Safety testing of new drugs is critical, and this protocol describes how to use validated standards to assess the reproducibility, robustness, and potential limitations of a CRS platform. This technique has been validated with these reagents in 11 different labs and an international collaborative study with similar patterns of response observed, therefore improving efforts in harmonization and reproducibility. Applications of this technique extend towards improved CRS risk testing of novel therapeutic antibodies, and it will also help to be ready for the clinical management of potential side effects.
This work is to improve reproducibility and harmonization, and a visual demonstration enhances the chances of reproducibility. Begin by reconstituting the contents of the reference reagent ampules in one milliliter of sterile distilled water and allowing 5 to 10 minutes of rehydration. Then, mix each antibody solution properly before transferring it to a sterile capped tube.
Next, dilute each reconstituted antibody and test antibody to 10 micrograms per milliliter in sterile PBS. Take a sterile, non-TC-treated, U-bottom polypropylene 96-well microtitre plate and add 100 microliters of the desired diluted antibody solution to the designated wells of the plate. Then, incubate the plate overnight at four degrees Celsius before performing the solid phase assay.
After collecting a minimum of 30 milliliters of peripheral whole blood into a heparinized tube, invert it several times to ensure proper mixing of the blood sample with sodium heparin. Set aside 15 milliliters of the whole blood sample in a separate tube for use later in the aqueous phase whole blood assay. Dilute the remaining 15 milliliters of whole blood by adding an equal volume of either PBS or serum-free RPMI 1640 media.
Gently layer the diluted blood on the top of 15 milliliters of a density gradient medium in a 50-milliliter tube. Centrifuge the tube using a swing-out rotor with no brake and a reduced acceleration at 500 G and room temperature for 20 minutes to separate the blood into its different components. After centrifugation, the density gradient will separate as a top layer of plasma, followed by a thin layer of buffy coat containing PBMCs and a bottom layer containing RBCs and polymorphonuclear granulocytes, including neutrophils and eosinophils.
Carefully remove the upper plasma layer, followed by the next layer, to harvest the PBMCs. For washing, resuspend the harvested buffy coat in 10 milliliters of PBS or serum-free RPMI 1640 media. Centrifuge the tube begin at 500 G for 10 minutes to pellet the cells.
Discard the supernatant and repeat the washing again. Then, resuspend the resultant pellet in two milliliters of complete RPMI media. Count cells using a hemocytometer and adjust the PBMC concentration to 1 million cells per milliliter in complete RPMI.
For the aqueous phase whole blood cytokine release assay, take the whole blood sample previously set aside, add 190 microliters of the sample to the wells of a 96-well U-bottom polystyrene plate, heat-treatment antibodies and reference reagents pre-diluted to 100 micrograms per milliliter in PBS, and add 10 microliters of the desired diluted antibody to the designated wells containing 190 microliters of whole blood. Incubate the plate for 48 hours in a humidified incubator at 37 degrees Celsius. For the solid phase PBMC cytokine release assay, obtain the previously prepared coated plates.
Using a multi-channel pipette, discard the antibody solution from the coated plates. After filling a reagent reservoir with PBS, wash the plate three times with 200 microliters of PBS to remove unbound monoclonal antibodies. Add 200 microliters of the previously prepared PBMC suspension to each well.
Incubate the plate for 48 hours in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. After the 48-hour incubation with control and test monoclonal antibodies, centrifuge the plates at 400 G for five minutes. Collect the cell-conditioned medium or plasma without disturbing the cell pellet.
Freeze the collected supernatant or plasma at minus 20 degrees Celsius. The PBMC solid phase assay results demonstrated that the positive control antibodies induced significantly high levels of cytokines when compared to the matched isotype controls. It was also observed that anti-CD28 superagonist induced a significantly higher-fold change of interleukin-2, or IL-2 release, than its matched isotype, whereas anti-CD3 and anti-CD52, while still inducing IL-2 expression, resulted in lower-fold changes.
In the whole blood aqueous phase assay, the level of detectable cytokines was relatively less than in the solid phase assay, but was characterized by a greater sensitivity to stimulation by anti-CD52.
