Stability testing of HPCs and MNCs from apheresis products.

Background: Hematopoietic progenitor cells (HPCs) and mononuclear cells (MNCs) are critical components of cell-based therapies, including bone marrow transplantation and regenerative treatments. Evaluation of the characteristics of these products during collection, storage, and transport is essential for maintaining cell viability and functionality. In this study, we evaluated the functional and molecular stability of samples collected for the evaluation of fresh HPC and MNC products.

New insights on potency assays from recent advances and discoveries in CAR T-cell therapy.

This review explores recent advances in the characteristics and manufacturing of CAR T-cell products. Traditional potency assays have been designed based on well-established CAR T-cell functionalities. However, the advent of innovative tools and methodologies has revealed a broader spectrum of important CAR T-cell characteristics that correlate with function.

Dendritic Cell Cancer Vaccines: Clinical Production for Cancer Immunotherapy.

Dendritic cell (DC) cancer vaccines are used to circumvent the problem that DCs in patients with cancer usually do not mature properly in the cancer environment. Peripheral DCs are fewer in number and hard to isolate cleanly. Instead, autologous DCs can be matured from monocyte precursors obtained by apheresis and elutriation from peripheral blood.

Dendritic Cell Cancer Vaccines: A Focused Review.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells to activate both CD4+ and CD8+ T lymphocytes. When immature, they take up and process antigens efficiently. When mature, they express high levels of MHC class I and II molecules and costimulatory molecules on their surface and secrete both IL-12 and IL-15 that can activate and steer T cells.

Topical TLR7 agonist and radiotherapy in patients with metastatic breast cancer.

Background: Toll-like receptor (TLR) agonists and radiation therapy hold promise for cancer immunotherapy. We conducted a phase I/II trial combining topical imiquimod (IMQ, a TLR-7 agonist) and local radiotherapy (RT) in patients with metastatic breast cancer accompanied by longitudinal transcriptional analysis of tumor biopsies.

Methods: The primary objective of the trial (NCT01421017) was to assess systemic responses by immune-related response criteria (irRC) after an 8-week cycle of topical IMQ and concurrent local RT (cohort 1).

Talicabtagene autoleucel for relapsed or refractory B-cell malignancies: results from an open-label, multicentre, phase 1/2 study.

Background: In low-income and middle-income counties (LMICs), the outcome of relapsed or refractory B-cell malignancies is poor due to the absence of effective therapies. We report the results of a phase 1/2 study of a novel humanised anti-CD19 4-1BB chimeric antigen receptor (CAR) T-cell therapy, talicabtagene autoleucel, for patients with relapsed or refractory B-cell malignancies.

Methods: This open-label, multicentre, phase 1/2 study was done at six tertiary cancer centres in India.

Immunophenotype of CAR T cells and apheresis products predicts response in CD22 CAR T cell trial for B cell acute lymphoblastic leukemia.

Although CAR T cell therapy is increasingly used to treat relapsed B cell acute lymphoblastic leukemia (ALL), 20%-30% of patients do not respond, and few clinical predictors of response have been established, especially in the pediatric population. A deeper analysis of CAR T cell infusion products, along with the apheresis product used as the starting material for CAR T cell manufacturing, provides valuable insights for predicting clinical outcomes. We analyzed infusion products and CD4/8-selected T cell starting materials from pediatric and young adult patients on a single-center study with relapsed/refractory B cell ALL who were undergoing treatment with CD22 CAR T cells and evaluated differences between T cells from responders and non-responders (NCT023215612).

Universal new blood cell elution method with extensive phenotyping.

Background/objectives: No erythrocyte elution method developed is uniformly successful or allows elution/phenotyping together. We previously developed an elution method using deionised formamide. We modified it to be universal for various cell types and call it modified formamide-method (Fm-method).

Efficient manufacturing of CAR-T cells from whole blood: a scalable approach to reduce costs and enhance accessibility in cancer therapy.

Background: Chimeric antigen receptor T (CAR-T) cells have significantly advanced the treatment of cancers such as leukemia and lymphoma. Traditionally, T cells are collected from patients through leukapheresis, an expensive and potentially invasive process that requires specialized equipment and trained personnel. Although whole blood collections are much more technically straightforward, whole blood starting material has not been widely utilized for clinical CAR-T cell manufacturing, in part due to lack of manufacturing processes designed for use in a good manufacturing practice (GMP) environment.

Regression of renal cell carcinoma by T cell receptor-engineered T cells targeting a human endogenous retrovirus.

Background: We discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T cell-mediated killing. Here, we described the cloning of a novel T cell receptor (TCR) targeting a CT-RCC HERV-E-derived antigen specific to ccRCC and characterized antitumor activity of HERV-E TCR-transduced T cells (HERV-E T cells).

Methods: We isolated a CD8 T cell clone from a patient with immune-mediated regression of ccRCC post-allogeneic stem cell transplant that recognized the CT-RCC-1 HERV-E-derived peptide in an HLA-A11-restricted manner.

T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function.

T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas).

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

Background: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity.

CAR-T cell expansion platforms yield distinct T cell differentiation states.

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown.

Expanding the reach of commercial cell therapies requires changes at medical centers.

The clinical application of cell therapies is becoming increasingly important for the treatment of cancer, congenital immune deficiencies, and hemoglobinopathies. These therapies have been primarily manufactured and used at academic medical centers. However, cell therapies are now increasingly being produced in centralized manufacturing facilities and shipped to medical centers for administration.

Manufacture of CD22 CAR T cells following positive versus negative selection results in distinct cytokine secretion profiles and γδ T cell output.

Chimeric antigen receptor T cells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, T cell selection, removes contaminating cell types that can potentially suppress T cell expansion and transduction. While positive selection of CD4/CD8 T cells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients.

Emerging Biomarkers for Monitoring Chimeric Antigen Receptor T-Cell Therapy.

Background: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment of hematologic malignancies and holds promise for solid tumors. While responses to CAR T-cell therapy have surpassed other available options for patients with refractory malignancies, not all patients respond the same way. The reason for this variability is not currently understood.

Healthcare center-based cell therapy laboratories supporting off-site manufactured cell therapies: The experiences of a single academic cell therapy laboratory.

Background: Healthcare center-based cell therapy laboratories (HC CTLs) evolved from solely processing hematopoietic stem cells for transplantation to manufacturing various advanced cellular therapies. With increasing interest in cellular therapy applications, off-site manufactured products are becoming more common. HC CTLs play a critical role in supporting these products by shipping out cellular starting material (CSM) for further manufacturing and/or receiving, storing, and distributing final products.

Immune determinants of CAR-T cell expansion in solid tumor patients receiving GD2 CAR-T cell therapy.

Chimeric antigen receptor T cells (CAR-Ts) have remarkable efficacy in liquid tumors, but limited responses in solid tumors. We conducted a Phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.

Assessment and comparison of viability assays for cellular products.

Background Aims: Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products.

Methods: Viability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated.

Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study.

Background Aims: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media.

Methods: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements.