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.

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.

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.

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.

Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.

Background Aims: Reference genes are an essential part of clinical assays such as droplet digital polymerase chain reaction (ddPCR), which measure the number of copies of vector integrated into genetically engineered cells and the loss of plasmids in reprogrammed cells used in clinical cell therapies. Care should be taken to select reference genes, because it has been discovered that there may be thousands of variations in copy number from genomic segments among different individuals. In addition, within the same person in the context of cancer and other proliferative disorders, substantial parts of the genome also can differ in copy number between cells from diseased and healthy people.

Longitudinal transcriptional analysis of peripheral blood leukocytes in COVID-19 convalescent donors.

Background: SARS-CoV2 can induce a strong host immune response. Many studies have evaluated antibody response following SARS-CoV2 infections. This study investigated the immune response and T cell receptor diversity in people who had recovered from SARS-CoV2 infection (COVID-19).

Genome-wide profiling of retroviral DNA integration and its effect on clinical pre-infusion CAR T-cell products.

Background: Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.

Identification of genomic determinants contributing to cytokine release in immunotherapies and human diseases.

Background: Cytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS.

Scaling up and scaling out: Advances and challenges in manufacturing engineered T cell therapies.

Engineered T cell therapies such as CAR-T cells and TCR-T cells have generated impressive patient responses in previously incurable diseases. In the past few years there have been a number of technical innovations that enable robust clinical manufacturing in functionally closed and often automated systems. Here we describe the latest technology used to manufacture CAR- and TCR-engineered T cells in the clinic, including cell purification, transduction/transfection, expansion and harvest.

Point-of-care cell therapy manufacturing; it's not for everyone.

The use of cellular therapies to treat cancer, inherited immune deficiencies, hemoglobinopathies and viral infections is growing rapidly. The increased interest in cellular therapies has led to the development of reagents and closed-system automated instruments for the production of these therapies. For cellular therapy clinical trials involving multiple sites some people are advocating a decentralized model of manufacturing where patients are treated with cells produced using automated instruments at each participating center using a single, centrally held Investigational New Drug Application (IND).

Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control.

Background: Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available.

Antigen Experienced T Cells from Peripheral Blood Recognize p53 Neoantigens.

Purpose: The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens.

Experimental Design: PBLs from patients with a mutated tumor were sorted for antigen-experienced T cells and stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol.

Eliminating transmissibility of bovine spongiform encephalopathy by dry-heat treatment.

Bovine spongiform encephalopathy (BSE) prion is more resistant to heat inactivation compared to other prions, but the effect of heat inactivation has been reported to differ depending on the BSE-contaminated tissue state or heating type. We aimed to evaluate the secure level of inactivation of original BSE transmissibility by dry-heating. Cattle tissues affected with BSE were subjected to dry-heat treatment for 20 min at various temperatures ranging from 150 to 1000 °C.

Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers.

BSE infectivity survives burial for five years with only limited spread.

The carcasses of animals infected with bovine spongiform encephalopathy (BSE), scrapie or chronic wasting disease (CWD) that remain in the environment (exposed or buried) may continue to act as reservoirs of infectivity. We conducted two experiments under near-field conditions to investigate the survival and dissemination of BSE infectivity after burial in a clay or sandy soil. BSE infectivity was either contained within a bovine skull or buried as an uncontained bolus of BSE-infected brain.

A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus-associated Epithelial Cancers.

Purpose: Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas.

Patients And Methods: The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers.

Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.

Immunotherapy using either checkpoint blockade or the adoptive transfer of antitumor lymphocytes has shown effectiveness in treating cancers with high levels of somatic mutations-such as melanoma, smoking-induced lung cancers and bladder cancer-with little effect in other common epithelial cancers that have lower mutation rates, such as those arising in the gastrointestinal tract, breast and ovary. Adoptive transfer of autologous lymphocytes that specifically target proteins encoded by somatically mutated genes has mediated substantial objective clinical regressions in patients with metastatic bile duct, colon and cervical cancers. We present a patient with chemorefractory hormone receptor (HR)-positive metastatic breast cancer who was treated with tumor-infiltrating lymphocytes (TILs) reactive against mutant versions of four proteins-SLC3A2, KIAA0368, CADPS2 and CTSB.

T-cell Responses to "Hotspot" Mutations and Unique Neoantigens Expressed by Human Ovarian Cancers.

This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers. Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures.

Long-Duration Complete Remissions of Diffuse Large B Cell Lymphoma after Anti-CD19 Chimeric Antigen Receptor T Cell Therapy.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) can induce complete remissions (CRs) of diffuse large B cell lymphoma (DLBCL). The long-term durability of these remissions is unknown. We administered anti-CD19 CAR T cells preceded by cyclophosphamide and fludarabine conditioning chemotherapy to patients with relapsed DLBCL.

Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study.

Background: Uveal melanoma is a rare tumour with no established treatments once metastases develop. Although a variety of immune-based therapies have shown efficacy in metastatic cutaneous melanoma, their use in ocular variants has been disappointing. Recently, adoptive T-cell therapy has shown salvage responses in multiple refractory solid tumours.

Characterization of an Immunogenic Mutation in a Patient with Metastatic Triple-Negative Breast Cancer.

The administration of autologous tumor-infiltrating lymphocytes (TILs) can mediate durable tumor regressions in patients with melanoma likely based on the recognition of immunogenic somatic mutations expressed by the cancer. There are limited data regarding the immunogenicity of mutations in breast cancer. We sought to identify immunogenic nonsynonymous mutations in a patient with triple-negative breast cancer (TNBC) to identify and isolate mutation-reactive TILs for possible use in adoptive cell transfer.

Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels.

Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells.