The authors would like to acknowledge the contributions of several individuals and organizations to this work. The Cell and Gene Therapy Laboratory and the Penn Translational and Correlative Studies Laboratory provided valuable assistance with process development and preparation for IND submissions. Melissa Varghese and Amanda DiNofia contributed to the process development and preparation for IND submissions that underly this manuscript. This work was supported by an Acceleration&#160;Grant of the Cell and Gene Therapy Collaborative of the Children&#39;s Hospital of Philadelphia. The authors would also like to thank Miltenyi Biotec for their technical and research support. Figure 1 is covered by copyright &#169; 2023 Miltenyi Biotec B.V. &#38; Co. KG; all rights reserved.

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

<ol>
	<li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tisagenlecleucel+in+children+and+young+adults+with+B-cell+lymphoblastic+leukemia.">Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia.</a> New England Journal of Medicine. 378 (5), 439-448 (2018).</li><li>Shah, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+anti-CD20,+anti-CD19+CAR+T+cells+for+relapsed+B+cell+malignancies:+A+phase+1+dose+escalation+and+expansion+trial.">Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: A phase 1 dose escalation and expansion trial.</a> Nature Medicine. 26 (10), 1569-1575 (2020).</li><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+for+sustained+remissions+in+leukemia.">Chimeric antigen receptor T cells for sustained remissions in leukemia.</a> New England Journal of Medicine. 371 (16), 1507-1517 (2014).</li><li>Grupp, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+for+acute+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.</a> New England Journal of Medicine. 368 (16), 1509-1518 (2013).</li><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+humanized+CD19-targeted+chimeric+antigen+receptor+(CAR)-modified+T+cells+in+children+and+young+adults+with+relapsed/refractory+acute+lymphoblastic+leukemia.">Efficacy of humanized CD19-targeted chimeric antigen receptor (CAR)-modified T cells in children and young adults with relapsed/refractory acute lymphoblastic leukemia.</a> Blood. 128 (22), 217 (2016).</li><li>Mock, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+manufacturing+of+CAR-T+cells+for+adoptive+immunotherapy+using+CliniMACS+Prodigy.">Automated manufacturing of CAR-T cells for adoptive immunotherapy using CliniMACS Prodigy.</a> Cytotherapy. 18 (8), 1002-1011 (2016).</li><li>Fern&#225;ndez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-compliant+manufacturing+of+NKG2D+CAR+memory+T+cells+using+CliniMACS+Prodigy.">GMP-compliant manufacturing of NKG2D CAR memory T cells using CliniMACS Prodigy.</a> Frontiers in Immunology. 10 (10), 2361 (2019).</li><li>Zhu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closed-system+manufacturing+of+CD19+and+dual-targeted+CD20/19+chimeric+antigen+receptor+T+Cells+using+CliniMACS+Prodigy+device+at+an+academic+medical+center.">Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T Cells using CliniMACS Prodigy device at an academic medical center.</a> Cytotherapy. 20 (3), 394-406 (2018).</li><li>Zhang, W., Jordan, K. R., Schulte, B., Purev, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clinical+grade+CD19+chimeric+antigen+receptor+T+cells+produced+using+automated+CliniMACS+prodigy+system.">Characterization of clinical grade CD19 chimeric antigen receptor T cells produced using automated CliniMACS prodigy system.</a> Drug Design, Development and Therapy. 12 (12), 3343-3356 (2018).</li><li>Abou-El-Enein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+manufacturing+of+CAR+T+cells+for+cancer+immunotherapy.">Scalable manufacturing of CAR T cells for cancer immunotherapy.</a> Blood Cancer Discovery. 2 (5), 408-422 (2021).</li><li>Miltenyi Biotec. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CliniMACS Prodigy User Manual. , (2021).</li><li>Ghassemi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+manufacturing+of+non-activated+potent+CAR+T+cells.">Rapid manufacturing of non-activated potent CAR T cells.</a> Nature Biomedical Engineering. 6 (2), 118-128 (2022).</li><li>U.S. Department of Health and Human Services, Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs) guidance for industry. , (2020).</li><li>U.S. Department of Health and Human Services, Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Considerations for the development of chimeric antigen receptor (CAR) T cell products draft guidance for industry. , (2022).</li></ol>

S.K., S.G., and Y.W. have received research support from Miltenyi Biotec.

CAR-T cell therapy has emerged as a promising treatment approach for B-cell and other malignancies. However, traditional CAR-T cell manufacturing methods have several limitations, such as high cost, labor-intensive production, and open steps that increase the risk of contamination. Recently, several semi-automated platforms, including the Miltenyi CliniMACS Prodigy (the &#34;processor&#34;), have emerged to address these limitations. The T cell transduction (TCT) process, integrated into the processor described in this m...

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.

<table><tbody><tr><td>12 x 75 borosilicate tubes</td><td>Charles River</td><td>TL1000</td><td></td></tr><tr><td>20 mL Reagent Bag</td><td>Miltenyi Biotec</td><td>170-076-631</td><td></td></tr><tr><td>50 mL Conical Tube</td><td>Fisher</td><td>05-539-10</td><td></td></tr><tr><td>150 mL Transfer Set</td><td>Fenwal</td><td>4R2001</td><td></td></tr><tr><td>2,000 mL Transfer Set</td><td>Fenwal</td><td>4R2041</td><td></td></tr><tr><td>7AAD</td><td>Fisher Scientific</td><td>BDB559925</td><td></td></tr><tr><td>Alcohol Prep</td><td>Tyco/Healthcare</td><td></td><td></td></tr><tr><td>Bag Access</td><td>Medline</td><td>2300E-0500</td><td></td></tr><tr><td>CD19 APC-Vio770 REAfinity</td><td>Miltenyi Biotec</td><td>130-113-643</td><td></td></tr><tr><td>CD19 CAR Detection Reagent Biotin</td><td>Miltenyi Biotec</td><td>130-129-550</td><td></td></tr><tr><td>CD19 PE</td><td>BD</td><td>555413</td><td></td></tr><tr><td>CD3 APC</td><td>BD</td><td>340440</td><td></td></tr><tr><td>CD4 VioBright FITC REAfinity</td><td>Miltenyi Biotec</td><td>130-113-229</td><td></td></tr><tr><td>CD45 VioBlue REAfinity</td><td>Miltenyi Biotec</td><td>130-110-637</td><td></td></tr><tr><td>CD8 APC-Vio770 REAfinity</td><td>Miltenyi Biotec</td><td>130-110-681</td><td></td></tr><tr><td>Cellometer Reference Beads 10um</td><td>Nexcelom</td><td>B10-02-020</td><td></td></tr><tr><td>Cellometer Reference Beads 15um</td><td>Nexcelom</td><td>B15-02-010</td><td></td></tr><tr><td>Cellometer Reference Beads 5um</td><td>Nexcelom</td><td>B05-02-050</td><td></td></tr><tr><td>Cellometer Slides</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td></td></tr><tr><td>CliniMACS CD4 GMP MicroBeads</td><td>Miltenyi Biotec</td><td>276-01</td><td>The CD4 reagent</td></tr><tr><td>CliniMACS CD8 GMP MicroBeads</td><td>Miltenyi Biotec</td><td>275-01</td><td>The CD8 reagent</td></tr><tr><td>CliniMACS PBS/EDTA Buffer</td><td>Miltenyi Biotec</td><td>130-021-201</td><td>The buffer</td></tr><tr><td>DMSO</td><td>Origen</td><td>CP-10</td><td></td></tr><tr><td>Freezing Bag 50 mL</td><td>Miltenyi Biotec</td><td>200-074-400</td><td></td></tr><tr><td>Freezing Vial, 1.8 mL</td><td>Nunc</td><td>12565171N</td><td></td></tr><tr><td>Freezing Vial, 4.5 mL</td><td>Nunc</td><td>12565161N</td><td></td></tr><tr><td>Human AB serum</td><td>Valley Biomedical</td><td></td><td>Sterile filtered, heat inactivated</td></tr><tr><td>Human Serum Albumin 25%</td><td>Grifols</td><td>68516-5216-1</td><td></td></tr><tr><td>Human Serum Albumin 5%</td><td>Grifols</td><td>68516-5214-1</td><td></td></tr><tr><td>MACS GMP Recombinant Human IL-2</td><td>Miltenyi Biotec</td><td>170-076-148</td><td>The cytokines</td></tr><tr><td>MACS GMP T Cell TransAct</td><td>Miltenyi Biotec</td><td>200-076-202</td><td>The activation reagent</td></tr><tr><td>MycoSeq Mycoplasma Detection Kit</td><td>Life Technologies</td><td>4460623</td><td></td></tr><tr><td>Needles, Hypodermic 14G</td><td>Medline</td><td>SWD200573</td><td></td></tr><tr><td>Needles, SlideSafe 18G</td><td>BD</td><td>B-D305918</td><td></td></tr><tr><td>Pipet tips, 2-200 &amp;mu;L, individually wrapped</td><td>Eppendorf</td><td>022492209</td><td></td></tr><tr><td>Pipet tips, 50-1000 &amp;mu;L, individually wrapped</td><td>Eppendorf</td><td>022492225</td><td></td></tr><tr><td>Pipets 10 mL</td><td>Fisher</td><td>13-678-27F</td><td></td></tr><tr><td>Pipets 25 mL</td><td>Fisher</td><td>13-675-30</td><td></td></tr><tr><td>Pipets 5 mL</td><td>Fisher</td><td>13-678-27E</td><td></td></tr><tr><td>Plasmalyte-A</td><td>Baxter</td><td>2B2544X</td><td>The electrolyte solution</td></tr><tr><td>Prodigy TS520 Tubing Set</td><td>Miltenyi Biotec</td><td>170-076- 600</td><td>The tubing set</td></tr><tr><td>Sterile Field</td><td>Medline</td><td>NON21001</td><td></td></tr><tr><td>Streptavidin PE-Vio770</td><td>Miltenyi Biotec</td><td>130-106-793</td><td></td></tr><tr><td>Syringe 1 mL</td><td>BD</td><td>309628</td><td></td></tr><tr><td>Syringe 10 mL</td><td>BD</td><td>302995</td><td></td></tr><tr><td>Syringe 3 mL</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Syringe 30 mL</td><td>BD</td><td>302832</td><td></td></tr><tr><td>Syringe 50 mL</td><td>BD</td><td>309653</td><td></td></tr><tr><td>TexMACS GMP Medium</td><td>Miltenyi Biotec</td><td>170-076-306</td><td>The medium</td></tr><tr><td>Triple Sampling Adapter</td><td>Miltenyi Biotec</td><td>170-076-609</td><td></td></tr><tr><td>Viral Vector</td><td>CHOP Clinical Vector Core</td><td></td><td>huCART19</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biological Safety Cabinet</td><td>The Baker Co</td><td></td><td></td></tr><tr><td>Cellometer Auto 2000</td><td>Nexcelom</td><td></td><td></td></tr><tr><td>CliniMACS Prodigy</td><td>Miltenyi Biotec</td><td>200-075-301</td><td>The processor</td></tr><tr><td>Controlled Rate Freezer</td><td>Planer/Kryosave</td><td></td><td></td></tr><tr><td>Endosafe nexgen-PTS150K</td><td>Charles River</td><td></td><td></td></tr><tr><td>Mettler Balance</td><td>Mettler</td><td></td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Fisher Sci</td><td></td><td></td></tr><tr><td>SCD Sterile Tubing Welder</td><td>Terumo</td><td></td><td></td></tr><tr><td>Sebra Tube Sealer</td><td>Sebra</td><td></td><td></td></tr><tr><td>Varitherm</td><td>Barkey</td><td></td><td>The dry thaw device</td></tr><tr><td>XN-330 Hematology Analyzer</td><td>Sysmex</td><td></td><td></td></tr></tbody></table>

chimeric antigen receptor t cell manufacturing on an automated cell processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Results from the initial three CAR-T manufacturing runs of the NCT05480449 trial are presented below in Table 3. The starting material, vector, culture cytokines, and AB serum concentrations were kept consistent for each run. Products were harvested on day 7 or 8. The average daily cell growth was 46% (increase in total cell count), indicating that the TCT process was effective in promoting cell expansion. These results suggest that the processor can produce consistent and reproducible CAR-T cell product...

Watch this Scientific Journal Video about Advancements in CAR-T Cell Manufacturing and Gene Therapy Production at JoVE.com

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

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

chimeric-antigen-receptor-t-cell-manufacturing-on-an-automated-cell

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2.4K Views.  Children’s Hospital of Philadelphia. Het voordeel van ons protocol is dat het een semi-automatisch, gesloten systeem is dat toch flexibiliteit biedt. De arbeidsintensieve stappen worden vervangen door de automatisering, die wordt uitgevoerd door een computer, en omdat het wordt uitgevoerd door een computer, kan de gebruiker zijn protocol aanpassen. Ook dit gebeurt allemaal in een gesloten systeem.We ontdekten dat het soort stamcellaboratoria dat vaak in ziekenhuizen aanwezig is, de mogelijkheid heeft om CAR-T-cellen van k...

Video: Auteur in de schijnwerpers: vooruitgang in de productie van CAR-T-cellen en de productie van gentherapie 

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

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 ...

Cancer Research

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Immunology and Infection

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

Bioengineering

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

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.

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

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