Grafting of protein L-binding activity onto recombinant antibody fragments

Muzard, Julien; Adi-Bessalem, Sonia; Juste, Matthieu; Laraba-Djebari, Fatima; Aubrey, Nicolas; Billiald, Philippe

doi:10.1016/j.ab.2009.02.035

Cited by 20 publications

(24 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3b and Table II). In agreement with the results obtained by Muzard et al (2009) in a comparable study on FR-grafting using scFv fragments this clearly demonstrates that the FR1 grafting strategy is successful but may induce subtle changes in antigen-binding kinetics and affinity.…”

Section: Discussionsupporting

confidence: 90%

See 1 more Smart Citation

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

Boes

Spiegel

Delbrück

et al. 2011

Biotech & Bioengineering

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 90%

“…To overcome these limitations we adapted a strategy first published by Muzard et al (2009). To generate an scFv with a high affinity to protein L, Muzard and co-workers transferred the FR1 region of a protein L-binding variable light chain.…”

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

View full text Add to dashboard Cite

show abstract

“…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. These modifications had little effect on either antigen binding activities or antibody yields.…”

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

View full text Add to dashboard Cite

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.

show abstract

“…First, the scFv4C1op amino acid sequence was engineered to produce the following changes. The VH (EVHLVE) and VL (DVLMTQSPLSLPVS-LGDQASIS) regions of scFv4C1 were replaced by the N-terminal regions of Db9C2 (QVQLQQ and DVQMTQSPASLSVS-GQTVTIT, respectively), plus the E17Q substitution in the VL framework 1 allowing the promotion of PpL binding activity (34). Second, from the previous amino acid sequence, a synthetic gene was performed according to GeneArt.…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, the replacement of some parts of the VL scFv4C1 N-terminal region by those of scFv9C2 made it possible for the first time to purify the Db4C1op fragment in a pure and homogeneous manner thanks to the protein L affinity. In addition, the flag peptide was suppressed to reduce the immunogenicity of the fragment, which is critical for therapeutic applications (34). The beginning of the VH scFv4C1 domain was also modified because a different expression vector (pSW1) was used in place of the original one (pHEN).…”

Section: Discussionmentioning

confidence: 99%

Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

Tommaso

Juste

Martin‐Eauclaire

et al. 2012

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Currently, the only known treatment for scorpion envenomation is serotherapy with a heterologous polyclonal antibody displaying low specificity. Results: The injection of a diabody mixture allowed mice to survive in a test that mimics severe envenomation with the crude venom. Conclusion:The diabody mixture displayed better protective power than any other known remedy. Significance: This could herald the next generation of antivenoms.

show abstract

Grafting of protein L-binding activity onto recombinant antibody fragments

Cited by 20 publications

References 24 publications

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

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

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

Contact Info

Product

Resources

About