Lars Linden

Preclinical Antitumor Efficacy of BAY 1129980—a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody–Drug Conjugate for the Treatment of Non–Small Cell Lung Cancer

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non–small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody–drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893–904. ©2017 AACR.

Biophysical and Sequence-Based Methods for Identifying Monovalent and Bivalent Antibodies with High Colloidal Stability

In vitro antibody discovery and/or affinity maturation are often performed using antibody fragments (Fabs), but most monovalent Fabs are reformatted as bivalent IgGs (monoclonal antibodies, mAbs) for therapeutic applications. One problem related to reformatting antibodies is that the bivalency of mAbs can lead to increased antibody self-association and poor biophysical properties (e.g., reduced antibody solubility and increased viscosity). Therefore, it is important to identify monovalent Fabs early in the discovery and/or optimization process that will display favorable biophysical properties when reformatted as bivalent mAbs. Here we demonstrate a facile approach for evaluating Fab self-association in a multivalent assay format that is capable of identifying antibodies with low self-association and favorable colloidal properties when reformatted as bivalent mAbs. Our approach (self-interaction nanoparticle spectroscopy, SINS) involves immobilizing Fabs on gold nanoparticles in a multivalent format (multiple Fabs per nanoparticle) and evaluating their self-association behavior via shifts in the plasmon wavelength or changes in the absorbance values. Importantly, we find that SINS measurements of Fab self-association are correlated with self-interaction measurements of bivalent mAbs and are useful for identifying antibodies with favorable biophysical properties. Moreover, the significant differences in the levels of self-association detected for Fabs and mAbs with similar frameworks can be largely explained by the physicochemical properties of the complementarity-determining regions (CDRs). Comparison of the properties of the CDRs in this study relative to those of approved therapeutic antibodies reveals several key factors (net charge, fraction of charged residues, and presence of self-interaction motifs) that strongly influence antibody self-association behavior. Increased positive charge in the CDRs was observed to correlate with increased risk of high self-association for the mAbs in this study and clinical-stage antibodies. We expect that these findings will be useful for improving the development of therapeutic antibodies that are well suited for high concentration applications.

Abstract 855: Increased in vitro potency and in vivo efficacy of FGFR2-targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344

Targeted Thorium-227 Conjugates (TTCs) consist of the alpha emitter Thorium-227 (227Th) coupled, by a 3, 2-HOPO chelator, to a tumor specific antibody. The alpha particles release high energy over a short range (2- 10 cell diameters), resulting in a potent local irradiation of the tumor with limited damage to surrounding tissue. Here, we describe the in vitro and in vivo evaluation of an FGFR2 targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344. FGFR2 (fibroblast growth factor receptor 2) is a receptor tyrosine kinase and overexpression of FGFR2 has been described in different cancers, while its expression in healthy human tissues is moderate to low. This renders FGFR2 an attractive antigen to explore the concept of targeted alpha therapy (TAT). The mode-of-action of TTCs is based on the induction of clustered DNA double strand breaks and G2 cell cycle arrest. We hypothesized that combination of FGFR2-TTC with inhibitors of DNA damage response (DDRi9s) may enhance potency and efficacy. The ataxia telangiectasia and Rad3-related protein (ATR) kinase is a central mediator of DDR. ATR kinase responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Inhibition of ATR kinase activity induces cell death especially in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Therefore, we investigated whether the combination of the FGFR2-TTC with the ATRi BAY 1895344 results in enhanced tumor sensitivity in vitro and in vivo. In in vitro cytotoxicity assays, the combination of FGFR2-TTC and BAY 1895344 resulted in increased potency of the FGFR2-TTC on three different cancer cell lines (KATO III (gastric), MFM-223 (triple negative breast cancer), SUM52-PE (triple negative breast cancer)). Mechanistic analysis demonstrated that the combination treatment resulted in reduced levels of G2 arrest and increased levels of DNA damage in comparison to single agent treatments. The combination was further evaluated in vivo using the MFM-223 breast cancer xenograft model. An increased anti-tumor efficacy of a low dose of FGFR2-TTC (100 kBq/kg) was observed in combination with BAY 1895344 compared to animals treated with vehicle. The presented data support the mechanism-based rationale for combining DNA damage induction by FGFR2-TTC with DNA damage repair inhibition using ATRi BAY 1895344. Our findings warrant further exploration of TTCs in combination with BAY 1895344 for cancer therapy. Citation Format: Katrine Wickstroem, Urs B. Hagemann, Antje M. Wengner, Anette Sommer, Alexander Kristian, Christine Ellingsen, Roger M. Bjerke, Jenny Karlsson, Olav B. Ryan, Lars Linden, Bertolt Kreft, Dominik Mumberg, Hanno Wild, Karl Ziegelbauer, Alan Cuthbertson. Increased in vitro potency and in vivo efficacy of FGFR2-targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 855.

Abstract 3091: Response of C4.4A-positive patient-derived xenograft models of ESCC, HNSCC and bladder cancer to BAY1129980, a C4.4A-targeted antibody drug conjugate

C4.4A (LYPD3) is a cancer- and metastasis-associated transmembrane cell surface protein which is expressed at high frequency and density in multiple tumor types including squamous and non-squamous non-small cell lung carcinoma ((NSCLC), head & neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC) and bladder cancer. C4.4A expression is restricted to a limited number of tissues (e.g. suprabasal layer of skin) making C4.4A an attractive target for the treatment of cancer with a C4.4A-targeted antibody-drug conjugate (ADC). BAY 1129980 (C4.4A-ADC), is an ADC consisting of a fully human C4.4A-targeting monoclonal antibody (technology licensed from BioInvent) conjugated via a novel, non-cleavable alkyl hydrazide linker to a novel, highly potent auristatin W, an antimitotic agent (technology licensed from Seattle Genetics, Inc.). This C4.4A-ADC has been previously shown to be efficacious in C4.4A positive cell line-derived and PDX models of NSCLC. Here we present new preclinical efficacy data of C4.4A-ADC in patient-derived xenograft (PDX) models of ESCC, HNSCC and bladder cancer. Models were selected based on tumor C4.4A levels as determined by mRNA levels and immunohistochemistry (IHC), the latter of which allowed ranking of models according to H-score, percentage of C4.4A positivity, and staining intensity (0 to 3+) in the cell membrane. Representative C4.4A-positive models were selected for in vivo efficacy studies (n=7 each): 4 HNSCC, 4 ESCC and 2 bladder cancers. C4.4A-ADC was administered as one cycle (Q4Dx3) at doses of 7.5 and 15 mg/kg, and efficacy was assessed up to 4 weeks post treatment for optimum tumor growth inhibition (TGI). In ESCC models a response to C4.4A ADC was seen in ES0190 (TGI of 77%; 15mg/kg) and in ES0195 (59%). In HNSCC models an ADC effect on tumor growth was observed in HN10847 (46%), HN9619 (34%), and HN10321 (22%). Finally, both bladder models tested were sensitive to treatment, with a transient response seen in BL0597 (41%) and a strong and complete tumor growth control in BL5001 (93%,) which was superior to cisplatin. The data show that C4.4A-positive PDX models of ESCC, HNSCC and bladder cancer can respond to C4.4A-ADC. C4.4A expression served as a marker for preselection of the models. Nevertheless, other factors may affect response and sensitivity of these tumor models, such as sensitivity to tubulin inhibition, ADC uptake and intracellular processing. In summary, these data support further exploration of the potential of BAY 1129980 in HNSCC, ESCC and bladder cancer in addition to NSCLC. A Phase 1 clinical trial of BAY 1129980 is ongoing (NCT02134197). Citation Format: Joerg Willuda, Carol Pena, Christoph Kneip, Patricia E. Carrigan, Hans-Georg Lerchen, Lars Linden, Bertolt D. Kreft. Response of C4.4A-positive patient-derived xenograft models of ESCC, HNSCC and bladder cancer to BAY1129980, a C4.4A-targeted antibody drug conjugate [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 3091. doi:10.1158/1538-7445.AM2017-3091

Abstract 1754: Identification of BAY 94-9343, a mesothelin antibody-drug conjugate (ADC): Characterization and anti-tumor activity in mesothelin-positive preclinical tumor models

Monoclonal antibodies have proven to be very effective in the treatment of various cancers, including solid tumors. For example, HERCEPTIN® and Erbitux® are successfully used to treat HER2-positive breast cancer and EGFR-positive colorectal cancer, respectively. Conjugation of cytotoxic drugs to antibodies represents a promising approach to improve cancer therapy. Antibody-drug conjugates (ADCs) are able to deliver highly potent toxophores to tumors while at the same time reducing systemic toxicity. Promising efficacy and tolerability profiles of ADCs have been observed in clinical trials including Hodgkin lymphoma (brentuximab vedotin) and breast cancer (trastuzumab-DM1), thus, development of new ADCs targeting tumor- associated antigens has potential to identifiy novel cancer therapeutics. Mesothelin, a glycoprotein expressed in mesothelial cells found in the membrane lining of the peritoneal and pleural cavities, is overexpressed in all mesotheliomas as well as many ovarian and pancreatic cancers. Due to its limited expression on normal tissues and higher expression in a number of tumor types, mesothelin represents an attractive ADC target. BAY 94-9343 consists of a fully human anti-mesothelin IgG1 antibody conjugated to the potent tubulin-binding drug DM4 with an average of 3.2 drug molecules per antibody. The resulting ADC bound to human recombinant mesothelin with high affinity (Kd = 15nM) leading to antigen-dependent internalization and potent cytotoxicity (nanomolar range in vitro IC50) in tumor cells that express mesothelin either endogenously or exogenously, but not in mesothelin-negative cells. In vivo, BAY 94-9343 demonstrated dose-dependent, mesothelin-specific anti-tumor efficacy in subcutaneous and orthotopic xenograft models at doses between 2.5 and 10 mg/kg using a Q3Dx3 schedule. Endogenously expressing mesothelin tumor models included sc and orthotopic OVCAR3 (ovarian), sc BxPC-3 (pancreatic) and sc NCI-H226 (mesothelioma). Furthermore, in mesothelin-positive patient-derived preclinical tumor models of both platinum-resistant ovarian cancer and gemcitabine-resistant pancreatic cancer, BAY 94-9343 exhibited high anti-tumor efficacy leading to partial and complete tumor regressions with a 10mg/kg Q3Dx3 dosing schedule. This ADC was well tolerated in mice at 10mg/kg (Q3Dx3) without any evidence of body weight loss, compared to either cisplatin or gemcitabine treatments. In summary, BAY 94-9343 is a mesothelin-targeted ADC with promising preclinical anti-tumor activity for mesothelin-positive tumors. 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 1754. doi:10.1158/1538-7445.AM2011-1754