Christoph Schatz

Therapeutic Mechanism and Efficacy of the Antibody Drug-Conjugate BAY 79-4620 Targeting Human Carbonic Anhydrase 9

Carbonic anhydrase IX (CAIX) is a cell surface glycoprotein that is expressed in many different tumors and yet restricted in normal tissues to the gastrointestinal tract. It is upregulated by hypoxia and correlates with tumor grade and poor survival in several tumor indications. Monoclonal antibodies (mAb) with single digit nanomolar binding affinity for CAIX were derived by panning with the recombinant ectodomain of CAIX against the MorphoSys HUCAL Gold library of human Fabs. Highest affinity Fabs were converted to full-length IgGs and subjected to further characterization based upon their avidity and selectivity for CAIX, their capacity to undergo internalization in CAIX-expressing cell lines, and their selective localization to CAIX-positive human xenografted tumors when administered to mice as fluorescent conjugates. Through this selection process, the 3ee9 mAb was identified, which upon conjugation to monomethyl auristatin E through a self-immolative enzyme-cleavable linker yielded the potent and selective CAIX antibody–drug conjugate CAIX-ADC (BAY 79-4620). In preclinical human xenograft models in mice representing several tumor indications, BAY 79-4620 showed potent antitumor efficacy and in some models showed partial and complete tumor shrinkage even following a single dose. The mechanism of action was shown by histology to involve the sequelae of events typical of antitubulin agents. Efficacy in murine preclinical models correlated semiquantitatively, with CAIX expression levels as determined by immunohistochemistry and ELISA. These preclinical data collectively support the development of BAY 79-4620 for the treatment of cancer patients with CAIX overexpressing tumors. Mol Cancer Ther; 11(2); 340–9. ©2011 AACR.

Anetumab ravtansine inhibits tumor growth and shows additive effect in combination with targeted agents and chemotherapy in mesothelin-expressing human ovarian cancer models

Despite the recent advances in the treatment of ovarian cancer, it remains an area of high unmet medical need. Epithelial ovarian cancer is associated with high levels of mesothelin expression, and therefore, mesothelin is an attractive candidate target for the treatment of this disease. Herein, we investigated the antitumor efficacy of the mesothelin-targeting antibody-drug conjugate (ADC) anetumab ravtansine as a novel treatment option for ovarian cancer in monotherapy and in combination with the antitumor agents pegylated liposomal doxorubicin (PLD), carboplatin, copanlisib and bevacizumab. Anetumab ravtansine showed potent antitumor activity as a monotherapy in ovarian cancer models with high mesothelin expression. No activity was seen in mesothelin-negative models. The combination of anetumab ravtansine with PLD showed additive anti-proliferative activity in vitro, which translated into improved therapeutic in vivo efficacy in ovarian cancer cell line- and patient-derived xenograft (PDX) models compared to either agents as a monotherapy. The combination of anetumab ravtansine with the PI3Kα/δ inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell lines in vitro, showing increased apoptosis in response to the combination treatment. In vivo, the combination of anetumab ravtansine with copanlisib resulted in more potent antitumor activity than either of the treatments alone. Likewise, the combination of anetumab ravtansine with carboplatin or bevacizumab showed improved in vivo efficacy in the ST081 and OVCAR-3 models, respectively. All combinations were well-tolerated. Taken together, these data support the development of anetumab ravtansine for ovarian cancer treatment and highlight its suitability for combination therapy with PLD, carboplatin, copanlisib, or bevacizumab.

Abstract 5859: HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in preclinical models of trastuzumab and T-DM1 resistance

The human epidermal growth factor receptor 2 (HER2) is encoded by the proto-oncogene c-erbB-2 and initiates downstream signaling pathways leading to cell proliferation and tumorigenesis. HER2 is overexpressed in several cancer (Ca) types and is one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Several HER2-targeting antibodies as well as antibody-drug conjugates are either approved or are in clinical development. Prolonged treatment with monoclonal antibodies and antibody drug conjugates have resulted in development of resistance and so there is still an unmet medical need for drugs of new mechanism of action targeting this important receptor system. We describe herein the generation of a high energy, alpha-particle emitting HER2 targeted thorium-227 antibody-chelator conjugate. HER2-TTC consists of the humanized HER2 targeting IgG1 antibody (trastuzumab) covalently linked via an amide bond to a 3,2-hydroxypyridino-based chelator moiety, enabling efficient radiolabeling with the alpha particle emitting radionuclide thorium-227 (Th-227). HER2-TTC was prepared at high radiochemical yield and purity. When tested for binding to recombinant HER2, HER2-TTC was shown to retain comparable binding affinity to trastuzumab. In vitro cytotoxicity experiments were performed on 8 cell lines with different HER2 expression levels (from 7 000 - 500 000 mAbs bound/ cell as determined by FACS) of breast, ovarian, gastric and lung cancer origin. HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range. In vivo biodistribution and anti-tumor efficacy of HER2-TTC was evaluated in the dose range 100-500 kBq/kg at a protein dose of 0.14 mg/kg and i.v. injection in the s.c. KPL-4 breast and Calu-3 lung model previously described to be resistant to trastuzumab. The biodistribution study demonstrated specific tumor accumulation of HER2-TTC in both models with a maximum of 77 and 50 %ID/g 227Th at t = 168 h post dose (decay corrected to T0), respectively. Significant antitumor efficacy was shown for HER2-TTC in the JIMT-1 s.c. breast Ca xenograft model resistant to trastuzumab and T-DM1. The promising preclinical anti-tumor activity supports the development of the targeted alpha therapeutic HER2-TTC for the treatment of trastuzumab and T-DM1 resistant patients. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Alexander Kristian, Christine Ellingsen, Dessislava Mihaylova, Solene Geraudie, Bard Indrevoll, Uta Wirnitzer, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in preclinical models of trastuzumab and T-DM1 resistance [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 5859. doi:10.1158/1538-7445.AM2017-5859

Abstract 5857: HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model

In 2015, the estimated incidence of new breast cancer (Ca) cases in the US was 234.190 and number of deaths 40.730. Human epidermal growth factor receptor 2 (HER2) is encoded by the proto-oncogene c-erbB-2 and initiates downstream signaling pathways leading to cell proliferation and tumorigenesis. HER2 is overexpressed in several cancer types and has emerged as one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Given that 20% of breast Ca patients are HER2 positive and 70% of patients with metastatic disease will develop bone metastases and associated morbidities, there is still an unmet medical need for improved therapies targeting HER2. Radium-223 (Ra-223) is a novel targeted alpha therapeutic for treatment of patients with castration-resistant prostate cancer and bone metastases. Localized high energy alpha particle emission induces double-stranded DNA breaks and cellular apoptosis. Thorium-227 (Th-227) is the immediate precursor for Ra-223 which, in contrast to Ra-223, can be complexed by chelating agents at high affinity, allowing targeted delivery to tumor cells via antibodies. We describe herein the generation of a novel HER2-targeted Th-227 conjugate (HER2-TTC). HER2-TTC consists of the humanized HER2 targeting IgG1 antibody trastuzumab covalently linked via an amide bond to a 3,2-hydroxypyridino-based chelator moiety, enabling efficient radiolabeling with the alpha particle emitting radionuclide Th-227. Anonymized samples of consenting breast cancer patients were analyzed by Immunohistochemistry (IHC). The IHC data demonstrated HER2 positive expression in breast tumor and matched bone metastases, supporting the preclinical evaluation of the anti-tumor efficacy of HER2-TTC in the BT-474 orthotopic bone mouse model. HER2-TTC was prepared at high radiochemical yield and purity. When tested for binding to recombinant HER2, HER2-TTC was shown to retain comparable binding affinity to trastuzumab. In vitro cytotoxicity experiment of HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range on breast cancer cell line BT-474 (430 000 mAbs bound/ cell as determined by FACS). Anti-tumor efficacy of HER2-TTC was evaluated at 250 and 500 kBq/kg at a protein dose of 0.14 mg/kg. X-ray imaging, serum bone formation marker PINP, micro CT 3D reconstruction imaging and histological analysis demonstrated significantly reduced bone lesions and tumor induced bone remodeling. The promising preclinical anti-tumor activity supports the development of the HER2-TTC as a novel targeted alpha therapeutic for the treatment of patients with HER2 positive bone metastatic disease. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Christine Ellingsen, Alexander Kristian, Dessislava Mihaylova, Steinar R. Uran, Mari Suominen, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model [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 5857. doi:10.1158/1538-7445.AM2017-5857

Abstract DDT02-01: In vitro and in vivo characterization of a novel anti-fibroblast growth factor receptor (FGFR) 2 antibody (BAY 1179470) for the treatment of gastric cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gastric cancer is the second most common cause of cancer-related mortality worldwide, thus new treatment options are urgently needed. In a subset of gastric cancers, over-expression of fibroblast growth factor receptor 2 (FGFR2), a receptor tyrosine kinase, has been described and may represent a potential therapeutic target for the treatment of FGFR2-positive gastric cancer patients. To this end, we have generated a fully human anti-FGFR2 antibody (BAY 1179470) using the BioInvent Phage Display library. BAY 1179470 binds to a unique FGFR2-specific epitope that is present in all FGFR2 isoforms. Upon binding to FGFR2, BAY 1179470 induces receptor dimerization, internalization and degradation, resulting in significant tumor growth inhibition in vivo in cell line-based and patient-derived gastric cancer models overexpressing FGFR2. Additive anti-tumor efficacy in vivo was achieved by combining BAY 1179470 with either cisplatin or paclitaxel. BAY 1179470 is fully cross-reactive with FGFR2 orthologues of mouse, rat, pig, cynomolgus monkey and rhesus macaque. No significant safety findings have been seen in animal studies. Thus, BAY 1179470 represents a novel anti-FGFR2 antibody with high anti-tumor activity in gastric cancer models and an excellent preclinical safety profile. BAY 1179470 is currently being tested in a first-in-man study in all-comers ([NCT01881217][1]) in Japan. Citation Format: Charlotte Kopitz, Anette Sommer, Stefanie Hammer, Axel Harrenga, Beatrix Stelte-Ludwig, Frank Dittmer, Frank Reetz, Ekkehard May, Ruprecht Zierz, Sabine Wittemer-Rump, Christoph Schatz, H. T. Huynh, Karl Ziegelbauer, Bertolt Kreft. In vitro and in vivo characterization of a novel anti-fibroblast growth factor receptor (FGFR) 2 antibody (BAY 1179470) for the treatment of gastric 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 DDT02-01. doi:10.1158/1538-7445.AM2014-DDT02-01 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01881217&atom=%2Fcanres%2F74%2F19_Supplement%2FDDT02-01.atom

Abstract 3614: In vivo efficacy of the carbonic anhydrase IX (CA9)-targeted antibody-drug conjugate BAY 79-4620 is superior to that of microtubule inhibitors in preclinical models of NSCLC, gastric and colorectal cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BAY 79-4620 is a novel antibody drug conjugate (ADC) consisting of a fully human monoclonal antibody directed against carbonic anhydrase IX (CA9) conjugated to the auristatin derivative monomethyl auristatin E (MMAE) and is currently in Phase I clinical testing. CA9 is overexpressed in a range of tumor types, including gastric cancer, non-small cell lung cancer (NSCLC), pancreatic cancer and colorectal cancer. CA9 is a hypoxia-inducible protein regulated by HIF-1α, and its expression has been linked to higher aggressiveness of tumors and is predictive of poor prognosis in several cancers. In this study, we first compared the internalization rates of the ADC BAY 79-4620 and of the naked antibody, which were found to be similar in several cell lines, indicating that CA9 internalization in vitro is not affected by the conjugated toxophore. It has previously been shown that the cytotoxicity of BAY 79-4620 depends on both CA9 expression and sensitivity of tumor cells to tubulin inhibitors. The anti-tumor efficacy of BAY 79-4620 was therefore compared to that of free toxophore MMAE as well as paclitaxel. At cumulative doses corresponding to a fraction of that of paclitaxel (calculated as toxophore equivalents), BAY 79-4620 showed equivalent or superior efficacy to paclitaxel and MMAE in xenograft models of human NSCLC and gastric carcinoma. Cumulative BAY 79-4620 doses corresponding to only 30% of the highest dose of free MMAE given showed equivalent or superior efficacy in xenograft models of non-small cell lung carcinoma. Moreover, BAY 79-4620 also showed efficacy in patient-derived NSCLC models that are resistant to paclitaxel and/or cisplatin. Finally, regrowing CRC tumors after an initial response to BAY 79-4620 responded well to a second cycle of BAY 79-4620 treatment. This indicates that no resistance through clonal selection of CA9 negative tumor cells was induced and that repeated treatment cycles should be possible for cancer patients. These results show the superiority of the CA9-targeted approach over the systemic administration of tubulin inhibitors, component of treatment regimens commonly used for the treatment of NSCLC and gastric cancer. Efficacy of BAY 79-4620 was particularly high in models expressing high levels of CA9, making BAY 79-4620 a promising agent for the treatment of CA9-positive NSCLC and gastric cancer. 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 3614. doi:10.1158/1538-7445.AM2011-3614

Abstract 4478: BAY 80-6946, a highly potent and efficacious class I PI3K inhibitor, induces complete tumor regression or tumor stasis in rat xenograft tumor models with PIK3CA mutant or PTEN deletion

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We report on BAY 80-6946, a highly selective and potent pan class I PI3K inhibitor currently in Phase I clinical trials. The PI3K pathway is aberrantly activated in many tumors either by growth factor receptor tyrosine kinases, or by the genetic mutation and amplification of key pathway components. BAY 80-6946 was evaluated in a panel of human xenograft tumor models. This compound was administered every second (Q2D) day or once weekly using intravenous (i.v.) bolus dosing. BAY 80-6946 was well tolerated with a MTD of 10 mg/kg (Q2D) in athymic rats. In the rat KPL-4 breast tumor model (PIK3CAH1047R and HER2O/E (protein levels)), BAY 80-6946 showed robust and dose-dependent tumor growth inhibition with complete tumor regressions (10/10 CRs) observed at the 3.0 and 6.0 mg/kg doses. The animals remained tumor-free until termination of the study on day 65. BAY 80-6946 also demonstrated complete tumor stasis in the human H460 large cell lung carcinoma and HCT116 colorectal carcinoma models (bearing PIK3CA (E545K and H1047R respectively) along with mutations in KRAS), In U87MG human glioma tumor model (PTEN-deletion) dosed Q2D schedule partial regressions (PRs) were observed. The robust in vivo anti-tumor activities for this class I pan PI3K inhibitor correlated very well with the potent and sustained pharmacodynamic inhibition of p-Akt in tumors, which allowed several i.v. dosing schedules including a once weekly schedule. Thus, BAY 80-6946 given at 6mg/kg, BID once weekly demonstrated similar efficacy as given every other day (Q2D) at 6 mg/kg. Further investigation of pharmacodynamic biomakers revealed that i.v. bolus injection of BAY 80-6946 1 and 6 mg/kg produced a significant dose-dependent reduction in [18F]FDG uptake measured by decrease in SUV in rat H460 tumors supporting the clinical exploration of [18F]FDG imaging to monitor pharmacodynamic drug activity in patients. In conclusion, BAY 80-6946 is a highly efficacious and well tolerated PI3K inhibitor in preclinical rat tumor models expressing HER2, PIK3CA mutant and PTEN-deletion. BAY 80-6946 is currently in phase I clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4478.

Abstract 2577: In vitro and in vivo efficacy of the anti-MN immunoconjugate BAY 79-4620, MN-IC, in MN (CAIX) expressing preclinical tumor models

BAY 79-4620 is a novel immunoconjugate consisting of a fully human monoclonal antibody directed against MN (carbonic anhydrase IX; CAIX) conjugated with the auristatin derivative MMAE currently in Phase I testing. CAIX is overexpressed in a range of tumor types, such as gastric cancer, non-small cell lung cancer, pancreatic cancer or colorectal cancer. CAIX expression is regulated by HIF-1α, making this protein a marker associated with tumor hypoxia. Expression of this protein has been linked to higher aggressiveness of tumors and is predictive of poor prognosis in several cancers. We report on the pharmacological profile of a novel immunoconjugate directed against MN (CAIX) conjugated with the auristatin derivative MMAE to a fully human anti-MN (CAIX) monoclonal antibody (BAY 79-4620). In vitro studies showed the specific binding and internalization of BAY 79-4620 into CAIX-expressing tumor cells. Intracellular release of the tubulin-inhibiting toxophore MMAE led to mitotic arrest and specific cell kill of tumor cells, with EC50s in the low nanomolar range in most CAIX-positive tumor cell lines tested. In CAIX-negative tumor cell lines, a cytotoxic effect of BAY 79-4620 was seen at only very high doses. Cytotoxicity of this immunoconjugate depends on both CAIX expression and sensitivity of tumor cells to tubulin inhibitors. In vivo activity of the CAIX targeted Mab-MMAE conjugate resulted in higher efficacy (minimum effective dose (MED) of 0.625mg/kg in the HeLa-MaTu model, Q4Dx3 dosing schedule) compared to the systemic administration of either free MMAE toxophore (MED not achieved) or unconjugated anti-CAIX antibody which lacked efficacy in all models tested. Tumor regressions (in 80% of the animals) were achieved at doses of immunoconjugate as low as 1.25mg/kg while higher doses up to 10 mg/kg resulted in complete tumor eradication (in 90% of the animals treated). Efficacy in the HT29 model tested was schedule independent when comparing single dose, Q7Dx2 and Q4Dx3 schedules. The in vivo mode of action of the immunoconjugate was confirmed by tubulin staining in the tumor sections. Treatment with the immunoconjugate was found to be less toxic than that with free MMAE. The maximum tolerated dose of BAY 79-4620 in mice was 60mg/kg, which in terms of toxophore delivered exceeds the LD50 of MMAE (1mg/kg). In summary, these results demonstrate that targeted delivery of MMAE resulted in higher efficacy of BAY 79-4620 in tumor models with high CAIX expression. The immunoconjugate BAY 79-4620 currently in Phase I testing is a promising novel agent for the treatment of gastric, non-small cell lung, pancreatic, colorectal and other MN (CAIX)-positive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2577.

Abstract 2704: Efficacy and candidate biomarker evaluation for the anti-MN immunoconjugate BAY 79-4620, MN-IC in MN (CAIX) positive preclinical xenograft models

BAY 79-4620 is a novel immunoconjugate consisting of a fully human monoclonal antibody directed against MN (carbonic anhydrase IX, CAIX) conjugated with the auristatin derivative MMAE currently in Phase I clinical testing. MN is overexpressed in a range of tumor types, such as gastric cancer, non-small cell lung cancer, pancreatic cancer or colorectal cancer. CAIX expression is regulated by HIF-1α and therefore associated with tumor hypoxia. Here we report on the correlation of tumor CAIX antigen expression levels, as determined by a newly developed CAIX IHC staining protocol, and the efficacy of BAY 79-4620 in six preclinical tumor models. Further, CAIX levels in xenograft tumors were compared to those in clinical samples of human tumors. Efficacy of BAY 79-4620 in tumor models increased with their CAIX expression levels. HeLa-MaTu, HT29 and PC3mm2 models characterized by the highest expression of CAIX showed 56-92 % tumor growth inhibition, when treated with 1 mg/kg BAY 79-4620. Hs746T, the tumor model with the lowest CAIX level, did not show tumor growth inhibition at 1 mg/kg MN-IC, but responded to higher doses. Analysis of CAIX expression by IHC using human tumor samples (gastric, CRC, NSCLC and breast) demonstrated that tumor cell CAIX expression varied both within and between tumor types. IHC scoring revealed that 95% of colon cancer tumors, 55% of NSCLC tumors, and 35% of breast cancer tumors assayed had ≥10% of cells scoring 2+ or 3+ by IHC. In preclinical models analysis of serum CAIX extracellular domain (ECD) by ELISA revealed an initial rise in plasma CAIX ECD as early as 2 days after 30 mg/kg BAY 79-4620 followed by a decrease to below-baseline levels. In two out of three models tested, changes or increased levels of the apoptosis marker CK18 were observed following BAY 79-4620 administration. Changes in serum CAIX ECD levels as well as CK18 will be explored as an early pharmacodynamic (PD) response markers in clinical trials for BAY 79-4620. In summary, these results demonstrate that targeted delivery of MMAE resulted in higher efficacy of BAY 79-4620 in tumor models with high CAIX expression. Responsive xenograft models were representative of clinical samples with respect to CAIX expression scored by IHC. Quantification of MN (CAIX) by IHC is a candidate stratification biomarker for the immunoconjugate BAY 79-4620 currently in Phase I testing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2704.