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Radium-223, a bone-specific alpha particle therapy, increased survival of patients with metastatic castration-resistant prostate cancer (mCRPC) and bone metastases (Parker et al., NEJM 2013) 177Lu-PSMA-617 is a small molecule radioligand that delivers beta-particle radiation to cancer cells expressing prostate-specific membrane antigen (PSMA). Despite improving overall survival of patients with heavily pretreated mCRPC (Sator et al., NEJM 2021), responses to 177Lu-PSMA-617 are often not durable and many patients develop progressive disease presumably due to microscopic osseous lesions. Compared to beta-particle radiation, alpha particles have a shorter range and higher linear energy transfer, making them more suitable for treating microscopic lesions. In this study, we evaluated preclinical antitumor efficacy and potential beneficial combination effects of radium-223 combined with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model mimicking progressive prostate cancer micrometastases in bone. LNCaP prostate cancer cells were inoculated into the right tibiae of male NOD.scid mice. Six weeks after inoculation, the mice were randomized to treatment groups based on serum prostate-specific antigen (PSA) and treated with two injections (Q4Wx2, i.v.) of vehicle, radium-223 (330 kBq/kg), 177Lu-PSMA-617 (10 MBq) or their combination 177Lu-PSMA-617 was given 24 hours after radium-223. PSA and bone formation marker procollagen type I intact N-terminal propeptide (PINP) were measured from blood samples and tumor-induced abnormal bone growth was determined by radiography. Terminal blood samples were analyzed for hematologic status at sacrifice. The combination treatment of radium-223 and 177Lu-PSMA-617 demonstrated robust antitumor efficacy, as indicated by lower serum PSA (mean change relative to pretreatment levels: 63.9 % vs 631 % (vehicle control), p=0.026), decreased tumor-induced abnormal bone area in tumor-bearing tibiae (p In conclusion, these preclinical results show that the combination of radium-223 and 177Lu-PSMA-617 is well-tolerated and efficacious in inhibiting osseous tumor growth and improving response. The combination of radium-223 and 177Lu-PSMA-I&T is currently being investigated in patients with mCRPC in a phase 1/2 trial (AlphaBet, NCT05383079). Citation Format: Arne Scholz, Matias Knuuttila, Justyna Zdrojewska, Mari I. Suominen, Esa Alhoniemi, Christoph A. Schatz, Sabine Zitzmann-Kolbe, Sanna-Maria Käkönen, Urs B. Hagemann. Radium-223 demonstrates increased antitumor activity in combination with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model of bone metastatic prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5043.

Patients with advanced prostate cancer have few treatment options. While androgen receptor (AR) inhibitors are the standard of care for the systemic treatment, patients often develop resistance despite an initial response. Thus, novel therapeutic approaches are required. Prostate-specific membrane antigen (PSMA; FOLH1) is highly expressed in prostate cancer with limited expression in normal tissues. Therefore, PSMA presents an attractive target for radionuclide therapy and PSMA-targeted therapies have already shown efficacy in the clinical setting. Here, we evaluated the efficacy of 225Ac-pelgifatamab, a PSMA-targeted actinium-225 conjugate, in combination with the AR inhibitor darolutamide in androgen-dependent and -independent prostate cancer models. AR inhibitors are known to upregulate PSMA expression, therefore providing a rationale for combining 225Ac-pelgifatamab with darolutamide and potentially resulting in an enhanced treatment effect. In vitro, darolutamide induced the expression of PSMA in androgen-dependent VCaP and androgen-independent 22Rv1 cells by more than 10-fold and 2-fold, respectively. The efficacy of darolutamide in combination with 225Ac-pelgifatamab was synergistic in these cells with combination indexes of 0.12 and 0.47, respectively. In the androgen-dependent, patient-derived ST1273 model, a single i.v. injection of 225Ac-pelgifatamab at 75 kBq/kg resulted in a treatment/control (T/C) ratio of 0.22 on day 30, while twice daily (BID) oral (p.o.) treatment with 100 mg/kg darolutamide showed a T/C ratio of 0.29. However, the combination of darolutamide and 225Ac-pelgifatamab resulted in enhanced antitumor efficacy with a T/C ratio of 0.008. Furthermore, 5/10 mice remained tumor-free 86 days after the 225Ac-pelgifatamab injection. In the androgen-independent cell line-derived 22Rv1 model, darolutamide (100 mg/kg, p.o., BID) monotherapy was not efficacious with a T/C of 0.84 on day 16 but a single i.v. dose of 225Ac-pelgifatamab at 150 kBq/kg resulted in a T/C ratio of 0.69. Remarkably, the combination of darolutamide and 225Ac-pelgifatamab resulted in further enhanced efficacy with a T/C ratio of 0.34. Flow cytometric analysis of isolated 22Rv1 tumors showed a 50-fold increase in PSMA expression upon darolutamide treatment compared with vehicle. Taken together, these results suggest that upregulation of PSMA expression contributes to the higher efficacy observed when darolutamide is combined with 225Ac-pelgifatamab. In conclusion, our results provide a proof of concept for investigating the combination of 225Ac-pelgifatamab with darolutamide in the clinical setting. Citation Format: Christoph A. Schatz, Bernard Haendler, Sabine Zitzmann-Kolbe, Aasmund Larsen, Hartwig Hennekes, Carsten H. Nielsen, Maria Z. Alfsen, Alan Cuthbertson, Stefanie Hammer, Arne Scholz, Urs B. Hagemann. PSMA-targeted actinium-225 conjugate (225Ac-pelgifatamab) potentiates the antitumor efficacy of darolutamide in androgen-dependent and -independent prostate cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5047.

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.

Targeted α-particle–emitting radionuclides have great potential for the treatment of a broad range of cancers at different stages of progression. A platform that accurately measures cancer cellular sensitivity to α-particle irradiation could guide and accelerate clinical translation. Here, we performed high-content profiling of cellular survival following exposure to α-particles emitted from radium-223 (223Ra) using 28 genetically diverse human tumor cell lines. Significant variation in cellular sensitivity across tumor cells was observed. 223Ra was significantly more potent than sparsely ionizing irradiation, with a median relative biological effectiveness of 10.4 (IQR: 8.4–14.3). Cells that are the most resistant to γ radiation, such as Nrf2 gain-of-function mutant cells, were sensitive to α-particles. Combining these profiling results with genetic features, we identified several somatic copy-number alterations, gene mutations, and the basal expression of gene sets that correlated with radiation survival. Activating mutations in PIK3CA, a frequent event in cancer, decreased sensitivity to 223Ra. The identification of cellular and genetic determinants of sensitivity to 223Ra may guide the clinical incorporation of targeted α-particle emitters in the treatment of several cancer types. Significance: These findings address limitations in the preclinical guidance and prediction of radionuclide tumor sensitivity by identifying intrinsic cellular and genetic determinants of cancer cell survival following exposure to α-particle irradiation. See related commentary by Sgouros, p. 5479

Mesothelin-targeted thorium-227 conjugate (MSLN-TTC) is a targeted alpha therapeutic consisting of a MSLN-targeting antibody anetumab covalently attached to a 3,2-HOPO chelator complexed with the alpha emitter thorium-227. It has potent in vivo activity in cell and patient-derived xenograft (PDX) models in monotherapy and in combination with DNA damage repair inhibitors. We evaluated the antitumor efficacy of MSLN-TTC in a mesothelin overexpressing ovarian PDX model (ST206B) in monotherapy and in combination bevacizumab or the multikinase inhibitor regorafenib which both have anti-angiogenic activity. The biodistribution of MSLN-TTC was studied in parallel. Athymic mice were transplanted s.c. with ST206B-derived tumor fragments and treated with MSLN-TTC (250 kBq/kg, Q7Dx2, 0.43 mg/kg) alone or in combination with bevacizumab (1.25 or 5 mg/kg, i.p., Q5D; or 5 mg/kg, single dose) or regorafenib (7.5, 15, or 30 mg/kg, QD, p.o. for 28 days). The biodistribution of the respective treatment groups was analyzed using a germanium detector. For molecular analyses, tumors were stained for γ-H2AX, Ki67, CD31 and α-SMA. MSLN-TTC monotherapy showed high in vivo potency at 2 × 250 kBq/kg with a Treated/Control (T/C) ratio of 0.28 (n=5) on day 29. Monotherapy treatment with bevacizumab and regorafenib showed dose-dependent antitumor activity with T/C ratios ranging from 0.31 (regorafenib 30 mg/kg) to 0.33 (bevacizumab 5 mg/kg, single dose). The in vivo efficacy of MSLN-TTC was further enhanced by combination therapy with bevacizumab (T/C 0.21; n=5) or regorafenib (T/C 0.15; n=5). Combination of MSLN-TTC and regorafenib (30 mg/kg) resulted in more complete and partial responses (3/4 CRs; 1/4 PR) as compared to MSLN-TTC (2/3 PRs; 1/3 PD) at day 141. All treatments were well-tolerated based on body weight assessment; a reversible myelosuppression of white blood cells was observed for MSLN-TTC monotherapy and combination with bevacizumab or regorafenib did not worsen it. In the biodistribution study, tumor-specific accumulation of MSLN-TTC was observed over 336 h (14 days) with lower accumulation in the MSLN-TTC + bevacizumab group (1.25 mg/kg, Q5D, p Citation Format: Urs B. Hagemann, Christine Ellingsen, Claudia Kamfenkel, Dieter Zopf, Jesper Fonslet, Carsten Nielsen, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. MSLN-targeted thorium-227 conjugate demonstrates increased antitumor activity in combination with bevacizumab and regorafenib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5355.

Targeted thorium-227 conjugates (TTCs) represent a new class of targeted alpha therapy (TAT). TTCs consist of an antigen targeting moiety, which is covalently attached to a 3,2-HOPO chelator, enabling specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. TTCs have demonstrated potent preclinical in vivo activity in monotherapy and in combination with DNA damage inhibitors. In the present work, the immunostimulatory effects of the mesothelin (MSLN) targeted thorium-227 conjugate (MSLN-TTC) was studied in immunocompetent mice, bearing human MSLN-transfected MC38 cells, in monotherapy and in combination with the PD-L1 inhibitor (PD-L1i) atezolizumab. Surface expression of DAMPs (calreticulin, HMGB1, HSP70 and HSP90), immunoinhibitory markers (PD-L1 and CTLA-4) and immunostimulatory markers (IFNAR, B7H2 and ICOS-L) was analyzed on murine, human MSLN-transfected MC38 colorectal cancer cells (MC38-hMSLN cells) after a 3-day exposure to MSLN-TTC. In vivo, C57/Bl6 mice were inoculated s.c. with MC38-hMSLN cells and treated with a single i.v. dose of non-radiolabeled MSLN-conjugate (0.14 mg/kg), isotype control-TTC (250 kBq/kg; 0.14 mg/kg), MSLN-TTC (250 kBq/kg; 0.14 mg/kg), PD-L1i (1.5 mg/kg; Q3D4; i.p.) or MSLN-TTC/PD-L1i combination. Tumors, lymph nodes, spleen and plasma were analyzed for MSLN and molecular markers. Tumor-free survivors were re-challenged with MC38-hMSLN or vector cells 125 d post-treatment. In a parallel arm of the experiment, CD8+ T cells were depleted using YTS169.4 antibody. MSLN-TTC treatment resulted in a specific dose-dependent increase of IFNAR, PD-L1 and HMGB1 in MC38-hMSLN cells in vitro. MSLN-TTC (T/C 0.38; Day 22) and PD-L1i (T/C 0.0.42; Day 22) monotherapies showed specific antitumor activity (T/C of radiolabeled isotype control was 0.76, Day 22) and their combination was synergistic (T/C 0.08; Day 22) with increased numbers of tumor free survivors. All three treatments increased HMGB1 levels in plasma. Induction of DSBs was observed in MSLN-TTC (+/- PD-L1i) treated tumors. Re-challenge of tumor-free survivors treated with MSLN-TTC, PD-L1i or MSLN-TTC/PD-L1i resulted in rejection of MC38-hMSLN, but not vector-cells. Infiltration of CD8+ T cells into tumors and their increase in tumor-draining lymph nodes were observed in MSLN-TTC, PD-L1i and MSLN-TTC/PD-L1i-treated groups but not in the vehicle group. Depletion of CD8+ T cells decreased the efficacy of PD-L1i and to a lower extent, MSLN-TTC and MSLN-TTC/PD-L1i. These results suggest that the efficacy of MSLN-TTC may involve activation of CD8+ T cells. These findings support the hypothesis that upon binding to antigen-positive cancer cells, TTC treatment induces DNA double strand breaks, leading to apoptosis and potentially immunogenic cell death, resulting in activation of the adaptive immune system. Citation Format: Urs B. Hagemann, Pascale Lejeune, Lisa Bartnitzky, Kathleen Stadelmann, Veronique Cruciani, Roger M. Bjerke, Sandra Berndt, Claudia Kamfenkel, Manuela Brand, Manuela Steinbach, Stefan Stargard, Christoph A. Schatz, Jenny Karlsson, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. Targeted thorium-227 conjugates demonstrate synergistic activity in combination with PD-L1 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2257.

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.

Targeted alpha therapy (TAT) agents are able to deliver high linear energy transfer alpha-radiation selectively to tumors. The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a TAT approach for mCRPC consisting of a human anti-PSMA antibody covalently linked to the chelator moiety (3,2 HOPO) radiolabeled with the alpha emitter thorium-227. PSMA-TTC has shown strong anti-tumor activity in PSMA-positive prostate cancer models (Hammer et al. AACR 2017/2018). Androgen receptor (AR) antagonists like enzalutamide have been effective in improving overall survival in CRPC patients, however, not all patients respond to these therapies and those responding develop resistance and progression of disease. Herein we evaluate in prostate cancer models combination treatments of PSMA-TTC with enzalutamide and the novel AR antagonist darolutamide, which has recently completed a clinical phase 3 trial. AR antagonists induced PSMA levels in LNCaP and C4-2 prostate cancer cells in vitro resulting in increased sensitivity to growth inhibition by PSMA-TTC. In vivo, the combination of PSMA-TTC with enzalutamide was tested in the hormone- and enzalutamide-sensitive patient-derived prostate cancer model ST1273 (South Texas Accelerated Research Therapeutics, San Antonio, Texas). A single i.v. injection of PSMA-TTC at 250 kBq/kg (0.14 mg/kg) resulted in stable disease in 50% and partial response in 38% of the animals 40 days after dosing. Daily treatment with enzalutamide (30 mg/kg po for 28d) resulted in stable disease in 86% and partial response in 14% of the animals. Combining PSMA-TTC and enzalutamide at the mentioned concentrations achieved tumor reduction in all animals, with partial response in 67% and complete response in 33% of the animals 40 days after start of treatment. No significant adverse effects on body weight were detected compared to vehicle treated animals in any of the groups. Additionally, the combination of PSMA-TTC with darolutamide was tested in the enzalutamide resistant patient-derived prostate cancer model KUCaP-1 (provided by Prof. O. Ogawa, University of Kyoto, Japan). PSMA-TTC monotherapy at 150 kBq/kg (0.43 mg/kg) injected twice at an interim of two weeks resulted in 79% tumor growth inhibition and darolutamide treatment (200 mg daily) achieved 60% tumor growth inhibition compared to vehicle treated animals. The combination of PSMA-TTC with darolutamide increased tumor growth inhibition compared to vehicle to 85% and, importantly, 77% of the animals showed stable disease or partial response until 57 days after start of treatment. In summary, combining PSMA-TTC with AR antagonists shows promising preclinical data in patient-derived prostate cancer models with increased response rates even in an enzalutamide-resistant model. Clinical investigation of this targeted alpha pharmaceutical investigational agent alone and in combination is warranted. Citation Format: Stefanie Hammer, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Aasmund Larsen, Chrstine Ellingsen, Oliver von Ahsen, Jenny Karlsson, Roger M. Bjerke, Olav B. Ryan, Pascale Lejeune, Hartwig Hennekes, Alan Cuthbertson, Dominik Mumberg. Preclinical activity of PSMA-TTC (BAY 2315497) in combination with androgen receptor antagonists in prostate cancer models [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 3726.

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.

The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a targeted alpha therapy approach for metastatic castration resistant prostate cancer that has shown strong anti-tumor activity in PSMA-positive prostate cancer models (Hammer et al. AACR 2017/2018/2019). PSMA-TTC consists of a fully human IgG1 PSMA-targeted antibody covalently linked to a 3,2-HOPO chelator moiety, stably complexing the alpha emitter thorium-227. As this chelator can also complex zirconium-89, positron emission tomography (PET) imaging using the zirconium-89 labeled variant of the PSMA antibody-chelator conjugate could support clinical development of PSMA-TTC by enabling monitoring of organ distribution. Herein, we describe radiolabeling of the PSMA antibody-chelator conjugate with zirconium-89 and its quality control using bioanalytical assays. In addition, the zirconium-89 labeled PSMA antibody-chelator conjugate was injected into mice bearing LNCaP xenograft tumors and biodistribution was assessed by measurement of radioactivity in tumor and organs as well as PET imaging at different time points after injection. The PSMA antibody-chelator conjugate was successfully labeled with zirconium-89 at high yield, purity and specific activity. Target binding was confirmed by determination of the immunoreactive fraction. Biodistribution experiments revealed high, long lasting uptake of zirconium-89 in PSMA-positive LNCaP tumors after single injection of the zirconium-89 labeled conjugate. An uptake of 40-50% of the injected dose per gram of tumor was measured at 24, 48, 72, 168 and 240h after injection. The PET images showed that the zirconium-89 labeled PSMA antibody-chelator conjugate provided an excellent image contrast for the delineation of LNCaP xenografts between 48 and 168 h after administration. Other organs showed only very little background and tumor-to-heart ratio was greater than 10 when PET imaging was performed between 48 and 168 h after injection. These results indicate that the zirconium-89 labeled PSMA antibody-chelator conjugate shows promise for PET imaging studies. These properties may allow its use to support clinical development of PSMA-TTC. Citation Format: Stefanie Hammer, Sabine Zitzmann-Kolbe, Felix Oden, Joerg Jannsen, Eva Bickel, Andre Mueller, Lothar Everz, Urs B. Hagemann, Jenny Karlsson, Olav B. Ryan, Hartwig Hennekes, Patricia E. Cole, Dominik Mumberg. Preclinical analysis of biodistribution and PET imaging of a zirconium-89 labeled PSMA-targeted antibody-chelator conjugate [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 1141.

Targeted Thorium-227 Conjugates (TTCs) consist of the alpha particle emitter thorium-227 bound with high affinity by a 3,2-HOPO chelator covalently attached to a tumor-specific antibody. Thorium-227 has a half-life of 18.7 days and decays to the alpha particle emitter 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 mechanism of action for alpha emitters is based on the induction of clustered DNA double strand breaks and G2 cell cycle arrest. In principle, inhibitors of DNA damage response (DDR) should sensitize cancer cells to TTCs. Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein known to be overexpressed in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers. This study describes the evaluation of combination treatment with anti-MSLN targeted thorium-227 conjugate (MSLN-TTC) in combination with DDR inhibitors. In vitro cytotoxicity experiments were performed on a set of cancer cell lines from different tissue origins, including Ovcar-3 (ovarian), NCI-H226 (mesothelioma), Capan-2 (pancreas) and mesothelin transfected HT29 (colon) cells. DDR inhibitors targeting Poly [ADP-ribose] polymerase 1 (PARP1), ataxia telangiectasia and Rad3-related (ATR), Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) included olaparib, BAY 1895344, AZD0156, and VX-984, respectively. Synergistic activity of MSLN-TTC was observed in vitro for all four DDR inhibitors with ATRi BAY 1895344 exhibiting the strongest increase in potency. We further evaluated the in vivo anti-tumor efficacy of MSLN-TTC monotherapy and combination treatment with DDR inhibitors ATRi BAY 1895344 or PARPi olaparib in Ovcar-3 xenografts in nude mice. Monotherapy of the MSLN-TTC yielded a dose dependent and specific tumor growth inhibition as compared with a radiolabeled isotype control. In the combination treatment an increased anti-tumor effect of MSLN-TTC at the lowest dose was observed when combined with a non-efficacious dose of either ATRi BAY 1895344 or olaparib, with a more pronounced effect for the combination of MSLN-TTC with ATRi BAY 1895344, resulting in tumor stasis. The present data support the rationale for combining the MSLN-TTC with DDRi's based on their individual mode of actions and bears strong potential for future cancer therapies in tumor indications characterized by overexpression of mesothelin, such as mesothelioma, ovarian, pancreatic, lung or triple-negative breast (TNBC) cancers. Citation Format: Katrine Wickstroem, Urs B. Hagemann, Antje M. Wengner, Alexander Kristian, Christine Ellingsen, Gerhard Siemeister, Roger M. Bjerke, Jenny Karlsson, Olav B. Ryan, Lars Linden, Bertolt Kreft, Dominik Mumberg, Hanno Wild, Karl Ziegelbauer, Alan Cuthbertson. Synergistic effect of MSLN-TTC in combination with DNA damage response inhibitors [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 849.

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.

Targeted Thorium-227 Conjugates (TTCs) consist of the alpha emitter Thorium-227 (227Th) coupled, by a 3, 2-HOPO chelator, to a tumor specific antibody. The alpha particles release high energy over a short range (2- 10 cell diameters), resulting in a potent local irradiation of the tumor with limited damage to surrounding tissue. Here, we describe the in vitro and in vivo evaluation of an FGFR2 targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344. FGFR2 (fibroblast growth factor receptor 2) is a receptor tyrosine kinase and overexpression of FGFR2 has been described in different cancers, while its expression in healthy human tissues is moderate to low. This renders FGFR2 an attractive antigen to explore the concept of targeted alpha therapy (TAT). The mode-of-action of TTCs is based on the induction of clustered DNA double strand breaks and G2 cell cycle arrest. We hypothesized that combination of FGFR2-TTC with inhibitors of DNA damage response (DDRi's) may enhance potency and efficacy. The ataxia telangiectasia and Rad3-related protein (ATR) kinase is a central mediator of DDR. ATR kinase responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Inhibition of ATR kinase activity induces cell death especially in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Therefore, we investigated whether the combination of the FGFR2-TTC with the ATRi BAY 1895344 results in enhanced tumor sensitivity in vitro and in vivo. In in vitro cytotoxicity assays, the combination of FGFR2-TTC and BAY 1895344 resulted in increased potency of the FGFR2-TTC on three different cancer cell lines (KATO III (gastric), MFM-223 (triple negative breast cancer), SUM52-PE (triple negative breast cancer)). Mechanistic analysis demonstrated that the combination treatment resulted in reduced levels of G2 arrest and increased levels of DNA damage in comparison to single agent treatments. The combination was further evaluated in vivo using the MFM-223 breast cancer xenograft model. An increased anti-tumor efficacy of a low dose of FGFR2-TTC (100 kBq/kg) was observed in combination with BAY 1895344 compared to animals treated with vehicle. The presented data support the mechanism-based rationale for combining DNA damage induction by FGFR2-TTC with DNA damage repair inhibition using ATRi BAY 1895344. Our findings warrant further exploration of TTCs in combination with BAY 1895344 for cancer therapy. Citation Format: Katrine Wickstroem, Urs B. Hagemann, Antje M. Wengner, Anette Sommer, Alexander Kristian, Christine Ellingsen, Roger M. Bjerke, Jenny Karlsson, Olav B. Ryan, Lars Linden, Bertolt Kreft, Dominik Mumberg, Hanno Wild, Karl Ziegelbauer, Alan Cuthbertson. Increased in vitro potency and in vivo efficacy of FGFR2-targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344 [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 855.

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

Prostate-specific membrane antigen (PSMA, FOLH1) is a type II transmembrane glycoprotein of the M28 peptidase family that acts as a glutamate carboxypeptidase on various substrates. PSMA is well established as a target antigen in prostate cancer due to its high and specific overexpression on the surface of prostate cancer cells at all tumor stages, including metastatic and hormone-refractory disease. Several PSMA targeting antibodies and ligands are currently in clinical development or compassionate use therapeutically or as imaging agents. Targeted alpha therapy (TAT) has an established clinical profile with the successful transition of Ra223, an alpha-particle emitter, from bench to bedside in prostate cancer. Thorium-227 is the immediate precursor for Ra223 via alpha-particle emission. We herein describe the generation of a novel TAT, a high energy, alpha-particle emitting PSMA-targeted thorium-227 conjugate (PSMA-TTC). PSMA-TTC consists of a fully human PSMA targeting IgG1 antibody covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with thorium-227 (227Th). PSMA-TTC was prepared in high radiochemical yield and purity and tested for binding affinity to PSMA target (ELISA) as well as PSMA expressing cell lines (FACS). In vitro cytotoxicity experiments were carried out on prostate CA cell lines with different PSMA levels (from 3.000 to 150.000 mAbs bound/ cell). In vivo biodistribution and anti-tumor efficacy were analyzed after i.v. injection of 100-500 kBq/kg at protein doses of 0.14 mg/kg to mice bearing s.c. prostate cancer xenograft models. Additionally, anti-tumor efficacy was evaluated in a PSMA expressing orthotopic bone xenograft model (LNCaP-Luc) monitored by bioluminescence imaging, micro CT and x-ray. PSMA-TTC retains binding affinities to PSMA target and PSMA positive cancer cells similar to the PSMA antibody. Strong in vitro potency and selectivity of PSMA-TTC was shown on different PSMA positive cells. Biodistribution studies in C4-2 xenografts demonstrated specific tumor uptake of PSMA-TTC with a maximum of 50 % of ID/g at t = 72h post dose administration. Selective significant antitumor efficacy was shown for PSMA-TTC in s.c. prostate CA xenograft models with high (C4-2) and medium/low (22Rv1) PSMA protein levels at doses of 250 and 500 kBq/kg. Furthermore, statistically significant prevention of tumor growth was observed after treatment with PSMA-TTC at a dose of 100 kBq/kg in an orthotopic bone xenograft model (LNCaP-Luc). The promising preclinical antitumor activity of PSMA-TTC supports its development for the treatment of patients with metastatic prostate cancer. Citation Format: Stefanie Hammer, Aasmund Larssen, Christine Ellingsen, Solene Geraudie, Derek Grant, Baard Indrevoll, Oliver von Ahsen, Alexander Kristian, Urs B Hagemann, Jenny Karlsson, Roger M Bjerke, Olav B Ryan, Dominik Mumberg, Bertolt Kreft, Alan Cuthbertson. Preclinical pharmacology of the PSMA-targeted thorium-227 conjugate PSMA-TTC: a novel targeted alpha therapeutic for the treatment of prostate 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 5200. doi:10.1158/1538-7445.AM2017-5200

The 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 (Ca) types and is one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Several HER2-targeting antibodies as well as antibody-drug conjugates are either approved or are in clinical development. Prolonged treatment with monoclonal antibodies and antibody drug conjugates have resulted in development of resistance and so there is still an unmet medical need for drugs of new mechanism of action targeting this important receptor system. We describe herein the generation of a high energy, alpha-particle emitting HER2 targeted thorium-227 antibody-chelator conjugate. 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 thorium-227 (Th-227). 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 experiments were performed on 8 cell lines with different HER2 expression levels (from 7 000 - 500 000 mAbs bound/ cell as determined by FACS) of breast, ovarian, gastric and lung cancer origin. HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range. In vivo biodistribution and anti-tumor efficacy of HER2-TTC was evaluated in the dose range 100-500 kBq/kg at a protein dose of 0.14 mg/kg and i.v. injection in the s.c. KPL-4 breast and Calu-3 lung model previously described to be resistant to trastuzumab. The biodistribution study demonstrated specific tumor accumulation of HER2-TTC in both models with a maximum of 77 and 50 %ID/g 227Th at t = 168 h post dose (decay corrected to T0), respectively. Significant antitumor efficacy was shown for HER2-TTC in the JIMT-1 s.c. breast Ca xenograft model resistant to trastuzumab and T-DM1. The promising preclinical anti-tumor activity supports the development of the targeted alpha therapeutic HER2-TTC for the treatment of trastuzumab and T-DM1 resistant patients. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Alexander Kristian, Christine Ellingsen, Dessislava Mihaylova, Solene Geraudie, Bård Indrevoll, Uta Wirnitzer, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in preclinical models of trastuzumab and T-DM1 resistance [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 5859. doi:10.1158/1538-7445.AM2017-5859

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