Carl Borrebaeck

Recombining germline-derived CDR sequences for creating diverse single- framework antibody libraries

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 × 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Antibodies in diagnostics – from immunoassays to protein chips

Antibodies are used extensively as diagnostic tools in a wide array of different analyses. Monoclonal and recombinant antibodies provide a never ending source of molecules and can produce endless possibilities for novel genetic constructs. Antibodies are still very much in vogue and are now also being used in microarray analysis of the proteome using protein chips. Here, recent opportunities presented by antibodies as diagnostic tools are reviewed.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Gene Family Clustering Identifies Functionally Associated Subsets of Human In Vivo Blood and Tonsillar Dendritic Cells

Human dendritic cells (DCs) are a distinct but heterogeneous lineage of APCs operating as the link between innate and adaptive immune responses, with the function to either maintain tolerance or trigger immunity. The DC lineage consists of several subpopulations with unique phenotypes; however, their functional characteristics and transcriptional similarities remain largely unknown. To further characterize the phenotypes and transcriptomes of the subsets, we purified myeloid CD16+, blood DC Ag 1+ (BDCA1+), and BDCA3+ DC populations, as well as plasmacytoid CD123+ DCs, from tonsillar tissue and peripheral blood. Transcriptional profiling and hierarchical clustering visualized that BDCA1+ DCs clustered with BDCA3+ DCs, whereas CD16+ DCs and CD123+ DCs clustered as distinct populations in blood. Differential expression levels of chemokines, ILs, and pattern recognition receptors were demonstrated, which emphasize innate DC subset specialization. Even though highly BDCA1+ and BDCA3+ DC-specific gene expression was identified in blood, the BDCA1+ DCs and BDCA3+ DCs from tonsils displayed similar transcriptional activity, most likely due to the pathogenic or inflammatory maturational signals present in tonsillar tissues. Of note, plasmacytoid DCs displayed less plasticity in their transcriptional activity compared with myeloid DCs. The data demonstrated a functionally distinct association of each of the seven subsets based on their signatures, involving regulatory genes in adaptive and innate immunity.

Exploiting sequence space : Shuffling in vivo formed complementarity determining regions into a master framework

A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.

Design of high-density antibody microarrays for disease proteomics: Key technological issues.

Antibody-based microarray is a novel proteomic technology setting a new standard for molecular profiling of non-fractionated complex proteomes. The first generation of antibody microarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein atlases, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody microarray set-ups have, however, proven to be challenging. In this mini-review we discuss key technological issues that must be addressed in a cross-disciplinary manner before true global proteome analysis can be performed using antibody microarrays.

Multiplexed Lipid Dip-Pen Nanolithography on Subcellular Scales for the Templating of Functional Proteins and Cell Culture

Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.

Detection of pancreatic cancer using antibody microarray-based serum protein profiling

The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv antibody microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of <12 months. Taken together, the data suggest that antibody microarray analysis of complex proteomes will be a useful tool to define disease associated protein signatures.

High-throughput proteomics using antibody microarrays: an update.

Antibody-based microarrays are a rapidly emerging technology that has advanced from the first proof-of-concept studies to demanding serum protein profiling applications during recent years, displaying great promise within disease proteomics. Miniaturized micro- and nanoarrays can be fabricated with an almost infinite number of antibodies carrying the desired specificities. While consuming only minute amounts of reagents, multiplexed and ultrasensitive assays can be performed targeting high- as well as low-abundance analytes in complex nonfractionated proteomes. The microarray images generated can then be converted into protein expression profiles or protein atlases, revealing a detailed composition of the sample. The technology will provide unique opportunities for fields such as disease diagnostics, biomarker discovery, patient stratification, predicting disease recurrence and drug target discovery. This review describes an update of high-throughput proteomics, using antibody-based microarrays, focusing on key technological advances and novel applications that have emerged over the last 3 years.

Serum proteome profiling of metastatic breast cancer using recombinant antibody microarrays

The driving force behind oncoproteomics is to identify biomarker signatures associated with a particular malignancy. Here, we have for the first time used large-scale recombinant scFv antibody microarrays in an attempt to classify metastatic breast cancer versus healthy controls, based on differential protein expression profiling of whole serum samples. Using this multiplexed and miniaturised assay set-up providing pM range sensitivities, breast cancer could be classified with a specificity and sensitivity of 85% based on 129 serum analytes. However, by adopting a condensed 11 analyte biomarker signature, composed of nine non-redundant serum proteins, we were able to distinguish cancer versus healthy serum proteomes with a 95% sensitivity and specificity, respectively. When a subgroup of patients, not receiving anti-inflammatory drugs, was analysed, a novel eight analyte biomarker signature with a further improved predictive power was indicated. In a longer perspective, antibody microarray analysis could provide a tool for the development of improved diagnostics and intensified biomarker discovery for breast cancer patients.