Christoph Roesli

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.

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.

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.

In vivo biotinylation of the vasculature in B-cell lymphoma identifies BST-2 as a target for antibody-based therapy

The discovery of accessible markers of lymphoma may facilitate the development of antibody-based therapeutic strategies. Here, we describe the results of a chemical proteomic study, based on the in vivo biotinylation of vascular proteins in lymphoma-bearing mice followed by mass spectrometric and bioinformatic analysis, to discover proteins expressed at the tissue-blood border of disseminated B-cell lymphoma. From a list of 58 proteins, which were more than 10-fold up-regulated in nodal and extranodal lymphoma lesions compared with their levels in the corresponding normal host organs, we validated BST-2 as a novel vascular marker of B-cell lymphoma, using immunochemical techniques and in vivo biodistribution studies. Furthermore, targeting BST-2 with 2 independent monoclonal antibodies delayed lymphoma growth in a syngeneic mouse model of the disease. The results of this study delineate a strategy for the treatment of systemic B-cell lymphoma in humans and suggest that anti-BST-2 antibodies may facilitate pharmacodelivery approaches that target the tumor-stroma interface.

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.

Comparative analysis of the membrane proteome of closely related metastatic and nonmetastatic tumor cells.

The identification of proteins that are preferentially expressed on the membrane of metastatic tumor cells is of fundamental importance in cancer research. Here, we report the systematic comparison of the membrane proteome of two closely related murine teratocarcinoma cell lines (F9B9 and F9DR), of which only one (F9DR) is capable of forming liver metastases in vivo. The proteomic methodology used in this study featured the surface protein biotinylation on tumor cells followed by protein purification on streptavidin resin and relative quantification of corresponding tryptic peptides by mass spectrometric procedures. The study allowed the identification of 998 proteins and the determination of their relative abundance. Proteins previously known to be associated with metastatic spread were found to be either up-regulated (e.g., synaptojanin-2) or down-regulated (e.g., Ceacam1) in F9DR cells. A dramatic increase in abundance at the cell membrane was observed for a broad variety of proteins (e.g., high-mobility group protein B1), which were mainly thought to reside in intracellular compartments, a finding that was confirmed using confocal laser scanning microscopy and immunochemical analysis of cell cultures. Furthermore, we showed by microautoradiographic analysis that certain target proteins can readily be reached by intravenously administered radiolabeled antibodies. Finally, we showed that the most promising antigens for antibody-based pharmacodelivery approaches are strongly and selectively expressed on the surface of tumor cells in three different syngeneic mouse models of liver metastases. Taken together, our results indicate that the expression of intracellular proteins on the membrane of metastatic cells is a feature much more common than previously expected.

Identification of the surface-accessible, lineage-specific vascular proteome by two-dimensional peptide mapping

The formation of blood vessels (angio genesis) and of lymphatic vessels (lymphangiogenesis) actively contributes to cancer progression and inflam mation. Thus, there has been a quest for identifying the molecular mechanisms that control lymphatic and blood vessel formation and function. Membrane and extracellular matrix proteins can serve as suitable tar gets for imaging and/or therapeutic targeting;however, conventional proteomic technologies often fail to iden tify them systematically due to insolubility in water and low abundance of membrane proteins. To circumvent this problem, we applied a gel‐free proteomics meth odology termed two‐dimensional peptide mapping (2D‐ PM) to cultured blood vascular (BECs) and lymphatic (LECs) endothelial cells. 2D‐PM comprises biotinylation of surface‐accessible proteins, their selective en richment, separation by HPLC, and analysis by mass spectrometry. We identified 184 proteins that were specifically or predominantly expressed by LECs and 185 proteins specifically expressed by BECs, whereas 377 additional proteins were equally detected in both cell types. For representative proteins, the differential, lineage‐specific expression was confirmed by Western analyses of cultured cells and by differential immuno fluorescence analyses of tissue samples. Our results identify the surface‐accessible, vascular lineage‐specific proteome, and they also reveal 2D‐PM as a powerful technology for the large‐scale screening of lineage‐ specific protein expression.— Roesli, C., Mumprecht, V., Neri, D., Detmar, M. Identification of the surface‐ accessible, lineage‐specific vascular proteome by two dimensional peptide mapping. FASEB J. 22, 1933–1944 (2008)

A Proteomic Approach for the Identification of Vascular Markers of Liver Metastasis

Vascular proteins expressed at liver metastasis sites could serve as prognostic markers or as targets for pharmacodelivery applications. We employed a proteomic approach to define such proteins in three syngeneic mouse models of liver metastasis. Vascular structures were biotinylated in vivo by a terminal perfusion technique, followed by mass spectrometric analysis of accessible biotinylated proteins. In this manner, we identified 12 proteins for which expression was selectively associated with liver metastasis, confirming this association by tissue immunofluorescence or in vivo localization with radiolabeled antibodies. In summary, our findings identify vascular proteins that may have prognostic or drug-targeting use in addressing liver metastases, a common issue in many advanced cancers.

Proteomic identification of vanin-1 as a marker of kidney damage in a rat model of type 1 diabetic nephropathy

At present, the urinary albumin excretion rate is the best noninvasive predictor for diabetic nephropathy (DN) but major limitations are associated with this marker. Here, we used in vivo perfusion technology to establish disease progression markers in an animal model of DN. Rats were perfused with a reactive ester derivative of biotin at various times after streptozotocin treatment. Following homogenization of kidney tissue and affinity purification of biotinylated proteins, a label-free mass spectrometry-based proteomic analysis of tryptic digests identified and relatively quantified 396 proteins. Of these proteins, 24 and 11 were found to be more than 10-fold up- or downregulated, respectively, compared with the same procedure in vehicle-treated rats. Changes in the expression of selected differentially regulated proteins were validated by immunofluorescence detection in kidney tissue from control and diabetic rats. Immunoblot analysis of pooled human urine found that concentrations of vanin-1, an ectoenzyme pantetheinase, distinguished diabetic patients with macroalbuminuria from those with normal albuminuria. Uromodulin was elevated in the urine pools of the diabetic patients, regardless of the degree of albuminuria, compared with healthy controls. Thus, in vivo biotinylation facilitates the detection of disease-specific changes in the abundance of potential biomarker proteins for disease monitoring and/or pharmacodelivery applications.

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trials.