Guenther Adolf

Target Binding Properties and Cellular Activity of Afatinib (BIBW 2992), an Irreversible ErbB Family Blocker

Deregulation of the ErbB (proto-oncogene B of the avian erythroblastosis virus AEV-H strain) receptor network is well recognized as an oncogenic driver in epithelial cancers. Several targeted drugs have been developed, including antibodies and small-molecule kinase inhibitors, each of them characterized by distinct patterns of ErbB receptor interactions. Understanding the precise pharmacological properties of these compounds is important for optimal use in clinical practice. Afatinib [BIBW 2992; N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide] is an ATP-competitive anilinoquinazoline derivative harboring a reactive acrylamide group. It was designed to covalently bind and irreversibly block enzymatically active ErbB receptor family members. Here, we show by X-ray crystallography the covalent binding of afatinib to wild-type epidermal growth factor receptor (EGFR) and by mass spectrometry the covalent interaction with EGFR, EGFRL858R/T790M, human epidermal growth factor receptor 2 (HER2), and ErbB-4. Afatinib potently inhibits the enymatic activity of ErbB-4 (EC50 = 1 nM) and the proliferation of cancer cell lines driven by multiple ErbB receptor aberrations at concentrations below 100 nM. N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butanamide (BI 37781), a close analog of afatinib lacking the acrylamide group and thus incapable of covalent bond formation, had similar potency on cells driven by EGFR or EGFRL858R, but less or no detectable activity on cells expressing EGFRL858R/ T790M HER2 or ErbB-4. These results stress the importance of the acrylamide group and show that afatinib differs from approved ErbB targeting agents by irreversibly inhibiting the kinase activity of all ErbB family members. They provide a mechanistic rationale for the distinct pharmacological features of this compound and explain the clinical activity seen in some patients who are resistant to antibody or kinase inhibitor therapy because of secondary mutations or ErbB receptor “reprogramming.”

Effective Immunoconjugate Therapy in Cancer Models Targeting a Serine Protease of Tumor Fibroblasts

Purpose: Invasion and metastasis of malignant epithelial cells into normal tissues is accompanied by adaptive changes in the mesenchyme-derived supporting stroma of the target organs. Altered gene expression in these nontransformed stromal cells provides potential targets for therapy. The present study was undertaken to determine the antitumor effects of an antibody-conjugate against fibroblast activation protein-α, a cell surface protease of activated tumor fibroblasts. Experimental Design: A novel antibody-maytansinoid conjugate, monoclonal antibody (mAb) FAP5-DM1, was developed to target a shared epitope of human, mouse, and cynomolgus monkey fibroblast activation protein-α, enabling preclinical efficacy and tolerability assessments. We have used stroma-rich models in immunodeficient mice, which recapitulate the histotypic arrangement found in human epithelial cancers. Results: Treatment with mAb FAP5-DM1 induced long-lasting inhibition of tumor growth and complete regressions in xenograft models of lung, pancreas, and head and neck cancers with no signs of intolerability. Analysis of chemically distinct conjugates, resistance models, and biomarkers implicates a unique mode of action, with mitotic arrest and apoptosis of malignant epithelial cells coupled to disruption of fibroblastic and vascular structures. Conclusions: We show that mAb FAP5-DM1 combines excellent efficacy and tolerability and provides a first assessment of the mode of action of a novel drug candidate for tumor stroma targeting, thus encouraging further development toward clinical testing of this treatment paradigm.

Beyond Trastuzumab: Second-Generation Targeted Therapies for HER-2-positive Breast Cancer

Growth factor receptors of the ErbB family play key roles in transmitting mitogenic and anti-apoptotic signals in epithelial cells. Aberrant activation of these pathways by a variety of mechanisms, including receptor over-expression due to gene amplification and activating mutations in receptors or downstream signal transducers, contributes to tumourigenesis, invasion and tumour angiogenesis. Consequently, these pathways have been the focus of intense drug discovery activities for a number of years, resulting in several approved and development-stage therapeutic agents. These include monoclonal antibodies as well as low-molecular-weight kinase inhibitors. In particular, trastuzumab, a monoclonal antibody specific for the human epidermal growth factor receptor (HER-2) receptor, has provided a major therapeutic advance for patients with HER-2-positive breast cancer, and the drug has often been heralded as the first example of personalised cancer medicine. Unfortunately, as a consequence of the side-effect profile of trastuzumab, a proportion of patients are not eligible for treatment; in addition, primary and acquired resistance mechanisms limit its efficacy. Further research into the mechanisms of resistance suggests that inhibition of additional members of the ErbB family, in particular the epidermal growth factor receptor (EGFR), also known as HER1, may maximise inhibition of the signalling pathways with a resultant improvement in efficacy. This chapter focuses on small-molecule inhibitors of both the HER-2 and EGFR/HER1 kinases that bind to their targets either reversibly (lapatinib, XL647, AEE788) or irreversibly (neratinib, pelitinib, BIBW 2992). The data reviewed here indicate that such inhibitors will be a useful addition to currently available treatment options for women with HER-2-positive breast cancer.

Abstract A249: BI 853520, a potent and highly selective inhibitor of protein tyrosine kinase 2 (focal adhesion kinase), shows efficacy in multiple xenograft models of human cancer.

PTK2/FAK is a widely expressed non-receptor tyrosine kinase located mainly at focal adhesions. Sensing upstream signals from growth factor receptors as well as integrins, the enzyme contributes to activation of multiple downstream signaling pathways involved in the regulation of cell survival, proliferation and motility. PTK2 protein expression is elevated in many human cancers, and dysregulated PTK2 is known to mediate anchorage-independent growth of malignant cells. Inhibition of PTK2 activity thus may interfere with tumor growth and metastasis formation. BI 853520 inhibited the enzymatic activity of PTK2 at low nanomolar concentrations. In a selectivity screen of 264 tyrosine and serine/threonine kinases, BI 853520 inhibited only 4 enzymes at a concentration of 1,000 nM by more than 50%. In contrast to PTK2 inhibitors described previously, the compound showed more than 1,000-fold selectivity against closely related kinases such as PYK2/PTK2B. In human PC-3 prostate carcinoma cells, PTK2 is highly expressed and phosphorylated on Tyr 397 . Treatment of PC-3 cells with BI 853520 inhibited autophosphorylation with an EC 50 of 1 nM. Colony formation of PC-3 cells in a soft agar matrix was inhibited with an EC 50 of 3 nM. In contrast, the same cell line growing in conventional monolayer culture showed growth inhibition only at 1,000-fold higher concentration. Similar results were obtained for other carcinoma cell lines. BI 853520 administered to immunodeficient nude mice showed good oral bioavailability (F = 90%) and a half-life of 5 hours. Daily oral treatment at dose levels up to 100 mg/kg over a period of several weeks was well tolerated by the animals. Treatment with BI 853520 resulted in suppression of PTK2 autophosphorylation in tissue samples from both human tumor xenografts and mouse skin. BI 853520 was tested for efficacy in a panel of 18 human tumor xenograft models representing multiple indications, including carcinomas of the lung, ovary, pancreas and prostate as well as sarcomas. At daily oral doses of 50 mg/kg the compound was highly active in a subset of models, including the PC-3 prostate carcinoma, resulting in strong suppression of tumor growth (TGI > 90% compared to controls) or tumor regression in individual animals. Significant inhibition of tumor growth was observed at doses as low as 6 mg/kg, indicating a substantial therapeutic window and opportunities for combination therapy. Another subset of models showed intermediate drug sensitivity (TGI 40 − 80%), whereas several models were resistant to treatment (non-significant TGI). This database is currently being explored to identify biomarkers predicting sensitivity to BI 853520 based on genetic aberrations, gene expression signatures and phosphoproteome analysis of xenograft tumors. In conclusion, BI 853520 is a novel, highly selective PTK2 kinase inhibitor that demonstrates anti-tumor activity in vitro and in vivo. Clinical development of BI 853520 has recently been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A249.

Abstract 1082: BI 811283, a potent inhibitor of the mitotic kinase Aurora B, shows dose- and schedule-dependent efficacy in human cancer xenograft models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. The essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for therapeutic intervention. BI 811283, a potent inhibitor of this key mitotic regulator, inhibits proliferation of a wide range of cultured human cancer cells at low nanomolar concentrations by inducing polyploidy, senescence and apoptosis. Methods. BomTac:NMRI-Foxn1nu mice were grafted subcutaneously with NCI-H460 non-small cell lung carcinoma (mutant KRAS, wild-type p53), HCT 116 colon carcinoma (mutant KRAS, wild-type p53) or BxPC-3 pancreas adenocarcinoma cells (wild-type KRAS, mutant p53). Treatment was initiated when the tumors had reached a volume of ∼50 mm3. BI 811283 was injected intravenously once or twice weekly as a single bolus or b.i.d. Alternatively, the compound was administered once-weekly by a continuous 24 h infusion via subcutaneously implanted osmotic mini-pumps. Multiple dose levels and dosing schedules were evaluated. Results. In models of human non-small cell lung cancer, colon carcinoma and pancreas carcinoma, multiple cycles of treatment with BI 811283 at total weekly doses of 20 to 75 mg/kg resulted in dose-dependent inhibition of tumor growth or tumor regression. Continuous s.c. infusion at 20 mg/kg over 24 h once-weekly was clearly superior to all bolus injection schedules delivering weekly doses up to 75 mg/kg. Furthermore, regression of large tumors (350 mm3) was induced in the HCT 116 colon carcinoma model. Biomarker analyses of HCT 116 tumors revealed that therapeutic doses of BI 811283 inhibited phosphorylation of histone H3, a direct substrate of Aurora B. Histological examination showed an accumulation of enlarged, multinucleated cells in accordance with the expected mechanism of action. Conclusions. BI 811283 has demonstrated potent antitumor activity in multiple cancer models at well-tolerated doses; treated tumors show hallmarks of Aurora B inhibition. Continuous infusion over 24 h provides a superior therapeutic index compared with bolus administration. The compound is currently under investigation 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 1082.

Abstract 1080: Molecular and cellular pharmacology of BI 811283, a potent inhibitor of Aurora B kinase

Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. These essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for anticancer drug development. Methods. BI 811283 was profiled in enzymatic kinase assays as well as in proliferation assays on various human cancer cell lines. Cell cycle status was assessed by DNA content analysis (Cellomics ArrayScan, FACScalibur). Histone H3 phosphorylation was determined by immunofluorescence (Cellomics ArrayScan). Apoptosis was detected by Western blotting for cleaved PARP and microscopic enumeration of DAPI-stained cells showing nuclear fragmentation. Senescent cells were identified by staining for SA-s-Gal activity. Results. BI 811283 inhibited human Aurora B kinase activity with an IC 50 value of 9 nM, Aurora A and C kinases with 70 nM and 17 nM, respectively. In a panel of 46 additional kinases representative of the human kinome, BI 811283 at 1000 nM inhibited 7/46 kinases by more than 50%. EC 50 values for inhibition of proliferation of >20 human cancer cell lines were in the range of 2 to 14 nM. In the non-small cell lung cancer cell line NCI-H460, treatment with BI 811283 resulted in a rapid ( 80%, paralleled by a marked increase in cell volume. An increase of cleaved poly (ADP-ribose) polymerase and a concomitant increase in the fraction of cells with nuclear fragmentation from Conclusions. BI 811283 is a potent and selective Aurora kinase inhibitor that inhibits proliferation of cancer cells independent of tissue origin or oncogenome status. Treated cells exhibit a polyploid phenotype characteristic for Aurora B inhibition and show hallmarks of senescence as well as a slow onset of apoptosis in a small fraction of cells. In vivo activity of BI 811283 has been demonstrated in multiple cancer xenograft models in nude mice (see accompanying poster). Phase I clinical trials are ongoing. 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 1080.

Abstract A208: Pharmacodynamic properties and antitumor efficacy of BI 836845, a fully human IGF ligand neutralizing antibody.

Insulin-like growth factor (IGF) signaling is thought to play an important role in the development of tumors and resistance to existing cancer therapies. The proliferative and pro-survival signals driven by the IGF ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 activates the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. To target IGF signaling for cancer therapy we have developed BI 836845, a fully human IgG1 antibody derived from an antibody phage display library that can bind to and neutralize the functions of both IGF-1 and IGF-2. Surface plasmon resonance (Biacore) analysis demonstrated that BI 836845 has an affinity (K D ) of 0.07 nM and 0.8 nM for human IGF-1 and IGF-2, respectively. BI 836845 also shows comparable affinities to mouse as well as rat IGF-1 and IGF-2, allowing a comprehensive pre-clinical characterization of the pharmacodynamic properties of the antibody in these species. Cell-based ELISA assays were used to demonstrate that BI 836845 potently neutralizes the ability of IGF-1 and IGF-2 to phosphorylate the human IGF-1R. Similarly, BI 836845 inhibits phosphorylation of IR-A driven by IGF-2. BI 836845 was also shown to completely and potently inhibit IGF bioactivity in human serum samples ex vivo. Pharmacodynamic effects of BI 836845 were studied in growing rats treated intravenously once weekly with 20, 60, or 200 mg/kg antibody. Inhibition of IGF bioactivity as determined by ex vivo IGF-1R phosphorylation potential in the plasma was observed at all dose levels, despite a corresponding increase in total IGF-1 levels. An increase in total IGF-1 levels has also been seen with IGF-1R targeted antibodies in clinical studies and is thought to be due to blockade of a growth hormone mediated physiological feedback mechanism. Treatment with BI 836845 resulted in a marked dose dependent reduction of rat body weight gain at all dose levels, consistent with the known role for IGFs in normal growth and development. BI 836845 potently inhibits the in vitro proliferation of cell lines derived from various cancers, with mesenchymal-derived cancer cell lines being particularly sensitive (EC 50 values in the low nanomolar range). In a model of Ewing s sarcoma, using immunodeficient nude mice subcutaneously transplanted with human RD-ES cells, twice-weekly treatment with BI 836845 resulted in partial inhibition of tumor growth. Treatment in combination with rapamycin resulted in improved efficacy at tolerated doses. In conclusion, BI 836845 is a potent IGF ligand neutralizing antibody whose pharmacological profile warrants further investigation in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A208.