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). Maintain T cells at a concentration of 106 T cells/mL during expansion. Culture T cells at 37 &#176;C, 20% O2, and 95% humidity with 5% CO2.</li>
	<li>After overnight stimulation, add lentiviral supernatant to activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection (MOI) of 3&#8722;5. 
	NOTE: The CD19-BB&#950; CAR lentivirus plasmid consists of a CD8 hinge, 4-1BB costimulatory domain, and CD3&#950; signaling domain21. CD19-BB&#950; lentiviral supernatant was generated as previously described21.</li>
	<li>On day 3, collect a representative aliquot of cells for cryopreservation. Prior to cryopreservation, remove the magnetic beads by gentle pipetting and magnetic separation. Prepare freezing medium containing phosphate-buffered saline (PBS) with 0.5% dimethyl sulfoxide (DMSO) and store in 4 &#176;C until use.
	<ol>
		<li>Centrifuge T cells at 300 x g for 5 min. Discard the supernatant and add 5 mL of PBS. Centrifuge cells at 300 x g for 5 min and discard the PBS.</li>
		<li>Resuspend the cell pellet in 1 mL of cold cryopreservation medium. Freeze T cells in a chilled freezing container and store at -80 &#176;C for 48 h. Transfer the frozen cells to liquid nitrogen.</li>
	</ol>
	</li>
	<li>Wash the rest of the T cells once in 5 mL of PBS to eliminate residual vector. Centrifuge at 300 x g for 5 min. Decant the PBS and resuspend the cell pellet in T cell culture medium at a concentration of 0.5 x 106 cells/mL.</li>
	<li>Split the T cells into two cultures, designated for day 5 and day 9. Count T cells by flow cytometry using counting beads (Table of Materials) and monoclonal antibodies to human CD4 and CD8, as well as a viability dye (Table of Materials).
	<ol>
		<li>To measure T cell concentration, prepare a master mix containing 500 &#181;L of PBS, 5 &#181;L of counting beads, 10 &#181;L of 7-Amino-actinomycin D cell viability solution, 4 &#181;L of CD4-FITC and 4 &#181;L of CD8-APC. Add 40 &#181;L of T cells to the master mix and measure cell concentration by flow cytometry based on number of live T cells/bead counts. Refeed to maintain the cultures at a concentration of 0.5 x 106 cells/mL every other day.</li>
	</ol>
	</li>
	<li>On day 5, count and cryopreserve day 5 cultures as described in steps 1.3 and 1.5.</li>
	<li>On day 7, wash 0.5 x 106 T cells in PBS and resuspend in 100 &#181;L of fluorescence activated cell sorting (FACS) buffer. Detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry.</li>
	<li>On day 9, count day 9 cultures and cryopreserve as described in step 1.3.</li>
</ol>
2. Phenotypic Assessment of T Cell Differentiation
<ol>
	<li>Prepare a master mix containing pre-titrated antibodies for anti-CD3&#8211;BV605 (clone OKT3), anti-CD14&#8211;Pacific Blue (PB) (clone HCD14), anti-CD19&#8211;PB (clone HIB19), anti-CD4&#8211;BV510 (clone OKT4), anti-CD8&#8211;H7APC (clone SK1), anti-CCR7&#8211;FITC (clone 150503), anti-CD45RO&#8211;PE (clone UCHL1), anti-CD27&#8211;PE-Cy7 (clone 1A4CD27), anti-CD95&#8211;PerCP-Cy5.5 (Clone DX2), and anti-CAR19-APC.</li>
	<li>Prepare individual fluorescence minus one (FMO) controls for anti-CD45RO&#8211;PE, anti-CCR7&#8211;FITC, anti-CD27&#8211;PE-Cy7 and anti-CD95&#8211;PerCP-Cy5.5 to distinguish positively stained cells from background.</li>
	<li>Prepare dead cell staining solution by diluting live/dead stock reagent (Table of Materials) 1:10,000 in PBS.</li>
	<li>Thaw day 3, day 5, and day 9 T cells that were previously cryopreserved. Centrifuge 1 x 106 T cells from each group at 300 x g for 3 min. Discard the supernatant. Wash the cells once with PBS. Centrifuge at 300 x g for 3 min and discard the PBS.</li>
	<li>Mix T cells with dead staining solution for 15 min at room temperature (RT), protected from the light.</li>
	<li>Add 1 mL of FACS buffer to quench the dead cell staining dye. Centrifuge at 300 x g for 3 min, discard the supernatant and resuspend the cell pellet in 100 &#181;L of FACS buffer containing the antibody cocktail described in step 2.1 and 2.2. Incubate for 1 h at 4 &#176;C.</li>
	<li>Add 1 mL of FACS buffer and centrifuge at 300 x g for 3 min to wash off unbound antibody. Repeat three times with FACS buffer.</li>
	<li>Resuspend cells in 1% paraformaldehyde (PFA) and store at 4 &#176;C.</li>
	<li>As CD45RO expression decreases after fixation, analyze the samples by flow cytometry after immunostaining. To assess differentiation, gate in the following order: singlets (FSC-H vs FSC-A), live CD3+ T cells (Dump [live-dead violet, CD14-PB and CD19-PB vs CD3-BV605], CD4-BV510 vs CD8-APC-H7). In CD4+ and CD8+ subsets, gate on CD45RO-PE vs CCR7-FITC to define na&#239;ve-like T cells (CD45RO-CCR7+), Tcm (CD45RO+CCR7+), Tem (CD45RO+CCR7-), and Tte (CD45RO-CCR7-). To identify Tscm, gate on CD27+ T cells in na&#239;ve-like T cells population. In this compartment, Tscm are CD95+ and Tn are CD95-.</li>
</ol>
3. In Vitro Functional Analysis
<ol>
	<li>CAR T cell proliferation and cytokine secretion
	<ol>
		<li>Verify CAR expression as well as cell viability as described in steps 1.5 and 1.7, by flow cytometry.</li>
		<li>Wash 5 x 106 T cells from each group (day 3, day 5 and day 9) with PBS and resuspend in a 1 &#181;M solution of carboxyfluorescein diacetate succinimidyl ester (CFSE) in PBS for 3.5 min at RT.</li>
		<li>Immediately add 10 mL of PBS containing 10% fetal bovine serum (FBS) to quench the reaction.</li>
		<li>Centrifuge the solution at 300 x g for 3 min. Discard the supernatant and repeat this wash step three times. Count the cells at the conclusion of CFSE staining using a Coulter counter (Table of Materials).</li>
		<li>Harvest CFSE-stained cells (unstimulated), resuspend in 1% PFA and store at 4 &#176;C for analysis by flow cytometry.</li>
		<li>Incubate the desired number of CFSE-stained CAR T cells with irradiated K562-CD19 (target) as well as K562-wild type (control) cells at a ratio of 1:1 for 120 h in cytokine free culture medium and culture conditions as described in step 1.1.</li>
		<li>After 24 h, centrifuge the culture vessel at 300 x g for 5 min. Collect 120 &#181;L of cellular supernatant. Assess activation-dependent production of IL2, IFN&#947;, TNF&#945;, GM-CSF, and other inflammatory cytokines (IL1&#946;, IL4, IL5, IL6, IL8, and IL10) by Luminex analyses in accordance with the manufacturer&#39;s recommendations.</li>
		<li>Replace the volume of supernatant that was collected in step 3.1.7 with an equivalent volume (120 &#181;L) of fresh medium.</li>
		<li>On day 3 (i.e., after 96 h), count and re-feed at a concentration of 0.5 x 106 cells/mL using bead-based flow cytometry as previously in step 1.5.</li>
		<li>On day 5, count the live CD3+ cells and calculate the fold change of live T cells relative to the live T cell count at day 0.</li>
		<li>Perform a comprehensive analysis of CFSE dilution in dividing CAR T cells using FlowJo software to highlight successive rounds of cell division. 
		NOTE: Proliferation assays are well described in the FlowJo manual.</li>
	</ol>
	</li>
	<li>Cytotoxicity assay 
	NOTE: The ability of CART19 cells to kill target cells expressing CD19 is evaluated using a 51Cr release-assay.
	<ol>
		<li>Label the target cells by mixing 5 x 105 K562-CD19, K562-wild type control cells, or NALM6 leukemia cells with 50 &#181;L of Na251CrO4 and 0.5 mL of RPMI supplemented with 10% FBS for 90 min in the incubator at 37 &#176;C.</li>
		<li>Centrifuge cells at 300 x g for 2.5 min. Discard the radioactive supernatant in appropriate disposal bins and wash the target cells in 5 mL of PBS. Repeat the wash steps twice.</li>
		<li>Resuspend the target cells in phenol red-free medium containing 5% FBS. Use this medium for the rest of the procedure to reduce background.</li>
		<li>After evaluating CAR expression and cell viability by flow cytometry (as described in section 5.1), mix CAR T cells with labeled target cells at effector:target (E:T) ratios of 10:1, 3:1 and 1:1, in triplicate. Transfer to a 96-well U bottom plate.</li>
		<li>In parallel, include target cells alone, and target cells with 1% sodium dodecyl sulfate (SDS), to determine spontaneous (S) and maximum (M) 51Cr release, respectively.</li>
		<li>Centrifuge cells at 300 x g for 5 min and incubate for 4 h or 20 h in a 37 &#176;C incubator with 5% CO2.</li>
		<li>After the designated time, centrifuge the culture vessel at 300 x g for 5 min. Collect 35 &#181;L of cellular supernatant and transfer to a reader plate. Avoid bubbles. Let the plate dry overnight.</li>
		<li>Seal the plate with a standard plate seal and count with a liquid scintillation counter. Chromium abundance in the supernatant provides a proxy of target cell killing. Calculate the percentage of specific lysis as follows: 100 x (counts per minute [cpm] experimental release &#8211; cpm S release)/ (cpm M release &#8211; cpm S release).</li>
	</ol>
	</li>
</ol>
4. In Vivo Functional Analysis
<ol>
	<li>Obtain 6&#8722;10-week-old NOD-SCID &#947;c&#8211;/&#8211; (NSG) mice, which lack an adaptive immune system, and assign them to treatment/control group randomly.</li>
	<li>Inject animals intravenously via tail vein with 1 x 106 NALM6 cells in 0.1 mL sterile PBS.</li>
	<li>After 5&#8722;7 days, confirm tumor engraftment by bioluminescence imaging (BLI). Inject 150 mg/kg of D-luciferin to mice which have been anesthetized with isoflurane (volume is dependent on body mass).</li>
	<li>Measure bioluminescence values using an imaging system. Quantify total flux using the corresponding software by drawing rectangles of identical area around mice, reaching from head to 50% of the tail length. Subtract the background for each image individually.</li>
	<li>After establishing leukemia, inject 3 x 106 day 9 CART19 cells, 0.5 x 106 day 3 CAR T cells, or corresponding non-transduced (NTD) human T cells via tail vein in a volume of 100 &#181;L of sterile PBS/Ca2+. 
	NOTE: As the bioactivity of cells harvested at various intervals during the culture process is different, the chosen concentration of day 3 cells is lower than day 9.</li>
	<li>To determine disease progression, measure the bioluminescence values twice a week as described above in step 4.3.</li>
	<li>To determine CAR T cell engraftment, collect 75 &#181;L blood via retro-orbital bleeding in an EDTA-coated tube. Transfer 50 &#181;L of blood to absolute counting tubes.</li>
	<li>Stain blood with antibodies against CD45, CD4, CD8 and CAR for 30 min at RT. Add 400 &#181;L of 1x FACS lysing solution to the tubes and vortex thoroughly. After staining, analyze surface marker expression by flow cytometry. 
	NOTE: Other surface markers can be used to assess the differentiation and exhaustion status of the T cells in blood.</li>
	<li>To measure cytokines levels in blood, centrifuge blood at 1,200 x g for 30 min at 4 &#176;C to separate serum from the upper layer of blood.</li>
	<li>Collect serum and measure the cytokines with a designated reader plate according to the manufacturer&#39;s instructions.</li>
</ol>

<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>

The authors have nothing to disclose.

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><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 ...

manufacturing-chimeric-antigen-receptor-car-t-cells-for-adoptive

Research

JoVE Journal

Cancer Research

14.9K Views.  Perelman School of Medicine at the University of Pennsylvania. We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Video: Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Immunology and Infection

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Bioengineering

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in ...

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Author Spotlight: Advancements in CAR-T Cell Manufacturing and Gene Therapy Production 

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

Medicine

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Source: Ghassemi, S., et al. <a href="https://app.jove.com/v/59949">Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy.</a> J. Vis. Exp. (2019)
This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. T Cell Activation, Transduction, ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

Source: Riccione, K., et al. <a href="https://app.jove.com/v/52397">Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care.</a> J. Vis. Exp. (2015).
This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host genome and produce surface-bound CARs targeting the tumor antigen.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...