Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

This protocol describes how to apply a pretargeted methodology to the targeted radiotherapy of cancer xenographs in the mouse model. Pretargeted radioimmunotherapy or PRIT allows us to inject the antibody and radioligand separately, and let the two combine in vivo using click chemistry. While we present PRIT using a xenograph model of colorectal cancer, this technique can be applied to any surface antigen targeting antibody.

It is important to select an antigen that does not internalize rapidly or shed extensively as these can reduce the efficacy of the approach. To synthesize huA33-TCO first prepare a 125 microliter solution of 40 milligrams per milliliter TCO-NHS in dry methylformamide in a 1.7 milliliter microcentrifuge tube. This solution can be aliquoted and frozen at 80 degree celsius for use in future experiments.

In a separate 1.7 milliliter microcentrifuge tube prepare a five milligram per milliliter solution of huA33 in one milliliter of phosphate buffered saline. Using small aliquots of 0.1 molar sodium carbonate, adjust the pH of the antibody solution to between 8.8 and 9.0. Use either pH paper or a pH meter with a microelectrode to monitor the pH, and exercise care that the pH does not exceed 9.0.

Add a volume corresponding to 40 molar equivalence of TCO-NHS to the antibody solution. To prevent precipitation of the hydrophobic TCO-NHS, add it slowly and with agitation. Allow the reaction to incubate at 25 degrees celsius on a thermal mixer for one hour with mild agitation.

Before purifying the huA33-TCO immunoconjugate using a pre-packed disposable size exclusion desalting column, equilibrate the size exclusion column as described by the supplier to remove any preservatives present in the column during storage which usually involves washing the column five times with a volume of PBS that corresponds to the volume of the column. Add the reaction mixture to the size exclusion column, noting the volume of the reaction mixture. After the reaction mixture has entered the column add an appropriate amount of PBS to bring the total volume of solution added to the column up to 2.5 milliliters.

Collect the product using two milliliters of PBS as the eluent. Measure the concentration of the huA33-TCO using a UV vis spectrophotometer monitoring the wavelength at 280 nanometers. If a solution with a higher concentration of immunoconjugate is desired concentrate the huA33-TCO solution using a centrifugal filter unit with a 50, 000 molecular weight cutoff following manufacturer's instructions.

Store the completed huA33-TCO immunoconjugate at 4 degree celsius in the dark. If it is to be used more than four days in the future store it at 80 degrees celsius in the dark. In a 1.7 milliliters centrifuge tube add 200 microliters of 0.25 molar ammonium acetate buffer adjusted to pH 5.5.

Add the desired amount of Lutetium-177 chloride to the buffer solution. The amount added will be dependent on the number of subjects in the experiment, and the radioactive doses being administered. Add Tetrazine PEG7-DOTA in DMSO to the radioactive mixture.

The amount added is dependent on the number of subjects being tested. Allow the solution to incubate at 37 degrees celsius for 20 minutes. Verify the radiolabeling is complete using radio-instant-thin-layer chromatography with 50 millimolar EDTA pH 5.5 as the mobile phase.

The labeled Lutetium-177-DOTA-PEG7-Tetrazine will remain at the baseline while free Lutetium-177 will be coordinated by the EDTA and travel with the solvent front. It is critical that the placement of the subcutaneous tumors does not interfere with your restraint technique. I recommend placing them on the flank side that is opposite to your restraining hand.

Since I restrain with my left hand I have chosen to place the tumors on the right flank. Sort female athymic nude mice with colorectal cancer xenograph, so that the average tumor volume in each cohort is roughly equal. To do so, list the animal identification numbers, ear notch pattern and tumor volume in three separate columns of a spreadsheet program.

Sort the data from smallest to largest tumor volume. In a fourth column assign each animal a cage number. Cycle through the cages in a snake-like pattern until all the animals are assigned a cage.

Once the cages are assigned sort the data by cage number. To inject the immunoconjugate draw doses of 150 microliters of huA33-TCO solution in syringes pretreated with 1%bovine serum albumin in PBS, and store these syringes on ice. To inject the radioligand, first draw doses in 150 microliters of 0.9%sterile saline containing 1.1 molar equivalence of the radiolabeled Tetrazine-PEG7-DOTA relative to the amount of huA33-TCO administered.

Use calipers to measure the longest side of the oblong tumor as well as the width which is perpendicular to the length. Weigh each mouse on a balance to track weight gain or weight loss over time. Finally, calculate the volume using the formula for volume of an ellipsoid assuming that the height is approximately equal to the width.

Shown here is the biodistribution of an in vivo pretargeting with huA33-TCO and Lutetium-177-DOTA-PEG7-Tetrazine in athymic nude mice bearing subcutaneous SW1222 human colorectal caner tumors. All injection intervals produce high activity concentrations in the tumor tissue as well as low activity concentrations in healthy organs. The 24 hour injection interval affords the highest tumoral uptake at 120 hours post injection.

Based on these findings a 24 hour interval was chosen for the subsequent longitudinal therapy study. Shown here is longitudinal therapy study of five groups of mice bearing subcutaneous SW1222 tumors depicted in average tumor volume as a function of time, and tumor volume normalized to initial volume as a function of time. There is a stark difference in the response of the experimental cohorts as compared to the control groups.

While the tumors in the mice receiving only one component of the pretargeted radioimmunotherapy strategy continued to grow unchecked, the tumors of the mice receiving the full pretargeted radioimmunotherapy regimen stopped growing and ultimately shrank. Importantly, no toxic side-effects were observed, and all animals maintained a weight within 20%of their initial mass. Strikingly, the mice of the experimental cohorts had a perfect record of survival at the end of the investigation.

When attempting this procedure it is of the utmost importance to accurately and reproducibly measure the tumors. When working with radioactivity consult your institution's radiation safety officer to develop safe protocols and facilities. Having proper safety controls in place will limit your exposure and avoid contamination.

Pretargeting affords a lower radiation dose to background organs than traditional radioimmunotherapy. By sharing this protocol we hope that others may be able to test their own models and ideas that will lead to new and exciting advances for patient treatment.