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Background: The aim of this study was to investigate dosimetry data and clinical variables to predict hematological toxicity in non-Hodgkin lymphoma (NHL) patients treated with [177Lutetium]Lu-liloto-mab satetraxetan. Material and methods: A total of 17 patients treated with [177Lu]Lu-lilotomab satetraxetan in a first-in-human phase 1/2a study were included. Absorbed dose to the red marrow was explored using SPECT/CT-imaging of the lumbar vertebrae L2–L4 over multiple time points. Percentage reduction of thrombocytes and neutrophils at nadir compared to baseline (PBN) and time to nadir (TTN) were chosen as indicators of myelosuppression and included as dependent variables. Two models were applied in the analysis, a multivariate linear model and a sigmoidal description of toxicity as a function of absorbed dose. A total of 10 independent patient variables were investigated in the multivariate analysis. Results: Absorbed dose to the red marrow ranged from 1 to 4 Gy. Absorbed dose to the red marrow was found to be the only significant variable for PBN for both thrombocytes and neutrophils. The sigmoid function gave similar results in terms of accuracy when compared to the linear model. Conclusion: Myelosuppression in the form of thrombocytopenia and neutropenia in patients treated with [177Lu]Lu-lilotomab satetraxetan can be predicted from the SPECT/CT-derived absorbed dose estimate to the red marrow. The present work was financially supported by the South-Eastern Norway Regional Health Authority.

Purpose 177Lu-lilotomab satetraxetan targets the CD37 antigen and has been investigated in a first-in-human phase 1/2a study for relapsed non-Hodgkin lymphoma (NHL). Tumor dosimetry and response evaluation can be challenging after radioimmunotherapy (RIT). Changes in FDG PET/CT parameters after RIT and correlations with tumor-absorbed doses has not been examined previously in patients with lymphoma. Treatment-induced changes were measured at FDG PET/CT and ceCT to evaluate response at the lesion level after treatment, and correlations with tumor-absorbed doses were investigated. Methods Forty-five tumors in 16 patients, with different pre-treatment and pre-dosing regimens, were included. Dosimetry was performed based on multiple SPECT/CT images. FDG PET/CT was performed at baseline and at 3 and 6 months. SUVmax, MTV, TLG, and changes in these parameters were calculated for each tumor. Lesion response was evaluated at 3 and 6 months (PET3months and PET6months) based on Deauville criteria. Anatomical changes based on ceCT at baseline and at 6 and 12 months were investigated by the sum of perpendiculars (SPD). Results Tumor-absorbed doses ranged from 35 to 859 cGy. Intra- and interpatient variations were observed. Mean decreases in PET parameters from baseline to 3 months were ΔSUVmax-3months 61%, ΔMTV3months 80%, and ΔTLG3months 77%. There was no overall correlation between tumor-absorbed dose and change in FDG PET or ceCT parameters at the lesion level or significant difference in tumor-absorbed doses between metabolic responders and non-responders after treatment. Conclusion Our analysis does not show any correlation between tumor-absorbed doses and changes in FDG PET or ceCT parameters for the included lesions. The combination regimen, including cold antibodies, may be one of the factors precluding such a correlation. Increased intra-patient response with increased tumor-absorbed doses was observed for most patients, implying individual variations in radiation sensitivity or biology. Trial registration ClinicalTrials.gov Identifier (NCT01796171). Registered December 2012

Patients with non-Hodgkin lymphoma (NHL) who are treated with rituximab may develop resistant disease, often associated with changes in expression of CD20. The next-generation β-particle–emitting radioimmunoconjugate 177Lu-lilotomab-satetraxetan (Betalutin) was shown to up-regulate CD20 expression in different rituximab-sensitive NHL cell lines and to act synergistically with rituximab in a rituximab-sensitive NHL animal model. We hypothesized that 177Lu-lilotomab-satetraxetan may be used to reverse rituximab resistance in NHL. Methods: The rituximab-resistant Raji2R and the parental Raji cell lines were used. CD20 expression was measured by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was measured by a bioluminescence reporter assay. The efficacies of combined treatments with 177Lu-lilotomab-satetraxetan (150 or 350 MBq/kg) and rituximab (4 × 10 mg/kg) were compared with those of single agents or phosphate-buffered saline in a Raji2R-xenograft model. Cox regression and the Bliss independence model were used to assess synergism. Results: Rituximab binding in Raji2R cells was 36% ± 5% of that in the rituximab-sensitive Raji cells. 177Lu-lilotomab-satetraxetan treatment of Raji2R cells increased the binding to 53% ± 3% of the parental cell line. Rituximab ADCC induction in Raji2R cells was 20% ± 2% of that induced in Raji cells, whereas treatment with 177Lu-lilotomab-satetraxetan increased the ADCC induction to 30% ± 3% of that in Raji cells, representing a 50% increase (P < 0.05). The combination of rituximab with 350 MBq/kg 177Lu-lilotomab-satetraxetan synergistically suppressed Raji2R tumor growth in athymic Foxn1nu mice. Conclusion: 177Lu-lilotomab-satetraxetan has the potential to reverse rituximab resistance; it can increase rituximab binding and ADCC activity in vitro and can synergistically improve antitumor efficacy in vivo.

[Fifteen patients with different pre-dosing (non-radioactive lilotomab) regimens were included and the cohort was divided into low and high non-radioactive lilotomab pre-dosing groups for some of the analyses.Significant decreases in tMTV and tTLG were observed at 3 months for patients receiving tTAD ≥ 200 cGy compared to patients receiving tTAD 200 cGy (p = .03 for both). All non-responders had tTAD 200 cGy. Large variations in tTAD were observed in responders. Reduction intTAD of ≥ 200 cGy may prove valuable to ensure clinical response, but further studies are needed to confirm this in a larger patient population. Furthermore, this work indicates that higher baseline tumor burden (up to 585 cm

The aim of this study was to investigate dosimetry data and clinical variables to predict hematological toxicity in non-Hodgkin lymphoma (NHL) patients treated with [A total of 17 patients treated with [Absorbed dose to the red marrow ranged from 1 to 4 Gy. Absorbed dose to the red marrow was found to be the only significant variable for PBN for both thrombocytes and neutrophils. The sigmoid function gave similar results in terms of accuracy when compared to the linear model.Myelosuppression in the form of thrombocytopenia and neutropenia in patients treated with [

Purpose The aim of this study was to explore the β-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

Introduction: CD37 has shown promise as a new therapeutic target for B-cell malignancies. CD37 is highly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Alpha-emitting radionuclides have demonstrated potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm) causing localized and irreparable DNA damage while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-generating radioisotope 212Pb for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Approximately 90,000 cases of CLL and NHL are expected annually in the US. Standard of care includes chemotherapy combined with anti-CD20 antibodies (obinutuzumab and rituximab) and Bruton’s tyrosine kinase inhibitor ibrutinib. While these therapies are initially effective, most patients inevitably relapse. Materials and methods: The efficacy and tolerability of a single dose 212Pb-NNV003 treatment were evaluated in disseminated models using CD37-expressing human CLL (MEC-2) and Burkitt’s lymphoma (Daudi) cells. Comparison to therapeutic doses of ibrutinib was investigated in the MEC-2 model. R2G2 or CB17-SCID mice were injected intravenously with 2.5×106 MEC-2 or 10×106 Daudi cells on day 0, followed by intravenous injection of 212Pb-NNV003 (2.5, 5 or 7.5 µCi in Daudi model and 5, 10, 15 or 20 µCi in MEC-2 model) or daily oral administration of ibrutinib (12.5 or 25 mg/kg) starting on day 2 (n=10-12 per group). 212Pb-cetuximab (unspecific isotype control), unlabelled NNV003 and NaCl were used as controls. Results: In vitro testing of the antibody showed that MEC-2 cells were 5 times more resistant to ibrutinib than Daudi cells. Treatment of mice injected intravenously with Daudi cells with 2.5 μCi 212Pb-NNV003 resulted in survival of 100% of the mice 100 days after start of treatment, while 100 days after start of treatment with 100 μg obinutuzumab only 40% of the mice were still alive. Control mice had a median survival of 7-8 weeks. Treatment of mice injected intravenously with MEC-2 cells with 15 μCi 212Pb-NNV003 resulted in survival of 90-100% for more than 25 weeks in two independent experiments. Ibrutinib treated mice with intravenous MEC-2 cells presented a median survival of only 4.3-4.4 weeks, comparable to the unlabelled NNV003 and saline groups (4.1 and 4.4 weeks). Mice receiving non-specific 212Pb-cetuximab presented a median survival of 7-9 weeks. Conclusion: The study shows that a single injection 212Pb-NNV003 is safe and effective for the treatment of CD37 positive CLL and NHL in preclinical models, with promising efficacy in both ibrutinib-resistant and sensitive CLL models. Treatment with 212Pb-NNV003 was superior to both ibrutinib and obinutuzumab. Further clinical testing is warranted. Citation Format: Jostein Dahle, Amal Saidi, Tania Stallons, Helen Heyerdahl, Ada Repetto-Llamazares, Julien Torgue. Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5432.

Purpose: 177Lu-lilotomab satetraxetan, a novel CD37 directed radioimmunotherapy (RIT), has been investigated in a first-in-human phase 1/2a study for relapsed non-Hodgkin lymphoma (NHL). Absorbed dose for all tumor tissue in the body is a crucial parameter in RIT which has traditionally been challenging to calculate. The aim of this study was to investigate the correlations between baseline FDG PET/CT and posttreatment SPECT/CT parameters, absorbed dose-response relationships and clinical responses.Materials and methods: A total of 15 patients with different pre-treatment and pre-dosing regimens were included. 177Lu-lilotomab satetraxetan was administered at dosage levels of 10, 15 or 20 MBq/kg. Total radioimmunoconjugate tumor volume (tRTV), total radioimmunoconjugate lesion uptake (tRLU) and total tumor absorbed dose (tTAD) were calculated from posttreatment SPECT/CT. The measured uptake values and absorbed doses were normalized for dosage when appropriate. For some of the analyses, the cohort was divided into low (arm 1) and high (arm 4+5) non-radioactive lilotomab pre-dosing groups. tMTV and tTLG were calculated from FDG PET/CT performed at baseline, 3 and 6 months after RIT, and the percent change for these parameters calculated (∆tMTV3months, ∆tTLG3months and ∆tMTV6months, ∆tTLG6months). Clinical responses were evaluated at 6 months.Results: tTMV and tRTV were significantly correlated (p higher at PET3months for patients receiving tTAD≥200cGy compared to patients receiving lower tumor absorbed doses (p=.03 for both). Also, significant decreases in ∆tMTV3months, ∆tTLG3months and ∆tMTV6months, ∆tTLG6months were observed with increasing tTAD in the high lilotomab patient group. Similarly, responders (patients with complete remission and partial remission) had higher mean tTAD compared to non-responders (stable disease and progressive disease). This was statistically significant in the high lilotomab group. Across the entire population, all non-responders had tTAD < 200cGy, and all patients with tTAD ≥ 200cGy were responders.Conclusion: This work indicates that 177Lu-lilotomab satetraxetan targets FDG avid lesions, and that increasing baseline (tMTV) does not have a decreasing effect on the total tumor absorbed dose (tTAD). The patient group receiving a higher amount of lilotomab pre-dosing demonstrated an absorbed dose–response relationship. Similar results were not observed in the low lilotomab group, which were expected since an overall very good response rate could mask such a relationship for this group. Regardless of pre-dosing, a mean absorbed dose to the total tumor tissue (tTAD) limit of 200cGy may prove valuable to separate clinical non-responders from responders.

For patients with indolent non-Hodgkin lymphoma who fail initial anti-CD20–based immunochemotherapy or develop relapsed or refractory disease, there remains a significant unmet clinical need for new therapeutic approaches to improve outcomes and quality of life. 177Lu-lilotomab satetraxetan is a next-generation single-dose CD37-directed radioimmunotherapy (RIT) which was investigated in a phase 1/2a study in 74 patients with relapsed/refractory indolent non-Hodgkin B-cell lymphoma, including 57 patients with follicular lymphoma (FL). To improve targeting of 177Lu-lilotomab satetraxetan to tumor tissue and decrease hematologic toxicity, its administration was preceded by the anti-CD20 monoclonal antibody rituximab and the “cold” anti-CD37 antibody lilotomab. The most common adverse events (AEs) were reversible grade 3/4 neutropenia (31.6%) and thrombocytopenia (26.3%) with neutrophil and platelet count nadirs 5 to 7 weeks after RIT. The most frequent nonhematologic AE was grade 1/2 nausea (15.8%). With a single administration, the overall response rate was 61% (65% in patients with FL), including 30% complete responses. For FL with ≥2 prior therapies (n = 37), the overall response rate was 70%, including 32% complete responses. For patients with rituximab-refractory FL ≥2 prior therapies (n = 21), the overall response rate was 67%, and the complete response rate was 24%. The overall median duration of response was 13.6 months (32.0 months for patients with a complete response). 177Lu-lilotomab satetraxetan may provide a valuable alternative treatment approach in relapsed/refractory non-Hodgkin lymphoma, particularly in patients with comorbidities unsuitable for more intensive approaches. This trial was registered at www.clinicaltrials.gov as #NCT01796171.

Lutetium (177Lu) lilotomab satetraxetan (177Lu-lilotomab) is a novel anti-CD37 radioimmunoconjugate (RIC) currently in Phase 2b clinical trial for treatment of non-Hodgkin lymphoma (NHL). The aim of the current study was to investigate tolerability and anti-tumor efficacy of multiple dosing of 177Lu-lilotomab in vivo. Nude mice with subcutaneous (s.c.) NHL (Ramos) xenografts were given 2, 3 or 4 weekly injections of 300 MBq/kg 177Lu-lilotomab or NaCl. Treatment tolerability was assessed by body weight, hematology and histopathological evaluation. A separate experiment in mice without xenografts was performed to collect long term toxicity data. Mice in this study were dosed more conservatively with the intention that all mice should survive until end of experiment and were administered 2 or 4 weekly injections of 200 MBq/kg 177Lu-lilotomab or NaCl. Total accumulated activity of 900 MBq/kg 177Lu-lilotomab given as three weekly injections was tolerated by nude mice with s.c. Ramos xenografts, which is 50 % higher than the maximum tolerated activity (MTA) of a single injection of 530-600 MBq/kg. In the long-term toxicity animals, the highest accumulated activity tested, 800 MBq/kg, was also well tolerated. Radioactivity-dependent transient reduction in white blood cell and platelet counts occurred in all treated groups, with nadir 1-3 weeks after last injection. This toxicity was radioactivity dependent and consistent with histopathological changes in hemolymphopoietic tissues. Significant tumor growth delay measured as time to reach 4 times intial tumor volume was observed in all groups given multiple injections of 300 MBq/kg 177Lu-lilotomab compared to NaCl control group (p

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.

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.

177Lu-lilotomab satetraxetan is a novel antibody radionuclide conjugate (ARC) currently in a phase 1/2a first-in-human dosage escalation trial for patients with relapsed CD37+ indolent non Hodgkin lymphoma (NHL). The aim of this study was to investigate biodistribution and absorbed doses to organs at risk. Methods: A total of seven patients treated with 177Lu-lilotomab satetraxetan were included for dosimetry. Patients were grouped based on two different pre-dosing regimens (with and without pre-dosing with 40 mg lilotomab) and were treated with different levels of activity per body weight (10, 15 and 20 MBq/kg). All patients were pre-treated with rituximab. Serial planar and SPECT/CT-images were used to determine time activity curves and patient specific masses for organs with 177Lu-lilotomab satetraxetan uptake. Doses were calculated with OLINDA/EXM. Results: Organs with distinct uptake of 177Lu-lilotomab satetraxetan, in addition to red bone marrow and tumors, were liver, spleen and kidneys. Largest uptake was found in the spleen, where doses ranged from 1.54 to 3.60 mGy/MBq. The liver received 0.70 to 1.15 mGy/MBq. The kidneys received the lowest dose of the source organs investigated; 0.16 to 0.79 mGy/MBq. No statistical significant differences in soft tissue absorbed doses for the two pre-dosing regimens were found. Whole body (WB) dose ranged from 0.08 to 0.17 mGy/MBq. Conclusion: The biodistribution study for patients treated with 177Lu-lilotomab satetraxetan revealed highest physiological uptake in liver and spleen, besides red marrow. For all dosage levels investigated, doses were found modest when compared to commonly assumed tolerance limits. The LYMRIT37-01 study is sponsored by Nordic Nanovector ASA. Johan Blakkisrud is supported by grants from the South-Eastern Norway Regional Health Authority. Harald Holte and Arne Kolstad were both in part supported by grants from the Norwegian Cancer Society.

Acute myeloid leukaemia is a disease of the haematopoietic stem and progenitor cells distinguished by an impairment of proliferation, propagation and differentiation of the haematopoietic lineage[1]. AML is a heterogenous disease, where patient stratification and personalised therapeutic strategies are of importance[2]. The transmembrane protein CD37 has been associated with regulation of cell to cell interaction and proliferation in both B and T cells. Mice knockout studies have suggested that CD37 is not crucial for B cell development, however, it is highly expressed in malignant and suboptimal conditions and thus represent a good target for B-cell malignancies[3]. Pereira et al. reported that their anti-CD37 antibody-drug conjugate was a potential therapeutic in AML[4]. Their study revealed anti-leukemic effect in primary AML material and cell lines bothin vivoandin vitro. An investigation of CD37 expression in AML patients is needed to understand the relevance of CD37 immunotherapy. To investigate the relevance of targeted therapy of CD37 the chimeric mAb NNV003 anti-CD37 antibody was evaluated in AML samples. Single-cell time-of-flight mass cytometry (CyTOF), an established tool to analyse the immune signalling in bone marrow and peripheral blood, was used to investigate CD37 expression in 59 AML patient samples. A panel was designed to categorise hierarchical landscape of AML and the leukemic stem cell population (LSCs). CyTOF identified CD37 expression exclusively in the B-cell population of our 59 patients, whereas the AML blast population was negative for CD37 expression. Flow cytometry, however; identified 5/59 patients with CD37 expression in the leukemic blast population. Although previous studies show that an anti-CD37 drug conjugate showed anti-leukemic effectin vivoandin vitro, our results do not support that it is a promising therapeutic biomarker. We have uncovered that malignant expression of CD37 is not a feature of the AML patients investigated. We do not consider CD37 to be a promising and good target for precision therapy of AML. Kayser, S. and M.J. Levis, Advances in targeted therapy for acute myeloid leukaemia. Br J Haematol, 2018. 180(4): p. 484-500.Patel, J.P., et al., Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med, 2012. 366(12): p. 1079-89.Payandeh, Z., et al., Anti-CD37 targeted immunotherapy of B-Cell malignancies. Biotechnol Lett, 2018. 40(11-12): p. 1459-1466.Pereira, D.S., et al., AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML. Mol Cancer Ther, 2015. 14(7): p. 1650-60. Disclosures Heyerdahl: Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Dahle:Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Gjertsen:Novartis:Consultancy;KinN Therapeutics AS:Current equity holder in private company;Pfizer Inc:Consultancy;BerGenBio AS:Consultancy, Research Funding;Alden Cancer Therapy AS:Current equity holder in private company.Mccormack:KinN Therapeutics AS:Current equity holder in private company.

Red marrow (RM) is often the primary organ at risk in radioimmunotherapy; irradiation of marrow may induce short- and long-term hematologic toxicity. 177Lu-lilotomab satetraxetan is a novel anti-CD37 antibody-radionuclide conjugate currently in phase 1/2a. Two predosing regimens have been investigated, one with 40 mg of unlabeled lilotomab antibody (arm 1) and one without (arm 2). The aim of this work was to compare RM-absorbed doses for the two arms and to correlate absorbed doses with hematologic toxicity. Methods Eight patients with relapsed CD37+ indolent B-cell non-Hodgkin lymphoma were included for RM dosimetry. Hybrid SPECT and CT images were used to estimate the activity concentration in the RM of L2-L4. Pharmacokinetic parameters were calculated after measurement of the 177Lu-lilotomab satetraxetan concentration in blood samples. Adverse events were graded according to the Common Terminology Criteria for Adverse Events, version 4.0. Results The mean absorbed doses to RM were 0.9 mGy/MBq for arm 1 (lilotomab+) and 1.5 mGy/MBq for arm 2 (lilotomab-). There was a statistically significant difference between arms 1 and 2 (Student t test, P = 0.02). Total RM-absorbed doses ranged from 67 to 127 cGy in arm 1 and from 158 to 207 cGy in arm 2. For blood, the area under the curve was higher with lilotomab predosing than without (P = 0.001), whereas the volume of distribution and the clearance of 177Lu-lilotomab satetraxetan was significantly lower (P = 0.01 and P = 0.03, respectively). Patients with grade 3/4 thrombocytopenia had received significantly higher radiation doses to RM than patients with grade 1/2 thrombocytopenia (P = 0.02). A surrogate, non-imaging-based, method underestimated the RM dose and did not show any correlation with toxicity. Conclusion Predosing with lilotomab reduces the RM-absorbed dose for 177Lu-lilotomab satetraxetan patients. The decrease in RM dose could be explained by the lower volume of distribution. Hematologic toxicity was more severe for patients receiving higher absorbed radiation doses, indicating that adverse events possibly can be predicted by the calculation of absorbed dose to RM from SPECT/CT images.

177Lu-lilotomab satetraxetan is a novel antibody radionuclide conjugate (ARC) currently tested in a phase 1/2a first-in-human dosage escalation trial for patients with relapsed CD37+ indolent non-Hodgkin’s lymphoma. The aim of this work was to develop dosimetric methods and calculate tumor absorbed radiation doses for patients treated with 177Lu-lilotomab satetraxetan. Methods: Patients were treated at escalating injected activities (10, 15 and 20 MBq/kg) of 177Lu-lilotomab satetraxetan and with different pre-dosing; with or without 40 mg unlabeled lilotomab. Eight patients were included for the tumor dosimetry study. Tumor radioactivity concentrations were calculated from Single Photon Emission Computer Tomography (SPECT) acquisitions at multiple time points, and tumor masses were delineated from corresponding Computer Tomography (CT) scans. Tumor absorbed doses were then calculated using the OLINDA sphere model. To perform voxel dosimetry, the SPECT/CT data and an in-house developed MATLAB program were combined to investigate the dose rate homogeneity. Results: Twenty-six tumors in 8 patients were ascribed a mean tumor absorbed dose. Absorbed doses ranged from 75 cGy to 794 cGy with a median of 268 cGy across different dosage levels and different pre-dosing. A significant correlation between the dosage level and tumor absorbed dose was found. Twenty-one tumors were included for voxel dosimetry, and parameters describing dose-volume coverage calculated. The investigation of intra-tumor voxel doses indicates that mean tumor dose is correlated to these parameters. Conclusion: Tumor absorbed doses for patients treated with 177Lu-lilotomab satetraxetan are comparable to doses reported for other radioimmunotherapy compounds. Although the inter-tumor variability was considerable, a correlation between tumor dose and patient dosage level was found. Our results indicate that mean dose may be used as the sole dosimetric parameter on the lesion level.

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 Each year more than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US. The standard of care is immuno-chemotherapy with alkylating agents in combination with an anti-CD20 monoclonal antibody (mAb). While immuno-chemotherapy regimens are initially effective in inducing responses, most patients inevitably relapse and the same therapies show decreasing efficacy with repeated administration. In recent years, CD37 has been considered as a target for B-cell malignancies. CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Therapies targeting CD37 expressing cells may become a useful alternative to CD20 targeting agents. Alpha-emitting radionuclides have demonstrated good potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm). The absorbed energy cause irreparable DNA double-strand breaks and localized cytotoxicity while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-emitting radioisotope 212Pb. Materials and Methods Dose-dependent efficacy and tolerability of a single-dose 212Pb-NNV003 treatment was evaluated using escalating activity levels in human disseminated models of Burkitt's lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. A study of the tolerability of 212Pb-NNV003 was performed in R2G2 mice without tumor prior to the efficacy studies. Results 212Pb-NNV003 displays a favorable toxicity profile in tumor-free mice single intravenous dose injections up to 15 µCi showing 100 % survival 4 weeks post-injection. No acute hematological toxicity was observed and animals who received 5, 10 or 15 μCi doses of 212Pb-NNV003 presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered by 20 days after injection. A single intravenous dose of 10, 15 or 20 µCi of 212Pb-NNV003 lead to 70 %, 90 % and 100 % of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection (Figure 1). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively. A single intravenous dose of 2.5, 5 and 7.5 µCi 212Pb-NNV003 lead to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection (Figure 2). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively. Conclusion The results of preclinical studies suggest that TAT using 212Pb-NNV003 is a safe and effective method for the treatment of CD37 positive CLL and NHL. Disclosures Saidi: Orano Med: Employment. Maaland:Nordic Nanovector: Employment. Torgue:Orano Med: Employment, Membership on an entity's Board of Directors or advisory committees. Heyerdahl:Nordic Nanovector: Employment, Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties.

Background CD37 is an internalizing transmembrane glycoprotein widely expressed on mature B-cells and B-cell malignancies. The next generation anti-CD37 radioimmunoconjugate (RIC) 177Lu-lilotomab satetraxetan (Betalutin®), containing the beta-emitting radionuclide lutetium-177, is currently being tested as one-time injection therapy in a clinical phase 2b trial for follicular lymphoma (FL) and phase 1 trial for diffuse large B-cell lymphoma (DLBCL). We recently reported the systems biology analysis of germinal center like (GCB) and activated B-cell like (ABC) DLBCL cell lines to CD37 targeting radioimmunotherapy (RIT) (Melhus, et al., PF642 at EHA23, Stockholm, 2018). 177Lu-Lilotomab satetraxetan showed generally promising activity against DLBCL cell lines, but treatment resistance was evident in a subset of cell lines. Resistance was not attributable to differences in CD37 expression nor correlated to genetic hallmarks of aggressiveness such as aberrations in TP53, BCL2 and MYC. In the present study we aimed at identifying resistance reversing drug combinations with 177Lu-lilotomab satetraxetan in two RIT resistant aggressive ABC-DLBCL cell lines. Materials and methods U-2932 and RIVA cells were treated for 18 hours with 177Lu-lilotomab satetraxetan (600 MBq/mg; 1 or 0.5 µg/ml), washed, and seeded on micro-well plates pre-printed with a drug library of 384 approved anti-cancer compounds at 10, 100, and 1000 nM (screen). For validation experiments, cells were treated with three different doses of 177Lu-lilotomab satetraxetan as described above and seeded on pre-printed plates with gradients of candidate hits ranging from 1-1280 nM. Cell viability was monitored at days 3 to 6 post seeding using a cell viability assay (RealTime-Glo™). Drug combinations were scored using Bliss and Chou-Talalay theorems. Results Compounds scoring in both cell lines included inhibitors targeting cell cycle kinases that regulate transition through mitosis, such as CDK1/2, AURKA/B, PLK1, as well as enzymes with important roles in DNA integrity surveillance and repair, such as topoisomerases. If the compounds also have or are being tested in clinical trials for treatment of non-Hodgkin lymphoma (NHL) they were considered candidates for further studies. PLK1 inhibitors BI2536 and GSK461364 potently inhibited cell proliferation at < 20 nM, with additional, but not synergistical benefit of combination with 177Lu-Lilotomab satetraxetan. The AURKA/B inhibitor Alisertib synergistically inhibited cell proliferation in combination treatment, but showed adverse effect at concentrations >160 nM, possibly owing to inhibition of a secondary target. The pan-CDK inhibitor JNJ-7706621 showed little effect alone but strongly synergized with 177Lu-lilotomab satetraxetan in inhibition of proliferation at doses > 80 nM. U-2932 and RIVA cells arrested in G2-phase with elevated DNA damage (flow cytometry; P-γ-H2AX and DNA staining) 18 hrs after treatment with 177Lu-lilotomab satetraxetan. Resistance reversal by anti-mitotics may thus underlie concomitant radiation during inhibitor mediated M-phase arrest of cells that eventually evaded G2-phase arrest. Conclusion In summary, combinatorial drug screening identified cell cycle kinase inhibitors as promising partners for combination treatment of aggressive DLBCL with 177Lu-lilotomab satetraxetan, warranting further exploration in pre-clinical models. Figure. Figure. Disclosures Rødland: Nordic Nanovector ASA: Patents & Royalties, Research Funding. Melhus:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Syljuåsen:Nordic Nanovector ASA: Patents & Royalties, Research Funding. Patzke:Nordic Nanovector ASA: Employment, Patents & Royalties.

Purpose: The purpose of this study was to investigate the effect of α-particle-emitting {sup 227}Th-trastuzumab radioimmunotherapy on tumor vasculature to increase the knowledge about the mechanisms of action of {sup 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{sup 3} (mean ± SEM) were treated with a single injection of either {sup 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 {sup 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 k{sub ep}, the rate constant of diffusion from the extravascular extracellular space to the plasma (P

The aim of the present study was to compare the biodistribution, normal tissue toxicity and therapeutic effect of the alpha-particle emitting 227 Th-trastuzumab and the beta-particle emitting 177 Lu-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 227 Th-trastuzumab (200, 400, 600 or 1000 kBq/kg), 177 Lu-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 227 Th-trastuzumab and 40 MBq/kg of 177 Lu-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 227 Th-trastuzumab and 200 MBq/kg of 177 Lu-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 177 Lu-trastuzumab, indicating a relative biological effect (RBE) of 2.8 for 227 Th-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 177 Lutrastuzumab than with 227 Th-trastuzumab, which indicates that the therapeutic index was three times higher for 177 Lutrastuzumab than for 227 Th-trastuzumab. Conclusion: In this xenograft model the RBE was higher for 227 Th-trastuzumab than for 177 Lu-trastuzumab, while the therapeutic index of 177 Lu-trastuzumab was superior to that of 227 Th-trastuzumab.

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.

International audience; Current preclinical dosimetric models often fail to take account of the complex nature of absorbed dose distribution typical of in vitro clonogenic experiments in targeted radionuclide therapy. For this reason, clonogenic survival is often expressed as a function of added activity rather than the absorbed dose delivered to cells/cell nuclei. We designed a multi-cellular dosimetry model that takes into account the realistic distributions of cells in the Petri dish, for the establishment of survival curves as a function of the absorbed dose. General-purpose software tools were used for the generation of realistic, randomised 3D cell culture geometries based on experimentally determined parameters (cell size, cell density, cluster density, average cluster size, cell cumulated activity). A mixture of Monte Carlo and analytical approaches was implemented in order to achieve as accurate as possible results while reducing calculation time. The model was here applied to clonogenic survival experiments carried out to compare the efficacy of Betalutin®, a novel 177Lu-labelled antibody radionuclide conjugate for the treatment of non-Hodgkin lymphoma, to that of 177Lu-labelled CD20-specific (rituximab) and non-specific antibodies (Erbitux) on lymphocyte B cells. The 3D cellular model developed allowed a better understanding of the radiative and non-radiative processes associated with cellular death. Our approach is generic and can also be applied to other radiopharmaceuticals and cell distributions.

The aim of this study was to investigate the role of serotonin and protein 53 (p53) status of the cells in the radiation-induced bystander effects (RIBE).The radiation-induced bystander response was investigated in human MCF-7 breast cancer cells and human HCT116 colorectal cancer cells employing medium-transfer experiments and micronuclei (MN) induction as an end-point. Irradiated cell conditioned medium (ICCM) from cells exposed to α-particle or γ-radiation was filtered and transferred to unirradiated cells 2 h following irradiation. MCF-7 cells were irradiated with 0.5 Gy α-particles, while HCT116 p53(+/+) and HCT116 p53(-/-) cells were irradiated with 0.5 Gy γ-radiation.Bystander MCF-7 cells, recipient of ICCM from 0.5 Gy α-particle irradiated MCF-7 cells grown in high serotonin conditions showed a modest but significant increase in MN, while MCF-7 cells receiving ICCM with low serotonin levels did not show any bystander effect. Added serotonin (100 ng/ml) led to a bystander effectin HCT116 p53(-/-) cells recipient of ICCM from 0.5 Gy γ-irradiated HCT116 p53(+/+) cells, but had no effect when the ICCM was from γ-irradiated HCT116 P53(-/-) cells.The results indicate that serotonin levels in the medium play a role in the RIBE and that there may be an interaction between the role of serotonin and the p53 status of the irradiated cells.

The objective of this study was to demonstrate the radiation-induced bystander effect (RIBE) in human F11 fibroblasts using micronucleus (MN) formation as an end-point and to investigate transcriptional responses towards low doses of α-particle radiation in irradiated and bystander fibroblasts employing genome-wide microarray analysis.The MN frequency was investigated in unirradiated bystander cells recipient of growth medium from α-particle irradiated (0, 0.1, 0.5 and 2 Gy) immortalized human F11 fibroblasts. The irradiated conditioned medium (ICM) was transferred from irradiated to bystander fibroblasts 2 h after irradiation. Microarray analysis was performed in 0.1 Gy α-particle irradiated cells, unirradiated bystander cells and appropriate controls at different time points after ICM transfer. Microarray data analysis was performed at the single-gene level and gene-set level.MN were induced in unirradiated fibroblasts recipient of ICM harvested from 0.1 Gy α-particle irradiated cultures, but not from cultures irradiated with 0.5 or 2 Gy. Fibroblasts irradiated with 0.1 Gy showed repression of 26 genes 4 h after being irradiated. However, no significantly altered genes were found in bystander fibroblasts at any of the time points used in this study. Gene-set enrichment analysis showed that pathways such as ribosome, protein export, proteasome and protein 53 (p53) signaling were enriched in irradiated cells. However, bystander cells were enriched only in the lysosome pathway 24 h after receiving ICM, indicating persistence of the signals received from the irradiated cells.MN formation was induced in bystander fibroblast receiving medium from 0.1 Gy α-particle irradiated fibroblasts, but this was accompanied with only minor transcriptional responses.

Purpose 177Lu-lilotomab satetraxetan is an antibody-radionuclide conjugate therapy for non–Hodgkin lymphoma, which is currently in a phase 1/2-study. The primary toxicity is transient myelosuppression. The aim of this work was to determine if a linear model including red marrow absorbed dose and various clinical factors can be used to predict myelosuppression. Methods Patient characteristics and dosimetric data for 17 patients were included. All patients had no or minor bone marrow infiltration. The percentage reduction of neutrophils and platelets at nadir compared to baseline was used as to indicate myelosuppression. Platelets and neutrophils were analysed separately. The Common Terminology Criteria version 4.0 were used to grade thrombocytopenia and neutropenia. A total of six factors believed to affect bone marrow reserve were tested: Red marrow absorbed dose, age at treatment, baseline cell-counts, history of prior external beam radiation therapy, total number of previous chemotherapy treatments (including rituximab) and elapsed time since the last chemotherapy treatment. Predictors were found using ordinary least square fitting, testing all possible subsets of variables. A significance level of 0.05 was used. A leave 1-out analysis was used to validate the final model. Results The typical patient, as described by the mean and standard deviation, was 69 (+/−10) years of age at treatment, had previously been treated 2.8 (+/−1.8) times with chemotherapy and might have previously received external beam radiation therapy (5/17 patients). Elapsed time since the last chemotherapy was 20.9 (+/−17.2) months. Mean red marrow absorbed dose was 1.45 (+/−0.45) Gy. Of the sub-sets of predictors investigated, only a model consisting solely of the red marrow absorbed dose was significant (p = 0.048 for platelets and 0.027 for neutrophils). Model coefficients indicate total loss of both platelets and neutrophils at 3 Gy. The leave-1 out analysis resulted in correct prediction of CTCAE-grading +/−1 grade for 76.5 % (13/17) and 88.2 % (15/17) of the patients for thrombocytopenia and neutropenia, respectively. Conclusions A linear model with red marrow absorbed dose as the only parameter predicted myelosuppression in 177Lu-lilotomab satetraxetan treated patients.

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.

7,8-dihydro-8-oxo-guanine (8-oxoG) is a mutagenic DNA lesion that is induced by ultraviolet A (UVA) radiation. 8-oxoG results in increased frequency of GC-->TA transversion mutations. UVA-induced mutant frequency was measured in the guanine phosphoribosyl transferase (gpt) gene of Chinese hamster ovary cells (AS52) that were stably transfected to overexpress the hOGG1 protein, the human DNA repair glycosylase for 8-oxoG. This mutant frequency was compared with ultraviolet A-induced mutant frequency in AS52 cells stably transfected with the same vector without the hOGG1 gene. The mutant frequency was significantly decreased in the hOGG1 overexpressing cells irradiated with 300 and 400 kJ/m2 ultraviolet A radiation, corresponding to 25% and 10% cell survival, respectively. The hOGG1 overexpressing cells repaired oxidative DNA lesions three times faster than the vector only cells as measured by a semi-automated alkaline elution assay using FPG enzyme, the bacterial OGG1 analogue, to cut DNA at oxidative base modifications. Thus, the lower mutation frequency in UVA-induced mutations of the hOGG1 overexpressing cells may be related to the increased repair of 8-oxoG. No GC-->TA mutations were detected in the UVA-irradiated hOGG1 overexpressing cells. The results suggest a link between the 8-oxoG lesion and UVA-induced mutagenesis. We propose that hOGG1 has a role in maintaining genomic stability in mammalian cells after oxidative stress.

Confluent layers of MDCK II cells were treated with four different photosensitizers (a purified version of hematoporphyrin derivative [Photofrin], tetra(3-hydroxyphenyl)porphine [3-THPP], meso-tetra(4-sulphonatophenyl)porphine [TPPS4] and ALA-induced Protoporphyrin IX) and irradiated with blue light, with UVA without exogenous photosensitizers, or incubated with the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone and 2-deoxy-D-glucose. Necrotic and apoptotic cells were detected about 4 h later by fluorescence microscopy. Dead cells appeared in distinct clusters in the confluent layers. The number of dead cells in these clusters was determined by manual counting and image analysis. Forty-one of the 43 experimental distributions of dead cells in clusters were found to be significantly different from a Monte Carlo simulation of the distribution of independently inactivated cells. However, a Monte Carlo simulation model, assuming that each dead cell increased the probability of inactivation of adjacent cells, fitted 34 of the 43 observed distributions of dead cells in clusters, indicating a significant bystander effect for all the investigated treatments. The bystander-effect model parameter, defined as a cell's increase in probability of dying when it has dead neighbors, was significantly lower for 3-THPP-PDT and TPPS4-PDT than for Photofrin-PDT, ALA-PDT and treatment with metabolic inhibitors.

Pheomelanin and pheomelanin precursors have been implicated as risk factors for induction of melanoma by ultraviolet radiation. The pheomelanin precursor, 5-S-cysteinyldopa, has been shown to sensitise DNA to oxidative damage by ultraviolet radiation. We here show that 5-S-cysteinyldopa significantly protects melanocytes from membrane damage (permeability) induced by ultraviolet A radiation. Thus, 5-S-cysteinyldopa, may at the same time sensitise DNA and protect membranes from damage induced by ultraviolet radiation.

Ultraviolet irradiation of cells can induce a state of genomic instability that can persist for several cell generations after irradiation. However, questions regarding the time course of formation, relative abundance for different types of ultraviolet radiation, and mechanism of induction of delayed mutations remain to be answered. In this paper, we have tried to address these questions using the hypoxanthine phosphoribosyl transferase (HPRT) mutation assay in V79 Chinese hamster cells irradiated with ultraviolet A or B radiation. Delayed HPRT(-) mutations, which are indications of genomic instability, were detected by incubating the cells in medium containing aminopterin, selectively killing HPRT(-) mutants, and then treating the cells with medium containing 6-thioguanine, which selectively killed non-mutant cells. Remarkably, the delayed mutation frequencies found here were much higher than reported previously using a cloning method. Cloning of cells immediately after irradiation prevents contact between individual cell clones. In contrast, with the present method, the cells are in contact and are mixed several times during the experiment. Thus the higher delayed mutation frequency measured by the present method may be explained by a bystander effect. This hypothesis is supported by an experiment with an inhibitor of gap junctional intercellular communication, which reduced the delayed mutation frequency. In conclusion, the results suggest that a bystander effect is involved in ultraviolet-radiation-induced genomic instability and that it may be mediated in part by gap junctional intercellular communication.

Background Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. Method We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. Results Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. Conclusions As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy. © 2004 Wiley-Liss, Inc.

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.

Madison Darby canine kidney II (MDCK II) cells were seeded out at two different densities and incubated with 125 micrograms/mL of the photosensitizer meso-tetra(4-sulfonatophenyl)porphine (TPPS4) for 18 h, washed and irradiated with blue light. Four hours later the cells were studied by fluorescence microscopy. Apoptotic cells were detected by virtue of the distinct condensation and fragmentation of chromatin, and necrotic cells were detected by uptake of propidium iodide. In addition apoptosis was measured by the TdT assay. The fraction of apoptotic cells and the fraction of necrotic cells were determined for both cell densities at various levels of survival. With < 55% total cell death the apoptotic fraction was significantly higher for cells in confluent monolayers than for cells growing in microcolonies at equitoxic doses. Confluent cells were 2.9 times more sensitive than cells in microcolonies partly due to a 1.5 times higher uptake of TPPS4 in monolayer cells. The difference in mode of cell death for the different cell densities was not related to any observable difference in subcellular localization pattern of TPPS4 at equitoxic doses of photodynamic treatment.