Background Engineered T cell therapies have revolutionized treatment of relapsed refractory haematological malignancies, however the cost of treatment for autologous products remains a significant challenge to their widespread use. The high cost is driven largely by the need for personalized manufacturing of autologous cell products. A non-conventional class of T cells, the gamma/delta T cell, can be safely transplanted into an unrelated recipient without inducing graft-versus host disease,1 making them an ideal candidate for mass-manufactured off-the-shelf T cell therapies. We have previously described a novel method of directing conventional alpha/beta T cells towards tumour targets by co-opting the T cell receptor using the T cell Antigen Coupler (TAC) receptor.2 Here, we describe the use of TAC receptors to engineer antigen-specific reactivity into gamma/delta T cells, resulting in highly potent anti-tumor cytotoxicity.
Methods Engineered gamma/delta T cells were manufactured by activating PBMCs with Zoledronate and IL-2. The TAC transgene was introduced into T cells using either VSV-G pseudotype lentivirus or GALV-psuedotyped gamma-retrovirus vectors.Through optimization studies, we determined transduction was highest 24 hours post-activation for lentivirus and 72 hours post-activation for gamma-retrovirus. Cultures were fed with IL-2 supplemented media every 2 – 3 days and enriched on Day 14 to >99% gamma/delta T cell purity using CD4/CD8 magnetic-activated cell sorting depletion (Miltenyi Biotec).
Results Both methods of gene transfer tested for our pilot study yielded excellent gene transduction (40% - 70%). Using lentivirus-engineered gamma/delta T cells, we demonstrated that the TAC receptor re-directs gamma/delta T cells to attack tumors in an antigen-specific manner. The presence of the TAC receptor did not interfere with lysis of tumor cells via the natural tumor-reactive gamma/delta T cell receptors. Importantly, TAC-engineered gamma/delta T cells displayed robust cytotoxicity at very low effector:target ratios (<1) and caused regression of human tumor xenografts that were otherwise resistant to non-engineered gamma/delta T cells. Curiously, gamma/delta T cell manufacturing was sensitive to the quality of the lentivirus product, where products with low titers were associated with outgrowth of conventional alpha/beta T cells. Outgrowth of alpha/beta T cells was not observed with gamma-retroviruses. We are presently evaluating the anti-tumor activity of gamma-retrovirus-engineered gamma/delta T cells.
Conclusions Off-the-shelf engineered gamma/delta T cells represent a strategy to reduce manufacturing cost and may represent the next generation of engineered T cell therapies.TAC receptors provide a robust tool for directing gamma/delta T cells to attack tumors that are otherwise resistant to gamma/delta T cells and should be evaluated further.
Acknowledgements This work was supported by the Samuel Family Foundation, the Ontario Centres of Excellence and Triumvira Immunologics.
Ethics Approval The study was approved by McMaster’s Animal Research Ethics Board, AUP#19-02-10.
Arruda LCM, Gaballa A, Uhlin M. Impact of γδ T cells on clinical outcome of hematopoietic stem cell transplantation: systematic review and meta-analysis. Blood Adv 2019;3(21):3436–3448.
Helsen CW, Hammill JA, Lau VWC, et al. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity. Nat Commun 2018;9(1):3049.
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