CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies

Abstract

The development of genetic modification techniques has led to a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. intensive efforts are being performed to develop cancer-targeted therapies to avoid the elimination of non-cancerous cells. One of the most promising approaches is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapy’s time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the sub-population of Tcm and Tscm in CAR-T cells, we investigated the production of a long-term stable and efficient cytotoxic CAR-T cell by modifications in the cell activation-dependent production using Phytohemagglutinin (PHA). PHA, a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19-specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared with aCD3/aCD28-amplified CAR-T cells. Two different types of CARs (aCD19 scFv CD8-(CD28 or 4-1BB)-CD3z-EGFRt)-expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm–Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells.

Phytohemagglutinin-Activated CAR-T Cells: Prolonged Persistence and Enhanced Anti-Tumor Response in CD19-Specific Acute Lymphoblastic Leukemia

“…The presence of a persistent antigen increases the proportion of TEM cell populations, as shown in studies on chronic infections; this also holds true for autoimmune diseases where self-antigens are continuously present (Devarajan, P. & Chen, 2013). The results of our in vitro and in vivo research suggest that TemEARLY cells generated after activation with PHA can persist in the bloodstream of patients as memory cells and provide a significant defense mechanism against recurring cancer (Gulden et al, 2023).…”

“…Therefore, it appears that CAR-T cells activated with PHA can be expanded more rapidly and easily before being administered to patients in clinical trials. In both types of T cells, the TemEARLY cell population is significantly higher with PHA activation (Gulden et al, 2023). TemEARLY cells have a range of impressive functions and dominate in target tissues.…”

Immunological Difference between Cord and Peripheral Blood: Review Article

“…Newborn CD4+CD45RA+ naïve T cells convert more quickly than their adult counterparts into CD4+CD45RO+ memory T cells in response to phytohemagglutinin and interleukin-2 (IL-2). This suggests that UCB naïve T cells have a higher potential for memory T cell transformation (7) . However, UCB cells produce significantly less IFNγ and TNF⍺ as well as IL interleukin-2 than adult PB lymphocytes, probably as a result of decreased expression of NFAT-dependent genes.…”

Nanosensor Chemical Cytometry: Advances and Opportunities in Cellular Therapy and Precision Medicine

“…Depending on the average size of the target cells, the height and width of the microchannel should be precisely controlled, allowing the user to induce the strongest and reliable cellular lensing in the channel. In addition to instrumental aspects, the use of adequate pretreatment reagents such as phytohemagglutinin (PHA), concanavlin A (Con A) or PMA, which are cytokine signaling proteins should be prepared to observe the therapeutic behavior of target immune cells since they enhance cellular molecular efflux analysis enabling more accurate biochemical sensing in NCC. − Surface functionalization of the nanosensor array is also another pretreatment protocol required for reliable NCC. Target cells can be in contact with the nanosensor array during analysis within channels, necessitating the use of nontoxic nanosensor materials.…”