98 NX-0255, a small molecule CBL-B inhibitor, expands and enhances tumor infiltrating lymphocytes (TIL) for use in adoptive cancer immunotherapy

Background Adoptive cell transfer (ACT) of TIL effects durable responses in patients with melanoma and some epithelial tumors. It is thought that poor in vitro cell expansion and inefficient T-cell migration to the tumor limits the broader application of this approach. The E3 ubiquitin ligase, Casitas B-lineage lymphoma b (CBL-B) is expressed in T-cells where it functions as a regulator of immune cell activation, in part by requiring CD28 co-stimulation in addition to T-cell receptor activation. We have developed NX-0255, a highly potent small molecule inhibitor of CBL-B, demonstrating its ability to increase T-cell derived cytokine secretion and proliferation in the presence or absence of co-stimulation. Here, we investigated the effects of NX-0255 on the ex vivo growth and characteristics of human TIL to create drug-enhanced TIL (DeTIL-0255) as an ACT product for treating patients with cancer. Methods TIL from ovary, colon, lung, head and neck, breast, and vulva carcinomas were cultured with IL-2 and compared in two experimental groups: NX-0255 without IL-2, or NX-0255 in combination with IL-2. Following 22 days of culture, cell number, and phenotype were assessed by flow cytometry and single-cell transcriptomics. Results Culturing of TIL in the presence of NX-0255 alone resulted in the expansion of cells, with numbers comparable to that of conventionally cultured TIL with IL-2. The addition of NX-0255 in combination with IL-2 significantly increased the number of cells expanded compared to TIL (n=16, p=0.004). Flow cytometric analysis demonstrated that DeTIL-0255 were significantly less exhausted compared to TIL, as shown by the significant reduction of CD8+ T-cells expressing PD-1 (p=0.02), and co-expressing PD-1+TIM-3+ (p=0.03) and PD-1+LAG-3+ (p=0.03).Furthermore, upon stimulation, the functional capacity of DeTIL-0255 was differentially enhanced, with significant increases in the absolute numbers of CD8+ T-cells expressing intracellular perforin (p=0.001), granzyme-B (p=0.005) and CD107a (p=0.01) when comparing DeTIL-0255 to TIL. An increase of CD8+ T-cells expressing CD137/4-1BB, a biomarker of CD8+ T-cell tumor reactivity was also observed (p=0.03). TCR repertoire and single-cell sequencing analysis demonstrated that DeTIL-0255 had increased TCR diversity and enhanced expression of genes associated with stemness (CD127+,CCR7+,CD62L+) and cytotoxicity (GNLY+,GZMB+,NKG7+). Conclusions Collectively, these data suggest that DeTIL-0255 increases the proportion and absolute numbers of less exhausted CD8+ memory T-cells, enhancing cytolytic T-cell function. Adoptive transfer of DeTIL-0255 may increase persistence and exhibit broader functional activity than conventional TIL, potentially conferring improved anti-tumor activity. Taken together, these data support the clinical development of DeTIL-0255 for the treatment of patients with cancer.