Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These ...approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566–73.. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein.
Bispecific antibodies (bsAbs) enable dual binding of different antigens with potential synergistic targeting effects and innovative therapeutic possibilities. The formation of bsAbs is, however, ...often dependent on complex engineering strategies with a high risk of antibody chain mispairing leading to contamination of the final product with incorrectly assembled antibody species. This study demonstrates formation of bsAbs in a generic and conceptually easy manner through fusion of single-domain antibodies (sdAbs) onto IgG scaffolds through flexible 10 amino acid linkers to form high-quality bsAbs with both binding functionalities intact and minimal product-related impurities. SdAbs are attractive fusion partners due to their small and monomeric nature combined with antigen-binding capabilities comparable to conventional human antibodies. By systematically comparing a comprehensive panel of symmetric αPD-L1×αHER2 antibodies, including reversely mirrored antigen specificities, we investigate how the molecular geometry affects production, stability, antigen binding and CD16a binding. SdAb fusion of the heavy chain was generally preferred over light chain fusion for promoting good expression and high biophysical stability as well as maintaining efficient binding to both antigens. We find that N-terminal sdAb fusion might sterically hinder antigen-binding to the Fv region of the IgG scaffold, whereas C-terminal fusion might disturb antigen-binding to the fused sdAb. Our work demonstrates a toolbox of complementary methods for in-depth analysis of key features, such as in-solution dual antigen binding, thermal stability, and aggregation propensity, to ensure high bsAb quality. These techniques can be executed at high-throughput and/or with very low material consumption and thus represent valuable tools for bsAb screening and development.
In a search for novel immunostimulating substances we detected that culture supernatants of the gram-positive phytopathogenic bacterium,
Rhodococcus fascians, were able to induce cytokine release ...(TNF
α) from mouse peritoneal macrophages. Monoclonal antibodies were generated against the active principle, and were employed for its isolation and partial characterization as a high molecular (MW>100 kDa) glycoprotein. In addition, methods practicable for its biotechnological preparation and several ELISA variants for its determination were developed.
Simple separation of DNA in antibody purification Christensen, Peter Astrup; Danielczyk, Antje; Stahn, Renate ...
Protein expression and purification,
10/2004, Letnik:
37, Številka:
2
Journal Article
Recenzirano
Producing monoclonal antibodies includes their efficient and simple purification. Growing hybridoma cells in media containing Prolifix, an alternative plant-based substitute for serum, provides ...supernatants containing large amounts of antibodies and defined low molecular weight additives. Antibodies can easily be separated from these compounds by fast ultrafiltration. However, DNA originating from lysed cells is present in substantial amounts and must be removed for most antibody applications. The present communication provides a fast, cheap, and efficient separation method by precipitating the DNA from a phosphate buffered solution with manganese chloride. Resulting antibodies have a high purity and an unchanged bioactivity. The method is especially valuable for antibodies which lose bioactivity by interactions with chromatographic matrices (as, for example, Sepharose) and can be used for various antibody isotypes.
The Thomsen-Friedenreich antigen (TF; CD176, Galβ1-3GalNAcα-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found ...in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of α-anomeric TF (TFα) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for α- and β-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TFα antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TFα- and TFβ-specific antibodies. The active antigen was identified as Galβ1-3GalNAc-, which was α-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TFα are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TFα structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.
Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been ...associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.
The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable ...loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.
A new monoclonal antibody to the Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope; Gal beta 1-3GalNAc-) has been developed that is specific for both anomeric forms of this disaccharide ...(TF alpha and TF beta, including related structures on glycolipids), and not assay restricted. We demonstrate that this avid antibody (A78-G/A7) is well suited for immunohistochemistry on paraffin-embedded and cryosectioned tissues, immunoblotting, ELISA techniques, and hemagglutination. Immunohistochemistry on paraffin sections does not require proteolytic or microwave pretreatment. The binding characteristics of this antibody are largely independent of variations in pH (6.0-8.2) and temperature (4-37 degrees C). Immunoblotting with KG-1 (human acute myelogenous leukemia) cells revealed a series of TF-active glycoproteins with a main band at about 155 kDa. Immunoprecipitation was performed using a new technique applicable to IgM-type antibodies.
