Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity.

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.

Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

A new helper phage for improved monovalent display of Fab molecules.

Phage display technology is a powerful tool for the identification of novel antibodies for drug discovery. Phage display libraries have been constructed with massive diversity, but their use may be hindered by limited antibody display levels when rescued with the M13KO7 helper phage. Variants of M13KO7 have been constructed previously that increase the levels of display of rescued phage, but all produce phage that display multiple copies of the antibody fragment on their surface and have reduced titer and infectivity. In this study, we describe a new helper phage, XP5, which increased the display level of Fab molecules more than two-fold compared to phage rescued with M13KO7. XP5 uses a combination of ribosome binding site spacing alterations and rare codon clusters to reduce the expression of pIII from the helper phage. This reduction in pIII expression leads to an increase in the incorporation of pIII-Fab fusions during phage rescue. The rescued phage displayed a single copy of the Fab molecule, preventing any avidity effects during the selection process. This also suggests that the percentage of the population of phage displaying a Fab molecule is increased when rescued with XP5. Additionally, the phage titers and infectivity are comparable to libraries rescued with M13KO7. After two rounds of panning we observed a nearly 5-fold increase in the number of antigen binding Fab molecules compared to panning conducted with the same library rescued with M13KO7. The nature of the mutations in XP5 makes it a universal substitute for M13KO7 in pIII-based phage display, compatible with most phagemids and bacterial strains.

Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide.

Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like beta-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF beta-chain (HGF beta) and competitively inhibited binding to Met with an IC(50) of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC(50) of approximately 20 microM. The 2D (1)H-NMR structure of HB10 revealed a beta-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF beta mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF beta domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.

Pharmacoproteomics of a metalloproteinase hydroxamate inhibitor in breast cancer cells: dynamics of membrane type 1 matrix metalloproteinase-mediated membrane protein shedding.

Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90alpha, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.

The canonical methionine 392 of matrix metalloproteinase 2 (gelatinase A) is not required for catalytic efficiency or structural integrity: probing the role of the methionine-turn in the metzincin metalloprotease superfamily.

Matrix metalloproteinases (MMPs) are an important family of extracellular proteases that process a variety of biologically significant molecules. MMPs are members of the metzincin superfamily of >770 zinc endopeptidases, which includes astacins, serralysins, adamalysins, leishmanolysins, and snapalysins. Metzincins are characterized by an absolutely conserved methionine residue COOH-terminal to the third histidine in the consensus sequence HEXXHXXGXX(H/D), where the histidine residues chelate a catalytic zinc ion. The canonical methionine is part of a tight 1,4-beta-turn that loops the polypeptide chain beneath the catalytic zinc ion, forming a hydrophobic floor to the Zn(2+) ion binding site. The role of this methionine is uncertain, but its absolute conservation indicates an essential catalytic or structural function. To investigate this hypothesis, we replaced Met-392 that forms the Met-turn of human MMP-2 (gelatinase A) by site-directed mutagenesis. The catalytic competence of leucine and serine mutants was assessed. (M392L)MMP-2 and (M392S)MMP-2 cleaved the physiological substrates gelatin, native type I collagen, and the chemokine monocyte chemoattractant protein-3 with similar efficiency to wild-type MMP-2. These mutants also cleaved two quenched fluorescent peptide substrates with a k(cat)/K(m) comparable to wild-type MMP-2 and underwent 4-aminophenylmercuric acetate-induced autoactivation with similar kinetics. (M392L)MMP-2 and (M392S)MMP-2 were inhibited by tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -4 and by the zinc chelators 1,10-phenanthroline and a synthetic hydroxamate inhibitor, Batimastat, similar to the wild-type protein, indicating an unaltered active site topography. A tryptic susceptibility assay also suggested that (M392L)MMP-2 and (M392S)MMP-2 were correctly folded. These results challenge the dogma that this methionine residue and the Met-turn, which are absolutely conserved in all of the subfamilies of the metzincins, play an essential role in catalysis or active site structure.

Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.

Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates.

By proteolytic modification of low abundant signaling proteins and membrane receptors, proteases exert potent posttranslational control over cell behavior at the postsecretion level. Hence, substrate discovery is indispensable for understanding the biological role of proteases in vivo. Indeed, matrix metalloproteinases (MMPs), long associated with extracellular matrix degradation, are increasingly recognized as important processing enzymes of bioactive molecules. MS is now the primary proteomic technique for detecting, identifying, and quantitating proteins in cells or tissues. Here we used isotopecoded affinity tag labeling and multidimensional liquid chromatography inline with tandem MS to identify MDA-MB-231 breast carcinoma cell proteins shed from the cell surface or the pericellular matrix and extracellular proteins that were degraded or processed after transfection with human membrane type 1-MMP (MT1-MMP). Potential substrates were identified as those having altered protein levels compared with the E240A inactive MT1-MMP mutant or vector transfectants. New substrates were biochemically confirmed by matrix-assisted laser desorption ionization-time-of-flight MS and Edman sequencing of cleavage fragments after incubation with recombinant soluble MT1-MMP in vitro. We report many previously uncharacterized substrates of MT1-MMP, including the neutrophil chemokine IL-8, secretory leukocyte protease inhibitor, pro-tumor necrosis factor alpha, death receptor-6, and connective tissue growth factor, indicating that MT1-MMP is an important signaling protease in addition to its traditionally ascribed roles in pericellular matrix remodeling. Moreover, the high-throughput and quantitative nature of isotope-coded affinity tag labeling combined with tandem MS sequencing is a previously undescribed degradomic screen for protease substrate discovery that should be generally adaptable to other classes of protease for exploring proteolytic function in complex and dynamic biological contexts.

Collagen binding properties of the membrane type-1 matrix metalloproteinase (MT1-MMP) hemopexin C domain. The ectodomain of the 44-kDa autocatalytic product of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage.

Up-regulation of the collagenolytic membrane type-1 matrix metalloproteinase (MT1-MMP) leads to increased MMP2 (gelatinase A) activation and MT1-MMP autolysis. The autocatalytic degradation product is a cell surface 44-kDa fragment of MT1-MMP (Gly(285)-Val(582)) in which the ectodomain consists of only the linker, hemopexin C domain and the stalk segment found before the transmembrane sequence. In the collagenases, hemopexin C domain exosites bind native collagen, which is required for triple helicase activity during collagen cleavage. Here we investigated the collagen binding properties and the role of the hemopexin C domain of MT1-MMP and of the 44-kDa MT1-MMP ectodomain in collagenolysis. Recombinant proteins, MT1-LCD (Gly(285)-Cys(508)), consisting of the linker and the hemopexin C domain, and MT1-CD (Gly(315)-Cys(508)), which consists of the hemopexin C domain only, were found to bind native type I collagen but not gelatin. Functionally, MT1-LCD inhibited collagen-induced MMP2 activation in fibroblasts, suggesting that interactions between collagen and endogenous MT1-MMP directly stimulate the cellular activation of pro-MMP2. MT1-LCD, but not MT1-CD, also blocked the cleavage of native type I collagen by MT1-MMP in vitro, indicating an important role for the MT1-MMP linker region in triple helicase activity. Similarly, soluble MT1-LCD, but not MT1-CD or peptide analogs of the MT1-MMP linker, reduced the invasion of type I collagen matrices by MDA-MB-231 cells as did the expression of recombinant 44-kDa MT1-MMP on the cell surface. Together, these studies demonstrate that generation of the 44-kDa MT1-MMP autolysis product regulates collagenolytic activity and subsequent invasive potential, suggesting a novel feedback mechanism for the control of pericellular proteolysis.

Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors.

The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptase-gamma and matriptase-2. Matrix metalloproteinases (MMPs) showed a restricted expression pattern in both normal and cancerous breast tissues with most expressed at low levels. However, of the several MMPs expressed in significant quantities, the carcinoma samples showed only slightly elevated amounts other than for MMP-28 which was strongly elevated. To discover new protease substrates we developed a novel yeast two-hybrid approach we term 'inactive catalytic domain capture' (ICDC). Here, an inactive mutant protease catalytic domain lacking the propeptide was used as a yeast two hybrid bait to screen a human fibroblast cDNA library for interactor proteins as a substrate trap. Wnt-induced signaling protein-2 (WISP-2) was identified by ICDC and was biochemically confirmed as a new MMP substrate. In another approach we used isotope-coded affinity tag (ICAT) labeling with tandem mass spectrometry to quantitate the levels of secreted or shed extracellular proteins in MDA-MB-231 breast carcinoma cell cultures in the presence or absence of membrane type 1-MMP (MT1-MMP) overexpression. By this proteomic approach we identified and biochemically confirmed that IL-8, the serine protease inhibitor SLPI, the death receptor-6, pro-TNF-alpha and CTGF are novel substrates of MT1-MMP. The utility and quantitative nature of ICAT with MS/MS analysis as a new screen for protease substrate discovery based on detection of cleaved or shed substrate products should be readily adaptable to other classes of protease for assessing proteolytic function in a cellular context.