Development of a closed CAR-T manufacturing process

The field of immunotherapy has emerged as a promising new type of treatment for cancer with the approval of the first two CAR-T therapies. The clinical success of T-cell based immunotherapies necessitates a robust manufacturing process for these products to be consistently produced at commercial scale. Our CAR-T workflow combines unit operation specific solutions for thaw of an apheresis unit, wash, CD3 selection, T-cell activation, lentiviral transduction, incubator- and reactor-based expansion culture, harvest, formulation, cryopreservation and thaw of CAR-T product. We have evaluated the impact of both serum-containing and xeno-free culture media, commercially available T-cell selection and activation reagents, closed small-scale culture vessel options, alternative solutions to enhance transduction, and the specific timing of process steps to develop a modular platform process that is robust and flexible for the varied needs of CAR-T developers. Frozen apheresis units are processed using the SmartWash protocol on the SepaxTM 2 and T-cells are isolated with EasySepTM Release CD3 Positive Selection Kit. The cells are then activated with ImmunoCult CD3/CD28/CD2 T-cell activator before being transduced 24 hours later using the SepaxTM 2. Expansion of Tcells are carried out in two stages: incubator-based culture before going into the XuriTM Cell Expansion System W25 with a perfusion feeding regime. Cultured cells are then harvested and washed in Plasmalyte-A with human serum albumin and formulated with CryoStor® CS10 using the FlexCell protocol on the Sefia™ Cell Processing System. The final cell products are cryopreserved using the VIA Freeze controlled-rate freezer. We have also accessed a point-of-care thawing strategy using the VIA Thaw. Our CAR-T process achieves greater than 1.0E10 expanded T-cells with \u3e80% eGFP transduction efficiency across an 8-day manufacturing process

Mechanisms Underlying the Immune Response Generated by an Oral Vibrio cholerae Vaccine

Mechanistic details underlying the resulting protective immune response generated by mucosal vaccines remain largely unknown. We investigated the involvement of Toll-like receptor signaling in the induction of humoral immune responses following oral immunization with Dukoral, comparing wild type mice with TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. Although all groups generated similar levels of IgG antibodies, the proliferation of CD4+ T-cells in response to V. cholerae was shown to be mediated via MyD88/TLR signaling, and independently of Trif signaling. The results demonstrate differential requirements for generation of immune responses. These results also suggest that TLR pathways may be modulators of the quality of immune response elicited by the Dukoral vaccine. Determining the critical signaling pathways involved in the induction of immune response to this vaccine would be beneficial, and could contribute to more precisely-designed versions of other oral vaccines in the future

Detection of influenza A and B neutralizing antibodies in vaccinated ferrets and macaques using specific biotin-streptavidin conjugated antibodies

Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100×TCID50 of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6×TCID50 resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100×T

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

<div><p>Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.</p></div

HT-FC allows intratumoral analysis of stromal and cancer cell subsets within primary ccRCC samples.

<p>(<b>A</b>) Heatmap showing expression of each of the 363 antibodies in four subpopulations of ccRCC samples: CD45⁺ immune cells, CD45⁻CD31⁺CD34⁺ vascular endothelial cells, CD45⁻TE7⁺ fibroblasts, and CD45⁻TE7⁻CD31⁻CD34⁻ cancer cells. Antibodies are simply arranged in alphabetical order on the vertical axis and the four populations for each sample are ordered across the top. A low resolution overview demonstrates a surprisingly reproducible “fingerprint” of tumor cell subpopulations from one sample to the next. (<b>B</b>) Supervised hierarchical clustering reveals clusters of antigens corresponding to specific cell subsets within tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105602#pone.0105602.s011" target="_blank">Table S6</a> for details). (<b>C</b>) Principal components analysis of the entire data set further illustrates how effectively the cell surface profile delineates the 4 distinct cell populations within primary ccRCC samples. Red: immune cells; Green: endothelial cells; Blue: fibroblasts; Orange: cancer cells.</p

HT-FC allows phenotypic segregation and identification of cell samples from diverse lineages.

<p>Unsupervised hierarchical clustering of percent-positive marker expression values generated on 119 samples was performed. Colours indicate emergence of biologically related samples into clusters based on surface marker profiles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105602#pone.0105602.s002" target="_blank">Figure S2</a> for a magnified image of the dendrogram.</p