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Purpose: Androgen receptor (AR) inhibitors are well established in the treatment of castration-resistant prostate cancer and have recently shown efficacy also in castration-sensitive prostate cancer. Although most patients respond well to initial therapy, resistance eventually develops, and thus, more effective therapeutic approaches are needed. Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and presents an attractive target for radionuclide therapy. Here, we evaluated the efficacy and explored the mode of action of the PSMA-targeted thorium-227 conjugate (PSMA-TTC) BAY 2315497, an antibody-based targeted alpha-therapy, in combination with the AR inhibitor darolutamide. Experimental Design: The in vitro and in vivo antitumor efficacy and mode of action of the combination treatment were investigated in preclinical cell line–derived and patient-derived prostate cancer xenograft models with different levels of PSMA expression. Results: Darolutamide induced the expression of PSMA in androgen-sensitive VCaP and LNCaP cells in vitro, and the efficacy of darolutamide in combination with PSMA-TTC was synergistic in these cells. In vivo, the combination treatment showed synergistic antitumor efficacy in the low PSMA-expressing VCaP and in the high PSMA-expressing ST1273 prostate cancer models, and enhanced efficacy in the enzalutamide-resistant KUCaP-1 model. The treatments were well tolerated. Mode-of-action studies revealed that darolutamide induced PSMA expression, resulting in higher tumor uptake of PSMA-TTC, and consequently, higher antitumor efficacy, and impaired PSMA-TTC–mediated induction of DNA damage repair genes, potentially contributing to increased DNA damage. Conclusions: These results provide a strong rationale to investigate PSMA-TTC in combination with AR inhibitors in patients with prostate cancer.

Purpose Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle–emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344. Methods and Materials The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice. Results In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC–mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. Conclusions The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.

Targeted α therapy (TAT) offers the potential for the targeted delivery of potent α-particle-emitting radionuclides that emit high linear energy transfer radiation. This leads to a densely ionizing radiation track over a short path. Localized radiation induces cytotoxic, difficult-to-repair, clustered DNA double-strand breaks (DSBs). To date, radium-223 (223Ra) is the only TAT approved for the treatment of patients with metastatic castration-resistant prostate cancer. Thorium-227 (227Th), the progenitor nuclide of 223Ra, offers promise as a wider-ranging alternative due to the availability of efficient chelators, such as octadentate 3,2-hydroxypyridinone (3,2-HOPO). The 3,2-HOPO chelator can be readily conjugated to a range of targeting moieties, enabling the generation of new targeted thorium-227 conjugates (TTCs). This review provides a comprehensive overview of the advances in the preclinical development of TTCs for hematological cancers, including CD22-positive B cell cancers and CD33-positive leukemia, as well as for solid tumors overexpressing renal cell cancer antigen CD70, membrane-anchored glycoprotein mesothelin in mesothelioma, prostate-specific membrane antigen in prostate cancer, and fibroblast growth factor receptor 2. As the mechanism of action for TTCs is linked to the formation of DSBs, the authors also report data supporting combinations of TTCs with inhibitors of the DNA damage response pathways, including those of the ataxia telangiectasia and Rad3-related protein, and poly-ADP ribose polymerase. Finally, emerging evidence suggests that TTCs induce immunogenic cell death through the release of danger-associated molecular patterns. Based on encouraging preclinical data, clinical studies have been initiated to investigate the safety and tolerability of TTCs in patients with various cancers.

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.

// Urs B. Hagemann 1 , Dessislava Mihaylova 1 , Steinar R. Uran 1 , Joergen Borrebaek 1 , Derek Grant 1 , Roger M. Bjerke 1 , Jenny Karlsson 1 and Alan S. Cuthbertson 1 1 Thorium Conjugate Research, Bayer AS, Oslo, Norway Correspondence to: Urs B. Hagemann, email: urs.hagemann@bayer.com Keywords: targeted alpha therapy (TAT), alpha particles, radioimmunotherapy, thorium-227, renal cell carcinoma Received: January 25, 2017 Accepted: March 13, 2017 Published: April 07, 2017 ABSTRACT The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 ( 227Th ). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of 227Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).

Purpose: Prostate-specific membrane antigen (PSMA) is an attractive target for radionuclide therapy of metastatic castration-resistant prostate cancer (mCRPC). PSMA-targeted alpha therapy (TAT) has shown early signs of activity in patients with prostate cancer refractory to beta radiation. We describe a novel, antibody-based TAT, the PSMA-targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) consisting of the alpha-particle emitter thorium-227 complexed by a 3,2-HOPO chelator covalently linked to a fully human PSMA-targeting antibody. Experimental Design: PSMA-TTC was characterized for affinity, mode of action, and cytotoxic activity in vitro. Biodistribution, pharmacokinetics, and antitumor efficacy were investigated in vivo using cell line and patient-derived xenograft (PDX) models of prostate cancer. Results: PSMA-TTC was selectively internalized into PSMA-positive cells and potently induced DNA damage, cell-cycle arrest, and apoptosis in vitro. Decrease in cell viability was observed dependent on the cellular PSMA expression levels. In vivo, PSMA-TTC showed strong antitumor efficacy with T/C values of 0.01 to 0.31 after a single injection at 300 to 500 kBq/kg in subcutaneous cell line and PDX models, including models resistant to standard-of-care drugs such as enzalutamide. Furthermore, inhibition of both cancer and cancer-induced abnormal bone growth was observed in a model mimicking prostate cancer metastasized to bone. Specific tumor uptake and efficacy were demonstrated using various PSMA-TTC doses and dosing schedules. Induction of DNA double-strand breaks was identified as a key mode of action for PSMA-TTC both in vitro and in vivo. Conclusions: The strong preclinical antitumor activity of PSMA-TTC supports its clinical evaluation, and a phase I trial is ongoing in mCRPC patients (NCT03724747).

The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a novel targeted alpha therapy (TAT) approach which is currently under investigation in a FiH trial in patients with metastatic castration resistant prostate cancer (mCRPC; NCT03724747). It consists of the alpha emitter thorium-227 complexed to a 3,2-HOPO chelator conjugated to a PSMA targeting antibody delivering radiation to PSMA expressing tumor cells and their microenvironment resulting in the induction of potentially cytotoxic DNA double-strand breaks (DBS). Combining with the PARP inhibitor olaparib may act synergistically via inhibition of DDR pathways. Here we evaluated the efficacy of PSMA-TTC in combination with olaparib that demonstrated a positive outcome in patients with homologous recombination repair-mutated mCRPC. We explored potential synergies of the combination treatment in prostate cancer models in vitro and in vivo and studied the mode of action by protein and RNA analysis. In vitro, synergy of the combination of PSMA-TTC with olaparib with a combination index (CI) of 0.7 was observed in three cell lines, namely LNCaP, 22Rv1 and MDA-PCa2b. with the first two (LNCaP and 22Rv1) carrying mutations in BRCA2. Only moderate synergy with a CI of 0.8 was seen in BRCA2 wt VCaP cells. In vivo, dose dependent anti-tumor activity with single i.v. injections of PSMA-TTC at 150 and 300 kBq/kg was seen in the LNCaP prostate cancer model resulting in a tumor/control (T/C) ratio of 0.3 and 0.09 at day 28, respectively. In contrast, olaparib given at 50 mg/kg QD did not show activity at day 28. The 300 kBq/kg PSMA-TTC dose combined with olaparib showed the most pronounced anti-tumor activity (T/C=0.04) and significantly increased the number of animals with complete tumor regressions compared to high dose PSMA-TTC alone (6 CRs vs 2 CRs, p = 0.0397, Chi-square) on day 28. At the end of the consecutive surveillance period on day 117, 7/8 (87.5%) animals remained tumor-free (putatively cured) in the combination group vs 4/10 (40%) mice in the PMSA-TTC monotherapy group (Chi-square test, P = 0.0400). Additive or synergistic toxicological effects for the combination treatment were not seen based on body weight loss and fatal tox rate. At the 117-day time point both the 300 kBq/kg PSMA-TTC monotherapy and combination with olaparib group demonstrated 0.3% and 9.3% body weight gain indicating good tolerability. Changes in markers of DNA damage response following treatment will be presented. In summary, combination of PSMA-TTC with olaparib shows synergistic preclinical anti-tumor activity and support further clinical investigation of this combination treatment in prostate cancer patients. Citation Format: Christoph A. Schatz, Stefanie Hammer, Antje Wengner, Urs B. Hagemann, Dominik Mumberg, Arne Scholz. PSMA-Targeted Thorium Conjugate (BAY 2315497) and olaparib combination show synergistic anti-tumor activity in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1393.

Purpose: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. Experimental Design: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. Results: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. Conclusions: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is the first targeted alpha therapy in clinical development for the treatment of patients suffering from MSLN-positive mesothelioma and ovarian cancer (NCT03507452). It consists of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. The main mode of action of MSLN-TTC is the induction of clustered DNA double-strand breaks upon alpha decay of thorium-227, resulting in cell death. The preclinical efficacy of MSLN-TTC has been demonstrated previously. The tumor control achieved by external beam radiation is partly driven by immune-stimulatory effects (Vatner et al; Frontiers in Oncology 2014). The essential mechanisms can be attributed to different pathways, including (a) induction of immunogenic cell death and (b) activation of the STING-pathway resulting in the secretion of pro-inflammatory cytokines (Vanpouille-Box C et al; NatComm 2017). We therefore investigated whether the targeted alpha therapy MSLN-TTC is also able to induce both of these pathways in vitro. The human ovarian cancer MSLN-expressing cell line OVCAR-3 was exposed to MSLN-TTC resulting in a dose dependent upregulation of markers of immunogenic cell death, e.g. calreticulin and HMGB-1. Further, exposure of OVCAR-3 cells to MSLN-TTC resulted in the secretion of pro-inflammatory cytokines, including IFN-β, IL-6 and IP-10. The efficacy of MSLN-TTC was evaluated in a syngeneic subcutaneous tumor model. As MSLN-TTC is not cross-reactive to murine MSLN, an MC38 cell line stably transfected with human MSLN was established (MC38-hMSLN). In vitro, MSLN-TTC induced specific reduction of MC38-hMSLN cell viability. Following single dose administration to MC38-hMSLN tumor-bearing mice, MSLN-TTC induced dose dependent antitumor activity with complete tumor eradication in 10 out of 36 treated animals. Interestingly, when tumor-free animals were re-inoculated with MC38-hMSLN cells 121 days post treatment, no tumor growth was observed. In contrast, tumors grew in animals inoculated with B16F10 cells, suggesting the development of an immune memory response against MC38-hMSLN cells. In summary, the data presented demonstrate that MSLN-TTC is able to induce immunogenic cell death and secretion of pro-inflammatory cytokines in vitro. Further, MSLN-TTC monotherapy evokes an immune-stimulatory effect in vivo. Citation Format: Urs B. Hagemann, Pascale LeJeune, Jenny Karlsson, Christoph A. Schatz, Alan S. Cuthbertson, Hartwig Hennekes, Karl Ziegelbauer, Dominik Mumberg. MSLN-TTC (BAY 2287411) induces immunogenic cell death and secretion of pro-inflammatory cytokines in vitro and triggers an immune memory effect against a mouse tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3926.

Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is a targeted alpha therapy consisting of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. We previously demonstrated the potent in vivo activity of MSLN-TTC in cell line and patient-derived xenograft models, and further, demonstrated its combination potential with DNA damage repair inhibitors. MSLN-TTC is currently in clinical development for the treatment of patients with MSLN-positive mesothelioma and ovarian cancer (NCT03507452). Development of cellular resistance mechanisms are often co-evolutionary to the treatment regimen given to the respective individual. As such, it is well described that ovarian cancer patients have a decreased overall survival when tested positive for markers of acquired drug resistance (ADR), e.g. phospho-glycoprotein (P-gp) (Herzog TJ et al; Oncotarget 2016). However, MSLN-TTC should also be effective independent of P-gp expression, as the major mode of action of MSLN-TTC is the induction of irreparable DNA double-strand breaks upon alpha decay by thorium-227, resulting in apoptotic cell death. To confirm this hypothesis, we first determined the involvement of P-gp in cellular resistance mechanisms in ovarian cancer. To this end, immunohistochemical staining of ovarian cancer patient samples for P-gp was performed. Expression of P-gp was observed in 15% (7/48) primary and relapsing specimens. We then evaluated the potency of MSLN-TTC in MSLN-expressing cell line models in vitro using the isogenic cell line pair OVCAR-8 (P-gp negative) and NCI-RES-ADR (P-gp positive) in comparison to the standard of care (SOC) therapies cisplatin, paclitaxel, doxorubicin and vinorelbine. Whereas paclitaxel and doxorubicin showed a 100- and 10-fold decrease in in vitro potency on the P-gp positive cell line NCI-RES-ADR vs the P-gp negative cell line OVCAR-8, the potency of MSLN-TTC on the two isogenic cell lines remained unaffected. To further confirm and explore these observations, in vivo xenograft models were conducted. Similar to the in vitro results, the in vivo efficacy of MSLN-TTC was not affected by the P-gp status in the OVCAR-8 vs NCI-RES-ADR xenograft model. In contrast, limited efficacy of paclitaxel and doxorubicin was observed in the P-gp-positive MSLN-expressing NCI-RES-ADR and Hela-MATU-ADR models. Similar, vinorelbine showed no efficacy in the Hela-MATU-ADR xenograft. In summary, these data demonstrate that the efficacy of MSLN-TTC is independent of the P-gp status. Further, these data suggest that MSLN-TTC may be active in patients relapsing after standard therapy. Citation Format: Urs B. Hagemann, Sabine Zitzmann-Kolbe, Alexander Kristian, Carolyn Sperl, Christoph A. Schatz, Roger M. Bjerke, Alan S. Cuthbertson, Hartwig Hennekes, Karl Ziegelbauer, Dominik Mumberg. MSLN-TTC (BAY 2287411) demonstrates increased activity in comparison to standard of care chemotherapy in models of acquired drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3937.

Targeted alpha therapy (TAT) agents deliver high linear energy transfer (LET) alpha-radiation selectively to tumors. The first TAT to be approved is radium-223 which prolongs overall survival in metastatic castration resistant prostate cancer (mCRPC) patients with symptomatic bone metastasis. Radium-223 shows a selective uptake in newly formed bone matrix such as bone metastasis and binds to hydroxyapatite. The PSMA targeted thorium-227 conjugate PSMA-TTC represents another TAT approach in mCRPC. It consists of a fully human PSMA IgG antibody covalently linked to the chelator moiety (3,2 HOPO). This antibody-chelator conjugate is radiolabeled with thorium-227, which decays with a half-life of 18.7 days to radium-223 via alpha-particle emission. Herein we describe tumor targeting and anti-tumor activity of PSMA-TTC in two PSMA positive patient derived xenograft (PDx) models of prostate cancer with different characteristics. In vivo biodistribution and anti-tumor efficacy were analyzed after i.v. injection of PSMA-TTC at radioactive doses from 75-500 kBq/kg and protein doses of either 0.14 or 0.43 mg/kg to tumor bearing mice. Initially, the PDx model KuCap1 (provided by Prof. O. Ogava, University of Kyoto, Japan), a prostate cancer model resistant to the second generation antiandrogen enzalutamide, was analyzed. In this model PSMA-TTC showed strong dose dependent tumor growth inhibition starting at a single dose of 75 kBq/kg. Moreover, after a single i.v. administration of PSMA-TTC at 300 kBq/kg 9 out of 10 mice (90%) showed either stable disease or tumor regression for at least 33 days after treatment. The observed activity was highly selective, as injection of a radiolabeled control conjugate at 300 kBq/kg showed only limited tumor growth inhibition. Next, PSMA-TTC was tested in the hormone- and enzalutamide-sensitive prostate cancer PDx model ST1273 (South Texas Accelerated Research Therapeutics, San Antonio, Texas). A single i.v. injection of PSMA-TTC resulted in significant tumor accumulation of thorium-227 for more than 3 weeks, whereas a radiolabeled isotype control conjugate did not show tumor uptake. In addition, single i.v. administration of PSMA-TTC showed strong dose dependent anti-tumor activity while limited tumor growth inhibition was observed for a radiolabeled isotype control conjugate. Single doses of either 250 or 500 kBq/kg resulted in a 100 % response rate 4 weeks after treatment, with all animals showing partial or even complete regression of tumor growth. No significant effects on body weight were detected compared to vehicle treated animals. In summary, PSMA-TTC shows strong anti-tumor activity in patient derived prostate cancer models which were either sensitive or resistant to standard of care drugs. These data warrant further clinical investigation of this targeted alpha pharmaceutical investigational agent. Citation Format: Stefanie Hammer, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Aasmund Larsen, Christine Ellingsen, Oliver von Ahsen, Jenny Karlsson, Roger M. Bjerke, Olav B. Ryan, Pascale Lejeune, Hartwig Hennekes, Alan Cuthbertson, Karl Ziegelbauer, Dominik Mumberg. Preclinical activity of PSMA-TTC, a targeted alpha therapeutic in patient-derived prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 844.

Targeted Thorium-227 Conjugates (TTCs) represent a new class of targeted alpha therapy. In this compound family a 3,2-HOPO chelator, which binds thorium-227 with high affinity, is covalently attached to an antibody. This enables the specific delivery of the alpha particle emitter thorium-227 to tumor cells. Thorium-227 has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life of 11.4 days), a calcium-mimetic used in the treatment of CRPC [Henriksen et al. J Nucl Med, 2003]. The high linear energy transfer from the alpha-emitter thorium-227 induces clustered DNA double-strand breaks. Its short penetration range of 2-10 cell diameters limits the damage to the normal tissue surrounding the tumor. We present the preclinical evaluation of a mesothelin targeted thorium-227 conjugate (MSLN-TTC), the first TTC that will enter clinical development in MSLN-positive solid tumor indications, based on the fully human anti-MSLN monoclonal antibody anetumab. MSLN is a 40 kDa membrane-anchored glycoprotein with prominent overexpression in mesothelioma, ovarian, pancreatic, lung and breast cancer. In normal tissue, MSLN is confined mainly to the mesothelial cells of pleura, peritoneum and pericardium. In vitro, the mode of action of MSLN-TTC in cellular assays was demonstrated to induce DNA double strand breaks, leading to cell cycle arrest and subsequent reduced cell viability. In vivo, MSLN-TTC demonstrated potent tumor growth inhibition administered as a single-dose in cell- and patient-derived xenograft tumor models. Similar anti-tumor activity to single dose application was observed when the MSLN-TTC was applied at fractionated doses. A trend for dependence of anti-tumor activity on MSLN expression levels in preclinical tumor models was observed. Biodistribution studies evaluated the tumor accumulation of MSLN-TTC in xenograft models. These studies served to develop a mechanistic PK/PD model, which was used to predict the efficacious dose in humans. The initiation of clinical investigation of the MSLN-TTC in mesothelin positive cancers' is planned for 2018. Citation Format: Urs B. Hagemann, Alexander Kristian, Christine Ellingsen, Katrine Wickstroem, Anne Mobergslien, Jenny Karlsson, Roger M. Bjerke, Christoph Schatz, Christoph Kneip, Joachim Schuhmacher, Liv-Ingrid Oedegaardstuen, Hartwig Hennekes, Anna Tafuri, Dominik Mumberg, Hanno Wild, Karl Ziegelbauer, Alan S. Cuthbertson. Mesothelin targeted thorium-227 conjugate (MSLN-TTC): Preclinical evaluation of a new targeted alpha therapeutic in mesothelin-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 850.

Ovarian cancer remains an area of high unmet medical need, with 239,000 patients newly diagnosed per year. Here we describe the mesothelin-targeting antibody drug-conjugate anetumab ravtansine as a novel treatment option for ovarian cancer. Internalization of anetumab ravtansine and co-localization with lysosomal markers in ovarian cancer cells was accompanied by rapid resynthesis of mesothelin, which may allow consecutive anetumab ravtansine treatment cycles. The strong antitumor activity of anetumab ravtansine was preserved during repeated treatment cycles in the OVCAR-3 ovarian cancer in vivo model. Mechanistically, treatment with anetumab ravtansine caused mitotic arrest characterized by increased pHH3 signal and monopolar spindle structures. Interestingly, anetumab ravtansine monotherapy also induced DNA damage as indicated by focal γH2AX signals. Ultimately, the cells engaged in apoptotic cell death. Strong in vitro monotherapy activity was demonstrated in a set of 11 ovarian cancer cell lines with IC50s between 3 and 90nM. The in vivo efficacy of anetumab ravtansine dosed at 2.5 mg/kg Q3Dx3 i.v. anetumab ravtansine was evaluated in 9 human ovarian cancer models with varying mesothelin expression levels. Strong in vivo efficacy at this moderate dose with T/C Citation Format: Christoph A. Schatz, Maria Quanz, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Beatrix Stelte-Ludwig, Sven Golfier, Charlotte Kopitz, Cem Elbi, Karl Ziegelbauer, Dominik Mumberg. Anetumab ravtansine has single-agent activity in mesothelin-expressing human ovarian cancer models and potentiates the activity of chemotherapeutics and targeted agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1780.

In 2015, the estimated incidence of new breast cancer (Ca) cases in the US was 234.190 and number of deaths 40.730. Human epidermal growth factor receptor 2 (HER2) is encoded by the proto-oncogene c-erbB-2 and initiates downstream signaling pathways leading to cell proliferation and tumorigenesis. HER2 is overexpressed in several cancer types and has emerged as one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Given that 20% of breast Ca patients are HER2 positive and 70% of patients with metastatic disease will develop bone metastases and associated morbidities, there is still an unmet medical need for improved therapies targeting HER2. Radium-223 (Ra-223) is a novel targeted alpha therapeutic for treatment of patients with castration-resistant prostate cancer and bone metastases. Localized high energy alpha particle emission induces double-stranded DNA breaks and cellular apoptosis. Thorium-227 (Th-227) is the immediate precursor for Ra-223 which, in contrast to Ra-223, can be complexed by chelating agents at high affinity, allowing targeted delivery to tumor cells via antibodies. We describe herein the generation of a novel HER2-targeted Th-227 conjugate (HER2-TTC). HER2-TTC consists of the humanized HER2 targeting IgG1 antibody trastuzumab covalently linked via an amide bond to a 3,2-hydroxypyridino-based chelator moiety, enabling efficient radiolabeling with the alpha particle emitting radionuclide Th-227. Anonymized samples of consenting breast cancer patients were analyzed by Immunohistochemistry (IHC). The IHC data demonstrated HER2 positive expression in breast tumor and matched bone metastases, supporting the preclinical evaluation of the anti-tumor efficacy of HER2-TTC in the BT-474 orthotopic bone mouse model. HER2-TTC was prepared at high radiochemical yield and purity. When tested for binding to recombinant HER2, HER2-TTC was shown to retain comparable binding affinity to trastuzumab. In vitro cytotoxicity experiment of HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range on breast cancer cell line BT-474 (430 000 mAbs bound/ cell as determined by FACS). Anti-tumor efficacy of HER2-TTC was evaluated at 250 and 500 kBq/kg at a protein dose of 0.14 mg/kg. X-ray imaging, serum bone formation marker PINP, micro CT 3D reconstruction imaging and histological analysis demonstrated significantly reduced bone lesions and tumor induced bone remodeling. The promising preclinical anti-tumor activity supports the development of the HER2-TTC as a novel targeted alpha therapeutic for the treatment of patients with HER2 positive bone metastatic disease. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Christine Ellingsen, Alexander Kristian, Dessislava Mihaylova, Steinar R. Uran, Mari Suominen, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model [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 5857. doi:10.1158/1538-7445.AM2017-5857

FGFR2 is a transmembrane tyrosine kinase receptor, consisting of three extracellular N-terminal immunoglobulin-like domains which are involved in ligand-binding as well as in receptor dimerization. Ligand-independent activation of FGFR2 signaling either via genomic amplification, gene fusion events, mRNA overexpression, or mutations has been observed e.g. in gastric cancer, colorectal cancer (CRC), and triple-negative breast cancer (TNBC). As such, FGFR2 has been described to be involved in cancer progression, promotion of oncogenesis, neoangiogenesis, as well as resistance to targeted therapies. Overexpression of FGFR2 and relatively low levels of cell surface expression of FGFR2 in normal human tissues renders FGFR2 an attractive candidate to explore targeted alpha therapy (TAT). We describe the generation of a high energy, alpha-particle emitting FGFR2 targeted thorium-227 conjugate (FGFR2-TTC). The FGFR2-TTC consists of a fully human FGFR2 binding IgG1 antibody (BAY 1179470) cross-reactive with mouse FGFR2, covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with the alpha particle emitting thorium-227 (227Th). In vitro cytotoxicity experiments with FGFR2-TTC demonstrated potency in the sub-picomolar range compared to a non-targeting control-TTC and a correlation between decrease in cell viability and increasing number of anti-FGFR2 antibodies bound per cell (ABC counts) in a panel of FGFR2-positive cancer cell lines. Upon treatment of cells with FGFR2-TTC, the DNA damage response marker protein γH2AX was up-regulated indicating that the mode-of-action involves induction of DNA double strand breaks. Furthermore, induction of the immunogenic cell death marker calreticulin was observed Biodistribution studies of the FGFR2-TTC in mouse models, evaluated by whole body autoradiography and acquisition of gamma-spectra specific for thorium-227, demonstrated specific accumulation of thorium-227 in FGFR2-positive tumors and very limited signal in murine organs and tissues. FGFR2-TTC exhibited in vivo tumor growth inhibition after a single dose in mouse xenograft models of CRC (NCI-H716) and gastric cancer (SNU-16). In addition, FGFR2-TTC showed anti-tumor activity in the aggressive murine syngeneic orthotopic 4T1 TNBC model. In summary, FGFR2-TTC has been established as a promising targeted alpha therapy (TAT) for efficacious and selective delivery of alpha emitter-based radiotherapy in several FGFR2-positive cancer indications. Further exploration for cancer therapy may thus be of interest. Citation Format: Urs B. Hagemann, Anette Sommer, Alexander Kristian, Ellen Wang, Åsmund Larsen, Uta Wirnitzer, Heidrun Ellinger-Ziegelbauer, Steffen Sandmann, Thorsten Poethko, Jenny Karlsson, Roger M. Bjerke, Lars Linden, Bertolt Kreft, Hanno Wild, Alan S. Cuthbertson. Preclinical activity of the FGFR2-targeted thorium-227 conjugate in preclinical models of colorectal, gastric and triple-negative 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 5199. doi:10.1158/1538-7445.AM2017-5199

Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein, involved in mediating cell-cell adhesion, metastatic spread, promotion of cell proliferation and resistance to apoptosis. Overexpression of MSLN is most prominent in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers, while in healthy tissue, MSLN is confined mainly to the mesothelial cells of the peritoneum and pericardium. Several MSLN-targeting approaches are currently being investigated, including antibody drug conjugates. We describe herein a high energy, alpha-particle emitting MSLN Targeted Thorium Conjugate (MSLN-TTC). Thorium-227 (227Th) has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life 11.4 days), a calcium-mimetic used in the treatment of CRPC [1]. The MSLN-TTC comprises an anti-mesothelin monoclonal antibody covalently linked via an amide bond to a chelator moiety possessing high affinity for thorium-227. We present data from in vitro cytotoxicity assays demonstrating selective cell killing on MSLN positive cell-lines as well as in vivo efficacy in a mouse orthotopic bone xenograft model using NCI-H226 luciferase labeled cells. Experimental procedures: MSLN-TTC was prepared in high radiochemical yields and purity. In vitro cytotoxicity experiments were performed on the mesothelin-positive cell lines Ovcar-3 (ovarian), NCI-H226 (lung mesothelioma) and mesothelin-transfected HT29 (HT29MSLN/colorectal) cells. An in vivo model was established by orthotopic intratibial inoculation of luciferase-transfected NCI-H226 cells in athymic mice. Development of bone disease was monitored by luciferase activity and the extent of bone lesions determined by x-ray imaging and microCT. Results: MSLN-TTC induced specific in vitro cytotoxicity via induction of DNA double strand breaks as determined by phosphorylated histone protein H2AX. MSLN-TTC demonstrated statistical significant in vivo potency administered as a single dose of either 250 or 500 kBq/ kg in the orthotopic bone xenograft model. Animals treated with MSLN-TTC showed a) significantly reduced disease in the bone metastatic lesions b) decreased metastatic disease in the lungs and c) significant reduction in osteolytic/ osteoblastic bone lesions as evidenced by X-Ray and microCT compared to the vehicle control group. Furthermore, no significant loss in body weight was observed during the course of the study demonstrating that the MSLN-TTC was well tolerated. The data presented support the further investigation of the MSLN-TTC in bone metastatic disease. References: 1. Henriksen, G., et al., Targeting of osseous sites with alpha-emitting 223Ra: comparison with the beta-emitter 89Sr in mice. J Nucl Med, 2003. 44(2): p. 252-9. Citation Format: Urs B. Hagemann, Else-Marie Hagelin, Katrine Wickstroem, Kristine Sponheim, Roger Smeets, Jenny Karlsson, Roger M. Bjerke, Mari I. Suominen, Yvonne Konkol, Jenni Bernoulli, Jukka Rissanen, Jussi Halleen, Liv-Ingrid Oedegaardstuen, Alan Cuthbertson. A novel high energy alpha-pharmaceutical: In vitro and in vivo potency of a mesothelin-targeted thorium-227 conjugate (TTC) in a model of bone disease. [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 591.

Introduction Clinically, bone is a very common site of metastatic spread in many cancers. In breast, and in particular cases of advanced estrogen receptor positive (ER+) cancers, the propensity of bone involvement is 85%. Similarly in lung cancer, 30-40% of patients with advanced disease develop bone metastases, and as recent advances in lung cancer therapies improve survival, the number of patients living with bone metastases is expected to increase. At the same time there is a paucity of especially ER+ and osteoblastic animal models available for the nonclinical evaluation of new treatment strategies. We present herein the development of four mouse models of breast and lung cancer suitable for screening of new therapies. Experimental procedures Human breast cancer cell lines BT-474 and MFM-223 and non-small cell lung cancer (NSCLC) cell lines NCI-H226luc and NCI-H322 were used. BT-474 is ER+, i.e. luminal B subtype and MFM-223 is basal subtype with androgen receptor (AR) expression. H226 originates from squamous cell carcinoma and H322 from adenocarcinoma of the lung. The different cell lines were inoculated in the tibia of female nude or NOD.scid mice. Half of the BT-474 inoculated mice had a s.c. slow release 17-beta estradiol pellet implanted. The formation of bone lesions was monitored by X-ray imaging. For H226 transfected with luciferase, tumor growth was also followed by bioluminescence imaging (BLI). Finally, tumor growth and type of bone lesion, i.e. ostelytic or oestoblastic, was confirmed by histology. Results Development of bone lesions was successful in 100% and 90% of animals, with or without hormonal supplementation respectively, four weeks after inoculation of BT-474 cells. Bone lesions were detected earlier in mice with estradiol pellet and were of lytic type. In contrast, bone lesions in mice without hormonal supplementation were strongly osteoblastic. For MFM-223, bone lesions were observed 4-6 weeks after inoculation and the success rate was 60% in nude mice and 70% in NOD.scid mice. For both lung cancer cell lines, 100% of the mice developed bone lesions and were detectable already two weeks after inoculation. H226luc cells developed osteoblastic-mixed lesions and H322 cells induced lytic lesions. Very interestingly, H226luc cells also formed lung metastases in all animals, as evidenced by BLI. Some lung metastases were also found in H322 inoculated mice. Conclusions Two new osteoblastic models are added to the current scarce selection and altogether four new bone lesion models representing different subtypes of breast and lung cancer were successfully established. The different types of bone reaction in these models offer a platform for studying the underlying pathways resulting in response to treatment in osteoblastic vs. osteolytic tumor microenvironment. Citation Format: Mari I. Suominen, Urs B. Hagemann, Yvonne Konkol, Jenni Bernoulli, Katja M. Fagerlund, Roger M. Bjerke, Jenny Karlsson, Jussi M. Halleen, Alan Cuthbertson. New models of breast and lung cancer bone metastases for preclinical efficacy testing. [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 640.

Introduction: The sialoadhesin receptor CD33 is a well-established target in the treatment of acute myeloid leukemia (AML). CD33 is expressed on leukemic blasts in AML and possibly also on leukemic stem cells in some patients (1). Thorium-227, an alpha-particle emitting radionuclide with a half-life of 18.7 days, is being studied for use as a possible cytotoxic agent. We have developed a novel Targeted Thorium Conjugate (TTC), comprising an anti-CD33 monoclonal antibody conjugated to a chelator for the efficient delivery of high-energy alpha-particles. We present data from in vitro cell cytotoxicity assays as well as biodistribution and efficacy data from preclinical animal models of AML. Experimental procedures: Antibody conjugates were prepared and labeled with 227Th at room temperature giving high radiochemical yields and purity. In vitro cell cytotoxicity experiments were carried out on HL-60 cells and the multi-drug resistant cell line KG-1 by measuring cell viability and monitoring apoptosis. In vivo efficacy and biodistribution were performed in a sub-cutaneous model of AML in athymic nude (nu/nu) mice using HL-60 cells. The 227Th-anti-CD33 conjugate was further tested in a disseminated model of AML using HL-60 cells, injected intravenously 5 days prior to treatment in CB.17 SCID mice with hind-leg paralysis as endpoint. Results: The 227Th-anti-CD33 conjugate was a potent and specific inducer of in vitro cell cytotoxicity independent of MDR-status. Mechanistic studies demonstrated cell arrest in G2 phase with cytotoxicity associated with apoptotic pathways. In vivo biodistribution in a sub-cutaneous model demonstrated a high tumor to blood ratio (TBR ∼ 6) at day 7 post injection based on measured 227Th-activity. Significant efficacy was achieved in two models of AML using human HL-60 cells. In athymic nude mice, a single injection of 227Th-anti-CD33 conjugate at 700 kBq/kg resulted in complete regression of established sub-cutaneous tumors. Importantly, no significant loss in body weight was observed during the course of the study, demonstrating that the dose was well tolerated. In parallel, statistically significant dose dependency of 227Th-anti-CD33 conjugate was demonstrated in a disseminated model of AML with median survival times (MST) of 90 days and 116 days for animals receiving 50 and 150 kBq/kg. Around 80% of animals dosed with either 2 × 150 or 1 × 300 kBq/kg where still alive at the end of the study with no gross pathological findings observed. Conclusions: The data presented are supportive of the further investigation of the 227Th-anti-CD33 conjugate for the treatment of AML. References: (1) Walter RB et al, Blood 2012, Vol 119, Number 26 (“Acute myeloid leukemia stem cells and CD33-targeted immunotherapy”) Citation Format: Urs B. Hagemann, Joergen Borrebaek, Adam O'Shea, Ellen Wang, Katrine Wickstroem, Roger M. Bjerke, Jenny Karlsson, Olav B. Ryan, Alan Cuthbertson. In Vivo efficacy of a novel anti-CD33 targeted thorium conjugate (TTC) in mouse models of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2462. doi:10.1158/1538-7445.AM2015-2462

Tumor microenvironments posses complex chemokine networks that contribute to the extent and phenotype of the host infiltrate. Malignant cells may gain functional chemokine receptors, often as a consequence of oncogenic mutations, allowing them to respond to distant chemokine gradients during metastasis. The chemokine receptor CCR4 was highly expressed in human renal cell carcinoma, RCC, biopsies and RCC patient plasma had abnormal levels of CCR4 ligands. However, during pre-clinical evaluation of CCR4 as a target in RCC, we found that both a small molecule CCR4 inhibitor and an anti-CCR4 antagonistic antibody had unexpected and novel anti-tumor activity in the mouse RCC RENCA model. CCR4 antagonists did not reduce the proportion of infiltrating leukocytes in the tumor microenvironment but altered the phenotype of myeloid cells, increased NK cells and Th1 cytokine levels, as well as reducing splenic MDSC infiltrate, and blood chemokine levels. Although the most prominent changes were in the myeloid compartment, anti-tumor activity was lost in athymic mice, supporting a role for the adaptive immune system in CCR4 antagonist action. CCR4 antagonists, alone, or in combination with other immune modulators, may be of interest in human cancers with high levels of tumor CCR4 and abnormal plasma CCR4 ligand levels. Citation Format: Chiara Berlato, Moddasar N. Kahn, Tiziana Schioppa, Richard Thompson, Eleni Maniati, Monica Canosa, Hagen Kulbe, Chris Sheldon, Keith Wreggett, Urs Hagemann, Alexander Duncan, Laura Fletcher, Robert W. Wilkinson, Thomas Powles, Sergio Quezada, Frances Balkwill. Antagonists of the chemokine receptor CCR4 reverse the tumor-promoting microenvironment of renal cancer. [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 1076. doi:10.1158/1538-7445.AM2014-1076