Targeting Cbl-b in cancer immunotherapy

Abstract

Cancer immunotherapy with immune-checkpoint blockade has improved the outcomes of patients with various malignancies, yet a majority do not benefit or develop resistance. To address this unmet need, efforts across the field are targeting additional coinhibitory receptors, costimulatory proteins, and intracellular mediators that could prevent or bypass anti-PD1 resistance mechanisms. The CD28 costimulatory pathway is necessary for antigen-specific T cell activation, though prior CD28 agonists did not translate successfully to clinic due to toxicity. Casitas B lymphoma-b (Cbl-b) is a downstream, master regulator of both CD28 and CTLA-4 signaling. This E3 ubiquitin ligase regulates both innate and adaptive immune cells, ultimately promoting an immunosuppressive tumor microenvironment (TME) in the absence of CD28 costimulation. Recent advances in pharmaceutical screening and computational biology have enabled the development of novel platforms to target this once 'undruggable' protein. These platforms include DNA encoded library screening, allosteric drug targeting, small-interfering RNA inhibition, CRISPR genome editing, and adoptive cell therapy. Both genetic knock-out models and Cbl-b inhibitors have been shown to reverse immunosuppression in the TME, stimulate cytotoxic T cell activity, and promote tumor regression, findings augmented with PD1 blockade in experimental models. In translating Cbl-b inhibitors to clinic, we propose specific gene expression profiles that may identify patient populations most likely to benefit. Overall, novel Cbl-b inhibitors provide antigen-specific immune stimulation and are a promising therapeutic tool in the field of immuno-oncology.

Keywords: CTLA-4 antigen; drug therapy, combination; immunotherapy; review; therapies, investigational.

Figure 1

(A) T cell stimulation via CD28 costimulatory signaling cascade. (B) Depiction of intracellular Cbl-b signaling in the tumor microenvironment. CD28 costimulation inhibits downstream Cbl-b signaling, while CTLA4 stimulation promotes Cbl-b activity. On activation, Cbl-b ubiquinates several key proteins that inhibit effector function, promoting an immunosuppressive phenotype. *Red blocked arrows indicate direct ubiquitination. APC, antigen presenting cell; Cbl-b, Casitas B lymphoma-b; MHCII, major histocompatibility complex II.

Figure 2

(A) Comparison of T cell-inflamed gene set enrichment score (Tinfl) versus CBLB gene expression across select TCGA tumor samples. Red box highlights tumor samples postulated to receive greatest benefit from Cbl-b inhibition versus ICI therapy±Cbl-b inhibition (purple box). Median expression values denoted by orange (higher CBLB), blue (lower CBLB), gray (lower Tinfl), and transparent (higher Tinfl). TMB, tumor mutational burden. Spearman’s correlation coefficient and p value are shown at the left corner of the figure. Methods: Gene set enrichment analysis (GSEA) was performed using normalized and log2-transformed RNAseq gene expression data from the TCGA. All analyses were performed using Bioconductor packages in R (V.4.0.3). (B) Heatmap of various IO targets (right) based on quartile expression of TCGA metastatic melanoma samples. CD274 (PDL1) data were added to bottom of heatmap for visual reference. Methods: Unsupervised hierarchical clustering performed across tumor samples; quartile expression calculated across all samples per gene. All analyses were performed using R (V.4.0.3). (C) Expression of fifteen IO targets versus CBLB expression in TCGA metastatic melanoma samples. Vertical (purple) lines denote CBLB quartiles; horizontal (dashed) lines denote IO target quartiles per facet. Methods: Faceted scatterplot with colors denoting quartile expression of each gene in R (V.4.0.3). Cbl-b, Casitas B lymphoma-b; ICI, immune checkpoint inhibitor; IO, immuno-oncology.