Evaluation of an 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH_1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH_1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[131I]iodobenzoate (iso-[131I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [131I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[131I]SGMIB-VHH_1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[131I]SGMIB-VHH_1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc2-iso-[131I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[131I]SGMIB-VHH_1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[131I]SGMIB-VHH_1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted.

mobile phase A is 25 mM sodium phosphate buffer and 1.5 M ammonium sulfate (pH=7.0); mobile phase B is 25 mM sodium phosphate buffer and 2-propanol (80% v/v sodium phosphate buffer and 20% v/v 2-propanol) (pH=7.0). The column was eluted with 0 to 100% B in 20 min at a flow rate of 0.5 mL/min. Analysis of the iso-SGMIB-VHH_1028 conjugate was done by HPLC using an Agilent PL Multisolvent 20 column eluted with pure Milli-Q ® water as the mobile phase. Disposable PD-10 desalting columns for gel filtration were purchased from GE Healthcare (Piscataway, NJ). Radioactivity levels were measured using a CRC-7 dose calibrator (Capintec, Pittsburgh, PA) for higher activities, and either an LKB 1282 (Wallac, Finland) or a Perkin Elmer Wizard II (Shelton, CT) automated gamma counter for lower activities. Mass spectra were obtained using an Advion (Ithaca, NY) ExpressionL CMS LC-MS System attached to an Agilent 1260 infinity HPLC, like the system described above. This mass spectrometer has the capability of determining molecular weights of compounds directly from TLC plates (Plate Express), and by ESI, atmospheric pressure chemical ionization (APCI), and atmospheric solids analysis probe (ASAP).

VHH_1028.
All experiments described in this section were performed by Cereius, Inc.
VHH_1028 was designed to enhance its compatibility for drug development and targeted radionuclide therapy by being engineered to ensure that no lysine residues were present in its CDR loops (Fig. S1). VHH_1028 was purified using Protein A chromatography to high purity (Fig. S1). VHH_1028 has a calculated molecular weight of 12,844 Da and an isoelectric point of 9.01.
VHH_1028 was recombinantly encoded in the pD2610-v5 plasmid vector (ATUM) with the signal peptide azurocidin and cloned into the NEB ® 5α competent E. coli (New England Biosciences). E. coli were inoculated in 2xYT media (ThermoFisher Scientific) with 45 µg/mL kanamycin and grown overnight. The E.coli plasmids were then harvested using the EndoFree Plasmid Maxi Kit and protocol (Qiagen). The plasmids were complexed to ExpiFectamine ® reagent (ThermoFisher) and then transiently transfected into ExpiCHO-S mammalian cells (ThermoFisher Scientific) at a concentration of 1 µg/mL per tissue culture volume. ExpiCHO-S cells were then maintained under standard conditions of 37°C and 8% CO2 for 10 days before the protein was harvested. For harvesting, cells were isolated out of culture by centrifugation at 4000 rcf for 15 min. The resulting supernatant containing secreted VHH was then purified using 1 mL Pierce TM Protein A chromatography cartridges (Thermo Fisher Scientific).
Purity of the protein was assessed using SDS-PAGE gels (BioRad) under reducing and non-reducing conditions. Purity was likewise assessed via size exclusion HPLC at 220 nm on an AdvanceBio SEC 120A column (Agilent Technologies). Before undertaking further experiments, protein purity was determined to be greater than 95% by both methods. Endotoxin levels were next determined utilizing the Endosafe ® Nexgen PTX (Charles River) to ensure endotoxin levels were lower than <0.50 EU/mg. Last, the mass of the protein was verified using ESI-LCMS under reducing conditions with PNGase F on a 6530 Accurate-Mass A-TOF MC/MS (Agilent Technologies at ATUM). The resulting protein was then formulated in PBS at a concentration of 2 mg/mL and stored at -80°C.
The affinity, specificity, and cross-reactivity of VHH_1028 was first examined in a series of colorimetric, indirect ELISA assays. Briefly, ELISA plates for each antigen were created by preparing each antigen at a concentration of 5 µg/mL, adding 50 µL to each well in a 96-well Immulon 4 HBX plate (Thermo Fisher Scientific), covering the plates, and then incubating at room temperature for four hours. After four hours, the plates were The binding affinity of VHH_1028 for the human HER2 receptor was assessed using two different recombinant antigen display modalities sourced from two different vendors.
In both instances, a sub-nanomolar affinity for human HER2 was evidenced regardless of vendor or recombinant antigen display method. Specifically, EC50 against the HER2-Fc fusion was found to be 0.77 ± 0.19 nM, while the EC50 was 0.69 ± 0.18 nM for the R&D Systems antigen comprising only the extracellular domain of the HER2 receptor (Fig. S2).
VHH_1028 did not exhibit any non-specific affinity for any of the other receptors in the human ErbB family other than the low background associated with IgG Fc fragment. Next, the cross-reactivity of VHH_1028 was evaluated against 5 animal species variants of HER2 (Fig. S2). VHH_1028 showed no affinity for either mouse or rat HER2.
However, it demonstrated strong affinity for HER2 receptors in canine and two species of non-human primate. The EC50 values were 1.09 nM, 1.19 nM, and 1.18 nM for canine, cynomolgus, and rhesus species, respectively. A full breakdown of all affinity values for all molecular targets also can be found in Fig S2. Statistical Analysis. Data are presented as mean ± standard deviation. Statistical significance for the biodistribution experiments was evaluated using a paired two-tailed Student t-test in GraphPad Prism 8. Differences with a P value < 0.05 were considered to be statistically significant.
Statistical and data analysis for the therapeutic efficacy studies was performed as follows: for each group, the average body weight and the standard deviation were calculated and plotted versus time. Normalized average body weight was calculated by setting the initial average body weight on the day of treatment (Day 0) to 100%. Animals were excluded at the day of death. Two-tailed paired t tests were conducted for the normalized average body weight between each group, and tests were only conducted for days when alive animals were present in each group. Average tumor volumes and standard deviations were calculated for each group and plotted versus time. When animals died or were euthanized, their tumor volume at the day of death was carried forward after the day of death for the calculation of average tumor volume. Two-tailed paired t tests were conducted for the average tumor volumes between each group, and tests were only conducted for days when animals remained alive in each group.
Differences with a P value < 0.05 were considered statistically significant. For survival data, log-rank (Mantel-Cox) test, log-rank test for trend, and Gehan-Breslow-Wilcoxon test were conducted. Maximum tumor response waterfall plot was generated for the multiple-dose study by recording the maximum tumor response (growth or inhibition) for each mouse during the experimental period.