ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.

Approach for Half-Life Extension of Small Antibody Fragments That Does Not Affect Tissue Uptake.

The utility of antigen-binding antibody fragments is often limited by their short half-lives. Half-life extension of such fragments is usually accomplished by attachment or binding to high-molecular-weight carriers that reduce the renal elimination rate. However, the higher hydrodynamic radius results in greater confinement in the vascular compartment and, thus, lower tissue distribution. We have developed a chemically controlled drug delivery system in which the drug is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; upon subcutaneous injection, the t1/2,ß of the released drug acquires the t1/2 of linker cleavage. In the present work, we compared the pharmacokinetics of an anti-TNFα scFv, the same scFv attached to 40 kDa PEG by a stable linker, and the scFv attached to hydrogel microspheres by a self-cleaving linker. We also developed a general approach for the selective attachment of ß-eliminative linkers to the N-termini of proteins. In rats, the scFv had a t1/2,ß of 4 h and a high volume of distribution at steady state (Vd,SS), suggesting extensive tissue distribution. The PEG-scFv conjugate had an increased t1/2,ß of about 2 days but showed a reduced Vd,SS that was similar to the plasma volume. In contrast, the tissue-penetrable scFv released from the hydrogel system had a t1/2,ß of about 2 weeks. Thus, the cleavable microsphere-scFv conjugate releases its protein cargo with a prolonged half-life comparable to that of most full-length mAbs and in a form that has the high tissue distribution characteristic of smaller mAb fragments. Other antigen-binding antibody fragments should be amenable to the half-life extension approach described here.

A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development.

A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies. BIOLOGICAL SIGNIFICANCE: The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML.

A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experiments.

The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS-ß-Ala-(L-Asp)(3)-biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo-NHS-LC-biotin, NHS-ß-Ala-(L-Asp)(3)-biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature.

Chemical proteomic and bioinformatic strategies for the identification and quantification of vascular antigens in cancer.

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels ("vascular targeting") is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation.

Characterization of a novel interaction between vasodilator-stimulated phosphoprotein and Abelson interactor 1 in human platelets: a concerted computational and experimental approach.

OBJECTIVE: The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. METHODS AND RESULTS: Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de/PlateletWeb.php), we performed a motif search of all sequences and identified potential target sites of class I EVH1 domains in human platelet proteins. Performing affinity purification with VASP-EVH1 domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 domain-specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. CONCLUSIONS: The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, as they offer the potential to analyze signal transduction beyond the scope of established pathways.