Cancer immunotherapy using immune checkpoint inhibitors has significantly improved the prognosis for patients with various malignancies. However, most patients either do not benefit or eventually develop resistance. To address this unmet need, researchers are actively seeking additional synergistic inhibitory receptors, agonist proteins, and intracellular mediators to prevent or bypass anti-PD1 resistance mechanisms. 

Casitas B lymphoma-b (Cbl-b) is an E3 ubiquitin ligase that plays a crucial role in regulating effector T cell function. In the absence of CD28 costimulation, Cbl-b is considered a key inhibitor of T cell activation. Through complex interactions with signal transducers, Cbl-b inhibits T cell activity and promotes immune tolerance in both the innate and adaptive immune systems. 

Currently, NX-1607 (also known as Cbl-b-IN-3), developed by Nurix Therapeutics, is the only Cbl-b inhibitor that has entered clinical research. Throughout this study, NX-1607 and Cbl-b-IN-3 will be used interchangeably to refer to the same compound. Our studies with Cbl-b-IN-3 have revealed its significant potential to enhance T and NK cell activation, underscoring the broader implications of Cbl-b inhibition in immuno-oncology therapies.

The Cbl-b protein was purified in-house at ICE Bioscience using an E. coli expression system. The purified protein demonstrated high purity and exhibited good activity (Figure 1).

Figure 1. Purified Cbl-b protein at ICE Bioscience (SDS-PAGE data).

Small molecule inhibitors designed to target Cbl-b typically bind between the TKB domain and the LHR domain in its inactive conformation, stabilizing the protein in this non-active closed state. We used SPR analysis to evaluate the binding of Cbl-b-IN-3 to Cbl-b. As shown in Figure 2, Cbl-b-IN-3 bound with greater affinity to Cbl-b than to c-Cbl.

Figure 2. The Cbl-b protein was immobilized on the surface of a sensor chip, and small molecules at various concentrations were passed over the chip’s surface. As they interacted with the immobilized protein, these interactions caused a shift in the resonant angle, which could be monitored. The data were fitted using a 1:1 kinetic binding model. 

The displacement assay, HTRF assay, and ELISA all confirmed the dose-dependent inhibition of Cbl-b by Cbl-b-IN-3 in vitro (Figure 3).

Figure 3. (A) Principle of the displacement assay: The biotin-tagged Cbl-b protein interacts with Cbl-b inhibitors conjugated to a fluorescent group. When the fluorescently labeled inhibitor binds to the Cbl-b protein, the addition of a streptavidin-labeled Tb detection reagent generates a high time-resolved fluorescence resonance energy transfer (TR-FRET) signal. If another inhibitor competes for binding, it displaces the fluorescently labeled inhibitor, significantly reducing the TR-FRET signal. (B) Titration of Cbl-b using a displacement assay. (C) Principle of the ELISA assay: Biotin-tagged Cbl-b is phosphorylated by SRC-ZAP70 in the presence of ATP. When added to an E2-coated ELISA plate, it binds to the ubiquitin-conjugating enzyme E2. After washing to remove unbound components, streptavidin-labeled HRP (horseradish peroxidase) is added. Upon the addition of TMB (3,3’,5,5’-tetramethylbenzidine), a high level of chemiluminescence is detected. If Cbl-b is inhibited by a compound, it will not bind to E2. After washing, all unbound and inhibited Cbl-b is removed, resulting in a very low chemiluminescence value. (D) Measurement of Cbl-b activity using the ELISA. (E) Measurement of Cbl-b activity using an HTRF assay.

The effect of Cbl-b-IN-3 on immune cells was measured using ICE Bioscience’s cell-based assays. We have also developed HiBiT cell lines for screening Cbl-b PROTAC molecules. 

CD25 and CD69 are surface markers that are often used to assess T cell activation. In CD3-stimulated human T cells, inhibition of Cbl-b with Cbl-b-IN-3 promoted CD25 and CD69 expression (Figure 4A). 

IL-2 and IFN-γ are crucial cytokines for T cell activation and the broader immune response. Inhibition of Cbl-b with Cbl-b-IN-3 promoted secretion of IL-2 and IFN-γ in both CD3 antibody-stimulated human T cells (Figure 4B) and mouse T cells (Figure 4D). Cbl-b-IN-3 also promoted the release of IL-2 in CD3 antibody-stimulated human whole blood (Figure 4C). 

Anti-CD3/28 antibodies are often used in vitro to stimulate T cell activation and proliferation. Incubation of CellTrace™ Violet-labeled T cells with anti-CD3/28 antibodies and IL-2 in the presence of Cbl-b-IN-3 significantly enhanced T cell proliferation in a dose-dependent manner (Figure 4E). 

Figure 4. (A) For surface marker detection, Pan-T cells were isolated from PBMCs and incubated with an anti-CD3 antibody in the presence of a Cbl-b inhibitor. CD25 and CD69 expression was detected by FACS. (B to D) Isolated Pan-T cells or blood were incubated with an anti-CD3 antibody in the presence of Cbl-b-IN-3. Cytokine release in the supernatant was detected by ELISA. (E) CellTrace™ Violet-labeled T cells were incubated with anti-CD3/28 antibodies and IL-2 in the presence of a Cbl-b inhibitor. T cell proliferation was detected by flow cytometry.

Several mechanisms within the tumor microenvironment (TME) can impair T cell responses that might otherwise control tumor growth. Notably, the accumulation of adenosine and prostaglandin E2 (PGE2) exerts a direct inhibitory effect on the early stages of T-cell activation, thereby facilitating tumor progression. Additionally, within the TME, TGF-β acts as a suppressor of T-cell proliferation, diminishes their effector functions, and hinders the maturation of T helper cells. 

Adenosine and PGE2 suppressed T cell activation by inhibiting IL-2 and IFN-γ release in isolated T cells from PBMCs. However, a concurrent increase in the levels of IFN-γ and IL-2 cytokines was observed after treatment with Cbl-b-IN-3 combined with adenosine (Figure 5A) or PGE2 (Figure 5B). TGF-β induced suppression of human primary T cell proliferation, and Cbl-b-IN-3 reversed this effect by enhancing cell proliferation (Figure 5C).

Figure 5. Isolated T cells from PBMCs were stimulated with anti-CD3/28 antibodies in the absence or presence of adenosine, PGE2, and TGF-β with or without Cbl-b-IN-3. Levels of IL-2 and IFN-γ were determined. For TGF-β-induced suppression, cell proliferation was detected via the CTG assay.

T-cell exhaustion can result from prolonged exposure of tumor cells to antigens, often occurring rapidly following tumorigenesis. This condition is typically marked by the high expression of inhibitory receptors such as CTLA-4, PD-1, TIM-3, LAG-3, and 2B4 on the T-cell surface. Additionally, it involves the loss of effector functions, including the production of cytokines like IFN-γ, IL-2, and TNF-α, and a reduction in the T-cells’ proliferative capacity. This state is characterized by a weakened T-cell response to antigens, rendering them less effective in providing T-cell help or eliminating target cells. 

We induced exhausted T cells by stimulating them with an anti-CD3/CD28 activator. After 10 days, the increase in PD-1 expression and decrease in IL-2 release confirmed the induction (Figure 6A). The inhibition of Cbl-b with Cbl-b-IN-3 enhances T cell responses to dendritic cells (DCs), promoting the release of IFN-γ (Figure 6B) and TNF-α (Figure 6C) in mixed lymphocyte reaction (MLR) assays.

Figure 6. Exhausted T cells were induced by stimulating them with an anti-CD3/CD28 activator for 10 days. The expression of PD-1 and the release of IL-2 (A) were detected. The activation effect of the Cbl-b inhibitor on the exhausted T cells was assessed by measuring the secretion of IFN-γ (B) and TNF-α (C) in the cell supernatant in a mixed lymphocyte reaction (MLR) assay where exhausted T cells were co-cultured with monocyte-derived dendritic cells. The Cbl-b inhibitor enhances T-cell activation in combination with a PD-1 antibody.

Tumors often develop resistance to the surveillance of the body’s innate immune cells, including natural killer (NK) cells. The E3 ubiquitin ligase Cbl-b has been recognized as a crucial regulator that restricts T cell activation and, more recently, NK cell activation. Consequently, NK cells deficient in Cbl-b exhibit an enhanced ability to combat tumor cells. 

As shown in Figure 7, Cbl-b-IN-3 enhanced NK cell responses to target cells, leading to increased release of TNF-α and IFN-γ from NK cells when co-cultured with K562 cells.

Figure 7. The NK cells isolated from PBMCs were pre-treated with a Cbl-b inhibitor and then co-cultured with K562 cells. The TNF-α (A) and IFN-γ (B) in the cell supernatant were detected to assess the activation of NK cells after Cbl-b inhibition.

Using our ready-to-use assays, we studied the effects of NX-1607 on T cells and NK cells under various conditions. The increase in CD25 and CD69 expression, along with the secretion of IL-2 and IFN-γ in CD3-stimulated human T cells, indicates the activation of T cells by NX-1607. NX-1607 can reverse the suppression of T cell activation exerted by adenosine, PGE2, and TGF-β. Additionally, NX-1607 can activate exhausted T cells in an MLR assay and enhance NK cell activation in a co-culture system. These results suggest a positive role for Cbl-b-IN-3 in cancer immunotherapy. 

In addition to T cell and NK cell functional assays, scientists at ICE Bioscience have developed HiBiT cell lines to better screen Cbl-b PROTAC molecules by measuring their degradation effects. These HiBiT cell lines enable high-throughput screening in the early stages of drug discovery.