David R. Stover

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.

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 2047: Discovery and molecular characterization of AGS-15/SLITRK6 as a novel target for antibody-mediated therapeutic development in bladder cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Discovery efforts utilizing suppression subtractive hybridization identified AGS-15/SLITRK6 as a differentially expressed gene in bladder cancer. SLITRK6 encodes a cell surface type I transmembrane protein, a member of the SLITRK family of factors with neuronal function activities. Comprehensive SLITRK6 RNA expression analysis in patient specimens revealed limited restricted normal tissue expression and high frequent expression in bladder cancers, as well as expression in subsets of breast, lung and several other cancer types. Tissue immunohistochemical analysis validated SLITRK6 protein limited expression in normal tissues and high expression levels in cancers. SLITRK6 expression profiling in cancer cell lines and Agensys proprietary patient-derived xenografts (PDXs) identified model systems in relevant cancer indications for further target characterization and antibody therapeutics development. Genomic analyses, including whole exome next generation sequencing, chromosomal aberrations and gene copy number assessments, were performed to evaluate SLITRK6 gene status in model cell lines and PDXs. Thus, we discovered and characterized AGS-15/SLITRK6 as a novel target with high levels and selective expression in tumors, particularly in bladder cancer. This cell surface antigens attractive expression profile is a major determinant of AGS-15/SLITRK6 as a suitable and preferential candidate for antibody drug conjugate therapeutic targeting in bladder cancer treatment and, potentially, in several other cancer indications. Citation Format: Yuriy Shostak, Suzanne Said, Deanna L. Russell, Michael D. Mattie, Mi Sook Chang, Ashley Christensen, Karen Morrison, Kendall Morrison, David Stover, Pia Challita-Eid. Discovery and molecular characterization of AGS-15/SLITRK6 as a novel target for antibody-mediated therapeutic development in bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2013-2047

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys

PurposeNectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models.ProceduresThe biodistribution of [89Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [18F]AGS-22M6 in cynomolgus monkeys was evaluated.ResultsA positive correlation was demonstrated between tumor nectin-4 expression and [89Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [18F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues.Conclusions[89Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

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 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

Abstract 4566: AGS-1C4D4: A fully human anti-PSCA antibody inhibits tumor formation and metastasis in orthotopic models of pancreatic cancer

Prostate stem cell antigen (PSCA) is a cysteine-rich cell surface glycoprotein expressed in about 50% of pancreatic and prostate cancers. AGS-PSCA is a hybridoma-derived fully human IgG1κ monoclonal antibody (MAb) targeting PSCA previously reported to have anti-tumor efficacy in prostate and pancreatic tumor models. AGS-1C4D4 is a CHO-derived antibody generated from the same gene as AGS-PSCA, with similar specificity and binding affinity to PSCA (Kd = 2.0 × 10-10M). Since hybridoma and CHO-derived MAbs displayed disparate glycosylation patterns by mass spectroscopy, we further evaluated their MAb effector functions. AGS-1C4D4 was found to have more potent ADCC activity in vitro on PSCA-expressing pancreatic cell lines, HPAC and Panc0203, using human PBMCs from multiple normal donors. However, both MAbs were similarly effective in mediating CDC on PSCA-expressing recombinant B300.19 cells. Deglycosylation greatly reduced the relative ADCC and CDC activities of both MAbs compared to their intact versions. Additional studies to elucidate the role of the effector functions of AGS-1C4D4 are currently being conducted in vivo using intact and deglycosylated MAbs and will be presented. While neither intact antibody had any direct cytotoxic activity on HPAC cells in vitro, both MAbs significantly inhibited tumor formation, local invasion and metastases to distant sites in orthotopic HPAC xenograft tumor models in vivo. In addition, AGS-1C4D4 significantly inhibited the growth and metastasis of established orthotopic HPAC tumors in combination with Gemcitabine. AGS-1C4D4 is currently being evaluated in a Phase 2 clinical study for pancreatic cancer in combination with Gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4566. doi:10.1158/1538-7445.AM2011-4566

Abstract 2832: Development of AGS-22M6E, a novel antibody drug conjugate (ADC) targeting Nectin-4 for the treatment of solid tumors

Nectin-4 is a type I transmembrane antigen identified by Agensys to be highly expressed at the RNA level in a significant number of bladder and breast cancer specimens. Analysis of protein expression carried out in an extensive panel of tumor specimens by immunohistochemistry confirmed strong to moderate expression levels of Nectin-4 in bladder, breast and pancreatic cancers (40-60%). A subset of lung and ovarian cancers (up to 30%) also showed significant expression levels of this protein. We have generated an antibody-drug conjugate (ADC) targeting Nectin-4, AGS-22M6E. The highly potent microtubule disrupting drug monomethyl auristatin E (MMAE) was conjugated via valine-citrulline cleavable linker to a fully human IgG1k antibody specific for Nectin-4. AGS-22M6E was characterized for binding affinity, species crossreactivity and cytotoxicity in vitro. AGS-22M6E binds to cell surface Nectin-4 with high affinity and crossreacts with Nectin-4 orthologs of cynomolgus monkey, rat and murine origin. Upon binding to its target on the cell surface AGS-22M6E is internalized and induces cell death in vitro in a dose dependent manner. In vivo efficacy of AGS-22M6E was tested in SCID mice on xenograft models of breast, bladder, pancreatic and lung cancers in subcutaneous and orthotopic modalities. Treatment of well established tumor xenografts with the ADC resulted in significant growth inhibition or complete eradication of tumors in various models covering multiple cancer indications. Pharmacokinetics of AGS-22M6E in non-tumor bearing mice was also evaluated and will be discussed. Overall, these data validate AGS-22M6E as an attractive therapeutic drug candidate for multiple solid tumor indications and support further clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2832. doi:10.1158/1538-7445.AM2011-2832