First name
Yongping
Last name
Wang

Title

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor.

Year of Publication

2023

Date Published

08/2023

ISSN Number

1940-087X

Abstract

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 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 (cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate the feasibility and discuss the advantages and disadvantages of using a 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.

DOI

10.3791/65488

Alternate Title

J Vis Exp

PMID

37607096
Featured Publication
No

Title

Immune Reconstitution Following TCRαβ/CD19-Depleted Hematopoietic Cell Transplantation for Hematologic Malignancy in Pediatric Patients.

Year of Publication

2021

Number of Pages

169.e1-169.e9

Date Published

2021 Feb

ISSN Number

2666-6367

Abstract

TCRαβ/CD19-depleted HCT has been used with excellent outcomes in pediatric patients with hematologic malignancies, and several studies have demonstrated rapid immune reconstitution in the nonmalignant setting. However, immune recovery following TCRαβ/CD19-depleted hematopoietic cell transplantation (HCT) for malignancy remains incompletely elucidated. Furthermore, the majority of studies to date have used haploidentical and matched unrelated donors. Here we report results of immune reconstitution following TCRαβ/CD19-depleted HCT for hematologic malignancy in 51 pediatric patients with hematologic malignancies, the majority of whom received grafts from unrelated donors. Grafts were from matched unrelated (n = 20), mismatched unrelated (n = 20), and haploidentical (n = 11) donors. The median CD34 cell dose was 10.2 × 10/kg (range, 4.54 to 20 × 10/kg), and the median TCRαβ cell dose was 2.53 × 10/kg (range, 0 to 44.9 × 10/kg). Conditioning was myeloablative with either busulfan or total body irradiation, cyclophosphamide, and thiotepa. Thirty-three patients also received rabbit antithymocyte globulin. No prophylactic post-transplantation immune suppression was routinely given. Forty-three patients received rituximab on day +1 for recipient positive Epstein-Barr virus serology. Forty-nine patients (96%) engrafted with a median time to neutrophil recovery of 13 days (range, 8 to 30 days). Thirty-seven patients (73%) are alive at a median follow-up of 25 months (range, 6 to 50 months). Nine patients (18%) developed grade II-IV acute graft-versus-host disease (GVHD), and 5 patients (11%) developed extensive chronic GVHD. Twenty-six patients (51%) experienced viral reactivation. T cell reconstitution was rapid with significant numbers of CD3, CD4, and CD8 T cells present on first assessment at 4 months post-HCT, and significant numbers of naïve CD4 T cells were present by 8 months post-HCT. Chronic GVHD was associated with delayed T cell recovery; however, T cell reconstitution was not affected by underlying diagnosis, donor source, TCRαβ T cell dose, conditioning regimen, or use of antithymocyte globulin. B cell recovery mirrored T cell recovery, and i.v. Ig was discontinued at a median of 8 months (range, 4 to 22 months) post-HCT in patients alive and relapse-free at last follow-up. Immune reconstitution is rapid following TCRαβ/CD19-depleted HCT in pediatric patients with hematologic malignancies. Donor graft source, haploidentical or unrelated, did not affect immune reconstitution. Viral reactivation is common in the first 100 days post-HCT, indicating that improved T cell defense is needed in the early post-HCT period.

DOI

10.1016/j.jtct.2020.10.006

Alternate Title

Transplant Cell Ther

PMID

33830028

Title

Unrelated donor α/β T cell- and B cell-depleted HSCT for the treatment of pediatric acute leukemia.

Year of Publication

2022

Number of Pages

1175-1185

Date Published

2022 Feb 22

ISSN Number

2473-9537

Abstract

Unrelated donor (URD) hematopoietic stem cell transplant (HSCT) is associated with an increased risk of severe graft-versus-host disease (GVHD). TCRαβ/CD19 depletion may reduce this risk, whereas maintaining graft-versus-leukemia. Outcome data with TCRαβ/CD19 depletion generally describe haploidentical donors, with relatively few URDs. We hypothesized that TCRαβ/CD19-depletion would attenuate the risks of GVHD and relapse for URD HSCT. Sixty pediatric and young adult (YA) patients with hematologic malignancies who lacked a matched-related donor were enrolled at 2 large pediatric transplantation centers between October 2014 and September 2019. All patients with acute leukemia had minimal residual disease testing, and DP typing was available for 77%. All patients received myeloablative total body irradiation- or busulfan-based conditioning with no posttransplant immune suppression. Engraftment occurred in 98%. Four-year overall survival was 69% (95% confidence interval [CI], 52%-81%), and leukemia-free survival was 64% (95% CI, 48%-76%), with no difference between lymphoid and myeloid malignancies (P = .6297 and P = .5441, respectively). One patient (1.7%) experienced primary graft failure. Relapse occurred in 11 patients (3-year cumulative incidence, 21%; 95% CI, 11-34), and 8 patients (cumulative incidence, 15%; 95% CI, 6.7-26) experienced nonrelapse mortality. Grade III to IV acute GVHD was seen in 8 patients (13%), and 14 patients (26%) developed chronic GVHD, of which 6 (11%) had extensive disease. Nonpermissive DP mismatch was associated with higher likelihood of acute GVHD (odds ratio, 16.50; 95% CI, 1.67-163.42; P = .0166) but not with the development of chronic GVHD. URD TCRαβ/CD19-depleted peripheral HSCT is a safe and effective approach to transplantation for children/YAs with leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02323867.

DOI

10.1182/bloodadvances.2021005492

Alternate Title

Blood Adv

PMID

34872106

Title

Partially CD3-depleted unrelated and haploidentical donor PSCT has favorable GVHD and survival rates in pediatric hematologic malignancy.

Year of Publication

2019

Date Published

2019 Nov 22

ISSN Number

1523-6536

Abstract

<p>Most children who may benefit from stem cell transplantation lack matched related donors. Alternative donor transplantations with unrelated (URD) or partially matched related donors (PMRD) carry increased risks of graft-versus-host-disease (GVHD) and mortality compared to matched related donor transplants. We hypothesized a strategy of partial CD3/CD19-depletion for URD or PMRD peripheral stem cell transplantation (PSCT) would attenuate risks of GVHD and mortality. We enrolled 84 pediatric patients with hematologic malignancies at the Children's Hospital of Philadelphia and the Children's Hospital of Wisconsin between April 2005 and February 2015 (NCT00579124: https://clinicaltrials.gov/ct2/show/NCT00579124; NCT01071226: https://clinicaltrials.gov/ct2/show/NCT01071226). Two (2.4%) experienced primary graft failure. Relapse occurred in 23 patients (27.4%; cumulative incidence 26.3%), and 17 patients (20.2%) experienced non-relapse mortality (NRM). Grade III-IV acute GVHD was observed in 18 patients (21.4%), and chronic GVHD was observed and graded as limited in 24 (35.3%) and extensive in 8 (11.7%). Three-year OS was 61.8% (95% CI 50.2 -71.4%) and EFS 52.0% (95% CI 40.3 - 62.4%). Age ≥15 years was associated with decreased OS (p=0.05) and EFS (p=0.05). Relapse was more common in children in second complete remission (p=0.03). Partially CD3-depleted alternative donor PSCT NRM, OS, and EFS compare favorably with previously published studies of T cell-replete PSCT. Historically, T cell-replete PSCTs have had a high ratio of extensive compared to limited chronic GVHD, which may explain the comparatively low relapse and NRM rates in our study despite similar overall rates of chronic GVHD. Partial T cell-depletion may expand donor options for children with malignant transplant indications lacking matched related donors by mitigating but not eliminating chronic GVHD.</p>

DOI

10.1016/j.bbmt.2019.11.022

Alternate Title

Biol. Blood Marrow Transplant.

PMID

31765697

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