IntroductionNovel immunotherapeutic strategies engaging human Natural Killer (huNK) cells have emerged as a promising treatment paradigm; however, their accurate preclinical evaluation requires reliable mouse models capable of supporting functional huNK cell engraftment.MethodsWe developed and characterized robust in vivo NK-humanized mouse models using immunodeficient human Interleukin-15 (huIL-15) transgenic (Tg) mice. In vitro amplified huNK cells were used as the source of immune cells. Multiple humanized mouse models were evaluated with optimized protocols. Fcγ receptor knocked-out (FcγR-KO) mice were employed to exclusively model antibody-dependent cellular cytotoxicity (ADCC) potential of huNK cells. A resting period prior to adoptive transfer was also introduced and assessed.ResultsOur NK-humanized mouse models were developed using in vitro amplified huNK cells, which facilitated the production of large number of viable and functional huNK cells and allowed the engraftment of greater cohort of mice. We identified several key parameters improving the long-term persistence and proliferation of functional huNK cells that maintain important functional cell surface markers. FcγR-KO mice exclusively modeled the ADCC potential of huNK cells without the confounding contribution of mouse immune cells. The introduction of a pre-transfer resting period improved tolerability and circumvented potential donor-dependent toxicity issues. Engrafted huNK cells were successfully mobilized in vivo by different NK-based therapeutics, including an engineered IL-2 and an ADCC-enhanced monoclonal antibody.DiscussionThe NK-humanized huIL-15 Tg mouse models developed here provide a versatile and translationally relevant platform for the evaluation and selection of NK cell-based drug candidates, improving clinical translatability and offering a robust framework for preclinical assessment of novel NK cell immunotherapies.

Multiple myeloma (MM), the second most common hematologic malignancy, remains incurable, highlighting the need for durable therapies. Natural killer (NK) cell engagers (NKCEs) represent a promising alternative to T cell therapies, offering potent anti-tumor activity with limited cytokine release. SAR445514 (SAR'514) is a trifunctional NKCE that co-engages NKp46 and FcγRIIIa to activate NK cells while targeting B cell maturation antigen (BCMA) on MM cells. After exploring several molecular formats with varied BCMA and NKp46 valency, we selected SAR'514, a monovalent format with enhanced antibody-dependent cellular cytotoxicity (ADCC). SAR'514 has potent and selective anti-tumor activity in vitro and in vivo, outperforming other FcγRIIIa-immune cell engagers, while inducing minimal cytokine release compared to T cell engagers targeting the same antigen. Ex vivo, SAR'514 efficiently activates NK cells from MM patients and induces cytotoxicity against autologous malignant cells, including those resistant to standard therapies. These findings support further development of SAR'514.

Human γδ T cells demonstrate remarkable and diverse antitumor properties driven by TCR-dependent activation. Their non-alloreactive nature and pivotal role in cancer immunity position them as attractive targets for immunotherapies. However, upon infiltrating tumors, due to mechanisms induced by the tumor microenvironment’s immune evasion strategies, these cells frequently become exhausted, greatly weakening the efficacy and antitumor potential of novel immunotherapeutic treatments. While being extensively characterized in CD8+ T cells, research on γδ T cell exhaustion remains scarce. There is a growing need for comprehensive models to investigate the reinvigoration properties of exhausted γδ T cells. This review synthesizes current strategies and models for evaluating novel immunotherapies aimed at rejuvenating exhausted γδ T cells. It explores a progression of approaches, from ex vivo studies and in vivo murine models to emerging in vitro systems. The advantages and limitations of these models are discussed to provide a comprehensive understanding of their potential in advancing therapeutic research. Furthermore, recent findings suggesting in vitro exhaustion phenotypes closely mirror those observed ex vivo highlight opportunities for preclinical innovation. By refining these models, researchers can better optimize the immunotherapies targeting this unique T cell subset.

Unconventional T cells such as gamma delta (γδ) T cells have garnered significant interest in cancer immunotherapy due to their potent anti-tumor properties including strong cytotoxic capabilities in hematological malignancies and their ability to recognize a broad range of tumor antigens independent of the classical major histocompatibility complex (MHC) molecules. Given the interest in engaging these immune cell populations in cancer therapy, we established a new in vivo humanized mouse model through the adoptive transfer of human γδT cells to support this emerging area of therapeutic interest. This model was created by transplanting γδT cells sorted from PBMC donors into Fc-resolv huIL15 NOG (functional knock-out of murine Fcγ receptors to minimize the confounding impact of mouse immune cells) mice, genetically engineered to express human IL-15. We demonstrate that various γδT cell populations, including Vδ1 and Vδ2, can be successfully maintained over a long period of time, preserving the expression of critical functional biomarkers such as NKG2D, CD69 or CD16, under various engraftment conditions. Additionally, we showed that hu γδT cells display functional engagement in vivo, achieving high efficacy with epcoritamab (CD20-CD3 Ab) against disseminated tumors in the hu γδT cell adoptive transfer mouse model. Lydia Blot, Lopes Joaquim, Emerson Serres, Vanessa Croize, Hervé Barret, Pauline Rettman, Clément Barjon, Dorothee Bourges, Antoine Lhermitte, Valéria Fantin, Sukhvinder Sidhu, Loreley Calvet. Development of a humanized mouse model for γδT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 33.

Recent advancements in cancer immunotherapies have highlighted the potential of Vγ9Vδ2 T cells as key players in the immune response against cancer. These cells, initially recognized for their broad activity in combating infections and tumors, are now emerging as promising candidates for targeted immunotherapeutic strategies, including T cell Engagers (TCEs). However, like other immune cells, Vγ9Vδ2 T cells can enter a state of dysfunction and exhaustion within the tumor microenvironment, driven by factors such as TCR overstimulation, immunosuppressive cytokines (e.g., TGF-β), and hypoxia. While the mechanisms of T cell exhaustion have been extensively studied in in vivo CD8+ T cells, the lack of a murine Vγ9Vδ2 subset complicates the investigation and generation of large numbers of exhausted Vγ9Vδ2 T cells. To address this, we present a novel in vitro protocol for rapidly generating exhausted Vγ9Vδ2 T cells through co-culture with Zoledronate-activated human tumor cells. We characterized the resulting cells using phenotypic, metabolomic, and transcriptomic analyses, comparing their profiles to published data on in vivo-exhausted cells. Furthermore, we measured the reactivation potential of these exhausted Vγ9Vδ2 T cells using anti-CD3 monoclonal antibodies, demonstrating that CD3 stimulation can partially reverse exhaustion and restore anti-tumor effector functions. Furthermore, exhausted Vγ9Vδ2 T cells retained some cytotoxic activity upon stimulation with T Cell Engagers, underscoring the versatility and applicability of our in vitro exhaustion model. This model offers a robust platform for the evaluation of novel immunotherapies targeting Vγ9Vδ2 T cells, facilitating preclinical assessments before transitioning to in vivo studies.

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

Multiple Myeloma (MM) is the second most prevalent hematopoietic malignancy, representing 10% of total blood cancers. Despite the emergence of new therapies, it is still an incurable disease. Safe, potent and innovative approaches with long lasting beneficial effects are required. BCMA (B Cell Maturation Antigen) is a cell surface receptor selectively expressed on normal and malignant plasma cells and promote cell proliferation and survival upon binding of its ligands APRIL (A PRoliferation Inducing Ligand) and BAFF (B cell Activating Factor). BCMA expression is highly prevalent on myeloma tumor cells and is maintained after standard of care treatments such as anti-CD38 therapies, or even BCMA-targeting agents (Cohen AD et al. J Clin Invest 2019). The ability of NK cells to intrinsically kill tumor cells, leaving healthy cells unharmed, with minimal pro-inflammatory cytokine release induction as compared to T cell-based therapies makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. We developed SAR’514, a trifunctional NK Cell Engager (NKCE) that activates NK cells through a dual engagement of NKp46 and CD16a, two major NK cell activating receptors highly expressed on NK cells in MM patients, and which redirects the activated NK cells to engage and kill BCMA+ tumor cells. We demonstrated that SAR’514 NK dual engagement was more potent than the single NK engagement with NKp46 or CD16a as well as the combination of NKp46 and CD16a engagement. SAR’514 leads to NK cell activation, degranulation and release of effector cytokines only in the presence of BCMA+ tumor cells. This antitumor activity is associated with very low IL-1β, IL-6, TNFα and IFNγ cytokine release as compared to a T cell engager targeting the same BCMA antigen, in PBMC and in whole blood settings in the presence of BCMA+ tumor target cells. In addition, the in vivo anti-tumor activity of an anti-murine NKp46 surrogate NKCE molecule was investigated in huFcgR transgenic mice engrafted with the EL4-huBCMA murine thymoma model. SAR’514 induced a significant mouse survival at 0.5 to 5 mg/kg with an overall survival of 90% as compared to the control group in which only 20% of mice survived. Importantly, SAR’514 exhibits ex vivo efficacy using bone marrow mononuclear cells (BMMC) from MM patients in an autologous setting showing an active and efficient primary MM cell killing against MM cells from patients that have failed diverse therapies, including standard of care treatments. In summary, these results demonstrate the efficacy of SAR’514 for controlling MM tumors in vivo and ex vivo, and provide consistent support for its clinical development. Citation Format: Alexandre Tang, Laurent Gauthier, Jochen Beninga, Benjamin Rossi, Nicolas Gourdin, Audrey Blanchard-Alvarez, Céline Amara, Jacqueline Courta, Alexandra Basset, Dorothée Bourges, Alexandre Desjonqueres, Emmanuelle Menoret, Catherine Pellat-Deceunynck, Philippe Moreau, Yannis Morel, Marielle Chiron, Eric Vivier, Angela Virone-Oddos. The novel trifunctional anti-BCMA NK cell engager SAR’514 has potent in-vitro, in-vivo and ex-vivo anti-myeloma effect through dual NK cell engagement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2960.

Over the past decade, an increasing number of immunotherapies aiming to improve the ability of the immune system to effectively eradicate tumor cells have been developed. Among them, targeting effector T cell subsets of the immune system with bispecific antibodies, called T Cell Engagers (TCEs), represents an attractive strategy. TCEs are designed to specifically direct cytotoxic T cells towards tumor cells, thereby inducing a strong activation leading to the lysis of tumor cells. New strategies for targeting specific T-cell subsets are currently being explored. In this study, we investigated the activity of different TCEs on both conventional alpha beta (αβ) T cells and unconventional gamma delta (γδ) T cells. We generated TCE molecules based on camelid single-domain antibodies (VHHs) that target the tumor-associated antigen CEACAM5 (CEA), together with particular T-cell receptor chains (TCRs) or a CD3 domain. The in vitro biological activity of the TCEs against the colon carcinoma cell line LS174T was measured using fresh and cultured human Vγ9Vδ2 and αβ T cells. We showed that Vγ9Vδ2 T cells display stronger antitumor activity in vitro than αβ T cells when activated with a CD3xCEA TCE. Furthermore, restricting T cell activation to Vγ9Vδ2 T cells limits the production of pro-tumor factors and pro-inflammatory cytokines, which are often associated with toxicity in patients. Taken together, these results suggest that Vγ9Vδ2γδ T cell-specific TCEs may represent safe, novel, specific, and effective molecules for improving antitumor immunotherapies.

xCT is a cystine/glutamate antiporter that exports intracellular glutamate for extracellular cystine. In the intracellular space, cystine is converted into cysteine which is subsequently used for glutathione (GSH) synthesis (a major antioxidant species). ARID1A is part of the SWI-SNF remodeling complex that binds on the xCT promoter and as such controls its gene expression. ARID1A deficiency which is highly prevalent in many cancers results in downregulation of xCT, impaired GSH biosynthesis and subsequent ROS induction, raising potential rationale to target xCT in ARID1A deficient cancers. By using a selective nanomolar range xCT inhibitor, we first showed that cell lines deficient for ARID1A were significantly more sensitive to the inhibition of xCT. Effects on proliferation could be reversed by N-acetylcysteine supplementation in the culture medium. In vivo, the xCT inhibitor confirmed its drug-like properties showing significant target engagement in an ARID1A deficient ovarian cell line xenograft model together with good PK and tolerability profiles. Altogether these studies have provided evidence that a specific drug-like inhibitor of xCT can be developed. This proprietary compound was used to confirm that in vitro the deficiency of ARID1A in ovarian cancer predicts sensitivity to xCT inhibition. Further in vivo pharmacological studies are required to confirm a specific molecular context predicting sensitivity towards xCT inhibition. Citation Format: Christophe Henry, Philippe Péron, Anne-Marie Blanchet, Arielle Genevois-Borella, Marc Trellu, Dorothée Bourges, Philippe Lienard, Alexandra Basset, Erwan Jouannot, Christophe Philippo, Laurent Debussche, Jürgen Moll. Pharmacological effects of selective xCT inhibition in ARID1A mutated cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 130.

Novel immunotherapeutic strategies targeting Natural Killer (NK) cells using monoclonal or multi-specific antibodies and immune modulator molecules are under development and there is a pressing need to have suitable mouse models for the evaluation of these therapeutics. To date, there are no validated in vivo mouse models that support the development of fully functional human NK cells. In recent years, the adoptive transfer of human peripheral mononuclear blood cells or CD34+ stem cells into highly immunodeficient mouse strains mostly allowed the evaluation of T cell-based therapies. However, these humanized mouse models have limitations and do not show an optimal development of innate immune cells such as NK, dendritic or myeloid cells. This is mostly due to the poor interspecies cross-reactivity of key factors necessary for cell development and maturation, like cytokines and growth factors.Here, we report the development and characterization of robust in vivo models that allow the engraftment and maintenance of fully functional human NK (huNK) cells, using several strains of immunodeficient transgenic mice for human cytokines (IL-15). The optimal parameters (source of immune cells, pre-conditioning regimen, route of administration) were identified. These models were characterized and validated by flow cytometry, and in vivo efficacy studies using an anti-CD20 Ab.In summary, we developed several murine models that exhibit significant numbers of functional huNK cells maintained over time in vivo. Different sources of human immune cell (CD34+ HSC, huNK cells) were used to implant immunodeficient huIL15 transgenic mice. HuNK cells were monitored by flow cytometry in all tested models. We showed huNK engraftment in both irradiated and non-irradiated models and confirmed the maintenance of expression of important functional NK markers on these cells. Efficacy studies performed in disseminated models showed promising results as efficacy of an anti-CD20 Ab, rituximab, was improved in presence of huNK cells, thus confirming the engagement of huNK cells in vivo in these models. Overall, we have developed several models that can sustain fully functional huNK cells over a period of several weeks that can be proficiently recruited in vivo by various NK-based therapeutics. Citation Format: Pauline Rettman, Dorothée Bourges, Laure-Marie Meyer, Ravi Rangara, Anna Ponchet-Lac, Nicolas Moindrot, Sukhvinder Sidhu, Céline Nicolazzi. Development of NK humanized mice models for the in vivo evaluation of NK cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4247.

How the immune system maintains peripheral tolerance under inflammatory conditions is poorly understood. Here we assessed the fate of gastritogenic T cells following inflammatory activation in vivo. Self‐reactive T cells (A23 T cells) specific for the gastric H+/K+ ATPase α subunit (HKα) were transferred into immunosufficient recipient mice and immunised at a site distant to the stomach with adjuvant containing the cognate HKα peptide antigen. Activation of A23 T cells by immunisation did not impact on either immune tolerance or protection from gastric autoimmunity in wild‐type BALB/c mice. However, increased presentation of endogenously derived HKα epitopes by dendritic cells (DCs) in the gastric lymph node of IE‐H+/K+β transgenic mice (IEβ) reduces A23 T‐cell tolerance to gastric antigens after inflammatory activation, with subsequent development of gastritis. While HKα‐specific A23 T cells from immunised wild‐type mice were poorly responsive to in vitro antigen specific activation, A23 T cells from immunised IEβ transgenic mice were readily re‐activated, indicating loss of T‐cell anergy. These findings show that DCs of gastric lymph nodes can maintain tolerance of pathogenic T cells following inflammatory stimulation and that the density of endogenous antigen presented to self‐reactive T cells is critical in the balance between tolerance and autoimmunity.

Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed “cross-presentation.” The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.