Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The overall goal of the following experiment is to pre targett cancerous tissue using a bio orthogonal reaction between Tet INE and trans cyclo octine for pet imaging. This is achieved by injecting a tumor bearing mouse with a trans cyclo tine labeled antibody following an interval period during which the antibody slowly accumulates at the tumor and clears from the blood. A tetra zine radio ligand is injected into the same animal.

The radio ligand clicks with a TCO labeled antibody at the tumor, and because it is a small molecule, it clears from the blood rapidly. The resulting small animal PET imaging shows clear delineation of the tumor with high contrast and high tumor to background activity ratios. The main advantage of this technique over existing methods like PET imaging with directly labeled antibodies, is that it provides high contrast images that only a fraction of the radiation dose to the patient.

The implications of this technique extend towards therapy because pre-targeting techniques can deliver very high amounts of radiation to tumors, while at the same time the doses deliver to healthy organs and healthy tissues is going to be very low. I'll be demonstrating this technique along with Catherine Kaza, a technician in our laboratory. The synthesis reaction begins by dissolving seven milligrams of NH two BN Notta in 600 microliters of 0.1 molar sodium bicarbonate at pH 8.1.

Check the pH and use small aliquots of 0.1 molar sodium carbonate to adjust the pH to 8.1 if needed. Next, add this solution to 0.5 milligrams of TZ NHS in a micro centrifuge tube. Allow this reaction to incubate for half an hour at room temperature with gentle agitation.

The TZ NHS stock can be dissolved in DMF or DMSO after half an hour. Purify the product by reverse phase C 18 HPLC to remove the unreactive NH two BN notta, which will register at 254 nanometers. The TZ NHS and TZ BN Notta are best monitored at 525 nanometers.

The retention time of the sample will vary with equipment, but using a zero to 100 to 100 to zero acetone nitrile to water gradient over 25 minutes, and an analytical 4.6 by 250 millimeters C 18 column, the retention times will be 16.5 15 and 10 minutes for the three compounds respectively. Freeze the collected HPLC eent in liquid nitrogen and then wrap the tube in opaque foil oil. Now, place the frozen tube in a lyophilized vessel and place it on a lyophilizer overnight.

To remove the HPLC mobile phase to verify the purity of the completed TZ bn no top precursor, use proton NMR analytical HPLC and or ESI mass spectrometry. Begin this reaction by preparing a 2.7 millimolar solution of T-C-O-N-H-S in DMF in a micro centrifuge tube in a second micro centrifuge tube. Repair a two milligrams per milliliter solution of HUA 33 in one milliliter of PBS pH 7.4.

Then using small additions of 0.1 molar sodium carbonate, adjust the pH of the solution to between 8.8 and 9.0, but not higher. Next, add a volume of T-C-O-N-H-S that corresponds to eight molar equivalents of the activated ester. Gently mix the solution with a few inversions and wrap it in foil.

Then set the tube up to incubate at room temperature for about an hour with mild agitation. After an hour, use a pre-packed disposable size exclusion desalting column to purify the immunoconjugate from the reaction. Add one milliliter of reaction mixture to the column and subsequently add 1.5 milliliters of 0.9%sterile saline.

Then elute the humanized HUA 33 TCO product with two milliliters of 0.9%saline. Measure the concentration of the product with a UV vis spectrometer. If the concentration is lower than desired, concentrate the product with a 50, 000 molecular weight centrifugal filter.

Begin by preparing a 0.5 milligrams per milliliter solution of TZ BN notta in a micro centrifuge tube. In a second micro centrifuge tube, add 10 microliters of the prepared TZ BN Notta solution to 400 microliters of 0.2 molar ammonium acetate buffer at pH 5.5. Be sure to measure and record the amount of radioactivity your sample before and after each of the upcoming steps.

This is essential to accurate calculation of radio chemical yields and record keeping. Now, add 2000 micro curies of copper 64 to the TZ BN Notta solution. Incubate this mixture at room temperature for 10 minutes with light agitation.

To purify the product use reverse phase C 18 HPLC dilute the products using a five to 95 to 95 to five acetone nitrile to water gradient over 15 minutes with 0.1%TFA. The radio labeled tz, BN nota will elute at 9.8 minutes while the free copper 64 will elute with the solvent front at two to four minutes after using a rotary evaporator to remove the HPLC eant, dissolve the radio labeled product in 0.9%sterile saline, then proceed to use the product as soon as possible. This procedure begins with diluting an Eloqua of the prepared HEA 33 TCO solution to a concentration of 0.5 milligrams per milliliter in 0.9%sterile saline.

Then inject 200 microliters of the solution into the tail vein of the xenograft bearing mouse. Let the humanized a 33 TCO circulate and accumulate in the tumor for 24 hours. After 24 hours, repair the copper 64 labeled TZ BN Notta at a concentration of 1.5 MILLICURIES per milliliter in 0.9%sterile saline.

Then use a tail vein injection to administer 200 microliters of the prepared radioligand solution to the mouse at the desired time. Anesthetize the mouse and place it on the bed of a small animal pet scanner. An anesthesia should be maintained by delivery via a nose cone, and the anesthetized state should be confirmed using a toe pinch.

Also to prevent drying, apply a thin layer of ophthalmic ointment. Now acquire pet data using a static scan with 60 million coincident events at 350 to 700 kilo electron volts, and with a coincidence timing of six nanoseconds after imaging wait for the mouse to regain consciousness before returning it to its cage, potentially with other mice. A thymic nude mice bearing a 33 antigen expressing SW 1222 colorectal cancer.

Xenografts were examined using the described protocol. Both acute biodistribution and PET imaging experiments reveal that the pre-targeting strategy is capable of delineating the colorectal cancer xenograft with excellent image contrast, and high tumor to background activity ratios. Over the course of several hours, the radio ligand clears in the feces and the tumor to background activity ratios become quite high.

Some variables in this pre-targeting approach required optimizing as they would for any imaging strategy. The administration of 300 micrograms of antibody TCO conjugate instead of 100 micrograms resulted in increased radioactivity visible in the mouse's heart, as did decreasing the interval from 24 to 12 hours. In both cases, the click reaction still occurred at the tumor illustrated quite clearly by the tumoral uptake at early time points.

However, the click reaction is clearly also occurring in the blood as well, which is not desired. A delicate balance of all of the pre-targeting variables must be achieved When attempting this procedure. It is very important that the mass of the TCO Immunoconjugate as well as the the time interval between the injections are optimized.

Don't forget that working with radioactivity can be extremely hazardous and precautions such as wearing appropriate PPE and making sure the radiation is shielded at all times should be taken during the experiment.