No abstract

Chimeric antigen receptor (CAR) T-cell therapy has been used with unprecedented clinical responses in relapsed/refractory B-ALL or B-NHL, MM patients. However, CAR-T therapy has not yet been proven effective in other hematological malignancies, such as T cell lymphoma and leukemia (T-NHL/T-ALL) and acute myeloid leukemia (AML). Developing CAR-T cells for patients with T-cell malignancies remains challenging as most CARs targeting T-lineage antigens, such as CD5, CD7, and CD3, target themselves, potentially resulting in fratricide of CAR-T cells. Thus, there is a need for technical approaches to alleviate the fratricide of CAR-T to treat T-cell malignancies successfully. As part of the approach, we studied whether the affinity of CAR and cognate antigen expression of CAR-T cells influence the intensity of CAR-T`s fratricide and hypothesized that if a single chain Fragment variable (scFv) with a low intensity of fratricide is applied to T-lineage antigen-downregulated CAR-T cells, the effector function of CAR-T cell targeting T cell antigen can be maximized. For this, we screened full human scFvs targeting CD5, a pan T-cell marker, tested the affinity of scFvs for CD5 antigen, and generated anti-CD5 CAR-T cells (CAR5). We evaluated the intensity of in vitro fratricide in CAR5 based on cell expansion and cytokine secretion in vitro and tested their anti-tumor activity in a CD5+ T-ALL xenograft model (Jurkat). In addition, to minimize CAR recognition to a cognate antigen expressed in CAR-T cells, we also generated CD5-downregulated CAR5s (CD5-KD CAR5) using a short hairpin RNA cassette integrated into an anti-CD5 CAR vector to test if the downregulation of CD5 in CAR5s can reduce the fratricide. We found that a CAR5 clone (C7CAR5) with the lowest level of fratricide in vitro, showed superior anti-tumor activity compared to other CAR5s in vivo and that CD5-downregulated C7 CAR5 (CD5-KD C7CAR5) further reduced the fratricide, maximizing the anti-tumor activity. In conclusion, we demonstrate that high-affinity CAR may not always show superior functionality in T cell malignancy CAR-T therapy, explaining one or more strategies to alleviate the fratricide are required. These studies provide the foundation for developing CD5 CART products to treat relapsed T-cell malignancies. Citation Format: Lee Young Ho, Seung Rok Yu, Jeong Hoon Jeong, Hyeong Ji Lee, Hyeon Jeong Cho, Hyung Cheol Kim. Mitigating the CD5 CAR-CD5 interaction enhances the functionality of CD5 CAR-T cells by alleviating the T-cell fratricide. [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 4086.

CD19 targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two in one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19 targeting CAR T cells in the context of four different checkpoint combinations PD1/TIM3, PD1/LAG3, PD1/CTLA4 and PD1/TIGIT and found that CAR T with PD1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD1 downregulation only, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD1 enhances short term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD1/TIGIT downregulated CAR T cells generated from diffuse large B cell lymphoma patient derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistence in vivo compared with conventional CD19 targeting CAR T cells. Overall, our results demonstrate that the cell intrinsic PD1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint mediated resistance in CAR T therapy.

T cells expressing chimeric antigen receptors (CAR-T cells) have shown unprecedented clinical responses against hematological malignancies. However, some patients relapse after CAR-T cell therapy due to antigen-negative escape variants. Additionally, CAR-T cell therapies showed limited clinical efficacy in solid tumors with high antigen heterogeneity. To overcome this, we metabolically labeled the glycans on cancer cells to redirect CAR-T cell cytotoxicity regardless of the endogenous antigen expression status of the cancer cells. We found that modifying cancer cells with N-azidoacetylmannosamine and bicyclo[6.1.0]non-4-yne-fluorescein isothiocyanate can elicit selective and durable cytotoxicity of anti-FITC CAR-T cells. Furthermore, we demonstrated that dinitrophenyl-conjugated sialic acid (Sia-DNP) generated DNP-modified glycans on cancer cells in situ that could be effectively targeted by anti-DNP CAR-T cells to eradicate established tumors in xenograft models. Our study illustrates that metabolic glycan labeling using unnatural sugars can be combined with CAR-T cell therapy to provide novel cancer immunotherapy for solid tumors that lack viable target antigens.