Chimeric antigen receptor (CAR) T-cell therapies have achieved remarkable success in treating B-cell malignancies, including acute lymphoblastic leukemia (B-ALL). However, despite high remission rates, relapse due to antigen escape remains a significant challenge. To overcome this, designing CAR T-cells targeting multiple cancer antigens simultaneously is a promising strategy. NKG2D ligands (NKG2DL) are eight stress-induced ligands expressed by cancer cells but largely absent on healthy cells.
We hypothesized that simultaneous targeting of NKG2DL (using the NKG2D extracellular domain) and CD19 can prevent CD19 antigen escape and improve long-term remission rates in B-ALL patients. We developed three tandem CARs targeting both CD19 and NKG2DL and demonstrated that two tandem candidates were highly effective against both CD19+ and CD19- cancer cell lines. Importantly, when compared to CD19 CAR T-cells, tandem CAR T-cells exhibited comparable cytokine secretion, cytolytic activity and proliferation levels when incubated with cancer cells expressing CD19 and were still effective when incubated with cancer cells lacking CD19. Moreover, T-cells transduced with the selected CD19/NKG2DL tandem CAR were functional against CD19+ primary B-ALL samples and controlled tumor growth in a highly challenging xenograft model representing a CD19- B-ALL relapse.
These findings provide proof-of-concept that NKG2D-based tandem CARs offer a promising approach to overcome antigen escape and enhance anti-tumor efficacy in B-cell malignancies.
Copyright © 2025 Bolsée, Violle, Jacques-Hespel, Nguyen, Lonez and Breman.

αβ T cell receptors (TCR) co-recognise peptide (p) antigens that are presented by major histocompatibility complex (MHC) molecules. While marked variations in TCR-p-MHC docking topologies have been observed from structural studies, the co-recognition paradigm has held fast. Using HLA-DQ2.5-peptide tetramers, here we identify a TRAV12-1+-TRBV5-1+ G9 TCR from human peripheral blood that binds HLA-DQ2.5 in a peptide-agnostic manner. The crystal structures of TCR-HLA-DQ2.5-peptide complexes show that the G9 TCR binds HLA-DQ2.5 in a reversed docking topology without contacting the peptide, with the TCR contacting the β1 region of HLA-DQ2.5 and distal from the peptide antigen binding cleft. High-throughput screening of HLA class I and II molecules finds the G9 TCR to be pan-HLA-DQ2 reactive, with leucine-55 of HLA-DQ2.5 being a key determinant underpinning G9 TCR specificity excluding other HLA-II allomorphs. Consistent with the functional assays, the interactions of the G9 TCR and HLA-DQ2.5 precludes CD4 binding, thereby impeding T cell activation. Collectively, we describe a naturally selected αβTCR from human peripheral blood that deviates from the TCR-p-MHC co-recognition paradigm.
© 2025. The Author(s).

Background/Objectives: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug's commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug's clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene's functional activity. Methods: Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay's adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. Results: Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay's specificity also in this context. Conclusions: We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug's mechanism of action (MoA) in the same assay.

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. Funding Source This work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx Declaration of interests The anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada

Inadequate antigen-specific T cells activation hampers immunotherapy due to complex antigen presentation. In addition, therapeutic in vivo T cell expansion is constrained by slow expansion rates and limited functionality. Herein, we introduce a model fusion protein termed antigen-presenting cell-mimic fusion protein (APC-mimic), designed to greatly mimicking the natural antigen presentation pattern of antigen-presenting cells and directly expand T cells both in vitro and in vivo. The APC-mimic comprises the cognate peptide-human leukocyte antigen (pHLA) complex and the co-stimulatory marker CD80, which are natural ligands on APCs. Following a single stimulation, APC-mimic leads to an approximately 400-fold increase in the polyclonal expansion of antigen-specific T cells compared with the untreated group in vitro without the requirement for specialized antigen-presenting cells. Through the combination of single-cell TCR sequencing (scTCR-seq) and single-cell RNA sequencing (scRNA-seq), we identify an approximately 600-fold monoclonal expansion clonotype among these polyclonal clonotypes. It also exhibits suitability for in vivo applications confirmed in the OT-1 mouse model. Furthermore, T cells expanded by APC-mimic effectively inhibits tumor growth in adoptive cell transfer (ACT) murine models. These findings pave the way for the versatile APC-mimic platform for personalized therapeutics, enabling direct expansion of polyfunctional antigen-specific T cell subsets in vitro and in vivo.
© 2024 The Authors.