Antibody-drug conjugates (ADCs) are considered as promising cancer treatment modalities that combine the selectivity of antibodies and the cytotoxic properties of payloads using chemical linkers. However, despite their success, ADCs still suffer from drawbacks, e.g., systemic toxicity, which limit their potential clinical applications. The systemic toxicity of an ADC is mainly related to the stability of its linker, and to the selectivity of its antibody towards the targeted antigen expressed on cancer cells. Lu/BCAM (Lutheran/basal cell adhesion molecule) is a member of the immunoglobulin superfamily and is a receptor for laminin, a protein that facilitates cell adhesion, migration, and invasion. A growing number of studies show that BCAM plays an essential role in tumor progression and is overexpressed on epithelial cancers e.g., skin cancer. Here we describe GENA-111, a human monoclonal anti-BCAM IgG4 (S228P) antibody that binds to human BCAM with high affinity, and that is significantly internalized by BCAM-positive tumor cells. We also describe the GENA-111-auristatin F ADC, wherein the GENA-111 antibody has been armed with an auristatin F derivative using a new linker technology and a stabilized thiol maleimide conjugation. This new linker technology comprises a cleavable peptidic sequence that facilitates multidrug attachment and the production of ADCs with tailored drug-to-antibody ratios (DARs). Cytotoxic drugs are rapidly and selectively released from the linker by the carboxypeptidase activity of Cathepsin B. In vitro cytotoxicity examination showed the potent cytotoxic effects of this GENA-111-auristatin F ADC on BCAM-expressing tumor cells, with a positive correlation between cytotoxicity and BCAM expression. Moreover, this ADC was also shown to significantly reduce the growth of tumor cells, including A431, T47D, and Huh7. The GENA-111-auristatin F ADC was evaluated in a xenograft mouse model established by subcutaneous injection of A431 cells, a BCAM positive human skin cancer cell line. The results of this study will be presented and discussed. Taken together, our data suggest that an ADC targeting BCAM e.g., GENA-111-auristatin F, might be a promising treatment strategy for BCAM positive epithelial cancer patients. Citation Format: Hyunkyung Yu, Nathalie Bellocq, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Bu-Nam Jeon, Léo Marx, Mathilde Pantin, Hyunjin Yoo, Seungmin Byun, Joo-Yeon Chung, Mi Young Cha, Patrick Garrouste, Frédéric Lévy. The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer [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 1760.

Antibodies are an attractive therapeutic modality for cancer treatment as they allow the increase of the treatment response rate and avoid the severe side effects of chemotherapy. Notwithstanding the strong benefit of antibodies, the efficacy of anti-cancer antibodies can dramatically vary among patients and ultimately result in no response to the treatment. Here, we have developed a novel means to regioselectively label the Fc domain of any therapeutic antibody with a radionuclide chelator in a single step chemistry, with the aim to study by SPECT/CT imaging if the radiolabeled antibody is capable of targeting cancer cells in vivo. A Fc-III peptide was used as bait to bring a carbonate electrophilic site linked to a metal chelator and to a carboxyphenyl leaving group in close proximity with an antibody Fc nucleophile amino acid (K317), thereby triggering the covalent linkage of the chelator to the antibody lysine, with the concomitant release of the carboxyphenyl Fc-III ligand. Using CHX-A''-DTPA, we radiolabeled trastuzumab with indium-111 and showed in biodistribution and imaging experiments that the antibody accumulated successfully in the SK-OV-3 xenograft tumour implanted in mice. We found that our methodology leads to homogeneous conjugation of CHX-A''-DTPA to the antibody, and confirmed that the Fc domain can be selectively labeled at K317, with a minor level of unspecific labeling on the Fab domain. The present method can be developed as a clinical diagnostic tool to predict the success of the therapy. Furthermore, our Fc-III one step chemistry concept paves the way to a broad array of other applications in antibody bioengineering.

Abstract Clinical development of therapeutic antibodies relies on robust and thorough preclinical evaluation of multiple pharmacodynamic and pharmacokinetic parameters of antibody candidates. While current methods are available for in vitro characterization of antibodies, in vivo determination of tissue distribution, tumor accumulation and retention and potential off-target effects cannot be performed non-invasively in vivo over a prolonged period of time. Here, we used AbYlinkTM, a novel site-selective labeling method of native antibodies, enabling the single-step covalent conjugation of a radionuclide chelator to the Fc domain of any IgG antibody isotype. Given the selectivity of this reaction for the Fc region of the immunoglobulin, labeling of the antibody at this location should not compromise binding to the antigen. Chelators that are covalently attached to the antibody allows for the labeling with radioisotopes such as 68Ga, 177Lu, 111In and 89Zr, and for the sensitive and quantitative molecular in vivo measurement by Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT). AbYlinkTM also permits the conjugation of fluorescent dyes to antibodies with the aim to measure antibody pharmacodynamic parameters by intravital microscopy. In this work, we have validated the technology by labeling several antibodies and ADCs with different payloads. Importantly, antibodies conjugated with AbYlinkTM retained the same affinity to the antigen as the original, unconjugated antibodies, contrary to the randomly labelled antibodies. We will also present data on the specific radiolabeling of commercially available trastuzumab with 111Indium and the SPECT images obtained after injection of mice bearing HER2-positive tumors with 111In-trastuzumab. The present data suggest that AbYlinkTM can be used to support preclinical drug discovery tools to assist in the selection of therapeutic antibody candidates and/or for pharmacodynamic assessment of commercial antibodies. Furthermore, the specificity of the chemical conjugation suggests that AbYlinkTM can help to control the chelator distribution on the antibody drug conjugates. Citation Format: Viktoriia Postupalenko, Léo Marx, David Viertl, Natalia Gasilova, Mathilde Plantin, Nadège Gsponer, Alexandre Johanssen, Thibaut Denoel, Gerrit Hagens, Jean-Manuel Segura, Frédéric Levy, Patrick Garrouste, John Prior, Margret Schottelius, Niklaus Schaefer, Origene Nyanguile. AbYlinkTM: A site-selective labeling method for preclinical imaging of therapeutic antibodies [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 1304.

Abstract Efficient induction of cell death is essential for efficacy of cancer therapies, and may be enhanced in combination therapies that promote apoptosis. Small molecule mimetics of the pro-apoptotic protein SMAC antagonize Inhibitor of Apoptosis Proteins (IAPs). Debio 1143 is a potent oral SMAC mimetic currently assessed in clinical trials in combination with chemo- and radiotherapy in different cancer indications. We have conducted a “one vs. many” drug combination screen in which Debio 1143 is combined pairwise with a panel of 128 candidate partner agents in order to identify drug combinations that inhibit growth of lung adenocarcinoma cells. Several synergistic Debio 1143 combinations were selected for further analysis. We confirmed that Debio 1143 synergistically inhibited growth in combination with taxanes paclitaxel and docetaxel, topoisomerase inhibitor SN-38, and the bromodomain inhibitor JQ1. The combination of Debio 1143 with any of these agents further inhibited clonogenic colony formation and induced apoptosis more than either agent alone. In two Debio 1143-sensitive cell lines Debio 1143 also induced formation of the cell-death inducing complex - the ripoptosome. Interestingly, while Debio 1143 broadly reduced protein levels of cIAP1 across cell lines, specifically the combination with JQ1 also reduced cIAP2 and XIAP levels and inhibited the canonical NF-κB pathway while inducing the non-canonical NF-κB pathway. Overall, these data support utility of several combinations of Debio 1143 with other agents, especially bromodomain inhibitors, in lung adenocarcinoma and other cancers. Specifically, these findings indicate that the synergy between JQ1 and Debio 1143 may derive from JQ1 contributing on different levels to key mechanisms relevant for SMAC mimetic antitumor activity. Citation Format: Casey G. Langdon, Norbert Wiedemann, Matthew A. Held, James T. Platt, Ramanaiah Mamillapalli, Pinar Iyidogan, Nicholas Theodosakis, Frederic Levy, Denis Robichon, Claudio Zanna, Gregoire Vuagniaux, Mel Sorensen, Shaomeng Wang, Marcus W. Bosenberg, David F. Stern. Debio 1143 synergizes with taxanes, topoisomerase and bromodomain inhibitors to inhibit growth of lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2529. doi:10.1158/1538-7445.AM2015-2529

Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here.

Background: Drug resistance is a major problem in cancer therapy that may be addressed by the combination of drugs simultaneously targeting multiple critical nodes of the signalling networks controlling growth and survival of cancer cells. The members of the Inhibitor of apoptosis protein (IAP) family are frequently overexpressed in most cancer types contributing to tumour cell survival and resistance to cancer therapy. The oral monovalent IAP inhibitor Debio 1143/AT-406 is currently in early clinical development. The aim of the study was to evaluate the activity of Debio 1143 as a single agent and in drug combinations in in vitro and in vivo lung cancer models of different histotypes. Materials and Methods: Drug sensitivity was assessed in clonogenic assays on 3-D cultures of patient-derived cancer xenografts of different lung histotypes. In vitro high-throughput combination screening (HTS) of 6 human lung adenocarcinoma cell lines was used to identify synergistic drug combinations for Debio 1143. Synergy was assessed using an AUC-based curve shift analysis method and selected synergistic combinations were further studied in tumour xenograft mouse models. Results: As a single agent Debio 1143 showed differential anti-proliferative activity in vitro in a majority of patient-derived cancer xenografts of small cell and squamous histology, whereas adenocarcinoma-derived samples were less responsive. However, HTS on 6 lung adenocarcinoma cell lines revealed synergy of Debio 1143 with several standard-of-care compounds. Synergy of the combination of Debio 1143 with docetaxel was further confirmed in vivo in mouse xenografts. Interestingly, while in vitro A549 cells were insensitive to Debio 1143 alone, a marked in vivo anti-tumour activity was observed on A549 xenografts. Conclusion: The IAP inhibitor Debio 1143 has single agent activity across 3D cultures of patient-derived xenografts of different lung cancer histotypes, as well as anti-tumour activity in NSCLC tumour xenograft mouse models. These findings provide a rationale for the combination of the IAP inhibitor Debio 1143 with standard-of-care compounds in different lung cancer histotypes and are the basis for ongoing clinical trials in several cancer types. Citation Format: Casey G. Langdon, Norbert Wiedemann, Mathew A. Held, James T. Platt, Frederic Levy, Denis Robichon, Claudio Zanna, Gregoire Vuagniaux, Mel Sorensen, Shaomeng Wang, Marcus W. Bosenberg, David F. Stern. Debio 1143, an oral antagonist of the inhibitor of apoptosis proteins, synergistically enhances the effects of multiple standard of care agents in human lung cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5441. doi:10.1158/1538-7445.AM2014-5441

Abstract Background: Drug resistance is a major problem in cancer therapy, which can be addressed by simultaneously targeting multiple critical nodes of the signalling networks controlling growth and survival of cancer cells. One such approach is to target heat shock protein 90 (HSP90), a chaperone for many potent oncogenic proteins involved in proliferation, survival, invasion, metastasis and angiogenesis. Pharmacologic inhibition of HSP90 results in the proteasomal degradation of client oncoproteins thereby eliminating their oncogenic activity. The oral HSP90 inhibitor Debio 0932 displays favorable pharmacologic features. The goal of this study was to identify novel synergistic drug combinations for Debio 0932 in non-small cell lung cancer (NSCLC) and renal cell cancer (RCC) which would support the clinical development of Debio 0932 in those two indications. Material and Methods: For NSCLC, an in vitro high-throughput combination screen was performed using 6 human NSCLC cell lines bearing various genetic alterations, where Debio 0932 was combined pairwise with 128 commercially available oncology compounds in a cell viability assay. Some additional compounds not included in the panel were also tested independently. For RCC, combination of Debio 0932 with several selected standard-of-care drugs was analyzed on a panel of 8 human RCC cell lines. Synergy was assessed by using an AUC-based curve shift analysis method or according to the Chou-Talalay equation. A selection of synergistic drug combinations was further studied using tumor xenograft mouse models of human NSCLC and RCC. Results: In NSCLC, anti-proliferative synergism with Debio 0932 was observed in vitro in combination with the standard-of-care drugs docetaxel, paclitaxel or gemcitabine, as well as with mTOR inhibitors. In RCC cell lines, combinations of Debio 0932 with RCC standard-of-care drugs also displayed anti-proliferative synergy. The synergy observed in vitro was further confirmed in mouse xenografts of human NSCLC and RCC cell lines, where the drug combinations caused marked anti-tumor activity that was superior to either monotherapy. Conclusion: Several synergistic drug combinations were identified for the HSP90 inhibitor Debio 0932 in NSCLC and RCC. These findings underline the feasibility of using in vitro high-throughput screening for the discovery of novel drug combinations with increased in vivo anti-tumor efficacy . Furthermore, they provide a rationale for the combination of Debio 0932 with standard-of-care drugs in NSCLC and RCC and are the basis for ongoing clinical trials in several cancer types. Citation Format: Casey G. Langdon, Norbert Wiedemann, Hélène Maby-El Hajjami, Mathew A. Held, James T. Platt, Grégoire Vuagniaux, Marcus W. Bosenberg, David F. Stern, Frédéric Lévy. Identification of synergistic drug combinations with the oral HSP90 inhibitor Debio 0932 in non-small cell lung cancer and renal cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2014-1792

Abstract Background: Drug resistance is a major problem in cancer therapy. The combination of drugs targeting simultaneously multiple critical nodes of the signalling networks controlling growth and survival of cancer cells is necessary to achieve long-lasting responses. The members of the Inhibitor of apoptosis protein (IAP) family are key negative regulators of programmed cell death. Their frequent overexpression in most cancer types contributes to tumor cell survival and resistance to cancer therapy making IAPs attractive therapeutic targets. The oral monovalent SMAC mimetic, Debio 1143/AT-406, functions as an antagonist of multiple IAP proteins (cIAP1/2 and XIAP) and is currently in clinical development for cancer treatment. The goal of this study is to identify novel synergistic combination partners with Debio 1143 in non-small cell lung cancer (NSCLC). Material and Methods: An in vitro high-throughput combination screen was performed using 6 human NSCLC cell lines bearing various genetic alterations. Debio 1143 was pairwise combined with 128 commercially available oncology compounds in a cell viability assay. To identify supra-additive drug combinations, the dose-response curves were analysed using the Bliss independence method. Synergistic combinations were further studied using tumor xenograft mouse models. Results: Among several interesting combinations, we observed strong synergism of Debio 1143 combined with docetaxel or paclitaxel, two standard-of-care drugs in NSCLC. The beneficial effect of the combination between Debio 1143 and taxanes was further validated in multiple mouse cancer xenografts where the combination caused marked anti-tumour activity that was superior to either monotherapy. Conclusion: These findings underline the feasibility of using in vitro high-throughput screening for the discovery of novel drug combinations with increased anti-tumour efficacy in vivo. Furthermore, they provide a rationale for the combination of the SMAC mimetic Debio 1143 with taxanes and are the basis for ongoing clinical trials in several cancer types. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A16. Citation Format: Casey G. Langdon, Norbert Wiedemann, Mathew A. Held, James T. Platt, Frédéric Lévy, Claudio Zanna, Grégoire Vuagniaux, Mel Sorensen, Shaomeng Wang, Marcus W. Bosenberg, David F. Stern. A drug combination screen identifies taxanes as synergistic agents with the oral IAP inhibitor Debio 1143 in non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A16.

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate-1 (BCSC-1) as a novel tumor suppressor in melanoma. BCSC-1 expression is decreased in human MM, and its ectopic expression in MMderived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC-1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC-1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.

Reference EPFL-CONF-171519View record in Web of Science Record created on 2011-12-16, modified on 2017-05-12

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

The level of intracellular proteins is mainly regulated through modifications by ubiquitin ligases that target them for degradation. Members of the NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin-1 interacting protein 4), possess up to four WW domains for specific association with PY motif-containing substrates. We have identified sorting nexin 9 (SNX9), a protein involved in endocytic processes, as a new substrate of Itch. Itch ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using truncated proteins, we found that the interaction with SNX9 is mediated by the proline-rich domain (PRD) of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. Furthermore, this effect is specific for Itch, as NEDD4, a related PRD-containing E3 ligase, does not bind SNX9. SNX18, a second member of the SNX family containing an SH3 domain, was also found to bind to Itch. Our results indicate that the pool of substrates of NEDD4 family E3 ubiquitin ligases extends beyond proteins containing PY motifs.

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4(+) T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.