Abstract 573: Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models

Abstract

Adoptive cell transfer (ACT) involving autologous tumor-specific lymphocytes (TIL) has shown promising results in the treatment of advanced cancer. Poor in vitro cell expansion, inefficient T-cell migration and T cell exhaustion in the tumor microenvironment greatly limits the broader application of this approach. The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is highly expressed in T cells, where it limits cell activation following TCR engagement, therefore functioning as an important intracellular checkpoint that limits T cell mediated anti-tumor responses. We have developed NX-0255 a highly potent small molecule inhibitor of CBL-B that increases TCR-mediated activation of T cells in the presence or absence of CD28 co-stimulation. Here we used the OT-1 ACT/E.G7-OVA lymphoma and the Pmel-1 ACT/B16 melanoma tumor models to investigate whether adding NX-0255, during the in vitro expansion of tumor-specific T cells would have favorable effects on their ability to reject tumors following adoptive transfer in vivo. OT-1 CD8+ T cells were expanded in vitro using anti-CD3 stimulation and either NX-0255 alone, IL-2 alone, or NX-0255 combined with IL-2 and subsequently transferred in vivo to mice bearing established E.G7-OVA. ACT of OT-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 demonstrated superior anti-tumor activity against E.G7-OVA lymphoma tumors, resulting in significant improvement in survival when compared to mice receiving OT-1 CD8+ T cells cultured with NX-0255 alone or IL-2 alone. Importantly, OT-1 CD8+ T cells cultured in the presence of NX-0255 or the combination of NX-0255 plus IL-2 were found at an increased frequency in the blood and in the tumor and persisted longer in circulation when compared to cells cultured with IL-2 alone. A lower percentage of the tumor-infiltrating OT-1 cells were triple-positive for exhaustion markers PD1+TIM3+LAG3+ expression when cultured with NX-0255 alone or the combination of NX-0255 and IL-2 compared to culture with IL-2 alone. Similarly, ACT of Pmel-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 provides a robust and durable anti-tumor response compared to mice receiving Pmel-1 CD8+ T cells cultured with IL-2 alone in the aggressive B16-OVA model. Flow cytometry analysis showed that Pmel-1 CD8+ T cells cultured in the presence of NX-0255 plus IL-2 were found at an increased frequency in the blood and showed an increased central-memory phenotype (CD44+CD62L+) after adoptive transfer when compared to cells cultured with IL-2 alone. Collectively, these data suggest that adding the CBL-B inhibitor, NX-0255, during the in vitro expansion of tumor-specific T cells increases the frequency and absolute numbers of less exhausted CD8+ memory T-cells, increasing their in vivo persistence and ability to infiltrate the tumor. Citation Format: Marilena Gallotta, Jose Gomez Romo, Alexandra Borodovsky, Austin Tenn-McClellan, Jennifer Stokes, Jennifa Gosling, Gwenn M. Hansen, Arthur Sands, Ryan Rountree, Cristiana Guiducci. Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor 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 573.

Small molecule inhibitors for cancer immunotherapy and associated biomarkers – the current status

“…NX-1607 is being assessed in tumor types known to be responsive to checkpoint blockade (NCT05107674). Interestingly, NX-1607 is also tested in diffuse large B cell lymphoma (DLBCL, post-Richter transformation), which is supported by preclinical data showing increased rituximab antibody-dependent cellular cytotoxicitydriven efficacy by CBL-B inhibition (48). Dose escalation data suggest a dose-proportional increase in exposure of NX-1607.…”

361 Universal expansion of CBL-B-inhibited tumor infiltrating lymphocytes, DeTIL-0255, from women with ovarian cancer: Process validation

“…Ex vivo inhibition of CBL-B with the small molecule inhibitor NX-0255 increases the expansion of stem-like TIL with enhanced in vivo tumor cytotoxicity and persistence compared to conventional TIL expanded in IL-2 alone. 6,7 Here we present our pre-clinical and early manufacturing experience of drug enhanced TIL therapy (DeTIL-0255) in OC. Methods Tumor tissue from N=21 consenting patients undergoing resection for OC across multiple US clinical sites was fragmented and cultured with IL-2 and NX-0255 under research (N=20) or clinical scale (N=1) manufacturing conditions.…”