Heike Petrul

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.

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 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.