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class="block-views-blockheader"> <div class="view-content"> <div class="fixed-element field field--name-field-paragraph"> <div class="view-row"> <div class="paragraph-view-type-banner flex"> <div class="bgr-image-header"> <img src="https://www.reactionbiology.com/wp-content/uploads/2023/07/banner-FINAL-option-2.jpg" alt="T Cell Killing Assays"> </div> <div class="container center"> <div class="field field--name-field-headline-big field--type-string field--label-hidden field__items"> <div class="field__item">Immuno-Oncology Platform</div> </div> </div> </div> </div> </div> </div> </div> </section> <div id="main-content"> <div id="block-proquinase-breadcrumbs" class="color-scheme-turquoise block block-system block-system-breadcrumb-block"> <div class="container"> <nav class="breadcrumb" role="navigation" aria-labelledby="system-breadcrumb"> <ol> <li><a href="https://www.reactionbiology.com">Home</a></li> <li>Services</li><li><a href="https://www.reactionbiology.com/services/immuno-oncology-platform/">Immuno-Oncology Platform</a></li><li><a href="https://www.reactionbiology.com/services/immuno-oncology-platform/immuno-oncology-assays/">Immuno-Oncology Assays</a></li> <li>T Cell Killing Assays</li> </ol> </nav> </div> </div> <div class="view-container view-simple-page-conent"> <div class="container"> <article id="post-2647" class="post-2647 page type-page status-publish hentry" itemtype="https://schema.org/CreativeWork" itemscope=""> <div class="entry-content entry-content-servises "> <h1>T Cell Killing Assays</h1> <div class="text-mit-bild-text"> <div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"> <p>T cell killing assays measure a drug’s potency to help T cells kill tumor cells. T cell killing assays are a valuable tool for immuno-oncology discovery projects for in vitro assessment of new drug candidates to:</p> <ul> <li>Understand the immune-modulating action of new immunotherapeutics</li> <li>Optimize the potency of new drugs</li> <li>Select the most promising drug candidates for animal studies.</li> </ul> <p>Cytotoxic T cells can recognize and kill target cancer cells. New therapies are designed to facilitate T cell activation and increase effector function while suppressing inhibitory mechanisms. Another approach is helping T cells to move in the proximity of their target cells. Both approaches can be investigated with our T cell killing assays. We are also open to exploring further avenues of modulating the immune response with our assays.</p> <p>Our T cell killing assays can be used in single substance or drug combination modality.</p> </div> <div class="contact-buttons"> <div class="color-scheme-turquoise block block-block-content block-block-content9594f627-1f8b-4f13-b65e-979c19e399c0"> <a href="https://www.reactionbiology.com/contact/contact-us"> <div class="field field--name-field-icon field--type-image field--label-hidden field__items"> <img decoding="async" src="https://www.reactionbiology.com/wp-content/themes/Divi-child/images/icons-email.svg" alt="E-Mail" title="E-Mail" typeof="foaf:Image"> </div> <div class="field field--name-field-text field--type-string field--label-hidden field__items"> Contact an expert </div> </a> </div> <div class="color-scheme-turquoise block block-block-content block-block-contentc2ce2ae7-66e6-4f1b-87fb-ded5e6bcfb90"> <a class="newsletter-icon-box" href="#"> <div class="field 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id="teaser-boxes-5_anchor" class="paragraph weiss black paragraph--type--paragraph-container paragraph--view-mode--default"><div id="tcka-id" class="paragraph text-right images-1 headline-right contact-buttons- paragraph--type--text-with-image paragraph--view-mode--default"><div class="container center"><div class="text-wrapper"><h3>T Cell Killing Strategies of Immunotherapies</h3> <div class="text-mit-bild-text"> <div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"> <p>Various strategies are used to enable tumor cell killing by cytotoxic T cells:</p> <ul> <li>Blockage of immune checkpoint inhibition</li> <li>T cell activation via binding to anti-CD3 and other stimulating receptors</li> <li>Physically engaging T cells and tumor cells</li> <li>Creation of T cells expressing tumor cell-specific T cell receptors (CAR-T)</li> </ul> <p>Reaction Biology’s killing assays were created to test the efficacy of these immunotherapy strategies on T cells.</p> </div> </div> <div class="text-mit-bild-bild field field--name-field-images field--type-image field--label-hidden field__items"> <div class="text-mit-bild-bild-rahmen"> <div class="field__item"><img loading="lazy" decoding="async" class="alignnone size-full wp-image-2665" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/220512_Mehdi_T-cell-killing-scheme-01.png" alt="" width="800" height="495" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/220512_Mehdi_T-cell-killing-scheme-01.png 800w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220512_Mehdi_T-cell-killing-scheme-01-300x186.png 300w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220512_Mehdi_T-cell-killing-scheme-01-768x475.png 768w" sizes="(max-width: 800px) 100vw, 800px" /></div> </div> </div> </div></div></div><div id="tcka-id2" class="paragraph paragraph--type--tab-container paragraph--view-mode--default"><div class="container center"><h2>T Cell Killing Assays Available at Reaction Biology</h2> <div class="tab-container"><ul class="tab-navigation"><li class="tab-active" data-id="#tab-tcka-id221">Assay options</li><li data-id="#tab-tcka-id222">ADCC assay performed with Jurkat T cells stably expressing FcγRIII receptor and an NFAT response element</li><li data-id="#tab-tcka-id223">Study example: Complement Dependent Cell Cytotoxicity (CDC) assay</li><li data-id="#tab-tcka-id224">Study example: Anti-CD3+anti-PD1 treatment</li><li data-id="#tab-tcka-id225">Study example: BiTE induced killing of lymphoma cells</li></ul><div class="field field--name-field-paragraph field--type-entity-reference-revisions field--label-hidden field__items"><div id="tab-tcka-id221" class="tab-content-active paragraph paragraph--type--tab-text-with-image paragraph--view-mode--default"><div class="field field--name-field-title field--type-string field--label-hidden field__items">Assay options</div><div class="tab-content"><div class="tab-content-wrapper"><div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"><p>Reaction Biology offers a versatile suite of immuno-oncology assay setups. Since every project is different, we enable a customized study layout with various options for immune cells, target cells, or readouts for our customers to choose from.</p> <p>Please <a href="https://www.reactionbiology.com/contact/contact-us">inquire</a> about further customization requirements to tailor an assay specific to your research needs.</p> <p><u>Options for immune cells</u></p> <ul> <li>PBMCs</li> <li>isolated T cells</li> </ul> <p><u>Options for target cells</u></p> <ul> <li>human or murine tumor cells</li> <li>tumor cells stably expressing tumor-associated antigens or luciferase</li> <li>2D or 3D cell culture</li> </ul> <p><u>Options for readout</u></p> <ul> <li>quantification of luciferase-expressing tumor cells</li> <li>flow cytometry</li> <li>live-cell imaging and high-content imaging</li> <li>Bliss factor determination of drug combination therapies</li> </ul> <p><u>Therapy options</u></p> <ul> <li>T cell killing enhanced by T cell activation</li> <li>T cell killing mediated by T cell engagement</li> <li>further therapy options are available upon request</li> </ul> </div></div></div></div><div id="tab-tcka-id222" class=" paragraph paragraph--type--tab-text-with-image paragraph--view-mode--default"><div class="field field--name-field-title field--type-string field--label-hidden field__items">ADCC assay performed with Jurkat T cells stably expressing FcγRIII receptor and an NFAT response element</div><div class="tab-content"><div class="tab-content-wrapper"><div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"><p><strong>Assay principle</strong></p> <p>Components of the cell-mediated immune system target and kill virus-infected or other diseased (e.g., cancer) cells. When FcR-bearing effector cells identify a target cell that has been opsonized by antibodies, the ADCC pathway is activated. Once the effector cells have been activated by binding, the target cells will be killed by the cell-mediated ADCC immune defense mechanism.</p> <p><strong>Goal</strong></p> <p>Antibody-dependent cell-mediated target killing of virus-infected or other diseased cells.</p> <p><strong>Setup</strong></p> <p>Effector cells (Jurkat T cells) cultured for ADCC bioassays are engineered with stably expressing FcγRIIIa and have an NFAT response element that promotes the expression of firefly luciferase. Upon antibody binding to target antigens on the cell surface of the target cells (tumor cells, virus-infected cells, or other diseased cells), the Fc portion of the target-bound antibody binds to the FcγRIIIa receptors on the cell surface of the effector cell. Through multiple cross-links, the binding ensures precise cell-to-cell recognition, which ultimately results in ADCC activation. The result of activating this pathway leads to the death of the target cells.</p> <p><img loading="lazy" decoding="async" class="alignnone size-full wp-image-2669" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped.png" alt="" width="2000" height="991" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped.png 2000w, https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped-300x149.png 300w, https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped-1024x507.png 1024w, https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped-768x381.png 768w, https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped-1536x761.png 1536w, https://www.reactionbiology.com/wp-content/uploads/2023/07/NK-cell-killing-assay-image-cropped-1080x535.png 1080w" sizes="(max-width: 2000px) 100vw, 2000px" /></p> <p><strong>Example Data</strong></p> <p><img loading="lazy" decoding="async" class="alignnone wp-image-2670" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/ADCC_Rituximab.png" alt="" width="300" height="307" /><img loading="lazy" decoding="async" class="alignnone wp-image-2668" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/ADCC_Anti-CD20.png" alt="" width="300" height="301" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/ADCC_Anti-CD20.png 706w, https://www.reactionbiology.com/wp-content/uploads/2023/07/ADCC_Anti-CD20-300x300.png 300w, https://www.reactionbiology.com/wp-content/uploads/2023/07/ADCC_Anti-CD20-150x150.png 150w" sizes="(max-width: 300px) 100vw, 300px" /></p> <p>ADCC Reporter Bioassay was used to measure the Fc effector function of Rituximab to demonstrate assay specificity.</p> </div></div></div></div><div id="tab-tcka-id223" class=" paragraph paragraph--type--tab-text-with-image paragraph--view-mode--default"><div class="field field--name-field-title field--type-string field--label-hidden field__items">Study example: Complement Dependent Cell Cytotoxicity (CDC) assay</div><div class="tab-content"><div class="tab-content-wrapper"><div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"><p><strong>Introduction</strong></p> <p>TNF is involved in the pathology of several inflammatory and autoimmune diseases, and the use of neutralizing antibodies or engineered TNFR fusion proteins to block TNF/TNFR binding has proven to be a viable strategy for providing clinical benefits in inflammatory diseases. TNF-producing cells could be targeted for destruction, which could help reduce the inflammatory burden even more. TNF-targeted antibodies can activate effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) to destroy mTNF-expressing inflammatory cells.</p> <p><strong>Goal</strong></p> <p>Complement-dependent cytotoxicity (CDC) assays are conducted to evaluate new therapeutics during the drug development phase. The CDC assay is used for safety profiling by screening therapeutic candidates to ensure that the compound does not induce complement events and to look for other unexpected effects. Fresh blood that is less than four hours old is used in this assay, as it generates better responses and results.</p> <p><strong>Example Data</strong></p> <p><img loading="lazy" decoding="async" class="size-medium wp-image-2671 aligncenter" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/CDC_Assay-297x300.png" alt="" width="297" height="300" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/CDC_Assay-297x300.png 297w, https://www.reactionbiology.com/wp-content/uploads/2023/07/CDC_Assay.png 710w" sizes="(max-width: 297px) 100vw, 297px" /></p> <p>CDC activity of infliximab assessed using the CDC assay developed by Reaction Biology.</p> </div></div></div></div><div id="tab-tcka-id224" class=" paragraph paragraph--type--tab-text-with-image paragraph--view-mode--default"><div class="field field--name-field-title field--type-string field--label-hidden field__items">Study example: Anti-CD3+anti-PD1 treatment</div><div class="tab-content"><div class="tab-content-wrapper"><div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"><p><strong>Killing of luciferase-expressing tumor cells via PBMCs stimulated with anti-CD3 treatment enhanced by immune-checkpoint inhibition</strong></p> <p><strong>Assay principle</strong></p> <p>Engaging the T-cell receptor complex leads to activation of resting T cells and induction of cytokine release. In this study example, we determined the potency of drugs enhancing T cell activation by co-cultivating PBMCs with tumor cells in the presence of anti-CD3. Further, we modulated the T cell’s killing capacity with the immune checkpoint inhibitor anti-PD1.</p> <p><strong>Setup</strong></p> <p><img loading="lazy" decoding="async" class="alignnone size-full wp-image-2675" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01.png" alt="" width="2000" height="334" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01.png 2000w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01-300x50.png 300w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01-1024x171.png 1024w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01-768x128.png 768w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01-1536x257.png 1536w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_anti-CD3_anti-PD1_setup-01-1080x180.png 1080w" sizes="(max-width: 2000px) 100vw, 2000px" /></p> <p>Tumor cells stably expressing luciferase were co-cultured with PBMCs at an E:T (effector to target cell ratio) of 6:1 or a range of E:T ratios, in the presence of anti-CD3 at low concentrations to induce killing at a suboptimal level. In a second experiment, the checkpoint inhibitor anti-PD1 was added at different concentrations (7x semi-log). The co-culture was incubated for 4 days after which the viability of the tumor cells was measured by luciferase activity.</p> <p><strong>Study results</strong></p> <p>T cell stimulation via anti-CD3 antibody resulted in activation of T cells and killing of tumor cells (A). This effect was enhanced by drug combination testing with anti-PD1 antibodies (B).</p> <p><strong><img loading="lazy" decoding="async" class="size-full wp-image-2674 aligncenter" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/220509_study_example_T-cell-activation-CD3-PD1_0.png" alt="" width="600" height="460" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/220509_study_example_T-cell-activation-CD3-PD1_0.png 600w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220509_study_example_T-cell-activation-CD3-PD1_0-300x230.png 300w" sizes="(max-width: 600px) 100vw, 600px" /></strong></p> <p>PBMCs from a healthy donor were co-cultured with cancer cell lines HT29 and COLO205 stably transduced with Firefly luciferase (FLuc).</p> <p>A. An E:T ratio of 6:1 was used with the addition of semi-log dilutions of anti-human CD3 (OKT3).<br /> B. Anti-human CD3 (OKT3) at a concentration of 0,68 ng/ml was used to stimulate T cells in a killing assay with different E:T ratios. Anti-PD1 antibody Nivolumab treatment slightly enhanced tumor cell killing.</p> <p>The % viability was calculated using tumor cells alone as maximal viability (100%) and Staurosporine treatment as maximal killing (0%).</p> </div></div></div></div><div id="tab-tcka-id225" class=" paragraph paragraph--type--tab-text-with-image paragraph--view-mode--default"><div class="field field--name-field-title field--type-string field--label-hidden field__items">Study example: BiTE induced killing of lymphoma cells</div><div class="tab-content"><div class="tab-content-wrapper"><div class="clearfix text-formatted field field--name-field-text field--type-text-long field--label-hidden field__items"><p><strong>Combination therapy to enhance BiTE-induced T cell killing of luciferase-labeled B-cell lymphoma cell line OCI-LY1</strong></p> <p><strong>Assay principle</strong></p> <p>Bi-specific antibodies contain two binding sites to engage T cells and tumor cells resulting in tumor cell killing by (i) bringing the cells in close proximity, and (ii) stimulating cytokine release in T cells.</p> <p>In this study example, we determine the capacity of a bi-specific engaging molecule to facilitate tumor cell killing by T cells and the combinatorial potential with other clinically relevant agents.</p> <p><strong>Setup</strong></p> <p><strong><img loading="lazy" decoding="async" class="alignnone wp-image-2672 size-large" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01-1024x155.png" alt="" width="1024" height="155" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01-1024x155.png 1024w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01-300x45.png 300w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01-768x116.png 768w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01-1536x233.png 1536w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01-1080x164.png 1080w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220524_Mehdi_T-cell_BiTE_setup-01.png 2000w" sizes="(max-width: 1024px) 100vw, 1024px" /></strong></p> <p>CD19-expressing B cell lymphoma cells stably expressing firefly luciferase are co-cultured with PBMCs at an E:T (effector to target cell ratio) of 2:1. We tested the bi-specific T cell engager Blinatumomab recognizing CD19 and CD3 at different concentrations (7x semi-log), with or without additional compounds. The co-culture was incubated for 4 days, after which the viability of the tumor cells was measured by luciferase activity.</p> <p><strong>Study results</strong></p> <p>BiTE Blinatumomab and the small molecule Lenalidomide act synergistically to kill CD19-positive B cell lymphoma cells, whereas the combination with S63845 acts antagonistically.<img loading="lazy" decoding="async" class="alignnone size-full wp-image-2673" src="https://www.reactionbiology.com/wp-content/uploads/2023/07/220609_BiTE_T-cell-killing_study-results.png" alt="" width="1470" height="485" srcset="https://www.reactionbiology.com/wp-content/uploads/2023/07/220609_BiTE_T-cell-killing_study-results.png 1470w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220609_BiTE_T-cell-killing_study-results-300x99.png 300w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220609_BiTE_T-cell-killing_study-results-1024x338.png 1024w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220609_BiTE_T-cell-killing_study-results-768x253.png 768w, https://www.reactionbiology.com/wp-content/uploads/2023/07/220609_BiTE_T-cell-killing_study-results-1080x356.png 1080w" sizes="(max-width: 1470px) 100vw, 1470px" /></p> <p>A. PBMCs from a healthy donor were incubated with the B cell lymphoma cell line OCI-LY1 at a 2:1 E:T ratio and treated with a range of 7 concentrations of Blinatumomab. Luciferase expression of live tumor cells was measured after 4 days, and viability was calculated using Staurosporine treated cells as maximal cell death control and untreated target cells as maximal viability control. An IC50 of 0,19 ng/ml was found for Blinatumomab. The combinatorial effects of Blinatumomab with Lenalidomide and MCL1-inhibitor S63845 are shown as examples.</p> <p>B. Using the bliss factor scoring, we determined which drug combinations produce additive, synergistic, or antagonistic effects for killing tumor cells.</p> </div></div></div></div></div></div></div></div></div> </div> <footer id="main-footer" aria-label="Site" itemtype="https://schema.org/WPFooter" itemscope=""> <div class="region region-footer"> <div class="color-scheme-turquoise block block-block-content" id="block-letsworktogether"> <div class="bgr-image-footer"> <img src="https://www.reactionbiology.com/wp-content/themes/Divi-child/images/lets-discover-footer.jpg " alt=""> </div> <div class="footer-wrapper"><div class="field field--name-field-link field--type-link field--label-hidden field__items"><div class="field field--name-field-text field--type-string field--label-hidden field__items">Let’s discover together.</div> <div class="textwidget"><p><a href="/contact/contact-us">Contact us</a></p> </div> </div></div> </div> <div id="et-footer-nav"> <div class="container"> <ul id="menu-footer-menu" class="bottom-nav"><li id="menu-item-37" class="menu-item menu-item-type-post_type menu-item-object-page menu-item-37"><a href="https://www.reactionbiology.com/terms-and-conditions/">Terms and Conditions</a></li> <li id="menu-item-38" class="menu-item menu-item-type-post_type menu-item-object-page menu-item-38"><a href="https://www.reactionbiology.com/privacy-policy/">Privacy Policy</a></li> <li id="menu-item-39" class="menu-item menu-item-type-post_type menu-item-object-page menu-item-39"><a href="https://www.reactionbiology.com/sitemap/">Site Map</a></li> <li id="menu-item-7757" class="menu-item menu-item-type-post_type menu-item-object-page menu-item-7757"><a href="https://www.reactionbiology.com/contact/contact-us/">Contact Us</a></li> <li id="menu-item-41" class="menu-item menu-item-type-post_type menu-item-object-page menu-item-41"><a href="https://www.reactionbiology.com/company/about-us/">Company</a></li> <li id="menu-item-42" class="menu-item menu-item-type-post_type menu-item-object-page menu-item-42"><a href="https://www.reactionbiology.com/faq/">FAQ</a></li> </ul> </div> </div>  </div> <div id="footer-bottom"> <div class="container clearfix"> <ul class="et-social-icons"> <li class="et-social-icon et-social-facebook"> <a href="https://www.facebook.com/ReactionBiology" class="icon"> <span>Facebook</span> </a> </li> <li class="et-social-icon et-social-linkedin"> <a href="https://www.linkedin.com/company/reaction-biology-corporation" class="icon"> <span>Google</span> </a> </li> <li class="et-social-icon et-social-twitter"> <a href="https://twitter.com/ReactionBiology" class="icon"> <span>Twitter</span> </a> </li> </ul><div id="footer-info">© <span class="year">2024</span> <strong>Reaction Biology</strong>. 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