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Next-generation Trop-2–targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti–Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site–mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2–expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10−12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10−8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2–expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2–targeted therapy.

Supplementary Online Material

Trop-2 drives the recruitment of Akt to the cell membrane via its PH domain.

Supplementary Methods: Protein microarrays,Multiplex Western blotting,Confocal time-lapse microscopy, Western blotting, Immunohistochemistry, 3D structure analysis; Supplementary Table 3:Breast cancer case series clinical and pathological parameters; the file also contains legends for Supplementary Figures 1-6.

Trop-2 proteome profiling.

Purpose: Inhibition of AKT is a key target area for personalized cancer medicine. However, predictive markers of response to AKT inhibitors are lacking. Correspondingly, the AKT-dependent chain of command for tumor growth, which will mediate AKT-dependent therapeutic responses, remains unclear.Experimental Design: Proteomic profiling was utilized to identify nodal hubs of the Trop-2 cancer growth–driving network. Kinase-specific inhibitors were used to dissect Trop-2–dependent from Trop-2–independent pathways. In vitro assays, in vivo preclinical models, and case series of primary human breast cancers were utilized to define the mechanisms of Trop-2–driven growth and the mode of action of Trop-2–predicted AKT inhibitors.Results: Trop-2 and AKT expression was shown to be tightly coordinated in human breast cancers, with virtual overlap with AKT activation profiles at T308 and S473, consistent with functional interaction in vivo. AKT allosteric inhibitors were shown to only block the growth of Trop-2–expressing tumor cells, both in vitro and in preclinical models, being ineffective on Trop-2–null cells. Consistently, AKT-targeted siRNA only impacted on Trop-2–expressing cells. Lentiviral downregulation of endogenous Trop-2 abolished tumor response to AKT blockade, indicating Trop-2 as a mandatory activator of AKT.Conclusions: Our findings indicate that the expression of Trop-2 is a stringent predictor of tumor response to AKT inhibitors. They also support the identification of target-activatory pathways, as efficient predictors of response in precision cancer therapy. Clin Cancer Res; 22(16); 4197–205. ©2016 AACR.

Trop-2-associated functions driven by Akt-phosphorylation

Proteomic analysis of the Trop-2 signal transduction network

This video shows MTE 4-14 cells cotransfected with Akt PH domain-EGFP (green) and Trop-2-mRFP1 (red). Recruitment and colocalization of Akt PH domain-EGFP with Trop-2-mRFP1 at the cell membrane was found to be quantitative and dynamic. The time lapse covers a period of about 27 min.

Trop-2-dependent Akt signaling.

Supplemental Figure 4: Trop-2-independent cell growth reduction by inhibitors of mTOR, NF-kB and JNK; Supplementary Figure 5: Dose-response curves for signaling pathway inhibitors; Supplementary Figure 6: Correlated expression of Trop-2, phospho-T308 Akt and phospho-S473 Akt in primary breast cancer

Next-generation Trop-2–targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti–Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site–mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2–expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10−12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10−8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2–expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2–targeted therapy.

Nanoscopic, millisecond-lasting membrane platforms are modeled to constitute main functional signaling units. However, multiple signaling events in cell growth induction, including receptor Tyr-kinase (RTK) stimulation, calcium signaling, cytoplasmic kinase recruitment, operate over much broader space-time frames. Through dynamic confocal/super-resolution/electron-microscopy on living cells, we discovered that signaling competence is acquired through growth factor-induced coalescence of activated growth inducers (RTK, CD9, CD81, CD98, Co-029, CD316, Trop-1, Trop-2, PI3K, PI4K), and downstream effectors (Grb-2, PKCα, ERK, Akt, Src, Syk, ILK, ezrin) into confined segments of the cell membrane (‘docks’), that recursively assemble over hundreds of seconds. Average size (25±5 μm; ~80,000 individual analyses) and lifetime (222±25 sec) of docks extend by orders of magnitude previously recognized space-time frames for cell signaling. Activated/phosphorylated kinases clustered at membrane docks (99% P-Erb-B4; 91% P-Ret; 90% P-PKCα; 90% P-Src). STED super-resolution microscopy revealed up to 10 µm2 signal transducer areas of effector colocalization. Docks >1.0 µm2 accounted for ~90% of colocalizations versus ≈1% of raft-sized domains (HGF, IGF-1>>SCF/VEGF), were inhibited by Tyr kinase and cytoskeleton inhibitors, and were shown to induce cancer cell proliferation. Citation Format: Saverio Alberti, Valeria Relli, Romina Tripaldi, Pasquale Simeone, Emanuela Guerra, Andrea Sacchetti, Paolo Ciufici, Antonino Moschella, Valeria Caiolfa, Moreno Zamai, Marco Trerotola. Growth factor-induced cell membrane macroplatforms drive protein kinase signaling for transformed cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2550.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca2+ signal. We reveal here that Trop-2 binds the cell membrane Na+/K+-ATPase, and that clustering of Trop-2 induces an intracellular Ca2+ rise followed by membrane translocation of PKCα, which in turn phosphorylates the Trop-2 cytoplasmic tail. This feed-forward signaling is promoted by the binding of Trop-2 to the PKCα membrane-anchor CD9. CRISPR-based inactivation of CD9 in CRC cells shows that CD9 is required by Trop-2 for recruiting PKCα and cofilin-1 to the cell membrane. This induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the β-actin cytoskeleton, and activation of Akt and ERK. The interaction between Trop-2 and CD9 was validated in vivo in murine models of CRC growth and invasion. Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of CRC patients, supporting a pivotal relevance in CRC malignant progression. Our findings demonstrate a previously unsuspected layer of cancer growth regulation, which is dormant in normal tissues, and is activated by Trop-2 in cancer cells.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca

Trop-2 is a type I transmembrane glycoprotein, which is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation. In its mature, glycosylated/functionally-competent form, Trop-2 associates with worse prognosis. A global quest was conducted for identifying decisive drivers of cancer metastasis. Cancer cell spheroids, wound healing and cell aggregation assays were utilized to assess cell-cell adhesion capacity. Immunofluorescence confocal microscopy and flow cytometry analysis were utilized to quantify expression of target signaling proteins, IHC analysis quantified the expression of target molecules in primary tumors and metastases. Pre-clinical models of orthotopic growth of colon cancer and metastatic diffusion to the liver were utilized. Xenotransplant transcriptome profiling analyzed transcription of EMT-related determinants. This led us to identify Trop-2 as a unique upregulated gene in colon cancer metastasis. Trop-2 was here shown to bind highly-expressed E-cadherin at the cell membrane. Trop-2 binding released E-cadherin from the cytoskeleton, inhibited cell-cell adhesion functions and activated signaling through β-catenin. This occurred in the absence of transcriptional downregulation of the CDH1 gene by EMT-associated transcription factors. The Trop-2/E-cadherin/β-catenin program was shown to lead to anti-apoptotic signaling, increased cell migration and enhanced cancer cell survival. In an orthotopic model of colon cancer, Trop-2 caused metastatic diffusion to the liver. The Trop-2-led E-cadherin-inactivation metastasis program was then found to induce metastatic relapse and shorter overall survival of colon cancer patients. Corresponding impact was indicated on breast, colon, ovary, uterus, stomach cancer metastatic diffusion, for broad impact on metastatic diffusion in human cancer. Acknowledgments:Support was provided by grants of Italian Ministry of Development (MI01_00424) and of University and Research (SCN_00558). M.T. was supported by the Programma Per Giovani Ricercatori “Rita Levi Montalcini” (Grant PGR12I7N1Z). Citation Format: Saverio Alberti, Emanuela Guerra, Valeria Relli, Rossano Lattanzio, Martina Ceci, Khouloud Boujnah, Valeria Garbo, Antonino Moschella, donato F. Altomare, Raffaella Depalo, Marco Trerotola. Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT [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 2851.

We recently reported that activation of Trop-2 through its cleavage at R87-T88 by ADAMIO underlies Trop-2-driven progression of colon cancer. However, the mechanism of action and pathological impact of Trop-2 in metastatic diffusion remain unexplored. Through searches for molecular determinants of cancer metastasis, we identified TROP2 as unique in its up-regulation across independent colon cancer metastasis models. Overexpression of wild-type Trop-2 in KM12SM human colon cancer cells increased liver metastasis rates in vivo in immunosuppressed mice. Metastatic growth was further enhanced by a tail-less, activated Delta cytoTrop-2 mutant, indicating the Trop-2 tail as a pivotal inhibitory signaling element. In primary tumors and metastases, transcriptome analysis showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 is through alternative mechanisms. Trop-2 can tightly interact with ADAM10. Here, Trop-2 bound E-cadherin and stimulated ADAM10-mediated proteolytic cleavage of E-cadherin intracellular domain. This induced detachment of E-cadherin from beta-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of beta-catenin signaling, which were further enhanced by the Delta cytoTrop-2 mutant. This Trop-2/E-cadherin/beta-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival. In patients with colon cancer, activation of this Trop-2-centered program led to significantly reduced relapse-free and overall survival, indicating a major impact on progression to metastatic disease. Recently, the anti-Trop-2 mAb Sacituzumab govitecan-hziy was shown to be active against metastatic breast cancer. Our findings define the key relevance of Trop-2 as a target in metastatic colon cancer. Funding Agencies|grants of Fondazione of the Cassa di Risparmio della Provincia di Chieti; Compagnia di San PaoloCompagnia di San Paolo [2489IT]; Italian Ministry of Development [MI01_00424]; Region Abruzzo (POR FESR) [C78C14000100005]; Oncoxx Biotech (Italian Ministry of University and Research, Smart Cities and Communities) [SCN_00558]; Programma Per Giovani Ricercatori "Rita Levi Montalcini", Italian Ministry of University and Research [PGR12I7N1Z]

Hundreds of proteins/genes have been linked to the metastatic phenotype. However, consistent markers of tumour aggressiveness and metastatic potential in breast cancer patients were not identified, not even including proteomic analysis and large-scale genome sequencing.Metastasis-associated genes were predicted to include not only drivers of the metastatic phenotype, but also secondary events, together with adaptive, counterbalancing changes. Thus, to identify candidates with a required role in metastatic diffusion, we looked for genes that were concordantly dysregulated across orthogonal cancer metastasis settings. This led us to identify Trop-2 as uniquely upregulated and associated to metastasis in experimental models of breast cancer, as well as in other solid tumors. We identified functional inactivation of E-cadherin by Trop-2 as the main motor of metastatic diffusion of such metastatic systems. Trop-2 binding to E-cadherin inactivated its cell-cell adhesion function, through release from the cytoskeleton, for activation of β-catenin transcriptional activity. This led to anti-apoptotic signaling, increased cell migration capacity and enhanced cancer cell survival. We showed that this mechanism led to metastatic diffusion of xenotransplants growing in immunosuppressed mice. An E-cadherin-inactivation metastasis program was then shown to be recapitulated in breast cancer patients, as well as in other solid tumors, over 24 independent case series, encompassing 13,042 primary tumours. Aggressive triple-negative breast cancers were shown to be driven toward global relapse by Trop-2 overexpression, through E-cadherin inactivation and activation of β-catenin transcriptional activity. No disease recurrence was observed in control cases over +12 years of follow-up. These finding lead to a novel paradigm of a Trop-2-driven, E-cadherin-inactivation program as a main metastasis driver in solid tumors. This may open far-reaching perspectives in diagnostic procedures and anti-cancer therapies. Citation Format: Saverio Alberti, Marco Trerotola, Valeria Relli, Rossano Lattanzio, Martina Ceci, Khouloud Boujnah, Valeria Garbo, Antonino Moschella, Patrizia Querzoli, Massimo Pedriali, Laura Antolini, Emanuela Guerra. Trop-2 inactivation of E-cadherin drives triple negative breast cancer relapse [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-04.

This abstract was withdrawn by the authors.

Non-small cell lung cancers (NSCLCs) represent 85% of lung tumors. NSCLCs encompass multiple cancer types, such as adenocarcinomas (LUADs), squamous cell cancers (LUSCs), and large cell cancers. Among them, LUADs and LUSCs are the largest NSCLC subgroups. LUADs and LUSCs appear sharply distinct at the transcriptomic level, as well as for cellular control networks. LUADs show distinct genetic drivers and divergent prognostic profiles versus LUSCs. Therapeutic clinical trials in NSCLC indicate differential LUAD versus LUSC response to treatments. Hence, LUAD and LUSC appear to be vastly distinct diseases at the molecular, pathological, and clinical level. Abandoning the notion of NSCLC may critically help in developing novel, more effective subtype-specific molecular alteration-targeted therapeutic procedures.

Simple Summary Although targeted therapies represent a promising approach in advanced biliary tract cancer (BTC), large groups of patients do not harbor actionable mutations, especially in extrahepatic cholangiocarcinoma (eCCA). In this review, we provide a timely appraisal of the evolving landscape of eCCA, especially focusing on recent studies on molecular characterization and molecularly targeted treatments in this aggressive malignancy. Abstract Biliary tract cancers (BTCs) include a heterogenous group of aggressive malignancies with limited therapeutic options. According to their anatomical location, these hepatobiliary tumors are usually classified into intrahepatic cholangiocarcinoma (iCCA), extrahepatic cholangiocarcinoma (eCCA), and gallbladder cancer (GBC). Unfortunately, BTCs are often diagnosed when already metastatic, and although the advent of genomic sequencing has led to a deeper understanding of iCCA pathogenesis, very little data are currently available about the molecular landscape of eCCA. Moreover, despite novel systemic treatments emerging in BTC, the grim prognosis of eCCA patients has not changed in the past decade, and no targeted therapies have been approved so far. The aim of the current review is to provide an overview regarding molecular features and potential targeted therapies in eCCA, together with novel therapeutic approaches and future directions of translational and clinical research on this highly aggressive disease that poses many unanswered questions.

Trop-2 is overexpressed in most human cancers, suggesting selective pressure for a key, conserved function. Here we show that Trop-2 stimulates cancer cell growth through the activation of a constitutively expressed, yet otherwise dormant, growth-control module. We discovered that crosslinking of membrane Trop-2 with specific Abs leads to a cytoplasmic Ca2+ raise through interaction with the Na+/K+-ATPase α1 subunit and mobilization of the intracellular stores. This triggers a feed-forward loop through Trop-2-dependent activation and membrane recruitment of PKCα;, which in turn phosphorylates the Trop-2 cytoplasmic tail at two target sites, activating the molecule to stimulate its downstream signaling targets Akt and ERK. Our findings indicate that the Trop-2-triggered cell growth operates through binding and extensive crosstalk with CD9, CD81, CD82 and CD151 through PKCα. Detailed analysis of CD9 and CD81 indicates that they bind to the HIKE region of the Trop-2 intracellular tail. Correspondingly, we found that the HIKE region of Trop-2 mediates its anchoring to the β-actin cytoskeleton through direct interaction with the ERM protein ezrin. Consistently, the Trop-2-dependent dynamic remodeling of the cell cytoskeleton is discovered to occur through activation of myosin II and binding of annexins A1/A11, α-actinin and gelsolin. Systematic drug screening, gene expression silencing and site-directed mutagenesis revealed that cytoskeleton disassembly, HIKE deletion and CD9 inhibition revert the growth of Trop-2-expressing cancer cells to that of their Trop-2-null counterparts. On the other hand, these inhibitors have no effects on basal cell growth. This indicates that Trop-2-centered protein interactions and activations are an essential step for the Trop-2-dependent cancer growth. Tight co-expression of the key components of the Trop-2 growth-stimulatory complex is found in a large breast cancer case series, thus indicating strong clinical relevance. Hence, Trop-2 triggers a universal, but otherwise dormant, layer of cancer growth, that overrides basal cell growth regulatory mechanisms and sensitizes tumors to targeted anticancer therapies. Citation Format: Marco Trerotola, Valeria Relli, Romina Tripaldi, Andrea Sacchetti, Kristina Havas, Pasquale Simeone, Emanuela Guerra, Annalaura Aloisi, Rossana La Sorda, Rossano Lattanzio, Daniele Vergara, Isabelle Fournier, Michel Salzet, Mauro Piantelli, Saverio Alberti. Trop-2 activates a dormant Na+/K+-ATPase/PKCα/CD9/ezrin signaling axis to override the basal growth program of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 367. doi:10.1158/1538-7445.AM2017-367

Purpose: Inhibition of AKT is a key target area for personalized cancer medicine. However, predictive markers of response to AKT inhibitors are lacking. Correspondingly, the AKT-dependent chain of command for tumor growth, which will mediate AKT-dependent therapeutic responses, remains unclear. Experimental Design: Proteomic profiling was utilized to identify nodal hubs of the Trop-2 cancer growth–driving network. Kinase-specific inhibitors were used to dissect Trop-2–dependent from Trop-2–independent pathways. In vitro assays, in vivo preclinical models, and case series of primary human breast cancers were utilized to define the mechanisms of Trop-2–driven growth and the mode of action of Trop-2–predicted AKT inhibitors. Results: Trop-2 and AKT expression was shown to be tightly coordinated in human breast cancers, with virtual overlap with AKT activation profiles at T308 and S473, consistent with functional interaction in vivo. AKT allosteric inhibitors were shown to only block the growth of Trop-2–expressing tumor cells, both in vitro and in preclinical models, being ineffective on Trop-2–null cells. Consistently, AKT-targeted siRNA only impacted on Trop-2–expressing cells. Lentiviral downregulation of endogenous Trop-2 abolished tumor response to AKT blockade, indicating Trop-2 as a mandatory activator of AKT. Conclusions: Our findings indicate that the expression of Trop-2 is a stringent predictor of tumor response to AKT inhibitors. They also support the identification of target-activatory pathways, as efficient predictors of response in precision cancer therapy. Clin Cancer Res; 22(16); 4197–205. ©2016 AACR.

Genome-wide association studies of complex physiological traits and diseases consistently found that associated genetic factors, such as allelic polymorphisms or DNA mutations, only explained a minority of the expected heritable fraction. This discrepancy is known as “missing heritability”, and its underlying factors and molecular mechanisms are not established. Epigenetic programs may account for a significant fraction of the “missing heritability.” Epigenetic modifications, such as DNA methylation and chromatin assembly states, reflect the high plasticity of the genome and contribute to stably alter gene expression without modifying genomic DNA sequences. Consistent components of complex traits, such as those linked to human stature/height, fertility, and food metabolism or to hereditary defects, have been shown to respond to environmental or nutritional condition and to be epigenetically inherited. The knowledge acquired from epigenetic genome reprogramming during development, stem cell differentiation/de-differentiation, and model organisms is today shedding light on the mechanisms of (a) mitotic inheritance of epigenetic traits from cell to cell, (b) meiotic epigenetic inheritance from generation to generation, and (c) true transgenerational inheritance. Such mechanisms have been shown to include incomplete erasure of DNA methylation, parental effects, transmission of distinct RNA types (mRNA, non-coding RNA, miRNA, siRNA, piRNA), and persistence of subsets of histone marks.

Trop-2 is an epithelial transmembrane glycoprotein that transduces a calcium signal and activates a growth-signaling network that converges on AKT. Trop-2 is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation, and in its mature, glycosylated/functionally-competent form associates with worse prognosis. Trop-2 extracellular domain contains an N-terminal cysteine-rich globular region followed by a cysteine-less region as a connecting “stem” to the transmembrane domain. Trop-2 molecules engage in homophylic interactions between adjacent cells and establish multimeric complexes with tight junction proteins, which may hinder accessibility by therapeutic antibodies. Up to now, Trop-2-targeted approaches have employed anti-Trop-2 monoclonal antibodies (mAb) which essentially recognize a single immunodominant epitope poised between the globular and stem regions. Such mAb have limited or no therapeutic efficacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specificity towards the globular versus stem regions. Hybridoma diversity was maximized by immunization with soluble human Trop-2 extracellular region produced in different transformed mammalian cell lines (human 293 and murine L) and in insect cells/baculovirus expressing system. These were expected to provide native folding of Trop-2 together with a broad spectrum of differential glycosylation. Trop-2-binding hybridomas were further selected by multiple rounds of flow cytometry analysis using live 293 cells expressing different Trop-2 extracellular portions. Two classes of mAb were identified, that bound the stem versus the globular region. These mAb efficiently bound Trop-2 expressing cancer cells and were able to inhibit cell growth in vitro. In vivo the naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of distinct tumors, including colon, ovary and prostate cancers. Notably, they showed differential efficacy for established tumors versus isolated-cell models of metastatic dissemination, consistent with our strategy of maximizing differential accessibility of Trop-2 according to growth mode. Most remarkably, we demonstrated in vivo synergy of these anti-Trop-2 mAb, paving the way for game-changing anti-cancer mAb therapy. The differential efficacy of the OX-G64 and OX-S55 anti-Trop2 mAb against different tumor histotypes and growth stages further allows to exploit their cancer-killing potential in pathological stage-tailored therapeutic approaches. Citation Format: Emanuela Guerra, Marco Trerotola, Valeria Relli, Chiara Pedicone, Antonella D' Amore, Francesca Dini, Silvia Fratarcangeli, Saverio Alberti. Novel anti-Trop-2 monoclonal antibodies with unique binding specificities show therapeutic synergy against most human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2017-4588

e14002 Background: Trop-2 is a calcium signal transducer that drives tumor cell growth. Trop-2 is overexpressed in the majority of carcinomas, where it associates with worse prognosis. The Trop-2 extracellular domain contains a N-terminal cysteine-rich globular domain followed by a stem-like cysteine-less region that connects to the transmembrane domain. Trop-2 engages in homophylic interactions between adjacent cells and interacts with tight-junction proteins, which may severely affect accessibility by therapeutic monoclonal antibodies (mAb). Available anti-Trop-2 mAb mostly recognize a single immunodominant epitope between the globular and stem regions, and have limited or no therapeutic efficacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specificity towards the globular versus stem regions. Methods: Balb-C mice were immunized with soluble human Trop-2 produced in human 293 and murine L cell lines and in baculovirus expression system. Domain-targeted anti-Trop-2 mAb were selected by flow cytometry using live 293 transfectants.Therapeutic potential was assessed in human cancer xenografts in murine models. mAb mode of action was investigated by Western blot, live-cell imaging and in vitro ADCC and cancer cell growth inhibition assays. Results: mAb were identified that were differentially directed against the Trop-2 stem versus globular regions. These mAb efficiently bound Trop-2 expressing cancer cells and inhibited cell growth in vitro. In vivo, naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of colon, ovary, breast and prostate cancers as xenografts in nude mice. NSG mice and in vitro mechanism profiling indicated efficient ADCC, together with efficient internalization for ADC development. Differential efficacy for established tumors versus isolated-cell models of metastatic dissemination was shown. Most remarkably, OX-G64/OX-S55 co-administration demonstrated sinergic growth inhibition in vivo. Conclusions: The OX-G64 and OX-S55 anti-Trop-2 mAb are novel domain-targeted, sinergic therapeutic mAb for game-changing anti-cancer immunotherapy.

Trop-2 is a calcium signal transducer that drives tumor growth. Anti-Trop-2 antibodies with selective reactivity versus Trop-2 maturation stages allowed to identify two different pools of Trop-2, one localized in the cell membrane and one in the cytoplasm. Of note, membrane-localized/functional Trop-2 was found to be differentially associated with determinants of tumor aggressiveness and distinct breast cancer subgroups. These findings candidated Trop-2 states to having an impact on cancer progression. We tested this model in breast cancer. A large, consecutive human breast cancer case series (702 cases; 8 years median follow-up) was analyzed by immunohistochemistry with anti-Trop-2 antibodies with selective reactivity for cytoplasmic-retained versus functional, membrane-associated Trop-2. We show that membrane localization of Trop-2 is an unfavorable prognostic factor for overall survival (1+ versus 0 for all deaths: hazard ratio, 1.63; P = 0.04), whereas intracellular Trop-2 has a favorable impact on prognosis, with an adjusted hazard ratio for all deaths of 0.48 (high versus low; P = 0.003). A corresponding impact of intracellular Trop-2 was found on disease relapse (high versus low: hazard ratio, 0.51; P = 0.004). Altogether, we demonstrate that the Trop-2 activation states are critical determinants of tumor progression and are powerful indicators of breast cancer patients survival.

The main non-small-cell lung cancer (NSCLC) histopathological subtypes are lung adenocarcinomas (LUAD) and lung squamous cell carcinomas (LUSC). To identify candidate progression determinants of NSCLC subtypes, we explored the transcriptomic signatures of LUAD versus LUSC. We then investigated the prognostic impact of the identified tumor-associated determinants. This was done utilizing DNA microarray data from 2,437 NSCLC patients. An independent analysis of a case series of 994 NSCLC was conducted by next-generation sequencing, together with gene expression profiling from GEO (https://www.ncbi.nlm.nih.gov/geo/). This work led us to identify 69 distinct tumor prognostic determinants, which impact on LUAD or LUSC clinical outcome. These included key drivers of tumor growth and cell cycle, transcription factors and metabolic determinants. Such disease determinants appeared vastly different in LUAD versus LUSC, and often had opposite impact on clinical outcome. These findings indicate that distinct tumor progression pathways are at work in the two NSCLC subtypes. Notably, most prognostic determinants would go inappropriately assessed or even undetected when globally investigating unselected NSCLC. Hence, differential consideration for NSCLC subtypes should be taken into account in current clinical evaluation procedures for lung cancer.