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

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% CO2.

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 molecules (Nanogam®Sanquin) were diluted with 2% FCS in PBS to 20 µg / mL and incubated with 2 × 105 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 receiver expression was expressed as mean fluorescent intensity (MFI).

Radiolabeling of NNV003 and IgG control for animal studies

NNV003 antibody (IgG1, mouse variable regions, κ, and human constant region, κ; Nordic Nanovector) was conjugated to TFP-N-sucDf (ABX GmbH) and subsequently radiolabeled with 89Zr as described previously22. To date, several 89Zr-labeled antibodies are produced according to this methodology and were evaluated in animals and patients without any sign of toxicity16,17,23,24. 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 89Zr oxalate (Perkin Elmer). RCP of [89Zr]Zr-N-sucDf-NNV003 was determined by trichloroacetic acid precipitation test17. Furthermore, NNV003 antibody was conjugated to p-SCN-Bn-DOTA (Macrocyclics) and subsequently radiolabeled using 177Lu chloride (Perkin Elmer) as described previously4.5.

Non-specific IgG control molecule was conjugated with a 50-fold molar excess of p-SCN-Bn-DTPA (Macrocyclics) as described previously25. Radiolabeling of IgG-DTPA was performed using 111In chloride (Mallinckrodt) by incubation for 1–2 h in ammonium acetate pH 5.5. Radiochemical purity of [111In]DTPA-IgG was assessed by instant thin-layer chromatography using 0.1 M citrate buffer pH 6.0 as eluent.

[89Zr]Zr-N-sucDf-NNV003 quality control

[89Zr]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 a 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 × 106 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 [89Zr]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 [89Zr]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.

Animal studies

All experiments were performed in accordance with relevant guidelines and regulations. All methods were reported in accordance with recommendations in the ARRIVE guidelines. Animal studies involving [89Zr]Zr-N-sucDf-NNV003 were approved by the Institutional Animal Care and Use Committee of the University Medical Center Groningen. Male BALB / c OlaHsd-Foxn1no mice (Envigo) 8–10 weeks of age were inoculated with 10 × 106 either REC1 or RAMOS cells. Murine NK cells were depleted to enhance tumor take-rate. This was achieved by administering the mice anti-asial GM1 treatment on 1 day before and 4, 11, 18 and 25 days after tumor inoculation. When tumors measured a volume of at least 200 mm3 (~ 14 days post inoculation for REC1 tumors and ~ 19 days for RAMOS tumors), mice received intravenous injections of 10 μg (~ 5 MBq) [89Zr]Zr-N-sucDf-NNV003 supplemented with either 0, 15 or 90 µg of unlabeled NNV003 and co-injected with an equal total protein dose (~ 1 MBq) of [111In]In-DTPA-IgG control (n = 5–6 mice per group). By co-injection of [89Zr]Zr-N-sucDf-NNV003 and [111In]In-DTPA-IgG, tumor uptake and biodistribution results 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 / cm3 tissue density, and heart using AMIDE medical image data examiner software v1.0.4. Tracer uptake was quantified as SUVmean and SUVmax, 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 [177Lu]Lu-DOTA-NNV003 were approved by the Norwegian Animal Research Authority. Biodistribution of [177Lu]Lu-DOTA-NNV003 was studied in the RAMOS tumor model. Female Hsd: Athymic Nude-Foxn1no mice (Envigo) 7–11 weeks of age were subcutaneously injected with 100 RAML 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) [177Lu]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, fixed 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.

Production and stability testing of clinical grade [89Zr]Zr-N-sucDf-NNV003

NNV003 was conjugated to TFP-N-sucDf at a 1: 2 molar ratio and subsequently radiolabeled with GMP-grade 89Zr. Quality control was performed on both NNV003-N-sucDf intermediate product and [89Zr]Zr-N-sucDf-NNV003 final product. This included analysis on appearance, yield, purity, concentration, pH, radiochemical purity, residual solvents, sterility, endotoxin content and IRF. Analytical procedures were validated to demonstrate suitability for use in quality control testing of NNV003-N-sucDf and [89Zr]Zr-N-sucDf-NNV003. The production processes for NNV003-N-sucDf and [89Zr]Zr-N-sucDf-NNV003 were validated according to GMP guidelines by the production of three consecutive validation batches.

NNV003-N-sucDf intermediate product was stored in sterile vials (BioPure) at – 80 ° C. Stability of NNV003-N-sucDf was analyzed at 0, 1, 3 and 6 months after production. [89Zr]Zr-N-sucDf-NNV003 final product was stored in sterile vials (BioPure) at 2–8 ° C and stability was analyzed at 0 and 96 h. Stability of [89Zr]Zr-N-sucDf-NNV003 in the syringe at RT was analyzed at 0 and 4 h. Stability tests consisted of quality control according to release specifications.

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 [89Zr]Zr-N-sucDf-NNV003 and [177Lu]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.

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