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.

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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JoVE Journal

Cancer Research

1.1K 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 ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...