The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. Ó2016 AACR.
Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.
Despite its high incidence and mortality rate, the molecular mechanisms underlying the oncogenesis and progression of prostate cancer are poorly understood. Recent studies suggest that hereditary prostate cancer is a complex disease involving multiple susceptibility genes, different models of Mendelian inheritance, incomplete penetrance and varying population ethnicity frequencies. 1,2 Recently a novel candidate susceptibility gene, ELAC2 (HPC2), was identified on chromosome 17p. 3 Two pedigree-specific high-risk variants, 1641insG and Arg781His, and 2 common moderate-risk variants, Ser217Leu and Ala541Thr, were identified. The results on ELAC2 mutations presented in the literature so far are not always consistent with a major role for ELAC2 as a prostate cancer susceptibility gene. 4 -6 However, association of the common missense variants in ELAC2 with sporadic prostate patients has been described in several independent studies. 5,[7][8][9] The ELAC2 gene encodes a protein of 826 amino acids and is a member of an uncharacterized, conserved gene family; orthologs have been identified in bacteria, S. cerevisiae, S. pombe and C. elegans. 3 Alignment of the sequences revealed a conserved histidine-motif, reminiscent of the one found in metallo-beta-lactamases. A recently published study on the ElaC gene product from E. Coli identifies it as a binuclear zinc phosphodiesterase. 10 The family shares amino acid similarity with the PSO2/SNM1 family of DNA interstrand crosslink repair proteins. Furthermore, a potential ATP/GTP-binding motif suggests that ELAC2 may possess ATPase/GTPase activity. The function of this protein has yet to be elucidated, and the functional significance of the cancer-associated missense mutations is not known.In our study, we investigate the properties of the protein encoded by the candidate prostate cancer susceptibility gene, ELAC2, and demonstrate a physical interaction with components of the ␥-tubulin complex.
CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent mono-methyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.
Ionizing radiation leads to rapid stabilization and activation of the p53 tumor suppressor. Previous reports demonstrate that murine p19ARF cooperates with p53 in the cellular response to gamma irradiation. Here, we show that endogenous ARF sequentially interacts with p53 and MDM2 following irradiation of primary human and mouse embryonic fibroblasts. Shortly after irradiation, p14ARF binds p53 independently of MDM2. As nuclear pools of p53 decline, endogenous p14ARF co-immunoprecipitates with MDM2 and is localized within the nucleolus. Interestingly, p14ARF nucleolar localization during this response is abrogated in cells lacking functional p53. Taken together, our data suggest that human and murine ARF contribute to the mammalian DNA damage response.
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