Zili An

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.

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody–Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML

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 monomethyl 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. Mol Cancer Ther; 14(7); 1650–60. ©2015 AACR.

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.

Discoidin Domain Receptor 1 Contributes to Tumorigenesis through Modulation of TGFBI Expression

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

The discovery and preclinical development of ASG-5ME, an antibody drug conjugate targeting SLC44A4 positive epithelial tumors including pancreatic and prostate cancer

Here, we report the development of an antibody–drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line – and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679–87. ©2016 AACR.

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 574: AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status

FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients. Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis. In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration. Citation Format: Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, Rene Hubert, Mary Brodey, Hector Avina, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Donate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 574.

Abstract 2650: Ags67e, an anti-cd37 monomethyl auristatin e antibody (mmae) drug conjugate as a potential therapeutic for non-hodgkin's lymphoma, chronic lymphocytic leukemia and acute myeloid leukemia

We have developed AGS67E, an antibody drug conjugate that targets CD37, a tetraspanin highly expressed on malignant B cells, for the potential treatment of non-Hodgkin9s lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). AGS67E is a fully human anti-CD37 monoclonal IgG2 antibody conjugated to the potent microtubule-disrupting agent, MMAE, via reduced cysteines and the protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl. AGS67E exhibits potent in vitro binding, internalization and cytotoxicity on a variety of NHL, CLL and AML models and patient-derived samples, including CD34+CD38- leukemic stem cells. AGS67E also demonstrates potent anti-tumor responses, including complete tumor regressions in a variety of NHL, CLL and AML xenografts, including Rituxan refractory models and patient-derived samples. In general, CD37 was highly expressed across all models and a strong correlation was observed between the in vitro and in vivo efficacy of AGS67E. To confirm binding of AGS67E in a variety of normal and patient-derived NHL, CLL and AML samples, we developed flow cytometry and immunohistochemistry (IHC) assays which have confirmed reported CD37 expression data in NHL & CLL. In normal hematopoietic cells, AGS67E bound strongly to B cells and to a much lesser extent to monocytes, T cells, neutrophils and NK cells. AGS67E also bound with high and similar affinity to cynomolgus monkey B cells and was equally cytotoxic to these and human B cells. In other normal tissues, AGS67E binding was only evident where lymphoid structures were apparent such as in the spleen and lymph node. With respect to CD37 expression in NHL, CLL and AML, AGS67E was found to bind to >80% of NHL and 100% of CLL and AML samples. Taken together, our findings suggest that AGS67E may serve as a potential therapeutic for NHL, CLL and AML. To our knowledge, this body of work is also the first demonstration that CD37 is well expressed and potentially drug-able in AML. Citation Format: Daniel S. Pereira, Claudia Guevara, Alla Verlinsky, Cyrus Virata, J Hsu Ssucheng, Zili An, Chungying Zhang, Nick Dinh, Hector Avina, Lisa Do, Sher Karki, Joseph Abad, Peng Yang, Jimmy Ou, Karen Morrison, Sing-Ju Moon, Faisal Malik, Liqing Jin, Michael Choi, Christina Wu, Banmeet Anand, Scott Cooper, Ingrid Joseph, Xiao-Chi Jia, Kendall Morrison, Pia Challita-Eid, Fernando Donate, Thomas Kipps, John Dick, David Stover. Ags67e, an anti-cd37 monomethyl auristatin e antibody (mmae) drug conjugate as a potential therapeutic for non-hodgkin9s lymphoma, chronic lymphocytic leukemia and acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2014-2650

Abstract 1086: Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. It is activated by binding to its ligand, collagen and plays a key role in cell survival, adhesion, migration and invasion. Although DDR1 is present in several normal tissues, it is overexpressed in various cancer types, including lung, colon, ovary and breast tumors as well as in gliomas, where it is known to be associated with poor prognosis. The significance of DDR1 in cancer was illustrated using shRNA silencing, which impaired tumor cell growth, both in vitro and in vivo. Using microarray analysis of tumor cells with DDR1 knockdown, we identified an upregulation of TGFBI expression, which was subsequently confirmed at the protein level. TGFBI is a TGFβ induced extracellular matrix protein secreted by tumor cells and has been reported to act as either a tumor promoter or suppressor, depending upon tumor type. Exogenous addition of recombinant TGFBI to tumor cells inhibited clonogenic growth, recapitulating shRNA data. When grown in vivo as xenografts, the DDR1 knockdown cell lines demonstrated a similar phenotype of reduced growth and tumors exhibited an increase in TGFBI protein levels. In summary, our data suggests that DDR1 expression levels influences tumor growth in part through modulation of TGFBI expression. Ongoing studies will help to understand how DDR1 and TGFBI are linked and contribute to tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1086. doi:1538-7445.AM2012-1086