Daulet Satpayev

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.

Pet imaging of cytotoxic human T cells using an 89Zr-labeled anti-CD8 minibody.

Meeting abstracts Major challenges to advancing the new wave of cancer immunotherapies are to select patients who will respond to single immunotherapies, identify those who need more tailored combination regimens, and then, determine early during treatment whether the therapy is working. “

Abstract LB-188: Sensitivity of 89Zr-labeled anti-CD8 minibody for PET imaging of infiltrating CD8+ T cells

Background- The ability to monitor CD8 positive tumor infiltrating lymphocytes (TILs) in vivo is important for evaluating response to immunotherapies and assisting in the development of more effective immune cell targeted single and combination therapies. “ImmunoPET” imaging of tumor infiltrating T cells can provide a specific and sensitive modality to aid selection of patients for specific immunotherapy regimens and determine whether the therapy is working. Here, we report initial results to define the number of CD8+ T cells that can be detected with 89 Zr-Df-IAB22M2C, an anti-CD8 immunoPET probe, using different animal models Methods- IAB22M2C, a humanized anti-CD8 minibody, was conjugated with desferrioxamine (Df) and radiolabeled with 89 Zr. NOD scid mice were implanted with varying ratios of CD8+ T and tumor cell admixtures either intramuscularly (IM) without Matrigel or subcutaneously (SC) with Matrigel. One or six days later, CD8+ T-cells were visualized with 89 Zr-Df-IAB22M2C. The same probe was used to detect CD8+ T cells in NSG TM mice engrafted with human PBMCs for 1 and 4 weeks to monitor the temporal progression of Graft versus Host Disease (GvHD). Results - CD8+ T cells implanted in the muscles of mice were imaged one day later and SC implanted Matrigel plugs imaged 6 days later. Both approaches yielded similar results and indicated that the lower limit of detection was between 1.6 and 4 million CD8+ T cells in a volume of ∼480 mm 3 in the presence of normal tissue background activity. The sensitivity of detection increased 10-fold when ex vivo radiolabeled CD8+ T cells were implanted SC with Matrigel. NSG TM mice engrafted with human PBMCs provide a reliable model for xenogeneic T cell driven Graft versus Host Disease (GvHD). Human CD8 + T cells were readily detectable in the spleens of mice 1 week post PBMC engraftment using 89 Zr-Df-IAB22M2C. As GvHD progressed 4 weeks later, expansion and trafficking of the engrafted T cells to extra-lymphoid tissues including lungs could be followed. Terminal biodistribution showed a 2-3 fold increase in radioactivity uptake in lungs by week 4 post-engraftment; a result that was confirmed by IHC analysis. T cell enumeration and IHC analyses are in progress to further define the sensitivity range using an optimal dose and specific activity of 89 Zr-Df-IAB22M2C. Conclusion- These studies show that the lower limit of CD8+ T cell detection by 89 Zr-Df-IAB22M2C is between 1.6-4.0 million cells in the presence of normal tissue background activity and that the probe can be used to monitor CD8+ T cell trafficking in a GvHD model in vivo. 89 Zr-Df-IAB22M2C has sensitivity properties that may enable the detection of CD8+ T cells in human tumors. Clinical trials with 89 Zr-Df-IAB22M2C in melanoma patients will commence later this year. Citation Format: Tove Olafsen, Ziyue Karen Jiang, Jason Romero, Charles Zamilpa, Filippo Marchioni, Green Zhang, Michael Torgov, Daulet Satpayev, Jean M. Gudas. Sensitivity of 89 Zr-labeled anti-CD8 minibody for PET imaging of infiltrating CD8+ T cells. [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 LB-188.

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