After CART19 infusion in 16 pts, except for peak of CART/ lymphocytes had statistical differences between Ab and beads group [18.4%（3.12-34.3%）vs. 26.5%（23.9-63.5%）,p=0.05]. Other cell characteristics including peak CART absolute count and occurrence time, CD4-to-CD8 ratio, cell expansion, and start time of detectable CART had no differences. Interested, in Ab group, 5/9 pts were continuously detected CART more than 3 months and 7/9 pts obtained complete remission (CR) in 3 months. But in beads group, 0/4 pts had detectable CART19 more than 3 months and 2/4 pts obtained CR in 3 months. Due to 3/7 pts didn't reach evaluation time for CART persistence duration and clinical efficacies in beads group, these two indicators were not been statistically analyzed.
Conclusion:
Ab group produced higher CAR-transduction efficiency and significant CD8-biased CART cells although lower cell expansion ex vivo, but beads group produced higher peak of CART/lymphocytes in vivo after CART infusion. Although CART persistence and clinical efficacies for 3/7 pts in beads group didn't reach the evaluation time, we supposed Ab group having more excellent results for r/r DLBCL pts because 5/9 pts had detectable CART cells more than 3 months and 7/9 pts obtained CR in 3 months which were remarkably higher than our overall data. The follow-up results are expected to be updated. Overall, different T-Cell activation approaches impact the resulting CART-Cell products and possible clinical outcomes.