Name: magnetic fluorescent bead-based dual-reporter flow analysis of pdl1-vaxx peptide vaccine-induced antibody blockade of the pd-1/pd-l1 interaction
Uploaded: 2023-07-07T13:00:00.000+00:00
Duration: 10 min 18 s
Description: 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

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

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	<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. 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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><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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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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Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.1K Views.  The Ohio State University Wexner Medical Center. Ons project onder leiding van professor Pravin Kaumaya is gericht op het ontwikkelen van B-cel epitooppeptidevaccins die kunnen worden gebruikt om het immuunsysteem te stimuleren om de kanker beter te bestrijden. Wij zijn van mening dat peptidevaccins die gericht zijn op immuuncontrolepunten zelfs kunnen worden gecombineerd met andere therapieën om de resultaten voor de patiënt te verbeteren. We wilden het blokkerende vermogen van onze anti-PD-L1-antilichamen testen in een traditionele micr...

Video: Auteur in de schijnwerpers: Magnetische fluorescerende kralen-gebaseerde dual-reporter flow-analyse van PDL1-Vaxx peptidevaccin-geïnduceerde antilichaamblokkade van de PD-1/PD-L1-interactie 

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 ...

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment.
The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

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 ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Immunology and Infection

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

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 ...

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

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