Despite breakthroughs achieved with cancer checkpoint blockade therapy (CBT), many patients do not respond to anti–programmed cell death-1 (PD-1) due to primary or acquired resistance. Human tumor profiling and preclinical studies in tumor models have recently uncovered transforming growth factor–β (TGFβ) signaling activity as a potential point of intervention to overcome primary resistance to CBT. However, the development of therapies targeting TGFβ signaling has been hindered by dose-limiting cardiotoxicities, possibly due to nonselective inhibition of multiple TGFβ isoforms. Analysis of mRNA expression data from The Cancer Genome Atlas revealed that TGFΒ1 is the most prevalent TGFβ isoform expressed in many types of human tumors, suggesting that TGFβ1 may be a key contributor to primary CBT resistance. To test whether selective TGFβ1 inhibition is sufficient to overcome CBT resistance, we generated a high-affinity, fully human antibody, SRK-181, that selectively binds to latent TGFβ1 and inhibits its activation. Coadministration of SRK-181-mIgG1 and an anti–PD-1 antibody in mice harboring syngeneic tumors refractory to anti–PD-1 treatment induced profound antitumor responses and survival benefit. Specific targeting of TGFβ1 was also effective in tumors expressing more than one TGFβ isoform. Combined SRK-181-mIgG1 and anti–PD-1 treatment resulted in increased intratumoral CD8+ T cells and decreased immunosuppressive myeloid cells. No cardiac valvulopathy was observed in a 4-week rat toxicology study with SRK-181, suggesting that selectively blocking TGFβ1 activation may avoid dose-limiting toxicities previously observed with pan-TGFβ inhibitors. These results establish a rationale for exploring selective TGFβ1 inhibition to overcome primary resistance to CBT.
Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.
IntroductionRenal cell carcinoma (RCC) is characterised by high lethality in advanced stages. Vastly resistant to radio- and chemotherapy, RCCs respond to targeted therapies such as tyrosine kinase inhibitors, mTOR antagonists and immune checkpoint inhibitors. However, limited response rates and emerging resistance mechanisms demand new treatment strategies.Scaffolding protein NEDD9 (neural precursor cell expressed, developmentally down regulated 9) is frequently overexpressed in various cancer types and associated with tumour aggressiveness. In vivo,high NEDD9 expression is associated with adverse clinical outcome and in vitro, NEDD9 promotes aggressiveness in RCC cells. Thus, we systematically characterised the role of NEDD9 in RCC both in vitro and in vivo with the goal to establish basis for new treatment strategies.Material and methodsUsing RNAi, we generated NEDD9-proficient and -deficient syngeneic (RENCA) and xenograft (786-O) tumour models via subcutaneous and orthotopic transplantations as well as tail vein injections. Tumour growth was monitored dynamically using calliper and MRI imaging. Extensive in vitro studies were performed to analyse NEDD9 and its associated oncogenic signalling cascades including Western blot, proliferation, migration and gene expression analyses. Tissue microarrays (TMAs) of 92 RCC patients were stained for NEDD9 and analysed using automated quantitative analysis (AQUA) technology to associate NEDD9 protein expression with clinical outcome.Results and discussionsNEDD9 is highly expressed in RCC cells and NEDD9 depletion significantly impairs migration and proliferation in both human and murine RCC cells. This is accompanied by reduced signalling of oncogenic pathways, particularly activation of ERK1/2. NEDD9 tumour promoting role was confirmed in vivo where NEDD9-deficient murine RCC cells exhibited significantly reduced tumour growth after subcutaneous and orthotopic syngeneic transplantation as well as inhibited lung metastatic seeding capacity after tail vein injection.In human RCC cells, only NEDD9 down-regulation was sufficient to completely abolish tumour growth in subcutaneous, orthotopic and lung seeding xenograft models. In line with our in vitro and in vivo results, we found high NEDD9 expression in human RCC tissue to be significantly associated with shorter overall survival.ConclusionWe show for the first time a crucial role for NEDD9 in RCC tumour progression in vivo, which potentially offers new therapeutic approaches.
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