A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo.

The alternatively spliced extra-domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra-domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody-based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (K(D) = 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody-based targeted anti-cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over-expression is detectable not only in solid tumors, but also in hematological malignancies.

Identification of new accessible tumor antigens in human colon cancer by ex vivo protein biotinylation and comparative mass spectrometry analysis.

One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody-based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry-based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty-seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody-based imaging and therapeutic biomolecular strategies.

Quantitative recovery of biotinylated proteins from streptavidin-based affinity chromatography resins.

The strong interaction between streptavidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for the facile derivatization of a variety of molecules (in particular, proteins) with biotin have been introduced, in order to allow their efficient recovery, immobilization and detection with streptavidin-based reagents. However, when desired, the release of biotinylated proteins from the streptavidin-based reagents remains a major problem, due to the extraordinary stability of this complex. This chapter presents a protocol developed in our laboratory for the quantitative elution of biotinylated proteins from streptavidin sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method is shown by the recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin.

Ligand-based vascular targeting of disease.

This review illustrates the basic principles of ligand-based vascular targeting and presents some of the most advanced results obtained in this field, not only in terms of biopharmaceuticals, which are currently being investigated in clinical and preclinical studies, but also in terms of enabling technologies that facilitate target and ligand discovery. Whereas most of the vascular targeting research activities have so far concentrated on tumoral angiogenesis, the development of non-oncological applications has recently gained momentum and is likely to become an important area of modern pharmaceutical research.

The extra-domain A of fibronectin is a vascular marker of solid tumors and metastases.

One of the most promising new avenues for the development of more selective and efficacious cancer therapies relies on the antibody-mediated targeted delivery of bioactive agents (e.g., cytokines) to the tumor environment. The identification of quantitative differences in the expression of accessible vascular proteins in metastatic lesions and host organs facilitate the development of antibody-based strategies, which should be highly efficient and selective, considering the fact that an over-exuberant neovasculature is a characteristic feature of aggressive cancers, and that tumor blood vessels are readily accessible for i.v. administered therapeutic agents. Metastasis is the main cause of death in cancer. The availability of metastasis-specific antigens accessible from the bloodstream will allow a selective delivery of therapeutic agents to metastatic lesions using antibodies as vehicles. Using a combination of vascular biotinylation of 129Sv mice bearing F9 liver metastases and mass spectrometry, we have identified 435 accessible proteins in metastasis and host organ specimens, of which 117 were exclusively detected in metastases. In particular, we found that the alternatively spliced extra-domain A (EDA) of fibronectin is strongly expressed in the neovasculature of liver metastases, while being undetectable in most normal organs. A human antibody to EDA was used to show EDA expression in the neovasculature of metastases and primary tumors of human cancer patients and to target mouse liver metastases and subcutaneous tumors in vivo. Human antibody fragments specific to the EDA domain of fibronectin promise to serve as general vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to metastatic sites.

In vivo protein biotinylation and sample preparation for the proteomic identification of organ- and disease-specific antigens accessible from the vasculature.

Targeted delivery of bioactive molecules to diseased organs or tissues by means of binding molecules specific to markers of diseases represents a promising area of pharmaceutical intervention. The availability of markers of pathology, ideally accessible from the vasculature, is crucial for such strategies. To this aim, here we present a protocol based on terminal perfusion of mice with a reactive ester derivate of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ or tissue extracts are (i) purified on streptavidin resin in the presence of strong detergents, (ii) digested on the resin and (iii) subjected to proteomic analysis. This technology is applicable to comparative proteomic investigations of differentially expressed, accessible proteins in numerous animal models having different physiological and pathological processes.

A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer.

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature.

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography-tandem mass spectrometry for identification. In the present study, in vivo biotinylation procedure led to the identification of hundreds of proteins in different mouse organs, including some showing a restricted pattern of expression in certain body tissues. Furthermore, biotinylation of mice with F9 subcutaneous tumors or orthotopic kidney tumors revealed both quantitative and qualitative differences in the recovery of biotinylated proteins, as compared to normal tissues. This technology is applicable to proteomic investigations of the differential expression of accessible proteins in physiological and pathological processes in animal models, and to human surgical specimens using ex vivo perfusion procedures.

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping.

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.

Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells: A transcriptomic and proteomic study.

Hypoxia is a characteristic feature of many human pathologies, including cancer. The sustained proliferation rate of tumor cells leads to alterations of the tumor microenvironment, that progressively becomes more acidic, nutrient-deprived, and hypoxic. The reduced partial pressure of oxygen triggers the onset of an adaptive response, aimed at increasing the local oxygen concentration by several complementary actions. Although directly exposed to the blood stream, endothelial cells lining the vascular lumen in tumors also can be exposed to hypoxia and therefore can contribute to the onset of the adaptive response that leads to tumor angiogenesis. Aiming at getting a detailed insight into the oxygen-dependent regulation of the transcriptional program of vascular endothelial cells and at identifying new relevant markers that may be used as targets for therapeutic intervention in tumor angiogenesis, we have performed a broad-range transcriptomic analysis, using the Affymetrix HG-U133A Gene Chips, of mRNA expression levels in human umbilical cord vein endothelial cells (HUVEC), exposed in vitro to hypoxia for different time periods. The transcriptomic analysis was complemented by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA levels and alternative splicing for some selected extracellular matrix protein genes, and by a proteomic analysis, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and tandem mass spectrometry for protein separation and identification, of hypoxic and normoxic HUVEC whole-cell lysates and subcellular fractions. Our analysis confirmed previous findings on genes whose expression is regulated by oxygen concentration but also identified new genes (e.g., CXCR4, claudin 3, CD24, tetranectin, Del-1, procollagen lysyl hydroxylase 1 and 2) which are transcriptionally upregulated in hypoxic conditions.

Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution.

The interaction between streptavidin and biotin is one of the most widely used tools in chemistry and biology. However, the release of biotinylated proteins from streptavidin resins remains a major problem, due to the extraordinary stability of this complex. We present a new protocol for the quantitative elution of biotinylated proteins from streptavidin Sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method was demonstrated by the quantitative recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin.

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.