Purification of antibodies using protein L-binding framework structures in the light chain variable domain

Nilson, Bo; Lögdberg, Lennart; Kastern, William; Björck, Lars; Åkerström, Bo

doi:10.1016/0022-1759(93)90273-a

Cited by 99 publications

(52 citation statements)

References 29 publications

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“…Then the cells were washed once in RPMI and twice in PBS. The cells were lysed and cleared by adding BC3, an anti-MUC1 mAb (IgM subclass), and Protein L Gel (Pierce) to immunoprecipitate BC3 antigen complex (80). The precleared lysates (200 μl) were immunoprecipitated using 25 μg P9 for 1 hour at 4°C, followed by adding 25 μl Protein L Gel for 2 hours.…”

Section: Methodsmentioning

confidence: 99%

“…TRAMP-C1 prostate cancer cells were treated with or without 25 μg/ml mAb P9 for 3 and 6 hours, respectively, and then were harvested, washed twice in cold PBS, lysed in lysis buffer, and cleared by adding IgM and Protein L Gel (Pierce) (80). The precleared lysates (200 μl) were immunoprecipitated using P9 or anti-Hsp90 Abs (BD Biosciences) for 1 hour at 4°C and were followed by adding 25 μl Protein L Gel for P9 or…”

Section: Methodsmentioning

confidence: 99%

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PIM-1–specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis

Hu¹,

Li²,

Vandervalk³

et al. 2009

J. Clin. Invest.

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Provirus integration site for Moloney murine leukemia virus (PIM1) is a proto-oncogene that encodes a serine/ threonine kinase with multiple cellular functions. Overexpression of PIM-1 plays a critical role in progression of prostatic and hematopoietic malignancies. Here we describe the generation of a mAb specific for GST-PIM-1, which reacted strongly with most human and mouse cancer tissues and cell lines of prostate, breast, and colon origin but only weakly (if at all) with normal tissues. The mAb binds to PIM-1 in the cytosol and nucleus as well as to PIM-1 on the surface of human and murine cancer cells. Treatment of human and mouse prostate cancer cell lines with the PIM-1-specific mAb resulted in disruption of PIM-1/Hsp90 complexes, decreased PIM-1 and Hsp90 levels, reduced Akt phosphorylation at Ser473, reduced phosphorylation of Bad at Ser112 and Ser136, and increased cleavage of caspase-9, an indicator of activation of the mitochondrial cell death pathway. The mAb induced cancer cell apoptosis and synergistically enhanced antitumor activity when used in combination with cisplatin and epirubicin. In tumor models, the PIM-1-specific mAb substantially inhibited growth of the human prostate cancer cell line DU145 in SCID mice and the mouse prostate cancer cell TRAMP-C1 in C57BL/6 mice. These findings are important because they provide what we believe to be the first in vivo evidence that treatment of prostate cancer may be possible by targeting PIM-1 using an Ab-based therapy.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

PIM-1–specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis

Hu¹,

Li²,

Vandervalk³

et al. 2009

J. Clin. Invest.

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show abstract

“…Genetic engineering may also allow Ab-fragments to acquire enhanced ability to bind to Protein L [111][112][113]. Boes et al [113] and Muzard et al [114] demonstrated that Protein L binding activity could be transferred from high-affinity Protein L binding antibodies to antibodies or scFv fragments that normally did not react with the ligand.…”

Section: Protein L and Its Use In Antibody Fragment Bioprocessingmentioning

confidence: 99%

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Rodrigo¹,

Gruvegard²,

Alstine

2015

Antibodies

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Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria-suggesting its consideration as a key unit operation in antibody fragment processing.

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“…Protein L, an Ig-binding protein from Peptostreptococcus magnus, interacts with framework region 1 (FR1) in the variable region of certain kappa light chain (LC (k) ) subtypes and thus binds to representatives of all antibody classes (IgG, IgM, IgA, IgE, and IgD) as well as their Fab and scFv derivatives (Nilson et al, 1992). However, it does not bind to lambda light chains (LC (l) ), nor to human LC (k) subtype II, and in murine antibodies it binds only to LC (k) subtype I (Nilson et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco

Boes

Spiegel

Delbrück

et al. 2011

Biotech & Bioengineering

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Complex multimeric proteins such as dimeric and secretory immunoglobulin A (IgA) can be difficult to produce in heterologous systems, although this has been achieved using several platforms including plants. As well as topical mucosal applications, dimeric IgA (dIgA), and secretory IgA (sIgA) can be used in tumor and anti-viral therapy, where their more potent cell-killing properties may increase their efficacy compared to current drugs based on IgG. However, the development of therapeutic IgA formats is hampered by the need to co-express four different polypeptides, and the inability to purify such molecules using conventional protein A or protein G affinity chromatography. The light chain (LC)-specific affinity ligand protein L is a potential alternative, but it only recognizes certain kappa light chain (LC(κ)) subtypes. To overcome these limitations, we have adapted a framework-grafting approach to introduce LCs that bind protein L into any IgA. As a model, we used the chimeric anti-human chorionic gonadotropin (hCG) antibody cPIPP, since this contains a murine LC((κ)) subtype that does not bind protein L. Grafting was achieved by replacing selected framework region 1 (FR1) residues in the cPIPP LC(κ) variable domain with corresponding residues from LC(κ) subtypes that can bind protein L. The grafted antibody variants were successfully purified by protein L affinity chromatography. These modifications affected neither their antigen-binding properties nor the yields achieved by transient expression in tobacco plants. Our results therefore show that LC FR1 grafting can be used as generic strategy for the purification of IgA molecules.

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Purification of antibodies using protein L-binding framework structures in the light chain variable domain

Cited by 99 publications

References 29 publications

PIM-1–specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis

PIM-1–specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco

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