An optimized procedure for efficient phage display of antibody fragments with a low folding efficiency

We recently developed an efficient bacterial expression system for phagemid-coded antigen-binding fragments of antibody (Fabs) without the use of a helper bacteriophage. This system is characterized by an unusually long cultivation at a low temperature and gentle induction of Fab expression without the addition of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG). This method allows for a high yield production of Fabs fused with phage gene III coat protein, even when the protein is defective in its folding ability. With this cultivation procedure, we aimed here at improving the production and selection efficiency of filamentous bacteriophages displaying functional Fabs on their surface (Fab-phages) that have high affinity but low folding ability. The Fab components of the Fab-phages used were clonally related but differed in their affinity and folding ability. The production of the functional Fab-phages was quantitatively evaluated under various culture conditions. With conventional phage particle preparation, the production of functional Fab-phages was significantly biased according to the folding ability of the displayed Fabs, and affinity-based biopanning was therefore unsuccessful. In contrast, with the present procedure employing cultivation at 25 °C for 16 h without IPTG induction, functional Fab-phages were produced without any such dependence on folding ability. With this optimized library, affinity-based biopanning was successful. Especially noteworthy, bead-based biopanning accurately discriminated between high affinity Fab-phages and Fab-phages with low or middling affinity. In obtaining Fab-phages with high affinity but low folding ability, these optimized procedures for both cultivation and selection were essential.     

IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb

IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 cDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8+ T and NK1.1(high) cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44(high)CD8+ population, and the increased population was maintained for >300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.     

A peptide isolated by phage display binds to ICAM-1 and inhibits binding to LFA-1

A mixed phage library containing random peptides from four to eight residues in length flanked by cysteine residues was screened using a recombinant soluble, form of human ICAM-1, which included residues 1–453, (ICAM-1_1–453). Phage bound to immobilized ICAM-1_1–453 were eluted by three methods: (1) soluble ICAM-1_1–453, (2) neutralizing murine monoclonal antibody, (anti-ICAM-1, M174F5B7), (3) acidic conditions. After three rounds of binding and elution, a single, unique ICAM-1 binding phage bearing the peptide EWCEYLGGYLRYCA was isolated; the identical phage was selected with each method of elution. Attempts to isolate phage from non-constrained (i.e., not containing cysteines) libraries did not yield a phage that bound to ICAM-1. Phage displaying EWCEYLGGYLRCYA bound to immobilized ICAM-1_1–453 and to ICAM-1_1–185, a recombinant ICAM-1, which contains only the two amino-terminal immunoglobulin domains residing within residues 1–185. This is the region of the ICAM-1 that is bound by LFA-1. The phage did not bind to proteins other than ICAM-1. The phage bound to two ICAM-1 mutants, which contained amino acid substitutions that dramatically decreased or eliminated the binding to LFA-1. Studies were also performed with the corresponding synthetic peptide. The linear form of the synthetic EWCEYLGGYLRCYA peptide was found to inhibit LFA-1 binding to immobilized ICAM-1_1–453 in a protein-protein binding assay. By contrast, the disulfide, cyclized, form of the peptide was inactive. The EWCEYL portion of the sequence is homologous to the EWPEYL sequence found within rhinovirus coat protein 14, a nonintegrin protein that binds to ICAM-1. Taken together, the results suggests that the EWCEYLGGYLRCYA sequence is capable to binding to immobilized ICAM-1. Phage display appears to represent a new approach for the identification of peptides that interfere with ICAM-1 binding to β2 integrins. © 1996 Wiley-Liss, Inc.     

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.     

Analysis of antibody A6 binding to the extracellular interferon gamma receptor alpha-chain by alanine-scanning mutagenesis and random mutagenesis with phage display

The monoclonal antibody A6 binds a conformational epitope comprising mainly the CC' surface loop on the N-terminal fibronectin type-III domain of the extracellular interferon gamma receptor (IFNgammaR). The crystal structure of an A6 Fab-IFNgammaR complex revealed an interface rich in the aromatic side chains of Trp, Tyr, and His residues. These aromatic side chains appear to interact with both polar and hydrophobic groups at the interface, a property which, in general, may be advantageous for ligand binding. To analyze these interactions in more detail, the affinities of 19 A6 alanine-scanning mutants for the IFNgammaR have been measured, using engineered A6 single chain variable region fragments, and a surface plasmon resonance biosensor. Energetically important side chains (DeltaG(mutant) - DeltaG(wt) > 2.4 kcal/mol), that form distinct hot spots in the binding interface, have been identified on both proteins. These include V(L)W92 in A6, whose benzenoid ring appears well situated for a pi-cation (or pi-amine) interaction with the side chain of receptor residue K47 and simultaneously for T-stacking onto the indole ring of W82 in the receptor. At another site, energetically important residues V(H)W52 and V(H)W53, as well as V(H)D54 and V(H)D56, surround the aliphatic side chain of the hot receptor residue K52. Taken together, the results show that side chains distributed across the interface, including many aromatic ones, make key energetic contributions to binding. In addition, the receptor CC' loop has been subjected to random mutagenesis, and receptor mutants with high affinity for A6 have been selected by phage display. Residues previously identified as important for receptor binding to A6 were conserved in the clones isolated. Some mutants, however, showed a much improved affinity for A6, due to changes at Glu55, a residue that appeared to be energetically unimportant for binding the antibody by alanine-scanning mutagenesis. An E55P receptor mutant bound A6 with a 600-fold increase in affinity (K(D) approximately 20 pM), which is one of the largest improvements in affinity from a single point mutation reported so far at any protein-protein interface.     

Identification of residues of the IFNAR1 chain of the type I human interferon receptor critical for ligand binding and biological activity

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a closed form, in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.     

Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease

Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.     

Interferon gamma is recognised by importin alpha/beta: enhanced nuclear localising and transactivation activities of an interferon gamma mimetic

Interferon (IFN) gamma's ability to localise in the nucleus and function in gene activation has been known for some time, although the role of the conventional nuclear transporting importin molecules is unclear. Here, we demonstrate for the first time the direct recognition of IFNgamma and an IFNgamma mimetic peptide by IMPalpha and the IMPalpha/beta heterodimer, where the IFNgamma mimetic shows higher affinity. Significantly, this correlates well both with in vivo ability to target green fluorescent protein to the nucleus in transfected cells as determined by quantitative confocal laser scanning microscopy, as well as GAS promoter activity of a luciferase reporter. This has important implications for IFNgamma's anti-viral action, and the potential use of the IFNgamma mimetic in antiviral therapies.     

Role of the N-terminus of epidermal growth factor in ErbB-2/ErbB-3 binding studied by phage display

Epidermal growth factor (EGF) binds with high affinity to the EGF receptor, also known as ErbB-1, but upon replacement of the N-terminal linear region by neuregulin (NRG) 1 or transforming growth factor (TGF) alpha sequences it gains in addition high affinity for ErbB-2/ErbB-3 heterodimers. However, these chimeras weakly bind to ErbB-3 alone. To further dissect the ligand binding selectivity of the ErbB network, we have applied the phage display technique to examine the role of the linear N-terminal region in EGF for interaction with ErbB-2/ErbB-3 heterodimers. A library of EGF variants was constructed in which residues 2, 3, and 4 were randomly mutated, followed by selection for binding to intact MDA-MB-453 cells that overexpress ErbB-2 and ErbB-3 but lack ErbB-1. Analysis of the selected phage EGF variants revealed clones with high binding affinity to ErbB-2/ErbB-3 while maintaining high affinity to ErbB-1. In these variants, Trp (or alternatively His) was almost exclusively present at position 2, while specific combinations of hydrophobic, basic, and small residues were found at positions 3 and 4. The mitogenic activity of the phage EGF variants corresponded with their relative binding affinity. Two of the selected EGF variants, EGF/WVS and EGF/WRS, were further characterized as recombinant proteins. In contrast to previously characterized chimeras of EGF with NRG-1 or TGF-alpha, these variants did not only show high binding affinity for ErbB-2/ErbB-3 heterodimers but also for ErbB-3 alone. These data show that the linear N-terminal region of EGF-like growth factors is directly involved in binding to ErbB-3.     

An aspartate conserved among G-protein receptors confers allosteric regulation of alpha 2-adrenergic receptors by sodium

The residue involved in sodium regulation of G-protein-coupled receptors has been identified by site-directed mutagenesis of the alpha 2-adrenergic receptor gene. Mutation of Asp-79 to Asn-79 entirely eliminates allosteric regulation of ligand binding by monovalent cations without perturbing the selectivity of adrenergic binding or allosteric modulation of that binding by amiloride analogs. The high degree of conservation of this aspartate residue in all G-protein-coupled receptors, without even a conservative change to glutamate, underscores the probable importance of this allosteric regulation.     

Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library

We have identified a peptide substrate for molecular chaperone Hsp40 Ydj1 by utilizing the combination of phage display library screening and isothemol titration calirimetry (ITC). The initial peptide substrate screening for Hsp40 Ydj1 has been carried out by utilizing a 7-mer phage display library. The peptide sequences from the bio-panning were synthesized and object to the direct affinity measurement for Hsp40 Ydj1 by isothemol titration calirimetry studies. The peptide which has the measurable affinity with Ydj1 shows enriched hydrophobic residues in the middle of the substrate fragment. The peptide substrate specificity for molecular chaperone Hsp40 has been analyzed. Published: October 1, 2004.     

Shp2E76K mutant confers cytokine-independent survival of TF-1 myeloid cells by up-regulating Bcl-XL

Shp2 has been known to mediate growth factor-stimulated cell proliferation, but its role in cell survival is less clear. Gain-of-function Shp2 mutants such as Shp2E76K are associated with myeloid leukemias. We found that Shp2E76K could transform cytokine-dependent human TF-1 myeloid cells into cytokine independence and further characterized the Shp2E76K-induced cell survival mechanism in this study. Expression of Shp2E76K suppressed the cytokine withdrawal-induced intrinsic/mitochondrial apoptosis pathway, which is controlled by the Bcl-2 family proteins. Analysis of Bcl-2 family proteins showed that Bcl-XL and Mcl-1 were up-regulated in Shp2E76K-transformed TF-1 (TF-1/Shp2E76K) cells. Knockdown of Bcl-XL but not Mcl-1 with short hairpin RNAs prevented Shp2E76K-induced cytokine-independent survival. Roscovitine, which down-regulated Mcl-1, also did not prevent cytokine-independent survival of TF-1/Shp2E76K cells, whereas the Bcl-XL inhibitor HA14-1 did. Ras and mitogen-activated protein kinases Erk1 and Erk2 (Erk1/2) were constitutively activated in TF-1/Shp2E76K cells, whereas little active Akt was detected under cytokine-free conditions. Shp2E76K-induced Bcl-XL expression was suppressed by Mek inhibitors and by a dominant-negative Mek1 mutant but not by the phosphoinositide 3-phosphate inhibitor LY294002 and the Akt inhibitor API-2. Inhibition of Erk1/2 blocked cytokine-independent survival of TF-1/Shp2E76K cells, whereas inhibition of Akt had a minimal effect on cytokine-independent survival of TF-1/Shp2E76K cells. These results show that Shp2E76K can evoke constitutive Erk1/2 activation in TF-1 cells. Furthermore, Shp2E76K induces cytokine-independent survival of TF-1 cells by a novel mechanism involving up-regulation of Bcl-XL through the Erk1/2 pathway.     

Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1

Integrins alpha(1)beta(1) and alpha(2)beta(1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha(1)beta(1) (alpha(1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999) J. Biol. Chem. 274, 24906-24913). Here, we compared the binding of the recombinant A-domain of alpha(2)beta(1) (alpha(2)A) to type I collagen with that of alpha(1)A using surface plasmon resonance and showed that alpha(2)A exhibited only one detectable class of binding sites in type I collagen, with a K(D) of approximately 10 microm at approximately 3 binding sites per collagen molecule. We further demonstrated that alpha(1)A and alpha(2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha(1)A- and alpha(2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha(1)A and alpha(2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha(1)A and alpha(2)A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of alpha(1)A and alpha(2)A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha(1)A and alpha(2)A.     

A novel binding site in collagen type III for integrins alpha1beta1 and alpha2beta1

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both alpha(1)I and alpha(2)I with high affinity and effectively inhibit the binding of alpha(1)I and alpha(2)I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.     

Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.