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Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1–resistant population was generated. T-DM1–resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non–cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)–positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243–53. ©2017 AACR.

Supplementary Figure S1: N87 and N87-TM cells are similarly sensitive to unconjugated DM1-SMe; Supplementary Figure S2: Trastuzumab binding to N87 and N87-TM cells; Supplementary Figure S3: Generation and characterization of T-DM1-resistant HCC1954-TM and BT474-TM cells; Supplementary Figure S4: Relative resistance profiles of N87-TM to N87 cells to T-ADCs, unconjugated payloads and standard-of-care chemotherapeutics; Supplementary Figure S5: Distribution of T-ADCs in N87-TM tumors; Supplementary Figure S6: N87-TM cells are cross-resistant to a T-ADC conjugated to a cleavable linker with a cysteine-capped auristatin analog; Supplementary Figure S7: N87-TM cells internalize T-ADC into non-low pH intracellular compartments; Supplementary Figure S8: CAV1-GFP expression causes intracellular caveolae formation capable of internalizing T-ADCs in N87 cells; Supplementary Figure S9: CAV1 knockdown is not sufficient to re-sensitize N87-TM cells to T-DM1; Supplementary Figure S10: Non-cleavable and cleavable T-ADCs co-localize with CAV1 similarly in N87 and N87-TM cells; Supplementary Table S1. Cytotoxicity values of Trastuzumab and isotype-control ADCs in HT29 cells, a HER2-negative cell line

Gastrointestinal malignancies, including colorectal cancer (CRC) remain an area of high unmet need. Here we demonstrate tumor selective and potent in vitro and in vivo efficacy with PF-07062119, a novel T cell engaging CD3 bispecific against tumors expressing the Guanylyl Cyclase C (GUCY2C) receptor, a target expressed in more than 90% of CRC, and in other gastrointestinal cancers. Additionally, to address immune evasion mechanisms, combinations of the GUCY2C-CD3 bispecific are explored with immune checkpoint blockade therapy, as well as with chemotherapeutic and anti-angiogenic agents that could enhance immune infiltration into tumors. Our preclinical in vivo data demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ϵ bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T cell mediated anti-tumor activity in several human xenograft models of CRC, using adoptive transfer of human T cells, including those with KRAS and BRAF mutations, as well as in models with syngeneic tumors in immunocompetent human CD3 transgenic mice. PF-07062119 activity was further enhanced when combined with anti PD-1/PD-L1 treatment or in combination with chemotherapy or anti-angiogenic therapy. A combination of immunohistochemistry, flow cytometry and CyTOF analyses was used to demonstrate the mechanism of action of PF-07062119 in single agent and combination studies in vivo. Toxicity and pharmacokinetic studies were done in cynomolgus macaques and indicated a monitorable and manageable toxicity profile. These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in CRC and other gastrointestinal malignancies. A clinical Phase I study has been initiated in patients with CRC, gastric and esophageal adenocarcinomas (NCT04171141). Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Diane Fernandez, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Keith Kobylarz, Lindsay King, Jatin Narula, Kerry Kelleher, David Schaer, Cris Kamperschroer, Bernard Buetow, Cynthia Rohde, Allison Moreau, Gilbert Wong, Puja Sapra. A novel GUCY2C - CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2283.

Background: Guanylyl cyclase C (GUCY2C) is a regulator of intestinal homeostasis and is expressed in more than 90% of colorectal cancer (CRC), as well as in other gastrointestinal malignancies. Target expression in normal tissues is largely restricted to the apical side of intestinal epithelial tight junctions, which could allow for preferential uptake of GUCY2C-targeted biologics by tumors that have disrupted tight junction architecture. Here we demonstrate tumor-selective uptake and potent efficacy with a half-life extended, GUCY2C-CD3 bispecific molecule that recruits CD3-positive T cells to induce cytotoxicity of GUCY2C-expressing tumors in several CRC models in vivo. Additionally, to address immune evasion mechanisms, rational combinations of the bispecific are explored with immune checkpoint blockade agents, as well as by blocking angiogenesis, which has been reported to enhance T-cell infiltration into tumors. Methods: GUCY2C-CD3 mediated activity was evaluated in vivo in several cell line- and patient derived-xenograft models of CRC, using adoptive transfer of human T cells in established subcutaneous and orthotopic tumors. Immunohistochemistry was performed to demonstrate biodistribution of the drug and recruitment of activated T cells in GUCY2C-expressing tumors vs. normal tissues. We also performed CYTOF analyses on GUCY2C-CD3-treated tumors and tumor-infiltrating lymphocytes (TILs) to identify markers of immune evasion. Combination studies with anti PD-1/PD-L1 checkpoint blockade agents and with an anti VEGF-A antibody were performed using the adoptive transfer model. Results: GUCY2C-CD3 bispecific showed preferential biodistribution to GUCY2C-expressing xenograft tumors compared to normal tissue. The drug demonstrated potent dose-dependent efficacy in several CRC models with no signs of toxicity. Bispecific treated tumors not only showed recruitment of activated T cells to the tumor site, but also upregulated checkpoint mechanisms, such as PD-L1 on tumors and exhaustion markers on TILs. Consequently, anti PD-1/PD-L1 checkpoint blocking antibodies provided enhanced efficacy when combined with GUCY2C-CD3. Additionally, significant combination benefit was observed when GUCY2C-CD3 was combined with an anti VEGF-A blocking antibody. Conclusions: Our preclinical data demonstrate that a GUCY2C-CD3 bispecific can selectively target colorectal tumors, including those with KRAS or BRAF mutations, which are difficult to treat with currently approved therapies. This bispecific shows combination benefits with T-cell checkpoint blockade agents, as well as antiangiogenesis agents, indicating that single-agent activity with GUCY2C-CD3 can be further enhanced with mechanisms that address immune evasion. Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Lindsay King, Jatin Narula, Kerry Kelleher, Cynthia Rohde, Lioudmila Tchistiakova, Anhco Nguyen, Puja Sapra. A GUCY2c-CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A16.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented. Citation Format: Marc Isaac Damelin, Alex Bankovich, Jeff Bernstein, Justin Lucas, Liang Chen, Sam Williams, Albert Park, Jorge Aguilar, Elana Ernstoff, Manoj Charati, Russell Dushin, Amy Jackson-Fisher, Monette Aujay, Christina Lee, Hanna Ramoth, Milly Milton, Johannes Hampl, Sasha Lazetic, Virginia Pulito, Douglas Armellino, Edward Rosfjord, Magali Guffroy, Hadi Falahatpisheh, Lindsay King, Frank Barletta, Robert Stull, Marybeth Pysz, Paul Escarpe, David Liu, Orit Foord, Brenda Gibson, Eric Powell, Christopher O’Donnell, Xiaohua Xin, Hans Peter Gerber, Puja Sapra, Scott Dylla. A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions. [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 1220.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprised of the HER2-targeting antibody trastuzumab and DM1, a microtubule depolymerizing agent covalently attached via a non-cleavable thioether linker. T-DM1 has demonstrated clinical benefit for patients with metastatic breast cancer, however activity may be limited by inherent or acquired resistance during prolonged treatment periods. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 positive tumors, are not well understood. We used the HER2+ cell line N87 to develop a model of T-DM1 resistance utilizing a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1 resistant population of N87 cells (N87-TM) was generated. N87-TM cells displayed 103-fold resistance toward T-DM1 treatment compared to the parental N87 cells. The N87-TM cells were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug efflux pump (e.g. MDR1) protein expression compared to parental N87 cells. Comparative proteomic profiling suggested an enrichment in proteins (e.g. caveolin-1, CAV1) that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize ADCs into intracellular CAV1+ puncta and alter their trafficking to the lysosome compared to N87 cells. Intriguingly, T-ADCs utilizing auristatin payloads attached via an enzymatically cleavable linker (i.e. ValCit linker) overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice in vivo formed T-DM1 refractory tumors which remain sensitive to T-ADCs with cleavable-linked auristatin payloads. When comparing antigen positive patient-derived xenograft models that were refractory to T-DM1 yet responded to T-ADCs with ValCit linker-payloads, CAV1 was found to be a predictive protein biomarker identifying T-DM1 refractory tumors. These data implicate caveolae-mediated endocytosis in ADC biology and suggest that alterations in this pathway may impact a tumor's response profile to ADCs with non-cleavable linkers. We also propose CAV1 as a novel protein biomarker whose high tumoral expression predicts a refractory response to the T-DM1 ADC. Citation Format: Matthew S. Sung, Xingzhi Tan, Christine Hosselet, Michael Cinque, Erik Upeslacis, Jonathon Golas, Fang Wang, Bingwen Lu, Laurie Tylaska, Lindsay King, Jeremy Myers, Edward Rosfjord, Judy Lucas, Hans-Peter Gerber, Frank Loganzo. Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC. [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 2113.