Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

This method provides an important tool in the field of protein modification, enabling the facile and efficient generation of antibody drug conjugates. The main advantages of this technique are the ease of handling, the high yield of antibody production, the speed of the psychodision reaction, and the stability of the linkage formed. Begin this procedure with growing HEK suspension cells as described in the text protocol.

When the density of 2.5 million cells per milliliter is reached, prepare a fresh solution of the cyclopropene derivative of lysine, or CpK, in 1 molar sodium hydroxide. Add 2.5 milliliters of CpK to expression medium supplemented with antibiotics. Mix the solution well and add 250 microliters of one molar HCl, then sterilize the solution using a 22 micron filter.

Now centrifuge 125 million cells at the target density for five minutes at 500 x g. Following centrifugation, add the DNA-transfection reagent mixture to the expression medium containing CpK and use this medium to resuspend the cells. Six to seven days after addition of CpK, harvest the trastuzumab bearing CpK antibodies from the supernatant by centrifuging the cells for 15 minutes at 3, 000 x g.

Then, filter the supernatant. Now add 5x bead wash buffer in 250 milliliter conical tubes and mix with the supernatant and add the preequilibrated protein A resin. Place the conical tube on a roller for three hours at room temperature to pull down the antibody and the supernatant.

Following incubation, transfer protein A resin with the supernatant into a column and allow the liquid to flow through. Then add 25 milliliters of wash buffer and allow that to flow through. Add one milliliter of one molar phosphate buffer, 150 millimolar sodium chloride to the collection tube and elute the antibody with four milliliters of 1 molar sodium citrate pH 3.

The acidic solution is immediately neutralized as it comes off the column by the basic solution already present in the collection tube. Next, dilute the antibody with 10 milliliters of PBS. Concentrate the antibody to 5 to one milliliters by centrifugal filtration and exchange the buffer three times with PBS.

Purify the samples by FPLC using a butyl hydrophobic interaction column with zero to 100%gradient of low salt buffer in high salt buffer over 30 minutes. Collect all fractions and monitor the elution at 280 nanometers. Concentrate the fractions that contain antibody by centrifugal filtration and exchange the buffer three times with PBS.

The antibody can then be stored at four degrees Celsius. Monomethyl auristatin E, or MMAE, is highly toxic. Therefore, use gloves and goggles when handling MMAE derivatives.

If you use unmodified MMAE as a control, handle the solid product inside a fume hood when preparing the stock solution in the MSO. To conjugate the antibody with tetrazine bearing molecule, dilute 10 molar equivalence of tetrazine MMAE per antibody with 20 microliters of acetonitrile and 76 microliters of PBS in a small conical tube. Add one molar equivalent of trastuzumab bearing CPK.

Mix thoroughly and allow to react for three hours at room temperature to form trastuzumab linked to MMAE. Molecules other than MMAE potentially larger and more sterically hindered can be linked to the antibody using this protocol. Add the whole volume of the reaction onto the spin column and centrifuge at 1500 x g for one minute.

To analyze the conjugate, equilibrate the HPLC HIC column with 100%high salt buffer for five minutes. Then mix 15 microliters of a one milligram per milliliter solution of trastuzumab linked to MMAE with 15 microliters of 2X high salt buffer in a vial. Inject the mixture onto the HPLC column.

Elute in an isocratic one milliliter per minute flow with 100%high salt buffer for one minute. Follow this with a 15 minute gradient from 100 to zero percent of high salt buffer in low salt buffer. Monitor the elution at 280 nanometers.

Integrate the peaks on the chromatogram with retention times of 7.5, 9.2, and 11.5 minutes, which correspond to trastuzumab with zero, one, and two conjugated toxins, respectively. Now, calculate the DAR by adding up the areas of the peaks at 9.2 minutes, which has a weight of one, and 10.5 minutes, which has a weight of two, and divide by the total area of the three peaks. To analyze the conjugate by LC-MS, first, deglycosylate 30 microliters of one milligram per milliliter ADC and the unmodified antibody in non-reducing conditions using 250 units of PNGase F for at least six hours at 37 degrees Celsius.

Thaw the serum and filter it through a 22 micron filter. Then fill the external wells of a 96 well plate with water. In the central wells, mix in triplicate 90 microliters of serum with 10 microliters of one milligram per milliliter trastuzumab tetramethylrhodamine in PBS at a final concentration of 1 milligram per milliliter.

Place the plate in an incubator saturated with humidity at 37 degrees Celsius and four percent CO2. Every 24 hours throughout five days, pipette each well thoroughly to mix and take a five microliter aliquot. Flash freeze the aliquot with liquid nitrogen and store at 80 degrees Celsius.

Once all samples have been collected, thaw the aliquots and analyze using an indirect ELISA, as detailed in the text protocol. Alternatively, a commercial ELISA kit can be used to measure the concentration of the ADC. Two days prior to the assay, fill the outer wells of a 96 well plate with water.

Then lift cells with 05%trypsin in 5 millimolar EDTA. Centrifuge the cells three minutes at 250 x g and resuspend in new medium. Then seed 3, 000 cells in 100 microliters per well in 96 well plates and place them back into the incubator.

Two days after seeding the cells, prepare serial dilutions of all samples in triplicate with 1%DMSO in complete medium. Now add 100 microliters of each sample into each well and incubate for five days at 37 degrees Celsius and four percent CO2. On day five, measure the cell viability.

To this end, use a commercial kit to lyse the cells and measure the ATP released. Plot the percentage of signal with respect to control cells treated with 1%DMSO. ADCs can be incubated at 37 degrees Celsius for five days in serum and assayed for cytotoxicity to prove functional stability.

An antibody with a cyclopropene handle can be obtained using this procedure with yields above 20 milligrams per liter after protein A purification. The antibody produced can be rapidly and site-specifically conjugated to a toxin bearing a tetrazine. Upon conjugation of the antibody with a toxin, average drug to antibody ratios are greater than 1.9, as measured by hydrophobic interaction chromatography.

The identity of the ADC is confirmed by mass spectrometry. The dihydropyridizene bond generated is stable in human serum for over five days at 37 degrees Celsius. The antibody drug conjugate is potent and selectively kills cells with high levels of HER2, rather than cells with low levels of HER2.

Furthermore, the antibody without the toxin and the toxin modified with the tetrazine linker have very low toxicity. The toxin alone shows a non-HER2 dependent toxicity highlighting the need of targeting to achieve selectivity. Following this procedure, we can rapidly and efficiently prepare antibody drug conjugates for the treatment of tumors.

Potentially, any drug functionalized with a tetrazine can be linked to any antibody targeting a cancer cell biomarker. The implications of this technique extend to any protein conjugate intended for therapy or diagnosis.