89Zr-PET imaging to predict tumor uptake of 177Lu-NNV003 anti-CD37 radioimmunotherapy in mouse models of B cell lymphoma.

[177Lu]Lu-DOTA-NNV003, a radioimmunoconjugate targeting CD37, is developed as novel radioimmunotherapy (RIT) treatment for patients with B cell non-Hodgkin's lymphoma (NHL). Since patients are at risk for developing hematological toxicities due to CD37 expression on normal B cells, we aimed to develop 89Zr-labeled NNV003 for positron emission tomography (PET) imaging as a surrogate tool to predict [177Lu]Lu-DOTA-NNV003 RIT whole-body distribution and tumor uptake. NNV003 antibody was first radiolabeled with 89Zr. [89Zr]Zr-N-sucDf-NNV003 tumor uptake was evaluated by PET imaging of mice bearing human CD37-expressing REC1 B cell NHL or RAMOS Burkitt's lymphoma xenograft tumors followed by ex vivo analysis. Finally, CD37-targeting of [89Zr]Zr-N-sucDf-NNV003 and [177Lu]Lu-DOTA-NNV003 RIT were compared. [89Zr]Zr-N-sucDf-NNV003 accumulated in REC1 tumors over time, which was not observed for non-specific, 111In-labeled IgG control molecule. In RAMOS tumor-bearing mice, [89Zr]Zr-N-sucDf-NNV003 tumor uptake was higher than [111In]In-DTPA-IgG at all tested tracer protein doses (10 µg, 25 µg and 100 µg; P < 0.01), further confirming [89Zr]Zr-N-sucDf-NNV003 tumor uptake is CD37-mediated. [89Zr]Zr-N-sucDf-NNV003 and [177Lu]Lu-DOTA-NNV003 RIT showed similar ex vivo biodistribution and tumor uptake in the RAMOS tumor model. In conclusion, [89Zr]Zr-N-sucDf-NNV003 PET imaging can serve to accurately predict CD37-targeting of [177Lu]Lu-DOTA-NNV003. To enable clinical implementation, we established a good manufacturing practice (GMP)-compliant production process for [89Zr]Zr-N-sucDf-NNV003.

Anti-CD37 radioimmunotherapy with 177Lu-NNV003 synergizes with the PARP inhibitor olaparib in treatment of non-Hodgkin's lymphoma in vitro.

BACKGROUND AND PURPOSE: PARP inhibitors have been shown to increase the efficacy of radiotherapy in preclinical models. Radioimmunotherapy results in selective radiation cytotoxicity of targeted tumour cells. Here we investigate the combined effect of anti-CD37 ß-emitting 177Lu-NNV003 radioimmunotherapy and the PARP inhibitor olaparib, and gene expression profiles in CD37 positive non-Hodgkin's lymphoma cell lines. MATERIALS AND METHODS: The combined effect of 177Lu-NNV003 and olaparib was studied in seven cell lines using a fixed-ratio ray design, and combination index was calculated for each combination concentration. mRNA was extracted before and after treatment with the drug combination. After RNA-sequencing, hierarchical clustering was performed on basal gene expression profiles and on differentially expressed genes after combination treatment from baseline. Functional gene annotation analysis of significant differentially expressed genes after combination treatment was performed to identify enriched biological processes. RESULTS: The combination of olaparib and 177Lu-NNV003 was synergistic in four of seven cell lines, antagonistic in one and both synergistic and antagonistic (conditionally synergistic) in two, depending on the concentration ratio between olaparib and 177Lu-NNV003. Cells treated with the combination significantly overexpressed genes in the TP53 signalling pathway. However, cluster analysis did not identify gene clusters that correlate with the sensitivity of cells to single agent or combination treatment. CONCLUSION: The cytotoxic effect of the combination of the PARP inhibitor olaparib and the ß-emitting radioimmunoconjugate 177Lu-NNV003 was synergistic in the majority of tested lymphoma cell lines.

Targeted alpha therapy for chronic lymphocytic leukaemia and non-Hodgkin's lymphoma with the anti-CD37 radioimmunoconjugate 212Pb-NNV003.

Relapse of chronic lymphocytic leukaemia and non-Hodgkin's lymphoma after standard of care treatment is common and new therapies are needed. The targeted alpha therapy with 212Pb-NNV003 presented in this study combines cytotoxic α-particles from 212Pb, with the anti-CD37 antibody NNV003, targeting B-cell malignancies. The goal of this study was to explore 212Pb-NNV003 for treatment of CD37 positive chronic lymphocytic leukaemia and non-Hodgkin's lymphoma in preclinical mouse models.An anti-proliferative effect of 212Pb-NNV003 was observed in both chronic lymphocytic leukaemia (MEC-2) and Burkitt's lymphoma (Daudi) cells in vitro. In biodistribution experiments, accumulation of 212Pb-NNV003 was 23%ID/g and 16%ID/g in Daudi and MEC-2 tumours 24 h post injection. In two intravenous animal models 90% of the mice treated with a single injection of 212Pb-NNV003 were alive 28 weeks post cell injection. Median survival times of control groups were 5-9 weeks. There was no significant difference between different specific activities of 212Pb-NNV003 with regards to therapeutic effect or toxicity. For therapeutically effective activities, a transient haematological toxicity was observed. This study shows that 212Pb-NNV003 is effective and safe in preclinical models of CD37 positive chronic lymphocytic leukaemia and non-Hodgkin's lymphoma, warranting future clinical testing.

The therapeutic effectiveness of 177Lu-lilotomab in B-cell non-Hodgkin lymphoma involves modulation of G2/M cell cycle arrest.

Some patients with B-cell non-Hodkin lymphoma Lymphoma (NHL) become refractory to rituximab (anti-CD20 antibody) therapy associated with chemotherapy. Here, the effect of the anti-CD37 antibody-radionuclide conjugate lutetium-177 (177Lu)-lilotomab (Betalutin®) was investigated in preclinical models of NHL. In SCID mice bearing DOHH2 (transformed follicular lymphoma, FL) cell xenografts, 177Lu-lilotomab significantly delayed tumor growth, even at low activity (100 MBq/kg). In athymic mice bearing OCI-Ly8 (diffuse large B-cell lymphoma, DLBCL) or Ramos (Burkitt's lymphoma) cell xenografts, 177Lu-lilotomab activity had to be increased to 500 MBq/kg to show a significant tumor growth delay. Clonogenic and proliferation assays showed that DOHH2 cells were highly sensitive to 177Lu-lilotomab, while Ramos cells were the least sensitive, and U2932 (DLBCL), OCI-Ly8, and Rec-1 (mantle cell lymphoma) cells displayed intermediate sensitivity. The strong 177Lu-lilotomab cytotoxicity observed in DOHH2 cells correlated with reduced G2/M cell cycle arrest, lower WEE-1- and MYT-1-mediated phosphorylation of cyclin-dependent kinase-1 (CDK1), and higher apoptosis. In agreement, 177Lu-lilotomab efficacy in vitro, in vivo, and in patient samples was increased when combined with G2/M cell cycle arrest inhibitors (MK-1775 and PD-166285). These results indicate that 177Lu-lilotomab is particularly efficient in treating tumors with reduced inhibitory CDK1 phosphorylation, such as transformed FL.

Targeting B-cell malignancies with the beta-emitting anti-CD37 radioimmunoconjugate 177Lu-NNV003.

PURPOSE: The aim of this study was to explore the ß-emitting lutetium-177 labelled anti-CD37 antibody NNV003 (177Lu-NNV003, Humalutin®) for the treatment of non-Hodgkin's lymphoma in in vitro studies and in animal models. METHODS: Cytotoxicity of 177Lu-NNV003 was measured in REC-1 (mantle cell lymphoma) and DOHH-2 (diffuse large B cell lymphoma) cell lines. Biodistribution was studied in mice bearing subcutaneous DOHH-2 or MEC-2 (chronic lymphocytic leukaemia) xenografts. The therapeutic effect of a single injection of 177Lu-NNV003 was measured in mice intravenously or subcutaneously injected with REC-1 cells. Haematological and histopathological assessments were used to evaluate the toxic effect of 177Lu-NNV003. The immunotherapeutic effect of NNV003 was assessed by measuring binding to Fcγ receptors, activation of ADCC and ADCP. NNV003's immunogenicity potential was assessed using in silico immunogenicity prediction tools. RESULTS: 177Lu-NNV003 showed an activity dependent antiproliferative effect in all cell lines. Maximum tumour uptake in vivo was 45% of injected activity/g in MEC-2 tumours and 15% injected activity/g in DOHH-2 tumours. In mice injected intravenously with REC-1 cells, 177Lu-NNV003 (50-100 MBq/kg) improved survival compared to control groups (p < 0.02). In mice with subcutaneous REC-1 xenografts, 500 MBq/kg 177Lu-NNV003 extended survival compared to the control treatments (p < 0.005). Transient haematological toxicity was observed in all mice treated with radioactivity. NNV003 induced ADCC and ADCP and was predicted to have a lower immunogenicity potential than its murine counterpart. CONCLUSION: 177Lu-NNV003 had a significant anti-tumour effect and a favourable toxicity profile. These results warrant further clinical testing in patients with CD37-expressing B cell malignancies.

Modifications in dynamic contrast-enhanced magnetic resonance imaging parameters after α-particle-emitting ²²7Th-trastuzumab therapy of HER2-expressing ovarian cancer xenografts.

PURPOSE: The purpose of this study was to investigate the effect of α-particle-emitting (227)Th-trastuzumab radioimmunotherapy on tumor vasculature to increase the knowledge about the mechanisms of action of (227)Th-trastuzumab. METHODS AND MATERIALS: Human HER2-expressing SKOV-3 ovarian cancer xenografts were grown bilaterally in athymic nude mice. Mice with tumor volumes 253 ± 36 mm(3) (mean ± SEM) were treated with a single injection of either (227)Th-trastuzumab at a dose of 1000 kBq/kg body weight (treated group, n=14 tumors) or 0.9% NaCl (control group, n=10 tumors). Dynamic T1-weighted contrast-enhanced magnetic resonance imaging (DCEMRI) was used to study the effect of (227)Th-trastuzumab on tumor vasculature. DCEMRI was performed before treatment and 1, 2, and 3 weeks after therapy. Tumor contrast-enhancement curves were extracted voxel by voxel and fitted to the Brix pharmacokinetic model. Pharmacokinetic parameters for the tumors that underwent radioimmunotherapy were compared with the corresponding parameters of control tumors. RESULTS: Significant increases of kep, the rate constant of diffusion from the extravascular extracellular space to the plasma (P<.05), and kel, the rate of clearance of contrast agent from the plasma (P<.01), were seen in the radioimmunotherapy group 2 and 3 weeks after injection, compared with the control group. The product of kep and the amplitude parameter A, associated with increased vessel permeability and perfusion, was also significantly increased in the radioimmunotherapy group 2 and 3 weeks after injection (P<.01). CONCLUSIONS: Pharmacokinetic modeling of MRI contrast-enhancement curves evidenced significant alterations in parameters associated with increased tumor vessel permeability and tumor perfusion after (227)Th-trastuzumab treatment of HER2-expressing ovarian cancer xenografts.

Comparing high LET 227Th- and low LET 177Lu-trastuzumab in mice with HER-2 positive SKBR-3 xenografts.

UNLABELLED: The aim of the present study was to compare the biodistribution, normal tissue toxicity and therapeutic effect of the alpha-particle emitting 227Th-trastuzumab and the beta-particle emitting 177Lu-trastuzumab in mice with HER2- expressing SKBR-3 breast cancer xenografts. METHODS: Biodistributions of the two radioimmunoconjugates were determined at different time points after i.v. injection. Inhibition of tumor growth was measured after single injection of 227Th-trastuzumab (200, 400, 600 or 1000 kBq/kg), 177Lu-trastuzumab (40 or 200 MBq/kg) or saline. The toxicity profiles were compared by measurements of body weight,clinical chemistry and hematological parameters, as well as histological examination of tissue specimens. RESULTS: 400 kBq/kg of 227Th-trastuzumab and 40 MBq/kg of 177Lu-trastuzumab both resulted in an absorbed radiation dose to tumor of approximately 3 Gy. A significant anti-tumor effect and increased survival were observed at injected dosages of 400-1000 kBq/kg of 227Th-trastuzumab and 200 MBq/kg of 177Lu-trastuzumab as compared to the saline control. When compared at the same therapeutic effect level (100% prolonged growth delay as compared to control) the absorbed radiation dose of 227Th-trastuzumab was 3 times lower than with 177Lu-trastuzumab, indicating a relative biological effect (RBE) of 2.8 for 227Th-trastuzumab. In contrast, when compared at the same temporary decrease of WBC count (50% decrease in number of white blood cells as compared to control), the growth delay was 3 times longer with 177Lutrastuzumab than with 227Th-trastuzumab, which indicates that the therapeutic index was three times higher for 177Lutrastuzumab than for 227Th-trastuzumab. CONCLUSION: In this xenograft model the RBE was higher for 227Th-trastuzumab than for 177Lu-trastuzumab, while the therapeutic index of 177Lu-trastuzumab was superior to that of 227Th-trastuzumab.

Targeted alpha therapy with 227Th-trastuzumab of intraperitoneal ovarian cancer in nude mice.

UNLABELLED: The aim of the current study was to investigate the therapeutic effect of 227Th-radioimmunotherapy on intraperitoneally growing human bioluminescent HER2 positive ovarian cancer cells. METHODS: In vitro toxicity of 227Th-trastuzumab in bioluminescent SKOV3-luc-D3 ovarian cancer cells was assessed in a growth assay. The biodistribution of intraperitoneally administrated 227Th-trastuzumab in athymic nude mice without tumor cells was determined. For in vivo therapy, seventy female athymic nude mice were intraperitoneally inoculated with tumor cells 17 days prior to injection of single 227Th-trastuzumab doses of 1000 kBq/kg, 600 kBq/kg or 400 kBq/kg, or three injections with 400 kBq/kg 227Th-trastuzumab separated by 4 weeks. Two control groups were given either 20 µg unlabeled trastuzumab or 0.9% NaCl. In vivo bioluminescence imaging was performed weekly before and after onset of therapy. Tumor growth, survival and toxicity were compared. RESULTS: There was a statistically significant therapeutic effect of the 227Th-trastuzumab treatment both with respect to survival and tumor growth. The maximum tolerated dosage was 600 kBq/kg 227Th-trastuzumab. In the in vitro study, two hours incubation with 20 kBq/ml of 227Th-trastuzumab, followed by washing, and subsequent culture of the cells resulted in an average absorbed radiation dose of 6 Gy after 11 days and complete growth inhibition. CONCLUSION: Targeted alpha therapy with 227Th-trastuzumab of human SKOV3-luc-D3 cells growing intraperitoneally in nude mice was clearly superior to unlabeled trastuzumab therapy. The results warrant further studies of 227Th-radioimmunotherapy used as adjuvant treatment and for metastatic cancer.

Fractionated therapy of HER2-expressing breast and ovarian cancer xenografts in mice with targeted alpha emitting 227Th-DOTA-p-benzyl-trastuzumab.

BACKGROUND: The aim of this study was to investigate therapeutic efficacy and normal tissue toxicity of single dosage and fractionated targeted alpha therapy (TAT) in mice with HER2-expressing breast and ovarian cancer xenografts using the low dose rate radioimmunoconjugate (227)Th-DOTA-p-benzyl-trastuzumab. METHODOLOGY/PRINCIPAL FINDINGS: Nude mice carrying HER2-overexpressing subcutaneous SKOV-3 or SKBR-3 xenografts were treated with 1000 kBq/kg (227)Th-trastuzumab as single injection or four injections of 250 kBq/kg with intervals of 4-5 days, 2 weeks, or 4 weeks. Control animals were treated with normal saline or unlabeled trastuzumab. In SKOV-3 xenografts tumor growth to 10-fold size was delayed (p<0.01) and survival with tumor diameter less than 16 mm was prolonged (p<0.05) in all TAT groups compared to the control groups. No statistically significant differences were seen among the treated groups. In SKBR-3 xenografts tumor growth to 10-fold size was delayed in the single injection and 4-5 days interval groups (p<0.001) and all except the 4 weeks interval TAT group showed improved survival to the control groups (p<0.05). Toxicity was assessed by blood cell counts, clinical chemistry measurements and body weight. Transient reduction in white blood cells was seen for the single injection and 4-5 days interval groups (p<0.05). No significant changes were seen in red blood cells, platelets or clinical chemistry parameters. Survival without life threatening loss of body weight was significantly prolonged in 4 weeks interval group compared to single injection group (p<0.05) for SKOV-3 animals and in 2 weeks interval group compared with the 4-5 days interval groups (p<0.05) for SKBR-3 animals. CONCLUSIONS/SIGNIFICANCE: The same concentration of radioactivity split into several fractions may improve toxicity of (227)Th-radioimmunotherapy while the therapeutic effect is maintained. Thus, it might be possible to increase the cumulative absorbed radiation dose to tumor with acceptable toxicity by fractionation of the dosage.

Treatment of HER2-expressing breast cancer and ovarian cancer cells with alpha particle-emitting 227Th-trastuzumab.

PURPOSE: To evaluate the cytotoxic effects of low-dose-rate alpha particle-emitting radioimmunoconjugate (227)Th-p-isothiocyanato-benzyl-DOTA-trastuzumab ((227)Th-trastuzumab [where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]) internalized by breast and ovarian cancer cell lines in order to assess the potential of (227)Th-trastuzumab as a therapeutic agent against metastatic cancers that overexpress the HER2 oncogene. METHODS AND MATERIALS: Clonogenic survival and cell growth rates of breast cancer cells treated with (227)Th-trastuzumab were compared with rates of cells treated with nonbinding (227)Th-rituximab, cold trastuzumab, and X-radiation. Cell growth experiments were also performed with ovarian cancer cells. Cell-associated radioactivity was measured at several time points, and the mean radiation dose to cells was calculated. RESULTS: SKBR-3 cells got 50% of the mean absorbed radiation dose from internalized activity and 50% from cell surface-bound activity, while BT-474 and SKOV-3 cells got 75% radiation dose from internalized activity and 25% from cell surface-bound activity. Incubation of breast cancer cells with 2.5 kBq/ml (227)Th-trastuzumab for 1 h at 4°C, followed by washing, resulted in mean absorbed radiation doses of 2 to 2.5 Gy. A dose-dependent inhibition of cell growth and an increase in apoptosis were induced in all cell lines. CONCLUSIONS: Clinically relevant activity concentrations of (227)Th-trastuzumab induced a specific cytotoxic effect in three HER2-expressing cell lines. The cytotoxic effect of (227)Th-trastuzumab was higher than that of single-dose X-radiation (relative biological effectiveness = 1.2). These results warrant further studies of treatment of breast cancer and ovarian cancer with (227)Th-trastuzumab.

Experimental α-particle radioimmunotherapy of breast cancer using 227Th-labeled p-benzyl-DOTA-trastuzumab.

BACKGROUND: The aim of the present study was to explore the biodistribution, normal tissue toxicity, and therapeutic efficacy of the internalizing low-dose rate alpha-particle-emitting radioimmunoconjugate 227Th-trastuzumab in mice with HER2-expressing breast cancer xenografts. METHODS: Biodistribution of 227Th-trastuzumab and 227Th-rituximab in nude mice bearing SKBR-3 xenografts were determined at different time points after injection. Tumor growth was measured after administration of 227Th-trastuzumab, 227Th-rituximab, cold trastuzumab, and saline. The toxicity of 227Th-trastuzumab was evaluated by measurements of body weight, blood cell, and clinical chemistry parameters, as well as histological examination of tissue specimens. RESULTS: The tumor uptake reached peak levels of 34% ID/g (4.6 kBq/g) 3 days after injection of 400 kBq/kg of 227Th-trastuzumab. The absorbed radiation dose to tumor was 2.9 Gy, while it was 2.4 Gy to femur due to uptake of the daughter nuclide 223Ra in bone; the latter already explored in clinical phases I and II trials without serious toxicity. A significant dose-dependent antitumor effect was observed for dosages of 200, 400, and 600 kBq/kg of 227Th-trastuzumab but no effect of 400 and 600 kBq/kg 227Th-rituximab (non-tumor binding). No serious delayed bone marrow or normal organ toxicity was observed, but there was a statistical significant reduction in blood cell parameters for the highest-dose group of 227Th-trastuzumab treatment. CONCLUSION: Internalizing 227Th-trastuzumab therapy was well tolerated and resulted in a dose-dependent inhibition of breast cancer xenograft growth. These results warrant further preclinical studies aiming at a clinical trial in breast cancer patients with metastases to bone.

Assessment of long-term radiotoxicity after treatment with the low-dose-rate alpha-particle-emitting radioimmunoconjugate (227)Th-rituximab.

PURPOSE: 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. METHODS: 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. RESULTS: 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 (p < 0.05), 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. CONCLUSION: 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.