Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

Our project lead by Professor Pravin Kaumaya are focused on developing B-cell heptapeptide vaccines that can be used to boost the immune system to better fight against the cancer. We believe that peptide vaccines targeting immune checkpoint targets may even be combined with other therapies to improve patient outcomes. We wanted to test the blocking ability of our anti PD-L1 antibodies in a traditional microbead assay, where human recombinant PD-L1 protein was bound to MagPlex beads.

We found that, once the PD-L1 was bound to the beads, they had a low binding capacity for its PD-1 ligand. This prevented us using this avenue of testing blocking. When binding protein to beads inhibits binding to ligand or antibodies, pretreatment with the test antibodies in solution is an option, later adding ligand-coated beads to the target blocker mixture.

Their interaction may be examined. This may be a different path to study the same target-ligand binding interaction. We have developed the B-Vaxx, PD1-Vaxx, PDL1-Vaxx, and CTLA4-Vaxx cancer vaccines.

These vaccines target HER2, and several immune checkpoint markers are involved in various forms of cancer. B-Vaxx and PD1-Vaxx are in ongoing clinical trials in Australia and the US.We hope our immune checkpoint vaccines will benefit more patients. In previous microsphere experiments, we found that PD-1 interacted well with its ligand PD-L1.

Therefore, we re-engineered the protocol, pre-binding the anti PD-L1 peptide antibodies to biotinylated PD-L1 protein before adding the PD-1 tagged MagPlex beads. This allowed us to test our antibodies for their ability to block PD-1-PD-L1 interaction. We will continue to focus on combination immunotherapies.

Let's overcome treatment resistance. We also want to explore more effective combination therapies that better inhibit tumor growth, prevent metastasis, and prolong patient survival rates. Start by preparing stock solutions of all antibodies at 2, 000 micrograms per milliliter.

Then, for each antibody, label the dilution tubes with their respective concentrations, including the name of the antibody. Also include a blank tube for each antibody as PBS-TBN vehicle-only control. Next, add 75 microliters of PBS-TBN to all antibody dilution tubes labeled as 500 micrograms per milliliter and below, including the vehicle-only control tubes.

For each antibody, pipette 150 microliters of the stock solution into the tube labeled as 1, 000 micrograms per milliliter. To create a complete dilution series for each antibody, transfer 75 microliters from the 1, 000 micrograms per milliliter tube to the subsequent lower dilution in the series. Afterward, close the newly completed dilution tube and briefly vortex it.

Then, add 25 microliters of diluted antibodies into the designated wells of a 96-well microtiter plate, followed by adding 25 microliters of biotinylated recombinant human PD-L1 to every reaction well. Cover the microtiter plate with a foil or plastic adhesive seal and incubate at room temperature for one hour with shaking on an orbital plate shaker at 600 RPM. Next, dilute the recombinant human PD-1 coupled beads to a concentration of 50, 000 beads per milliliter with PBS-TBN.

Remove the 96-well microtiter reaction plate from the shaker and peel the adhesive plate seal. Add 50 microliters of the recombinant human PD-1 coupled bead mixture to each well. Seal the plate tightly and incubate for one hour in the dark at room temperature on an orbital shaker set at 600 RPM.

After incubation, carefully remove the adhesive plate seal. Then place the plate on the magnetic plate separator for two minutes to immobilize the beads. Confirm that the magnet and microtiter plate are securely connected.

Flip the plate over and discard the supernatant carefully. To wash the excess reaction reagents from the beads, remove the microtiter plate from the magnetic plate separator and add 150 microliters of PBS-TBN to each well. After removing the sealer and immobilizing the beads as demonstrated, confirm that the magnet and microtiter plate are secured and dump the supernatant.

Now, add 100 microliters of SAPE working solution to each reaction well and resuspend the beads by pipetting. Seal the plate and incubate it for one hour at room temperature in the dark, then carefully remove the adhesive plate sealer. Once the beads are immobilized as well as the magnet and plate are secured together, gently dump the supernatant by inverting the plate.

To wash the excess SAPE off the beads, remove the microtiter plate from the magnetic plate holder and add 150 microliters of PBS-TBN to each well to resuspend the beads. Then, immobilize the beads by placing the microtiter plate on a magnetic plate separator for two minutes. After confirming that the magnet and microtiter plate is secured, invert the plate and remove the supernatant.

Next, add 100 microliters of diluted Brilliant Violet 421 conjugated anti-human or anti-rabbit immunoglobulin G to their respective wells. Seal the plate and incubated for one hour in the dark at room temperature. Then, immobilize the beads and remove the adhesive plate sealer.

Confirm that the magnet and microtiter plate are secured and dump the supernatant. Wash the excess secondary antibodies from the beads by adding 150 microliters of PBS-TBN to each well to resuspend the beads. Next, place the microtiter plate on the magnetic plate separator for two minutes to immobilize the beads.

Confirm that the magnet and microtiter plate are securely together and then invert the plate and dump the supernatant. Once the fourth and final wash buffer is removed, take out the microtiter plate from the magnetic plate separator. Using a pipette, resuspend the beads in 100 microliters of PBS-TBN per well.

Finally, to determine the median fluorescent intensity of each reaction, read the plate on the dual reporter flow analysis system with the aspiration volume set to 50 microliters, minimum bead count to 100 beads, the timeout setting to 40 seconds, gating range to 7, 000 to 17, 000, and operating mode to the dual reporter. All four polyclonal anti-PD-L1 peptide antibodies blocked the recombinant human PD-L1 interaction with PD-1. The percentage of inhibition of the different anti PD-L1 peptide antibodies ranged from 48%to 74%at the maximum tested concentration of 1, 000 micrograms per milliliter.

The positive control monoclonal antibody atezolizumab achieved 92%blockade of the PD-1-PD-L1 interaction at the maximum tested concentration of four micrograms per milliliter. Comparative binding analysis of PDL1-Vaxx-induced candidate antibodies to recombinant human PD-L1 indicated that all four polyclonal anti PD-L1 peptide antibodies bound to recombinant human PD-L1 in a concentration-dependent manner. The anti-PDL1(130)antibody showed the highest RH PD-L1 binding signal of the four PDL1-Vaxx-induced antibody candidates.