In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400–800 μM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-NG-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 μM lovastatin for three days prior to addition of Tumor necrosis factor – α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

The cytolinker and scaffolding protein, plectin, has emerged as a potent driver of malignant hallmarks in many human cancers due to its involvement in various cellular activities contributing to tumorigenesis, including cancer cell proliferation, adhesion, migration, invasion, and signal transduction. Evidence shows that beyond plectin’s diverse protein interactome, its cancer-specific mislocalization to the cell surface enables its function as a potent oncoprotein. As such, therapeutic targeting of plectin, its protein interactors, and, in particular, cancer-specific plectin (CSP) presents an attractive opportunity to impede carcinogenesis directly. Here, we report on plectin’s differential gene and protein expression in cancer, explore its mutational profile, and discuss the current understanding of plectin’s and CSP’s biological function in cancer. Moreover, we review the landscape of plectin as a prognostic marker, diagnostic biomarker, and target for imaging and therapeutic modalities. We highlight how, beyond their respective biological importance, plectin’s common overexpression in cancer and CSP’s cancer-specific bioavailability underscore their potential as high-value druggable targets. We discuss how recent evidence of the potent anti-cancer effects of CSP therapeutic targeting opens the door for cell-surface mislocalized proteins as novel therapeutic targets.

Cancer-specific plectin (CSP) is a pro-tumorigenic protein selectively expressed on the cell surface of major cancers, including ovarian cancer (OC). Despite its assessable localization, abundance, and functional significance, the therapeutic efficacy of targeting CSP remains unexplored. Here, we generated and investigated the anticancer effects of a novel CSP-targeting monoclonal antibody, 1H11, in OC models. Its therapeutic efficacy as a monotherapy and in combination with chemotherapy was evaluated in vitro using two OC cell lines and in vivo by a subcutaneous ovarian cancer model. 1H11 demonstrated rapid internalization and high affinity and specificity for both human and murine CSP. Moreover, 1H11 induced significant and selective cytotoxicity (EC50 = 260 nM), G0/G1 arrest, and decreased OC cell migration. Mechanistically, these results are associated with increased ROS levels and reduced activation of the JAK2-STAT3 pathway. In vivo, 1H11 decreased Ki67 expression, induced 65% tumor growth inhibition, and resulted in 30% tumor necrosis. Moreover, 1H11 increased chemosensitivity to cisplatin resulting in 60% greater tumor growth inhibition compared to cisplatin alone. Taken together, CSP-targeting with 1H11 exhibits potent anticancer activity against ovarian cancer and is deserving of future clinical development.

Background: Pancreatic ductal adenocarcinoma (PDA) is the 3rd deadliest cancer, diagnosed typically in advanced stages, with only an 8% 5-year survival rate, thus demonstrating the need for novel therapeutic approaches that significantly enhance chemo- and/or immune-therapy. Our team previously identified a promising functional target for cancer therapy in PDA, cell surface plectin 1 (CSP1) that is aberrantly expressed on PDA cells and thus a cell surface-associated biomarker of cancer. CSP1 expression first becomes apparent in high grade dysplasias, remaining high in early and advanced cancers and in metastases. Our first-in-human imaging trial in PDA patients using a CSP1-targeted imaging agent revealed that CSP is an available target and accessible for binding, a potentially a target for cancer therapy. We hypothesized that a monoclonal antibody (mAb) against CSP1 could lead to novel pancreatic cancer treatment options, thus, we developed a therapeutic mAb, e.g., ZB131, representing a first-in-class antibody selectively targeting CSP1. Methods: ZB131 is a humanized mAb targeted against human plectin 1 (rhSec8) that also binds murine CSP1. ZB131 affinity and its effect on cancer cells including proliferation, cytotoxicity, and migration were tested in vitro using saturation binding, SRB-based survival assays, flow cytometry, and migration assays on various pancreatic cell types and homeostatic “normal” controls. In vivo validation was performed using two nu/nu mouse models bearing subcutaneous MiaPACA2 or Yapc PDA cells, and a syngeneic KPC-derived tumor model to also evaluate immune responses to tumors treated with ZB131 or IgG control. Results: ZB131 exhibits high specificity and high affinity (0.4±0.1nM) to CSP1, and functionally induces G0 growth arrest followed by necrotic cell death of PDA cells in culture, and is synergistic with gemcitabine resulting in a 50-fold decrease in IC50. In vivo, in subcutaneous xenograft models, ZB131 monotherapy decreased PDA tumor volume 5-fold as compared to controls, and in combination with cisplatin resulted in sustained tumor reduction with greater than 85% tumor necrosis. In subcutaneous syngeneic PDA models, ZB131 induced complete tumor regression within 35 days mediated by an anti-tumor immune response as upon tumor rechallenge, full tumor regression was again achieved without additional ZB131 therapy. Leukocyte complexity analysis of regressing PDA tumors versus controls revealed an ~3-fold increase in effector and central memory T cells. Conclusion: CSP1 is a first in class anti-cancer target expressed on the cell surface of PDA, as well as other cancers including ovarian, esophageal and head neck. ZB131, an anti-CSP1 mAb, induces tumor cell intrinsic cell death, as well as a robust anti-tumor T cell response leading to complete tumor regression indicating the potential therapeutic efficacy of ZB131 in late-stage cancers. Citation Format: Julien Dimastromatteo, Amanda Poisonnier, Samantha Perez, Lisa Coussens, Kimberly Kelly. Therapeutic targeting of cell surface plectin induces anti-cancer immune response and pancreatic cancer regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1558.

Cancer-associated fibroblasts (CAFs) execute diverse and complex functions in cancer progression. While reprogramming the crosstalk between CAFs and cancer epithelial cells is a promising avenue to evade the adverse effects of stromal depletion, drugs are limited by their suboptimal pharmacokinetics and off-target effects. Thus, there is a need to elucidate CAF-selective cell surface markers that can improve drug delivery and efficacy. Here, functional proteomic pulldown with mass spectrometry was used to identify taste receptor type 2 member 9 (TAS2R9) as a CAF target. TAS2R9 target characterization included binding assays, immunofluorescence, flow cytometry, and database mining. Liposomes conjugated to a TAS2R9-specific peptide were generated, characterized, and compared to naked liposomes in a murine pancreatic xenograft model. Proof-of-concept drug delivery experiments demonstrate that TAS2R9-targeted liposomes bind with high specificity to TAS2R9 recombinant protein and exhibit stromal colocalization in a pancreatic cancer xenograft model. Furthermore, the delivery of a CXCR2 inhibitor by TAS2R9-targeted liposomes significantly reduced cancer cell proliferation and constrained tumor growth through the inhibition of the CXCL-CXCR2 axis. Taken together, TAS2R9 is a novel cell-surface CAF-selective target that can be leveraged to facilitate small-molecule drug delivery to CAFs, paving the way for new stromal therapies.

Background: Cell surface plectin (CSP) is an abundant bioavailable target exclusively found on the surface of ovarian epithelial cancers while remaining entirely intracellular in pre-malignant tissue. Under current treatment options, 80% of women, even if remission is achieved, will experience recurrence of disease demonstrating an urgent need for more effective therapeutics. Herein, we investigate the anticancer effects of a CSP-targeting monoclonal antibody (ZB131) in in-vitro and in-vivo models of ovarian cancer (OC) as well as elucidate key signaling pathways it modulates. We demonstrate that treatment with ZB131 is efficacious both as a monotherapy and in combination with chemotherapy. Methods: A first-in-class CSP-targeting monoclonal antibody, ZB131, was developed and characterized for specificity and affinity via ELISA and kinetic binding assays. In-vitro efficacy of ZB131 was determined by Cell Titer-Glo assay and flow cytometry, impact on cell-cycle by flow cytometry, and on migration by scratch assay using multiple OC cell lines. ROS accumulation was evaluated by DCFDA assay. Phosphorylation changes of signaling kinases were measured by LiCor. In-vivo validation was performed in an OC xenograft model. The expression of proliferative markers was evaluated by IHC staining of tumor sections. Treatment toxicity was assessed from serum chemistry tests and histological evaluation of major organs. Similar in-vivo and in-vitro techniques were used to characterize ZB131's effect in combination with cisplatin. Results: ZB131 demonstrates high affinity (0.4±0.1nM) and specificity for both murine and human CSP. Treatment with ZB131 decreased cell proliferation, migration, induced G0/G1 growth arrest, and increased ROS accumulation followed by cell death in OC cell lines. ZB131 induced two coordinated events, decreased phosphorylation of cell proliferation cascades (AKT, PRAS40, ERK1/2, and GSK3alpha/beta) and increased phosphorylation of stress signaling pathways (JNK, c-Jun, p38, MSK1/2, and AMPK). In vivo, ZB131 therapy caused a 50% tumor growth reduction as well as decreased expression of cyclin D and Ki67 with no toxic side effects. Impressively, ZB131 synergizes with cisplatin resulting in a greater than 50-fold decrease in IC50 in-vitro and showed sustained tumor growth reduction with greater than 85% tumor necrosis over the course of treatment in-vivo. Conclusion: CSP is a novel highly abundant therapeutic target exclusively localized to the cell surface of ovarian, colorectal, lung, and pancreatic cancers which collectively account for more than half of all estimated cancer deaths. Our findings illustrate that CSP-targeting with ZB131 induces cytotoxicity by activating stress pathways and dampening proliferation and survival pathways. This study serves as the basis for informing a novel treatment strategy for CSP-positive tumors. Citation Format: Samantha Melinda Perez, Julien Dimastromatteo, Kimberly A. Kelly. A novel monoclonal antibody targeting cell-surface plectin has potent antitumor activity in ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 946.

3083 Background: Cancer-Specific Plectin (CSP) is a novel pro-tumorigenic target that is selectively expressed on the surface of tumors, while absent from benign tissue. CSP is highly expressed in many solid cancers, particularly pancreatic (PC), cholangiocarcinoma (CCA), and ovarian (OC). ZB131 (Zielbio), is a humanized anti-CSP IgG1 monoclonal antibody that binds specifically to CSP on the surface of tumor cells, rapidly internalizes and modulates key proliferative and cytoskeletal remodeling signaling pathways to decrease cancer cell growth and increase the recruitment of anti-tumor immune infiltrates. Methods: The dose escalation (3+3 design) and expansion are enrolling adult patients (pts) with advanced treatment-refractory solid tumors. ZB131 was administered IV over 60 minutes (without premedication) weekly. The starting dose of ZB131 is 0.3 mg/kg followed by 1,3, 9, 15 and 30 mg/kg. Primary objectives are safety, tolerability, and maximum tolerated dose/determination of Phase 2 dose. Secondary objectives are preliminary efficacy (RECIST v1.1), Pharmacokinetic (PK) and pharmacodynamic parameters in blood and paired tumor biopsies. Results: As of 31 Jan 2023, 24 pts had enrolled (9 PC, 6 CCA, 4 OC, 5 other). The median age was 59.5 yr (range, 35 – 84) with a median of 4 prior lines of therapy. The median ZB131 treatment duration is 5.3 weeks (range, 1 – 33). No dose-limiting toxicities were observed up to the dose of 30 mg/kg which is currently enrolling. Treatment-related adverse events (TRAEs) were grade 1/ 2: nausea (n=6), fatigue (n=5), anemia (n=3), diarrhea (n=2), flulike symptoms (n=2), and vomiting (n=2). Mild flu-like symptoms and nausea/vomiting were observed during or following infusion at doses of 9 mg/kg and above. Except for one episode of Gr 3 neutropenia at the 30 mg/kg dose, no other drug-related grade 3/4 TRAE’s were reported. Five pts had stable disease range, 12 – 33 weeks (2 PC, 1 CCA, 2 OC), with a 43% decrease in CA-125 observed in one OC pt. The PKs are linear and dose-proportional for all dose levels evaluated (estimated T½ of 6-9 days). No antidrug antibodies have been detected. Conclusions: Interim data from dose-escalation of this first-in-class, anti CSP antibody, demonstrates good tolerability with encouraging signs of activity and target engagement in heavily pretreated pts. Results support further clinical evaluation of ZB131 in dose-expansion cohorts including OC and including combination therapy with gemcitabine. Clinical trial information: NCT 05074472 .

Cholangiocarcinoma (CCA) is a lethal cancer with a high unmet need. The survival rate is low (5-year survival <20%), most non-palliative patients relapse within two years, and treatments in the advanced setting are limited. While FGFR2 and IDH1 inhibitors are effective in patients with select genomic alterations, they address only a small fraction (<20%) of biliary tract cancers. We now present the discovery and characterization of ZB131, a first in class humanized monoclonal antibody targeted to cancer specific plectin (CSP). CSP is a pro-tumorigenic protein exclusively expressed on the cancer cell surface. Previously, we demonstrated remarkable tumor regressions in preclinical murine models of pancreatic and ovarian cancer using a tool anti-CSP murine antibody. Here, we show that intra- and extra-hepatic CCA are 92% and 86% positive for membranous plectin staining by IHC, respectively, suggesting that anti-CSP therapy could be broadly effective against CCA. Indeed, in CCA cell lines in vitro, ZB131 (0.4nM binding affinity) arrests cell cycle, inhibits cell migration, and triggers intracellular ROS accumulation. In addition, both ZB131 and its murine version significantly suppressed tumor growth in 83% of the mice in a xenograft model and caused complete regression in the remaining 17%. Previous data from our group and others suggesting that CSP plays a role in chemoresistance led us to evaluate the efficacy of ZB131 in combination with gemcitabine. In vitro, we treated CSP-positive WITT cells with combinations of 0-500nM gemcitabine plus 0-1000nM ZB131 and found pockets of synergy or additivity. In a CCA xenograft model, the combination of 1mg/kg ZB131 and 100mg/kg gemcitabine showed 91% suppression in tumor growth compared with 74% in gemcitabine alone and 77% in huIgG plus gemcitabine at day 32. Collectively, our results suggest that ZB131 renders CCA tumors more chemosensitive and enhances the antitumor effects of gemcitabine. In contrast to the standard-of-care CCA treatment regimen, ZB131 showed remarkably low toxicity, likely because the antibody bypasses any non-malignant cells where plectin remains strictly cytoplasmic. Fc effector functional studies revealed a lack of antibody-dependent cell-mediated and complement-dependent cytotoxicity. Hemolysis and cytokine release studies suggested that ZB131 is compatible with human blood and unlikely to cause adverse cytokine release. Furthermore, in GLP toxicology studies, no signs of toxicity were observed at the highest dose evaluated of 100mg/kg/week, providing a >30-fold safety margin from the estimated human efficacious dose of 3mg/kg/week. In summary, ZB131 is an exciting new avenue for the treatment of CCA and other CSP-positive cancers with an excellent safety profile and strong antitumor activity in vitro and in murine models. Phase I clinical trials of ZB131 have been initiated. Citation Format: Samantha M. Perez, Randall Burton, Denise Krawitz, Christopher Sheth, Pina Cardarelli, Molly Owens, Brian Murphy, Matthew J. Reilley, Lindsey T. Brinton, Kimberly A. Kelly. First in class drug ZB131 shows efficacy in cholangiocarcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3591.