Efficient immunoaffinity chromatography of lymphocytes directly from whole blood

T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.

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“…1A). The described Streptag:Strep-Tactin interaction has already led to several research and clinical applications 30,[36][37][38][39][40] .…”

Section: Molecular Structure Composition and Function Of Expamers Nmentioning

confidence: 99%

Expamers: a new technology to control T cell activation

Poltorak

Graef

Tschulik

et al. 2020

Sci Rep

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“…Another method we used is the fragment antigen-binding (Fab) (antibody fragment) Traceless Affinity Cell Selection (TACS ® ) based on immunoaffinity chromatography. Antigen-specific strep-tagged Fab-fragments are reversibly bound to an agarose matrix via Strep-Tactin ® [ 3 ]. The heterogeneous cell suspension is loaded onto the Fab-labeled agarose matrix, where target cells are captured by clusters of antigen-specific Fab fragments with high avidity, while non-target cells pass through the matrix.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

Weiß

Gerdes²,

Berthold³

et al. 2021

Cells

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T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are “label-free”, while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells.

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“…These days, a wide range of cell isolation techniques are available, with density gradient centrifugation, cell filtering, and immunoaffinity methods as most prominent. Although density gradients are frequently used for the separation of plasma, platelets, peripheral blood mononuclear cells (PBMCs) or erythrocytes from other blood constituents [1], immunoaffinity techniques can be used in both liquid and tissue biopsies and enable enrichment of a wide variety of cell types and organelles [2,3]. This immune-based enrichment of specific cell types is based on antibodies, and their isolation from human in vivo specimens is thus limited to cell populations that have distinct surface markers [4].…”

Section: Introductionmentioning

confidence: 99%

FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations

Maes

Cools

Willems

et al. 2020

IJMS

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Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.

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Efficient immunoaffinity chromatography of lymphocytes directly from whole blood

Cited by 13 publications

References 31 publications

Expamers: a new technology to control T cell activation

Expamers: a new technology to control T cell activation

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations

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