Background
T-cell Non-Hodgkin Lymphoma (T-NHL) is a diverse group of aggressive malignancies which are minimally researched and have poorer patient outcomes than those with B-cell NHL (Stuver et. al., 2022, Foss et. al., 2011). Therapies are characterized by pan T cell cytotoxicity, poor response rates and the requirement for multiple levels of treatments resulting in toxicity and suppression of the immune system that can lead to life threatening infections (Bellei et. Al., 2018). Clinical trials are considered a first line option for advanced disease, speaking to the lack of viable treatment options. One possible target for T cells is the T-cell receptor (TCR). TCRs possess tremendous diversity owing to diverse variable β chain (Vβ) (n=48) and α chain (Vα) (n=46) gene segments, that recombine with D and J gene segments to form a functional TCR. T Multiplexed PCR of the TCR gene has been used to identify the presence of a distinct malignant clone and facilitate diagnosis of T-NHL (Savage et. al., 2008). Thus, the Vβ chains represent a therapeutic target that discriminates the clonally expanded malignant T cells from the vast majority of healthy T-cells. Lucero et. al. (2023), showed specific targeting of T cell receptor Vβ chains, for T-NHL patients with an expanded TCRVβ clone using a naked antibody approach. Similar modalities have used BiTE and CAR-T. One downside of the naked antibody and BiTE approach (Paul et. Al., 2021) is that they require patients to have functional effector NK and CD8 T cells, and these cells are often not present in advanced T-NHL diseases. The expanded Vβ chain could also be targeted by CAR-T cell therapy; however, this approach is hampered by concerns of toxicity and poor autologous T cell quality due to the patients' disease (Marofi et. al., 2021). Therefore, we propose CAR-NK cell therapy utilizing the TCRVβ chain (Fig 1A) for patients with advanced T-NHL against the clonally expanded TCRVβ which will allow for an off the shelf approach that bypasses the need for autologous effector cells.
Methods
To show proof of concept, TCRVβ13 CAR-NK cells were manufactured from an NK-92 cell line, HaNK, which expresses a high affinity CD16 V158 FcγRIIIa receptor (Jochems, et. al., 2016). CAR transduction was confirmed via flow cytometry. Along with the CAR, NK/T-cell lymphoma cell lines, EL4 (mouse) and Jurkat (human), were transduced through a lentiviral vector containing a plasmid to express our Vβ of interest, Vβ13 (EL4-V13 and Jurkat-V13, respectively) (Lucero, 2023). We performed In vitro cytotoxicity co-culture assays on NK/T-cell lymphoma cell lines, EL4-V13 and Jurkat-V13, to confirm functional activity in CAR-HaNK cells compared to wild type HaNK cells at three different effector to target ratios. From the in vitro cytotoxicity assay, assays on supernatants from each of the co-cultures for changes of the cytokines INF-gamma and TNF-alpha levels to assess functional NK activity are in progress.
Results
The in vitro co-culture cytotoxicity assays showed increased cytotoxicity of the TCRVβ CAR-HaNK cells in comparison to HaNK cells in the presence of the TCRVβ for both EL-4-V13 and Jurkat-V13 cell lines. Targeting of the EL-4 cell line without the transduced TCRVβ showed similar percent cytotoxicity in CAR-HaNK cells and HaNK cells, while EL-4-V13 cell line showed significant increase in cytotoxicity when co-cultured with CAR-NK cells in comparison to the HaNK cells. In the 1:1 co-culture of either CAR-HaNK or HaNK cells with EL-4-V13 there was a 27% increase in cytotoxicity in the CAR-NK in comparison to the HaNK cells (P<0.0001), the 5:1 showed 71% increase in CAR-NK (P<0.0001), and the 10:1 showed an 80.5% increase in CAR-HaNK (P<0.0001) (Fig 1B). Similarly, in the human cell line Jurkat-V13, co-cultures with CAR-HaNK cells showed an increase in cytotoxicity in the 1:1 of 30% (P=0.0169), the 5:1 of 37% (P=0.0074), and 10:1 of 57.7% (P<0.0001) when comparing to HaNK cells (Fig 1B).
Conclusions
The goal of these preclinical studies is to leverage advances in CAR development with the efficacy of targeting TCRVβ as a direct and safe therapeutic option for patients with T-NHL. If successful, this will propel both cell-directed therapies and a novel target to a disease characterized by few efficacious, durable and safe therapies.
Disclosures
Tyner:Kronos: Research Funding; Petra: Research Funding; Meryx: Research Funding; Incyte: Research Funding; Schrodinger: Research Funding; Tolero: Research Funding; Recludix Pharma: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Aptose: Research Funding; Acerta: Research Funding. Druker:Recludix Pharma, Inc.: Consultancy, Current holder of stock options in a privately-held company; The RUNX1 Research Foundation: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Membership on an entity's Board of Directors or advisory committees; Labcorp: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Astellas: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Incyte: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Syndax: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; AstraZeneca: Other: PI or Co-investigator on clinical trial(s) funded via contract with OHSU.; CureOne: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: PI or Co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Oregon Health & Science University: Current Employment, Patents & Royalties: #1518 (exclusive option agreement with CytoImage); #0606/Patent 6958335 (Novartis exclusive license); #2573; #0843; #0996; Therapy Architects, LLC: Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Adela, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Tolero: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Enliven Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; US Patent and Trademark Office: Patents & Royalties: Patents 6958335 (Novartis exclusive license), 4326534, 7416873, 7592142, 10473667, 10664967, 11049247; Gilead: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Celgene: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Bristol Myers Squibb: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; DNA SEQ: Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties: #2063 (licensed exclusively to Merck & Co); and #2524, Research Funding; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; Multicancer Early Detection (MCED) Consortium: Membership on an entity's Board of Directors or advisory committees; Beat AML LLC: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma, Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Cepheid: Membership on an entity's Board of Directors or advisory committees. Lydon:VB Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Bowers:VB Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.
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