Advancements in γδT cell engineering: paving the way for enhanced cancer immunotherapy

Comprising only 1-10% of the circulating T cell population, γδT cells play a pivotal role in cancer immunotherapy due to their unique amalgamation of innate and adaptive immune features. These cells can secrete cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and can directly eliminate tumor cells through mechanisms like Fas/FasL and antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike conventional αβT cells, γδT cells can target a wide variety of cancer cells independently of major histocompatibility complex (MHC) presentation and function as antigen-presenting cells (APCs). Their ability of recognizing antigens in a non-MHC restricted manner makes them an ideal candidate for allogeneic immunotherapy. Additionally, γδT cells exhibit specific tissue tropism, and rapid responsiveness upon reaching cellular targets, indicating a high level of cellular precision and adaptability. Despite these capabilities, the therapeutic potential of γδT cells has been hindered by some limitations, including their restricted abundance, unsatisfactory expansion, limited persistence, and complex biology and plasticity. To address these issues, gene-engineering strategies like the use of chimeric antigen receptor (CAR) T therapy, T cell receptor (TCR) gene transfer, and the combination with γδT cell engagers are being explored. This review will outline the progress in various engineering strategies, discuss their implications and challenges that lie ahead, and the future directions for engineered γδT cells in both monotherapy and combination immunotherapy.


Introduction
Immunotherapy has revolutionized cancer treatment, effectively integrating with established medical practices such as surgery and chemotherapy (1,2).This approach boosts the immune system's capability to target and eliminate malignant cells, thereby increasing antitumor efficacy and minimizing off-target effects (3).Within the realm of immunotherapy, various strategies have been developed, including the use of immune cells, checkpoint inhibitors, and cytokines.Notably, T cell-based therapies, particularly Chimeric Antigen Receptor (CAR) T cell therapy, have demonstrated significant success against blood cancers (4).In parallel, therapies utilizing NK cells, macrophages, and B cells are emerging as novel treatments for solid tumors and other malignancies (5)(6)(7).
Immune cells play crucial roles in the body's defense mechanisms, including T cells, which are central to cell-mediated immune responses; B cells, which produce antibodies and mediate humoral immunity; and NK cells, which can induce apoptosis in infected or malignant cells as part of the innate immune response (3).Among these immune cells, gdT cells stand out for their unique role in bridging innate and adaptive immunity (8)(9)(10).They target and kill cancer cells without the restriction of major histocompatibility complex (MHC) molecules, thus having a broader recognition on cancer cells, including those deficient in MHC class I. gdT cells are adept at secreting cytokines like interferon-g (IFN-g) and tumor necrosis factor-a (TNF-a), and they can directly eliminate tumor cells through mechanisms such as Fas/FasL and antibody-dependent cell-mediated cytotoxicity (ADCC) (11).Their ability to migrate to peripheral tissues and respond rapidly to target cells (12), coupled with their lack of involvement in graft-versus-host disease (GvHD) (13, 14), makes them ideal candidates for off-the-shelf cell therapy solutions.
Furthermore, gdT cells are crucial in orchestrating anti-tumor immune responses.They can act as professional antigen-presenting cells (APCs) or influence other APCs like dendritic cells, thereby enhancing the activation of abT cells and the overall immune response against tumors (15,16).Theproduction of cytokines, including IL-17 and IL-22, by gdT cells plays a vital role in shaping the tumor microenvironment (TME), thereby influencing tumor growth in various contexts (16)(17)(18)(19)(20)(21)(22)(23).This dual role highlights the complexity and importance of gdT cells in tumor immunology and fuels ongoing research into leveraging their therapeutic potential in novel cancer immunotherapies, such as adoptive cell therapy (ACT) (9,(24)(25)(26)(27).
Despite their significant therapeutic promise, the clinical application of gdT cells faces challenges.As a minor subset of T cells, they often struggle with in vivo survival and proliferation (28), limited persistence, and potential functional suppression upon infiltrating the complex TME (29, 30).To overcome these obstacles, recent advancements in gene-engineering technologies are paving the way for optimizing the therapeutic potential of gdT cells in cancer treatment (8,9,15,28,31,32).By genetically modifying these cells to express CARs or enhancing their native T cell receptors (TCRs), their specificity and cytotoxicity against tumor cells can be significantly bolstered.The use of genetic editing tools like CRISPR/Cas9 to knock out inhibitory receptors or to insert cytokine genes further enhances their proliferative and cytotoxic capacities.Concurrently, combination therapies are being explored to enhance the anti-tumor activity of gdT cells, including the use of bispecific antibodies, checkpoint blockade, and cytokine co-administration.
This review aims to deliver a comprehensive overview of cutting-edge approaches to augment gdT cell immunotherapy.It delves into the biological underpinnings and inherent advantages of gdT cells pertinent to their role in immunotherapeutic applications, as well as scrutinizes the forefront of gene-engineering methods being crafted to surmount existing barriers within gdT cell treatment modalities.Additionally, the synergy of gene-modified gdT cells with other treatment modalities is explored, informed by recent clinical research findings.These studies will shed light on the prospective trajectory of gdT cell immunotherapy, underscoring its potential to significantly enhance treatment outcomes for cancer patients.
2 Properties and functions of gdT cells 2.1 gdT cells ontogeny and gdTCRs diversity gdT cells are the first T cell lineage to develop in the thymus and can be observed in humans as early as 12.5 weeks of gestational age.However, once generated, these cells will expand and mature extrathymically, and their gene repertoire changes in response to age (33).gdT cells derive their name from their TCRs, which are made up of gamma and delta chains.Like abT cells, gdT cells undergo somatic V(D)J rearrangement, a process that generates diverse TCRs to respond to a wide range of antigens (34).However, in contrast to abTCRs, gdTCRs allow cross-reactivity with multiple ligands and each combination is associated with different functional avidities (35).Despite the fact that V(D)J rearrangement of gdT cells generates less diversity than abT cells, TCR d chains have a higher potential of diversity at the complementarity-determining region 3 (CDR3) junction and can provide information on a person's unique history of infection (33,36,37).

Tumor targeting mechanisms
gdT cells target real and perceived immunological insults through the production and release of soluble factors.One example of this is when gdT cells recognize pathogen specific antibodies and stress-induced antigens.In response, gdT cells will produce Th1 cytokines including IFN-g and TNF-a.Subsequently, gdT cells also release cytotoxic granules containing perforin and granzyme, further promoting pathogen degradation (38).Additionally, there is evidence in literature suggesting that Vg9Vd2 cells-a subset of gdT cells (discussed in the next section) can act as sensors of a dysregulated isoprenoid metabolism that target specifically cancer cells (39).Moreover, several recent studies have indicated that different subsets of gdT cells may have remarkably different functions in targeting tumor cells (40)(41)(42)(43).Therefore, it is important to understand the structure and subsets of gdT cells, which we describe in the next section.

gdT cell subsets
This section will focus on two main subsets of gdT cells: Vd1 and Vg9Vd2.
Additionally, Vd1 T cells have private TCR repertoires and significant TCR diversity that mainly originate from TRD repertoires (49).They also manifest features of adaptive immunity, including long-lasting functional memory in gdT cells and adaptive clonal expansion, particularly in response to viral infections (49)(50)(51).Studies demonstrate that Vd1 T cells recognize tumor antigens or cell stress signals through gdTCR and various activating receptors shared with NK cells.These include NK group 2 member D (NKG2D), natural cytotoxicity receptors (NCR, such as NKp30, NKp44, NKp46), and coactivating/adhesion DNAXactivating molecule (DNAM-1) (52-56).Their ligands are frequently expressed on stressed neoplastic cells, for instance, MHC class I chain-related protein A and B (MICA/B), UL16binding proteins (ULBP) 1-4 are common ligands for NKG2D.
Vd1+ T cells can be directly activated through NKG2D upon the expression of its ligand (e.g., MICA) on tumors, without the need for overt TCR stimulation as seen in abT cells (55, 57).Moreover, the expression of NCRs on Vd1 T cells is correlated with increased granzyme B and enhanced cytotoxicity against lymphoid leukemia cells (55).Evidence in the literature suggests that Vd1 T cells can recognize stress-induced antigens including non-classical MHC class I-like molecules, such as CD1 family (including CD1c, CD1d), MICA/B, ULBP molecules (including ULBP3), and annexin A2 (52-54, 58-62).Interestingly, Vd1+ T cells are less susceptible to activation-induced cell death (AICD) compared to Vd2+ T cells.Despite variations in their antigen recognition, both Vd2 and Vd1 T cells share similar cytotoxic mechanisms via the perforin/granzyme-B mediated secretory pathway and death receptor pathways such as TRAIL/TRAIL-R, Fas/FasL (38).
Recognition of CD1d is dependent on the presence of lipid and glycolipid on foreign antigens, suggesting that Vd1 T cells could recognize these antigens in a lipid-dependent manner (63).Bai et al.'s study directly demonstrates this principle of antigen presentation of MHC and lipid recognition by Vd1 T cells (64).However, the exact mechanism of CD1d recognition in Vd1 T cells is still unclear and remains an area of continued investigation.
Furthermore, MICA, a stress-induced antigen, triggers activation and expansion of Vd1 subset via NKG2D when it is expressed on the surface of tumor cells (52-54).These cells have also been shown to recognize ULBP3, a "kill me" signal, expressed on leukemic B cells, suggesting an additional mechanism through  With advances in innovative isolation techniques and deepening comprehension of Vd1 T cells, these cells hold high promise as a potential candidate for cancer immunotherapy, particularly as tissue-associated or tumor-infiltrating lymphocytes.Their manipulation using well-designed cell engagers or immune checkpoint inhibitors in situ represents an accessible and costeffective approach.In the future, the use of single cell sequencing/ proteomics techniques will be essential to dissect the heterogeneity and functional plasticity of Vd1 T cells shaped by the TME, thereby aiding their clinical implementation.

Vg9Vd2 gdT cells
Vg9Vd2 (Vd2) T cells are among the most studied subsets of gdT cells, partially because these cells represent the most abundant subset in peripheral blood (Figure 1).Vg9Vd2 cells are generally considered as the first line of defense, forming an essential part of the innate immunity.All Vg9Vd2 T cells consist of a public Vg9 chain and private Vd2 chain.However, Vd2 T cells can be further divided into two subclasses (Vg9+Vd2+ and Vg9-Vd2+) that exhibit distinct properties.Vg9+Vd2+ T cells exhibit innate characteristics, while Vg9-Vd2+ T cells show adaptive features and undergo pathogen-driven differentiation similar to conventional CD8+ T cells (44,65,66).
Similarly, the recognition of Vd2 cells is mediated by gdTCR or NK cell-activating receptors such as NKG2D and DNAM1.These cells are unique due to their semi-invariant property that allows recognition of specific antigens.Vd2+ TCRs are capable to recognize phosphoantigens (P-Ag), non-peptide antigens that accumulated in tumor cells due to their dysregulated mevalonate pathway (67).The activation of gd T cells is intricately linked to the recognition of P-Ag.This process heavily involves the proteins butyrophilin 2A1 (BTN2A1) and butyrophilin 3A1 (BTN3A1).BTN2A1 binds to Vg9+ gdTCRs.BTN3A1 acts as a critical mediator by presenting P-Ag to gd T cells through its intracellular B30.2 domain (68).This interaction is pivotal for initiating the downstream signaling pathways that lead to gd T cell activation and immune responses.Furthermore, Vg9Vd2 T cells have distinct patterns of development in fetus and adults.Fetal Vg9Vd2T cells are generated in the fetal thymus, while adult Vg9Vd2 T cells are developed after birth in response to environmental stimuli and expanded polyclonally by microbial P-Ag exposure (37, 69).The CD16+ Vd2 T cells can also mediate ADCC upon binding to tumorspecific antibodies, which is absent in Vd1 T cells (70).Additionally, these cells can function like professional APCs by phagocytosing and processing target antigens, then presenting them with MHC molecules.This process, in turn, induces CD4+ and CD8+ responses in abT cells (16,(71)(72)(73).
Recent studies suggest both subclasses of Vg9Vd2 T cells play key roles in the immune defense against pathogens and tumor cells.The number of Vg9Vd2 T cells increases dramatically during some infections and these cells display potent cytotoxic activity.During stimulation with non-peptidic antigens, Vg9Vd2 T cells can be activated via a dual mechanism involving the recognition of FcgRIIIa (CD16a) following the TCR-CD3 complex, which are cell surface antigens for T lymphocytes and NK cells (74).This activation schema belies a keystone role for Vg9Vd2 T cells in the defense of pathological infection as well as tumorigenesis.
3 Sources of gdT cells and their expansion strategies

Sources of gdT cells
The successful clinical application of gdT cell-based immunotherapy must address several challenges, starting with the selection of appropriate sources (Figure 2).Inconsistent effects of autologous gdT cells have prompted investigators to design standardized cell products.Because HLA-matching is not required, fully allogeneic mismatched or haplo-identical gdT cells sourced from healthy donors have emerged as an appealing approach with a commendable safety profile (75, 76).A thorough investigation into the donor's infection history can also benefit patient outcomes when used as a screening criterion.For instance, the reactivation of cytomegalovirus (CMV) in patients receiving HSCT can potentially induce the expansion of Vd2 neg gdT cell clones, which exhibit dual reactivity to CMV and acute myeloid leukemia (AML) (77-79).Another challenge lies in determining which gdT cell subset will be more effective for a specific tumor considering their differing characteristics, particularly their chemotaxis ability and tumor cytotoxicity.Up to now, the main sources of gdT cells include cord blood, peripheral blood, skin, and inducible pluripotent stem cells (iPSCs).
The developmental trajectory of gdT cells reveals that Vd1+ cells constitute the predominant population (approximately 50%) of gdT cells in cord blood at birth, while Vd2+ cells typically represent 25% (80).Over time, Vg9Vd2 T cells emerge as the predominant subset (over 75%) of the gdT cell population in peripheral blood by adulthood, with less than 10% being Vd1+ (80).Therefore, cord blood has been explored for its predominant expansion of Vd1+ cells, or occasional viable expansion of Vd2+ cells (81)(82)(83).However, there are several challenges associated with the in vitro expansion of gdT cells from cord blood, including a low number of gdT cells (less than 1% of cord blood lymphocytes), phenotypically and functionally immature gdT cells, and a poor response to IL-2 and phosphoantigen stimulation (80).In contrast, gdT cells isolated from peripheral blood mononuclear cells (PBMCs) are predominantly Vg9Vd2 (84).Due to their relative convenience and availability, PBMCs provide easy and stable access for expanding Vg9Vd2 T cells and viable Vd1+ cells such as Delta One T (DOT) cells.Additionally, owing to natural tissue tropism of Vd1+ cells, human tissues such as skin also provide an alternative source of Vd1+ cells through enzymatic digestion or other methods (85,86).Despite the roles of skin gdT cells in the cutaneous malignances such as melanoma, complex skin gdT cell subsets necessitate a thorough investigation for therapeutic strategies (87).
In addition to gdT cells derived from donors, these cells can also be generated from iPSCs (88, 89).Two companies, Century Therapeutics and CytoMed Therapeutics, have developed platforms that enrich gdT cells from healthy donor leukapheresis and then reprogram them into T cell-derived iPSCs (TiPSCs).TiPSCs are engineered with CAR expression, followed by directed differentiation into gd CAR-T cells (Figure 2) (90).During this process, the genome characterization of a single CAR-TiPSC clone enables the production of a highly uniform clonal gd CAR-T cell bank (> 95% CAR expression) and minimal DNA mutation caused by engineering (90, 91).This off-the-shelf platform provides an appealing source of gdT cells with several benefits: overcoming quantitative limitations of gdT, reducing the wait time for ex vivo expansion of gdT cells, and not relying on the ex vivo expansion efficiency of PBMC-derived gdT cells (92).Importantly, TiPSC-derived gdT cells retain cytotoxicity to solid and blood tumor through both gdTCR and NKG2D (92).However, this complex manufacturing process is time-consuming and needs more evaluation on potential risks.
Overall, most of the research is adopting PBMC and cord blood as the primary source of gdT cells.In contrast, investigations into skin-derived and iPSC-derived gdT cells are still in the preclinical stages.Our current understanding of the migration and colonization of gdT cells in peripheral tissues primarily relies on research conducted in mice.Further studies involving humans will significantly advance our comprehension of tissue-specific gdT cells, potentially expanding the applications of Vd1+ cells in immunotherapy.

Strategies to expand gdT cells: prerequisite for therapeutic infusion
The clinical-scale manufacturing of gdT cells requires robust and highly reproducible expansion methods that meet good manufacturing practice (GMP) standards.Current approaches mainly include cytokine only, synthetic p-Ag and bisphosphonate (BP) stimulation, antibody-based expansion, and feeder cell-based strategies as summarized in Table 1.Undoubtably, cytokine combinations strategies simplify the manufacturing process but often produce insufficient expansion.
p-Ag or BPs have been recognized as the most established approaches to selectively expand Vd2+ gdT cells (9).Zoledronic acid (ZOL), a BP, has been widely used to numerically expand Vg9Vd2 T cells in vivo and ex vivo.ZOL can be used alone or in combination with IL-2 to achieve these effects (115).ZOL (5 uM) and IL-2 (1000IU/ml) administration over 14 days has been reported to initiate an over 4000-fold proliferation and expansion of gdT cells (mainly Vg9Vd2) from PBMCs of both healthy donors and patients with advanced non-small cell lung cancer (116).However, the expansion folds and purities of gdT cells vary in different published results.
Current protocols for expanding Vd1+ T cells in vitro primarily rely on mitogenic plant lectins such as phytohemagglutinin (PHA) or concanavalin-A (ConA), which induce AICD in Vg9Vd2 T cells (93,117).To transition from the laboratory to the clinic, more  Process of engineering gdT cells.The process of engineering gdT cells involves several key steps.Common sources of gdT cells include the skin, cord blood, and peripheral blood mononuclear cells (PBMCs), with the allogeneic pathway involving isolation from a healthy donor and the autologous pathway involving isolation from the patient's own cells.After isolation, gdT cells are expanded and engineered through various strategies such as the use of chimeric antigen receptors (CARs), T cell receptor (TCR) transfer, and cell engager.Engineered gdT cells can also be derived from induced pluripotent stem cells (iPSCs).In the next step, gdT cells go through purification to develop "off-the-shelf" engineered gdT cells.Finally, the engineered gdT cell product is administered to patients as a form of immunotherapy.Created with BioRender.com.
The feeder cell-based method utilizing artificial antigenpresenting cells (aAPCs) has been explored to provide gdT cells with a sustained activation and costimulation signal.K562, a human chronic erythroleukemic cell line lacking MHC expression, is primarily used as aAPCs.These cells are engineered with costimulatory molecules (like CD80, CD86, CD137) and antigens (e.g., CMV antigen-pp65), allowing for the targeted expansion of specific gdT cell subsets (108, 118).Deniger et al. first activated and propagated polyclonal gdT cells utilizing K562-based aAPCs as irradiated feeders (108, 118).This method requires the additional labor-intensive manufacturing process of culturing feeder cells, yet it mitigates the AICD effects in gdT cells associated with prolonged antigen exposure.Additionally, methods of removing all residual feeder cells before infusion remains a hurdle to clinical implementation of this approach.To address this, several solutions have been proposed, such as gamma-irradiation of aAPCs and the transduction of aAPCs with an inducible suicide gene (107).The ex vivo aAPC expanded donor-derived gdT cells are under evaluation of safety and cell dose in a phase I/II trial (NCT05015426) in patients with high-risk acute leukemia (104).
In the future, efforts should focus more on eliminating the use of xenogeneic serum and feeder cells and integrating GMP/ pharmaceutical-grade reagents into the expansion process.An example of such a method is the protocol proposed by Bold et al. in a recently published article, which has shown better outcomes in terms of expansion and purity (119).Further efforts can be directed towards enhancing the rate of gdT cell expansion, optimizing the procedure, and lowering manufacturing costs.Besides assessing quantity, evaluating the quality of expanded gdT cells-such as memory and exhaustion phenotypes, is crucial for maximizing therapeutic efficacy and requires further investigations.

Engineering strategies: the advances and advantages of gdT cell-based immunotherapy
To date, the pharmaceutical industry has explored three primary categories of strategies for gdT cell engineering, which encompass: (1) CAR-T therapy; (2) antibody-based approaches, such as cell engagers or bispecific antibodies; and (3) engineering or transfer of TCRs.CAR-T therapy remains the predominant approach, while antibody-based strategies are gaining prominence due to several advantages.Research is ongoing to investigate combination of therapies aimed at maximizing the unique capabilities of gdT cells.Lists of engineering strategies and ongoing clinical trials are presented in Tables 2, 3, respectively.
4.1 gd CAR-T cell therapy: extend from but exceed the conventional ab CAR-T therapy CAR-T therapy, with its potential for HLA-independent tumor antigen recognition, has found its place as a key player in cancer immunotherapy.Traditionally, abT cells have been the main candidates for CAR development (150).However, despite their effectiveness, these cells present several limitations.They are susceptible to GvHD, can cause severe and potentially lethal toxicities, contribute to the development of cytokine release syndrome (CRS), and pose issues related to antigen escape (150).These challenges have spurred an interest in alternative solutions, with gdT cells showing potential to offset these limitations.
Given the wealth of limitations associated with abT cells, gdT cells are garnering interest as an alternative for CAR-T therapy.These cells do not instigate GvHD, curb antigen escape resulting in decreased relapse rates, and retain beneficial traits such as a less differentiated phenotype with enhanced antigen presentation capacity (125,151).With these advantages, gd CAR-T cells may have the potential to overcome the obstacles that have historically troubled conventional ab CAR-T therapy.
Interestingly, gd anti-CD19 CAR-T cells have been reported to produce fewer inflammatory cytokines compared to their ab counterparts, suggesting a potential decrease in cytokine-mediated side effects (90).
However, the optimization of CAR for highly specific antigen recognition remains vital.Recent studies have investigated the incorporation of ligands like NKG2DL and inhibitory receptor programmed cell death ligand 1 (PD-L1) into CAR constructs to improve safety or efficacy (158).Some attempts have even added T cell antigen coupling (TAC) components to gdT cells, thereby redirecting them to target tumors with reduced off-tumor toxicity compared to conventional CAR-T cells (159, 160) (Figure 3D).Adicet Bio is working on CAR designs that target tumor intracellular antigens using their TCR-Like monoclonal antibodies (TCRLs) technology (91).
Clinical trials are in progress for CAR-gdT cells targeting various antigens such as CD19 (NCT02656147, NCT05554939), CD20, NKG2DL, CD7, CD33, CD28, and CD123 (Table 3).While many of these trials have yet to disclose their results, some promising preliminary findings have been reported.For instance, Adicet Bio, Inc. is testing an allogeneic CD20 CAR+ Vd1 gdT cell  called ADI-001, designed for patients with refractory B cell malignancies (NCT04735471).Their early report shows a 71% overall response rate and 63% complete response rate among patients with aggressive B-Cell non-Hodgkin lymphoma, all without the presentation of GvHD (91).
One challenge with CAR-T therapy is its potential ineffectiveness in tumors exhibiting heterogeneity or low antigen expression.Dual-specific CARs, which target two antigens concurrently, are proposed as a potential solution, although this requires further investigation (161).Other research focuses on finetuning other CAR components, including the intracellular signaling and transmembrane domain, with construction of Boolean logic gates for combinatorial antigen sensing.Balancing the DNA length of dual-CAR plasmids and transduction efficiency necessitates further study.
Recent innovative extracellular designs aimed at enhancing safety also include the development of ON/OFF switches like the masked CAR.Here, the antigen-binding site of CAR is coupled with a masking peptide through a protease-sensitive linker.Activation of masked CAR-T cells occurs when tumor microenvironment proteases cleave this linker, causing the masking peptide to detach, and revealing the antigen-binding site (162) (Figure 3C).In essence, this provides a level of control, reducing risks associated with unregulated CAR-T activation (162).
To sum up, while there are promising advancements in the development of gdT cell-based CAR-T therapies, it is critical to continue fine-tuning these interventions for increasing specificity and safety.A combination of innovative design strategies and rigorous clinical trials may bring forth the next generation of cancer immunotherapies.The hope is for these novel treatments to cure more patients, more reliably, with fewer side effects, revolutionizing the approach to cancer treatment.

Co-stimulatory domain design and combinatorial strategies: emphasize the unique characteristics of gdT cells
Over the years, CARs have progressed through several generations, differentiated by the quantity and nature of their costimulatory domains, like CD28 and 4-1BB, which play a pivotal role in gdT cell activation and cytotoxic function (163) (Figure 3A).Initial designs of CAR gdT cells were largely based on pre-existing CAR-abT designs, failing to capitalize on the unique benefits of gdT cells due to a dearth of knowledge on the fundamental CAR signaling mechanisms in gdT cells.CAR-abT cells recognize tumor cells through the CAR pathway while completely bypassing the abTCR.Meanwhile, in CAR-gdT cells, the inherent gdTCR signal can synergize with logic-gated CARs, providing MHC-independent cytotoxicity and downstream CD3z signals.Besides, CAR-gdT cells retain multiple activating NK receptors alongside CAR and TCRgd, potentially enhancing recognition and activation.In the tumor immunoescape setting, CAR-gdT cells have been proved the ability to recognize antigen-negative tumor cells in CAR-independent manner (125).CARs designed for gdT cells can also incorporate gdT cell-specific signaling domains, such as NKG2D-DAP10, as an intracellular costimulatory domain for activation.Despite this development, contemporary research on CAR gdT cells predominantly employs second or third-generation designs.It has been observed, though, that single antigen recognition in these CARs leads to poor discrimination between tumor and healthy cells, contributing to on-target off-tumor toxicity.Furthermore, CAR-T cells exhibit strong limitations in treating T cell malignancies due to difficulties like lethal T cell aplasia and CAR-T cell fratricide stemming from shared target antigens (129).Even extending CAR T cell therapies to T cell acute lymphoblastic leukemia (T-ALL) has proven challenging, despite shared molecular commonalities with B cell acute lymphoblastic leukemia (B-ALL).
Moreover, CARs providing both CD3z stimulus and CD28 costimulation are prone to tonic signaling, leading to functional exhaustion and impaired CAR-T cell function.A unique construct called ADI-925 has been developed by Adicet Bio to help tackle this.It incorporates an enhanced intracellular DAP10 chimeric adaptor (CAd), 4-1BB, and a modified CD3z costimulation, designed to enhance tumor targeting through endogenous NKG2D receptors (121, 164) (Figure 3B).
Novel strategies are also emerging, employing Boolean logic gates (like AND, OR, AND NOT) enabling CAR-T cells to detect multiple antigens, reducing off-tumor toxicity and minimizing potential antigen escape (Figures 3E, F).Dual-targeting CAR gdT cells, like those targeting GD2 and PTK7 in preclinical studies for neuroblastoma, were developed to help avoid antigen escape through an OR-gate strategy) (165).Though promising, tandem bispecific OR-gate CAR-T cells may induce excessive CD3z signaling during co-stimulation, necessitating alternative strategies (165).
Bi-specific CARs with split co-stimulatory signals and a shared CD3z domain have emerged as another strategy, allowing for optimal CAR-T cell activation only when both antigens are simultaneously present (161, 166).Furthermore, ideas like chimeric costimulatory receptors (CCRs), also known as recognition-based logic-gated CAR, and non-signaling CARs (NSCARs) have been proposed to mitigate on-target off-tumor toxicity (123,129).CCRs, traditional CARs without CD3z signaling domain, provide co-stimulation whilst avoiding tonic CD3z signaling of gdT cells.Thus, these reduce on-target offtumor toxicity by separating co-stimulatory input from the primary TCR signal (129).Moreover, CCRs have the potential to target malignant cells while sparing healthy tissues in scenarios where the target antigen is broadly expressed (123, 129).Fisher et al. developed a co-stimulation-only CAR, wherein the CAR is fit only to provide co-stimulation, thereby restricting tonic signaling but still facilitating rapid downstream response upon activation (164).Concurrently, CAR-gdT cytotoxicity can be selectively triggered by both the CAR signal and the inherent gdTCR signal when encountering cancer cells (130).CCR can also function as a switch chimeric receptor combined with a second-generation CAR (Figure 3B).The switch receptor typically includes an inhibitory receptor (e.g.PD-1 or TIGIT) and an intracellular costimulatory signal (167).For instance, the PD-1-CD28 construct as anti-PD-L1 CCR can potentially convert the inhibitory signal into an activating one (167).Such a design can accelerate activation of CAR-T cells and improve their survival in the immunosuppressive tumor microenvironment (167,168).On the other hand, NSCARs capitalize on gdT cells' MHC-independent cytotoxic capacity while eliminating all CAR signaling domains (129).This results in antigen-specific tumor cell-targeting capability without influencing T cell activation, as demonstrated by Fleischer et al. with CD5-NSCAR-and CD19-NSCAR-engineered gdT cells, designed specifically for T-ALL and B-ALL relief (129).
Despite the promise of these technologies, factors like NSCAR shedding on gdT cells and antigen downregulation in target cells have somewhat limited their translational application in clinical therapies.Additionally, the necessity of intracellular signaling domains in CAR design is being reconsidered when applied to gdT cells.Deletion of these domains can potentially allow for the transduction of multiple NSCARs, due to a decrease in overall CAR size.
In conclusion, recent years have seen significant expansion in the approaches to T cell engineering, including innovations such as synNotch receptors, iCAR, and several others (158,169).However, the design and development of CARs for gdT cells haven't kept pace.A deeper understanding of gdT cell cytotoxicity mechanisms and further research into these novel CAR structures will be critical in achieving maximum safety and efficacy, thereby unlocking the full potential of CAR gdT cell therapies.

CAR transduction methods
The primary methodologies for CAR-T therapy involve permanent DNA-based transfection methods that include viral transduction (using lentiviruses or retroviruses) and non-viral transfection, typically utilizing transposon systems like Sleeping Beauty and Piggy Bac (170) (Table 2).While lentiviruses and retroviruses are commonly used, concerns about their safety, predominantly due to their immunogenic properties, and their complex and costly manufacturing processes may limit their utility.Despite these concerns, retrovirally-modified CAR-T cells have proven tolerable safety profiles in extensive clinical trials (171).However, the transduction of gdT cells has been challenged due to their relatively limited proliferation and susceptibility to AICD compared to that of abT cells (172).Gammaretroviruses necessitate active cell proliferation for the penetration of viral nucleic acids into the nucleus.This poses a challenge for the transduction of gdT cells compared to abT cells, demanding necessary specific proliferative stimuli for effective gdT cell transduction (172).
Simultaneously, advancements are being made in non-viral technologies to address some drawbacks associated with viral transductions, such as potential oncogenesis, immunogenicity, and high cost (170).Non-viral transposon vectors possess simpler manufacturing processes, cost efficiency, enhanced safety, stable integration of large sequence (>10 kb), but often face efficiency challenges (173).These non-viral integrative vectors rely on temporary cell pore formation or endocytosis, accomplished via various chemical or physical techniques, including electroporation and liposomes (174).
More recently, non-permanent gene transfer methods that utilize non-integrating gene delivery like mRNA-based CAR expression have started to gain traction (154).The utilization of mRNA in CAR-T cells allows for a "biodegradable" approach, in which the cell's potency is short-term.The use of mRNA electroporation was first applied in early stages of ab CAR-T development, but initial clinical trials indicated a lack of efficacy, potentially due to the poor quality and quantity of patient-derived autologous abT cells (NCT02623582).This led researchers to explore the use of allogeneic Vg9Vd2 T cells from healthy donors.Investigations revealed that after mRNA electroporation, CAR expression persisted for up to 120 hours, with peak expression at the 24-hour mark (175).Enhanced anti-AML activity of mRNAbased anti-CD123 gd CAR-T was observed both in vivo and in vitro (131).Despite these promising results, the transient nature of receptor expression means that further applications may need to employ strategies such as repeated or intratumoral injections to ensure therapeutic efficacy.Future advancements in CAR gdT cell therapy may favor non-viral integrating and lipid nanoparticles technological platforms (170).
In the domain of hematological malignancies, CAR gdT cell therapy holds formidable promise.However, the development of universal CAR gdT cells capable of effectively treating solid tumors remains a pressing need, necessitating ongoing research to overcome the physical and immunological challenges associated with solid tumor immunity.Given the unique stimulatory signals and recognition mechanisms of gdT cells, it is evident that the design of CARs for these cells needs to undergo revisions and refinements as our understanding of their biological mechanisms deepens.In essence, while there has been substantial progress in the field of CAR gdT cell therapy, future work that ensures the safety, efficacy, and broad applicability of this promising therapy modality, especially in the context of solid tumors, remains a critical need in the field.

Cell engagers or bispecific antibodies: easier ways to enhance gdT cells recognition
Cell engagers and bispecific antibodies have become an increasingly attractive immunotherapeutic method for enhancing the anti-cancer activity of gdT cells.Bispecific T cell engagers (bsTCEs) are specially designed antibodies, each having two separate binding areas aimed at individual components like tumor-associated antigens (TAAs) and the TCR complex (Vd2 or Vg9) (176).The flexibility of bsTCEs allows for varied applications, such as MHC-independent targeting of TAAs by gdT cells, immune checkpoint modulation, and controlling inflammatory and other signaling pathways (176).These functionalities provide several unique advantages, including their small molecular size and high versatility, eliminating the need for additional co-stimulatory signals for T cell activation, low picomolar range for the half-maximal effective concentration (EC50), effectiveness against both blood-borne and solid tumors, excellent safety profile, and efficient and cost-effective production (177).Most frequently, cell engagers incorporate a fragment-based design or lgG/lgG-like formats (136,137).Fragment-based designs principally modify constructs such as scFv (178), Fab (135), or single-domain antibodies (sdAbs, also known as V HH ) (176) into their binding regions (Figures 4A-C).sdAbs, originating from the variable domain of heavy-chain-only antibodies, have attracted attention because of their unique features, including small size, target specificity, and minor immunogenicity (179).Currently, cell engagers can be applied both as stand-alone therapies and in partnership with allogeneic gdT cells to generate readily available products.
The first CD3-targeting bsTCEs, exemplified by blinatumomab and Tebentafusp, yielded significant positive outcomes in B-cell malignancy and melanoma patients during clinical trials (NCT03070392) (180).However, adverse effects like CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) constrained their clinical usage (177).Further, CD3-targeting bsTCEs may unintentionally activate other CD3+ T cell subsets, which could depress tumor-specific immune responses (137).As a category of innate T cells, gdT cells present a logical choice for engagement to reduce CRS and off-tumor toxicity.
Bispecific antibodies (bsAbs) comprise a class of engineered antibodies with two distinct binding sites, setting them apart from traditional antibodies (181) (Figures 4D-F).These antibodies, as exemplified by anti-Vg9/CD123 bsAbs, selectively rally Vg9+ gdT cells, promoting cell conjugate formation between gdT cells and AML cells (136).As such, these cell engagers can enhance Vg9Vd2+ T cell cytotoxicity against B-cell lymphoma, particularly when accompanied by a co-stimulatory signal pair (178).ImCheck Therapeutics' humanized anti-BTN3A antibody, ICT01, serves as another example.It operates by recognizing three distinct BTN3A forms and prompting their activated conformation, thereby selectively activating Vg9Vd2 T cells in an antigen-independent manner (182).
In a phase I/II clinical trial, ICT01 showed tolerable safety profile and increased infiltration of Vg9Vd2 T cells into tumor tissue in patients with advanced solid tumors (NCT04243499).(Table 3) Besides, LAVA-1207 (PSMA-Vd2 bsTCEs) has shown a favorable safety profile and clinical symptom improvement (decreased PSA level) in a Phase 1/2a clinical trial involving metastatic castration-resistant prostate cancer (mCRPC) patients (N=20, NCT05369000) (137).
Notably, as cell engagers depend on the activation and migration of the patient's inherent gdT cell pool, initial Vg9d2T cell counts could be a useful predictor for clinical outcomes.Take, for instance, a melanoma patient with a high baseline count of circulating Vg9Vd2 T cells who showed considerable tumor infiltration of Vg9+ T cells post ICT01 administration (182).Cell engagers can also be combined with gdT cell-based therapies to develop easily available TAA-targeting gdT cell products (148).
In conclusion, while cell engagers and bispecific antibodies present significant potential compared to CAR-T therapy, their definitive superiority is yet to be determined.Like CAR-T therapy, cell engagers also encounter hurdles such as immune escape owing to loss of target antigen expression and an immunosuppressive tumor microenvironment.Further research is needed to modify cell engagers specifically for gdT cells, paving the way for effective treatments in the future.

TCRs engineering or transfer: a highly specific and reproducible manner
Harnessing natural receptors through the engineering or transfer of T cell receptors (TCRs) serves as an alternative approach to the use of synthetic ones.The transduction of cancer-specific TCRs is an appealing strategy for generating large volumes of readily available, antigen-specific T cells.Transferring cancer-specific abTCR engenders T cell specificity, simplifying procedures compared to isolating specific T cell subsets.However, the transgenic transfer of abTCRs to other abT cells runs the risk of triggering TCR competition and mispairing.Recognizing these limitations, gdT cells are appreciated as safe and ideal carriers for antigen-specific effector cells because TCR-a and -b chains can't pair with TCR-g and -d chains (138,184).To produce cytotoxic gdT cells capable of attacking tumor cells and secreting cytokines via ab and gdTCR-dependent activity, one can isolate tumor antigenspecific ab CD8+ cytotoxic T lymphocytes and clone their TCR ab genes (138) (Figure 4I).However, a notable reduction in gdTCR expression post abTCR transduction was observed, likely due to competition for limited CD3 molecules (138).
Van der Veken et al. demonstrated that abTCR -transduced gdT cells display sustained in vivo endurance and can elicit a recall response (139, 184).More so, infusing abTCRs from invariant natural killer T (iNKT) cells into gdT cells can create bi-potential T cells with NKT cell functionality (143) (Figure 4J).Other research endeavors are concentrated on transferring gdTCR to abT cells to leverage the superior understanding of their effects and memory function mechanisms (185).One product, GDT002, which contains Vg9Vd2TCR-expressing abT cells, allows abT cells to detect augmented phosphoantigens in stressed or malignant cells (185).An ongoing phase 1/2 study is investigating GDT002's safety and tolerability in patients with multiple myeloma.Furthermore, strategies for engineering TCRgd involve fusing with single-chain variable fragments (scFv) or Fab fragments from antibodies.For example, one study used CRISPR/Cas9 to fuse an anti-PD-L1 scFv to the TCRg or d chain in activated gdT cells, creating scFv-g/d-TCRgd cells that showcased anti-tumor capacity akin to traditional CAR-T cells (186) (Figure 4G).Alternatively, the Fab domain of an antibody can be connected to the C-terminal signaling domain of the g and d chains of the TCR, creating an antibody-TCR construct (187) (Figure 4H).The use of the TCR alongside endogenous costimulatory molecules can lower co-stimulation input compared to CAR constructs, thus diminishing cytokine release and mitigating the exhaustion phenotype (187).Anti-CD19 Fab -TCR-gdT cells or ET019003, for instance, have displayed similar anti-tumor actions against B-cell lymphoma as CAR-T cells in vivo (187).
Promisingly, a phase I clinical trial (NCT04014894) indicates that, aside from showing agreeable safety profiles, ET019003 has achieved an impressive clinical response rate (87.5%) among patients with relapsed or refractory diffuse large B-cell lymphoma (188).However, TCR gene transduction or engineering research has somewhat stagnated in recent years, possibly due to complex manufacturing processes involved.In summary, the exploration of novel therapeutic approaches incorporating gdT cells continues to expand, with significant potential for future cancer treatment innovations.

Combination therapy
CAR-T cell therapy has proven extremely promising for treating hematologic malignancies.However, distinct issues related to the immunosuppressive microenvironment of solid tumors require further refinement and personalization of this approach.A potential solution could be combination therapies that adequately address the complexity of solid malignancies.
The concurrent usage of CAR-T/bsTCE therapies and immune checkpoint inhibitors is recognized as a potentially effective strategy to overcome immune system suppression.Exhaustion status, marked by the upregulation of inhibitory receptors, can potentially compromise the therapeutic efficacy of CAR-T cells (189).In a murine model of bone metastatic prostate cancer, gd CAR-T cells persisted in the tumor-bearing tibia for approximately 21 days post-infusion.However, these cells exhibited an upregulation of PD-1 expression while simultaneously losing expression of activation markers (151).Consequently, the combination of therapies such as ICT01 and pembrolizumab, an anti-PD1 antibody, exhibited favorable safety profiles in a phase I clinical trial (NCT04243499).This suggests that the coadministration of CAR-T/bsTCE therapy with anti-PD-1/PD-L1 antibodies could potentially boost treatment benefits (151).
Chemotherapy and radiotherapy, owing to their immunesensitizing attributes, are plausible options for combination therapy with immunotherapy (190).Temozolomide (TMZ), a chemotherapy mainstay for glioblastoma (GBM), transiently heightens the expression of stress-associated antigens such as NKG2DL on tumor cells.Engineering gdT cells to express the methylguanine DNA methyltransferase (MGMT) can thus potentially confer TMZ resistance, enabling the engineered cells to operate efficiently despite the presence of therapeutic concentrations of chemotherapy.The amalgamation of TMZ and MGMT-modified autologous gdT cells, or drug resistant immunotherapy (DRI), showed improved survival outcomes in a model of high-grade gliomas compared to monotherapy (124).
In a phase I clinical trial, INB-200 (an example of DRI) displayed a favorable safety profile, extended progression-free survival (PFS), and presented no dose-limiting toxicities, CRS, or neurotoxicity in glioblastoma multiforme patients (NCT04165941).As it stands, most approved combination immunotherapies largely rely on a combination of immune checkpoint inhibitors (ICIs) and have emerged as first-line treatments for several major cancer types (191).The future of combination immunotherapies with gdT cells likely extends beyond ICI-based approaches, aiming for control and eradication of established tumors.Further research in this area will be instrumental in harnessing the full therapeutic potential of gdT cells.

Challenges and limitations
Tapping into the potential of genetically engineered gdT cells holds the promises of breakthroughs in cancer immunotherapy, albeit with scientific and technical hurdles.The multifaceted nature of gdT cell biology coupled with the complexities of genetic manipulation throws inevitable challenges in the way of optimizing therapeutic potential.
The extensive heterogeneity of gdT cells, which includes various subsets with distinct antigen recognition patterns, homing properties, and effector functionalities, presents a significant challenge in standardizing genetic engineering strategies (192).Additionally, our understanding of the gdTCR repertoire lags behind that of abTCRs (141).Although cell engagers and bispecific antibodies have shown potential to robustly activate gdT cells, effective signal optimization is still underway (178).Certain constraints of gene-engineering, such as the need for CD8 or other co-stimulators which gdT cells lack, and the intricate manufacturing processes involved, serve as significant obstacles (138).Although gene-transduction techniques, such as mRNA electroporation and lentiviral transduction, have seen noticeable advancements over the past years, the efficiency of integrating genes into gdT cells using either viral or non-viral vectors is yet to reach optimal levels.mRNA electroporation allows for rapid expression and poses fewer risks of insertional mutations, while also being associated with lower cellular toxicity.However, this method only provides transient expression of CARs, requiring multiple infusions of CAR T-cells and an extension of their cytotoxic lifespans from a therapeutic perspective (154).On the other hand, lentiviral transduction, often considered time-consuming, also carries the risk of damaging essential genes or regulatory sequences during the period required for expression (193)(194)(195)..
In comparison to ab CAR-T cells, gd CAR-T cells often present less complete clearance of tumor cells in vivo.This characteristic could be attributed to reduced persistence of gd CAR-T in the immunosuppressive microenvironment (125,196), necessitating multiple infusions and a large supply of gd CAR-T cells.Furthermore, CAR-T cells could potentially contribute to antigen loss in target cells, resulting in diminished antigen density (197).
While the introduction of bispecific T cell engagers has propelled cancer immunotherapy, especially against hematological malignancies by offering an easy and cost-effective treatment option, their efficacy remains undermined by co-triggering of immunosuppressive T cell populations, such as regulatory T cells (Tregs) (137).Even though the combination of CAR and bispecific gdT cell engagers has shown promising results towards improving anti-tumor efficacy and reducing cytotoxicity, the tumor cells' ability to evade the immune system strengthened by gdT cells is still under investigation (198).
Interestingly, gdT cells, under certain conditions, may also promote tumor growth (199,200).This trait might be influenced by the TME or interactions with other immune cells.gdT cells have been known to promote tumor growth by producing IL-17, a process influenced by factors such as TME-related metabolism, microbial products, and inflammatory cells (201,202).Considering the association of gdT cells with autoimmune diseases, a thorough investigation of their long-term clinical outcomes is essential when their activation or suppression is incorporated into treatments (203).
Implementing engineered gdT cell immunotherapy in a clinical setting presents its own set of challenges.Identifying suitable patients and healthy donors and creating standardized monitoring guidelines are crucial.Determining the correct dosage-whether based on body weight or the number of cells per infusion-and understanding its relation to treatment success is a significant hurdle.There is also a pressing need to address the risk of disease recurrence post-treatment, bolster the therapy's durability, and decide whether to opt for monotherapy (with a single or several doses) or a combination approach (204).In addition, as production is resource-intensive and coupled with strict regulatory, ethical, and safety considerations, and high costs.Thus, widespread access to this form of therapy is limited.To fully employ the potential of gdT cell therapies, extensive research and collaboration are necessary.
6 Conclusions and future perspectives gdT cell-based immunotherapies represent a promising frontier in cancer treatment, introducing innovative approaches to overcome the limitations of traditional therapies.The development of gene-engineering strategies, such as CAR T therapy, bispecific antibodies and cell engagers, and TCR gene transfer, has significantly advanced the efficacy of gdT cells, addressing their challenges in abundance, expansion, and targeting efficiency.Despite these strides, hurdles such as the nuanced understanding of gdT cell behaviors, targeting solid tumors effectively, and preventing post-treatment relapse persist.
The remarkable potential of gdT cell therapies lies in their ability to offer a paradigm shift in cancer treatment, utilizing their unique properties for more precise, potent, and personalized interventions.Their versatility in recognizing cancer cells without MHC restriction provides a substantial advantage in reducing the risk of immune escape and addressing tumor heterogeneity.
Looking ahead, research must focus on understanding gdT cells' metabolic needs and cytokine profiles within the tumor microenvironment to enhance their antitumor activity.Additionally, it is critical to develop strategies that improve the persistence of CAR gdT cells and maintain target antigen visibility, ensuring long-term therapeutic success.The exploration of Vd1 subsets and the creation of iPSC-derived gdT cells hold promise for developing universally applicable CAR gdT cell therapies.Furthermore, optimizing the engineering of gdT cells for safer and more efficient delivery, coupled with the strategic combination of these therapies with other treatments, will enhance efficacy and durability.
Emphasis should also be placed on designing therapies that reduce the risk of relapse and increase sustainability.Regulatory, manufacturing, and logistical challenges will need to be addressed to facilitate the clinical translation of these therapies.The ultimate goal is to harness the full therapeutic potential of gdT cells, offering new hope to patients with various types of cancer.
The future of gdT cell immunotherapy lies in the convergence of molecular biology, genetic engineering, and clinical research.As our understanding evolves, so will the potential of gdT cells as a powerful tool in the arsenal against cancer, paving the way for more effective, tailored, and sustainable cancer treatments.

FIGURE 1
FIGURE 1 Tumor targeting mechanisms of Vd1 and Vd2.Different gdT cells activation modes by tumor cells.The tissue resident Vd1 T cells recognize cancer cells via their specific Vd1 T cell receptors (TCRs), which bind Annexin A2 and lipid antigens presented by CD1.Besides, Vd1 T cells also use NKG2D and natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and NKp46 for tumor cell recognition.Vd2 T cells are predominant in the peripheral blood and can migrate into tumor tissues.Their specific Vd2 TCRs recognize BTN3A1 and BTN2A1 after the isopentenyl pyrophosphate (IPP) accumulation.CD16 expressed by Vd2 T cells can bind therapeutic antibodies to trigger Vd2-mediated antibody-dependent cell-mediated cytotoxicity (ADCC).In addition, both Vd1 and Vd2 T cells express natural killer receptors (NKRs), which recognize tumor cells by binding to MHC class I chain-related protein A and B (MICA/B), and UL16-binding proteins (ULBPs).Created with BioRender.com.
efforts have been made to avoid potentially hazardous components.Almeida et al. first developed a clinical-grade two-step method through combination of cytokines (IL-1b, IL-4, IL-21, and IFN-g) and anti-CD3 mAb (clone: OKT-3) to achieve the expansion of Vd1+ T cells (94).This method enables large-scale expansion (up to 2,000-fold) of Vd1+ T cells known as DOT cells (94).GDX012, based on DOT cells, has been granted orphan drug designation by FDA for AML treatment and is currently undergoing evaluation in a phase I trial (NCT05001451).Recently, Ferry et al. also apply only anti-CD3 mAb and IL-15 to stimulate abTCRand CD56-depleted PBMC, resulting in robust Vd1 cell expansion (97).

FIGURE 3
FIGURE 3 Established strategies for CAR-gdT cells.Single-antigen CAR recognition: (A) Conventional CARs are classified as first-, second-, third-, or fourth generation depending on their number of costimulatory domains.(B) Innate enhanced DAP10 chimeric adaptor (CAd), combined with 4-1BB and modified CD3z co-stimulation, enhances tumor targeting through endogenous NKG2D receptors.(C) The masked CAR (mCAR) incorporates a masking peptide.When proteases are present in the tumor microenvironment (TME), the linker is cleaved, releasing the masking peptide, and activating the CAR.This mechanism helps reduce on-target off-tumor toxicity.(D) A T cell antigen coupler (TAC) is also designed to reduce toxicity and promote more efficient anti-tumor response.It is comprised of a tumor-associated antigen (TAA) binding domain, CD3 binding domain, and CD4 co-receptor domain.Combinatorial antigen CAR recognition: (E) OR-gate CARs enable dual-targeting of antigens with separate single-chain variable fragment (scFv) domains.To prevent antigen escape, they can be designed to have two consecutive scFv domains connected to the standard CAR chassis.(F) AND-gate CARs are only activated when both antigens are present simultaneously, employing two separate receptors comprising the CD3z and costimulatory domains.A chimeric costimulatory receptors (CCR)-based AND-gate has its CD3z signaling domain from a gdTCR and can target multiple antigens which can enhance cytotoxicity and prevent tonic CD3z signaling.CCR can also be paired with a switch receptor which can be an inhibitor receptor such as programmed death-1 (PD-1) along with a costimulatory domain like CD28.Non-signaling CARs (NSCARs) do not possess signaling domains and utilize an antigen-specific tumor targeting mechanism.Created with BioRender.com.

FIGURE 4
FIGURE 4 Established strategies for engineering gdT cells.Cell engager designs: Fragment based cell engagers include tandem single-chain variable fragment (scFv), tandem variable heavy chain (VHH), and (scFv)2-Fab.(A) A tandem scFv antibody comprises two different scFvs joined by a linker.(B) Tandem VHH is depicted as a bispecific T cell engager (bsTCE) with an anti-CD1d VHH linked to an anti-Vd2 VHH.(C) An example of (scFv)2-Fab antibody, Her2/Vg9, is composed of an anti-Vg9 Fab domain and two anti-Her2 scFvs.This design selectively recruits gd T cells and enhances cytotoxicity.IgG based cell engagers encompass tandem VHH-Fc, bispecific antibodies (BsAb), and (scFv)2-Fc-Ag.(D) Tandem VHH-Fc antibodies involve two VHHs linked to a Fc domain.(E) One type of BsAb connects an anti-Vg9 domain and an anti-CD123 domain via Knobs-into-holes heterodimerization technology.(F) (scFv)2-Fc-Ag is shown as an anti-CD19 scFv connected to a BTN2A1/3A1 domain via an Fc linker.Engineering gdTCRs and transferring specific abT-TCR or NKT-TCRs into gdT cells: (G) One approach to engineering gdTCRs is to fuse an anti-programmed cell death ligand 1 (PD-L1) scFv to either the g or d chain of gdTCR to limit T cell exhaustion.(H) Another approach is an antibody-TCR, such as an anti-CD19 Fab domain linked to a gdTCR.(I) abTCRs and CD8 ab genes can be transferred to gdT cells to enable targeting specific tumor cells and avoid TCR mispairing.(J) Natural killer T (NKT) cell-derived abTCRs can also be transferred into gdT cells to enhance proliferation, IFN-g production, and antitumor effects.Created with BioRender.com.
As a result, autologous DRI-gdT cells (INB-400) have proceeded to a phase II clinical trial, and MGMT-modified allogeneic gdT cells (INB-410) are currently undergoing a phase Ib clinical trial (NCT05664243).The product INB-400/410, developed by IN8bio, has been granted FDA Orphan Drug Designation for the Treatment of Newly Diagnosed Glioblastoma.

TABLE 1
Comparison of different methods for gd-T cell expansion.

TABLE 2
Different strategies for engineering gdT cells.

TABLE 3
Summary of ongoing clinical trials of engineered gdT products.