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>

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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 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 ...

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

Research

JoVE Journal

Cancer Research

14.8K 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

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

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 the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

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 ...

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...