manufacturing chimeric antigen receptor t-cells using an automated cell processor

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating patients with refractory B-cell malignancies1,2,3,4,5. However, the traditional manufacturing methods for CAR-T cells are labor-intensive, time-consuming, and require highly trained technicians to carry out highly specialized steps. For example, the traditional manufacturing process of an autologous CAR-T cell product involves density gradient centrifugation, elutriation&#160;or magnetic separation to enrich T cells, activation, and transduction with a viral vector in a sterile flask, and expansion in a bioreactor prior to harvest and formulation. Various systems have emerged recently that aim to partially automate this process. For example, the Miltenyi CliniMACS Prodigy (hereafter referred to as the &#34;processor&#34;) is an automated cell processing device that can perform many of these steps in an automated fashion6,7,8,9. An in-depth discussion of traditional and automated CAR-T manufacturing methods is presented in a recent review article10.
The processor builds upon the functionality of the CliniMACS Plus, a U.S. Food and Drug Administration (FDA)-approved medical device for the processing of hematopoietic progenitor cells. The processor includes a cell cultivation unit that allows for&#160;automated washing, fractionation, and cultivation of cells (Figure 1). The T cell transduction (TCT) process is a preset program within the processor device that largely replicates manual CAR-T cell manufacture. TCT allows for customizable cell processing using a graphical user interface (the &#34;Activity Matrix,&#34; Figure 2). Because the processor automates many steps and consolidates the functionality of multiple devices into one machine, it requires less training and specialized troubleshooting skills from technologists. Because all steps are performed within a closed, single-use tubing set, the processor may be operated in facilities with less stringent air-handling infrastructure than would be considered acceptable for an open manufacturing process. For example, we are operating the processor in a facility certified as ISO class 8 (comparable to EU grade C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65488/65488fig01.jpg" /> 
Figure 1: CAR-T cell manufacturing using the T cell transduction system. Shown is the processor&#160;with the tubing set installed. The tubing set allows for connecting other components such as bags containing processing buffer, culture medium, and lentiviral vector via sterile welding. Once the leukapheresis product is added to the Application bag, it can be labeled with T Cell Selection Beads, passed through the Separation column, and then transferred into the Reapplication bag. Selected cells are then directed to the Cultivation unit of the instrument for culture and activated with the Activation reagent (see Table of Materials). The final product is collected in the Target cell bag. Throughout the process, it is possible to remove samples for quality control aseptically. Grey numbers inside of circles represent the numbered valves on the processor that direct the liquid path through the tubing set. Reproduced with permission from 11.&#160;<a href="https://www.jove.com/files/ftp_upload/65488/65488fig01large.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/65488/65488fig02.jpg" /> 
Figure 2: Activity Matrix. After T cell selection and activation, the remainder of the CAR-T cell manufacturing process is fully customizable. Activities&#160;can be added or deleted and scheduled for the appropriate day and time, and the culture volume after the activity can be specified (Volume). For example, the Transduction activity was configured to begin at 10:00 AM on Day 1, and the culture volume at the end of the activity was set as 100 mL. The Activity Matrix can be edited throughout the cultivation period. The status of the process can be monitored on the integrated screen of the processing device. <a href="https://www.jove.com/files/ftp_upload/65488/65488fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The aim of this manuscript is to provide a detailed walk-through of manufacturing CAR-T cells using the processor and additionally provide guidance on the in-process and product release testing that will likely be required by regulators to approve an investigational new drug (IND) application. The presented protocol stays close to the vendor&#39;s recommended approach and is the underlying protocol for IND 28617, which is currently being evaluated in a single-center investigator-initiated phase I/II clinical trial. This trial aims to determine the safety and efficacy of using this processor to manufacture humanized CD19-directed autologous CAR-T cells for patients with B cell acute lymphoblastic leukemia (B-ALL) or B-lineage lymphoblastic lymphoma (B-Lly) [NCT05480449]. The trial started in September 2022 and is planned to enroll up to 89 patients ages 0-29 years with B-ALL or B-Lly. We report some manufacturing results from the trial in the manuscript.
We would like to point out that although the manuscript is presented as a protocol with steps to follow, it should be considered a starting point for others to begin optimizing their own CAR-T cell manufacturing process. A non-comprehensive list of possible variations to the presented protocol includes: using fresh instead of cryopreserved T cells as starting material; using a different method of T cell enrichment or omitting it altogether; using different media and cytokine cocktails such as IL7/IL15 instead of IL2; varying the concentration of human AB serum or omitting it altogether; timing of transduction; using &#34;multi-hit&#34; transductions; varying agitation, culture volumes, and feeding schedule; using different methods of genetic transfer including electroporation of nucleic acids or non-lentiviral vectors; using a different final formulation buffer and/or cryoprotectant; and infusing CAR-T cells fresh instead of cryopreserving for infusion at a later time. These variations may have a significant impact on the cellular composition and potency of the therapeutic product.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Overall Process Step</td>
			<td>Process Day</td>
			<td colspan="4">Technical Details</td>
		</tr>
		<tr>
			<td>Cell Enrichment</td>
			<td rowspan="2">Day 0</td>
			<td colspan="4">Selection of CD4+/CD8+ T cells&#160;</td>
		</tr>
		<tr>
			<td>Cell Activation</td>
			<td colspan="4">T cell culture seeding and activation</td>
		</tr>
		<tr>
			<td>Cell Transduction</td>
			<td>Day 1</td>
			<td colspan="4">Lentiviral transduction (100 mL culture volume)&#160;</td>
		</tr>
		<tr>
			<td rowspan="12">Cell Expansion (followed by cell formulation)</td>
			<td>Day 2</td>
			<td colspan="4">--</td>
		</tr>
		<tr>
			<td>Day 3</td>
			<td colspan="4">Culture Wash (1 cycle); Shaker activated; Culture volume increases to 200 mL</td>
		</tr>
		<tr>
			<td>Day 4</td>
			<td colspan="4">--</td>
		</tr>
		<tr>
			<td>Day 5</td>
			<td colspan="4">Feed (50 mL); Culture volume reaches final volume of 250 mL</td>
		</tr>
		<tr>
			<td>Day 6</td>
			<td colspan="4">In-process sample; Media exchange (-125 mL / +125 mL)</td>
		</tr>
		<tr>
			<td>Day 7</td>
			<td colspan="4">Media exchange (-150 mL / +150 mL) or Harvest&#160;</td>
		</tr>
		<tr>
			<td>Day 8</td>
			<td colspan="4">In-process sample; Media exchange (-150 mL / +150 mL) or Harvest</td>
		</tr>
		<tr>
			<td>Day 9</td>
			<td colspan="4">Media exchange (-180 mL / +180 mL) or Harvest</td>
		</tr>
		<tr>
			<td>Day 10</td>
			<td colspan="4">In-process sample; Media exchange (-180 mL / +180 mL) or Harvest</td>
		</tr>
		<tr>
			<td>Day 11</td>
			<td colspan="4">Media exchange (-180 mL / +180 mL) or Harvest</td>
		</tr>
		<tr>
			<td>Day 12</td>
			<td colspan="4">Media exchange (-180 mL / +180 mL) or Harvest</td>
		</tr>
		<tr>
			<td>Day 13</td>
			<td colspan="4">Harvest</td>
		</tr>
	</tbody>
</table>
Table 1: Process timeline and overview. This table summarizes the TCT process steps employed in a current clinical trial [NCT05480449]. The process starts with T cell enrichment by&#160;CD4+/CD8+ selection,&#160;culture seeding,&#160;and activation on Day 0, followed by transduction on Day 1. Cells rest for 48 h, followed by a culture wash, an increase of the culture volume to 200 mL, and agitation using a shaking mechanism. On Day 6, the first in-process sample is taken. Cells are harvested once sufficient cells are available for at least three full doses of CAR-T cells (5 &#215; 106 CAR-T cells/kg if the patient is &#60;50 kg, otherwise 2.5 &#215; 108 CAR-T cells) and quality control testing (~2 &#215; 106 CAR-T cells); or once the culture reaches a total of 4-5 x 109 cells. Abbreviations: TCT = T cell transduction; CAR-T = chimeric antigen receptor T cells; MACS = magnetic-activated cell sorting.

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

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

The advantage of our protocol is that it is a semi-automated, closed system that still offers flexibility. The labor-intensive steps are replaced by the automation, which is run by a computer, and because it's run by a computer, the user can customize their protocol. This is also all done in a closed system.We found that the sort of stem cell laboratories that are commonly present in hospitals have the capability to produce clinical grade CAR-T cells at significantly lower cost than commercial alternatives. We think that our process could be easily adapted to make CAR-T cell specific for other targets, and also to manufacture other types of cell and gene therapies. Our team is poised to explore other CAR-T cell targets and new gene therapies.On a broader scale, cell and gene therapies are rapidly expanding, and we are excited to continue to support and innovate in this exciting field.

Watch this Scientific Journal Video about Manufacturing Chimeric Antigen Receptor T-Cells Using an Automated Cell Processor at JoVE.com

Manufacturing Chimeric Antigen Receptor T-Cells Using an Automated Cell Processor  

Begin by turning on the instrument and selecting the T-cell transduction process from the touchscreen interface. Click run to initiate the procedure and follow the onscreen instructions provided. On the perimeter input screen input the operator initials tubing set, lot number, and expiration date.Next, select full process on the process setup screen. Now, choose two vials of selection reagent for the CD4, CD8 selection method. Install the tubing set, ensuring that all lure connections are tightly set and complete the upper and lower integrity tests by following the instructions on the touchscreen.Now, attach the medium and processing buffer bags per the onscreen guidance and begin the automatic priming of the tubing set. Once the transfer cell product screen appears, transfer the thawed T-cell product to a 150 milliliter transfer bag and sterile weld it to the application bag of the tubing set. Next, use a quality control pouch to obtain a sample from the application bag and perform a cell count using a hematology analyzer.Connect the CD4 and CD8 reagent vials to the tubing set and start the selection process for T-cell enrichment. After enrichment, remove a sample of the selected cells from the reapplication bags quality control pouch. Enter the desired cell concentration and starting number.Now, attach a vial of the activation reagent according to onscreen instructions. Then, set the carbon dioxide concentration to 5%and the culture chamber temperature to 39 degrees Celsius. Establish the activity matrix using the enhanced feeding protocol as a starting point and modify individual steps to your specific optimized protocol.In this case, set the transduction activity time to 24 hours after seeding and the culture wash to 48 hours after transduction. Set the activate shaker time to begin 30 minutes after starting the culture wash. Delete any medium bag exchange and waste bag exchange activities as they are no longer required.Then, press okay on the screen to initiate cultivation. Allow the instrument to automatically pump the required volume from the reapplication bag into the culture chamber. Thaw and prepare the calculated vector volume in a 20 milliliter reagent bag with a cold medium to reach a final volume of 10 milliliters.Adjust the activity matrix to set the transduction time to two minutes into the future and press okay to initiate the transduction activity. Sterile weld the vector bag to the tubing set following the onscreen instructions. Modify the activity matrix according to the actual transduction start time with the culture wash scheduled 48 hours after transduction.Schedule the time of activate shaker to 30 minutes after the culture wash. Finally, ensure the media exchange occurs in the afternoon at 2:00 PM on day six. Press the sample button and follow the onscreen instructions to acquire a quality control sample from the active culture.Divide the sample into three one milliliter aliquots for various analyses. Prepare the final formulation buffer, saving a portion for the cryoprotectant preparation later. Next, adjust the activity matrix, setting the end of culture to two minutes in the future.Attach the final formulation buffer to the tubing set and begin harvest. Seal off and remove the target cell bag. This bag contains the CAR T-cells ready for infusion or cryo-preservation.RT runs of the clinical trial was 46%This indicates that the TCT process effectively promoted cell expansion. The cell viability of the final product was between 88 to 94%and the CD3 T-cell purity was 89 to 93%Endotoxin, mycoplasma, sterility, replication competent lentivirus, and residual leukemic cells were not detectable.

manufacturing-chimeric-antigen-receptor-t-cells-using-an-automated

Research

JoVE Journal

Medicine

405 ビュー.  Children’s Hospital of Philadelphia. まず、装置の電源を入れ、タッチスクリーンインターフェースからT細胞形質導入プロセスを選択します。「実行」をクリックして手順を開始し、画面の指示に従います。外周入力画面で、オペレーターのイニシャル、チューブセット、ロット番号、有効期限を入力します。次に、プロセス設定画面でフルプロセスを選択します。次に、CD4、CD8選択法用の選択試薬の...