Sabine Zitzmann-Kolbe

Identification and Characterization of a Peptide with Affinity to Head and Neck Cancer

Combination therapy has improved the quality of life for patients with squamous cell carcinomas of the head and neck (HNSCCs) but has not decisively changed prognosis. Targeted therapies, which enhance accumulation of the drug in the tumor, may be realized using tumor-specific binding peptides. This paper identifies and characterizes an HNSCC affine peptide. Methods: From a phage library comprising 109 different displayed peptides, 1 peptide was enriched after 5 in vitro selection rounds on HNO223 tumor cells. Subsequently, the gained peptide sequence H2N-SPRGDLAVLGHKY-CONH2 (HBP-1) was synthesized as an amide and labeled with 125I. In vitro studies for binding kinetics and competition were performed with 5 different HNSCC cell lines. Furthermore, the stability of the peptide was evaluated in human serum. The in vivo biodistribution of 131I-labeled peptide was determined in HNSCC tumor–bearing nude mice. The results were further validated in human HNSCC tumor tissue sections using fluorescence-labeled HBP-1. Competition experiments were performed to determine the binding sequence and validate the target. Results: The HBP-1 motif was enriched in 62% of all phages sequenced. Labeled 125I-HBP-1 showed binding to 5 different HNSCC cell lines and a maximum binding to HNO97 cells, with 11% of the applied dose per 106 cells and an inhibitory concentration of 50% of 38.9 nM. Stability experiments in human serum showed a half-life of 55 min. In 2 different HNSCC tumor xenografts, 131I-HBP-1 accumulated rapidly, with stable uptake until 45 min after intravenous application. Peptide immunohistochemistry of HNSCC tissue sections exhibited tumor staining by HBP-1, whereas normal tissue remained negative. Sequence mutation and competition experiments revealed that the intrinsic RGD motif in combination with the intrinsic LXXL motif is responsible for the binding ability of HBP1. The RGDLXXL sequence within this peptide is known and indicates that binding occurs via the αvβ6 rather than the αvβ3 integrin. Conclusion: Within the sequence of HBP-1 is a RGDLXXL motif, and most likely it is targeting the αvβ6 receptor of the integrin family of cell adhesion receptors. HBP-1 represents a promising lead structure for the development of targeted therapies or diagnostic procedures in patients with HNSCC.

Zn(II)-bis(cyclen) complexes and the imaging of apoptosis/necrosis.

In vivo cell-death imaging is still a challenging issue. Until now, only (99m)Tc-labeled HYNIC-rh-annexin A5 has been extensively studied in clinical trials. In the ongoing search for an alternative imaging agent, we synthesized a series of fluorescent zinc-cyclen complexes as annexin A5 mimics and studied structural variations on the uptake behavior of cells undergoing apoptosis/necrosis. The number of cyclen chelators was varied and the spacer separating cyclen from the central scaffold was modified. Five zinc-cyclen complexes were labeled with fluorescein for flow cytometric studies and one was labeled with (18)F for in vivo applications. Jurkat cells were treated with staurosporine to induce apoptosis/necrosis, incubated with the fluorescein-labeled zinc complexes and analyzed them by flow cytometry. Fluorescent annexin A5 and propidium iodide were applied as reference dyes. Flow cytometry revealed greater accumulation of zinc-cyclen complexes in staurosporine treated cells. The uptake was contingent on the presence of zinc and the fluorescence intensity was dependent on the number of zinc-cyclen groups. Confocal laser scanning microscopy showed the {bis[Zn(cyclen)]}(4+) complex distributed throughout the cytosol different to annexin A5. Owing to the structural similarity of the bis-cyclen ligands with CXCR4 binding bis-cyclam derivatives the zinc-cyclen complex uptake was challenged with the meta derivative of AMD3100. Lack of uptake depletion in staurosporine treated cells ruled out measurable CXCR4 interaction. PET imaging using the (18)F labeled zinc-cyclen complex revealed significantly higher uptake in an irradiated Dunning R3327-AT1 prostate tumor as compared to the contralateral control tumor. PET imaging of a HelaMatu tumor model additionally showed an increased uptake after taxol treatment. It could be demonstrated that the fluorescent zinc-cyclen complexes offer potential as new agents for flow cytometry and microscopic imaging of cell death. In addition, the (18)F labeled analogue holds promise for in vivo applications providing informations about cell death after radiation therapy and cytostatic drug treatment.

Challenges in optimizing a prostate carcinoma binding peptide, identified through the phage display technology

The transfer of peptides identified through the phage display technology to clinical applications is difficult. Major drawbacks are the metabolic degradation and label instability. The aim of our work is the optimization of DUP-1, a peptide which was identified by phage display to specifically target human prostate carcinoma. To investigate the influence of chelate conjugation, DOTA was coupled to DUP-1 and labeling was performed with 111In. To improve serum stability cyclization of DUP-1 and targeted d-amino acid substitution were carried out. Alanine scanning was performed for identification of the binding site and based on the results peptide fragments were chemically synthesized. The properties of modified ligands were investigated in in vitro binding and competition assays. In vivo biodistribution studies were carried out in mice, carrying human prostate tumors subcutaneously. DOTA conjugation resulted in different cellular binding kinetics, rapid in vivo renal clearance and increased tumor-to-organ ratios. Cyclization and d-amino acid substitution increased the metabolic stability but led to binding affinity decrease. Fragment investigation indicated that the sequence NRAQDY might be significant for target-binding. Our results demonstrate challenges in optimizing peptides, identified through phage display libraries, and show that careful investigation of modified derivatives is necessary in order to improve their characteristics.

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 1079: Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer

BAY 1163877 is an orally available, highly potent and selective pan fibroblast growth factor receptor (FGFR) inhibitor. In an ongoing Phase 1 clinical trial (NCT01976741) BAY 1163877 showed clinical responses at exceptional tolerability in patients suffering from different tumor types including urothelial bladder carcinoma or lung tumors, which were selected based on elevated FGFR1-3 mRNA expression. In the preclinical phase, the compound demonstrated significant single agent anti-tumor activity in various tumor models with different FGFR alterations leading to FGFR overexpression (e.g. FGFR gene amplifications or mutations). Genetic alterations of FGFRs can also be found in breast cancer with 7.5 - 17% of all tumors harboring a FGFR1 gene amplification. Elevated FGFR1 mRNA levels can be found in up to 22% of breast cancer cell lines as well as clinical samples. Other FGFR alterations include FGFR2 or FGFR4 gene amplifications as well as elevated FGFR mRNA levels, which were reported in all breast cancer subtypes. We therefore investigated BAY1163877 monotherapy in various breast cancer models. Due to the favorable clinical safety profile of BAY1163877, we also examined a combination treatment with early line antihormonal therapies in hormone receptor positive breast cancer. In vitro profiling of BAY 1163877 in a number of breast cancer cell lines showed a clear association of efficacy with expression levels of different FGFR isoforms. The efficacy was further investigated in several patient- or cell line-derived breast cancer in vivo models. For instance, BAY 1163877 alone dosed 38mg/kg twice daily induced tumor growth inhibition of greater than 90% in a subcutaneous mouse syngeneic 4T1 breast cancer model expressing elevated levels of FGFR2. Resistance to endocrine therapy appears associated with FGFR1 gene amplification and may explain the poor prognosis of FGFR1 overexpressing tumors treated with adjuvant tamoxifen. We therefore investigated the combination of the panFGFR-inhibitor BAY 1163877 with the clinically used antihormonal compound fulvestrant in selected luminal breast cancer PDX models. Some of these models showed resistance to antihormonal treatment in monotherapy but improved in vivo efficacy in combined treatment using BAY 1163877 and fulvestrant. These data may warrant further clinical investigation of BAY1163877alone or in combination with antihormonal therapy in patients with FGFR overexpressing breast cancer. Citation Format: Oliver Politz, Peter Ellinghaus, Sebastian Bender, Sylvia Gruenewald, Franziska Siegel, Marie-Pierre Collin, Sabine Zitzmann-Kolbe, Dominik Mumberg, Karl Ziegelbauer. Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer [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 1079. doi:10.1158/1538-7445.AM2017-1079