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Background: Patients with advanced-stage ovarian cancer face a poor prognosis because of recurrent peritoneal cavity metastases following surgery and chemotherapy. Alpha-emitters may enable the efficient treatment of such disseminated diseases because of their short range and highly energetic radiation. Radium-224 is a candidate α-emitter due to its convenient 3.6-day half-life, with more than 90% of the decay energy originating from α-particles. However, its inherent skeletal accumulation must be overcome to facilitate intraperitoneal delivery of the radiation dose. Therefore, 224Ra-labeled CaCO3microparticles have been developed. Objective: The antitumor effect of CaCO3microparticles as a carrier for 224Ra was investigated, with an emphasis on the ratio of activity to mass dose of CaCO3, that is, specific activity. Methods: Nude athymic mice were inoculated intraperitoneally with human ovarian cancer cells (ES-2) and treated with a single intraperitoneal injection of 224Ra-labeled CaCO3microparticles with varying combinations of mass and activity dose, or cationic 224Ra in solution. Survival and ascites volume at sacrifice were evaluated. Results: Significant therapeutic effect was achieved for all tested specific activities ranging from 0.4 to 4.6 kBq/mg. Although treatment with a mean activity dose of 1305 kBq/kg of cationic 224Ra prolonged the survival compared with the control, equivalent median survival could be achieved with 224Ra-labeled microparticles with a mean dose of only 420 kBq/kg. The best outcome was achieved with the highest specific activities (2.6 and 4.6 kBq/mg). Conclusion: Radium-224-labeled CaCO3microparticles present a promising therapy against cancer dissemination in body cavities.

Receptor-targeted image-guided Radionuclide Therapy (TRT) is increasingly recognized as a promising approach to cancer treatment. In particular, the potential for clinical translation of receptor-targeted alpha-particle therapy is receiving considerable attention as an approach that can improve outcomes for cancer patients. Higher Linear-energy Transfer (LET) of alpha-particles (compared to beta particles) for this purpose results in an increased incidence of double-strand DNA breaks and improved-localized cancer-cell damage. Recent clinical studies provide compelling evidence that alpha-TRT has the potential to deliver a significantly more potent anti-cancer effect compared with beta-TRT. Generator-produced 212Pb (which decays to alpha emitters 212Bi and 212Po) is a particularly promising radionuclide for receptor-targeted alpha-particle therapy. A second attractive feature that distinguishes 212Pb alpha-TRT from other available radionuclides is the possibility to employ elementallymatched isotope 203Pb as an imaging surrogate in place of the therapeutic radionuclide. As direct non-invasive measurement of alpha-particle emissions cannot be conducted using current medical scanner technology, the imaging surrogate allows for a pharmacologically-inactive determination of the pharmacokinetics and biodistribution of TRT candidate ligands in advance of treatment. Thus, elementally-matched 203Pb labeled radiopharmaceuticals can be used to identify patients who may benefit from 212Pb alpha-TRT and apply appropriate dosimetry and treatment planning in advance of the therapy. In this review, we provide a brief history on the use of these isotopes for cancer therapy; describe the decay and chemical characteristics of 203/212Pb for their use in cancer theranostics and methodologies applied for production and purification of these isotopes for radiopharmaceutical production. In addition, a medical physics and dosimetry perspective is provided that highlights the potential of 212Pb for alpha-TRT and the expected safety for 203Pb surrogate imaging. Recent and current preclinical and clinical studies are presented. The sum of the findings herein and observations presented provide evidence that the 203Pb/212Pb theranostic pair has a promising future for use in radiopharmaceutical theranostic therapies for cancer.

Prostate-specific membrane antigen (PSMA) is the most promising target for radioligand therapy of prostate cancer. The aim of this study was to prepare a small molecular ligand p-SCN-Bn-TCMC-PSMA (NG001) and compare it with the commonly used DOTA-based PSMA-617. The PSMA-targeting ability of the 212 Pb-labelled ligands was evaluated using PSMA-positive C4-2 human prostate cancer cells. Lead-212 is an in vivo generator of alpha particles by its daughter nuclides 212 Bi and 212 Po. NG001 was synthesized by conjugating the isothiocyanato group of p-SCN-Bn-TCMC to the amino group of a glutamate-urea-based PSMA-binding entity. Molecular size, chelator unit and chelator linking method are different in NG001 and PSMA-617. Both ligands were efficiently labelled with 212 Pb using a 224 Ra/212 Pb-solution generator in transient equilibrium with progeny. Lead-212-labelled NG001 was purified with a yield of 85.9±4.7% and with 0.7±0.2% of 224 Ra. Compared with [212 Pb]Pb-PSMA-617, [212 Pb]Pb-NG001 displayed a similar binding and internalization in C4-2 cells, with comparable tumour uptake in mice bearing C4-2 tumours, but almost a 2.5-fold lower kidney uptake. Due to the rapid normal tissue clearance and tumour cell internalization, any significant translocalization of 212 Bi was not detected in mice. In conclusion, the obtained results warrant further preclinical studies to evaluate the therapeutic efficacy of [212 Pb]Pb-NG001.

Simple Summary Currently, there is no treatment that can cure patients with late stage metastatic prostate cancer. Prostate-specific membrane antigen is a type of protein overexpressed on the membrane surface of most prostate cancer cells. The preclinical and clinical experiences in the rapidly evolving field of targeted alpha-particle radiation therapy for metastatic prostate cancer overexpressing prostate-specific membrane antigen are reviewed. Targeted alpha therapy employs radionuclides emitting highly energetic alpha-particles (cytotoxic payload) chelated to small molecules or monoclonal antibodies designed to target prostate-specific membrane antigen. In this review, we summarize the availability of therapeutic alpha-emitting radionuclides (terbium-149, astatine-211, bismuth-212 (lead-212), bismuth-213, radium-223, actinium-225, thorium-227), and the development of small molecules and antibodies targeting prostate-specific membrane antigen. The limitations in studies using animal models of human prostate cancer to accurately predict efficacy and toxicity in patients are addressed. We have attempted to also critically discuss hurdles related to logistical and supply aspects between different alpha-emitting prostate-specific membrane antigen-targeting radiopharmaceuticals. Lastly, we discuss the potentials, limitations, and future perspectives of prostate-specific membrane antigen-targeted alpha therapy. Abstract Bone, lymph node, and visceral metastases are frequent in castrate-resistant prostate cancer patients. Since such patients have only a few months’ survival benefit from standard therapies, there is an urgent need for new personalized therapies. The prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer and is a molecular target for imaging diagnostics and targeted radionuclide therapy (theragnostics). PSMA-targeted α therapies (PSMA-TAT) may deliver potent and local radiation more selectively to cancer cells than PSMA-targeted β− therapies. In this review, we summarize both the recent preclinical and clinical advances made in the development of PSMA-TAT, as well as the availability of therapeutic α-emitting radionuclides, the development of small molecules and antibodies targeting PSMA. Lastly, we discuss the potentials, limitations, and future perspectives of PSMA-TAT.

Lead-212 is recognized as a promising radionuclide for targeted alpha therapy for tumors. Many studies of 212Pb-labeling of various biomolecules through bifunctional chelators have been conducted. Another approach to exploiting the cytotoxic effect is coupling the radionuclide to a microparticle acting as a carrier vehicle, which could be used for treating disseminated cancers in body cavities. Calcium carbonate may represent a suitable material, as it is biocompatible, biodegradable, and easy to synthesize. In this work, we explored 212Pb-labeling of various CaCO3 microparticles and developed a protocol that can be straightforwardly implemented by clinicians. Vaterite microparticles stabilized by pamidronate were effective as 212Pb carriers; labeling yields of ≥98% were achieved, and 212Pb was strongly retained by the particles in an in vitro stability assessment. Moreover, the amounts of 212Pb reaching the kidneys, liver, spleen, and skeleton of mice following intraperitoneal (i.p.) administration were very low compared to i.p. injection of unbound 212Pb2+, indicating that CaCO3-bound 212Pb exhibited stability when administered intraperitoneally. Therapeutic efficacy was observed in a model of i.p. ovarian cancer for all the tested doses, ranging from 63 to 430 kBq per mouse. Lead-212-labeled CaCO3 microparticles represent a promising candidate for treating intracavitary cancers.

Alpha-particle emitting radionuclides continue to be the subject of medical research because of their high energy and short range of action that facilitate effective cancer therapies. Radium-224 (224Ra) is one such candidate that has been considered for use in combating micrometastatic disease. In our prior studies, a suspension of224Ra-labeled calcium carbonate (CaCO3) microparticles was designed as a local therapy for disseminated cancers in the peritoneal cavity. The progenies of224Ra, of which radon-220 (220Rn) is the first, together contribute three of the four alpha particles in the decay chain. The proximity of the progenies to the delivery site at the time of decay of the224Ra-CaCO3microparticles can impact its therapeutic efficacy. In this study, we show that the diffusion of220Rn was reduced in labeled CaCO3suspensions as compared with cationic224Ra solutions, both in air and liquid volumes. Furthermore, free-floating lead-212 (212Pb), which is generated from released220Rn, had the potential to be re-adsorbed onto CaCO3microparticles. Under conditions mimicking anin vivoenvironment, more than 70% of the212Pb was adsorbed onto the CaCO3at microparticle concentrations above 1 mg/mL. Further, the diffusion of220Rn seemed to occur whether the microparticles were labeled by the surface adsorption of224Ra or if the224Ra was incorporated into the bulk of the microparticles. The therapeutic benefit of differently labeled224Ra-CaCO3microparticles after intraperitoneal administration was similar when examined in mice bearing intraperitoneal ovarian cancer xenografts. In conclusion, both the release of220Rn and re-adsorption of212Pb are features that have implications for the radiotherapeutic use of224Ra-labeled CaCO3microparticles. The release of220Rn through diffusion may extend the effective range of alpha-particle dose deposition, and the re-adsorption of the longer lived212Pb onto the CaCO3microparticles may enhance the retention of this nuclide in the peritoneal cavity.

Lead-212 is a promising radionuclide for cancer therapy, but no primary

Objectives To investigate the therapeutic potential of the next-generation anti-CD37 radioimmunoconjugate 177 Lu-lilotomab satetraxetan (177 Lu-lilotomab) in combination with the anti-CD20 antibody rituximab for treatment of mice with non-Hodgkin's lymphoma (NHL) xenografts. Methods Nude mice with subcutaneous (s.c.) Burkitt's lymphoma Daudi xenografts and SCID mice intravenously (i.v.) injected with Mantle cell lymphoma Rec-1 cells were treated with either 177 Lu-lilotomab or rituximab alone or with the combination of both treatments. Tumour volume, body weight, blood counts and clinical status were monitored. CD20 expression was measured using flow cytometry with fluorescence-labelled rituximab. Results The combination of 177 Lu-lilotomab and rituximab was synergistic for treatment of nude mice with s.c. Daudi xenografts while it was additive for treatment of SCID mice with i.v. injected Rec-1 cells. Binding of rituximab to NHL cells in-vitro was increased by pretreatment with 177 Lu-lilotomab. Conclusions Treatment of mice with NHL xenografts with 177 Lu-lilotomab synergistically increased tumour suppression of subsequent anti-CD20 immunotherapy and improved survival. If the same effect is confirmed in a recently started clinical study, it could change the way radioimmunotherapy and CD20 immunotherapy would be used in the future.

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter 224 Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of 224 Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with 224 Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with 224 Ra - microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma.

Introduction Alpha-emitting radionuclides have gained considerable attention as payloads for cancer targeting molecules due to their high cytotoxicity. One attractive radionuclide for this purpose is 212 Pb, which by itself is a β-emitter, but acts as an in vivo generator for its short-lived α-emitting daughters. The standard method of preparing 212 Pb-labeled antibodies requires handling and evaporation of strong acids containing high radioactivity levels by the end user. An operationally easier and more rapid process could be useful since the 10.6h half-life of 212 Pb puts time constraints on the preparation protocol. In this study, an in situ procedure for antibody labeling with 212 Pb, using a solution of the generator nuclide 224 Ra, is proposed as an alternative protocol for preparing 212 Pb-radioimmunoconjugates. Methods Radium-224, the generator radionuclide of 212 Pb, was extracted from its parent nuclide, 228 Th. Lead-212-labeling of the TCMC-chelator conjugated monoclonal antibody trastuzumab was carried out in a solution containing 224 Ra in equilibrium with progeny. Subsequently, the efficiency of separating the 212 Pb-radioimmunoconjugate from 224 Ra and other unconjugated daughter nuclides in the solution using either centrifugal separation or a PD-10 desalting size exclusion column was evaluated and compared. Results Radiolabeling with 212 Pb in 224 Ra-solutions was more than 90% efficient after only 30min reaction time at TCMC-trastuzumab concentrations from 0.15mg/mL and higher. Separation of 212 Pb-labeled trastuzumab from 224 Ra using a PD-10 column was clearly superior to centrifugal separation. This method allowed recovery of approximately 75% of the 212 Pb-antibody-conjugate in the eluate, and the remaining amount of 224 Ra was only 0.9±0.8% (n=7). Conclusions The current work demonstrates a novel method of producing 212 Pb-based radioimmunoconjugates from a 224 Ra-solution, which may be simpler and less time-consuming for the end user compared with the method established for use in clinical trials of 212 Pb-TCMC-trastuzumab.

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with 212Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of 212Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of 212Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3–10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of 212Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of 212Pb-NG001.

Lead-212 is a promising radionuclide for cancer therapy, but no primary 212Pb activity standardization has been published. A need therefore exists for accurate estimation of injected doses of 212Pb activity in equilibrium with progeny, when it comes to preclinical and clinical trials. In this study, 212Pb activity was determined using a high purity germanium (HPGe) detector, which allowed the determination of geometry-specific calibration factors for commercially available reentrant ionization chambers (ICs) and sodium iodide (NaI) detectors.

To investigate the therapeutic potential of the next-generation anti-CD37 radioimmunoconjugateNude mice with subcutaneous (s.c.) Burkitt's lymphoma Daudi xenografts and SCID mice intravenously (i.v.) injected with Mantle cell lymphoma Rec-1 cells were treated with eitherThe combination ofTreatment of mice with NHL xenografts with

BACKGROUND/AIM Bone metastases are associated with increased morbidity and poor prognosis in a variety of cancers. The present study investigated the effects of targeted radionuclide therapy with α-emitting, bone-seeking radium-224 (224Ra) on osteolytic bone metastasis of MDA-MB-231(SA)-GFP human breast cancer cells injected intracardially into nude mice. MATERIALS AND METHODS Vehicle, ethylenediamine tetra (methylene phosphonic acid) (EDTMP) and 224Ra-solution (45, 91 or 179 kBq/kg) with EDTMP were intravenously administered to mice two days after cell injection. The bone-seeking EDTMP was added to the 224Ra-solution to improve bone targeting of 212Pb, which is a progeny of 224Ra. RESULTS Radium-224 solution treatment decreased in a dose-dependent manner the areas of osteolytic lesions in the hind limbs and the number of tumor foci in the whole skeleton, and extended survival. Paraplegia was not observed in 179 kBq/kg 224Ra-solution group. CONCLUSION Radium-224-solution containing chelated 212Pb is a promising candidate for the treatment of breast cancer patients with bone metastases.

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter 224Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of 224Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with 224Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with 224Ra-microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma.

Alpha-emitting radionuclides have gained considerable attention as payloads for cancer targeting molecules due to their high cytotoxicity. One attractive radionuclide for this purpose isRadium-224, the generator radionuclide ofRadiolabeling withThe current work demonstrates a novel method of producing

Introduction: Radium-224 (Ra-224) labeled calcium carbonate (CaCO3) microparticles were developed with the intent to treat micrometastases located in the abdominal cavity. The slowly degradable microparticles act as carriers for the α-emitter Ra-224 and ensure high intraperitoneal (IP) retention of the radioactivity without cellular targeting. This novel α-therapy has a short action range in tissue and is designed to confine the radiation exposure to the IP cavity, treating both linings of the peritoneal surfaces and liquid volumes. The distribution of the Ra-224 labeled microparticles was examined by planar gamma imaging, SPECT and CT. The ex vivo biodistribution of Ra-224 and its progeny Pb-212 was also determined. Experimental procedures: Fifteen female Wistar rats were infused IP with Ra-224-labeled microparticles (200-400 kBq, 100 mg CaCO3). The injections were performed by use of a perforated catheter (pigtail catheter), followed by a Plasmalyte flush to mimic the planned clinical administration. The Ra-224 labeled microparticles were imaged using both planar gamma imaging, SPECT and CT to evaluate the distribution over time in the abdominal region. Ex vivo biodistribution was performed 2, 24, 96, 168 and 336 hours after particle infusion and organs were harvested for activity measurements. The activity was determined at 2 hours and 48 hours after harvesting for all samples. This allowed differentiation between the biodistribution of Ra-224 and the biodistribution of its progeny Pb-212. Furthermore, 4 rats were injected IP with free Ra-224 and 2 rats were injected intravenously with free Ra-224 to achieve a baseline for the biodistribution. These control animals were euthanized at 24 and 168 hours, and 168 hours, respectively. Results: Based on both CT and SPECT images, the particles distributed to the entire peritoneum, albeit with local areas of high microparticle concentration. Over the course of the experiment there was, as expected, a modest but evident systemic leakage of Ra-224 from the particles in the peritoneal cavity. The amount was estimated by the bone uptake of radioisotopes Pb-212 and Ra-224, using IP administered free Ra-224 as reference. The retention of Ra-224 in the peritoneal cavity was found to be > 87% at 24 hours and > 77% at 168 hours. Redistribution of the progeny Pb-212 was observed as modest uptake in the kidneys. Conclusions: High peritoneal retention of both Ra-224 and its progeny Pb-212 after IP injection of Ra-224 labeled CaCO3 microparticles, was found in rats with high translational value to the clinical setting. SPECT imaging supported distribution of the Ra-224 labeled microparticles to the entire peritoneal lumen in the animals. SPECT and CT revealed some clusters of labeled microparticles. Due to the short range of the therapeutic relevant α-particles, the clusters are not expected to have an impact on therapeutic or safety aspects. Citation Format: Jesper Fonslet, Carsten H. Nielsen, Lotte K. Kristensen, Ida S. Jorstad, Kim Lindland, Roy H. Larsen, Øyvind S. Bruland, Andreas Kjaer, Tina B. Bønsdorff. Biodistribution of Ra-224 and its daughter Pb-212 after intraperitoneal infusion of Ra-224 labeled microparticles in rats [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 3922.

The biodistribution of the anti-CD37 radioimmunoconjugate (177)Lu-tetraxetan-tetulomab ((177)Lu-DOTA-HH1) was evaluated. Biodistribution of (177)Lu-tetraxetan-tetulomab was compared with (177)Lu-tetraxetan-rituximab and free (177)Lu in nude mice implanted with Daudi lymphoma xenografts. The data showed that (177)Lu-tetulomab had a relevant stability and tumor targeting properties in the human lymphoma model. The half-life of (177)Lu allowed significant tumor to normal tissue ratios to be obtained indicating that (177)Lu-tetraxetan-tetulomab could be suitable for clinical testing. The biological and effective half-life in blood was higher for (177)Lu-tetraxetan-tetulomab than for (177)Lu-tetraxetan-rituximab. The biodistribution of (177)Lu-tetraxetan-tetulomab did not change significantly when the protein dose was varied from 0.01 to 1 mg/kg. Dosimetry calculations showed that the absorbed radiation doses to normal tissues and tumor in mice were not significantly different for (177)Lu-tetraxetan-tetuloma b and (177)Lu-tetraxetan-rituximab. The absorbed radiation doses were extrapolated to human absorbed radiation doses. These extrapolated absorbed radiation doses to normal tissues for (177)Lu-tetraxetan-tetulomab at an injection of 40 MBq/kg were significantly lower than the absorbed radiation doses for 15 MBq/kg Zevalin, suggesting that higher tumor radiation dose can be reached with (177)Lu-tetraxetan-tetulomab in the clinic.

The anti-CD20 antibody rituximab labelled with the alpha-particle-emitting radionuclide (227)Th is of interest as a radiotherapeutic agent for treatment of lymphoma. Complete regression of human lymphoma Raji xenografts in 60% of mice treated with 200 kBq/kg (227)Th-rituximab has been observed. To evaluate possible late side effects of (227)Th-rituximab, the long-term radiotoxicity of this potential radiopharmaceutical was investigated.BALB/c mice were injected with saline, cold rituximab or 50, 200 or 1,000 kBq/kg (227)Th-rituximab and followed for up to 1 year. In addition, nude mice with Raji xenografts treated with various doses of (227)Th-rituximab were also included in the study. Toxicity was evaluated by measurements of mouse body weight, white blood cell (WBC) and platelet counts, serum clinical chemistry parameters and histological examination of tissues.Only the 1,000 kBq/kg dosage resulted in decreased body weight of the BALB/c mice. There was a significant but temporary decrease in WBC and platelet count in mice treated with 400 and 1,000 kBq/kg (227)Th-rituximab. Therefore, the no-observed-adverse-effect level (NOAEL) was 200 kBq/kg. The maximum tolerated activity was between 600 and 1,000 kBq/kg. No significant signs of toxicity were observed in histological sections in any examined tissue. There were significantly (p0.05), but transiently, higher concentrations of serum bile acids and aspartate aminotransferase in mice treated with either (227)Th-rituximab or non-labelled antibody when compared with control mice. The maximum tolerated dose to bone marrow was between 2.1 and 3.5 Gy.Therapeutically relevant dose levels of (227)Th-rituximab were well tolerated in mice. Bone marrow suppression, as indicated by decrease in WBC count, was the dose-limiting radiotoxicity. These toxicity data together with anti-tumour activity data in a CD20-positive xenograft mouse model indicate that therapeutic effects could be obtained with relatively safe dosage levels of the radioimmunoconjugate.

The radiobiological and radiochemical properties of radium-223 (223Ra, T1/2 = 11.4 d) render this alpha-emitting radionuclide promising for targeted cancer therapy. Together with its short-lived daughters, each 223Ra decay produces four alpha-particle emissions which enhance therapy effectiveness at the cellular level. In this paper, we review the recently published data reported for pre-clinical and clinical use of 223Ra in cancer treatment. We have evaluated two distinct chemical forms of 223Ra in vivo: 1) cationic 223Ra as dissolved RaCl2, and 2) liposome-encapsulated 223Ra. Cationic 223Ra seeks metabolically active osteoblastic bone and tumor lesions with high uptake and strong binding affinity based on its similarities to calcium. Based on these properties, we have advanced the clinical use of 223Ra for treating bone metastases from breast and prostate cancer. The results show impressive anti-tumor activity and improved overall survival in hormone-refractory prostate cancer patients with bone metastases. In other studies, we have evaluated the biodistribution and tumor uptake of liposomally encapsulated 223Ra in mice with human osteosarcoma xenografts, and in dogs with spontaneous osteosarcoma and associated soft tissue metastases. Results indicate excellent biodistributions in both species. In dogs, we found considerable uptake of liposomal 223Ra in cancer metastases in multiple organs, resulting in favorable tumor- to-normal soft tissue ratios. Collectively, these findings show an outstanding potential for 223Ra as a therapeutic agent.

International audience; 177Lu-DOTA-HH1 (177Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of 177Lu-HH1, 177Lu-DOTA-rituximab (177Lu-rituximab) and non-specific 177Lu-DOTA-IgG1 (177Lu-IgG1) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg 177Lu-HH1 as compared with mice treated with similar activities of 177Lu-rituximab or non-specific 177Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of 177Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg 177Lu-rituximab experienced severe radiation toxicity. The retention of 177Lu-rituximab in organs of the mononuclear phagocyte system was longer than for 177Lu-HH1, which explains the higher toxicity observed in mice treated with 177Lu-rituximab. In vitro internalization studies showed that 177Lu-HH1 internalizes faster and to a higher extent than 177Lu-rituximab which might be the reason for the better therapeutic effect of 177Lu-HH1.

The bone-seeking, α-particle-emitting radiopharmaceutical Alpharadin, 223RaCl2 (half-life = 11.4 days), is under clinical development as a novel treatment for skeletal metastases from breast and prostate cancer. This article summarizes the current status of preclinical and clinical research on 223RaCl2. Potential advantages of 223Ra to that of external beam irradiation and registered β-emitting bone seekers are discussed. Published data of 223Ra dosimetry in mice and a therapeutic study in a skeletal metastases model in nude rats have indicated significant therapeutic potential of bone-seeking α-emitters. This article provides short-term and long-term results from the first clinical single dosage trial. We also present data from a repeated dosage study of five consecutive injections of 50 kBq/kg body weight, once every 3rd week, or two injections of 125 kBq/kg body weight, 6 weeks apart. Furthermore, interim results are described for a randomized phase 2 trial involving 64 patients with hormone-refractory prostate cancer and painful skeletal metastases who received four monthly injections of 223Ra or saline as an adjuvant to external beam radiotherapy. Lastly, we present preliminary dose estimates for 223Ra in humans. Results indicate that repeated dosing is feasible and toxicity is low, and that opportunities are available for combined treatment strategies.

Purpose: The main goals were to study the safety and tolerability of the α-emitter radium-223 (223Ra) in breast and prostate cancer patients with skeletal metastases. In addition, pain palliation was evaluated. Experimental Design: Fifteen prostate and 10 breast cancer patients enrolled in a phase I trial received a single i.v. injection of 223Ra. Five patients were included at each of the dosages: 46, 93, 163, 213, or 250 kBq/kg and followed for 8 weeks. Palliative response was evaluated according to the pain scale of the European Organization for Research and Treatment of Cancer QLQ C30 questionnaire at baseline and at 1, 4, and 8 weeks after injection. Results: Weekly blood sampling during follow-up revealed mild and reversible myelosuppression with nadir 2 to 4 weeks after the injection. Importantly, for thrombocytes only grade 1 toxicity was reported. Grade 3 neutropenia and leucopenia occurred in two and three patients, respectively. Mild, transient diarrhea was observed in 10 of the 25 patients. Nausea and vomiting was more frequently observed in the highest dosage group. Serum alkaline phosphatase decreased with nadir averages of 29.5% in females and 52.1% in males. Pain relief was reported by 52%, 60%, and 56% of the patients after 7 days, 4, and 8 weeks, respectively. 223Ra cleared rapidly from blood and was below 1% of initial level at 24 hours. Gamma camera images indicated, in accordance with pretreatment 99mTc-MDP scans, accumulation of 223Ra in skeletal lesions. Elimination was mainly intestinal. Median survival exceeded 20 months. Conclusions: 223Ra was well tolerated at therapeutically relevant dosages. Phase II studies have therefore been initiated.

The alpha-particle-emitting radionuclide 223Ra (t(1/2) = 11.4 d) is of interest for use in targeted radionuclide therapy. In order to provide radium-labeled monoclonal antibodies, the development of a chelator binding radium in a stable fashion is required. As a part of the search for potentially useful radium chelators, the relative stability of 223Ra-chelates with linear and cyclic chelating agents was evaluated by means of competition extraction experiments.

We herein report a comparison of the radiolabels 177Lu and 125I bound to the monoclonal antibody HH1 that targets the CD37 antigen expressed on non-Hodgkin B-cell lymphomas.Mixtures of 177Lu and 125I-labeled HH1 antibody were co-injected into nude mice carrying Ramos xenografts and the biodistribution using the paired label format allowing tracer comparisons in each individual mouse.Products of the two radionuclides had very similar immunoractivity in vitro but showed different properties in vivo. Both products had relevant stability in blood and most normal tissues in nude mice carrying subcutaneous Ramos xenografts. However, both the tumor uptake and retention were significantly higher for 177Lu vs. 125I labeled HH1. The tumor to normal tissue ratios were several-fold improved for 177Lu compared to radioiodine labeled antibodies.The data presented herein support the evaluation of CD37 as a target for clinical 177Lu-based radioimmunotherapy against b-cell malignancies.

Betalutin (177Lu-satetraxetan-lilotomab) is an antibody radionuclide conjugate (ARC) in development for the treatment of Non-Hodgkin's lymphoma (NHL). The FACT-Lym is a validated health related quality of life instrument specifically designed to assess lymphoma patients. This questionnaire has been used previously to demonstrate significant reductions in the quality of life of patients undergoing treatment, likely as a result of treatment related toxicities (Hlubocky FJ, et al, Lymphoma 2013). Methods Patients with relapsed incurable NHL of follicular grade I-IIIA, marginal zone, mantle cell, lymphoplasmacytic and small lymphocytic subtypes who had received at least one prior treatment regimen were eligible for enrolment. All patients received a single dose of 15 MBq/kg of 177Lu-satetraxetan-lilotomab. The FACT-Lym instrument used in the study has 42 questions grouped into 5 subscales (physical, social/family, emotional and functional well-being and additional concerns). Questionnaires were completed by all subjects in the phase 2 stage of the study during screening, at month 3 and month 12. Responses were categorised on a 5-point Likert scale from "0- not at all" to "4-very much" with a higher score indicating a better quality of life. The analysis is conducted as part of a protocol specified interim analysis of efficacy, safety and quality of life. Results A total of 36 patients have been enrolled into this phase 1/2 study of which 15 patients who have undergone treatment in the phase 2 part are included in the current analysis. The median age of the patients was 68 years and the median number of prior regimens was 2 (range: 1-9). All 15 subjects have completed the screening assessment and 7 patients have completed the 3 month assessment, data from all subjects at both screening and the month 3 assessment will be presented at the annual meeting. To date 907 responses to questions have been collected including 622 questions at screening and 285 responses at 3 months post-treatment. At enrolment patients overall had a good health-related quality of life with a median baseline score of 121 and this was similar at 3 months post-treatment (median score: 132, p-value: 0.558). This maintenance of the patients quality of life was achieved while 5/7 patients (71%) showed a complete or partial response to treatment. Of the 7 patients who have so far completed the month 3 assessment, 3 had grade 3 thrombocytopenia or neutropenia (none had grade 4), the quality of life assessment of these patients (median score: 158) was similar to or higher than the 4 patients who did not have grade 3 thrombocytopenia/neutropenia (median score: 110). This indicates no negative impact on the health related quality of life when patients experienced haematological side effects associated with 177Lu-satetraxetan-lilotomab. When the 3-month assessment has been completed by all patients a subscale analysis and a correlation with ECOG performance status will be performed. Conclusion The health related quality of life as measured by the validated FACT-Lym questionnaire was maintained following treatment with Betalutin, while also achieving a durable tumour response in a significant proportion of the patients assessed. The treatment related hematological side effects experienced by patients were modest and appeared not to have a negative effect on the quality of life. Further analysis is required to confirm these findings when the 3 month assessment has been completed by all patients. Disclosures Kolstad: Nordic Nanovector: Other: Membership of Scientific Advisory Board. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership. Baylor Curtis:Nordic Nanovector: Employment. Østengen:Nordic Nanovector: Employment. Turner:Nordic Nanovector: Employment. Hartvig Larsen:Nordic nanovector: Equity Ownership. Holte:Amgen: Research Funding; Mundipharma: Research Funding.

Aim: To investigate the therapeutic effect of combined treatment with the anti-CD37 antibody radionuclide conjugate (ARC) 177Lu-satetraxetan-lilotomab (177Lu-p-SCN-Bn-DOTA-HH1, tradename Betalutin®, short name 177Lu-lilotomab) and the anti-CD20 immunotherapy with rituximab in a subcutaneous (s.c.) preclinical model of non-Hodgkin lymphoma (NHL). These studies build on previously presented data that showed that treatment with 177Lu-lilotomab increased binding of rituximab in NHL cells in-vitro and in-vivo. Materials and Methods:The therapeutic effect of the combination of 177Lu-lilotomab and rituximab was studied in nude mice with s.c. Burkitt's lymphoma (Daudi) xenografts using 250 MBq/kg 177Lu-lilotomab, 1 dose of 40 mg/kg rituximab, 4 doses of 10 mg/kg rituximab orNaCl. Treatmentswere given every 3 to 4 days (Table 1). Nine to 10 mice bearinga total of 16 to 18 tumors per group were used in the study. Tumor volume was measured every 2-3 days and weekly after study day 100. The therapeutic effect of the combination treatmentswas compared with the therapeutic effect of each of the treatments alone using Log-Rank and clustered cox-proportional hazards tests. Results:The effect of the combination treatment in mice with s.c. xenografts was better than that of each treatment alone (Figure 1 & 2). The median survival of mice given the combination treatment was longer (>157 days) than each of the treatments alone (31-60 days) and of the sum of both treatments alone (91-100 days), but the difference was not statistically significant (Table 2). The study is currently on-going and none of the mice given the combination treatment have any palpable tumors, so it is expected that the median survival of those mice eventually will double the sum of both treatments alone. Median time to quadruplicate tumor volume was 2 times longer for the combination treatments (>157 days) than in any of the treatments alone (25-49 days) and longer than the sum of both treatments alone (74-82 days) (Table 2). When using time to quadruplicate tumor volume as end-point, the 177Lu-lilotomab + 4 x rituximab combination was significantly different from either treatment alone (p < 0.01, clustered cox-proportional hazards model). Moreover, the median time to complete remission of tumors in mice given the combination treatment was 5 times shorter (13 days) than each of the treatments alone (>66 days) (p Conclusion:Treatment of NHL in-vitro and in-vivo with 177Lu-lilotomab results in an increased binding, tumor uptake and tumor suppression of anti-CD20 immunotherapy. If the same effect is confirmed in clinical studies, it could change the way ARC and CD20 immunotherapy would be used in the future. Disclosures Repetto-Llamazares: Nordic Nanovector ASA: Employment, Equity Ownership. Larsen:Nordic Nanovector ASA: Equity Ownership. O'Shea:Nordic Nanovector ASA: Employment, Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership.

177Lu-Satetraxetan-lilotomab (Betalutin®) is a novel CD37-binding IgG1 antibody labelled with the beta-emitter lutetium-177, in a ready-to-use formulation currently in Phase 1/2 clinical development for the treatment of Non-Hodgkins lymphoma. CD37 is an internalizing transmembrane antigen highly expressed on most B-cell malignancies, and is a promising therapeutic target. The optimisation of the pre-dosing regimen prior to administration of 177Lu-Satetraxetan-lilotomab may result in an improved safety and efficacy profile. The phase 1 stage of this study is designed with 4 arms to test different pre-dosing regimens (no pre-dosing, rituximab and two doses of lilotomab) on the effect of 177Lu-Satetraxetan-lilotomab. Methods: Patients with relapsed incurable NHL of follicular grade I-IIIA, marginal zone, mantle cell, lymphoplasmacytic and small lymphocytic subtypes and with platelet counts ≥ 150 x109/l were eligible for inclusion in the study. All patients received pre-treatment with rituximab (375 mg/m2) to deplete peripheral B cells and improve biodistribution of the labelled antibody. Pre-dosing with lilotomab (cold anti-CD37 antibody) was given in arms 1 and 4 or rituximab in arm 3, within 4 hours of 177Lu-Satetraxetan-lilotomab to block binding in the non-tumour tissue. The pre-treatment and pre-dosing regimen used in each arm is summarised below: Arm 1: rituximab day1 and 8, lilotomab (40 mg) plus Betalutin day 29 Arm 2: rituximab day 1 and 8, Betalutin day 29 Arm 3: rituximab day 1, rituximab plus Betalutin day 15 Arm 4: rituximab day 1, lilotomab (100 mg/m2) plus Betalutin day 15 The starting dose for Arm 1 was10 MBq/kg and was 15 MBq/kg for arm 2, 3 and 4 and in phase 2. Response was assessed by FDG PET/CT scans at 3 and 6 months post-treatment and then by CT scan up to 5 years after treatment. The results of the protocol specified interim analysis will be presented. Results: A total of36 patients have been enrolled into study, of which 24 are currently evaluable. Patients enrolled into the study had either follicular (n=20), mantel cell (n=2) or marginal zone (n=2) lymphoma. The number of prior therapies ranged from 1 to 8. The efficacy and safety results from patients enrolled into Arms 3 and 4 and treated with two different pre-dosing regimens will be presented for the first time. The most common toxicities observed were hematologic with all dose limiting toxicities (DLTs) being reversible and manageable and related to thrombocytopenia and neutropenia. At a dose of 15 MBq/kg, pre-dosing with 40 mg of lilotomab (Arm 1 and phase 2) reduced the incidence of hematological DLTs to 14% (2/14 patients) compared with 100% (2/2 patients) with no pre-dosing in Arm 2. No DLTs have been reported at 10 MBq/kg in either Arm 1 or 2. Fourteen serious adverse events (SAEs) were reported by 8 patients: Atrial fibrillation (n=2) and platelet count decreased (n=2) were the only SAEs reported by more than one patient. There have been no deaths and no secondary malignancies or other long-term safety events. The overall tumor response rate observed in 23 patients evaluable for efficacy was 57%, comprising 7/23 complete responses, 6/23 partial responses, 5/23 stable disease and 5/23 with progressive disease. In addition, one patient had a confirmed transformed lymphoma at 3 months. One patient is still in remission more than 3 years after treatment and two further patients are still in remission more than 2 years after treatment. Conclusions: Betalutin has the potential to be a novel, safe and effective therapy for B-cell malignancies with durable responses. Betalutin, a single dose, ready-to-use formulation, has a predictable and manageable safety profile which is improved by pre-dosing. Most AEs were haematological, all transient and reversible. Disclosures Kolstad: Nordic Nanovector: Other: Membership of Scientific Advisory Board. Illidge:Nordic Nanovector: Consultancy. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership. Baylor Curtis:Nordic Nanovector: Employment. Østengen:Nordic Nanovector: Employment. Turner:Nordic Nanovector: Employment. Hartvig Larsen:Nordic nanovector: Equity Ownership. Holte:Mundipharma: Research Funding; Amgen: Research Funding.

To minimize nontarget organ uptake in animals receiving radiolabeled amidobisphosphonates, the influence of pretreatment with cold 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APB, pamidronate) was studied. Three groups of animals were given pure 3-[125I]iodobenzamide-N-3-hydroxypropylidene-3,3-bisphosphonate (IBPB) and 3-[211At]astatobenzamide-N-3-hydroxypropylidene-3,3-bisphosphonate (ABPB) (control); co-injection of APB and IBPB/ABPB; and 1 h preinjection of APB followed by IBPB/ABPB, respectively. A significant reduction of uptake in normal tissue was observed, whereas the bone uptake remained constant at 35–50%ID/g tissue. This study suggests that co- or preinjection of pamidronate may reduce the normal organ radiation doses when using these radiohalogenated bisphosphonates for endoradiotherapeutic procedures.

A new bisphosphonate was synthesised using N-succinimidyl-3-[ 125 I]iodobenzoate and 3-amino-1-hydroxypropylidene as the reagents. After purification by reverse phase chromatography the product, 3-[ 125 I]iodobenzamide-N-3-hydroxypropylidene-3,3-bisphosphonate, was injected intravenously into nude mice and the radioactivity content in various normal tissues was measured. Both at 3 h and at 17 h after injection a highly selective bone uptake was observed. Average ratios femur/blood and skull/blood were 35.9 and 36.6 at 3 h and 111.6 and 128.0 at 17 h, respectively. While clearing was observed for all other tissues, the bone radioactivity content did not decrease significantly from 3 h to 17 h after injection, indicating a high stability of this bisphosphonate in bone tissue. This preliminary study implies that pharmacokinetics, biological activity and clinical applications of amidebisphosphonates in general, and the described compound in particular, should be further evaluated.

Normal tissue accumulation of 211 At must be minimized during targeted radiotherapy with 211 At-labeled compounds. Therefore, we investigated the ability of seven compounds to block normal organ uptake of [ 211 At]astatide in mice: potassium iodide, sodium thiocyanate, sodium perchlorate, sodium periodate, cysteine, 2,3-dimercapto-1-propanesulfonic acid, and meso -2,3-dimercaptosuccinic acid. The monovalent anions I − , SCN − , and ClO 4 − reduced 211 At uptake in stomach and thyroid, while thiocyanate and cysteine were the only compounds to significantly reduce activity levels in lungs and spleen. This study suggests that blocking agents may help reduce normal organ radiation doses in endoradiotherapeutic procedures with 211 At-labeled radiopharmaceuticals.

A paired-label study was performed in athymic mice bearing subcutaneous D-54 MG human glioma xenografts to compare the localization of human/mouse anti-tenascin chimeric antibody 81C6 labeled by reaction with N-succinimidyl 3-[211At]astatobenzoate and N-succinimidyl 3-[131I]iodobenzoate. Over the 48-h observation period, the distribution of 211At- and 131I-labeled antibody were quite similar in tumor and normal tissues except stomach. These data were used to calculate human radiation doses for both intravenously and intrathecal administered 211At-labeled chimeric 81C6 using a quality factor of 5 for alpha-emissions.

Radium-223 dichloride selectively targets osteoblastic bone metastases with high-energy, short-range alpha-particles following intravenous injection. It is the first targeted alpha-emitter to improve overall survival in randomized phase 2 and 3 studies and provides a novel treatment option with favorable safety profile in castrate-resistant prostate cancer patients with bone metastases.

The α-particle emitter 211 At was covalently coupled to 1.8 μm aminated monodisperse polymer particles (MDPP) and used to irradiate the intraperitoneal cavity in mice with disseminated tumour cells. Specific activity has previously been shown to influence the therapeutic efficacy of α-particle emitting compounds and the therapeutic efficacy of 211 At-MDPP with various specific activity was therefore investigated. Groups of mice (10 animals per group) were treated with intraperitoneal injections of 100 kBq of 211 At-MDPP with specific activities of 0.19, 0.55, 1.7, 5.0, IS, and 45 MBq/mg. A significantly prolonged survival was observed in the treated groups compared to the control group (from 19 to 26 days vs. 12 days, median). The difference in survival between the 211 At-MDPP treated groups was not significant, but some animals with short survival were observed in the groups that had received the 0.19, 15 and 45 MBq/mg preparations. K13 monoclonal antibody values, which are an indicator of tumour growth, were high in some animals in the 15 and 45 MBq/mg groups (day 7 values).

The monoclonal antibody against CD20, rituximab, alone, or as part of combination therapies, is standard therapy for non-Hodgkin's B-cell lymphoma. Despite significantly better clinical results obtained for beta-emitting radioimmunoconjugates (RICs), RICs targeting CD20 are not commonly used in medical practice, partly because of competition for the CD20 target. Therefore, novel therapeutic approaches against other antigens are intriguing. Here, the binding properties of a novel antibody against CD37 (tetulomab) were compared with those of rituximab. The therapeutic effect of (177)Lu-tetulomab was compared with (177)Lu-rituximab on Daudi cells in vitro. The biodistribution, therapeutic and toxic effects of (177)Lu-tetulomab and unlabeled tetulomab were determined in SCID mice injected with Daudi cells. The affinity of tetulomab to CD37 was similar to the affinity of rituximab to CD20, but the CD37-tetulomab complex was internalized 10-times faster than the CD20-rituximab complex. At the same concentration of antibody, (177)Lu-tetulomab was significantly more efficient in inhibiting cell growth than was (177)Lu-rituximab, even though the cell-bound activity of (177)Lu-rituximab was higher. Treatment with 50 and 100 MBq/kg (177)Lu-tetulomab resulted in significantly increased survival of mice, compared with control groups treated with tetulomab or saline. The CD37 epitope recognized by tetulomab was highly expressed in 216 out of 217 tumor biopsies from patients with B-cell lymphoma. This work warrants further pre-clinical and clinical studies of (177)Lu-tetulomab.

The potential of delivering a high and localized radiation dose using α-particle-emitting radioimmunoconjugates (RIC's) is investigated as treatment for some types of cancer. High specific activity of the RIC may for some uses be desirable, allowing enrichment of the radionuclide on membrane antigens on target cells. Radiation exposure during the radiolabelling procedures may, however, reduce the antigen binding ability of RIC's of high specific activity. The influence of α-particle dose on the cell binding fraction was therefore investigated for two monoclonal antibodies, i.e., TP-1 F(ab') 2 and TP-3 IgG. Samples of 125 I-labelled MoAb were added different amounts of [ 211 At]astatide to give accumulated doses ranging from 50 to 50,000 Gy. After the 211 At had decayed 15 half-lives, the cell binding fractions were measured. The results show that [ 211 At] astatide activities up to 25 kBq/μl, corresponding to an accumulated α-particle radiation dose of approximately 1,000 Gy, could be tolerated without significantly reducing the cell binding fraction, whereas higher doses gradually reduced the immunoreactivity. Although there may be differences in dose sensitivity of different radioimmunoconjugate due to differences in radionuclide conjugation methods, size of the antibody and structure of the antigen binding portion, the results presented here indicate that it is possible to produce radioimmunoconjugates of high specific activity with currently used methods of preparation. The potential problem of radiolysis should, however, be taken into consideration when planning procedures for preparation and use of α-particle-emitting radioimmunoconjugates.

Background Osteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo. Methods and Results A murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant. Conclusion Our results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies.

CD37 is an internalizing transmembrane antigen highly expressed on most B-cell malignancies, and is a promising therapeutic target. Betalutin® is a novel CD37-binding murine IgG1 antibody (HH1) labelled with the beta-emitter lutetium-177, in a ready-to-use formulation currently in Phase 1/2 clinical development. Following the completion of recruitment into Arms 1 & 2 of this phase I/II study, efficacy and safety data from patients (pts) receiving Betalutin are reported. Methods: Patients with relapsed incurable NHL of follicular grade I-IIIA, marginal zone, mantle cell, lymphoplasmacytic and small lymphocytic subtypes and with platelet counts ? 150 x109/l were eligible for inclusion in the study. Pts received rituximab (375 mg/m2) on day 1 and 8 to deplete normal B cells. On day 29 pre-dosing with 50mg HH1 (cold CD37 antibody) was administered before Betalutin injection (Arm 1 and phase 2). In Arm 2 Betalutin was administered without HH1 pre-dosing on day 29. The starting doses for Arm 1 and 2 were 10 MBq/kg and 15 MBq/kg respectively. Response was assessed by FDG PET/CT scans. Results: A total of 18 evaluable pts were enrolled into Arm 1/phase II (n = 15) and Arm 2 (n = 3) with a median age of 68 years and either follicular lymphoma (n = 17), or mantle cell lymphoma (n = 1). The number of prior therapies ranged from 1 to 8. An additional, 3 pts have been enrolled in the phase 2 part of the study, data from these pts will also be presented. The most common toxicities observed were hematologic with all DLTs being reversible and manageable. In Arm 1, at 20 MBq/kg (n = 3), G 3/4 neutropenia and/or thrombocytopenia were observed in all pts. Platelet transfusions were given to 2 pts. At 15 MBq/kg (n = 6), 2 DLTs were observed: one G 3 thrombocytopenia lasting >14 days and one G 4 neutropenia/ thrombocytopenia lasting >7 days. Both DLTs recovered without intervention. In Arm 2 with 15 MBq/kg (n = 2) G4 thrombocytopenia was observed in both pts and one patient also had G4 neutropenia lasting >7 days. This pt was hospitalised due to sepsis. No DLTs have been reported at 10 MBq/kg in either arm. Eleven serious adverse events (SAEs) were reported by 8 pts: Atrial fibrillation (n = 2) was the only SAE occurring in more than one pt (at 15 MBq/kg in Arm 1). No secondary malignancies or other long-term events were observed. The overall tumor response rate observed in 17 evaluable pts was 65%, comprising 4/17 complete responses, 7/17 partial responses, 3/17 stable disease and 3/17 progressive disease. In addition, one pt had a confirmed transformed lymphoma at 3 months. One pt is still in remission 3 years after treatment. Conclusions: Betalutin, a single dose, ready-to-use formulation, has a predictable and manageable safety profile. Most AEs were haematological, all transient and reversible. Promising efficacy and durable responses were observed. Betalutin has the potential to be a novel, safe and effective therapy for B-cell malignancies. Citation Format: Arne Kolstad, Ulf Madsbu, Matthew Beasley, Michael Bayne, Caroline Stokke, Tore Bach-Gansmo, Ayca Muftuler Løndalen, Jon Erik Holtedahl, Mona Elisabeth Revheim, Øyvind Bruland, Jostein Dahle, Laurie Baylor Curtis, Åse Østengen, Simon Turner, Nils Bolstad, Roy H. Larsen, Signe Spetalen, Martin Erlanson, Stine Nygaard Rudå, Harald Holte. Efficacy and safety results of Betalutin® (177Lu-DOTA-HH1) in a phase I/II study of patients with non-hodgkin B-cell lymphoma (NHL). [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 LB-252.

Immunotherapy (IT) with the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy has resulted in significantly improved response rate and survival in patients with various types of CD20 positive B-cell lymphoproliferative disorders. To be effective, rituximab depends on selective expression of a sufficient number of CD20 antigens per cell. Treatment with rituximab alone or in combination with chemotherapy can, however, result in disappearance of the CD20 expression, which may result in reduced clinical effect of subsequent CD20 targeted treatments. We have discovered that treatment of NHL in vitro and in vivo with the anti-CD37 antibody radionuclide conjugate (ARC) 177Lu-DOTA-HH1 (177Lu-HH1 or Betalutin™) results in an upregulation of the CD20 antigen expression, and therefore represents a rationale for a combination treatment with both agents. The in vitro expression of CD20 in Burkitt's Lymphoma, Daudi, cells 1-7 days after treatment with 177Lu-HH1 increased up to 120 % when compared with cells treated with unlabeled mAb, while Ramos (Burkitt's Lymphoma) and Rec-1 (Mantle Cell Lymphoma) cells showed 10 to 30 % increase, indicating a variation of the antigen upregulation in vitro with different cell lines. An upregulation of CD20 at the same order of magnitude was observed when cells where treated with similar absorbed radiation doses of external beam radiation. Treatment of nude mice with Ramos xenografts with 177Lu-HH1 resulted in a 3 times higher uptake of radiolabeled rituximab in tumor xenografts 5 days after start of treatment than in mice treated with unlabeled HH1 (p < 0.05) while uptake in normal organs was similar in both treatment groups (p > 0.05). SCID mice with intravenously injected Rec-1 cells were treated with NaCl, 100 mg rituximab, 40 MBq/kg 177Lu-HH1 or with the combination of 40 MBq/kg 177Lu-HH1 followed with 100 mg rituximab 5 days later. The combination of 177Lu-HH1 and rituximab resulted in significantly improved survival as compared with NaCl or rituximab alone, and a strong therapeutic gain as compared with 177Lu-HH1 alone (Table 1). In conclusion, 177Lu-HH1 treatment seems to improve uptake of rituximab and increase tumor suppression when used prior to anti-CD20 monoclonal antibody targeting in preclinical models. The reason for the upregulation of CD20 is probably related to the oxidative stress induced by the ARC-treatment, which will be evaluated in further studies. If the upregultation of CD20 is confirmed in clinical studies this effect could affect the way ARC and CD20 immunotherapy would be used in the future. Table 1. Therapy experiment groups and result Group Median ± SD Surviving fraction at the end of the study % Increase in symptom free survival compared to control NaCl + NaCl 64 ± 2 0.1 ---- NaCl + Rituximab 75 ± 10 0.3 15.4 177 Lu-HH1 + NaCl 92 ± 14 * 0.3 43.8 177 Lu-HH1 + Rituximab > 132 * 0.7 > 106.3 *Significantly different from NaCl + NaCl group (p < 0.01) Disclosures Repetto-Llamazares: Nordic Nanovector ASA: Employment, Equity Ownership. Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stokke:Nordic nanovector ASA: Equity Ownership. Generalov:Nordic Nanovector ASA: Employment. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership.

Background: CD37 is an internalizing transmembrane antigen highly expressed by normal B cells and on most of B-cell malignancies, and represents an interesting therapeutic target for the treatment of B-cell NHL. 177Lu-DOTA-HH1 (Betalutin®) is a novel CD37-targeting antibody radionuclide conjugate in clinical development. It consists of a CD37-binding murine IgG1 antibody HH1 labelled with the short-ranged beta-emitter lutetium-177 (T½ = 6.7 days) chelated to DOTA. 177Lu-DOTA-HH1 is delivered in a ready-to-use formulation. Efficacy and safety data of patients (pts) receiving 177Lu-DOTA-HH1 with HH1 pre-dosing, as well as new efficacy and safety data from pts receiving 177Lu-DOTA-HH1 without HH1 pre-dosing will be presented. Methods: Pts with relapsed incurable CD37 positive NHL of follicular grade I-IIIA, marginal zone, mantle cell, lymphoplasmacytic and small lymphocytic subtypes and with platelet counts ≥ 150 x109/l were eligible for inclusion in the study. In a 3+3 study design pts received rituximab (375 mg/m2) day 1 and 8 in order to deplete normal B cells. On day 29 pre-dosing with HH1 (50 mg, cold CD37 antibody) was administered before 177Lu-DOTA-HH1 injection (Arm 1). In Arm 2 177Lu-DOTA-HH1 was administered without HH1 pre-dosing on day 29. The starting doses for Arm 1 and 2 were 10 MBq/kg b.w. and 15 MBq/kg b.w, respectively. Pts enrolment has been completed (n=13) in Arm 1 with the dose-limiting toxicity (DLT) observed at 20 MBq/kg bw and a dose expansion cohort is currently open for enrollment at 15 MBq/kg with HH1 pre-dosing. Arm 2 is currently open for enrollment. Tumour response was assessed by FDG PET/CT scans (Cheson 2007), and pts will be followed for 5 years. Results: Arm 1:A total of13 (M/F 11/2) pts, median age 68 years, follicular lymphoma (n=12), and mantle cell lymphoma (n=1) have been enrolled since the study start in December 2012. The range of prior therapies was 1 to 8, where 5 of 13 pts were refractory to rituximab. The most common toxicities observed were hematologic and all DLTs were reversible and manageable. At 20 MBq/kg (n=3) G 3/4 neutropenia and/or thrombocytopenia were observed in all pts and platelet transfusions were required in 2 pts. At 15 MBq/kg (n=6) DLTs were: 1 G 3 thrombocytopenia lasting >14 days and 1 G 4 neutropenia/ thrombocytopenia lasting >7 days. The median time to nadir for platelets and neutrophils was 40 and 49 days, respectively. No pts experienced febrile neutropenia. Serious AEs were reported in 5 pts: at 10 MBq/kg pneumonia (possibly related) and pulmonary embolism (PE) unrelated, in the same pt, with history of PE; thrombocytopenia requiring platelet transfusions (2 pts) and epistaxis in 1 of them (20 MBq/kg), possibly related; transient atrial fibrillation (2 pts) at 15 MBq/kg, possibly related. No secondary malignancies or other long term events have been observed. Best overall tumor response observed across all dose levels were 4 complete and 3 partial remissions, 2 stable disease and 4 progression of disease (one pt had confirmed transformed lymphoma at 3 months). The duration of response (complete and partial remissions) ranged from 6 to more than 21 months. One patient is still in remission after 2 years. The median response duration has not yet been reached. Arm 2: Inclusion in this arm is ongoing. Data on efficacy and safety will be presented and compared with the pts receiving pre-dosing. Conclusions: 177Lu-DOTA-HH1, which is a single dose ready-to-use formulation, has a predictable and manageable safety profile. Most AEs were hematological in nature, all transient and reversible. Promising efficacy and durable responses have been observed. 177Lu-DOTA-HH1 has the potential to be a novel therapy for B-cell malignancies. Disclosures Kolstad: Nordic Nanovector ASA: Membership on an entity's Board of Directors or advisory committees, Research Funding. Bolstad:Nordic Nanovector ASA: Employment. Bruland:Nordic Nanovector ASA: Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership. Hartvig Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Two anti-osteosarcoma monoclonal antibodies (TP-3 IgG and TP-1 F(ab′)2) were labelled with the α-particle emitting radionuclide 211At and, for comparison of stability, with 125I using the N-succinimidyl-3-(trimethylstannyl)benzoate intermediate. The quality of the final preparations was measured with immunoreactivity analyses using intact osteosarcoma cells. Immunoreactivity was well retained with values in the range of 65% to 85% for 211At-labelled and 125I-labelled TP-3 IgG and approximately 60% for both 211At-labelled and 125I-labelled TP-1 F(ab)2. Tumour uptake and retention as well as normal tissue distribution in mice with osteosarcoma xenografts were measured. The uptake of the two radionuclides in tumour was similar, while there was a slight general increase in normal tissue activity at later points for the 211At-labelled MoAbs compared to the 125I-labelled MoAbs, probably caused by a minor release of free 211At from the MoAb preparations. The stable retention in tumor tissue demonstrated in this study indicates that 211At-labelled MoAbs may have potential in the treatment of tumours that allow a rapid uptake.

Cyclotron-produced 211At was distilled from a Bi metal target and coupled to N-succinimidyl-3-(trimethylstannyl)benzoate. The resulting N-succinimidyl-3-(211At)astatobenzoate was thereafter coupled to aminated monosized polymer particles with a diameter of 1.8 μm. The total time elapsed from the end of the cyclotron irradiation until the final product was prepared was about 2.5 hours. From 23 to 51% of the target activity at the end of bombardment was measured in the final conjugate. Solid-liquid extraction purification of the astatinated intermediate, using Sep-pak columns (Waters), gave more reproducible yields in the final conjugation step. The 211At-labelled particles were incubated with fetal calf serum, human serum and human full blood at room temperature. The 211At activity on the particles was measured before and after three times washing at 4, 24 and 48 hours. The stability was not significantly different from 100% for all media and for all time points. This indicates that 211At-labelled particles can be stable under in vivo conditions, and may thereby be a promising agent for intracavitary radiotherapy on freefloating cancer cells or surface fixed cells.

Radioimmunotherapy based on α-particle emitters has excellent properties as a treatment against micrometastatic and disseminated cancers because of the short path length (50 – 80 μm) and high linear energy transfer (∼ 100 keV/ μm). Alpha-particles produce clustered DNA double-strand breaks and highly reactive hydroxyl radicals when hitting biological tissue. Hence, targeted α-particle therapy offers the potential of selective tumor cell killing with low damage to surrounding normal tissue. The ideal applications for targeted α-therapy are in treating neoplastic cells in circulation or when cancer cells are present as free-floating cells or spread along compartment walls. This review will provide a brief overview of the most promising radionuclides for targeted α-therapy and compare their relative biological effectiveness (RBE) and normal tissue toxicity.

The alpha-emitter 211At was bound to polymer microspheres with a diameter of 1.8 microns. The distributions in mice of intraperitoneally injected 211At microspheres, 90Y silicate colloid, and 32P chromic phosphate colloid were compared. The microspheres with 211At spread rapidly in the peritoneal cavity and remained mainly on the intraperitoneal surfaces. Intraperitoneal injection of 90Y colloid resulted in high levels in intraperitoneal fat and the diaphragm, but 1 day after injection 8.5% of the injected dose per gram was found in blood and after 6 days 2.5% was observed in bone. The highest accumulation of 32P was found in liver and spleen. The injection of additional nonradioactive chromic phosphate colloid resulted in an even higher accumulation of 32P in spleen and liver. The same phenomenon was not observed with 211At microspheres. It is suggested that it is not only the particle size which is important in the distribution of intraperitoneally injected colloid, but the amount of colloid, the type of colloid, the addition or presence of other substances such as ascites, and the animal species might also influence the distribution. In conclusion, the intraperitoneal distribution of 211At-labeled microspheres in mice was favorable compared with 90Y and 32P colloid. These data must be viewed cautiously since the distribution might be different in other animal species or humans.

Purpose To determine whether the low-dose-rate α-particle–emitting radioimmunoconjugate 227 Th–1,4,7,10- p-isothiocyanato-benzyl- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)–rituximab can be used to inactivate lymphoma cells growing as single cells and small colonies. Methods and Materials CD20-positive lymphoma cell lines were treated with 227 Th-DOTA-rituximab for 1–5 weeks. To simulate the in vivo situation with continuous but decreasing supply of radioimmunoconjugates from the blood pool, the cells were not washed after incubation with 227 Th-DOTA-rituximab, but half of the medium was replaced with fresh medium, and cell concentration and cell-bound activity were determined every other day after start of incubation. A microdosimetric model was established to estimate the average number of hits in the nucleus for different localizations of activity. Results There was a specific targeted effect on cell growth of the 227 Th-DOTA-rituximab treatment. Although the cells were not washed after incubation with 227 Th-DOTA-rituximab, the average contribution of activity in the medium to the mean dose was only 6%, whereas the average contribution from activity on the cells' own surface was 78%. The mean dose rates after incubation with 800 Bq/mL 227 Th-DOTA-rituximab varied from 0.01 to 0.03 cGy/min. The average delay in growing from 10 5 to 10 7 cells/mL was 15 days when the cells were treated with a mean absorbed radiation dose of 2 Gy α-particle radiation from 227 Th-DOTA-rituximab, whereas it was 11 days when the cells were irradiated with 6 Gy of X-radiation. The relative biologic effect of the treatment was estimated to be 2.9–3.4. Conclusions The low-dose-rate radioimmunoconjugate 227 Th-DOTA-rituximab is suitable for inactivation of single lymphoma cells and small colonies of lymphoma cells.

Purpose To determine the relative biologic effects (RBE) of α-particle radiation from 227 Th-rituximab and of β-radiation from 90 Y-tiuexetan-ibritumomab (Zevalin) compared with external beam X-radiation in the Raji lymphoma xenograft model. Methods and Materials Radioimmunoconjugates were administered intravenously in nude mice with Raji lymphoma xenografts at different levels of activity. Absorbed dose to tumor was estimated by separate biodistribution experiments for 227 Th-rituximab and Zevalin. Tumor growth was measured two to three times per week after injection or X-radiation. Treatment-induced increase in growth delay to reach tumor volumes of 500 and 1,000 mm 3 , respectively, was used as an end point. Results The absorbed radiation dose-rate in tumor was slightly more than 0.1 Gy/d for the first week following injection of 227 Th-rituximab, and thereafter gradually decreased to 0.03 Gy/d at 21 days after injection. For treatment with Zevalin the maximum dose-rate in tumor was achieved already 6 h after injection (0.2 Gy/d), and thereafter decreased to 0.01 Gy/d after 7 days. The relative biologic effect was between 2.5 and 7.2 for 227 Th-rituximab and between 1 and 1.3 for Zevalin. Conclusions Both at low doses and low-dose–rates, the 227 Th-rituximab treatment was more effective per absorbed radiation dose unit than the two other treatments. The considerable effect at low doses suggests that the best way to administer low-dose–rates, α-emitting radioimmunoconjugates is via multiple injections.

Radioimmunotherapy (RIT) with the alpha-emitter 227Th is currently under evaluation. 227Th is conjugated to the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA. In this study, the binding of 227Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both Kd and the total number of antigens. Rael cells had more than three times as many antigens as the other two cell lines, and the functional Kd found for Rael cells was significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that 227Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.

Objective A reliable analysis of antibody binding may lead to more successful selection of the optimal antibodies. The most important parameters are affinity (equilibrium dissociation constant, Kd), the number of antigen sites on the cells (Bmax) and the on (ka) and off (kd) rate constants of binding. The affinity and the number of cellular binding sites are usually determined by equilibrium binding experiments and subsequent Scatchard analysis. The on and off rate constants are determined by kinetic binding experiments. However, it is necessary to perform two to three different types of experiment in order to determine these parameters. Methods We have developed an alternative one-step method based on a kinetic binding experiment and a mathematical description of antibody binding to antigen. The method was compared with kinetic and equilibrium binding methods. Results The results obtained using two different cell lines were in good agreement with results obtained with Scatchard analysis and kinetic binding experiments. Conclusion An alternative one-step method for determination of parameters describing binding of antibodies to antigens on cells has been developed. The method gives reliable estimates of affinity and number of antigens and in addition gives information on the kinetics of binding.

In this study, the feasibility of constructing radioimmunoconjugates by using the novel therapeutic candidate alpha-emitter, (227)Th, was evaluated. By use of the bifunctional chelator, p-SCN-benzyl-DOTA, (227)Th was conjugated to the two monoclonal antibodies, rituximab and trastuzumab. Their stability in 80% fetal bovine serum at 37 degrees C was measured. The immunoreactive fractions were determined by using CD20- and HER/2-positive cells, respectively. The overall labeling yield spanned from 6% to 17%. The radioimmunoconjugates demonstrated a relevant stability in serum and showed appropriate antigen-binding abilities.

α-emitting radionuclides are highly cytotoxic and are of considerable interest in the treatment of cancer. A particularly interesting approach is in radioimmunotherapy. However, α-emitting antibody conjugates have been difficult to exploit clinically due to the short half-life of the radionuclides, low production capability, or limited source materials. We have developed a novel technology based on the low-dose rate α-particle–emitting nuclide 227Th, exemplified here using the monoclonal antibody rituximab. In vitro, this radioimmunoconjugate killed lymphoma cells at Becquerel per milliliter (Bq/mL) levels. A single injection of 227Th-rituximab induced complete tumor regression in up to 60% of nude mice bearing macroscopic (32-256 mm3) human B-lymphoma xenografts at Becquerel per gram (Bq/g) levels without apparent toxicity. Therapy with 227Th-rituximab was significantly more effective than the control radioimmunoconjugate 227Th-trastuzumab and the standard β-emitting radioimmunoconjugate for CD20+ lymphoma90Y-tiuxetan-ibritumomab. Thorium-227 based constructs may provide a novel approach for targeted therapy against a wide variety of cancers.