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.

Higher spleen uptake was observed for [ 177 Lu]Lu-DOTA-NNV003 compared to [ 89 Zr]Zr-N-sucDf-NNV003 (5.6 vs. 2.1%ID/g; P < 0.05). Murine CD37 is expected to be expressed on B cells present in the spleen, however NNV003 is not cross-reactive with murine CD37. Furthermore, [ 89 Zr]Zr-N-sucDf-NNV003 spleen uptake was similar to [ 111 In]In-DTPA-IgG, indicating this uptake is not CD37-mediated. In mice, the spleen plays an important role in antibody pharmacokinetics due to its high blood flow and loose capillaries, but also due to the    , is currently in phase II for patients with anti-CD20 refractory follicular lymphoma and has previously been studied in phase I/IIa clinical trials in patients with relapsed, CD37-positive, indolent and aggressive NHL (ClinicalTrials.gov Identifier: NCT01796171, NCT02658968) 3 . These studies showed high RIT uptake in tumors, but also in red bone marrow, liver, spleen, and kidneys 9,10 . This may be explained by CD37 expression on mature, normal B cells in these tissues. Predosing with unlabeled lilotomab significantly reduced the absorbed radiation dose in healthy CD37expressing tissues due to 177 Lu-lilotomab satetraxetan 9 . Also, 177 Lu-lilotomab satetraxetan dosimetry, biodistribution, and tumor targeting were improved by lilotomab predosing compared with rituximab predosing or no predosing 3 . Informing clinicians on whether tumors are effectively targeted by [ 177 Lu]Lu-DOTA-NNV003 and gaining insight into the amount of cold antibody dose required to saturate CD37 expression in healthy tissues are essential to optimize future [ 177 Lu]Lu-DOTA-NNV003 RIT dose-regimens. We showed that [ 89 Zr]Zr-N-sucDf-NNV003 PET imaging can serve as a surrogate for [ 177 Lu]Lu-DOTA-NNV003 RIT whole-body distribution and represents a potentially attractive tool to assess distribution to tumors and healthy tissues.
The combined approach of RIT and diagnostics such as molecular imaging may support precise cancer therapy in both palliative and curative settings 19 . SPECT/CT imaging was routinely used for assessing biodistribution  www.nature.com/scientificreports/ and dosimetry of 90 Y-and 131 I-based RIT antibodies in the early 2000s 20,21 , but low quantities of γ-photons emitted by 177 Lu complicate quantification. As a therapeutic agent, even low dose pre-treatment imaging of 177 Lu may result in local toxicity, while this risk is limited for the low energy β + -rays of PET radioisotopes. In this respect, gallium-68 ( 68 Ga)/ 177 Lu is a commonly used theranostic pair for studying receptor expression or drug distribution. However, 68 Ga, given its relatively short physical half-life of 67.6 min, provides no insight in internalizing properties of an antibody. As internalization is an essential factor for both efficacy and toxicity of 177 Lu-based RIT agents, longer-lived PET radioisotopes such as 89 Zr may better reflect 177 Lu RIT in vivo behavior. In a recent study, response at the tumor lesion level after treatment with 177 Lu-lilotomab satetraxetan was evaluated by FDG PET/CT and did not correlate with tumor-absorbed dose 11 . They hypothesized that the combination regimen of radiolabeled and cold antibodies might preclude such a correlation. In our study, we were able to quantitatively visualize tumor uptake in the presence of unlabeled antibody using [ 89 Zr]Zr-N-sucDf-NNV003 PET imaging. In patients with relapsed B cell NHL, a pre-therapy scan with 89 Zr-ibritumomab tiuxetan was used to predict radiation dosimetry during 90 Y-ibritumomab tiuxetan therapy 14 . Importantly, 89 Zr-ibritumomab tiuxetan whole-body distribution was not affected by simultaneous 90 Y-ibritumomab tiuxetan therapy. These findings emphasize the potential of an [ 89 Zr]Zr-N-sucDf-NNV003 pre-therapy scan to predict CD37-targeting by  www.nature.com/scientificreports/

Materials and methods
Cell lines and flow cytometry experiments. Human CD37-expressing cell lines RAMOS (Burkitt's lymphoma) and REC1 (Mantle cell lymphoma) were obtained from the American Type Culture Collection. RAMOS and REC1 cell lines were tested and authenticated in July and October 2019 respectively using short tandem repeat profiling. Cells were cultured in Roswell Park Memorial Institute (RPMI) medium, supplemented with 10% fetal calf serum (FCS) and incubated at 37 °C in a humidified atmosphere with 5% CO 2 . CD37 expression by RAMOS and REC1 cells was determined by flow cytometry. Cells were harvested in 2% FCS in phosphate-buffered saline (PBS) and kept on ice prior to use. NNV003 and non-specific human IgG control molecule (Nanogam ® , Sanquin) were diluted with 2% FCS in PBS to 20 µg/mL and incubated with 2 × 10 5 cells/mL for 1 h at 4 °C. Bound NNV003 and control antibodies were detected using a phycoerythrin-conjugated goat anti-human IgG secondary antibody (SouthernBiotech; 2040-09) diluted 1:50 with 2% FCS in PBS and analyzed on a BD Accuri C6 flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v10 (Tree Star) and surface receptor expression was expressed as mean fluorescent intensity (MFI).
Radiolabeling of NNV003 and IgG control for animal studies. NNV003 antibody (IgG 1 , mouse variable regions, κ, and human constant region, κ; Nordic Nanovector) was conjugated to TFP-N-sucDf (ABX GmbH) and subsequently radiolabeled with 89 Zr as described previously 22 . To date, several 89 Zr-labeled antibodies are produced according to this methodology and were evaluated in animals and patients without any sign of toxicity 16,17,23,24 . In short, NNV003 was incubated with a twofold molar excess of TFP-N-sucDf at pH 9.0-9.5. After incubation for 1 h at room temperature (RT), pH was set to 4.0-4.5. Ethylenediaminetetraacetic acid (EDTA; Hospital Pharmacy UMCG) 25 mg/mL was added and incubated for 30 min at 35 °C to transchelate Fe(III) from the TFP-N-sucDf hydroxamate groups. NNV003-N-sucDf was subsequently purified using a Vivaspin-2 concentrator (Sartorius GmbH), aliquoted and stored at − 80 °C until use. On the day of tracer injection, NNV003-N-sucDf was radiolabeled using GMP-grade 89 Zr oxalate (Perkin Elmer). RCP of [ 89 Zr]Zr-N-sucDf-NNV003 was determined by trichloroacetic acid precipitation test 17 . Furthermore, NNV003 antibody was conjugated to p-SCN-Bn-DOTA (Macrocyclics) and subsequently radiolabeled using 177 Lu chloride (Perkin Elmer) as described previously 4,5 .
Non-specific IgG control molecule was conjugated with a 50-fold molar excess of p-SCN-Bn-DTPA (Macrocyclics) as described previously 25 . Radiolabeling of IgG-DTPA was performed using 111 In chloride (Mallinckrodt) by incubation during 1-2 h in ammonium acetate pH 5.5. Radiochemical purity of [ 111 In]In-DTPA-IgG was assessed by instant thin-layer chromatography using 0.1 M citrate buffer pH 6.0 as eluent.
[ 89 Zr]Zr-N-sucDf-NNV003 quality control. [ 89 Zr]Zr-N-sucDf-NNV003 purity and concentration were determined by size-exclusion high-performance liquid chromatography (SE-HPLC). A Waters SE-HPLC system was equipped with a dual-wavelength absorbance detector, in-line radioactivity detector and TSK-Gel SW column G3000SWXL 5 µm, 7.8 mm (Joint Analytical Systems GmbH). PBS (9.0 mM sodium phosphate, 1.3 mM potassium phosphate, 140 mM sodium chloride, pH 7.2; Hospital Pharmacy UMCG) was used as mobile phase at a flow of 0.7 mL/min. NNV003-N-sucDf IRF was determined on human CD37-expressing Burkitt's lymphoma RAMOS cells. Cells were harvested in PBS with 0.5% bovine serum albumin (BSA), diluted to 75 × 10 6 cells and 0.2 mL added per tube to a total of 5 tubes. CD37-specific binding sites were blocked in 2 tubes by incubation with 20 µg NNV003-N-sucDf for 15 min at RT. Subsequently, 8 ng [ 89 Zr]Zr-N-sucDf-NNV003 (~ 9000 counts per minute) was added to each tube and incubated for 1 h at RT. Tubes were counted in a calibrated well-type gamma counter (LKB instruments), subsequently spun down and washed with 0.5% BSA in PBS for three times, after which tubes were counted again. IRF was expressed as the average percentage of CD37-bound [ 89 Zr]Zr-N-sucDf-NNV003 as a fraction of the percentage of total activity added in non-blocked tubes corrected for non-specific binding in the blocked tubes. Acceptance criteria were set at ≤ 5% non-specific binding in blocked tubes and NNV003-N-sucDf IRF at ≥ 0.8. can be compared within the same animal, thereby providing valid results on target-specific uptake. Also, the number of animals required for these studies can be reduced using this strategy. Mice underwent microPET scanning at 1, 3 and 5 days post injection (pi), followed by ex vivo biodistribution.
MicroPET scans were performed using a Focus 220 rodent scanner (CTI Siemens). Scans were reconstructed using a 2-dimensional ordered-subset expectation maximization reconstruction algorithm with Fourier rebinning, 4 iterations, and 16 subsets. Data sets were corrected for decay, random coincidences, scatter, and attenuation. For in vivo quantification, regions of interest were drawn for tumor based upon ex vivo weight, assuming 1 g/cm 3 tissue density, and heart using AMIDE medical image data examiner software v1.0.4. Tracer uptake was www.nature.com/scientificreports/ quantified as SUV mean and SUV max , calculated from the mean or maximum activity in the region of interest and divided by the injected dose per gram body weight. For ex vivo biodistribution studies, relevant organs were collected, weighed and counted using a calibrated well-type gamma counter. Standards of injected tracer were included to correlate measured counts to the percentage of injected tracer activity. After correction for decay, ex vivo tissue uptake was expressed as the percentage of injected radioactivity dose per gram tissue (%ID/g) and standardized uptake value (SUV) by correcting for injected dose and mouse body weight. Animal experiments involving [ 177 Lu]Lu-DOTA-NNV003 were approved by the Norwegian Animal Research Authority. Biodistribution of [ 177 Lu]Lu-DOTA-NNV003 was studied in the RAMOS tumor model. Female Hsd:Athymic Nude-Foxn1 nu mice (Envigo) 7-11 weeks of age were subcutaneously injected with 100 µL RAMOS cell suspension from a donor mouse xenograft to enhance tumor take-rate. Mice received intravenous injections of 4-10 µg (0.5-0.9 MBq) [ 177 Lu]Lu-DOTA-NNV003 (IRF 74.4-81.7%), followed by tissue collection and ex vivo biodistribution analysis at 1 h, 6 h, 1 day and 3 days pi (n = 4 mice per group).
Ex vivo tissue preparation and immunohistochemistry. For ex vivo tissue analysis, formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were prepared. FFPE blocks were sliced into 4 µm tumor tissue sections, fixated on microscope slides and dried overnight at 60 °C. For CD37 immunohistochemistry, tumor tissue sections were deparaffinized in xylene and rehydrated. Heat-induced antigen retrieval was performed in 10 mM citrate (pH 6.0) for 15 min at 95-100 °C. Endogenous peroxidase was blocked by 10-min incubation with 10% hydrogen peroxide in PBS. Slides were incubated with rabbit anti-human CD37 antibody (Proteintech; 21044-1) or rabbit IgG antibody control (Abcam; ab172730) diluted to 0.8 µg/mL in 1% BSA in PBS for 1 h at RT. Thereafter, slides were incubated with Dako EnVision horseradish peroxidase system (Agilent Technologies) for 30 min at RT, followed by 10-min incubation with diaminobenzidine chromogen. Hematoxylin counterstaining was applied routinely. For histological analysis of tumors, hematoxylin/eosin staining was performed on subsequent tissue sections. Digital scans of slides were acquired by a Hamamatsu NanoZoomer 2.0-HT multi-slide scanner and analyzed with NanoZoomer Digital Pathology viewer software. Statistical analysis. Data were analyzed for statistical significance in GraphPad Prism v7.0 using the Mann-Whitney U test for non-parametric data followed by Bonferroni post-test correction for comparison of more than two groups. Ex vivo biodistribution of [ 89 Zr]Zr-N-sucDf-NNV003 and [ 177 Lu]Lu-DOTA-NNV003 were compared with Welch's t-test for unequal variances. Correlation was assessed by Spearman's rank-order correlation test. In vitro experiments were repeated at least 3 times. p values < 0.05 were considered significant.