The human colorectal carcinoma cell line, HCT116, previously demonstrated to express moderate amounts of CD44v6 (16) was purchased from ATCC (Manassas, VA, USA) and cultured in McCoy's modified Eagle's medium (Biochrom Kg, Berlin, Germany) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine and 1% antibiotics (100 IU penicillin and 100 µg/ml streptomycin (all from Biochrom Kg). The human squamous cell carcinoma cell line, UM-SCC-74B, previously shown to express a low amount of CD44v6 (24,25) was kindly provided by Professor T.E. Carey (University of Michigan, Ann Arbor, MI, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Biochrom Kg) with the same supplements, and 1% non-essential amino acids (Thermo Fisher Scientific, Uppsala, Sweden). The cells were incubated at 37°C in a 5% CO₂ atmosphere and cultured for no longer than three months.

AbD15179 was converted to the full-length human IgG1 isotype in collaboration with Bio-Rad AbD Serotec (Puchheim, Germany) as described below. Variable domain VH and VL gene fragments were subcloned into the pMORPH2_h_Ig vector series for human IgG1 expression (35,36) using MfeI/BlpI for insertion into the HC vector backbone and EcoRV/BsiWI for insertion into the LC vector backbone. These vectors carry the human Ig constant region and the human kappa constant region, respectively. Antibody expression was performed transiently using the HKB11 cells (ATCC) (37) in a humidified atmosphere at 37°C and 5% CO₂. The day prior to transfection, 100 ml HKB11 cells were seeded at 0.5×10⁶ viable cells/ml in a shake flask containing MAC1.0 medium supplemented with 1% FCS. Transfection was carried out by the addition of 100 µg of each expression construct followed by 400 µl Q-PEI transfection reagent (Polyplus, Illkirch, France). At 3 h post-transfection 100 ml ExCell-VPRO medium (Sigma-Aldrich, Stockholm, Sweden) containing 1% FCS and 1X GlutaMAX (Thermo Fisher Scientific) were added to the cells. Cell culture supernatants were harvested at day 6 post-transfection and subjected to protein A affinity chromatography. The purified antibody AbD15179_hIgG1, here referred to as AbN44v6, was re-buffered to either PBS, pH 7 (1.2 mg/ml), or 0.07 M sodium borate, pH 9.2 (3 mg/ml). The commercially available anti-CD20 full antibody, rituximab (cat. no. 494237), was purchased commercially from Apoteket AB (Luleå, Sweden).

The two beta emitters ¹⁷⁷Lu and ¹³¹I were selected for the study as they are highly relevant as therapeutic radionuclides in nuclear medicine, and share similar characteristics with regard to half-life, mean beta energy emission and mean range in vivo. The ¹⁷⁷Lu and ¹³¹I isotopes were purchased from PerkinElmer (Waltham, MA, USA) as ¹⁷⁷LuCl₃, pH 1, and 0.1 M ¹³¹I-NaOH, pH 12–14, respectively. For ¹⁷⁷Lu labeling, AbN44v6 and rituximab were chelated in 0.07 M sodium borate, pH 9.2, with CHX-A″DTPA at a molar ratio of 1:5 during an incubation period of 4 h at 37°C. The excess CHX-A″DTPA was separated from the antibodies using a NAP5 size exclusion chromatography column (GE Healthcare, Uppsala, Sweden) equilibrated with filtered 0.2 M ammonium acetate, pH 5.5, stored over Chelex 100. Subsequently, 1–30 MBq of ¹⁷⁷LuCl₃ was added to 10–375 µg of AbN44v6 and 4–40 MBq of ¹⁷⁷LuCl₃ was added to 100–200 µg of rituximab, and incubated for 1 h at room temperature. For in vitro experiments, the free ¹⁷⁷Lu was purified from labeled antibody using a NAP5 size exclusion chromatography column equilibrated with serum-free medium. For in vivo experiments, the ¹⁷⁷Lu-labeled AbN44v6 was purified using Nanosep 3K (Pall Norden AB, Lund, Sweden); the sample was added to four different filters and centrifuged for 35 min at 14,000 × g before washing twice with 200 µl of MQ water for 30 min at 14,000 × g.

The direct iodination of both AbN44v6 and rituximab with ¹³¹I or ¹²⁵I was performed by the addition of 40 µl (4 mg/ml) of Chloramine-T (CAT) to typically 20–375 µl of antibody (1.2 mg/ml) and 2–20 MBq of radioiodine for 60 sec on ice prior to terminating the reaction with 80 µl (4 mg/ml) Na₂SO5 (NBS).

To determine the labeling yield, purity and stability of the labeled antibody fragments, ITLC was performed on ¹³¹I, ¹²⁵I- and ¹⁷⁷Lu-labeled conjugates. Approximately 0.5 µl of the conjugate was placed on a chromatography strip (Biodex, Shirley, NY, USA) and placed into a 'running buffer' (70% acetone for ¹³¹I and ¹²⁵I or 0.2 M citric acid for ¹⁷⁷Lu), followed by measurements on a Cyclone Storage Phosphor System (PerkinElmer). The data were analyzed using the OptiQuant image analysis software (PerkinElmer). The purity of the samples taken immediately, as well as stored at 4°C for 48 h, was analyzed. An ethylenediaminetetraacetic acid (EDTA) challenge assessed the stability of the ¹⁷⁷Lu-labeled antibody using a 500-fold molar excess of EDTA and determined the stability by ITLC following incubation at 37°C for 48 h. The purification of both antibodies resulted in 100% purity for radio-iodination and >95% for ¹⁷⁷Lu-labeling.

In order to assess CD44v6-specific binding, in vitro specificity tests of the radioconjugates were performed. Approximately 2×10⁴ UM-SCC-74B or 3×10⁴ HCT116 cells/well were seeded in 48-well plates and incubated for 24 h prior to the addition of 20 kBq (25–30 nM) of ¹⁷⁷Lu-AbN44v6 or ¹²⁵I-AbN44v6 to each well. In selected wells, ≥20-fold molar excess of unlabeled AbN44v6 was added to the wells to block specific binding. The plates were incubated at 37°C for 24 h before the cells were washed and collected and the binding measured using a 1480 WIZARD gamma well-counter (Wallac Oy, Turku, Finland).

In order to assess cellular uptake and the retention of the radiolabeled conjugates, LigandTracer experiments were performed. The cells (5–10×10⁵) were seeded in 10-cm dishes and incubated for at least 24 h before running LigandTracer Yellow for ¹⁷⁷Lu/¹³¹I or LigandTracer Grey for ¹²⁵I (Ridgeview Instruments, Vänge, Sweden). Two different concentrations (3 and 10 nM) of radiolabeled antibodies were added, and the uptake was measured for 90 min for each concentration before removing the radiolabeled antibodies and replacing the medium to measure the dissociation rate. Analysis was performed using TraceDrawer, version 1.7 (Ridgeview Instruments).

In order to assess the potential therapeutic effects of the radioconjugates, in vitro therapy experiments were performed on 3D multicellular tumor spheroids, where the growth of tumor spheroids was followed over time. For liquid overlay 3D spheroid formation, 0.15 g of agarose was dissolved in 10 ml of PBS, pH 7.4, with 1% penicillin/streptomycin and 5% incomplete, serum-free DMEM for liquid overlay of two 96-well plates. For spheroid formation, 1×10³ UM-SCC-74B and 1–1.5×10³ HCT116 cells were seeded in 96-well plates pre-coated with 50 µl of agarose. The plates were incubated at 37°C in a 5% CO₂ atmosphere for 3 days prior to treatment with either ¹⁷⁷Lu/¹³¹I-AbN44v6 or rituximab. The concentrations of radio-labeled antibodies were added in the range of 10–100 kBq/well (5–140 nM per well for AbN44v6 and 30–111 nM per well for rituximab). The spheroids were followed for 10 days, obtaining images at day 3, 6 and 10 post-treatment using a Canon EOS 700D digital camera (Canon, Tokyo, Japan) mounted on an inverted Nikon Diaphot-TMD microscope (Nikon, Tokyo, Japan). The spheroid images were analyzed semi-automatically using ImageJ software version 1.48 (NIH, Bethesda, MD, USA). Outlines of the spheroid shape were drawn and the area of the spheroids was measured. Spheroid volume was then calculated using the formula V = 4/3 πr³, assuming all spheroids retained a spherical form for volume calculations.

The in vivo experiments were carried out in accordance with the current Swedish laws and regulations, designed to comply with ethical guidelines to reduce and refine animal experiments, while maintaining data quality, with permission granted by the Committee of Animal Research Ethics Uppsala. Nu/nu Balb/c mice (female; n=30; average weight, 18±2 g) were housed under standard conditions and fed ad libitum. One day prior to tracer injection, the animals were given 1% sodium iodide in drinking water to block the uptake of free ¹²⁵I in the thyroid (24,38–41). Each animal was administered approximately 200 kBq ¹⁷⁷Lu-AbN44v6 and 150 kBq ¹²⁵I-AbN44v6 in a total of 15 µg by an intravenous injection of a volume of 80 µl into the tail vein. Doses were selected according to previous studies (24,42). The animals were divided into 6 groups (n=5 per group) and euthanized by an intraperitoneal injection of a mixture of ketamine (10 mg/ml) and xylazine (1 mg/ml) at a dose of 0.2 ml per 10 g of body weight, followed by heart puncture at 3, 6, 24, 48, 96 and 168 h post-injection. The blood and organs of interest were excised, and the uptake of ¹⁷⁷Lu/¹²⁵I-AbN44v6 was measured in a gamma well-counter. Three injection standards of each radioconjugate were included in all gamma well-counter measurements. The uptake of each radionuclide was calculated as a percentage of injected activity/organ, and the tumor/organ ratios were calculated as activity/g_tumor divided by activity/g_organ.

SUV values [(organ activity/organ weight)/(administered activity/animal weight)] were calculated for the selected organs at the above-mentioned time-points. At time zero, SUV was assumed to be zero. For dosimetry, non-decay corrected values (SUVn) were obtained by multiplying the SUV values with the decay curve of ¹⁷⁷Lu and ¹³¹I. The organs were then integrated between 0 and 168 h using the EXCEL 2007 histogram method. The time interval of 0–168 h corresponds to approximately one half-life of the nuclides. An estimate of decays remaining after 168 h was made by integrating an exponential curve passing through the 96 and 168 h time-points. To calculate the absorbed dose in standard anatomical phantoms, the dosimetry system, Medical Internal Radiation Dose (MIRD), was used for the total number of decays in each main organ. The residence times (RT) were calculated and used as an input to the OLINDA software. The self-dose and the cross-fire dose, as well as the obtained doses in different organs and the effective dose, were calculated using the OLINDA software. To scale the animal data to humans, the SUV values were assumed to be invariant.

Statistical analyses were performed using GraphPad Prism software (version 6.2). All data are presented as the means ± SD unless otherwise stated. The Student's t-test was used to calculate statistical significance in the specific uptake of radiolabeled AbN44v6. Statistical significance was calculated using one-way ANOVA followed by Sidak's multiple comparisons test for defining differences between control, targeted and untargeted radiation in 3D RIT. Values of p≤0.05, p≤0.01, p≤0.001 and p≤0.0001 were considered to indicate statistically significant differences or highly statistically significant differences, as appropriate.

For labeling with ¹⁷⁷Lu, the specific activities varied between 50 and 150 kBq/µg of AbN44v6 with an average yield of 74±19%. The EDTA challenge resulted in 100% stability of ¹⁷⁷Lu-AbN44v6 after 48 h of incubation. For direct radioiodination, the specific activities for ¹²⁵I varied between 37 and 83 kBq/µg with an average yield of 87±5%. Specific activities for ¹³¹I were 80–83 kBq/µg with an average yield of 80±3%. Specific activities for ¹³¹I-rituximab varied between 30 and 124 kBq/µg with an average yield of 73±11%.

In order to assess the effects of labeling, the specific binding of conjugates, as well as to verify the CD44v6 expression levels in the cells, LigandTracer analyses and in vitro specificity assays were performed. Furthermore, the binding properties of ¹²⁵I-labeled AbN44v6 were compared to ¹³¹I-labeled AbN44v6 in order to determine whether ¹²⁵I-AbN44v6 could be used as a model for ¹³¹I-AbN44v6.

AbN44v6 was first labeled with ¹⁷⁷Lu using a CHX-A″-DTPA chelate. A clear cellular uptake was detected by HCT116 cells (a moderate CD44v6-expressing cell line) at both concentrations, followed by a slow dissociation phase of 5% loss/h (Fig. 1A, black curve). The affinity of ¹⁷⁷Lu-AbN44v6 was calculated using the HCT116 measurements obtained on LigandTracer, resulting in an estimated K_D in the low (1–10) nanomolar range. The signal-to-noise ratio of UM-SCC-74B, a low CD44v6-expressing cell line, was below the detection limit for the LigandTracer instrument (Fig. 1A, gray curve).

Radioiodination of AbN44v6 with either ¹²⁵I (Fig. 1B, black curve) or ¹³¹I (Fig. 1B, gray curve) resulted in near identical measurements on HCT116 cells with a clear uptake at both concentrations. A two-phased dissociation, where ~45% of the bound ligand detached during the first hour, was observed for both radioiodinated conjugates. The second dissociation phase steadied to a slower dissociation of 5 and 12% loss/h for ¹²⁵I-AbN44v6 and ¹³¹I-AbN44v6, respectively, which was similar to that of ¹⁷⁷Lu-AbN44v6. Furthermore, the selected negative control for in vitro 3D (spheroid) RIT, rituximab, was labeled with ¹³¹I and evaluated for binding to both cell lines (Fig. 1C). LigandTracer was unable to detect any binding on either cell line using the ¹³¹I-rituximab conjugate.

To determine the CD44v6 specificity of the radiolabeled AbN44v6, an in vitro specificity assay using both ¹⁷⁷Lu-AbN44v6 and ¹²⁵I-AbN44v6 on UM-SCC-74B and HCT116 cells (Fig. 1D and E) was performed. The uptake in blocked wells with an excess of unlabeled AbN44v6 decreased to an average of 8.6 and 2.8% for ¹⁷⁷Lu-AbN44v6 on UM-SCC-74B and HCT116 cells, respectively (p<0.0001). For ¹²⁵I-AbN44v6, the uptake in blocked wells with an excess of unlabeled AbN44v6 decreased to an average of 33.9 and 4.4% for UM-SCC-74B and HCT116 cells, respectively. This result indicated a retained biological function following both labeling procedures and antigen-antibody specificity.

The potential therapeutic effects of the radiolabeled antibodies were assessed in in vitro 3D tumor models (spheroid models). By the end of the assay (day 10), only UM-SCC-74B spheroids treated with 100 kBq of the targeted radiation differed significantly to untreated controls (p<0.05 and p<0.01 for (¹⁷⁷Lu- or ¹³¹I-AbN44v6, respectively) (Fig. 2A and B). A more pronounced therapeutic effect was observed on the HCT116 spheroids, which expressed a higher level of target antigen than the UM-SCC-74B spheroids. Furthermore, increasing activities resulted in more pronounced therapeutic effects (Fig. 2C and D). While the lowest activity (10 kBq/well) did not result in significant differences in growth progression of HCT116 spheroids of ¹³¹I-AbN44v6, activities exceeding 10 kBq/well differed significantly from the untreated controls for both radioconjugates. Furthermore, HCT116 spheroids treated with 100 kBq of ¹³¹I-AbN44v6 demonstrated a stunted growth throughout the assay, whereas all other treatments started to recover normal growth ratios by the end of the assay (day 10) (data not shown).

Since unbound radioconjugates present in the medium are expected to contribute to the total dose received by the spheroids, negative controls using radiolabeled rituximab assessed the damage caused by untargeted radiation. The extent of the impaired growth of radiolabeled rituximab differed both between the two cell lines and the two radio-nuclides. Radiolabeled rituximab accounted for the majority of the growth impairment observed in the UM-SCC-74B spheroids (Fig. 2B). Despite significant growth inhibition between UM-SC-74B controls and the highest added activity of targeted radiation (100 kBq), significance was not obtained between 100 kBq of targeted and non-targeted activity (data not shown). Conversely, the effects on HCT116 spheroid growth were mainly caused by targeted radiation, where significant therapeutic effects of the AbN44v6 radioconjugates compared to radiolabeled rituximab were demonstrated for added activities of ≥10 kBq/well and ≥30 kBq/well for ¹⁷⁷Lu-AbN44v6 and ¹³¹I-AbN44v6, respectively (Fig. 2D and Table I).

The biodistribution of ¹⁷⁷Lu-AbN44v6 can be seen in Fig. 3. Uptake in the dose-limiting organs of interest peaked at 3 h (kidneys), 6 h (liver) and 24 h (spleen) at approximately 10, 32 and 35% ID/g, respectively. By the final time-point, the uptake in the kidneys had diminished 10-fold (from 10 to 1%). Similarly, at 168 h post-injection (p.i.), the uptake in the liver and spleen had decreased to approximately 7 and 15% ID/g, respectively. As expected, the uptake in the blood peaked at the earliest time-point (20% ID/g), but decreased to 3% ID/g at 24 h p.i., an 85% decrease from the uptake 3 h p.i. At 168 h p.i., the activity in the blood was almost undetectable. ¹⁷⁷Lu-AbN44v6-uptake in most other organs followed the uptake in the blood and diminished rapidly over time. The biodistribution of ¹²⁵I-AbN44v6 can be seen in Fig. 4. A similar pattern was discernible for ¹²⁵I-AbN44v6 as for ¹⁷⁷Lu-AbN44v6. The primary difference in biodistribution profiles between the two radioconjugates was the prolonged circulation time of ¹²⁵I-AbN44v6. However, the uptake of ¹²⁵I-AbN44v6 in the liver and spleen was lower than its ¹⁷⁷Lu-labeled counterpart, despite the longer circulation time of the radio-iodine conjugate. All organs, including the kidneys, liver and spleen, followed the same pattern of uptake, and the clearance of ¹²⁵I-AbN44v6 as observed for the blood, resulting in rapidly decreasing values. The highest uptake of ¹²⁵I-AbN44v6 in all assessed organs was detected at either 3 or 6 h p.i. In dose-limiting organs, the uptake peaked at approximately 10% (kidneys), 15% (liver) and 12% (spleen) at 3 h p.i., decreasing by >50% at 24 h p.i. and 10-fold or more by the last time-point.

Integrated non-decay corrected iSUVn values are shown in Table II for ¹⁷⁷Lu- and ¹³¹I-labeled AbN44v6. The calculated residence times were used as input to the OLINDA software (Table III). The sum of the residence times used as an input represented <10% of the total amount of administered decays both for ¹⁷⁷Lu and ¹³¹I. An equal amount was entered in the remaining body to compensate for eventual missing activity. The organ doses and effective doses are shown in Tables IV and V for ¹⁷⁷Lu- and ¹³¹I-labeled AbN44v6, respectively. The effective dose was in the order of 0.1 mSv/MBq for both labels. Critical organs for ¹⁷⁷Lu-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were onsidered the critical organs for ¹³¹I-AbN44v6.