This work was supported by grants from the California Institute for Regenerative Medicine (CIRM) grant TR3-05641 and NIH grants P30 CA33572 (cores). D.W. is supported by NCI fellowship 1F99CA234923-01.

Our IRB does not require review of this protocol. We receive discarded fresh glioblastoma tumor samples from the City of Hope Pathology Department that are coded, and our laboratory cannot gain access to the key. We receive the specimens with data only on prior treatment and disease state at the time of biopsy/resection, with no identifying data.
1. Media Preparation
<ol>
	<li>Prepare neural stem cell media for culturing primary GBM cell lines: DMEM:F12, 1:50 B27, 5 &#181;g/mL heparin, and 2 mmol/L L-glutamine; supplemented with 20 ng/mL epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (FGF) twice a week (see Table of Materials).</li>
	<li>Prepare T cell media: X-VIVO 15 containing 10% fetal calf serum (FCS); supplemented with 70 IU/mL rhIL-2 and 0.5 ng/mL rhIL-15 every 48 h (see Table of Materials).</li>
	<li>Prepare co-culture media: take neural stem cell media without EGF and FGF supplement, and add 10% FCS.</li>
	<li>Prepare FACS staining solution (FSS): HBSS, 2% FCS, NaN3 (0.5 g/500 mL).</li>
</ol>
2. Preparation of GBM Tumor Cells
<ol>
	<li>Harvest low-passage GBM tumor spheres (TSs) by centrifugation at 300 x g for 4 min and discard supernatant. 
	NOTE: GBM tumor spheres (TSs) are generated from resected tumors as described previously22,23,24, and maintained in neural stem cell media, in incubators with 5% CO2 at 37 &#176;C.</li>
	<li>Pre-warm co-culture media in a 37 &#176;C water bath.</li>
	<li>Add 1 mL of cold accutase to GBM TSs, dissociate TSs by pipetting for 30-60 s, and stop dissociation by adding 5 mL of warm co-culture media. 
	NOTE: GBM TSs should not be kept on accutase for more than 5 min.</li>
	<li>Harvest GBM cells by centrifugation at 300 x g for 4 min, discard supernatant, and resuspend cells in 2 mL of co-culture media.</li>
	<li>Determine cell concentration using a cell viability counter. 
	NOTE: Cell viability must be &#62;70%.</li>
</ol>
3. Preparation of CAR T Cells
<ol>
	<li>Less than 24 h before the assay, take 100 &#181;L of cultured CAR T cells into a flow cytometry tube and add 2 mL of FSS. 
	NOTE: CAR T cells were generated as described previously11,21 and cultured in T cell media.</li>
	<li>Centrifugation at 300 x g for 4 min and discard supernatant.</li>
	<li>Add 2 mL of FSS to wash the cells, centrifuge at 300 x g for 4 min, and discard supernatant.</li>
	<li>Stain the cells with appropriate antibody to indicate CAR expression at 4 &#176;C for 30 min. 
	NOTE: For example, if an IL13R&#945;2-targeted CAR described previously25 is used, then stain with anti-IL13 antibody.</li>
	<li>Wash twice with 2 mL of FSS and analyze CAR exn using a flow cytometer.</li>
	<li>Determine the CAR% on T cells using the gating strategy shown in Figure 1B.</li>
	<li>At the day of co-culture, harvest all CAR T cells by centrifugation at 300 x g for 4 min, discard supernatant, and resuspend cells in 2 mL of co-culture media.</li>
	<li>Determine cell concentration using a cell viability counter. 
	NOTE: Cell viability must be &#62;70%.</li>
</ol>
4. Set up tumor &#8212; T Cell Co-culture
<ol>
	<li>Dilute tumor cells to a concentration of 0.16 million/mL with co-culture media.</li>
	<li>Based on CAR%, dilute CAR T cells to a concentration of 0.04 million CAR+ cells/mL with co-culture media. 
	NOTE: For example, if the CAR is 50% and T cell concentration is 0.4 million/mL, then make a 1:5 dilution to get the final concentration of 0.04 million CAR+ cells/mL.</li>
	<li>Pipette 100 &#181;L of diluted tumor cells into each well of a 96-well flat-bottom tissue culture plate.</li>
	<li>Pipette 100 &#181;L of diluted CAR T cells into each well that contains tumor cells and mix well. 
	NOTE: Each tumor-T cell co-culture will be analyzed at 4 time points. 4-6 replicates might be required at every time point based on different analysis, but &#60;3 replicates per time point is not recommended; see Table 1 for a representative platemap.</li>
	<li>Maintain the plate in a 37 &#176;C, 5% CO2 incubator.</li>
</ol>
5. Tumor Cell Rechallenge
NOTE: Rechallenge takes place at 2, 4 and 6 days post the initial co-culture setup (Figure 1A).
<ol>
	<li>Harvest and dissociate GBM TSs as described above (Steps 2.1-2.6).</li>
	<li>Resuspend GBM cells at a concentration of 0.64 million/mL.</li>
	<li>Determine the co-culture wells that need rechallenge (see Table 1) and carefully remove 50 &#181;L media from the top of each well.</li>
	<li>Add 50 &#181;L of GBM cell suspension into each well and mix well, then put the plate back into a 37 &#176;C, 5% CO2 incubator.</li>
</ol>
6. Harvest Samples and Flow Cytometric Analysis
NOTE: Samples will be harvested at 1, 3, 5 and 7 days post the initial co-culture setup, with 1, 2, 3 and 4 rounds of tumor challenge, respectively.
<ol>
	<li>Pre-warm 0.05% trypsin-EDTA solution in 37 &#176;C waterbath.</li>
	<li>Determine the wells that need harvesting and transfer the media into a new round-bottom 96-well plate.</li>
	<li>Pipette 50 &#181;L of trypsin-EDTA into the wells to digest remaining tumor cells at 37 &#176;C for 5 min.</li>
	<li>Under a microscope, confirm the cells have detached from the bottom.</li>
	<li>Pipette around the well bottom to resuspend detached cells, then transfer trypsin-EDTA containing detached cells to the corresponding wells of the round-bottom 96-well plate.</li>
	<li>Centrifuge the round-bottom 96-well plate at 300 x g, 4 &#176;C for 4 min, then discard supernatant.</li>
	<li>Add 200 &#181;L/well of FSS to wash the cells, centrifuge at 300 x g, 4 &#176;C for 4 min, then discard supernatant.</li>
	<li>Resuspend cells in 100 &#181;L/well FSS containing antibodies (see Table of Materials), and stain cells at 4 &#176;C for 30 min.</li>
	<li>Add 100 &#181;L/well FSS to cells, centrifuge at 300 x g, 4 &#176;C for 4 min, then discard supernatant.</li>
	<li>Add 200 &#181;L/well of FSS to wash the cells, centrifuge at 300 x g, 4 &#176;C for 4 min, then discard supernatant.</li>
	<li>Resuspend cells with 100-200 &#181;L/well of FSS with 500 ng/mL DAPI, then analyze samples by flow cytometry.</li>
</ol>
7. Functional and Phen Otypic Analysis of CAR T Cells
<ol>
	<li>Retrieve the data files from flow cytometer, and gate all live (DAPI-) cells (Figure 1C).</li>
	<li>Quantify tumor cells by gating the CD45- population and CAR T cells by gating the CD45+, CAR+ population (Figure 1C). 
	NOTE: If the tumor cells express CD45 (e.g. Raji lymphoma cells), anti-CD3 staining can be used to differentiate T cells from tumor cells.</li>
	<li>Plot tumor cell and CAR T cell number through the time course.</li>
	<li>Identify T cell activation by the co-expression of 4-1BB and CD69. 
	NOTE: These surface markers can be used to analyze T cell activation 6-24 h post initial co-culture.</li>
	<li>Identify T cell exhaustion by the expression of PD-1, LAG-3 and TIM-3.</li>
	<li>Identify T cell memory status by the expression of CD45RO and CD62L.</li>
</ol>

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The CAR construct described in this work have been licensed by Mustang Bio., Inc., for which S.J.F. and C.E.B. receive royalty payments. All other authors declare no potential conflicts of interest.

This repetitive tumor cell challenge assay provides a convenient approach to evaluate CAR T cell functional potency, using an in vitro setup that recapitulates the high tumor burden associated with in vivo tumor models. During this 7-day assay, CAR T cells undergo four rounds of tumor challenge (initial co-culture and 3x rechallenge), creating an environment where exhausted T cells may lose their capability to respond to tumor challenge despite a potent initial response. Tumor cell elimination and CAR T cell expansion represent two fundamental readouts that can be acquired from this assay, while the phenotypes of CAR T cells can be easily analyzed by staining surface or intracellular markers that indicate T cell activation, exhaustion and/or memory. This assay is also convenient to combine with non-biased analysis of CAR T cells transcriptome, proteome or phosphor-proteome21,26, and T cell polyfunctionality which has been adopted to predict clinical responses27. Furthermore, tumor cells can also be tested for their adaptive response against T cell immunity such as cytokine secretion28. Since samples are harvested at multiple time points, this assay allows for not only static, but also dynamic analysis of CAR T cell behavior when responding to excess number of tumor cells.
The assay is particularly powerful in comparing the effector potency of CAR T cells targeting the same antigen but with different designs and/or manufacturing processes. We have used this assay to identify that CD4+ CAR T cells were able to mediate superior long-term effector function than CD8+ cells21. It was also shown that in vivo CAR efficacy was correlated with the cytotoxicity at later time points (D5-D7) during this assay, further highlighting the necessity of using repetitive tumor challenge for in vitro CAR T cell evaluation. Although GBM cells were used as the example of target cells here, this assay could be easily adopted to a different T cell-tumor combination. Notably, CAR T cell behavior can also vary upon different antigen densities and engineering cancer cells to express various levels of targeted antigens provided the tool to investigate sequential molecular events upon CAR recognition29. Therefore, this assay can also be exploited to examine the activation pattern of certain CAR T cells against different targets.
Since the target cell viability and CAR T cell expansion are two upfront readouts of this assay, it is critical that all the co-cultures start with viable (&#62;70%) tumor and T cells. When performing the rechallenge processes, the action of aspirating media (step 5.3) should not disturb tumor and T cells in the bottom of the wells, in order not to introduce unnecessary variations of cells counts. When performing this assay on suspension target cell lines, it is recommended to harvest remaining cells by centrifugation before tumor cell rechallenge.
One limitation of this assay is that the setup parameters (E:T ratios of initial co-culture and rechallenge) needs to be adjusted based on different CAR-tumor combinations. For example, if the tumor cells express a considerable level of immuno-inhibitory molecules (e.g. PD-L1), a higher E:T ratio is recommended given that the overall killing efficiency is impaired compared with PD-L1-low tumor cells. Before applying the assay to new CAR-tumor combinations, a pilot study is recommended to determine the optimal conditions, which usually allows the most potent CAR T cells tested in the assay to eliminate &#62;80% of tumor cells at the end point. Since direct cell-cell contact is needed for CAR T cell-mediated killing, if the tumor cells were fluorescence-labeled, then the panel of flow cytometric analysis must be adjusted accordingly (e.g. if the tumor cells were GFP-labeled, then any FITC-conjugated antibodies should not be used).
The clinical activity of CAR T cells against B cell malignancies has led to two FDA-approved drugs. However, the response has shown substantial variation across different patients27,30,31, which was elucidated to correlate with the phenotypic properties of the CAR products30. This in vitro repetitive tumor challenge assay further provides an approach to functionally test the CAR effector potency in addition to the phenotypic characterizations and polyfunctionality cytokine production, and allows for higher-throughput screening of clinical products in comparison to in vivo tumor models while retaining the predictive fidelity. Overall, this assay could be used for T cell functional test for CAR T cell design, pre-clinical and clinical development.

Immunotherapy using chimeric antigen receptor (CAR)-engineered T cells has seen promising outcomes against B cell malignancies1,2,3,4, while the potential of targeting other tumors continues to be under rigorous investigation5,6,7. Great progress has been made to optimize the CAR construct, manufacturing process and patient pre-infusion regimens6,7,8, and novel synthetic biology approaches are expected to increase their persistence, safety and tumor specificity9,10. However, it has been difficult and labor-intensive to appropriately assess the therapeutic potential of CAR T cells in order to guide the best choice for clinical translation. The most established model thus far to evaluate CAR T cell function is in immunodeficient mice bearing human tumor xenografts, whereby CAR T cells are examined for antitumor efficacy and compared at titrated cell doses11,12,13,14,15. These in vivo murine studies are labor-intensive and time-consuming, especially when screening large numbers of parameters. Further, in vivo studies can be restrained by the accessibility of mouse strains, animal care facilities and animal-handling techniques. Therefore, there is a need to develop more convenient in vitro assays allowing for quick readouts of effector activity, which also faithfully reflect the in vivo antitumor function of these T cells.
Conventional methods to determine the cytotoxicity of T cells in vitro have focused on the detection of degranulation, cytokine production and the ability to lyse radioisotope-labeled target cells (i.e., chromium release assays). While these assays are informative for defining CAR T cell specificity and redirected target recognition, they often fail to reflect in vivo antitumor potential of engineered T cells12,13,16. In certain cases, in vitro killing activity in short term assays showed an inverse correlation with in vivo antitumor function16. Such inconsistency is likely the result of high effector:target (E:T) ratios used in these in vitro assays, and therefore the inability to differentiate CAR T cell products that are prone to exhaustion17. By contrast, during in vivo tumor eradication T cells usually respond against large tumor burdens, thereby requiring multiple rounds of killing and subsequently driving T cell differentiation and exhaustion18,19,20, which is one of the major barriers against effective tumor clearance by CAR T cells12,13. Meanwhile, most short-term in vitro killing assays also do not readout differences in T cell proliferation, whereas in CAR T cell treated patients the capacity for CAR T cell expansion is strongly correlated with clinical responses4. Thus, the appropriate in vitro assay would need to recapitulate conditions of high tumor burden, induction of T cell exhaustion, and allows for the readout of T cell expansion.
Here we describe a strategy to evaluate CAR T cells for repetitive tumor killing potential, with a simple in vitro co-culture assay. Different T cell effector activity parameters can be simultaneously examined, including target cell killing, CAR T cell expansion and memory- or exhaustion-associated phenotypes. The results generated from this assay correlate well with the in vivo antitumor effect of CAR T cells, and can be exploited to assess the potency of CAR T cell products. While we describe an assay to evaluate IL13Ra2-targeted CAR T cells against primary GBM lines21, it can be readily adapted to any CAR T cell platform.

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			<td height="40" style="height:40px;width:451px;">0.05% Trypsin/0.53mM EDTA in HBSS w/o Calcium and Magnesium</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">MT25051CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">1M Hepes</td>
			<td style="width:123px;">Irvine Scientific</td>
			<td style="width:344px;">9319</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">200 mM L-Glutamine</td>
			<td style="width:123px;">Cambrex Bio Science</td>
			<td style="width:344px;">17-605E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">4&#39;,6-diamidino-2-phenylindole, dihydrochloride (DAPI) - FluoroPure grade</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">D21490</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">Accutase</td>
			<td style="width:123px;">Innovative Cell Technologies</td>
			<td style="width:344px;">AT104</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:451px;">Aldesleukin Proleukin (rhIL-2)</td>
			<td style="width:123px;">Novartis Oncology</td>
			<td style="width:344px;">NDC 0078-0495-61</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD137, PE</td>
			<td>BD Biosciences</td>
			<td>555956</td>
			<td>4B4-1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD19, PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>557835</td>
			<td>SJ25C1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD3, PERCP</td>
			<td>BD Biosciences</td>
			<td>340663</td>
			<td>SK7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD4, FITC</td>
			<td>BD Biosciences</td>
			<td>340133</td>
			<td>SK3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD45, PERCP</td>
			<td>BD Biosciences</td>
			<td>340665</td>
			<td>2D1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD45RO, PE</td>
			<td>BD Biosciences</td>
			<td>561137</td>
			<td>UCHL1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD62L, APC</td>
			<td>BD Biosciences</td>
			<td>559772</td>
			<td>DREG-56</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD69, APC</td>
			<td>BD Biosciences</td>
			<td>340560</td>
			<td>L78</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-CD8, APC-Cy7</td>
			<td>BD Biosciences</td>
			<td>348793</td>
			<td>SK1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-IL-13, PE</td>
			<td>BD Biosciences</td>
			<td>340508</td>
			<td>JES10-5A2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-LAG-3, PE</td>
			<td>eBiosciences</td>
			<td>12-2239-41</td>
			<td>3DS223H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-PD-1, APC-Cy7</td>
			<td>BioLegend</td>
			<td>329921</td>
			<td>EH12.2H7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-TIM-3, APC</td>
			<td>eBiosciences</td>
			<td>17-3109-42</td>
			<td>F38-2E2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">B-27 Serum-Free Supplement (50X)</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">Corning 96 Well Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile (Product #3596)</td>
			<td style="width:123px;">Corning Life Sciences</td>
			<td style="width:344px;">3596</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Defined fetal bovine serum,HI IR</td>
			<td style="width:123px;">Hyclone Labs</td>
			<td style="width:344px;">SH30070.03IH</td>
			<td></td>
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			<td height="21" style="height:21px;width:451px;">DMEM F-12 50:50 (1X) w/o Glutamine</td>
			<td style="width:123px;">MediaTech, Inc.</td>
			<td style="width:344px;">15-090-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">DMEM High Gluc w/o L-Glu, Na Pyr 1 L</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">11960051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">DMEM-Ham&#39;s F12 50:50 Mixture with L-glutamine and 15 mM HEPES</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">MT10092CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">HBSS</td>
			<td style="width:123px;">Irvine Scientific</td>
			<td style="width:344px;">9224</td>
			<td></td>
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			<td height="21" style="height:21px;width:451px;">Heat-Inactivated FCS</td>
			<td style="width:123px;">Hyclone</td>
			<td style="width:344px;">SH30070.03</td>
			<td></td>
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			<td height="40" style="height:40px;width:451px;">Heparin Sodium(1,000 U/ml)</td>
			<td style="width:123px;">American Pharmaceutical</td>
			<td style="width:344px;">401811B</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:451px;">MACSQuant</td>
			<td style="width:123px;">Milteni Biotech Inc.</td>
			<td style="width:344px;">BIS013220&#160;</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">PBS 1X W/CA &#38; MG</td>
			<td>Irvine Scientific</td>
			<td>9236</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:451px;">PBS with 1MM EDTA (No Ca2+ or Mg2+)</td>
			<td style="width:123px;">VWR Scientific Products</td>
			<td style="width:344px;">PB15009A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Recombinant human EGF</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">236-EG-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Recombinant human FGF</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">233-FB</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">rhIL-15 Working Dilution (10 ng/&#181;L)</td>
			<td style="width:123px;">CellGenix, US Operations</td>
			<td style="width:344px;">tF0297</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:451px;">Sodium Azide (NaN3)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;">S8032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Sorvall ST40R</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:344px;">41693700</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:451px;">TC-HEPES BUFFER (1M) SOLN 100ML&#160;</td>
			<td style="width:123px;">IRVINE SCIENTIFIC CORP</td>
			<td style="width:344px;">6472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Tecnol Procedure Mask</td>
			<td style="width:123px;">Kimberly-Clark</td>
			<td style="width:344px;">47117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">X-VIVO 15</td>
			<td style="width:123px;">Lonza</td>
			<td style="width:344px;">BW04-744Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the assay described above, we have distinguished the differential effector potency and killing dynamics mediated by CD4+ and CD8+ CAR T cells. The results presented here illustrate the difference between CD4+ and CD8+ CAR T cells, generated from a healthy donor independent of our previous publication21.
Through a standard degranulation assay, we were able to observe that both CD4+ and CD8+ CAR T cells became equally activated against GBM cells that express the targeted antigen, as indicated by the expression of CD107a and intracellular cytokine (Figure 2A). However, using the repetitive tumor challenge assay, we found that CD4+ but not CD8+ CAR T cells exhibited the capability of multiple-round killing (Figure 2B). CD4+ CAR T cells also achieved better expansion in comparison to CD8+ cells during this assay (Figure 2C). The difference of expansion between CAR T cell subsets were only observed from D3 after two rounds of tumor challenge (1:12 E:T), while the difference of cytotoxicity was observed beginning at D5 after three rounds of tumor challenge (1:20 E:T), indicating that short-term assays failed to elucidate differences in the potency of different CAR T cell products. When the 1:1 mixed CD4+ and CD8+ CAR T cells were applied to this assay, they were found to outperform CD8+ but not CD4+ CAR T cells on long-term cytotoxicity (Figure 2B). The expansion of CD8+ CAR T cells was induced by the co-applied CD4+ cells, while CD4+ CAR T cell expansion was inhibited in the presence of CD8+ cells (Figure 2C).
To explain the mechanism that distinguishes the effector activity between CD4+ and CD8+ CAR T cells, we first confirm that 24 h after initial co-culture, both CD4+ and CD8+ CAR T cells were comparably activated (Figure 3A). Meanwhile, as indicated by CD45RO and CD62L expression, both CD4+ and CD8+ CAR T cells showed a transition from central memory (CD45RO+, CD62L+) to effector memory (CD45RO+, CD62L-) phenotype during repetitive tumor challenge (Figure 3B). Then we characterized the cells at D3 of this assay, a time before the CAR T cell subsets started to display functional difference. T cell exhaustion was marked by the co-expression of inhibitory receptors PD-1, LAG-3 and TIM-315,21, which showed that CD8+ CAR T cells were more prone to exhaustion compared with CD4+ CAR T cells (Figure 3C). Further, no difference was seen on CD8+ CAR T cell exhaustion in the presence/absence of co-applied CD4+ cells (Figure 3C), indicating that CD4-induced CD8+ CAR T cell expansion is not associated with a better effector function.
Together, the results from this assay identified that CD4+ CAR T cells outperformed CD8+ cells specifically in long-term cytotoxicity. Despite the similar short-term cytotoxicity (1-3 days, once rechallenged), CD4+ CAR T cells sustained effector function upon repetitive tumor challenge, while CD8+ cells became exhausted and failed to control tumor cell growth. When the two subsets were mixed, CD4+ CAR T cells facilitated the expansion of CD8+ CAR T cells, but the exhaustion status of CD8+ cells were not ameliorated, resulting in the lack of synergistic effect between the two groups. Similar difference between CD4+ and CD8+ CAR T cells was seen in an in vivo model as previously described21. Indeed, CD8+ CAR T cells were able to mediate short-term tumor clearance but followed with antigen-positive recurrence; in contrast, CD4+ CAR T cell treatment resulted in long-term tumor eradication21, which is reminiscent of their repetitive killing potential using the in vitro rechallenge assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59275/59275fig1.jpg" /> 
Figure 1: Schema and analysis strategy of repetitive challenge assay. (A) Schema and timeline of repetitive tumor challenge assay. For each well, CAR T cells were first co-cultured with PBT030-2 GBM cells (4,000 CAR+ cells, 16,000 tumor cells) and re-challenged with 32,000 tumor cells every other day (D2, D4 and D6). Analysis of tumor cell and CAR T cell number, as well as CAR T cell phenotype is carried out at D1, D3, D5 and D7. (B) Gating strategy to determine CAR% in T cells before setting up the co-culture. (C) Gating strategy of live cells, tumor cells and CAR T cells from the repetitive challenge assay. (D) Tumor cells number at different times of the rechallenge assay, co-cultured with untransduced T cells. Error bars = &#177;SEM. <a href="https://www.jove.com/files/ftp_upload/59275/59275fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59275/59275fig2.jpg" /> 
Figure 2: Repetitive challenge assay reveals differences in killing and proliferative potency between CD4+ and CD8+ CAR T cells. (A) Degranulation and intracellular cytokine staining of CD4+ and CD8+ CAR T cells after 5 h of co-culture with GBM cells (E:T=1:1). (B) CD4+, CD8+ or mixed (CD4:CD8=1:1) CAR T cells were applied to repetitive challenge assay, and viable tumor cells were quantified. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 compared with CD4+, using one-way ANOVA with Bonferroni&#39;s Multiple Comparison Tests. (C) CD4+ and CD8+ CAR T cell expansion during repetitive tumor challenge assay. Comparison of (left to right) CD4+ vs CD8+, single subset; CD4+ vs CD8+, within the &#34;mixed&#34; group; CD4+, single vs mixed; CD8+, single vs mixed. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 using an unpaired Student&#39;s t test. Error bars = &#177;SEM. <a href="https://www.jove.com/files/ftp_upload/59275/59275fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59275/59275fig3.jpg" /> 
Figure 3: Analysis of T cell phenotype during repetitive challenge assay. (A) 4-1BB and CD69 staining on CAR T cells at D1 of the assay. (B) CD45RO and CD62L staining of CAR T cells before applying to rechallenge assay (input), and at D3 and D7 of the assay. (C) Co-expression of PD-1, LAG-3 and TIM-3 on CAR T cells at D3 of the assay. (Top) Gating strategies to identify T cells expressing 1, 2 or 3 inhibitory receptors. (Bottom) Comparison of: CD4+ cells (single subset), CD8+ cells (single subset), CD8+ cells (within the &#34;mixed&#34; group). <a href="https://www.jove.com/files/ftp_upload/59275/59275fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/59275/59275table1.jpg" /> 
Table 1: Representative plate map of rechallenge setup.

Watch this Scientific Journal Video about In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function at JoVE.com

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

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 ...

in-vitro-tumor-cell-rechallenge-for-predictive-evaluation-chimeric

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Cancer Research

11.5K Views.  City of Hope Beckman Research Institute and Medical Center. 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.

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

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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.

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.

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

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 ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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 ...

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 &#181;m zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 &#181;m of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

The present protocol develops an image-based technique for rapid, non-destructive, and label-free regional cell density and viability measurement within 3D tumor aggregates. Findings revealed a cell-density gradient, with higher cell densities in core regions than outer layers in developing aggregates and predominantly peripheral cell death in HER2+ aggregates treated with Trastuzumab.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These ...

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 ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Evaluating Quantitative Tumor Cell Killing by CAR-Expressing Jurkat Cells

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...