◥Purpose: Prostate-specific membrane antigen (PSMA) is an attractive target for radionuclide therapy of metastatic castrationresistant 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 PSMApositive 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).

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

BackgroundCC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis.MethodologyUsing phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.SignificanceFor the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

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

<p>Figures and Figure Legends for Supplementary Figures 1, 2, 3, 4 and 5</p>