The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degradation of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide molecules, we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degradation. The homodimerized compound 15a was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different cell lines, prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the molecular mechanism of thalidomide analogues.

Small-molecule heterobifunctional degraders can effectively control protein levels and are useful research tools.

No abstract

The use of T-cell engagers (TCEs) to treat solid tumors is challenging, and several have been limited by narrow therapeutic windows due to substantial on-target, off-tumor toxicities due to the expression of low levels of target antigens on healthy tissues. Here, we describe TNB-928B, a fully human TCE that has a bivalent binding arm for folate receptor alpha (FRα) to selectively target FRα overexpressing tumor cells while avoiding the lysis of cells with low levels of FRα expression. The bivalent design of the FRα binding arm confers tumor selectivity due to low-affinity but high-avidity binding to high FRα antigen density cells. TNB-928B induces preferential effector T-cell activation, proliferation, and selective cytotoxic activity on high FRα expressing cells while sparing low FRα expressing cells. In addition, TNB-928B induces minimal cytokine release compared to a positive control TCE containing OKT3. Moreover, TNB-928B exhibits substantial ex vivo tumor cell lysis using endogenous T-cells and robust tumor clearance in vivo , promoting T-cell infiltration and antitumor activity in mouse models of ovarian cancer. TNB-928B exhibits pharmacokinetics similar to conventional antibodies, which are projected to enable favorable administration in humans. TNB-928B is a novel TCE with enhanced safety and specificity for the treatment of ovarian cancer.

The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide are all approved for the treatment of multiple myeloma. IMiDs bind cereblon (CRBN), a substrate adaptor for the CRL4 E3 ubiquitin ligase (CRL4CRBN). In multiple myeloma, IMiDs enhance the binding of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) to CRL4CRBN, leading to their ubiquitination and degradation. Depletion of IKZF1 and IKZF3 results in growth inhibition in multiple myeloma cells. In addition, IMiDs can block the physiologic function of CRBN what has been shown to contribute to the anti-proliferative effects as well as other properties of the drug. Recently, IMiDs were exploited for the generation of Proteolysis Targeting Chimeras (PROTACs). These molecules link the IMiD structure to another small molecule that binds a protein of interest (POI). Such IMiD-based PROTACs are capable to guide the CRBN-CRL4 E3 ligase to the POI, resulting in its ubiquitination and degradation. Here, we designed pomalidomide-based homobifunctional PROTACs and investigated their ability to induce self-directed CRBN ubiquitination and degradation (Figure 1A). We evaluated different attachment strategies, modifications and linker lengths and tested the effect of a series of homo-dimeric compounds on their potency to deplete CRBN protein levels. The homodimeric compound 15a with a linker length of 8 atoms was identified as the most potent CRBN degrader with a minimal remaining effect on IKZF1 (Figure 1B). Homodimeric PROTACs with longer linkers exhibited a weaker capability for CRBN degradation and had a more potent effect on IKZF1 protein levels. The effect of compound 15a on CRBN was blocked after pre-treatment with a proteasome inhibitor or MLN4924, a neddylation activating enzyem inhibitor that blocks Cullin E3 ligases. Co-immunoprecipitation revealed that 15a induces interaction of two CRBN molecules. The homo-PROTAC 15a was active at low concentrations below 100 nM and had long-lasting effects on the intracellular CRBN level (Figure 1B). Applying global proteome analyses in the multiple myeloma cell line MM1.S demonstrated that PROTAC 15a specifically induced degradation of CRBN and had only weak effects on IKZF1 and IKZF3 and no effect on the other members of the CRL4 ligase family, including DDB1 and CUL4A which are in close proximity to CRBN in the CRL4CRBN complex. CRBN inactivation by our compounds had no effect on proliferation of different multiple myeloma cell lines. Pre-treatment with Homo-PROTAC 15a prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide and lenalidomide on multiple myeloma cell growth. This was consistent with genetic inactivation of CRBN by CRISPR/Cas9. In conclusion, we generated the first chemical inhibitors of CRBN that can serve as a useful tool for future biomedical investigations on CRBN-related signaling. These compounds will also help to discriminate whether an IMiD effect depends on CRBN-mediated targeted degradation of neo-substrates or from blocking the physiologic function of CRBN. Furthermore, our data confirm the essential role of CRBN in IMiD activity in multiple myeloma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.