Supplementary Figure 4 from A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Supplementary Figure 1 from A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Supplementary Figure 3 from A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Supplementary Figure 5 from A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Supplementary Figure 6 from A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Supplementary Figure 2 from A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Abstract As of April 2021, there are five commercially available chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies. With the current transition of CAR T cell manufacturing from academia to industry, there is a shift toward Good Manufacturing Practice (GMP)–compliant closed and automated systems to ensure reproducibility and to meet the increased demand for patients with cancer. In this review, we describe current CAR T cell clinical manufacturing models and discuss emerging technologic advances that embrace scaling and production optimization. We summarize measures being used to shorten CAR T cell manufacturing times and highlight regulatory challenges to scaling production for clinical use. Significance: As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development. This review provides a critical appraisal of these technologies, which can be leveraged to scale and optimize the production of next-generation CAR T cells. See related video: https://aacrjournals.org/content/webinar-car-t-cell-manufacturing

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

Kriley, Isaac R. MD; Feldman, Steve A.; Tran, Eric; Black, Mary; Xu, Hui; Rosenberg, Steven A. MD, PhD; Goff, Stephanie MD Author Information

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (TN) and central memory (TCM) CD8+ T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8+ TN cells (CD4−CD62L+CD45RA+) and CD8+ TCM (CD4−CD62L+CD45RA−) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of TN and TCM we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.

It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F-1 and F-2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.

Chromogranins are the major components of the secretory granules of most neuroendocrine cells. Within the secretory pathway, chromogranins are involved in granulogenesis, and in sorting and processing of secretory proteincargo prior to, secretion. Once secreted, they have hormonal, autocrine, and paracrine activities. The chromogranin family includes chromogranins A (CgA) and B (CgB)and secretogranin II (Sgll, once called chromogranin C). The related “granins” NESP55, 7B2, secretogranin III/1B 1075 (SgIII), and secretogranin IV/HISL-19 antigen (SgIV), are also sometimes included when considering the chromogranins. While it is useful to consider the granin proteins as a family with many common features, it is also necessary to examine the distinct features and properties of individual members of the granin family to understand fully their functions, employ them efficiently as tissue, serum, and urinary markers for neuroendocrine neoplasia, and develop an evolutionary-biologic perspective on their contribution to mammalian physiology. Recent advances in chromogranin research include establishing the role of CgA in granulogenesis and the role of CgB in nuclear transcription; new biologic activities for CgA-, CgB-, and SgII-derived peptides; and new marker functions for granins and their proteolytically processed products in endocrine neoplasias

Journal of Developmental & Behavioral Pediatrics: September 1981 - Volume 2 - Issue 3 - p 97-104