Arthur B. Raitano

Monoclonal Antibodies to Six-Transmembrane Epithelial Antigen of the Prostate-1 Inhibit Intercellular Communication In vitro and Growth of Human Tumor Xenografts In vivo

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein highly expressed in primary prostate cancer, with restricted expression in normal tissues. In this report, we show STEAP-1 expression in prostate metastases to lymph node and bone and in the majority of human lung and bladder carcinomas. We identify STEAP-1 function in mediating the transfer of small molecules between adjacent cells in culture, indicating its potential role in tumor cell intercellular communication. The successful generation of two monoclonal antibodies (mAb) that bind to cell surface STEAP-1 epitopes provided the tools to study STEAP-1 susceptibility to naked antibody therapy. Both mAbs inhibited STEAP-1-induced intercellular communication in a dose-dependent manner. Furthermore, both mAbs significantly inhibited tumor growth in mouse models using patient-derived LAPC-9 prostate cancer xenografts and established UM-UC-3 bladder tumors. These studies validate STEAP-1 as an attractive target for antibody therapy in multiple solid tumors and provide a putative mechanism for mAb-induced tumor growth inhibition.

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells.

The cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-γ) were studied in five human pancreatic tumor cell lines. During a 48-h incubation, MIA PaCa-2 cells were most sensitive to rhTNF (56% cytotoxicity, 500 U/ml), T3M4 cells were most sensitive to rhIFN-γ (54% cytostasis, 250 U/ml), and ASPC-1 and COLO 357 cells were most sensitive to the combination of rhTNF and rhIFN-γ (56 and 55% cytotoxicity, respectively, 250 U/ml of each cytokine). The PANC-1 cells were relatively insensitive to either the individual or the combined effects of these cytokines. All five cell lines exhibited specific, highaffinity receptors for 125I-labeled rhTNF (480–8,610 sites/cell) and rhIFN-γ (2,0504,280 sitedcell). The MIA PaCa-2 cells, which were the most sensitive to the inhibitory effects of rhTNF, also possessed the largest number of 125I rhTNF receptors; all other cell lines had a relatively low number of binding sites and low sensitivity. In contrast, no direct correlation could be made between the number of IFN-y binding sites and inhibitory sensitivity in any of the cell lines. Incubation of COLO 357 cells at 37°C with either 125I rhTNF or 125I rhINF-γ led to internalization of the respective 125I-labeled ligand. Our findings document the presence of cytokine receptors in human pancreatic carcinoma cells and suggest that postreceptor events rather than differences in receptor number or affinity more likely govern the responsiveness of pancreatic cancer cells to TNF and IFN-7.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models

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. ©2016 AACR.

Development of ASG-15ME, a Novel Antibody–Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody–drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301–10. ©2016 AACR.

Long-term effect of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells

SummaryThe long-term cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-7) were studied in five human pancreatic carcinoma cell lines. The pancreatic tumor cell lines were heterogenous in their response to individual cytokines. During a 7-d incubation, MIA PaCa-2 cells were more sensitive to rhTNF than rhIFN-γ, whereas ASPC-1, T3M4, and COLO 357 cells were more sensitive to rhIFN-γ than rhTNF. PANC-1 cells were relatively insensitive to both cytokines. In a previous report, we demonstrated synergistic cytotoxic effects of rhTNF and rhIFN-γ during 48-h incubations in ASPC-1 and COLO 357 cells (Raitano et al,Pancreas, April, 1990). In this study, a 7-d treatment with both rhTNF and rhIFN-γ did not produce synergistic effects in any of the cell lines. However, a 24-h treatment with rhIFN-γ, followed by removal of the cytokine, markedly increased the long-term cytotoxic/cytostatic effects of rhTNF in ASPC-1, COLO 357, and T3M4 cells. In contrast, a similar pretreatment with rhTNF did not increase the long-term cytotoxic/cytostatic effects of rhIFN-γ in any of the cell lines. These data suggest that, in some human pancreatic carcinoma cell lines, rhIFN-γ may be especially useful in the long-term suppression of growth. Furthermore, brief pulses of rhIFN-γ may also be especially efficacious when followed by a subsequent prolonged exposure of cells to rhTNF.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppress tumor necrosis factor secretion in vitro.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppressed peripheral blood leukocyte secretion of tumor necrosis factor (TNF). The suppressive activities of all three fluids had similar characteristics: each was heat and acid stable, removed by adsorption on immobilized lectins, and abrogated the stimulatory effect of interferon‐γ. Inhibition of leukocyte TNF secretion was observed only when either serum or conditioned medium was added to leukocytes at the initiation of culture; delaying the addition by 2 h failed to suppress cytokine secretion. Suppression by all fluids was also found to be reversible by washing cells free of suppressive activity. Although serum, tumor, and fibroblast culture supernatants inhibited cytokine secretion, they failed to alter the cytotoxic activity of recombinant human TNF on murine L929 cells. This study suggests that factors which can inhibit TNF secretion are present in human blood and are secreted by both fibroblasts and tumor cells. These suppressive factors may play an important role in the regulation of TNF secretion and cytokine homeostasis.

Abstract 4393: ASG-5ME is a novel antibody drug conjugate (ADC) for treating prostate cancers

AGS-5 is a novel transmembrane antigen discovered by Agensys using transcriptional profiling that is highly expressed in a variety of epithelial tumors. A fully human IgG2k monoclonal antibody that binds to AGS-5 with high affinity was conjugated with the anti-microtubulin drug, monomethyl auristatin E (MMAE), via a conditionally labile valine-citrulline (vc) maleimidocaproyl linker to yield an average drug: antibody ratio of 3.7. Following cell surface binding, ASG-5ME is internalized and trafficked through the endocytic pathway, where proteolytic cleavage releases free active drug that subsequently binds and disrupts microtubules. We evaluated the expression of AGS-5 protein in a large number of prostate patient tumors using IHC. ASG-5ME was evaluated for its cell cytoxicity in vitro, and its anti-tumor activity was investigated on different established xenograft tumors, using both patient-derived and cell line models of prostate cancer. The pharmacokinetics of ASG-5ME in mice was also evaluated. Our studies indicated AGS-5 protein was expressed in more than 95% of primary prostate cancer specimens and distant metastases, and on more than 90% and 80% of pancreatic and gastric cancer specimens, respectively. ASG-5ME, bound with high affinity (Kd = 0.5 nM) to both human and cynomolgus monkey AGS-5 and mediated potent dose dependent cell cytotoxicity in vitro. ASG-5ME showed significant long term tumor regression and eradication of multiple established prostate cancer xenografts, including those grown orthotopically. Phamacokinetic (PK) analysis of ASG-5ME in mice indicated that the ADC was stabile with long T1/2 of ∼12 days. When considered together these data indicate that ASG-5ME is a promising therapeutic ADC for the treatment of prostate and other AGS-5 expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4393.

Abstract 2436: AGS-16M8F is a novel antibody drug conjugate (ADC) for treating renal and liver cancers

AGS-16 is a novel transmembrane antigen discovered by Agensys using transcription profiling and is expressed in kidney, liver and other cancers. A fully human IgG2k monoclonal antibody that binds to AGS-16 with high affinity was conjugated to the anti-microtubulin drug monomethyl auristatin F (MMAF) via a noncleavable maleimidocaproyl (mc) linker to yield the antibody drug conjugate, AGS-16M8F. AGS-16M8F was evaluated for binding affinity, species cross reactivity and cell cytotoxicity in vitro. Its’ anti-tumor activity was investigated using multiple established patient-derived and cell line models of renal clear cell carcinomas. The pharmacokinetics (PK) of AGS-16M8F was evaluated in mice. In addition, the expression of AGS-16 protein in patient kidney and liver cancer specimens was confirmed using IHC. AGS-16M8F bound with high affinity (Kd = 0.3 nM) to both human and cynomolgus monkey AGS-16 orthologs. Following cell surface binding, AGS-16M8F was internalized and trafficked to lysosomes leading to catabolism and release of active drug metabolite. AGS-16M8F mediated potent cell cytotoxicity in vitro and led to significant and prolonged growth inhibition or complete eradication of multiple established renal cancer xenograft models, including those grown orthotopically. Pharmacokinetic analysis of AGS-16M8F in mice demonstrated ADC stability. IHC analysis confirmed that AGS-16 was highly expressed in over 80% of renal clear cell cancers and 33% of hepatocellular carcinomas. When considered together these data indicate that AGS-16M8F is a promising therapeutic ADC for the treatment of renal and other AGS-16 expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2436.