James W. Stave

Antibody and Antigen Contact Residues Define Epitope and Paratope Size and Structure

A total of 111 Ag–Ab x-ray crystal structures of large protein Ag epitopes and paratopes were analyzed to inform the process of eliciting or selecting functional and therapeutic Abs. These analyses illustrate that Ab contact residues (CR) are distributed in three prominent CR regions (CRR) on L and H chains that overlap but do not coincide with Ab CDR. The number of Ag and Ab CRs per structure are overlapping and centered around 18 and 19, respectively. The CR span (CRS), a novel measure introduced in this article, is defined as the minimum contiguous amino acid sequence containing all CRs of an Ag or Ab and represents the size of a complete structural epitope or paratope, inclusive of CR and the minimum set of supporting residues required for proper conformation. The most frequent size of epitope CRS is 50–79 aa, which is similar in size to L (60–69) and H chain (70–79) CRS. The size distribution of epitope CRS analyzed in this study ranges from ∼20 to 400 aa, similar to the distribution of independent protein domain sizes reported in the literature. Together, the number of CRs and the size of the CRS demonstrate that, on average, complete structural epitopes and paratopes are equal in size to each other and similar in size to intact protein domains. Thus, independent protein domains inclusive of biologically relevant sites represent the fundamental structural unit bound by, and useful for eliciting or selecting, functional and therapeutic Abs.

Impact of Immunization Technology and Assay Application on Antibody Performance – A Systematic Comparative Evaluation

Antibodies are quintessential affinity reagents for the investigation and determination of a proteins expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost (“DNA immunization”; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.

Bacteriophage-Based Enrichment Coupled to Immunochromatographic Strip–Based Detection for the Determination of Salmonella in Meat and Poultry

Immunochemical-based methods for the detection of Salmonella in food can be complicated by the presence of closely related, immunocrossreactive non-Salmonella species in the sample that may cause false-positive results. To circumvent this problem, specific bacteriophages against immunocrossreactive, non-Salmonella bacteria were used in the sample enrichment step to suppress their growth and improve the performance of an immunochromatographic strip-based detection method for Salmonella. Cross-reactive bacteria were isolated from various food sources and were characterized with a panel of Salmonella somatic O antigen-specific monoclonal antibodies. These cross-reactive bacteria were primarily Citrobacter spp. and Escherichia coli with serology shared with Salmonella serogroups B, D, and F. These bacteria were used as hosts for the isolation of specific lytic bacteriophages. When formulated with the primary enrichment, the bacteriophage cocktail significantly reduced false positives with a broadly reactive immunochromatographic test strip. This was demonstrated in both artificially and naturally contaminated meat. False positives in naturally contaminated beef samples were reduced from 32 of 115 samples tested to zero. In raw meat and poultry with a relatively high bioburden (>10(5) CFU/g), the use of the bacteriophage-based enrichment procedure gave improved recovery of Salmonella compared with the conventional culture-based reference method. This was observed when coupled to either test strip-based or selective agar-based detection. The use of specific bacteriophages for the control of immunocrossreactive and competitive microflora during the food sample enrichment step provides a new approach for enhancing the performance of both immunological- and cultural-based detection methods.

Snorkel: an epitope tagging system for measuring the surface expression of membrane proteins.

Tags are widely used to monitor a protein’s expression level, interactions, protein trafficking, and localization. Membrane proteins are often tagged in their extracellular domains to allow discrimination between protein in the plasma membrane from that in internal pools. Multipass membrane proteins offer special challenges for inserting a tag since the extracellular regions are often composed of small loops and thus inserting an epitope tag risks perturbing the structure, function, or location of the membrane protein. We have developed a novel tagging system called snorkel where a transmembrane domain followed by a tag is appended to the cytoplasmic C-terminus of the membrane protein. In this way the tag is displayed extracellularly, but structurally separate from the membrane protein. We have tested the snorkel tag system on a diverse panel of membrane proteins including GPCRs and ion channels and demonstrated that it reliably allows for monitoring of the surface expression.

Abstract 4325: Monoclonal antibodies to transmembrane proteins.

Transmembrane proteins, including multipass transmembrane proteins like GPCRs and ion channels, are important targets for therapeutic monoclonal antibody (mab) discovery. Therapeutic antibodies to this class of proteins are generally targeted to extracellular domains displayed on the surfaces of living cells. Challenges associated with developing antibodies to this class of targets are small numbers of extracellular amino acids, membrane-dependent protein conformation, difficulty in expression at high levels, high amino acid sequence homology of human and mouse proteins, and post-translational modifications. DNA immunization strategies with full-length constructs and high throughput flow cytometry screening of mab binding to transfected and control cells was used to generate and identify large numbers of mabs to CXCR4 and ADORA2A (GPCRs) and CD20 (a tetraspan membrane protein). Panels of mabs were generated for all 3 targets with low numbers of hybridoma fusions. For each target the mab gene sequences were shown to be unique and contain levels of somatic hypermutation comparable to existing benchmark therapeutic antibodies. Epitope mapping with mutant proteins identified diverse patterns of reactivity including known and novel specificities. Functional assays including apoptosis and receptor modulation (calcium flux and cAMP modulation) further demonstrated that the technical approach generated diverse panels of antibodies that exhibit functional activity as good or better than existing benchmark therapeutic antibodies Citation Format: Michael C. Brown, Ross Chambers, Dale V. Onisk, Tony R. Joaquim, Lewis J. Stafford, Klaus Lindpaintner, Daniel Keter, James W. Stave. Monoclonal antibodies to transmembrane proteins. [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 4325. doi:10.1158/1538-7445.AM2013-4325