A Bispecific METxMET Antibody-Drug Conjugate with Cleavable Linker Is Processed in Recycling and Late Endosomes.

Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

A Biparatopic Antibody That Modulates MET Trafficking Exhibits Enhanced Efficacy Compared with Parental Antibodies in MET-Driven Tumor Models.

PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Neuroendocrine-derived peptides promote prostate cancer cell survival through activation of IGF-1R signaling.

BACKGROUND: Neuroendocrine (NE) cells promote the progression of prostate cancer to a castration-resistant state through the production of paracrine growth factors. We have demonstrated this principle using in vitro and in vivo proliferative endpoints; however, the contributions of NE-derived pro-survival factors and anti-apoptosis to this phenomenon have not been thoroughly investigated. METHODS: Here, we utilized conditioned-medium (CM) from LNCaP cells, engineered to undergo NE differentiation, and examined its effects on PC3 and LNCaP cell survival. RESULTS: Statistically significant changes in clonogenic survival, Annexin V staining, PARP cleavage and trypan blue positivity of approximately twofold were observed in the presence of NE-derived CM relative to control-CM for both LNCaP and PC3 cells. These changes were partially abrogated by antagonists of the neuropeptides neurotensin, bombesin, and PTHrP. Selective inhibitors of IGF-1R, EGFR or Src caused significant and nearly complete blockade of prostate cancer cell survival due to NE secretions. Similar increases in cell survival were observed for LNCaP or PC3 cells treated with NE-derived medium in the presence of docetaxel. Increased phosphorylation of IGF-1R, following treatment with NE-derived medium, was accompanied by decreased protein tyrosine phosphatase, receptor type F (PTPRF) mRNA, and protein levels. Overexpression of PTPRF decreased cell survival, the amplitude and duration of IGF-1R phosphorylation, and enhanced PARP cleavage in the presence of NE-derived medium. CONCLUSIONS: These data support the hypothesis that NE-derived factors act upon prostate cancer cells to stimulate pro-survival signaling and describe a novel mechanism of cross-talk between NE-derived factors and IGF-1R, mediated in part by PTPRF.

Inhibition of neurotensin receptor 1 selectively sensitizes prostate cancer to ionizing radiation.

Radiotherapy combined with androgen depletion is generally successful for treating locally advanced prostate cancer. However, radioresistance that contributes to recurrence remains a major therapeutic problem in many patients. In this study, we define the high-affinity neurotensin receptor 1 (NTR1) as a tractable new molecular target to radiosensitize prostate cancers. The selective NTR1 antagonist SR48692 sensitized prostate cancer cells in a dose- and time-dependent manner, increasing apoptotic cell death and decreasing clonogenic survival. The observed cancer selectivity for combinations of SR48692 and radiation reflected differential expression of NTR1, which is highly expressed in prostate cancer cells but not in normal prostate epithelial cells. Radiosensitization was not affected by androgen dependence or androgen receptor expression status. NTR1 inhibition in cancer cell-attenuated epidermal growth factor receptor activation and downstream signaling, whether induced by neurotensin or ionizing radiation, establish a molecular mechanism for sensitization. Most notably, SR48692 efficiently radiosensitized PC-3M orthotopic human tumor xenografts in mice, and significantly reduced tumor burden. Taken together, our findings offer preclinical proof of concept for targeting the NTR1 receptor as a strategy to improve efficacy and outcomes of prostate cancer treatments using radiotherapy.

LKB1 and Src: antagonistic regulators of tumor growth and metastasis.

In this issue of Cancer Cell, Carretero and colleagues report that Src and FAK signaling pathways are activated in lung cancers when the tumor suppressor LKB1 is deleted. These findings suggest the use of unique combinatorial therapies for treatment of lung cancers.