Where could gammadelta T cells take us in the treatment of cancer?

KEYWORDS:

• Adoptive cell therapy
• allogeneic cells
• bisphosphonates
• cancer immunotherapy
• CAR-T cells
• phosphoantigens
• γδ T cells

1. Introduction

In the last few years, a growing interest in the context of cancer immunotherapy has focused on a population of unconventional T lymphocytes, known as γδ T cells, which act as a bridge between innate and adaptive immune systems. This interest emerges thanks to their peculiar properties, which allow overcoming the limitations of their αβ T cells counterpart. Indeed, they recognize a wide range of antigens, such as lipids, phospho-antigens, and peptides, in an MHC-independent manner, according to different γδ T cell populations (Figure 1). Combining the power of a TCR with a variety of natural cytotoxicity receptors, and the capability of producing IFN-γ, granzyme and perforin, they can orchestrate adaptive immune responses even in an HLA-mismatched context [1]. These characteristics make γδ T cells sensitive to a wide range of tumor signals and alert to immune evasion mechanisms.

Figure 1. Features of γδ T cell subsets: antigen recognition and killing.

2. The new perspective on γδ T cells cancer immunotherapy

Initially, γδ T cell-cancer immunotherapy was mainly based on two approaches: in vivo activation and adoptive transfer of ex vivo expanded autologous γδ T cells. The in vivo activation requires drugs such as aminobisphosphonates (e.g. Zoledronate) which inhibit the farnesyl pyrophosphate synthase of the mevalonate pathway and up-regulate isoprenoid biosynthesis, thus leading to the accumulation of endogenous PAgs [2].

The ex vivo approach requires the isolation of γδ T cells from peripheral blood mononuclear cells, their activation induced directly by natural (e.g. IPP and HMBPP) or synthetic (e.g. BrHPP) phosphoantigens (PAgs) plus IL-2, and the adoptive transfer. Despite some initial enthusiasm, both of these approaches failed in their long-term efficacy for reasons related to the biological functions of these cells and their plasticity in the context of tumors, and the effect of treatment, such as the dose-limiting toxicities and the off-target proliferation of regulatory T cells [3].

Indeed, scientific interest has shifted to allogeneic adoptive transfer of γδ T cells and tumor-targeted activation using bispecific antibodies or γδ-CAR-T cells (Figure 2, Tables 1 and Table 2).

Figure 2. Novel strategies for γδ T cell-based immunotherapy.

Table 1. Ongoing γδ T cells-based clinical trials.

Clinical trial	Tumor target	Estimated enrollment	Setting	Clinical phase	Study reference
Ex-vivo expanded allogeneic γδ T cells transfer
Allogeneic γδ T cells immunotherapy in patients with relapsed or refractory non-Hodgkin’s lymphoma (NHL) and peripheral T cell lymphomas (PTCL)	Non-Hodgkin’s lymphoma; peripheral T cell lymphoma	10	2 cycles of 2 dosage escalated manner infusions at 14 days’ intervals	Early phase I	NCT04696705
γδ T cell against hematological malignancies after allo-HSCT	Acute myeloid leukemia; acute lymphoblastic leukemia; myelodisplastic syndrome; lymphoma	20	Dose escalation (5 dose increments) between 3 patient’s cohorts: negative MRD or SD positive MRD but not HR HR	Phase I/II	NCT04764513
γδ T cell immunotherapy for treatment of lung cancer	Lung cancer	30	Three experimental groups: Treated only with surgery Treated only with γδ T cells infusion Treated with both surgery and γδ T cells infusion	Phase I/II	NCT03183232
γδ T cell immunotherapy for treatment of liver cancer	Liver cancer	30	Three experimental groups: Treated only with surgery Treated only with γδ T cells infusion Treated with both surgery and γδ T cells infusion	Phase I/II	NCT03183219
γδ T cell immunotherapy for treatment of breast cancer	Breast cancer	100	Three experimental groups: Treated only with surgery Treated only with γδ T cells infusion Treated with both surgery and γδ T cells infusion	Phase I/II	NCT03183206
IRE plus γδ T cell against locally advanced pancreatic cancer	Pancreatic cancer	62	Two experimental groups: Treated only with IRE surgery Treated with both IRE surgery and γδ T cells infusion	Phase I/II	NCT03180437
Preliminary antileukemic activity of GDX012 in patients with MRD positive acute myeloid leukemia	Acute myeloid leukemia	3	GDX012 (allogeneic cell therapy enriched for Vδ1 + T cells) suspension for IV Infusion (single dose) following chemotherapy for lymphodepletion	Phase I	NCT05001451
Bispecific antibodies
LAVA-051 in patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia	Chronic lymphocytic leukemia; multiple myeloma; acute myeloid leukemia	102	Part 1: administration of LAVA-051 via intravenous infusion with dose escalation. Part 2: administration of LAVA-051 at the dose established in part 1 (dose expansion)	Phase I/II	NCT04887259
LAVA-1207 in patients with therapy refractory metastatic castration resistant prostate cancer	Prostate cancer	66	Part 1: administration of LAVA-1207 via intravenous infusion with dose escalation. Part 2: administration of LAVA-1207 at the dose established in part 1 (dose expansion)	Phase I/II	NCT05369000
γδ-CAR-T cells
ADI-001 anti-CD20 CAR-engineered allogeneic gamma delta (γδ) T cells in adults with B cell malignancies	Lymphoma; follicular lymphoma; mantle-cell marginal zone lymphoma; primary mediastinal B-cell lymphoma diffuse large B cell lymphoma non-Hodgkin lymphoma	78	Part 1a: administration of ADI-001 via infusion with ascending dose levels as a single dose to determine MTD or MAD Part 1b: administration of ADI-001 via infusion at the MTD/MAD to evaluate the safety of multiple doses (Part 1b). Part 2: administration of ADI-001 via infusion at the MTD/MAD to confirm recommended phase 2 dose (dose expansion).	Phase I	NCT04735471
TEG002 infusion in relapsed/refractory multiple myeloma patients	Multiple myeloma	26	Dose escalation segment, 3–9 patients per dose cohort will receive: dose level 1 (low), level 2 (medium), level 3 (high)	Phase I	NCT04688853

Abbreviations: MRD, minimal residual disease; HR, hematological relapse; SD, stable disease; IRE, irreversible electroporation; MTD, maximum-tolerated dose; MAD, maximum-assessed dose.

Table 2. Advantages and limitations of γδ T cells-based immunotherapy.

Approach	Advantage	Limitation
γδ CAR-T cell therapy	-Reduced risk of cytokines related syndrome -Lower risk of αβTCR -mediated off-target toxicity -Maintenance of antitumor activity -Possible multiple reinfusion -Preserved ability to act as APCs -Long lifespan	-Low rate of circulating γδ T cells
Bispecific antibodies	-Engagement of only γδ TCR, not all kinds of cytotoxic, immunoregoulatory, immunosuppressive T cells, like CD3 bispecific antibodies -Reduced cytokine storm and low toxicity	-Short half-life
Ex-vivo expanded allogeneic γδ T cells transfer	-Improvement of off-the-shelf immune cells to cancer patients without GVHD	-Circulating Vγ9Vδ2 inter-donor variability -Necessity of optimized cell expansion protocols

2.1. Allogeneic γδ T cells transfer

Several clinical trials are ongoing on allogeneic γδ T cells transfer, because of the low risk of graft-versus-host disease (GvHD); two recent studies have confirmed the safety, tolerability, and efficacy of ex-vivo expanded allogeneic γδ T cells, obtained from healthy donors, on patients with relapsed or refractory non-Hodgkin’s B cell lymphoma or peripheral T cell lymphoma, except for γδ T lymphoma (NCT04696705) and on patients with hematological malignancies after allogeneic hematopoietic stem cell transplantation (NCT04764513). Similarly, a more recent large phase I clinical trial from a total of 132 late-stage malignant liver and lung cancer patients with a total of 414 cell infusions validated the clinical safety of allogeneic Vγ9Vδ2 T cells (NCT03183232, NCT03183219, NCT03183206, and NCT03180437) [4]. Few studies are using Vδ1 T cells, due to the lack of specific expansion protocols. Based on the ‘Delta One T’ cells (DOT) procedure developed in Silva-Santos lab [5], GammaDelta Therapeutics has developed an allogeneic, non-engineered Vδ1 T cell therapy (GDX012) for the treatment of patients with acute myeloid leukemia with minimal residual disease, in a first-in-human phase I clinical trial (NCT05001451).

2.2. Bispecific antibodies

Vγ9Vδ2 T cell-mediated killing of pancreatic adenocarcinomas was enhanced by a bispecific antibody binding the Vγ9 chain and HER2 tumor antigen, significantly more than pAg-stimulated γδ T cells alone or a CD3/HER2 bispecific antibody [6]. A bispecific antibody that simultaneously binds to the Vγ9 chain and CD123 (acute myeloid leukemia target antigen) selectively recruits Vγ9⁺ γδ T cells rather than pan T cells, mediating cytotoxicity against acute myeloid leukemia blasts either in vitro and in vivo [7]. Recently, Van Diest et al. have generated a novel bispecific molecule by linking the extracellular domains of a tumor-reactive Vγ9Vδ2 TCR to a CD3-binding moiety (GABs), which can mimic the model action mediated by the Vγ9Vδ2 TCR. αβ T cells are thus redirected by GABs against a variety of hematopoietic and solid tumor cell lines, as well as primary acute myeloid leukemia. GABs also increase the infiltration of immune cells in a 3D bone marrow niche, while leaving healthy tissues intact and eradicating primary multiple myeloma cells. Finally, in a subcutaneous myeloma xenograft model, GABs significantly reduce tumor growth in vivo [8].

A T cell engager bispecific antibody also has the potential to improve the efficacy of adoptively transferred γδ T cells. Rui Yang et al. [9], have developed a Y-body-based bispecific antibody Vδ2 x PD-L1 that redirects Vγ9Vδ2 T cells toward PD-L1 positive tumor cells, inducing their killing. The combination of Vδ2 x PD-L1 with adoptively transferred Vγ9Vδ2 T cells inhibits the growth of existing tumor xenografts while increasing the number of Vγ9Vδ2 T cells in the tumor bed. Currently, an open-label phase I/IIa study (NCT04887259) conducted by Lava Therapeutics in patients with relapsed/refractory chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia evaluates the efficacy and safety of LAVA-051, a 27kD humanized bispecific single-domain antibody (VHH) that directly engages CD1d and the Vδ2-TCR chain of Vγ9Vδ2 T cells to mediate potent killing of CD1d-expressing tumor cells. LAVA-051 also engages type-1 NKT cells and has a high potency with a low risk of cytokine release syndrome, allowing for a potentially broad therapeutic window. Lava Therapeutics is also conducting a phase I/IIa clinical study to evaluate in patients with metastatic castration-resistant prostate cancer (NCT05369000) LAVA-1207, a Gammabody™ that activates Vγ9Vδ2 T cells upon crosslinking to prostate-specific membrane antigen (PSMA) to trigger the potent and preferential killing of PSMA⁺ tumor cells.

2.3. γδ-CAR-T cells therapy

First γδ-CAR-T cell was described by Rischer et al., who demonstrated that peripheral blood-derived γδ T cells transduced with recombinant retrovirus encoding G(D2)- or CD19-specific CARs and then expanded in vitro, efficiently recognized antigen-expressing tumor cell targets [10]. In 2020, Rozenbaum et al., expanded CD19-Vγ9Vδ2-CAR-T cells using Zoledronate/IL-2, demonstrating that, unlike standard CD19-αβ-CAR-T cells, γδ T cells bearing a CD19-CAR react in vitro and in vivo both against tumor cells expressing CD19 and against those that do not. This effect was amplified by Zoledronate, implying that these engineered γδ T cells can target leukemia cells even after antigen loss and retain pAg specificity via their TCR [11]. Moreover, another group used mRNA electroporation to engineer Vγ9Vδ2 T cells with a CAR based on the extracellular domain of NKG2D (NKG2DL), demonstrating an enhanced in vitro killing activity against a variety of solid tumor cell lines, including those resistant to Zoledronate treatment and their efficacy in vivo in limiting tumor progression [12]. Although γδ-CAR-T therapy was initially focused on the Vδ2 subset, allogeneic Vδ1-CAR-T cells have also been recently developed, supporting their potential use in the clinic. Allogeneic Vδ1-CAR-T cells, known as ADI-001, target malignant B cells via CD20 antigen and inhibit B-cell lymphoma xenografts in immunodeficient mice. A phase I study (NCT04735471) of ADI-001 has been initiated in patients diagnosed with B cell malignancies who have relapsed or are refractory to at least two prior regimens. ADI-001 was well tolerated, with a favorable safety profile and encouraging preliminary efficacy [13]. Moreover, Makkouk’s group has developed in a preclinical study Vδ1-CAR-T cell genetically modified to bind Glypican-3 (GPC-3) and secrete IL-15, for the treatment of hepatocellular carcinoma and potentially of other solid tumors that overexpress GPC-3. A single dose of GPC-3.CAR/sIL-15 Vδ1 T cells optimally control tumor growth in a HepG2 mouse model, with no evidence of xenogeneic GvHD [14]. Another recently implemented strategy, harnesses the overexpression of a high-affinity Vγ9Vδ2 TCR in αβ T cells. These next-generation CAR-T cells, so-called TEGs, combine the properties of αβ T cells, such as robust proliferation and longevity, with the ability of the Vγ9Vδ2 T cells to recognize butyrophilin (BTN) molecules on tumor cells in an MHC-independent manner. Thus, this strategy overcomes the diversity of natural Vγ9Vδ2 T cells and avoids the negative regulation of their TCR through innate receptors. Gadeta is currently investigating in a phase I study (NTR6541) the safety of TEG001 cell suspension, based on the high affinity of Vγ9Vδ2 TCR clone 5, for infusion in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, or multiple myeloma. TEG001 eradicates established primary leukemic blasts in a PDX leukemia model, while healthy hematopoietic compartments derived from human cord blood remain unharmed despite TEGs persistence up to 50 days after infusion, ensuring its safety and efficacy [15]. In addition, the second-generation Vγ9Vδ2 TCR-bearing TEG (GDT002) is currently being evaluated in a multicenter first-in-human phase I study testing safety, tolerability, and preliminary efficacy for the treatment of multiple myeloma (NCT04688853). Preclinical studies have demonstrated anti-tumor activity against multiple myeloma cell lines, as well as myeloma cells from primary bone marrow aspirates from both naive patients and patients who had relapsed/refractory to current standard treatment modalities.

Very recently, Wallet et al. have described an induced pluripotent stem cell (iPSC)-derived γδ CAR-T (γδ-CAR-iT), generated by reprogramming γδ T cells to produce pluripotent stem cells, which exhibits in vitro tumor cell killing, in the presence of IL-15. γδ-CAR-iT cells release significantly fewer IFN-γ and other inflammatory cytokines after activation, than conventional blood-derived αβ-CAR-T cells [16]. As per the latest updates, Kiromic BioPharma proposes new γδ T-cell therapeutic products, which will be evaluated in phase I clinical trials in between late 2022 and early 2023. The first one is allogeneic, non-viral, non-engineered Deltacel, based on expanded, enriched, and activated γδ T cells. The others are genetically engineered products Procel/KB-PD1 and Isocel/KB-ISO targeting respectively PD-L1⁺ and mesothelin isoform 2⁺ tumors.

3. Expert opinion

Currently, immunotherapeutic strategies based on γδ T cells tumor-targeting mechanisms might be key to obtaining more robust and consistent clinical responses. Together, these innovative γδ T cell-based immunotherapies can broaden the present repertoires of tumor-associated antigen recognition and reduce side effects. To ensure their successful clinical implementation, additional research is required on the immunosuppressive impact of the tumor microenvironment on γδ T cells, as well as clinical studies on the combination therapy with checkpoint inhibitors to solve the dysfunction or exhaustion status of chronically activated γδ T cells, reinvigorating their anti-tumor abilities and ensuring persistence.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was funded by grants from the Italian Ministry of Health (Grant No. GR 2016-02364931) to S Meraviglia and from the Ministry of Education and Research to F Dieli (Grant No. PRIN 2017-2017M8YMR8_001) and S Meraviglia (Grant No. PRIN 2017-2017ALPCM).