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/pubmed?term=((B-cell+epitope+peptide+cancer+vaccines%3A+a+new+paradigm+for+combination+immunotherapies+with+novel+checkpoint+peptide+vaccine%5BTitle%5D)+AND+%22Future+Oncology%22%5BJournal%5D)">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/pubmed?term=((Revolutionization+in+cancer+therapeutics+via+targeting+major+immune+checkpoints+PD-1%2C+PD-L1+and+CTLA-4%5BTitle%5D)+AND+%22Pharmaceuticals%22%5BJournal%5D)">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/pubmed?term=((Preclinical+studies+of+a+novel+human+PD-1+B-cell+peptide+cancer+vaccine+PD1-Vaxx+from+BALB%2Fc+mice+to+beagle+dogs+and+to+non-human+primates+%28cynomolgus+monkeys%29%5BTitle%5D)+AND+%22Frontiers+in+Oncology%22%5BJournal%5D)">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/pubmed?term=((Nivolumab%3A+Targeting+PD-1+to+bolster+antitumor+immunity%5BTitle%5D)+AND+%22Future+Oncology%22%5BJournal%5D)">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/pubmed?term=((Product+review+on+the+Anti-PD-L1+antibody+atezolizumab%5BTitle%5D)+AND+%22Human+Vaccines+%26+Immunotherapeutics%22%5BJournal%5D)">Product review on the Anti-PD-L1 antibody atezolizumab.</a> Human Vaccines & 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/pubmed?term=((Immune-related+adverse+events+associated+with+immune+checkpoint+blockade%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((A+paradigm+shift%3A+Cancer+therapy+with+peptide-based+B-cell+epitopes+and+peptide+immunotherapeutics+targeting+multiple+solid+tumor+types%3A+Emerging+concepts+and+validation+of+combination+immunotherapy%5BTitle%5D)+AND+%22Human+Vaccines+%26+Immunotherapeutics%22%5BJournal%5D)">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 & Immunotherapeutics. 11 (6), 1368-1386 (2015).</li>
<li>Guo, L., Kaumaya, P. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+prototype+checkpoint+inhibitor+B-cell+epitope+vaccine+%28PD1-Vaxx%29+en+route+to+human+Phase+1+clinical+trial+in+Australia+and+USA%3A+Exploiting+future+novel+synergistic+vaccine+combinations%5BTitle%5D)+AND+%22British+Journal+of+Cancer%22%5BJournal%5D)">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/pubmed?term=((Prevention+of+mammary+tumors+with+a+chimeric+HER-2+B-cell+epitope+peptide+vaccine%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((Conformational+HER-2%2Fneu+B-cell+epitope+peptide+vaccine+designed+to+incorporate+two+native+disulfide+bonds+enhances+tumor+cell+binding+and+antitumor+activities%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Phase+I+active+immunotherapy+with+combination+of+two+chimeric%2C+human+epidermal+growth+factor+receptor+2%2C+B-cell+epitopes+fused+to+a+promiscuous+Tcell+epitope+in+patients+with+metastatic+and%2For+recurrent+solid+tumors%5BTitle%5D)+AND+%22Journal+of+Clinical+Oncology%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((Immunogenicity+and+antitumor+efficacy+of+a+novel+human+PD-1+B-cell+vaccine+%28PD1-Vaxx%29+and+combination+immunotherapy+with+dual+trastuzumab%2Fpertuzumab-like+HER-2+B-cell+epitope+vaccines+%28B-Vaxx%29+in+a+syngeneic+mouse+model%5BTitle%5D)+AND+%22Oncoimmunology%22%5BJournal%5D)">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/pubmed?term=((A+newly+discovered+PD-L1+B_cell+epitope+peptide+vaccine+%28PDL1-Vaxx%29+exhibits+potent+immune+responses+and+effective+anti-tumor+immunity+in+multiple+syngeneic+mice+models+and+%28synergizes%29+in+combination+with+a+dual+HER-2+B-cell+vaccine+%28B-Vaxx%29%5BTitle%5D)+AND+%22Oncoimmunology%22%5BJournal%5D)">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>Angeloni, S., Das, S., De Jager, W., Dunbar, S. Luminex Corporation. The xMAP® 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® 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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreen%2C+MR+%22Estimation+of+cell+number+by+hemocytometry+counting%22">Estimation of cell number by hemocytometry counting</a>. 2019 (11), Cold Spring Harbor Protocols. (2019).</li>
<li>Gibbs, J., Vessels, M., Rothenberg, M. 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><tbody><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Activation Buffer: 0.1 M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 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% NaN&lt;sub&gt;3&lt;/sub&gt;)</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&amp;nbsp;</td><td>M2933</td><td></td></tr><tr><td>&lt;strong&gt;Coupling Reagents&lt;/strong&gt;</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.&amp;nbsp; (Austin, TX)</td><td>40-50016</td><td></td></tr><tr><td>EDC, 10 mg&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>sNHS solution, 250 &amp;micro;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% NaN&lt;sub&gt;3&lt;/sub&gt; (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>&lt;strong&gt;Instrumentation and Ancillary Lab Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>xMAP INTELLIFLEX (dual-reporter instrument)</td><td>Luminex Corp.&amp;nbsp; (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)&amp;nbsp;</td><td>Luminex Corp.&amp;nbsp; (Austin, TX)</td><td>CN-0269-01</td><td></td></tr><tr><td>Luminex Magnetic Tube Separator (or comparable)</td><td>Luminex Corp.&amp;nbsp; (Austin, TX)</td><td>CN-0288-01</td><td></td></tr><tr><td>MagPlex Microspheres (magnetic, fluorescent, 6.5-&amp;micro;m-diameter beads)</td><td>Luminex Corp.&amp;nbsp; (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>&lt;strong&gt;Peptides, Antibodies, &amp;amp; Detection Reagents&lt;/strong&gt;</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&amp;nbsp;</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&amp;nbsp;</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.

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

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 

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.

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 ...

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Cancer Research

1.2K Views.  The Ohio State University Wexner Medical Center. 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.

Video: 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 

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Immunology and Infection

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating the efficacy of PD-1/PD-L1 inhibitors. A previously established human PD-1/PD-L1 blockade assay utilizing Surface Plasmon Resonance (SPR) technology (first-generation PD-1/PD-L1 inhibitor SPR screening platform) demonstrated results comparable to those obtained through Homogeneous Time-Resolved Fluorescence (HTRF) and cell-based assays, with potential for large-scale screening. Herein, an optimized version of this assay (second-generation PD-1/PD-L1 inhibitor SPR screening platform) is presented, featuring a dual-step coupling process that combines amine and bio-streptavidin coupling to enhance PD-1 orientation control on the chip and reduce PD-1 protein consumption. The updated platform was successfully validated using the PD-1/PD-L1 inhibitor BMS-1166, showing blockade effects comparable to the previous SPR-based method and other established techniques such as ELISA. These results confirm the reliability of the approach. This optimized SPR screening platform offers a high-throughput and reliable tool for identifying novel PD-1/PD-L1 inhibitors, advancing cancer immunotherapy research, and highlighting the potential of SPR in immune checkpoint inhibitor screening.

This protocol describes a blockade assay for PD-1/PD-L1 inhibitors using surface plasmon resonance technology. It employs a dual-step immobilization strategy and a tailored buffer system to accurately measure response units, facilitating the assessment of blockade rates for compounds or biologics. Additionally, it supports the high-throughput identification of PD-1/PD-L1 inhibitors.

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Biology

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1&#8211;blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1&#8211;blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1&#8211;blocking antibodies in cancer patients.

We developed a simple flow cytometry assay for evaluating the binding of PD-1&#8211;blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The ...

Medicine

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells

Source: Strazza, M., et al. <a href="https://app.jove.com/v/58433">Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes.</a> J. Vis. Exp. (2018).
In this video, we demonstrate a technique to perform the co-immunoprecipitation of GFP-tagged wild-type and mutated PD-1 proteins using beads coated with anti-GFP antibodies. The bead-bound PD-1-GFP proteins are incubated with SHP2 proteins that interact with PD-1, enabling the identification of specific structural motifs in PD-1 that play a critical role in SHP2 interaction.

Source: Strazza, M., et al. Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes. J. Vis. Exp. (2018).
In this ...

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

Summary

Explore More Videos

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

In Vivo EPR Assessment of pH, pO₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells