Sandra Berndt

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

Antibody‐drug conjugates with pyrrole‐based KSP inhibitors as novel payload class

The number of cytotoxic payload classes successfully employed in antibody-drug conjugates (ADCs) is still rather limited. The identification of ADC payloads with a novel mode of action will increase therapeutic options and potentially increase the therapeutic window. Herein, we describe the utilization of kinesin spindle protein inhibitors (KSPi) as a novel payload class providing highly potent ADCs against different targets, for instance HER-2 or TWEAKR/Fn14. Aspects of technical optimization include the development of different linker attachment sites, the stabilization of ADC linkage to avoid payload deconjugation and finally, the tailor-made design of active metabolites with a long lasting intracellular exposure in the tumor matching the mode of action of KSP inhibition. These KSPi-ADCs are highly potent and selective in vitro and demonstrate in vivo efficacy in a broad panel of tumor models including complete regressions in a patient-derived urothelial cancer model.

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

Abstract 1210: Preclinical pharmacology and repeated dose toxicity of the novel agonistic TWEAK receptor binding antibody BAY-356

TWEAK receptor (TWEAKR, FN14) is a member of the tumor necrosis factor receptor superfamiliy and is highly expressed in a variety of human solid tumor types, and its overexpression is associated with poor prognosis and metastasis. To explore targeting of TWEAKR for cancer therapy we have generated the novel, anti-TWEAKR antibody BAY-356. Its potent agonistic activity leads to TWEAKR hyperactivation and subsequent induction of cell death in vitro and tumor growth inhibition in vivo. BAY-356 is a fully human aglycosylated antibody (Kd ∼ 10nM) that binds to a novel epitope within the TWEAKR ectodomain of various species as determined by BiaCore. In vitro, BAY-356 showed strong agonistic activity on TWEAKR-positive tumor cells, including activation of NFκB- and STAT1 pathways, increase of TWEAKR protein expression, increased IL-8 secretion, caspase 3/7 activation, and proliferation inhibition in a dose-dependent manner. BAY-356 inhibited tumor growth in several TWEAKR-positive tumor models (NCI-H1975, WiDr, ScaBER, and HN10321) with growth inhibition rates of 49-71% when treated with 3-10 mg/kg BAY-356 twice weekly for up to 3 weeks. The activity of BAY-356 was independent of ADCC activation. In a preventative syngeneic CT26-tumor model in Balb/c mice, BAY-356 induced complete responses. Anti-tumor activity of BAY-356 was associated with high tumor levels of TNF alpha protein. To investigate the toxicity of BAY-356, a repeated dose-toxicity study was performed in Cynomolgus monkeys. Animals were dosed with 10, 20, and 40 mg/kg by weekly intravenous injection for 4 weeks. Compound-related clinical findings consisted of an increase of the serum markers amylase and lipase from 10 mg/kg onwards, urea and creatinine from 20 mg/kg onwards and the transaminases ALT and GDPH at 40 mg/kg. Histopathological evaluation revealed focal ductular epithelial hyperplasia with periductular fibrosis in the exocrine pancreas (at 10 & 20 mg/kg), renal tubular hyperplasia and degeneration, Bowman capsule hyperplasia, and glomerulosclerosis in the kidney starting at 10 mg/kg and bile duct hyperplasia in liver at 20 mg/kg and higher. The HNSTD was set as the highest tested dose of 40 mg/kg. Immunohistochemical analysis of TWEAKR expression in these organs demonstrated a dose dependent induction and increase when compared to untreated controls which correlated with the histopathological findings. From these data it can be concluded that hyperactivation of TWEAKR signaling by BAY-356 leading to strong anti-tumor efficacy in various mouse models is invariably accompanied by target-mediated side-effects originating from enhanced TWEAKR induction in in particular in kidneys, pancreas, and liver of sensitive species such as Cynomolgus monkeys. Citation Format: Sandra Berndt, Christian Votsmeier, Ruprecht Zierz, Jakob Walter, Anna-Lena Frisk, Stefanie Hammer, Heiner Apeler, Bertolt Kreft. Preclinical pharmacology and repeated dose toxicity of the novel agonistic TWEAK receptor binding antibody BAY-356. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1210.

Abstract 2089: Therapeutic index prediction of the agonistic aglycosylated TWEAK receptor binding antibody BAY-356

BAY-356 is a novel aglycosylated anti-TWEAK receptor antibody with potent agonistic activity evaluated for cancer therapy. In order to predict an efficacious therapeutic dose of BAY-356 in man, we sought to determine a therapeutic index where the exposure related to therapeutic efficacy was compared with the exposure obtained following doses tested in toxicology studies. BAY-356 (1 mg/kg) was administered intravenously to healthy Cynomolgus monkeys and plasma concentrations measured in order to determine pharmacokinetic (PK) parameters. Using allometric scaling, the PK in humans was predicted. Efficacy data of BAY-356 in WiDr, HN10321, A253 and SCaBER xenograft models representing colorectal, head and neck squamous cell carcinoma and bladder cancer, were used to derive exposure-response models for each tumor model where the plasma exposure of BAY-356 is assumed to have an effect on the tumor size in a tumor growth model. NONMEM 7.3 was used in the estimations. The tolerability of BAY-356 was tested in monkeys at doses of 10, 20 and 40 mg/kg by weekly intravenous injections over 4 weeks. Treatment resulted in slight to moderate toxicity in liver, kidneys and pancreas and 10 mg/kg was set to be an exposure dose well tolerated and not to be exceeded by therapeutic exposure. A dosing strategy in humans predicted to result in the same exposure as 10 mg/kg weekly in monkey is 18 mg/kg every third weeks (Q3W). Using the estimated exposure-response in xenograft models, human tumor doubling times of 8 weeks (A253 and HN10321), 24 weeks (WiDr) and 12 and 24 weeks (SCaBER) in combination with human predicted PK parameters, tumor reduction over time in humans was predicted based on a human dosing strategy of 18 mg/kg BAY-356 Q3W. For the A253 tumor model, stable disease in humans was predicted while the results from HN10321 and SCaBER models predicted a 13-60% decrease in tumor size in humans following 12 weeks treatment. For WiDr, this dosing strategy was predicted to not be efficacious in humans. The results based on modelling of xenograft data indicate that 18 mg/kg BAY-356 dosed Q3W is predicted to be efficacious in humans for certain tumor cells. Citation Format: Anders Viberg, Eva Hanze, Lisa Dietz, Ruprecht Zierz, Sandra Berndt, Sabine Wittemer-Rump. Therapeutic index prediction of the agonistic aglycosylated TWEAK receptor binding antibody BAY-356. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2089.