Poster Presentation MIN Spring Retreat 2023

CD28 stimulation enhances CAR T cell differentiation, particularly in older patients, but CD28 abundance is predictive of CAR T cell product quality. (#108)

Palak H Mehta 1 , Shivali Savita Chinni 1 , Patrick Leung 1 , Aaron Harrison 2 , Hannah Hughes-Parry 3 4 , Misty R Jenkins 3 4 5 , Clare Y Slaney 2 4 , Michael H Kershaw 2 4 , Anthony Jaworowski 1 , Rachel M Koldej 4 6 , David S Ritchie 4 6 , Kylie M Quinn 1 7
  1. School of Health and Biomedical Sciences , Royal Melbourne University Of Technology, Melbourne, VIC, Australia
  2. Immune Innovation Laboratory , Peter MacCallum Cancer Foundation, Melbourne, VIC, Australia
  3. Immunology Division, Walter and Eliza Hall Institute , Melbourne, VIC, Australia
  4. Department of Medicine, University of Melbourne , Melbourne, VIC, Australia
  5. Peter MacCallum Cancer Foundation, Melbourne, VIC, Australia
  6. Australian Cancer Research Foundation Translational Laboratory , Royal Melbourne Hospital , Melbourne, VIC, Australia
  7. Department of Biochemistry, Biomedicine Discovery Institute, Monash University , Melbourne, VIC, Australia

Aim: Chimeric antigen receptor (CAR) T cell therapy (CTT) is an autologous T cell-based treatment for B cell-based malignancies. During CTT, the patient’s T cells must first be activated in vitro with either anti-CD3 or anti-CD3/CD28 monoclonal antibodies (mAb). Dysfunctional T cells, such as terminally differentiated CD28- T cells, could hinder the efficacy of CTT and are found at higher frequencies in older patients. This study aims to define the optimal activation protocol for CAR T cell generation with older donors and to test whether CD28 expression on patient PBMCs can predict CAR T cell product quality.

Methods: Samples from young (20-30yo) and aged (60+) healthy donors were used to generate CAR T cells using both anti-CD3 and anti-CD3/CD28 mAb activation protocols.  CD28 expression was assessed pre-activation and CAR T cell quality was tracked, in terms of yield, phenotype and function. Correlative analyses were performed to determine whether CD28 expression could predict CAR T cell product quality with either anti-CD3 or anti-CD3/CD28 mAb activation.

Results: Aged PBMC samples generated more differentiated CAR T cell products than young samples, and anti-CD3/CD28 mAb activation further exacerbated differentiation as compared to anti-CD3 mAb activation in both young and aged donors. Anti-CD3/CD28 mAb activation also generated a higher frequency of IFNg- and TNF-producing CAR T cells, increased killing capacity and augmented proliferation potential for both donor groups. Finally, we defined a novel metric of CD28 abundance with high predictive value for CAR T cell product quality.

Conclusions: In summary, anti-CD3/CD28 mAb activation protocols exacerbate T cell differentiation and may not be optimal for CAR T cell generation with aged samples. Accordingly, CD28 abundance on patient PBMCs can predict CAR T cell product quality and may be used to identify patients who would benefit from anti-CD3/CD28 mAb activation protocols.