Name: manufacturing chimeric antigen receptor (car) t cells for adoptive immunotherapy
Uploaded: 2019-12-17T12:00:00
Description: Watch this Scientific Journal Video about Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy at JoVE.com

This work was supported in part through funding provided by Novartis Pharmaceuticals through a research alliance with the University of Pennsylvania (Michael C. Milone) as well as St. Baldrick&#39;s Foundation Scholar Award (Saba Ghassemi).

All animal studies are approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
1. T Cell Activation, Transduction, and Expansion
<ol>
	<li>Activate fresh or cryopreserved primary human T cells by mixing with anti CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM HEPES, and IL2 (100 units/mL). Mai...

<ol>
	<li>Brentjens, R. J., 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=CD19-targeted+T+cells+rapidly+induce+molecular+remissions+in+adults+with+chemotherapy-refractory+acute+lymphoblastic+leukemia.">CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.</a> Science Translational Medicine. 5 (177), 177ra138 (2013).</li><li>Grupp, S. A., 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=Chimeric+antigen+receptor-modified+T+cells+for+acute+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.</a> The New England Journal of Medicine. 368 (16), 1509-1518 (2013).</li><li>Kalos, M., 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=T+Cells+with+Chimeric+Antigen+Receptors+Have+Potent+Antitumor+Effects+and+Can+Establish+Memory+in+Patients+with+Advanced+Leukemia.">T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia.</a> Science Translational Medicine. 3 (95), 95ra73 (2011).</li><li>Kochenderfer, J. N., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+B-cell+cancer+with+T+cells+expressing+anti-CD19+chimeric+antigen+receptors.">Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.</a> Nature Reviews. Clinical Oncology. 10 (5), 267-276 (2013).</li><li>Maude, S. L., 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=Chimeric+antigen+receptor+T+cells+for+sustained+remissions+in+leukemia.">Chimeric antigen receptor T cells for sustained remissions in leukemia.</a> The New England Journal of Medicine. 371 (16), 1507-1517 (2014).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+in+chronic+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.</a> The New England Journal of Medicine. 365 (8), 725-733 (2011).</li><li>Porter, D. L., 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=Chimeric+antigen+receptor+T+cells+persist+and+induce+sustained+remissions+in+relapsed+refractory+chronic+lymphocytic+leukemia.">Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.</a> Science Translational Medicine. 7 (303), 303ra139 (2015).</li><li>Maude, S. L., Teachey, D. T., Porter, D. L., Grupp, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+chimeric+antigen+receptor+T-cell+therapy+for+acute+lymphoblastic+leukemia.">CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.</a> Blood. 125 (26), 4017-4023 (2015).</li><li>Kochenderfer, J. N., 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=Lymphoma+Remissions+Caused+by+Anti-CD19+Chimeric+Antigen+Receptor+T+Cells+Are+Associated+With+High+Serum+Interleukin-15+Levels.">Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels.</a> Journal of Clinical Oncology. 35 (16), 1803-1813 (2017).</li><li>Bollard, C. M., Rooney, C. M., Heslop, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+therapy+in+the+treatment+of+post-transplant+lymphoproliferative+disease.">T-cell therapy in the treatment of post-transplant lymphoproliferative disease.</a> Nature Reviews. Clinical Oncology. 9 (9), 510-519 (2012).</li><li>Brestrich, G., 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=Adoptive+T-cell+therapy+of+a+lung+transplanted+patient+with+severe+CMV+disease+and+resistance+to+antiviral+therapy.">Adoptive T-cell therapy of a lung transplanted patient with severe CMV disease and resistance to antiviral therapy.</a> American Journal of Transplantation. 9 (7), 1679-1684 (2009).</li><li>Savoldo, B., 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=Treatment+of+solid+organ+transplant+recipients+with+autologous+Epstein+Barr+virus-specific+cytotoxic+T+lymphocytes+(CTLs).">Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs).</a> Blood. 108 (9), 2942-2949 (2006).</li><li>Berger, C., 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=Adoptive+transfer+of+effector+CD8++T+cells+derived+from+central+memory+cells+establishes+persistent+T+cell+memory+in+primates.">Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates.</a> The Journal of Clinical Investigation. 118 (1), 294-305 (2008).</li><li>Gattinoni, L., 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=A+human+memory+T+cell+subset+with+stem+cell-like+properties.">A human memory T cell subset with stem cell-like properties.</a> Nature Methods. 17 (10), 1290-1297 (2011).</li><li>Hinrichs, C. S., 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=Adoptively+transferred+effector+cells+derived+from+naive+rather+than+central+memory+CD8++T+cells+mediate+superior+antitumor+immunity.">Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (41), 17469-17474 (2009).</li><li>Klebanoff, C. A., 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=Central+memory+self/tumor-reactive+CD8++T+cells+confer+superior+antitumor+immunity+compared+with+effector+memory+T+cells.">Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (27), 9571-9576 (2005).</li><li>Wang, X., 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=Engraftment+of+human+central+memory-derived+effector+CD8++T+cells+in+immunodeficient+mice.">Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice.</a> Blood. 117 (6), 1888-1898 (2011).</li><li>Wang, X., 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=Comparison+of+naive+and+central+memory+derived+CD8++effector+cell+engraftment+fitness+and+function+following+adoptive+transfer.">Comparison of naive and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer.</a> Oncoimmunology. 5 (1), e1072671 (2016).</li><li>Fraietta, J., 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=Determinants+of+response+and+resistance+to+CD19+chimeric+antigen+receptor+(CAR)+T+cell+therapy+of+chronic+lymphocytic+leukemia.">Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.</a> Nature Medicine. 24 (5), 563-571 (2018).</li><li>Ghassemi, S., 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=Reducing+Ex+Vivo+Culture+Improves+the+Antileukemic+Activity+of+Chimeric+Antigen+Receptor+(CAR)+T+Cells.">Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells.</a> Cancer Immunology Research. 6 (9), 1100-1109 (2018).</li><li>Milone, M. C., 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=Chimeric+receptors+containing+CD137+signal+transduction+domains+mediate+enhanced+survival+of+T+cells+and+increased+antileukemic+efficacy+in+vivo.">Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 17 (8), 1453-1464 (2009).</li><li>Cieri, N., 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=IL-7+and+IL-15+instruct+the+generation+of+human+memory+stem+T+cells+from+naive+precursors.">IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.</a> Blood. 121 (4), 573-584 (2013).</li><li>Cui, G., 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=IL-7-Induced+Glycerol+Transport+and+TAG+Synthesis+Promotes+Memory+CD8+T+Cell+Longevity.">IL-7-Induced Glycerol Transport and TAG Synthesis Promotes Memory CD8 T Cell Longevity.</a> Cell. 161 (4), 750-761 (2015).</li><li>Xu, Y., 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=Closely+related+T-memory+stem+cells+correlate+with+in+vivo+expansion+of+CAR.CD19-T+cells+and+are+preserved+by+IL-7+and+IL-15.">Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15.</a> Blood. 123 (24), 3750-3759 (2014).</li><li>Singh, N., Perazzelli, J., Grupp, S. A., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+memory+phenotypes+drive+T+cell+proliferation+in+patients+with+pediatric+malignancies.">Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies.</a> Science Translational Medicine. 8 (320), 320ra323 (2016).</li></ol>

Here we describe approaches to measure the function and efficacy of CAR T cells harvested at varying intervals throughout ex vivo culture. Our methods provide comprehensive insight into assays designed to assess proliferative capacity as well as effector function in vitro. We describe how to measure CAR T cell activity following stimulation through the CAR and detail xenograft models of leukemia using CAR T cells harvested at day 3 vs day 9 of their logarithmic expansion phase.
There are inher...

Chimeric antigen receptors (CARs) provide a promising approach to redirect T cells against distinct tumor antigens. CARs are synthetic receptors that bind an antigen target. While their precise composition is variable, CARs generally contain 3 distinct domains. The extracellular domain directs binding to a target antigen and is typically comprised of a single chain antibody fragment linked to the CAR via an extracellular hinge. The second domain, commonly derived from the CD3&#950; chain of the T cell receptor (TCR) complex, promotes T cell activation following CAR engagement. A third costimulatory domain is included to enhance T cell function, engraftment, metabolism, and persistence. The success of CAR T cell therapy in various hematopoietic malignancies including B cell acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and multiple myeloma highlights the therapeutic promise of this approach1,2,3,4,5,6. The recent Food and Drug Administration (FDA) approvals for two CD19-specific CAR T cell therapies, tisagenlecleucel for pediatric and young adult ALL and axicabtagene ciloleucel for diffuse large B-cell lymphoma, reinforces the translational merit of CAR T cell Therapy.
CAR T-based approaches involve the isolation of T cells from peripheral blood, activation, genetic modification, and expansion ex vivo. Differentiation is an important parameter regulating CAR T cell efficacy. Accordingly, restricting T cell differentiation during ex vivo culture enhances the ability of the infused product to engraft, expand, and persist, providing long term immunosurveillance following adoptive transfer2,7,8,9. T cells consist of several distinct subsets including: na&#239;ve T cells (Tn), central memory (Tcm), effector memory (Tem), effector differentiated (Tte) and stem cell memory (Tscm). Effector differentiated T cells have potent cytolytic ability; however, they are short lived and engraft poorly10,11,12. In contrast, T cells with a less-differentiated phenotype including na&#239;ve T cells and Tcm exhibit superior engraftment and proliferative abilities following adoptive cell transfer13,14,15,16,17,18. The composition of the collected T cells in the premanufactured product can vary across patients and correlates with the therapeutic potential of CAR T cells. The proportion of T cells with a na&#239;ve-like immunophenotype in the starting apheresis product is highly correlated with both engraftment and clinical response19.
Culture duration is an important parameter influencing differentiation in CAR T cells prepared for adoptive transfer. We recently developed an approach to generate superior quality CAR T cells using an abbreviated culture paradigm20. Using our approach, we showed that limited culture gives rise to CAR T cells with superior effector function and persistence following adoptive transfer in xenograft models of leukemia. Here, we present the approaches to reliably generate CART19 cells (autologous T cells engineered to express anti-CD19 scFv attached to CD3&#950; and the 4-1BB signaling domains) and include a detailed description of the assays that provide insight into CAR T bioactivity and efficacy prior to adoptive transfer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti CD3/CD28 dynabeads</td>
			<td>Thermo Fisher</td>
			<td>40203D</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD8</td>
			<td>BD Biosciences</td>
			<td>555369</td>
			<td>RRID:AB_398595</td>
		</tr>
		<tr>
			<td>APC-H7 Mouse anti-Human CD8 Antibody</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>RRID:AB_1645481</td>
		</tr>
		<tr>
			<td>BD FACS Lysing Solution 10X Concentrate</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Trucount Absolute Counting Tubes</td>
			<td>BD Biosciences</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-human CD4 Antibody</td>
			<td>BioLegend</td>
			<td>317444</td>
			<td>RRID:AB_2561866</td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-human CD3 Antibody</td>
			<td>BioLegend</td>
			<td>317322</td>
			<td>RRID:AB_2561911</td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>Life Technolohgies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads,</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-Human CD197 (CCR7) Antibody</td>
			<td>BD Pharmingen</td>
			<td>561271</td>
			<td>RRID:AB_10561679</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD4</td>
			<td>BD Biosciences</td>
			<td>555346</td>
			<td>RRID:AB_395751</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human AB serum</td>
			<td>Valley Biomedical</td>
			<td>HP1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2 IS, premium grade</td>
			<td>Miltenyi</td>
			<td>130-097-744</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>28030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintillation counter, MicroBeta trilux</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Violet</td>
			<td>Molecular Probes</td>
			<td>L34964</td>
			<td></td>
		</tr>
		<tr>
			<td>Multisizer Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na251CrO4</td>
			<td>Perkin Elmer</td>
			<td>NEZ030S001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue anti-human CD14 Antibody</td>
			<td>BioLegend</td>
			<td>325616</td>
			<td>RRID:AB_830689</td>
		</tr>
		<tr>
			<td>Pacific Blue anti-human CD19 Antibody</td>
			<td>BioLegend</td>
			<td>302223</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD45RO Antibody</td>
			<td>BD Biosciences</td>
			<td>555493</td>
			<td>RRID:AB_395884</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-human CD95 (Fas) Antibody</td>
			<td>BioLegend</td>
			<td>305610</td>
			<td>RRID:AB_493652</td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-human CD27 Antibody</td>
			<td>Beckman Coulter</td>
			<td>A54823</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red-free medium</td>
			<td>Gibco</td>
			<td>10373-017</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure SDS Solution, 10%</td>
			<td>Invitrogen</td>
			<td>15553027</td>
			<td></td>
		</tr>
		<tr>
			<td>Via-Probe</td>
			<td>BD Biosciences</td>
			<td>555815</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Gibco</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin - K+ Salt</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td></td>
		</tr>
	</tbody>
</table>

manufacturing chimeric antigen receptor (car) t cells for adoptive immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Using the methods described above, we stimulated and expanded T cells for either 3 or 9 days (Figure 1A,B). We also analyzed their differentiation profile, as indicated by the gating strategy outlined in Figure 1C, by measuring the abundance of distinct glycoproteins expressed on the cell surface. We show a progressive shift towards effector differentiation over time during ex vivo culture (Figure 1...

Watch this Scientific Journal Video about Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy at JoVE.com

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

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Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...