An Enhanced and Scalable Process for the Purification of SIV Gag-Specific MHC Tetramer

Grimm, Karen M.; Trigona, Wendy L.; Heidecker, Gwendolyn J.; Joyce, Joseph G.; Fu, Tong-Ming; Shiver, John W.; Keller, Paul M.; Cook, James C.

doi:10.1006/prep.2001.1507

Cited by 5 publications

(5 citation statements)

References 28 publications

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“…3D culture systems, xenofree medium, and TFF are suitable for GMP-grade biologics. [35][36][37]39 Here we show that 3D xenofree cultures of umbilical cord mesenchymal stem cells and TFF of the conditioned supernatants produced substantially higher yields of exosomes than standard 2D culture and UC. Whereas exosomes isolated from both 2D and 3D cultures via both UC and TFF have similar size distributions and protein contents, 3D-TFF-exosomes are more efficient at siRNA delivery to neurons and at inducing mRNA silencing.…”

Section: Discussionmentioning

confidence: 59%

“…We then compared the gold standard method of exosome isolation, differential UC ( Figure 2C), with TFF, a scalable concentration and buffer exchange strategy used during large-scale manufacturing of biologics 35,36 and viruses 37 ( Figure 2D). TFF isolates exosomes according to their size, whereas differential UC relies on both vesicle size and sedimentation properties.…”

Section: D Culture and Tangential Flow Filtration Enhances Exosome Ymentioning

confidence: 99%

“…We combined the strengths of two production strategies to develop a robust and scalable strategy compatible with good manufacturing practices (GMPs) for exosome production from mesenchymal stem cells: (1) microcarrier-based three-dimensional (3D) cell culture is commonly used to grow adherent cells in bioreactors; 34 and (2) tangential flow filtration (TFF) is a method to concentrate proteins or viruses from large amounts of cell culture media. [35][36][37] The physicochemical characteristics of exosomes derived from two-dimensional (2D) or 3D cultures, as well as produced by differential UC or TFF, were compared. We show that both 3D culture and TFF improve the yield of exosomes to a cumulative extent of 140-fold.…”

Section: Introductionmentioning

confidence: 99%

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Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity

Haraszti

Miller

Stoppato³

et al. 2018

Molecular Therapy

352

298

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Exosomes can deliver therapeutic RNAs to neurons. The composition and the safety profile of exosomes depend on the type of the exosome-producing cell. Mesenchymal stem cells are considered to be an attractive cell type for therapeutic exosome production. However, scalable methods to isolate and manufacture exosomes from mesenchymal stem cells are lacking, a limitation to the clinical translation of exosome technology. We evaluate mesenchymal stem cells from different sources and find that umbilical cord-derived mesenchymal stem cells produce the highest exosome yield. To optimize exosome production, we cultivate umbilical cord-derived mesenchymal stem cells in scalable microcarrier-based threedimensional (3D) cultures. In combination with the conventional differential ultracentrifugation, 3D culture yields 20-fold more exosomes (3D-UC-exosomes) than two-dimensional cultures (2D-UC-exosomes). Tangential flow filtration (TFF) in combination with 3D mesenchymal stem cell cultures further improves the yield of exosomes (3D-TFF-exosomes) 7-fold over 3D-UC-exosomes. 3D-TFF-exosomes are seven times more potent in small interfering RNA (siRNA) transfer to neurons compared with 2D-UC-exosomes. Microcarrierbased 3D culture and TFF allow scalable production of biologically active exosomes from mesenchymal stem cells. These findings lift a major roadblock for the clinical utility of mesenchymal stem cell exosomes.

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Section: Discussionmentioning

confidence: 59%

Section: D Culture and Tangential Flow Filtration Enhances Exosome Ymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity

Haraszti

Miller

Stoppato³

et al. 2018

Molecular Therapy

352

298

View full text Add to dashboard Cite

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“…For instance, tetramers with higher ligand avidity have been successfully produced from biotinylated class I MHC monomers (44,45). Likewise, we have produced tetrameric BHAP by streptavidin binding of biotinylated monomers.…”

Section: Discussionmentioning

confidence: 99%

A Bifunctional Targeted Peptide that Blocks HER-2 Tyrosine Kinase and Disables Mitochondrial Function in HER-2-Positive Carcinoma Cells

et al. 2005

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The HER-2 oncoprotein is commonly overexpressed in a variety of human malignancies and has become an attractive antitumor target. A number of strategies to inhibit the HER-2 receptor tyrosine kinase are currently the focus of intensive preclinical and clinical research. In the present study, we have engineered a bifunctional peptide, BHAP, which consists of two modular domains: a HER-2-targeting/neutralizing domain and a mitochondriotoxic, proapoptotic domain. The chimeric peptide is biologically active and capable of selectively triggering apoptosis of HER-2-overexpressing cancer cells in culture, even those previously described as Herceptin resistant. Furthermore, BHAP slows down growth of HER-2-overexpressing human mammary xenografts established in SCID mice. This approach can be extended to the development of tailored targeted chimeric peptides against a number of overexpressed cellular receptors implicated in the development and progression of cancer. (Cancer Res 2005; 65(15): 6891-900)

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“…HLA-A2 tetramers were synthesized as described elsewhere [34], using the following epitopes: from the EBV lytic cycle protein, BMLF1, GLCTLVAML; CMV matrix phosphoprotein, pp65, NLVPMVATV; FLU matrix, M1, GILGFVFTL; HIV gag, SLYNTVATL; and HIV pol, IL-KEPVHGV. HLA-A3 tetramers containing the HIV gag epitope RLRPGGKKKHIV and HLA-B8 tetramers containing the HIV nef epitope FLKEKGGL were sythesized as described elsewhere [35].…”

Section: Donorsmentioning

confidence: 99%

Antigen Burden Is a Major Determinant of Human Immunodeficiency Virus–Specific CD8⁺T Cell Maturation State: Potential Implications for Therapeutic Immunization

et al. 2003

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The majority of untreated human immunodeficiency virus (HIV) type 1-infected individuals ultimately develop uncontrolled viremia and progressive disease. Cytotoxic T lymphocytes (CTLs) are known to play an important role in controlling HIV-1 replication, which has led to an increasing interest in augmenting conventional antiretroviral therapy with therapeutic vaccination. The successful development of a therapeutic vaccine will rely on the ability to correlate an aspect of the immune response with clinical outcome. In this study, the CD8(+) T cell maturation status of antigen-specific cells in models of well and poorly controlled virus infections were compared, to show that a memory phenotype predominates when antigen loads are absent or low. In HIV-1 infection, the emergence of memory CD8(+) T cells was found to occur only in individuals with highly suppressed viral replication for an extended duration. Such assessments of the immune response may provide a refined measure of virus control.

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An Enhanced and Scalable Process for the Purification of SIV Gag-Specific MHC Tetramer

Cited by 5 publications

References 28 publications

Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity

Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity

A Bifunctional Targeted Peptide that Blocks HER-2 Tyrosine Kinase and Disables Mitochondrial Function in HER-2-Positive Carcinoma Cells

Antigen Burden Is a Major Determinant of Human Immunodeficiency Virus–Specific CD8⁺T Cell Maturation State: Potential Implications for Therapeutic Immunization

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An Enhanced and Scalable Process for the Purification of SIV Gag-Specific MHC Tetramer

Cited by 5 publications

References 28 publications

Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity

Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity

A Bifunctional Targeted Peptide that Blocks HER-2 Tyrosine Kinase and Disables Mitochondrial Function in HER-2-Positive Carcinoma Cells

Antigen Burden Is a Major Determinant of Human Immunodeficiency Virus–Specific CD8+T Cell Maturation State: Potential Implications for Therapeutic Immunization

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Antigen Burden Is a Major Determinant of Human Immunodeficiency Virus–Specific CD8⁺T Cell Maturation State: Potential Implications for Therapeutic Immunization