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

Abstract

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.

Keywords: CAR-T; cancer; cellular therapy; engineering; immunotherapy; γδT cells.

Figure 1

Tumor targeting mechanisms of Vδ1 and Vδ2. Different γδT cells activation modes by tumor cells. The tissue resident Vδ1 T cells recognize cancer cells via their specific Vδ1 T cell receptors (TCRs), which bind Annexin A2 and lipid antigens presented by CD1. Besides, Vδ1 T cells also use NKG2D and natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and NKp46 for tumor cell recognition. Vδ2 T cells are predominant in the peripheral blood and can migrate into tumor tissues. Their specific Vδ2 TCRs recognize BTN3A1 and BTN2A1 after the isopentenyl pyrophosphate (IPP) accumulation. CD16 expressed by Vδ2 T cells can bind therapeutic antibodies to trigger Vδ2-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, both Vδ1 and Vδ2 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.

Figure 2

Process of engineering γδT cells. The process of engineering γδT cells involves several key steps. Common sources of γδT 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, γδT 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 γδT cells can also be derived from induced pluripotent stem cells (iPSCs). In the next step, γδT cells go through purification to develop “off-the-shelf” engineered γδT cells. Finally, the engineered γδT cell product is administered to patients as a form of immunotherapy. Created with BioRender.com.

Figure 3

Established strategies for CAR- γδT 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 CD3ζ 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 CD3ζ and costimulatory domains. A chimeric costimulatory receptors (CCR)-based AND-gate has its CD3ζ signaling domain from a γδTCR and can target multiple antigens which can enhance cytotoxicity and prevent tonic CD3ζ 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

Established strategies for engineering γδT 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-Vδ2 VHH. (C) An example of (scFv)2-Fab antibody, Her2/Vγ9, is composed of an anti-Vγ9 Fab domain and two anti-Her2 scFvs. This design selectively recruits γδ 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-Vγ9 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 γδTCRs and transferring specific αβT-TCR or NKT-TCRs into γδT cells: (G) One approach to engineering γδTCRs is to fuse an anti- programmed cell death ligand 1 (PD-L1) scFv to either the γ or δ chain of γδTCR to limit T cell exhaustion. (H) Another approach is an antibody-TCR, such as an anti-CD19 Fab domain linked to a γδTCR. (I) αβTCRs and CD8 αβ genes can be transferred to γδT cells to enable targeting specific tumor cells and avoid TCR mispairing. (J) Natural killer T (NKT) cell-derived αβTCRs can also be transferred into γδT cells to enhance proliferation, IFN-γ production, and antitumor effects. Created with BioRender.com.