Background
Casitas B-lineage lymphoma b (CBL-B) is an E3 ubiquitin ligase, which is highly expressed in peripheral T cells.1 CBL-B knockout T cells upregulate TCR-mediated T cell activation regardless of CD28,2 and have shown to increase IL-2 and IFN- levels.3 4 Loss of CBL-B can prevent CD8+ T cell exhaustion and increase immune cells infiltration in tumors that suppressed tumor growth in mouse models.5–7 The YF550-C1, which is our developed small molecule that targeted CBL-B protein, showed dramatically immunotherapy effects in cancer.
Methods
The CBL-B knockout T cells isolated from spleen of CBL-B knockout mice stimulated with anti-CD3 or anti-CD3/28 for 48 hours. Human PBMC or Pan T were stimulated with anti-CD3 or anti-CD3/28 in the presents of 8 titration doses of CBL-B inhibitors, and the culture medium were used for detection of IL-2 or IFN- by ELISA. Anti-CD3 was used to stimulate T cell activation in vivo, and YF550-C1 was orally administrated with three doses of YF550-C1 before stimulation. The activation was evaluated by measuring serum concentration of IL-2 and IFN-, or the ratio changes of CD69+ T cells. Syngeneic and PD-1 resistant PDX (patient-derived-xenograft) tumor mouse models was used to evaluate tumor growth.
Results
CBL-B deficiency suppressed tumor growth in mouse tumor models and enhanced cytokines release in T cells upon TCR engagement. (figure 1). YF550-C1 elevated IL-2 and IFN- in TCR stimulated human PBMCs. (figure 2). YF550-C1 alleviated PGE2-mediated immunosuppression of human T cell activation (figure 3). YF550-C1 enhanced cytokines release and T cells activation in in vivo anti-CD3 stimulated mouse model. (figure 4). Single-agent YF550-C1 treatment was sufficient to suppress tumor growth in syngeneic mouse models. (figure 5). Single-agent YF550-C1 treatment was sufficient to suppress PD-1/PD-L1 resistant NSCLC patient-derived tumor growth. (figure 6). Combination of YF550-C1 and anti-PD-L1 treatment showed synergy effect in mouse model. (figure 7)
Conclusions
YF550-C1 increases cell activation, inhibits T cell immunosuppression and enhances antitumor activity in multiple preclinical tumor models. YF550-C1 is a promising candidate for cancer treatment as monotherapy or in combination with PD-L1 blockade.
Acknowledgements
YF550-C1 was developed by Zhuhai Yufan Biotechnologies Co., Ltd and supported by MingMed Biotechnology Co., Ltd.
References
Gu H, et al. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nature immunology, 2002;3(12):1192–1199.
Bachmaier K, et al. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature, 2000;403(6766):211–6.
Han S, et al. Overproduction of IL-2 by Cbl-b deficient CD4(+) T cells provides resistance against regulatory T cells. Oncoimmunology, 2020;9(1):1737368.
Han S, et al. Overproduction of IFNgamma by Cbl-b-Deficient CD8+ T Cells Provides Resistance against Regulatory T Cells and Induces Potent Antitumor Immunity. Cancer Immunol Res, 2022;10(4):437–452.
Chiang JY, et al. Ablation of Cbl-b provides protection against transplanted and spontaneous tumors. Journal of Clinical Investigation, 2007;117(4):1029–1036.
Kumar J, et al. Deletion of Cbl-b inhibits CD8+ T-cell exhaustion and promotes CAR T-cell function. Journal for ImmunoTherapy of Cancer, 2021;9(1):e001688.
Loeser S, et al. Spontaneous tumor rejection by cbl-b-deficient CD8+ T cells. Journal of Experimental Medicine, 2007;204(4):879–891.
Ethics Approval
The animal studies were approved by institute of animal care union and committee in MingMed Biotechnology Co., Ltd. (Ethics No. IACUC-20221111–01).
Abstract 1400 Figure 1
CBL-B deficiency suppressed tumar growth in mouse tumor models and enhanced cytokines release in T cells upon TCR engagement
Abstract 1400 Figure 2
YF550-C1 elevated IL-2 and IFN-y in TCR stimulated human PBMCs
Abstract 1400 Figure 3
YF550-C1 alleviated PGE2-mediated immunosuppression of human T cell activation
Abstract 1400 Figure 4
YF550-C1 enhanced cytokines release and T cells activation in in vivo anti-CD3 stimulated mouse model
Abstract 1400 Figure 5
Single-agent YF550-C1 treatment was sufficient to suppress tumor growth in syngeneic mouse models
Abstract 1400 Figure 6
Single-agent YF550-C1 treatment was sufficient to suppress PD-1/PD-L1 resistant NSCLC patient-derived tumor growth
Abstract 1400 Figure 7
Combination of YF550-C1 and anti-PD-L1 treatment showed synergy effect in mouse model