Search

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background 1-5 . γδ T cells can sense conserved cell stress signals prevalent in transformed cells 2,3 , although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells 4 -recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8 ), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Competing Interests: Competing interests: JK is shareholder of Gadeta (www.gadeta.nl). JK, ZS, DB, AV, and EvD are authors on the following patent applications: 2013: WO 2013 /147606 A1 Combinatorial gamma 9 delta 2 T cell receptor chain exchange, (C. Grunder, JK); 2017: 20170319674 Use of antibodies for enrichment of engineered t cells with exogenous immune receptors and antibodies for use in depletion of engineered t cells (JK) ; 2017: WO/2017/2017/212074 Novel method for identifying delta T cell (or gamma T cell) receptor chains or parts thereof that mediate antitumor or anti-infectious response (JK and colleagues; 2017: WO/2017/212072 Human leukocyte antigen restricted gamma delta T cell receptors (JK and colleagues), 2017/ 62508807 (ZS & JK) Composition and methods for cell targeting therapies; 2018/ 6044506 (EvD, DB, JK): GABs as next generation of immune therapy

γδT cell receptors (γδTCRs) recognize a broad range of malignantly transformed cells in mainly a major histocompatibility complex (MHC)-independent manner, making them valuable additions to the engineered immune effector cell therapy that currently focuses primarily on αβTCRs and chimeric antigen receptors (CARs). As an exception to the rule, we have previously identified a γδTCR, which exerts antitumor reactivity against HLA-A*24:02-expressing malignant cells, however without the need for defined HLA-restricted peptides, and without exhibiting any sign of off-target toxicity in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse models. This particular tumor-HLA-A*24:02-specific Vγ5Vδ1TCR required CD8αα co-receptor for its tumor reactive capacity when introduced into αβT cells engineered to express a defined γδTCR (TEG), referred to as TEG011; thus, it was only active in CD8 + TEG011. We subsequently explored the concept of additional redirection of CD4 + T cells through co-expression of the human CD8α gene into CD4 + and CD8 + TEG011 cells, later referred as TEG011_CD8α. Adoptive transfer of TEG011_CD8α cells in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mice injected with tumor HLA-A*24:02 + cells showed superior tumor control in comparison to TEG011, and to mock control groups. The total percentage of mice with persisting TEG011_CD8α cells, as well as the total number of TEG011_CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4 + CD8 + double-positive TEG011_CD8α, which resulted in higher total counts of functional T cells in spleen and bone marrow. We observed that tumor clearance in the bone marrow of TEG011_CD8α-treated mice associated with better human T cell infiltration, which was not observed in the TEG011-treated group. Overall, introduction of transgenic human CD8α receptor on TEG011 improves antitumor reactivity against HLA-A*24:02 + tumor cells and further enhances in vivo tumor control., Competing Interests: DB, ZS, and JK are inventors on different patents with γδTCR sequences, recognition mechanisms, and isolation strategies. JK is cofounder and shareholder of Gadeta (www.gadeta.nl). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Johanna, Hernández-López, Heijhuurs, Scheper, Bongiovanni, de Bruin, Beringer, Oostvogels, Straetemans, Sebestyen and Kuball.)

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α-dependent tumor-specific allo-HLA-A*24:02-restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αβT cells from HLA-A*24:02 harboring individuals. αβT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA-A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse model to allow the preparation of a first-in-men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft-versus-host disease-like symptoms and extensive analysis of pathologic changes in NSG-A24:02 mice did not show any off-target toxicity of TEG011. However, loss of persistence of TEG011 in tumor-bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock-treated mice. In conclusion, TEG011 is well tolerated without harming HLA-A*24:02 + expressing healthy tissues, and TEG011 persistence seems to be crucial for long-term tumor control in vivo., (©2020 Society for Leukocyte Biology.)

γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2 + T cells compared with other tumor types. By reconstructing matched Vδ2 - TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies., (©2020 American Association for Cancer Research.)

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αβTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αβTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Vγ9Vδ2 T cell activation plays an important role in antitumor and antimicrobial immune responses. However, there are conditions in which Vγ9Vδ2 T cell activation can be considered inappropriate for the host. Patients treated with aminobisphosphonates for hypercalcemia or metastatic bone disease often present with a debilitating acute phase response as a result of Vγ9Vδ2 T cell activation. To date, no agents are available that can clinically inhibit Vγ9Vδ2 T cell activation. In this study, we describe the identification of a single domain Ab fragment directed to the TCR of Vγ9Vδ2 T cells with neutralizing properties. This variable domain of an H chain-only Ab (VHH or nanobody) significantly inhibited both phosphoantigen-dependent and -independent activation of Vγ9Vδ2 T cells and, importantly, strongly reduced the production of inflammatory cytokines upon stimulation with aminobisphosphonate-treated cells. Additionally, in silico modeling suggests that the neutralizing VHH binds the same residues on the Vγ9Vδ2 TCR as the Vγ9Vδ2 T cell Ag-presenting transmembrane protein butyrophilin 3A1, providing information on critical residues involved in this interaction. The neutralizing Vγ9Vδ2 TCR VHH identified in this study might provide a novel approach to inhibit the unintentional Vγ9Vδ2 T cell activation as a consequence of aminobisphosphonate administration., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Human Vγ9Vδ2 T cells respond to tumor cells by sensing elevated levels of phosphorylated intermediates of the dysregulated mevalonate pathway, which is translated into activating signals by the ubiquitously expressed butyrophilin A1 (BTN3A1) through yet unknown mechanisms. Here, we developed an unbiased, genome-wide screening method that identified RhoB as a critical mediator of Vγ9Vδ2 TCR activation in tumor cells. Our results show that Vγ9Vδ2 TCR activation is modulated by the GTPase activity of RhoB and its redistribution to BTN3A1. This is associated with cytoskeletal changes that directly stabilize BTN3A1 in the membrane, and the subsequent dissociation of RhoB from BTN3A1. Furthermore, phosphoantigen accumulation induces a conformational change in BTN3A1, rendering its extracellular domains recognizable by Vγ9Vδ2 TCRs. These complementary events provide further evidence for inside-out signaling as an essential step in the recognition of tumor cells by a Vγ9Vδ2 TCR., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Major limitations of currently investigated αβT cells redirected against cancer by transfer of tumor-specific αβTCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous α or βTCR chains. Therefore, the ability of a defined γ9δ2TCR to redirect αβT cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a γ9δ2TCR efficiently reprograms both CD4(+) and CD8(+) αβT cells against a broad panel of cancer cells while ignoring normal cells, and substantially reduces but does not completely abrogate alloreactivity. γ9δ2TCR-transduced αβT cells reduced colony formation of progenitor cells of primary acute myeloid leukemia blasts and inhibited leukemia growth in a humanized mouse model. Thereby, metabolites of a dysregulated mevalonate pathway are targeted and the additional application of widely used biphosphonates is crucial for in vivo efficacy most likely because of its modulating effect on cytokine secretion of γ9δ2TCR-transduced αβT cells. Expression of NKG2D ligands and F1-ATPase contributed to the activity of γ9δ2TCR-transduced αβT cells but were not mandatory. In summary, γ9δ2 TCRs are an attractive alternative to broadly redirect αβT cells against cancer cells with both an improved efficacy and safety profile compared with currently used αβTCRs.