Characterization Of Antibody Fragments Research Articles

Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts

Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosylation is needed. Besides the correctly expressed Fab molecule, a multitude of host cell impurities and product related impurities are present in the crude sample. The identification and characterization of the product-related impurities, such as modified Fab-molecules or free light chain, are of utmost importance. The objective of this work was to design a purification strategy to isolate and characterize Fab and related impurities. A three-dimensional chromatography method was established, consisting of two affinity steps (Protein G and Protein L) and subsequent cation exchange chromatography, followed by mass spectrometry analysis of the purified samples. The procedure was automated by collecting the eluted target species in loops and directly loading the samples onto the high-resolution cation exchange chromatography column. As an example, four different Fab molecules are characterized. All four samples contained mainly the correct Fab, while only one showed extensive N-terminal pyroglutamate formation of the Fab. In another case, we found a light chain variant with uncleaved amino acids from the lead molecule, which was not used for the formation of whole Fab as only correct Fab was found in that sample. Impurities with lower molecular weights, which were bound on the Protein L column, were observed in all samples, and identified as fragments of the light chain. In conclusion, we have devised a platform for characterizing Fab and Fab-related impurities, which significantly facilitated strain selection and optimization of cultivation conditions.

High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires

The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies.

Abstract 2138: Targeting cancer-associated fibroblasts using antibodies recognizing fibroblast activation protein-alpha.

Abstract An expanding body of work suggests that the tumor stroma plays an integral role in the initiation and development of epithelial cancers. Within the stroma, cancer-associated fibroblasts (CAFs) are involved in several functions that form integral aspects of tumor progression, including immune suppression, metabolism, and invasion and metastasis. Despite the demonstrated importance of CAFs within the tumor microenvironment, targeting CAFs in a therapeutic context remains a challenge. The work presented here details the isolation and characterization of antibody fragments targeting multiple epitopes of fibroblast activation protein-alpha (FAP), a membrane-bound protease selectively overexpressed on CAFs in numerous epithelial cancers. The combination of a minimalist antibody fragment library and yeast surface display allowed for the isolation of more than 50 unique antibodies recognizing the human or murine forms of FAP, with close to 30 exhibiting cross-reactivity. To narrow the pool of cross-reactive antibodies to a biologically relevant subset, the fragments were converted to an antibody-like format that could be secreted from yeast. Without purification, the soluble proteins were first used to investigate the abilities of individual fragments to recognize FAP transiently expressed on the surface of HEK293 cells. Proteins for which binding to FAP could be confirmed were then used in combination with antibody fragments displayed on the surface of yeast in order to determine which sets of fragments bind distinct epitopes of FAP. Flow cytometry-based assays revealed that the isolated fragments recognize two or more distinct epitopes of the antigen. A handful of fragments is currently being subjected to affinity maturation and conversion to IgGs, and work to further investigate the in vitro and in vivo characteristics of the proteins is ongoing. These proteins may allow for the ablation of cancer-associated fibroblasts or interference with the proteolytic activity of FAP in the tumor microenvironment, providing biological insights into the roles of CAFs and therapeutic approaches to cancer treatment. Furthermore, the general antibody isolation and characterization approaches used in this work are accessible to many single-investigator laboratories and should be applicable to the targeting of numerous cancer antigens. Citation Format: James A. Van Deventer, Karl D. Wittrup. Targeting cancer-associated fibroblasts using antibodies recognizing fibroblast activation protein-alpha. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2013-2138