The authors thank Sherry Dunbar PhD, MBA of Luminex Corporation (Austin, TX) for research support and Matthew Silverman PhD of Biomedical Publishing Solutions (Panama City, FL; mattsilver@yahoo.com) for scientific and writing assistance. This work was supported by awards to Pravin T. P. Kaumaya from the National Institutes of Health (R21 CA13508 and R01 CA84356) and Imugene Ltd, Sydney, Australia (OSU 900600, GR110567, and GR124326).

Flow Analysis of PDL1-Vaxx Antibody Blockade Using Magnetic Fluorescent Beads

<ol>
	<li>Kaumaya, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+epitope+peptide+cancer+vaccines:+a+new+paradigm+for+combination+immunotherapies+with+novel+checkpoint+peptide+vaccine.">B-cell epitope peptide cancer vaccines: a new paradigm for combination immunotherapies with novel checkpoint peptide vaccine.</a> Future Oncology. 16 (23), 1767-1791 (2020).</li><li>Pandey, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revolutionization+in+cancer+therapeutics+via+targeting+major+immune+checkpoints+PD-1,+PD-L1+and+CTLA-4.">Revolutionization in cancer therapeutics via targeting major immune checkpoints PD-1, PD-L1 and CTLA-4.</a> Pharmaceuticals. 15 (3), 335 (2022).</li><li>Guo, L., Overholser, J., Good, A. J., Ede, N. J., Kaumaya, P. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+studies+of+a+novel+human+PD-1+B-cell+peptide+cancer+vaccine+PD1-Vaxx+from+BALB/c+mice+to+beagle+dogs+and+to+non-human+primates+(cynomolgus+monkeys).">Preclinical studies of a novel human PD-1 B-cell peptide cancer vaccine PD1-Vaxx from BALB/c mice to beagle dogs and to non-human primates (cynomolgus monkeys).</a> Frontiers in Oncology. 12, 826566 (2022).</li><li>Brahmer, J. R., Hammers, H., Lipson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nivolumab:+Targeting+PD-1+to+bolster+antitumor+immunity.">Nivolumab: Targeting PD-1 to bolster antitumor immunity.</a> Future Oncology. 11 (9), 1307-1326 (2015).</li><li>Shah, N. J., Kelly, W. J., Liu, S. V., Choquette, K., Spira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+review+on+the+Anti-PD-L1+antibody+atezolizumab.">Product review on the Anti-PD-L1 antibody atezolizumab.</a> Human Vaccines &amp; Immunotherapeutics. 14 (2), 269-276 (2018).</li><li>Postow, M. A., Sidlow, R., Hellmann, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-related+adverse+events+associated+with+immune+checkpoint+blockade.">Immune-related adverse events associated with immune checkpoint blockade.</a> The New England Journal of Medicine. 378 (2), 158-168 (2018).</li><li>Kaumaya, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paradigm+shift:+Cancer+therapy+with+peptide-based+B-cell+epitopes+and+peptide+immunotherapeutics+targeting+multiple+solid+tumor+types:+Emerging+concepts+and+validation+of+combination+immunotherapy.">A paradigm shift: Cancer therapy with peptide-based B-cell epitopes and peptide immunotherapeutics targeting multiple solid tumor types: Emerging concepts and validation of combination immunotherapy.</a> Human Vaccines &amp; Immunotherapeutics. 11 (6), 1368-1386 (2015).</li><li>Guo, L., Kaumaya, P. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+prototype+checkpoint+inhibitor+B-cell+epitope+vaccine+(PD1-Vaxx)+en+route+to+human+Phase+1+clinical+trial+in+Australia+and+USA:+Exploiting+future+novel+synergistic+vaccine+combinations.">First prototype checkpoint inhibitor B-cell epitope vaccine (PD1-Vaxx) en route to human Phase 1 clinical trial in Australia and USA: Exploiting future novel synergistic vaccine combinations.</a> British Journal of Cancer. 125 (2), 152-154 (2021).</li><li>Dakappagari, N. K., Douglas, D. B., Triozzi, P. L., Stevens, V. C., Kaumaya, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+mammary+tumors+with+a+chimeric+HER-2+B-cell+epitope+peptide+vaccine.">Prevention of mammary tumors with a chimeric HER-2 B-cell epitope peptide vaccine.</a> Cancer Research. 60 (14), 3782-3789 (2000).</li><li>Dakappagari, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+HER-2/neu+B-cell+epitope+peptide+vaccine+designed+to+incorporate+two+native+disulfide+bonds+enhances+tumor+cell+binding+and+antitumor+activities.">Conformational HER-2/neu B-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities.</a> The Journal of Biological Chemistry. 280 (1), 54-63 (2005).</li><li>Kaumaya, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+active+immunotherapy+with+combination+of+two+chimeric,+human+epidermal+growth+factor+receptor+2,+B-cell+epitopes+fused+to+a+promiscuous+Tcell+epitope+in+patients+with+metastatic+and/or+recurrent+solid+tumors.">Phase I active immunotherapy with combination of two chimeric, human epidermal growth factor receptor 2, B-cell epitopes fused to a promiscuous Tcell epitope in patients with metastatic and/or recurrent solid tumors.</a> Journal of Clinical Oncology. 27 (31), 5270-5277 (2009).</li><li>Bekaii-Saab, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+immunotherapy+trial+with+two+chimeric+HER-2+B-cell+peptide+vaccines+emulsified+in+montanide+ISA+720VG+and+Nor-MDP+adjuvant+in+patients+with+advanced+solid+tumors.">Phase I immunotherapy trial with two chimeric HER-2 B-cell peptide vaccines emulsified in montanide ISA 720VG and Nor-MDP adjuvant in patients with advanced solid tumors.</a> Clinical Cancer Research. 25 (12), 3495-3507 (2019).</li><li>Kaumaya, P. T. P., Guo, L., Overholser, J., Penichet, M. L., Bekaii-Saab, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+and+antitumor+efficacy+of+a+novel+human+PD-1+B-cell+vaccine+(PD1-Vaxx)+and+combination+immunotherapy+with+dual+trastuzumab/pertuzumab-like+HER-2+B-cell+epitope+vaccines+(B-Vaxx)+in+a+syngeneic+mouse+model.">Immunogenicity and antitumor efficacy of a novel human PD-1 B-cell vaccine (PD1-Vaxx) and combination immunotherapy with dual trastuzumab/pertuzumab-like HER-2 B-cell epitope vaccines (B-Vaxx) in a syngeneic mouse model.</a> Oncoimmunology. 9 (1), 1818437 (2020).</li><li>Guo, L., Overholser, J., Darby, H., Ede, N. J., Kaumaya, P. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newly+discovered+PD-L1+B_cell+epitope+peptide+vaccine+(PDL1-Vaxx)+exhibits+potent+immune+responses+and+effective+anti-tumor+immunity+in+multiple+syngeneic+mice+models+and+(synergizes)+in+combination+with+a+dual+HER-2+B-cell+vaccine+(B-Vaxx).">A newly discovered PD-L1 B_cell epitope peptide vaccine (PDL1-Vaxx) exhibits potent immune responses and effective anti-tumor immunity in multiple syngeneic mice models and (synergizes) in combination with a dual HER-2 B-cell vaccine (B-Vaxx).</a> Oncoimmunology. 11 (1), 2127691 (2022).</li><li>. Luminex Corporation. The xMAP&#174; Cookbook, 5th edition Available from: <a target="_blank" href="https://info.luminexcorp.com/en-us/research/download-the-xmap-cookbook?utm_referrr=https%3A%2F%2Fwww.luminexcorp.com%2F">https://info.luminexcorp.com/en-us/research/download-the-xmap-cookbook?utm_referrr=https%3A%2F%2Fwww.luminexcorp.com%2F</a> (2022)</li><li>MagPlex&#174; Microspheres Documentation. Product information sheet. Luminex Corporation Available from: <a target="_blank" href="https://www.luminexcorp.com/magplex-microspheres/#overview">https://www.luminexcorp.com/magplex-microspheres/#overview</a> (2014)</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Estimation of cell number by hemocytometry counting. 2019 (11), (2019).</li><li>Selecting the Detection System - Colorimetric, Fluorescent, Luminescent Methods for ELISA Assays. Corning Inc Available from: <a target="_blank" href="https://www.corning.com/catalog/cls/documents/application-notes/CLS-DD-AN-458.pdf">https://www.corning.com/catalog/cls/documents/application-notes/CLS-DD-AN-458.pdf</a> (2019)</li></ol>

Pravin T.P. Kaumaya is a consultant to Imugene, Ltd.

The purpose of checkpoint-related cancer immunotherapy is to disrupt the interaction between checkpoint proteins and their important ligands in tumor survival and progression2. This research group is actively developing novel PD-1 and PD-L1 vaccines that elicit an antibody response that targets and interrupts the PD-1/PD-L1 checkpoint3,8,13,14. Previously, two variations of enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate the effects of anti-PDL1-peptide antibodies on the inhibition of the recombinant PD1/PD-L1 interaction14. (1) In the first variant, rhPD-L1 was coated on a microtiter plate, and then the plate was incubated with diluted anti-PDL1 vaccine candidate-induced antibodies. The inhibition of the recombinant PD-1/PD-L1 interaction by the antibodies was then evaluated by adding biotinylated rhPD-1 and quantifying the binding to the immobilized rhPD-L1 using a streptavidin-horseradish peroxidase conjugate and a colorimetric substrate. We defined this as the direct blockade assay. (2) In the second variant, PD-1 was coated on the microtiter plate. Biotinylated rhPD-L1 was pre-incubated with each of the anti-PLD1 candidate-induced polyclonal antibodies in separate reaction tubes. The rhPDL1/anti-PDL1 mixtures were then added to the plate wells containing immobilized rhPD-1 and allowed to react. Any rhPDL1 that reacted with the immobilized rhPD-1 in the presence of the potentially blockading PDL1-Vaxx-induced antibodies was detected with subsequent streptavidin-HRP and colorimetric substrate incubation. We defined this as the reverse blockade assay.
The reverse blockade of the recombinant PD-1/PD-L1 interaction by anti-PDL1-peptide antibodies showed inhibition of the signal (i.e., PD-1/PD-L1 blockade) in an antibody concentration-dependent manner14, while the direct blockade approach did not provide consistent results (not shown). The bead-based dual-reporter blockade assay was developed to verify the ELISA results and to investigate the blockade of the PD-1/PD-L1 interaction in a fluid phase, which eliminates the potential steric hindrance/binding epitope availability issues associated with immobilizing recombinant proteins on well bottoms. The microsphere analysis was directly correlated to the ELISA blocking results using the reverse blockade assay (Figure 2). Additionally, fluorescence-based immunoassays can provide improved assay sensitivity and a broadened dynamic range compared to colorimetric ELISAs18, and further, the multiplexed bead-based assay allows the concurrent performance of two independent immunoassays within a single reaction. The sulfo-NHS and EDC used for the covalent coupling of the microspheres to rhPD-1 might have led to the performance differences seen between the direct blockade and reversed blockade assays and the observed differences in sensitivity between the ELISA and Luminex bead-based recombinant PD-1/PD-L1 interaction assays. Further chemical and molecular level investigation is warranted to study the possible mechanisms responsible for these differences.
Both ELISA14 and the bead-based assays demonstrate that PDL1-Vaxx-induced anti-PDL1 antibodies can inhibit PD1/PD-L1 checkpoint complex formation. The peptide-based PDL1-Vaxx successfully induces anti-PDL1 antibodies that can block the PD-1/PD-L1 interaction. This approach may serve as a novel therapeutic strategy for treating cancer, as supported by preclinical animal studies3,13,14. Planned clinical trials will determine the efficacy of the PDL1-Vaxx for checkpoint immunotherapy and disease control in cancer patients.

In the immune system&#39;s T-cells, B-cells, and intracellular checkpoints, signaling pathways regulate immune activities. Some cancer cells protect themselves from immune attack by stimulating checkpoint targets, which inhibit immune function and promote neoplastic survival and proliferation. Oncologic immunotherapy by checkpoint inhibition uses antibodies to target and block the signaling checkpoints and, thus, restore the anti-neoplastic functions of the immune system1,2,3. Highly effective anti-cancer therapies currently include the monoclonal antibodies nivolumab, which targets programmed death protein 1 (PD-1)4, and atezolizumab, which targets programmed death ligand 1 (PD-L1)5. This approach has shown great clinical success in treating cancer patients. However, the clinical utility of current checkpoint inhibition strategies is mitigated by adverse events and treatment resistance, especially in single-agent therapy6. A combination of immunotherapy and more effective therapeutic strategies with lower toxicity is urgently needed in cancer treatment1,3,6.
Over the past 30 years, Dr. Kaumaya&#39;s laboratory has developed peptide cancer vaccines and peptide mimic-related agents for cancer therapy, some of which are in ongoing clinical trials1,2,7,8,9,10,11,12,13,14. For example, B-Vaxx with HER-2 combination immunotherapy has shown patient benefits against metastatic and/or recurrent solid tumors in clinical trials12. The laboratory&#39;s latest cancer vaccines are PD1-Vaxx2,13 and PDL1-Vaxx14, which have shown great advantages in preclinical studies, especially in combination treatment. The PD1-Vaxx has completed dose-escalation clinical trials in the US and Australia. The PD1-Vaxx will be combined with atezolizumab in the Phase 1b trial to start in May, 2023. This report focuses on evaluating the ability of PDL1-Vaxx-induced antibodies to block the PD-1/PD-L1 interaction.
The PDL1-Vaxx cancer vaccine is a novel B-cell peptide epitope vaccine with PD-L1 amino acids 130-147 linked to the promiscuous T-cell measles virus fusion (MVF) peptide via a GPSL peptide linker. Preclinical studies have shown that PDL1-Vaxx is highly immunogenic in stimulating anti-cancer antibody production in various animal models, prolongs survival, and reduces tumor burden14. These antibodies generated against the PD-L1 peptide can successfully block the PD1/PD-L1 interaction, thus resulting in anti-neoplastic activity. This report introduces an assay that analyzes the blockade of PD1/PD-L1 complex formation by PDL1-Vaxx-induced antibodies using a magnetic bead-based format with a dual-reporter readout on a flow cytometry instrument.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Activation Buffer: 0.1 M NaH2PO4, pH 6.2</td>
			<td>Millipore/Sigma</td>
			<td>S3139</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay/Wash Buffer: PBS-TBN (1x PBS, pH 7.4 + 0.1% BSA + 0.05 % (v/v) Tween-20; 0.05% NaN3)</td>
			<td>Millipore/Sigma</td>
			<td>P3563 (PBS+Tween20), A7888 (Bovine serum albumin), S8032 (sodium azide)</td>
			<td></td>
		</tr>
		<tr>
			<td>Coupling Buffer: 50 mM 2-morpholinoethanesulfonic acid (MES), pH 5.0</td>
			<td>MilliporeSigma&#160;</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>Coupling Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)</td>
			<td>ThermoFisher Scientific (Waltham, MA)</td>
			<td>77149</td>
			<td></td>
		</tr>
		<tr>
			<td>xMAP Antibody Coupling Kit (if desired), includes:</td>
			<td>Luminex Corp.&#160; (Austin, TX)</td>
			<td>40-50016</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC, 10 mg&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sNHS solution, 250 &#181;L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Activation/Coupling Buffer: 0.1 M 2-morpholinoethanesulfonic acid (MES), pH 6.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Buffer: 1x PBS, pH 7.4 + 0.1% BSA + 0.05 % (v/v) Tween-20; 0.05% NaN3 (PBS-TBN)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>ThermoFisher Scientific (Waltham, MA)</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrumentation and Ancillary Lab Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>xMAP INTELLIFLEX (dual-reporter instrument)</td>
			<td>Luminex Corp.&#160; (Austin, TX)</td>
			<td>INTELLIFLEX-DRSE-RUO</td>
			<td></td>
		</tr>
		<tr>
			<td>Low protein-binding round bottom 96-well plate</td>
			<td>ThermoFisher Scientific (Waltham, MA)</td>
			<td>07-200-761</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex Magnetic Plate Separator (or comparable)&#160;</td>
			<td>Luminex Corp.&#160; (Austin, TX)</td>
			<td>CN-0269-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex Magnetic Tube Separator (or comparable)</td>
			<td>Luminex Corp.&#160; (Austin, TX)</td>
			<td>CN-0288-01</td>
			<td></td>
		</tr>
		<tr>
			<td>MagPlex Microspheres (magnetic, fluorescent, 6.5-&#181;m-diameter beads)</td>
			<td>Luminex Corp.&#160; (Austin, TX)</td>
			<td>MC-1**** (varies by bead label)</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind microcentrifuge tubes</td>
			<td>ThermoFisher Scientific (Waltham, MA)</td>
			<td>022431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptides, Antibodies, &#38; Detection Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atezolizumab (humanized anti-PDL1 IgG1 monoclonal antibody), positive control</td>
			<td>Genentech/Roche (San Francisco, CA)</td>
			<td>n/a (prescription medications)</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated recombinant human PDL1&#160;</td>
			<td>Sino Biological (Wayne, PA)</td>
			<td>10084-H49H-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421-congugated donkey anti-human IgG</td>
			<td>Jackson Immunoresearch Laboratories Inc. (Westgrove, PA)</td>
			<td>709-675-149</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421-congugated donkey anti-rabbit IgG</td>
			<td>Jackson Immunoresearch Laboratories Inc. (Westgrove, PA)</td>
			<td>711-675-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human PD-1 (poly-histidine tagged)</td>
			<td>Acro Biosystems (Newark, DE)</td>
			<td>PD1-H5256&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-conjugated R-phycoerythrin (SAPE)</td>
			<td>Agilent (Santa Clara, CA)</td>
			<td>PJRS34-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trastuzumab (Herceptin, humanized anti-HER2 monoclonal antibody), negative control</td>
			<td>Genentech/Roche (San Francisco, CA)</td>
			<td>n/a (prescription medications)</td>
			<td></td>
		</tr>
	</tbody>
</table>

magnetic fluorescent bead-based dual-reporter flow analysis of pdl1-vaxx peptide vaccine-induced antibody blockade of the pd-1/pd-l1 interaction

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide (&#34;promiscuous&#34; T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

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

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 led by Professor Pravin Kaumaya are focused on developing B-cell epitope peptide 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 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, prebinding 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 that overcome treatment resistance. We also want to explore more effective combination therapies that better inhibit tumor growth, prevent metastasis, and prolong patient survival rates.

The assay was able to precisely quantify the inhibition of the PD-1/PD-L1 interaction by four unique polyclonal antibodies, generated against the rhPD-L1 vaccine peptides, that are being explored as potential cancer therapeutic agents. The schematic of this assay is provided in Figure 1. The amount of biotinylated rhPD-L1 that bound to rhPD-1-conjugated beads and the inhibition of this binding by the four PLD1-Vaxx-induced antibody candidates was measured in Reporter Channel 1 using a streptavidin-PE detection reagent that directly bound rhPD-L1 (Figure 2).
All four polyclonal anti-PDL1-peptide antibodies blocked the rhPD-L1 interaction with PD-1 that had been immobilized on microspheres to variable extents. The percentage of inhibition of the different anti-PDL1 peptide antibodies ranged from 48% to 74% at the maximum tested concentration of 1,000 &#181;g/mL. The positive control monoclonal antibody atezolizumab achieved 92% blockade of the PD-1/PD-L1 interaction at the maximum tested concentration14 of 4 &#181;g/mL (Figure 2). All of the experimental PDL1-Vaxx antibodies showed concentration-dependent inhibition of rhPD-L1 binding to rhPD-1 conjugated beads compared with trastuzumab, the negative control antibody that was not expected to interact with the PD-1/PD-L1 system.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65467/65467fig02.jpg" /> 
Figure 2: Blockade of rhPD-L1 interaction with rhPD-1 coupled to magnetic beads by anti-PDL1-peptide antibodies, as shown by a new fluorescent bead-based immunoassay. Recombinant human PD-1 was coupled to magnetic microspheres, and the beads were then incubated with biotinylated rhPD-L1 that had been pre-incubated with different anti-PDL1-peptide antibodies. A streptavidin-phycoerythrin detection reagent was used to bind the biotin and, thus, assess the relative quantity of rhPD-L1 that was available to bind to PD-1. Polyclonal antibodies raised in rabbits against the PDL1 peptide vaccines (anti-PDL1[36], anti-PDL1[50], anti-PDL1[95], and anti-PDL1[130]) were tested for inhibitory activity and showed 48%-74% blockade of recombinant PD-1/PD-L1 interactions at the highest concentration tested. Atezolizumab (a different anti-PDL1 monoclonal antibody) was used as the positive control. The unrelated commercial monoclonal antibody trastuzumab (anti-HER2) was used as a negative control. This figure is adapted from Guo et al.14. <a href="https://www.jove.com/files/ftp_upload/65467/65467fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65467/65467fig03.jpg" /> 
Figure 3: Comparative binding of different PDL1-Vaxx-induced antibodies to rhPD-L1 complexed to rhPD1-coated magnetic beads. Brilliant violet 421-conjugated secondary detection antibody was used to compare the binding of different rabbit polyclonal anti-PDL1-peptide antibodies to rhPD-L1 via rhPD-1 coated beads. The BV421 blue fluorescence signal was recorded in Reporter Channel 2 of the dual-reporter instrument; this signal correlates with the relative binding efficiency of the experimental anti-PDL1-peptide antibodies. Trastezumab (anti-HER2), a monoclonal antibody that targets a different checkpoint than PD-1/PD-L1, was used as a negative control. MFI represents the average bead median fluorescence intensity, which was measured in duplicate reaction wells per condition. This figure is adapted from Guo et al.14. <a href="https://www.jove.com/files/ftp_upload/65467/65467fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The relative abilities of the four experimental PDL1-Vaxx-induced candidate antibodies to bind rhPD-L1 were compared using a separate detection system (BV421-conjugated anti-rabbit IgG) that was evaluated on the instrument&#39;s second reporter channel. These results indicated that all four polyclonal anti-PDL1-peptide antibodies bound to rhPD-L1 in a concentration-dependent manner14 (Figure 3). The anti-PDL1(130) antibody showed the highest rhPDL1 binding signal of the four PDL1-Vaxx-induced antibody candidates.

Watch this Scientific Journal Video about Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction at JoVE.com

Checkpoint inhibitors are important targets in developing therapies for the battle against cancer. This report introduces a novel PDL1 peptide-based cancer vaccine, PDL1-Vaxx, which induces neutralizing polyclonal antibody production that blocks PD-1/PDL1 complex formation. This work also details the development and testing of a fluorescent bead-based assay for analyzing this activity.

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...

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