Camelid-derived Tcell engagers harnessing human γδ T cells as promising antitumor immunotherapeutic agents.

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as T cell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic T cells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αß) T cells and unconventional gamma delta (γδ) T cells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with T cell receptor chains or a CD3 domain. We show that Vγ9Vδ2 T cells display stronger in vitro antitumor activity than αß T cells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral T cells to Vγ9Vδ2 T cells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ T cell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies.

Sepsis-trained macrophages promote antitumoral tissue-resident T cells.

Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.

Phase I study of zoledronic acid combined with escalated doses of interleukine-2 for early in vivo generation of Vγ9Vδ2 T-cells after haploidentical stem cell transplant with posttransplant cyclophosphamide.

The presence of donor Vγ9Vδ2 T-cells after haploidentical hematopoietic stem cell transplant (h-HSCT) has been associated with improved disease-free survival. These cells kill tumor cells in a non-MHC restricted manner, do not induce graft-versus-host disease (GVHD), and can be generated by stimulation with zoledronic acid (ZA) in combination with interleukin-2 (IL-2). This monocentric phase I, open-label, dose-escalating study (ClinicalTrials.gov: NCT03862833) aimed at evaluating the safety and possibility to generate Vγ9Vδ2 T-cells early after h-HSCT. It applied a standard 3 + 3 protocol to determine the maximum tolerated dose (MTD) of increasing low-doses of IL-2 (5 days [d] per week, 4 weeks) in combination with a single dose of ZA, starting both the first Monday after d + 15 posttransplant. Vγ9Vδ2 T-cell monitoring was performed by multiparameter flow cytometry on blood samples and compared with a control cohort of h-HSCT recipients. Twenty-six patients were included between April 2019 and September 2022, 16 of whom being ultimately treated and seven being controls who received h-HSCT only. At the three dose levels tested, 1, 0, and 1 dose-limiting toxicities were observed. MTD was not reached. A significantly higher number of Vγ9Vδ2 T-cells was observed during IL-2 treatment compared with controls. In conclusion, early in vivo generation of Vγ9Vδ2 T-cells is feasible after h-HSCT by using a combination of ZA and repeated IL-2 infusions. This study paves the way to a future phase 2 study, with the hope to document lesser posttransplant relapse with this particular adaptive immunotherapy.

The antitumor activity of human Vγ9Vδ2 T cells is impaired by TGF-ß through significant phenotype, transcriptomic and metabolic changes.

Despite significant advances, the eradication of cancer remains a clinical challenge which justifies the urgent exploration of additional therapeutic strategies such as immunotherapies. Human peripheral Vγ9Vδ2 T cells represent an attractive candidate subset for designing safe, feasible and effective adoptive T cell transfer-based therapies. However, following their infiltration within tumors, γδ T cells are exposed to various regulating constituents and signals from the tumor microenvironment (TME), which severely alter their antitumor functions. Here, we show that TGF-ß, whose elevated production in some solid tumors is linked to a poor prognosis, interferes with the antigenic activation of human Vγ9Vδ2 T cells in vitro. This regulatory cytokine strongly impairs their cytolytic activity, which is accompanied by the induction of particular phenotypic, transcriptomic and metabolic changes. Collectively, these observations provide information for better understanding and targeting the impact of TME components to regulate the antitumor activity of human T cell effectors.

Development of ICT01, a first-in-class, anti-BTN3A antibody for activating Vγ9Vδ2 T cell-mediated antitumor immune response.

Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti­butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen­major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)­binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αß T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.

BTN2A1, an immune checkpoint targeting Vγ9Vδ2 T cell cytotoxicity against malignant cells.

The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

Anti-Tumor Efficacy of PD-L1 Targeted Alpha-Particle Therapy in a Human Melanoma Xenograft Model.

PD-L1 (programmed death-ligand 1, B7-H1, CD274), the ligand for PD-1 inhibitory receptor, is expressed on various tumors, and its expression is correlated with a poor prognosis in melanoma. Anti-PD-L1 mAbs have been developed along with anti-CTLA-4 and anti-PD-1 antibodies for immune checkpoint inhibitor (ICI) therapy, and anti-PD-1 mAbs are now used as first line treatment in melanoma. However, many patients do not respond to ICI therapies, and therefore new treatment alternatives should be developed. Because of its expression on the tumor cells and on immunosuppressive cells within the tumor microenvironment, PD-L1 represents an interesting target for targeted alpha-particle therapy (TAT). We developed a TAT approach in a human melanoma xenograft model that stably expresses PD-L1 using a 213Bi-anti-human-PD-L1 mAb. Unlike treatment with unlabeled anti-human-PD-L1 mAb, TAT targeting PD-L1 significantly delayed melanoma tumor growth and improved animal survival. A slight decrease in platelets was observed, but no toxicity on red blood cells, bone marrow, liver or kidney was induced. Anti-tumor efficacy was associated with specific tumor targeting since no therapeutic effect was observed in animals bearing PD-L1 negative melanoma tumors. This study demonstrates that anti-PD-L1 antibodies may be used efficiently for TAT treatment in melanoma.

Beyond CAR T cells: Engineered Vγ9Vδ2 T cells to fight solid tumors.

Despite recent significant progress in cancer immunotherapies based on adoptive cell transfer(s)(ACT), the eradication of cancers still represents a major clinical challenge. In particular, the efficacy of current ACT-based therapies against solid tumors is dramatically reduced by physical barriers that prevent tumor infiltration of adoptively transferred effectors, and the tumor environment that suppress their anti-tumor functions. Novel immunotherapeutic strategies are thus needed to circumvent these issues. Human peripheral blood Vγ9Vδ2 T cells, a non-alloreactive innate-like T lymphocyte subset, recently proved to be a promising anti-tumor effector subset for ACT-based immunotherapies. Furthermore, new cell engineering tools that leverage the potential of CRISPR/Cas technology open astounding opportunities to optimize their anti-tumor effector functions. In this review, we present the current ACT strategies based on engineered T cells and their limitations. We then discuss the potential of engineered Vγ9Vδ2 T cell to overcome these limitations and improve ACT-based cancer immunotherapies.

Emerging Challenges of Preclinical Models of Anti-tumor Immunotherapeutic Strategies Utilizing Vγ9Vδ2 T Cells.

Despite recent advances, the eradication of cancers still represents a challenge which justifies the exploration of additional therapeutic strategies such as immunotherapies, including adoptive cell transfers. Human peripheral Vγ9Vδ2 T cells, which constitute a major transitional immunity lymphocyte subset, represent attractive candidates because of their broad and efficient anti-tumor functions, as well as their lack of alloreactivity and easy handling. Vγ9Vδ2 T cells act like immune cell stress sensors that can, in a tightly controlled manner but through yet incompletely understood mechanisms, detect subtle changes of levels of phosphorylated metabolites of isoprenoid synthesis pathways. Consequently, various anti-tumor immunotherapeutic strategies have been proposed to enhance their reactivity and cytotoxicity, as well as to reduce the deleterious events. In this review, we expose these advances based on different strategies and their validation in preclinical models. Importantly, we next discuss advantages and limits of each approach, by highlighting the importance of the use of relevant preclinical model for evaluation of safety and efficacy. Finally, we propose novel perspectives and strategies that should be explored using these models for therapeutic improvements.

γ9δ2T cell diversity and the receptor interface with tumor cells.

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.

Contribution of the SYK Tyrosine kinase expression to human iNKT self-reactivity.

Invariant Natural Killer T (iNKT) cells are particular T lymphocytes at the frontier between innate and adaptative immunities. They participate in the elimination of pathogens or tumor cells, but also in the development of allergic reactions and autoimmune diseases. From their first descriptions, the phenomenon of self-reactivity has been described. Indeed, they are able to recognize exogenous and endogenous lipids. However, the mechanisms underlying the self-reactivity are still largely unknown, particularly in humans. Using a CD1d tetramer-based sensitive immunomagnetic approach, we generated self-reactive iNKT cell lines from blood circulating iNKT cells of healthy donors. Analysis of their functional characteristics in vitro showed that these cells recognized endogenous lipids presented by CD1d molecules through their TCR that do not correspond to α-glycosylceramides. TCR sequencing and transcriptomic analysis of T cell clones revealed that a particular TCR signature and an expression of the SYK protein kinase were two mechanisms supporting human iNKT self-reactivity. The SYK expression, strong in the most self-reactive iNKT clones and variable in ex vivo isolated iNKT cells, seems to decrease the activation threshold of iNKT cells and increase their overall antigenic sensitivity. This study indicates that a modulation of the TCR intracellular signal contributes to iNKT self-reactivity.

Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes.

BACKGROUND: Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, PDCD1 editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments. METHODS: Here we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to edit PDCD1 gene in human effector memory CD8+ T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validated PDCD1 editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain's sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR. RESULTS: Here we demonstrated the feasibility to edit PDCD1 gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent on PDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model. CONCLUSION: The use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

Identification of a transient state during the acquisition of temozolomide resistance in glioblastoma.

Drug resistance limits the therapeutic efficacy in cancers and leads to tumor recurrence through ill-defined mechanisms. Glioblastoma (GBM) are the deadliest brain tumors in adults. GBM, at diagnosis or after treatment, are resistant to temozolomide (TMZ), the standard chemotherapy. To better understand the acquisition of this resistance, we performed a longitudinal study, using a combination of mathematical models, RNA sequencing, single cell analyses, functional and drug assays in a human glioma cell line (U251). After an initial response characterized by cell death induction, cells entered a transient state defined by slow growth, a distinct morphology and a shift of metabolism. Specific genes expression associated to this population revealed chromatin remodeling. Indeed, the histone deacetylase inhibitor trichostatin (TSA), specifically eliminated this population and thus prevented the appearance of fast growing TMZ-resistant cells. In conclusion, we have identified in glioblastoma a population with tolerant-like features, which could constitute a therapeutic target.

Combined chemotherapy and allogeneic human Vγ9Vδ2 T lymphocyte-immunotherapies efficiently control the development of human epithelial ovarian cancer cells in vivo.

Epithelial ovarian cancer (EOC) represents 5% of human gynecologic cancers in the world, is heterogeneous and highly invasive with a dismal prognosis (5 year-survival rate <35%). Diagnosis of EOC is frequently made at advanced stages and, despite aggressive treatments combining surgery and chemotherapy, fatal relapse rapidly occurs and is accompanied by a peritoneal carcinosis. In this context, novel therapeutical advances are urgently required. Adoptive transfer(s) of immune effector cells, including allogeneic human Vγ9Vδ2 T lymphocytes, represent attractive targets for efficiently and safely tracking tissue-invading tumor cells and controlling tumor dissemination in the organism. Our study describes the establishment of robust and physiological orthotopic model of human EOC in mouse, that includes surgical resection (ovariectomy) and chemotherapy, which are ineluctably accompanied by a fatal peritoneal carcinosis recurrence. Through a complementary set of in vitro and in vivo experiments, we provide here a preclinical proof of interest of the antitumor efficiency of adoptive transfers of allogeneic human Vγ9Vδ2 T lymphocytes against EOC, in association with surgical debulking and standard chemotherapies (i.e., taxanes and platinum salts). Moreover, our results indicate that chemo- and immunotherapies can be combined to improve the antitumor efficiency of immunotherapeutic lines. Altogether, these results further pave the way for next-generation antitumor immunotherapies, based on local administrations of human allogeneic human Vγ9Vδ2 T lymphocytes, in association with standard treatments.

NKG2D Controls Natural Reactivity of Vγ9Vδ2 T Lymphocytes against Mesenchymal Glioblastoma Cells.

PURPOSE: Cellular immunotherapies are currently being explored to eliminate highly invasive and chemoradioresistant glioblastoma (GBM) cells involved in rapid relapse. We recently showed that concomitant stereotactic injections of nonalloreactive allogeneic Vγ9Vδ2 T lymphocytes eradicate zoledronate-primed human GBM cells. In the present study, we investigated the spontaneous reactivity of allogeneic human Vγ9Vδ2 T lymphocytes toward primary human GBM cells, in vitro and in vivo, in the absence of any prior sensitization. EXPERIMENTAL DESIGN: Through functional and transcriptomic analyses, we extensively characterized the immunoreactivity of human Vγ9Vδ2 T lymphocytes against various primary GBM cultures directly derived from patient tumors. RESULTS: We evidenced that GBM cells displaying a mesenchymal signature are spontaneously eliminated by allogeneic human Vγ9Vδ2 T lymphocytes, a reactivity process being mediated by γδ T-cell receptor (TCR) and tightly regulated by cellular stress-associated NKG2D pathway. This led to the identification of highly reactive Vγ9Vδ2 T lymphocyte populations, independently of a specific TCR repertoire signature. Moreover, we finally provide evidence of immunotherapeutic efficacy in vivo, in the absence of any prior tumor cell sensitization. CONCLUSIONS: By identifying pathways implicated in the selective natural recognition of mesenchymal GBM cell subtypes, accounting for 30% of primary diagnosed and 60% of recurrent GBM, our results pave the way for novel targeted cellular immunotherapies.

An X-ray Vision for Phosphoantigen Recognition.

Invariant Vγ9Vδ2 T cells respond to "phosphoantigen" metabolites through binding to the B30.2 domain of butyrophilin BTN3A. Yang et al. (2019) use molecular dynamic simulations based on X-ray structures of distinct B30.2 domain dimers to identify the asymmetric dimer as most active, which has implications for the inside-out signaling mechanism.

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human Vϒ9Vδ2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Towards Deciphering the Hidden Mechanisms That Contribute to the Antigenic Activation Process of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T cells represent a major unconventional γδ T cell subset located in the peripheral blood of adults in humans and several non-human primates. Lymphocytes that constitute this transitional subset can sense subtle level changes of intracellular phosphorylated intermediates of the isoprenoid biosynthesis pathway (phosphoantigens, pAg), such as isopentenyl pyrophosphate, during cell stress events. This unique antigenic activation process operates in a rigorous framework that requires the expression of butyrophilin 3A1 (BTN3A1/CD277) molecules, which are type I glycoproteins that belong to the B7 family. Several studies have further shown that pAg specifically bind to the intracellular B30.2 domain of BTN3A1 linked to the antigenic activation of Vγ9Vδ2 T cells. Here, we highlight the recent advances in BTN3A1 dynamics induced upon the binding of pAg and the contribution of the different subunits to this activation process. Recent reports support that conformational modifications of BTN3A1 might represent a key step in the detection of infection or tumorigenesis by Vγ9Vδ2 T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that target defined functions of this unique γδ T cell subset.

IL-21 Increases the Reactivity of Allogeneic Human Vγ9Vδ2 T Cells Against Primary Glioblastoma Tumors.

Glioblastoma multiforme (GBM) remains the most frequent and deadliest primary brain tumor in adults despite aggressive treatments, because of the persistence of infiltrative and resistant tumor cells. Nonalloreactive human Vγ9Vδ2 T lymphocytes, the major peripheral γδ T-cell subset in adults, represent attractive effectors for designing immunotherapeutic strategies to track and eliminate brain tumor cells, with limited side effects. We analyzed the effects of ex vivo sensitizations of Vγ9Vδ2 T cells by IL-21, a modulating cytokine, on their cytolytic reactivity. We first showed that primary human GBM-1 cells were naturally eliminated by allogeneic Vγ9Vδ2 T lymphocytes, through a perforin/granzyme-mediated cytotoxicity. IL-21 increased both intracellular granzyme B levels and cytotoxicity of allogeneic human Vγ9Vδ2 T lymphocytes in vitro. Importantly, IL-21-enhanced cytotoxicity was rapid, which supports the development of sensitization(s) of γδ T lymphocytes before adoptive transfer, a process that avoids any deleterious effect associated with direct administrations of IL-21. Finally, we showed, for the first time, that IL-21-sensitized allogeneic Vγ9Vδ2 T cells significantly eliminated GBM tumor cells that developed in the brain after orthotopic administrations in vivo. Altogether our observations pave the way for novel efficient stereotaxic immunotherapies in GBM patients by using IL-21-sensitized allogeneic human Vγ9Vδ2 T cells.