an ex vivo technique to manufacture chimeric antigen receptor t cells

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Activate fresh or cryopreserved primary human T cells by mixing them with anti-CD3/CD28 magnetic beads at a ratio of 3 beads per T cell in the wells of the culture dishes. Culture the T cells in X-VIVO 15 medium supplemented with normal human AB serum, L-glutamine, HEPES, and IL-2.
Activate the T cells at a concentration of 1 million T cells per milliliter during expansion, and culture them at 37 degrees Celsius with 20% oxygen, 5% carbon dioxide, and 95% humidity. After overnight stimulation, add lentiviral supernatant to the activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection between 3 and 5. Continue culturing the cells using the same conditions.
On day 3, collect a representative aliquot of the cells of cryopreservation. Prior to cryopreservation, remove the magnetic beads by gently pipetting and magnetic separation. Prepare the freezing medium which contains PBS with 0.5% DMSO and store it at 4 degrees Celsius until ready to use.
Next, centrifuge the T cells at 300 times g for 5 minutes. Discard the supernatant and add 5 milliliters of PBS. Centrifuge the cells again at 300 times g for 5 minutes and discard the PBS. Resuspend the pellet in 1 milliliter of cold cryopreservation medium. Freeze the T cells in a chilled freezing container and store at minus 80 degrees Celsius for 48 hours.
After this, transfer the frozen cells to liquid nitrogen. Wash the rest of the T cells once in 5 milliliters of PBS to eliminate any residual vector. Centrifuge at 300 times g for 5 minutes, then decant the PBS and resuspend the cell pellet in T cell culture medium at a concentration of 500,000 cells per milliliter.
Split the T cells into two cultures designed for day 5 and 9. Count the T cells by flow cytometry using counting beads and monoclonal antibodies to human CD4 and CD8 as well as a viability dye. Refeed the cultures every other day to maintain them at a concentration of 500,000 cells per milliliter.
On day 5, counter and cryopreserve the day 5 cultures as previously described. On day 7, wash 500,000 cells in PBS and resuspend them in 100 microliters of fluorescence-activated cell sorting buffer. Then detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry. On day 9, count and cryopreserved the day 9 cultures as previously described.

an-ex-vivo-technique-to-manufacture-chimeric-antigen-receptor-t-cells

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

EoE Sub Articles

eoe sub articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. T Cell Activation, Transduction, and Expansion
<ol>
	<li>Activate fresh or cryopreserved primary human T cells by mixing with anti-CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and interleukin 2 (IL2, 100 units/mL). Maintain T cells at a concentration of 106 T cells/mL during expansion. Culture T cells at 37 &#176;C, 20% O2, and 95% humidity with 5% CO2.</li>
	<li>After overnight stimulation, add lentiviral supernatant to activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection (MOI) of 3&#8722;5. 
	NOTE: The CD19-BB&#950; CAR lentivirus plasmid consists of a CD8 hinge, 4-1BB costimulatory domain, and CD3&#950; signaling domain. CD19-BB&#950; lentiviral supernatant was generated as previously described.</li>
	<li>On day 3, collect a representative aliquot of cells for cryopreservation. Prior to cryopreservation, remove the magnetic beads by gentle pipetting and magnetic separation. Prepare a freezing medium containing phosphate-buffered saline (PBS) with 0.5% dimethyl sulfoxide (DMSO) and store at 4 &#176;C until use.
	<ol>
		<li>Centrifuge T cells at 300 x g for 5 min. Discard the supernatant and add 5 mL of PBS. Centrifuge cells at 300 x g for 5 min and discard the PBS.</li>
		<li>Resuspend the cell pellet in 1 mL of cold cryopreservation medium. Freeze T cells in a chilled freezing container and store at -80 &#176;C for 48 h. Transfer the frozen cells to liquid nitrogen.</li>
	</ol>
	</li>
	<li>Wash the rest of the T cells once in 5 mL of PBS to eliminate the residual vector. Centrifuge at 300 x g for 5 min. Decant the PBS and resuspend the cell pellet in a T cell culture medium at a concentration of 0.5 x 106 cells/mL.</li>
	<li>Split the T cells into two cultures, designated for day 5 and day 9. Count T cells by flow cytometry using counting beads (Table of Materials) and monoclonal antibodies to human CD4 and CD8, as well as a viability dye (Table of Materials).
	<ol>
		<li>To measure T cell concentration, prepare a master mix containing 500 &#181;L of PBS, 5 &#181;L of counting beads, 10 &#181;L of 7-Amino-actinomycin D cell viability solution, 4 &#181;L of CD4-fluorescein isothiocyanate (FITC) and 4 &#181;L of CD8-allophycocyanin (APC). Add 40 &#181;L of T cells to the master mix and measure cell concentration by flow cytometry based on the number of live T cells/bead counts. Refeed to maintain the cultures at a concentration of 0.5 x 106 cells/mL every other day.</li>
	</ol>
	</li>
	<li>On day 5, count and cryopreserve day 5 cultures as described in steps 1.3 and 1.5.</li>
	<li>On day 7, wash 0.5 x 106 T cells in PBS and resuspend in 100 &#181;L of fluorescence-activated cell sorting (FACS) buffer. Detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry.</li>
	<li>On day 9, count day 9 cultures and cryopreserve as described in step 1.3.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti CD3/CD28 dynabeads</td>
			<td>Thermo Fisher</td>
			<td>40203D</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD8</td>
			<td>BD Biosciences</td>
			<td>555369</td>
			<td>RRID:AB_398595</td>
		</tr>
		<tr>
			<td>APC-H7 Mouse anti-Human CD8 Antibody</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>RRID:AB_1645481</td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads,</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human AB serum</td>
			<td>Valley Biomedical</td>
			<td>HP1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2 IS, premium grade</td>
			<td>Miltenyi</td>
			<td>130-097-744</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>28030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Multisizer Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Gibco</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

Take primary T cells, mixed with antibody-conjugated magnetic beads in an interleukin-2-supplemented medium, and incubate.
The antibodies bind to CD3 in the T cell receptor complex and the co-stimulatory receptor CD28, while interleukin-2 binds to its cognate receptor, causing T cell activation and proliferation.
Introduce lentiviral vectors with a transgenic RNA encoding a chimeric antigen receptor or CAR, and incubate.
Upon vector-cell membrane fusion, the viral RNA undergoes reverse transcription into DNA, integrating into the host genome.
The transduced T cells express surface-bound CARs.
Harvest the cells at defined intervals.
For cryopreservation, take an aliquot of cells, detach the bound beads by pipetting, and magnetically separate them.
Spin down the cells and resuspend them in a cryopreservation medium.
Freeze the cells and store them at a cryogenic temperature.
To assess CAR expression, take another aliquot of cells, spin down the cells, and resuspend them in a buffer.
Introduce a CAR-specific fluorescently-tagged antibody and detect CAR expression using flow cytometry.

48 ビュー. キメラ抗原受容体T細胞を作製するex vivo技術

ビデオ: キメラ抗原受容体T細胞を作製するex vivo技術 - 実験

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

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

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

同系マウス B 細胞リンパ腫モデル CD19 車 T 細胞の前臨床評価

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

JoVE Journal

がん研究

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

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

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

CRISPR/Cas9を使用してCAR-TセルのGM-CSFをノックアウト

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

Production of Human CRISPR-Engineered CAR-T Cells

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

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

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

記録

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells