Simone Greven

Preclinical efficacy of the auristatin-based antibody-drug conjugate BAY 1187982 for the treatment of FGFR2-positive solid tumors.

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody-drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line-based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331-9. ©2016 AACR.

Site-Specific Conjugation of Peptides and Proteins via Rebridging of Disulfide Bonds Using the Thiol–Yne Coupling Reaction

Herein, we describe an extension of our previously reported photomediated disulfide rebridging methodology to the conjugation of peptides and proteins. The methodology proved to be reproducible with various alkynes and different peptides. This study includes the first rebridging of the disulfide bond of a peptide through a thiol-yne reaction with a cyclooctyne. In all cases, the rebridging was proven by MS analyses and confirmed by the absence of olefinic protons on (1)H NMR spectra of the resulting products. Finally, this one-pot reduction thiol-yne conjugation was successfully applied to an antibody Fab fragment with a promising conversion, which set a good ground for the future syntheses of new protein and antibody conjugates.

Abstract 46: Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens

Antibody-drug conjugates (ADCs) are promising agents that are developed for targeted delivery of cytotoxic payloads to tumor cells. ADCs share a common design of antibody, linker, and cytotoxic payload. Despite significant efforts, the number of available payload classes with a differentiated mode-of-action that can successfully be employed to generate antibody-drug conjugates (ADCs) is still rather limited. So far, only ADCs with microtubule depolymerizing or DNA binding payloads have been approved. The identification of ADC payload classes with a novel mode-of-action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of the kinesin spindle protein (KSP/Eg5/KIF11) have generated interest due to their high anti-tumor activity. However, the transfer of the potency of small molecule KSP inhibitors (KSPis) to highly efficient clinical regimens with a sufficient therapeutic window remains challenging. Through the conjugation of a novel pyrrole subclass of KSPis to antibodies targeting different cancer antigens, we generated a panel of ADCs and characterized them both in vitro and in vivo. ADCs targeting either EGFR or TWEAKR/Fn14 showed strong and specific internalization and displayed specific and potent anti-proliferative efficacy in vitro. In cytotoxicity assays, these ADCs exhibited sub-nanomolar potency in antigen-positive cancer cell lines (EGFR/TWEAKR-pos. NCI-H292; TWEAKR-pos. BxPC3, LoVo) and more than 100-fold selectivity versus non-targeted control-ADC containing the same linker and the same payload. Furthermore, selective anti-tumor efficacy of EGFR- and TWEAKR-KSPi-ADCs was demonstrated in vivo using both cancer cell line-derived models of NSCLC (NCI-H292), urothelial cell carcinoma (UCC) (KU-19-19), and renal cell carcinoma (A498), as well as in the TWEAKR-positive patient-derived xenograft UCC model BFX469. At doses of 5-10 mg/kg qw or bw potent anti-tumor efficacy with treated-to-control ratios (T/C) between 0.16 to 0.28 as well as complete regressions were observed. In summary, KSP inhibitors have been established as a promising new payload class allowing the generation of highly potent and selective ADCs for the treatment of solid tumors. Citation Format: Anette Sommer, Sandra Berndt, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Sven Wittrock, Anne-Sophie Rebstock, Lisa Dietz, Christoph Mahlert, Simone Greven, Nils Griebenow, Yolanda Cancho-Grande, Rolf Jautelat, Heiner Apeler, Bertolt Kreft. Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens [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 46. doi:10.1158/1538-7445.AM2017-46

Abstract 4491: FGFR2-ADC potently and selectively inhibits growth of gastric and breast cancer xenograft models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antibody-drug conjugates (ADCs) represent a promising therapeutic approach for treatment of cancer. We have developed a novel ADC directed against fibroblast growth factor receptor 2 (FGFR2). FGFR2 is overexpressed in several cancer indications, such as gastric cancer and breast cancer, thus representing an interesting therapeutic target for the treatment of FGFR2 positive cancer patients with an ADC-based therapy. The FGFR2-ADC consists of the fully human anti-FGFR2-mAb BAY 1179470 conjugated via a stable linker to a novel auristatin cytotoxic agent (technology licensed from Seattle Genetics). The FGFR2-mAb BAY 1179470, which is cross-reactive with human, mouse, rat and monkey, induces internalization of FGFR2. Quantitative data on FGFR2 antibody bound per cell (ABC) were determined with the QuantiBrite assay using BAY 1179470. FGFR2-ADC has a potency in the single digit nM to sub nM range in a panel of FGFR2-positive cells lines (e.g., SNU-16, KatoIII, SUM52-PE, MFM-223) and shows more than 100-fold selectivity versus FGFR2-negative cell lines. High levels of FGFR2 on cancer cells correlate with internalization efficacy and cytotoxic activity in vitro. FGFR2-ADC is highly efficacious in monotherapy and results in tumor growth inhibition in the gastric cancer xenograft model SNU-16 and tumor regression in the breast cancer xenograft model MFM-223. At doses efficacious in mice, FGFR2-ADC is well tolerated. The pre-clinical efficacy and tolerability data obtained for FGFR2-ADC suggest a therapeutic index and support clinical testing. Citation Format: Anette Sommer, Carl F. Nising, Christoph Mahlert, Charlotte C. Kopitz, Hans-Georg Lerchen, Simone Greven, Beatrix Stelte-Ludwig, Joachim Schuhmacher, Ruprecht Zierz, Sabine Wittemer-Rump, Christoph Schatz, Frank Reetz, Heiner Apeler, Rolf Jautelat, Bertolt Kreft, Karl Ziegelbauer. FGFR2-ADC potently and selectively inhibits growth of gastric and breast cancer xenograft models. [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 4491. doi:10.1158/1538-7445.AM2014-4491

Abstract 3234: Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class

The number of cytotoxic payload classes with different modes-of-action which have been successfully employed in antibody-drug conjugates (ADC) is still rather limited. So far, only ADCs with microtubule inhibitors, DNA binding payloads or topoisomerase I inhibitors have been advanced into clinical testing. To this end, the identification of ADC payload classes with a novel mode of action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of kinesin spindle protein (KSP/Eg5) have generated interest due to their high antitumor potency. However, transferring the preclinical potency of small molecule KSP inhibitors (KSPis) into highly efficient clinical regimens with a sufficient therapeutic window has remained challenging. We have investigated a new pyrrole subclass of KSPis which showed subnanomolar potency against a large panel of tumor cell lines for their utility as a novel payload class in ADCs. Towards this goal different attachment sites for linkers have been explored in the KSPi molecule which were found compatible with cleavable and/or non-cleavable linkers. Subnanomolar potency and selectivity of ADCs with antibodies targeting either HER2, EGFR or TWEAKR could be demonstrated in vitro. For selected ADCs, the intracellular trafficking and metabolite formation was investigated and KSP inhibition was confirmed as the ADC mode of action. Depending on the linker composition differential profiles of the ADC metabolites with regard to efflux, cellular permeation, and bystander effect have been achieved. Moreover, specific accumulation in the tumor versus other tissues was demonstrated in biodistribution studies in vivo. In conclusion, KSP inhibitors have been established as a versatile new payload class for the generation of highly potent and selective ADCs. Citation Format: Hans-Georg Lerchen, Sven Wittrock, Nils Griebenow, Mario Lobell, Anne-Sophie Rebstock, Yolanda Cancho-Grande, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Anette Sommer, Sandra Berndt, Carsten Terjung, Heiner Apeler, Bertolt Kreft, Rolf Jautelat. Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class [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 3234. doi:10.1158/1538-7445.AM2017-3234

A study on the trastuzumab conjugation at tyrosine using diazonium salts

Herein, we report on the conjugation of trastuzumab with 2,5-difluorobenzene diazonium tetrafluoroborate. According to the amount of diazonium salt used, an average loading of 1.0 to 3.8 azo compounds per antibody was found. Tryptic digestion and subsequent MS–MS analyses allowed the identification of the main conjugation sites. Furthermore, additional conjugation reactions of trastuzumab with 4-formylbenzene diazonium hexafluorophosphate (FBDP) allows us to make a short study on the influence of the different reaction parameters on the outcome of the conjugation reaction, both in terms of antibody loading and of conjugation sites. In all cases, antibody conjugation was achieved with full selectivity towards tyrosine residues.