Universal Ready-to-Use Immunotherapeutic Approach for the Treatment of Cancer: Expanded and Activated Polyclonal γδ Memory T Cells

Abstract

In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.

Cancer immunotherapy with γδ T cells: many paths ahead of us

“…Since mice do not express γδ TCRs homologous to human Vδ2 (i.e., mouse γδ T cells are not activated by phosphoantigens), immunodeficient or humanized mice transplanted with human tumors and γδ T cells are frequently used as a preclinical in vivo model. 143 While both subsets can kill a broad range of malignant cells and show efficacy in xenograft models, 18 , 97 , 98 , 144 – 148 they display different patterns of NK receptor and accessory molecule expression, 82 and they also display related yet distinct cytotoxic hallmarks as revealed by recent single-cell RNAseq studies. 149 The expansion of Vδ1 T cells with a specific protocol involving a two-step process with selected cytokines including IL-15 in the second step has been defined as DOT (“delta one T cells”).…”

“… 97 Other protocols for expanding highly cytolytic γδ T cells mainly of the Vδ1 variety utilized mitogen phytohemagglutinin (PHA) plus IL-7 stimulation 150 or artificial antigen-presenting cells (APCs) expressing costimulatory molecules and CMV-pp65 antigens. 144 Furthermore, polyclonal γδ T cells expressing various TCR VγVδ elements and broad cytotoxic activity against various tumor cells have also been generated in the presence of CD137L-expressing artificial APC and IL-2 plus IL-21. 98 The most widely used protocol for selectively expanding Vδ2T cells relies on ZOL stimulation of PBMCs in the presence of IL-2, 100 but similarly efficient Vδ2T cell activation in vitro can be achieved with synthetic pAgs such as BrHPP 20 and HMBPP.…”

What Can Gamma Delta T Cells Contribute to an HIV Cure?

“…In both studies, expanded Vδ1 cells expressed a number of surface markers related to cytotoxic function, including NKp30, NKp44, NKp46, NKG2D, 2B4, DNAM-1, and CD94. Other approaches to expand Vδ1 cells include the use of artificial APCs (aAPCs) presenting CMVderived peptides (Polito et al, 2019). Vδ1 cells expanded by this approach recognize an array of tumor cells and virally infected cell lines, but the mechanism underlying this recognition was not assessed.…”

“…Advantages of γδT cell-based immunotherapy include: 1) Lack of MHC restriction: GMP-compliant protocols have been developed to generate large banks of expanded γδT cells, allowing for an "off-the-shelf " allogeneic product that is not reliant on MHC matching (Deniger et al, 2014;Almeida et al, 2016;Polito et al, 2019). The lack of MHC-restriction of γδT cells is a major advantage that should avoid issues of graft-vs.-host disease in γδT -based immunotherapy.…”

Epithelial Ovarian Cancer and the Immune System: Biology, Interactions, Challenges and Potential Advances for Immunotherapy

“…However, it has also been shown that specific subsets of γδ T-cells can directly promote cancer progression through secretion of immunosuppressive IL-17, while others can inhibit DC maturation or induce DC senescence [ 352 ]. A clinical grade protocol to isolate and expand large numbers of polyclonal γδ T-cells capable of gene-modification to enhance anti-tumour activity has been developed recently [ 356 ].…”