This work was supported by ProMab Biotechnologies.

The PBMCs used here were previously isolated from whole blood from the Stanford Hospital Blood Center according to the Institutional Review Board (IRB)-approved protocol (13942) using the Ficoll-Paque density gradient described before42. The participants&#39; informed consent was not applicable as the blood we used to collect the PBMCs was obtained commercially from the Stanford Hospital Blood Center.
1. Preparation of CAR mRNA
<ol>
	<li>Template preparation 
	NOTE: Either a linearized plasmid or a PCR fragment can serve as a template to synthesize CAR mRNA in vitro, provided they contain a proper 5&#39; SP6 or T7 promoter, a 5&#39; untranslated region (UTR), a CAR transgene, and a 3&#39;UTR with an appropriate poly(A) tail. In this study, we used a CAR plasmid DNA construct containing an SP6 promoter, a short human beta-globin 5&#39;UTR, a CAR transgene, and a 3&#39;UTR composed of two tandem beta-globin 3&#39;UTRs followed by a 112-bp poly(A) tail43. The UTRs, coding sequence, and poly(A) tail all affect RNA stability and translational efficiency43,44,45. A poly(A) tail of 120 bp has been shown to increase protein yield in cell culture compared to shorter poly(A) tails43.
	<ol>
		<li>Prepare CAR construct plasmid DNA using a plasmid prep kit according to the manufacturer&#39;s instructions. High-quality plasmid DNA is essential for generating a high yield of CAR mRNA.</li>
		<li>Linearize the CAR plasmid DNA with the restriction enzyme BglII. Choose a restriction enzyme that cuts after the poly(A) tail, preferentially close to the end of the poly(A) tail. Digest 10 &#181;g (or 10,000 ng) of the plasmid quantified by a spectrophotometer in a 100 &#181;L reaction mix containing 10 U of the enzyme, 10 &#181;L of digestion buffer, and nuclease-free water overnight at 37 &#176;C. 
		NOTE: Make sure the restriction enzyme does not cut within the CAR transgene and its flanking promotor, UTR, and poly(A) tail sequences. Complete linearization of the CAR plasmid is necessary for efficient CAR mRNA transcription.</li>
		<li>Add 200 &#181;g/mL protease K and 0.5% w/v SDS into the digestion mix and incubate at 50 &#176;C for 1 h.</li>
		<li>Purify the linearized CAR plasmid DNA. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, v/v) to the digestion reaction. Vortex vigorously for approximately 15 s. Spin the sample in a bench-top centrifuge at maximum speed for 5 min at room temperature (RT).</li>
		<li>Transfer the upper aqueous layer, which contains the DNA, to a new microcentrifuge tube. Avoid transfer of any of the middle and bottom layers, which contain protein and phenol impurities. Perform a second round of extraction with chloroform to reduce impurities.</li>
		<li>Add 1/10th volume of 7.5 M ammonium acetate or 3 M sodium acetate and mix gently. Add 2.5 volumes of ethanol to precipitate the DNA. Store the sample at &#8722;20 &#176;C for at least 30 min.</li>
		<li>Spin the sample at maximum speed for 15 min at 4 &#176;C to pellet the DNA. Remove the supernatant carefully. Add 500 &#181;L of 70% ethanol to wash the DNA pellet.</li>
		<li>Spin the sample again for 2 min at 4 &#176;C. Carefully remove the supernatant. Leave a small amount of solution in the tube.</li>
		<li>Repeat the spin for 2 min at 4&#176;C. Carefully remove the remaining supernatant. Air-dry the DNA pellet at RT for about 10 min in a sterile hood until no ethanol solution is visible at the bottom of the tube. 
		NOTE: Avoid overdrying the DNA pellet, as it might be difficult to dissolve.</li>
		<li>Add 20 &#181;L of nuclease-free water and pipet up and down gently to dissolve the DNA. Centrifuge the sample briefly at maximum speed. Measure DNA concentration with a spectrophotometer.</li>
		<li>Store the sample on ice for immediate use or at &#8722;20 &#176;C for future use. Load 100 ng of the purified DNA on an agarose gel and verify the size and integrity. The DNA should appear as a single, distinct band of the expected size. No extra or smeared bands should be observed. 
		&#8203;NOTE: The quality of the template DNA is critical for efficient mRNA transcription. Avoid using impure, fragmented, and degraded DNA as a template for mRNA transcription.</li>
	</ol>
	</li>
	<li>In vitro transcription (IVT)
	<ol>
		<li>Synthesize the CAR mRNA in vitro. Make sure to use nuclease-free reagents, tubes, and pipette tips throughout the procedures.</li>
		<li>Thaw the reagents listed in Table 1 to RT, except for the SP6 RNA polymerase mix, which should be stored at &#8722;20 &#176;C till use. Prepare the transcription reaction in the order shown in Table 1 at RT. Mix gently and incubate at 37 &#176;C for at least 2 h. 
		NOTE: The reaction can be scaled up or down based on the amount of mRNA needed. The reaction shown above typically yields at least 100 &#181;g (or 100,000 ng) of capped mRNA when using high-quality template DNA. Longer incubation, i.e., overnight, may increase mRNA yield.</li>
		<li>Add 2 &#181;L of RNase-free DNase I to the reaction, mix gently, and incubate for 15 min at 37 &#176;C to degrade the template DNA.</li>
		<li>Purify the transcribed CAR mRNA using an RNA Cleanup Kit (see Table of Materials). Follow the kit manufacturer&#39;s instructions for the purification. Elute the mRNA in nuclease-free water. 
		NOTE: The mRNA can also be purified using the phenol-chloroform extraction method shown above (step 1.1.4).</li>
		<li>Measure the mRNA concentration with a spectrophotometer. Load at least 200 ng of the mRNA on an agarose gel and verify its size and integrity. The mRNA should appear as a dominant band of the expected size, without any additional or smeared bands. Store the mRNA at &#8722;80 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Activation and electroporation of T Cell
<ol>
	<li>Suspend 1 x 106 cryopreserved human peripheral blood mononuclear cells (PBMCs) in 1 mL of CAR-T medium (AIM-V medium supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and 10 ng/ml of human recombinant IL-2). Activate the T cells with an equal number of prewashed Human T-Activator CD3/CD28 beads (see Table of Materials). Culture and expand the cells at 37 &#176;C in a humidified incubator with 5% CO2 for several days. 
	NOTE: The amount of PBMCs can be scaled up or down based on the amount of T cells required for subsequent experiments.</li>
	<li>Thaw prepared CAR mRNA on ice. Add 1 mL of CAR-T medium per well to two wells of a 12-well plate and equilibrate the medium by placing the plate in the incubator at 37 &#176;C.</li>
	<li>Transfer 5 x 106 T cells to a sterile tube and remove the beads by putting the tube onto a magnetic stand and carefully pipetting the cell suspension to a new tube.</li>
	<li>Centrifuge the tube at 300 x g for 5 min at RT. Discard the supernatant and resuspend the cell pellet in 1 mL of sterile PBS.</li>
	<li>Centrifuge the tube again. Discard the supernatant and resuspend the cells in 200 &#181;L of Neon buffer T (see Table of Materials).</li>
	<li>Pipet 100 &#181;L of cells to a new tube and add 5 &#181;g (or 5,000 ng) of CAR mRNA to the cells46. Supplement Neon buffer T to a total of 125 &#181;L and mix by pipetting up and down. 
	NOTE: The amount of CAR mRNA to be transfected by electroporation may be increased up to 8 &#181;g (or 8,000 ng) per 1 x 106 T cells without negatively affecting cell viability, and a higher amount of mRNA may lead to increased CAR expression46.</li>
	<li>Add 25 &#181;L of Neon buffer T to the remaining 100 &#181;L cells. This serves as the negative control.</li>
	<li>Electroporate the T cells. Use the Neon Pipette to aspirate the T cells/CAR mRNA mixture or the negative control T cells into a Neon 100 &#181;L tip and electroporate the cells with a 10 ms pulse of 1800 V.</li>
	<li>Immediately transfer the cells to the equilibrated culture medium in the 12-well plate (prepared at step 2.2) after the electroporation.</li>
	<li>Return the plate back to the incubator and expand the cells until ready for use. To ensure healthy and rapid growth of the CAR T cells, maintain cell density between 1&#8722;3 x 106 cells/mL with fresh medium until needed.</li>
</ol>
3. Assessment of CAR expression
<ol>
	<li>Mix the CAR T cells by pipetting up and down gently several times in each well, and transfer at least 1 x 105 cells to a 5 mL FACS tube.</li>
	<li>Add 3 mL of cold wash buffer (PBS containing 2 mM EDTA and 0.5% w/v BSA) to the tube. Centrifuge the tube at 500 x g for 5 min at RT. Remove the supernatant carefully.</li>
	<li>Resuspend the cell pellet in 100 &#181;L of wash buffer. Add 2 &#181;L of the CAR detection reagent allophycocyanin (APC)-conjugated goat anti-human IgG F(ab&#39;)2 antibody (see Table of Materials) and 2 &#181;L of a viability dye 7-AAD into the suspended cells. 
	NOTE: The CAR can also be detected using an appropriately labeled target antigen or an anti-tag antibody if the CAR contains a tag (i.e., FLAG). If an unlabeled antibody is used for CAR detection, incubation with an appropriate fluorescence-labeled secondary antibody is required after step 3.4.</li>
	<li>Mix the sample by gently shaking the tube and incubate on ice for 30 min. Avoid light during the incubation. Wash the cells with 3 mL of cold wash buffer, and centrifuge the tube at 500 x g for 5 min at RT.</li>
	<li>Discard the supernatant and resuspend the cells in 100 &#181;L of wash buffer. Analyze the cells using a flow cytometer immediately. Make sure the detection reagent&#39;s fluorescent label and the viability dye are properly compensated using appropriate positive and negative controls.</li>
	<li>Evaluate the CAR expression using the following gating strategy: Gate on the cells using the FSC-A vs SSC-A plot; Gate on the single cells using the FSC-A vs FSC-H plot; Gate on the live cells using the 7-AAD staining.</li>
	<li>Calculate the percentage of live T cells that were stained by the anti-human IgG F(ab&#39;)2 antibody. Compare the CAR-T cells to the negative control T cells, which should have minimal staining.</li>
</ol>
4. Analysis of CAR-T cell function
<ol>
	<li>Cytotoxicity analysis
	<ol>
		<li>Make sure the target tumor cells express the antigen in a conformation that can be recognized by the CAR. To confirm the antigen expression, analyze the tumor cells by flow cytometry using the parent antibody from which the CAR was derived. If the CAR cannot recognize the tumor cells, then the CAR-T cells will fail to kill the tumor cells.</li>
		<li>Prepare target tumor cells expressing the CAR&#39;s antigen on the cell surface. Trypsinize the adherent tumor cells with 0.05% trypsin-EDTA solution for 1 min at 37 &#176;C and prepare single cell suspension with appropriate culture medium at 1&#8722;4&#160;x 105 cells/mL. 
		NOTE: The tumor cells should be well-adherent to ensure accurate analysis in the subsequent steps. If the tumor cells adhere poorly, detach easily or grow in suspension, coat the wells of the plate used in the following step with an appropriate antibody, polycationic polymer, or biological material such as fibronectin or collagen to facilitate cell attachment.</li>
		<li>Prepare an E-Plate (see Table of Materials). Design the experimental setup. Add 50 &#181;L of pre-warmed tumor cell culture medium into each well and place the E-Plate into the Real-Time Cell Analysis (RTCA) instrument to measure background impedance. Measure the impedance at 1 min per measurement (or sweep) for 3 sweeps. 
		NOTE: The background impedance is used to set the cell index to zero. Without background impedance measurement, the wells cannot be compared to each other, rendering the results non-interpretable.</li>
		<li>Remove the E-Plate from the RTCA&#160;instrument and add 100 &#181;L of the tumor cell suspension to each well. Therefore, each well contains 1&#8722;4 x 104 cells in 150 &#181;L total volume. 
		NOTE: The number of tumor cells to be seeded in each well should be determined prior to the experiment. Seed the E-Plate with a range of cell numbers, then monitor the cell indexes over the next 24&#8722;48 hours. The desired number of cells maintains a steady increase in cell index for at least 24 hours without plateauing. Excessive cell seeding can accelerate nutrient depletion and medium acidification, resulting in early plateauing of the cell index. In addition, replicate wells should be included for each group to identify potential outlier measurements.</li>
		<li>Equilibrate the E-Plate at RT for 30 min. This step allows the tumor cells to settle and adhere to the bottom of the wells. 
		NOTE: This step is necessary for accurate cell index measurement. Skipping this step usually causes well-to-well variability in cell index measurements and uneven tumor cell adhesion within the wells, resulting in increased noise in the assay.</li>
		<li>Place the E-Plate back to the RTCA instrument and monitor the cell indexes for about 24 h, with sweeps taken every 5&#8722;15 min.</li>
		<li>The next day, prepare the CAR-T cell suspension. Suspend the CAR-T cells (the effector cells) from step 2.10 in RPMI 1640 medium plus 10% FBS to a concentration determined by the tumor cell (the target cell) and the desired effector:target (E:T) cell ratio. For example, if the tumor cell suspension was at a density of 2 x 105 cells/mL and an E:T ratio of 10:1 is desired, then the CAR-T cells should be suspended to 2 x 106 cells/mL. Include proper controls such as tumor cells alone (no effector cells) and control T cells (T cells electroporated without mRNA or with control mRNA). 
		NOTE: For optimal results, use the CAR-T cells 1-2 days after electroporation when the surface CAR expression is high. To explore the optimal E:T ratio, a range of E:T ratios (for example, 3:1 to 20:1) can be tested. Unless the CAR-T frequency is very low, i.e., under 10%, an E:T ratio of 10:1 or 20:1 is usually sufficient to achieve near-complete killing of the tumor cells.</li>
		<li>Pause the cell index recording and remove the E-Plate from the RTCA instrument.</li>
		<li>Tilt the plate slightly, remove 50 &#181;L of culture medium from each well carefully without touching the tumor cells, and add 100 &#181;L of the CAR-T cells or control T cells into each well.</li>
		<li>Return the E-Plate to the RTCA instrument and resume cell index monitoring for at least 24 h with sweeps taken every 5&#8722;15 min. 
		NOTE: The length of cell index monitoring depends on how quickly the CAR-T cells target the tumor cells. A 24 h incubation period is usually sufficient for the CAR-T cells to exert maximum cytotoxic effect.</li>
		<li>Stop the cell index monitoring, remove the E-Plate from the RTCA instrument, and analyze the cytotoxicity result. Calculate cytotoxicity as the percentage of tumor cells that were killed by the CAR-T cells. Calculate mean cytotoxicity of the CAR-T cells and control T cells using the RTCA software. To calculate the cytotoxicity manually, use the formula 
		&#8203;cytotoxicity = ((cell index of tumor cells) &#8722; (cell index of tumor cells plus effector cells)) / (cell index of tumor cells) &#215; 100%.</li>
	</ol>
	</li>
	<li>Cytokine secretion analysis
	<ol>
		<li>To analyze the cytokines such as IFN-&#978; secreted by the CAR-T cells during the cytotoxicity analysis, transfer the supernatant from the E-Plate to a round-bottom or V-shaped 96-well plate. 
		NOTE: If a time-course analysis of the cytokine secretion is desired, pause the cell index monitoring at step 4.1.10, collect a small portion of the supernatant from each well, return the E-Plate back to the RTCA instrument, and resume the monitoring.</li>
		<li>Centrifuge the 96-well plate at 300 x g for 5 min at RT. Carefully transfer the supernatants to a new 96-well plate without disturbing the cell pellets.</li>
		<li>Measure the cytokine levels in the supernatants using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer&#39;s instructions. The supernatants usually require dilution before measurement, sometimes over 100-fold, depending on the cytokines being analyzed.</li>
		<li>Analyze the data by interpolating the sample absorbance values against a standard curve generated from serially diluted standards. Prepare graphs with error bars representing the mean &#177; standard deviation (SD) using appropriate software. To compare the difference between treatments, a Student&#39;s t test can be performed. A p-value less than 0.05 is usually considered statistically significant. 
		NOTE: Data points that are clear outliers, likely due to experimental errors, should be excluded from the analysis.</li>
	</ol>
	</li>
</ol>

The authors Liang Hu, Robert Berahovich, Yanwei Huang, Shiming Zhang, Jinying Sun, Xianghong Liu, Hua Zhou, Shirley, Xu, Haoqi Li, and Vita Golubovskaya are employees of ProMab Biotechnologies. Lijun Wu is an employee and shareholder of ProMab Biotechnologies.

In this study, we describe a detailed nonviral, non-integrating approach for generating transient CAR-T cells and provide technical procedures for assessing CAR-T cell function. This approach avoids the use of conventional retroviral and lentiviral vectors for permanent and random CAR transgene delivery, instead leveraging electroporation to efficiently introduce in vitro modified CAR mRNA into T cells. This method enables high-level, transient CAR expression on the T cell surface, significantly reducing the ris...

Cancer is an increasingly important threat to human health, with an annually estimated 23.6 million newly diagnosed cases and 10 million deaths globally1. Surgical treatment combined with radiation and chemotherapy remains the gold standard of care for various types of localized noninvasive and invasive cancers2,3,4. Although the traditional systematic treatment has achieved tremendous success in managing early-stage cancers, it is very toxic and has limited effect on metastatic and hematological cancers5. Patients with these cancers undergo frequent treatments and endure significant toxicity in the hope of stabilizing and delaying disease progression. Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a revolutionary targeted therapy that demonstrates potent efficacy for B cell hematological cancers and offers hope to these desperate patients6.
CAR-T cells are engineered T lymphocytes expressing a CAR on the cell surface that specifically recognizes tumor-associated antigens (TAAs) and activates the cells without requiring antigenic presentation from major histocompatibility complex proteins (MHCs)7. Upon activation, the CAR-T cells secrete a panel of cytokines and lyse tumor cells independently of MHCs, thus enhancing the destruction of tumors even when MHCs are downregulated or lost due to the immunosuppressive tumor microenvironment8,9. CARs consist of at least three distinct domains&#8212;an extracellular single-chain fragment variant (scFv) containing the antigen-recognizing variable regions of a TAA-specific antibody, a transmembrane domain that anchors the CAR to the cell surface, and an intracellular domain that mediates adaptor protein recruitment and downstream signaling10. Based on variations in the intracellular domains, the CAR structures have evolved through five generations so far, with the first generation containing only the CD3&#950; activation domain and subsequent generations possessing one or more extra costimulatory domains from molecules such as 4-1BB and CD2811. These intracellular domains affect the CAR-T cell signaling mechanisms, proliferation, survival, and toxicity, which together determine the clinical antitumor efficacy. The clinical success of CAR-T cell therapy comes from second-generation CAR-T cells that specifically target CD19, a biomarker highly expressed on B-lineage cells, for treating B-cell leukemias and lymphomas12,13. To date, the Food and Drug Administration (FDA) has approved six second-generation CAR-T cell therapies targeting either CD-19 or the B-cell maturation antigen (BCMA) for relapsed or refractory hematological malignancies, including large B-cell lymphoma, mantle cell lymphoma, and multiple myeloma 6. The newer generations of CAR-T cells are currently undergoing intensive preclinical and clinical studies14,15.
Clinical production of CAR-T cells is a complex, expensive, and time-consuming process. Currently, the leukocytes used for CAR-T cell preparation come from the patients themselves to avoid allogeneic reactions, although donor-derived universal (or off-the-shelf) CAR-T cells are under active development and clinical evaluation16. Following isolation of the leukocytes from the patient&#39;s peripheral blood by leukapheresis, a foreign gene fragment encoding the CAR is introduced into the activated T cells using either viral or nonviral approaches17,18,19. Retroviruses and lentiviruses are the most common viral vectors for CAR gene delivery, which results in random and permanent integration of the CAR-encoding DNA into the T cell genome20. Nonviral approaches, including mRNA-lipid nanoparticles, transposon systems, and CRISPR/Cas9 genome editing, are less common21. Subsequently, the CAR-expressing T cells are expanded ex vivo on a large scale and then collected and reinfused back into the patients22,23. Manufacturing viral vectors that meet stringent Good Manufacturing Practice (GMP) standards are very difficult, complex, and costly, placing a significant burden on manufacturers, regulatory agencies, and patients, thereby limiting its widespread clinical application.
Despite the exciting success of CAR-T therapy, there is growing concern about its safety. Given the random nature of CAR transgene integration into the T cell genome during viral or transposon transduction, disruption of tumor suppressor genes or activation of oncogenes may occur, leading to malignant transformation of the T cells24,25. Since the FDA&#39;s approval of the first CAR-T cell therapy product in 2017, follow-up studies show that as many as 15% of patients who receive the therapy develop secondary primary malignancies (SPMs) such as T cell lymphoma (TCL), non-small cell lung cancer (NSCLC) and skin cancer26,27,28,29. Strikingly, CAR transgenes were highly detected in some SPMs30,31, and some cases of TCL were directly caused by abnormal expansion of the CAR-T cells24,32,33, indicating a direct role of the CAR-T products in inducing some SPMs. Other studies further identified CAR transgene insertions in critical genes such as TET234, JAK135, and PBX236 in the SPM tumor cells. In light of the accumulating evidence of T-cell malignancies following CAR-T cell therapy, the FDA recently concluded that a boxed warning highlighting a serious risk of T-cell malignancy is required for all currently approved CD19-directed and BCMA-targeted CAR-T cell therapies37. However, large-cohort studies following the long-term effects of CAR-T treatment report an incidence of SPMs ranging from 3.8% to 15% among treated patients26,27,28,38,39. The incidence is not significantly higher than that observed in blood cancer patients receiving traditional systematic treatment40,41, suggesting a relatively safe profile for CAR-T cell therapy. Nonetheless, there is an urgent need to develop safe and efficacious CAR-T cells that minimize the risk of SPMs through more cost-effective approaches.
Here, we describe a detailed protocol for a nonviral, non-integrating approach to generate CAR-T cells. The goal is to provide a straightforward, step-by-step guide for generating small-scale, effective CAR-T cells with transient CAR expression, thereby avoiding insertional mutagenesis and minimizing the risk of SPMs. We show that the electroporation of T cells with modified mRNA encoding a CAR specific to the TAA HER2 resulted in robust and transient expression of the anti-HER2 CAR on the T cell surface. While expressing the CAR, the T cells potently killed HER2-positive tumor cells and secreted a high level of IFN-&#978;. The protocol is described in four separate but continuous sections that include preparation of CAR mRNA, activation and electroporation of T cell, assessment of CAR expression, and analysis of CAR-T cell function. This approach is suitable for developing and producing transient CAR-T cells for both academic research and clinical cell therapies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >7-AAD viability dye&#160;</td>
			<td >Biolegend</td>
			<td >420404</td>
			<td ></td>
		</tr>
		<tr >
			<td >ACEA Novocyte flow cytometer</td>
			<td >Agilent</td>
			<td >NovoCyte 3000</td>
			<td></td>
		</tr>
		<tr >
			<td >AIM-V medium</td>
			<td >Gibco</td>
			<td >12055083</td>
			<td></td>
		</tr>
		<tr >
			<td >APC goat anti-human IgG F(ab&#39;)2 antibody</td>
			<td >Jackson ImmunoResearch Laboratories</td>
			<td >109-136-097</td>
			<td></td>
		</tr>
		<tr >
			<td >BglII Restriction Enzyme</td>
			<td >New England BioLabs (NEB)</td>
			<td >R0144L</td>
			<td></td>
		</tr>
		<tr >
			<td >3&#180;-O-Me-m7G(5&#39;)ppp(5&#39;)G RNA Cap Structure Analog</td>
			<td >NEB</td>
			<td >S1411S</td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td >Gibco</td>
			<td >11965092</td>
			<td></td>
		</tr>
		<tr >
			<td >Dynabeads Human T-Activator CD3/CD28 beads</td>
			<td >Gibco</td>
			<td >11131D</td>
			<td></td>
		</tr>
		<tr >
			<td >E-Plate 96</td>
			<td >Agilent</td>
			<td >5232376001</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >Sigma</td>
			<td >459844-4L</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >Lonza.com</td>
			<td >14-503F</td>
			<td></td>
		</tr>
		<tr >
			<td >HiScribe SP6 RNA Synthesis Kit</td>
			<td >New England BioLabs (NEB)</td>
			<td >E2070S</td>
			<td></td>
		</tr>
		<tr >
			<td >Human IL-2 Recombinant Protein</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td></td>
		</tr>
		<tr >
			<td >Millennium RNA Markers</td>
			<td >Invitrogen</td>
			<td >AM7150</td>
			<td></td>
		</tr>
		<tr >
			<td >Monarch RNA Cleanup Kit (500 &#956;g)</td>
			<td >NEB</td>
			<td >T2050L</td>
			<td></td>
		</tr>
		<tr >
			<td >N1-Methylpseudo-UTP</td>
			<td >Trilink</td>
			<td >N-1081-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Neon Transfection Instrument</td>
			<td >Invitrogen</td>
			<td >MPK5000</td>
			<td></td>
		</tr>
		<tr >
			<td >Neon Transfection System 100-&#956;L Kit</td>
			<td >Invitrogen</td>
			<td >MPK10096</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin (10000 U/mL)</td>
			<td >Gibco</td>
			<td >14-503F</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI 1640 Medium</td>
			<td >Gibco</td>
			<td >11875135</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin-EDTA (0.05%), phenol red</td>
			<td >Gibco</td>
			<td >25300120</td>
			<td></td>
		</tr>
		<tr >
			<td >UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td >Invitrogen</td>
			<td >15593049</td>
			<td></td>
		</tr>
		<tr >
			<td >xCELLigence Real-Time Cell Analysis (RTCA) instrument</td>
			<td >Agilent</td>
			<td >RTCA MP</td>
			<td></td>
		</tr>
		<tr >
			<td >ZymoPURE II Plasmid Midiprep Kit</td>
			<td >Zymo Research</td>
			<td >D4201</td>
			<td></td>
		</tr>
	</tbody>
</table>

a nonviral approach to generate transient chimeric antigen receptor t cells using mrna for cancer immunotherapy

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

We constructed a second-generation HER2-targeted CAR containing a scFv derived from the humanized anti-HER2 mouse monoclonal antibody (mAb) 4D547, a transmembrane region, and an intracellular 4-1BB costimulatory domain followed by the CD3&#950; activation domain (Figure 1A). The DNA sequence encoding the CAR contained a 5&#39; SP6 promoter to drive transcription of the CAR mRNA in vitro (Figure 1B). The CAR mRNA was approximately...

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

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

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

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Research

JoVE Journal

Cancer Research

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

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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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 ...

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 ...

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 ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

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 ...