Anti-idiotypic protein domains selected from protein A-based affibody libraries

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.     

Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A.

Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K(d)) in the range between 0.5 and 3 microm. One variant (Z(IgA1)) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2)) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.     

Electrophilic Affibodies Forming Covalent Bonds to Protein Targets

Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target. The proximity between cysteine, lysine, or histidine on the target protein drove covalent bond formation to the electrophile on the affibody. Covalent bonds did not form to a non-interacting point mutant of the target, and there was minimal cross-reactivity with serum, cell lysate, or when imaging at the cell surface. Electrophilic affibodies showed more stable protein imaging at the surface of mammalian cells, and the sensitivity of protein detection in an immunoassay improved by two orders of magnitude. Thus electrophilic affibodies combined good specificity with improved detection of protein targets.     

Phage-based molecular directed evolution yields multiple tandem human IgA affibodies with intramolecular binding avidity

Affibodies are a group of affinity proteins that are based on a 58-amino-acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. A single human IgA affibody with high IgA affinity has been generated by directed evolution. It remains interesting whether tandem IgA affibody proteins could increase binding capacity. Here, we report the generation of multiple tandem IgA affibodies by directed evolution using a combinatorial phage library displaying the IgA affibody A1 and/or A2 linked with three random amino acids. These affibodies exhibited markedly increased IgA binding capacity, as shown by enzyme linked immunosorbent assay, immunoblotting and surface plasmon resonance assays. We further showed that these tandem IgA affibodies displayed preferential binding to intact IgA molecules compared to individual IgA chain, suggesting intramolecular binding avidity. Our data demonstrates that artificial multiple tandem human IgA affibodies with relevant biological binding avidity were successfully yielded by phage-based molecular evolution. These results have broad implications for the design and development of binding proteins that target important biological molecules.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.     

Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies

CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily. It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GST-hCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NMR and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.     

Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

Background aimsKey obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells.MethodsWe delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3⁺Vα24⁺Vβ11⁺ iNKT cells.ResultsGene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4⁺ and CD8⁺ responder T cells.DiscussionThese data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.     

The NF-kappa B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes

We explored the role of the NF-kappa B pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-kappaBalpha (inhibitor of kappa B alpha factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-kappa B site around -840 decreased, while ectopic expression of I-kappa B kinase-beta (IKK beta) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKK beta, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-kappa B factors that bind to the NF-kappa B binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-kappa B activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-alpha. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKK beta and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.     

Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

The human CD81 (hCD81), the most recently proposed receptor of hepatitis C virus (HCV), can especifically bind to HCV envelope glycoprotein2 (E2). In this study, hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library (PVⅢ9aaCys). Eighteen of the 75 clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay (ELISA) and competitive inhibition test. Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA. Sequence comparison of the motif showed no amino acid homology with the native HCV E2. The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells, and induce HCV E2 specific immune response in vivo. These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules. Our findings cast new light on developing HCV receptor antagonists.     

Identification and expression of human CD81 gene on murine NIH/3T3 cell membrane

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). In this study, eukaryotic expression vector pCDM8-hCD81 containing hCD81 cDNA and pSV2neo helper plasmid was used to cotransfect with lipofectamine into murine fibroblast cell line NIH/3T3 to establish an hCD81-expressing cell line. Resistant cell clones were obtained 20 days after the selection with neomycin (600 micro/ml) and then cultured as monoclones. The expression of the transfected hCD81 gene in the cells was verified by RT-PCR and flow cytometry analyses. One of the selected cell clones showed obvious expression of hCD81 and was named NIH/3T3-hCD81. Competitive inhibition tests indicated that the binding of monoclonal anti-hCD81 (JS-81) to NIH/3T3-hCD81 cells was inhibited by recombinant HCV E2 protein, suggesting that the expressed hCD81 molecules on NIH/3T3-hCD81 cells maintain natural conformation of binding to HCV E2. The transfected NIH/3T3-hCD81 cells should be of great potential value in studies on HCV attachment and onset of infection.     

人CD46启动子真核表达载体的构建

为了构建人CD46（hCD46）启动子指导目的基因表达的真核表达载体,提取HeLa细胞基因组DNA,用PCR扩增出hCD46基因的启动子区域,序列分析结果表明其与GenBank中hCD46基因5’端某片段的同源性为99.9％。用此启动子替换pcDNA3EGFP中的CMV启动子,并在hCD46启动子和EGFP基因之间插入兔β-球蛋白基因第二内含子（RGI）,得到的重组表达载体转染CHO和SP2/0两种鼠源细胞,流式细胞术检测表明CHO细胞EGFP的表达量高于SP2/0细胞,表达特性与人体CD46相似;RGI可以增强EGFP的表达量,但不改变其表达的组织特异性,提示克隆的hCD46启动子可以用于研制模拟人体CD46基因表达特性的转基因小鼠。     

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.     

Lipoteichoic acid and muramic acid modulate the expression of CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells

The aim of this study is to evaluate the effect of lipoteichoic acid (LTA) and muramic acid (MA) on costimulatory molecules CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells. The interactions between these molecules strongly influence the immune response through the regulation of cytokine release which, on its own, is able to regulate the immunological response by a feedback mechanism. Our results show that LTA and MA regulate expression of CD86 on macrophages while the expression of CD80 remains unmodified. LTA and MA increase the expression of CD86 on THP-1 cells, a macrophage cell line. MA increased Jurkat T cells CD152 expression.     

Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells

CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-gamma production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.     

Mechanism for down-regulation of CD28 by Nef

SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.     

The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28

We investigated the role of protein kinase C theta (PKCtheta) in the activation of the NF-kappaB cascade in primary human CD4(+) lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca(2+)-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4(+) T cells. The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells. Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.     

Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site

Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRPalpha) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRPalpha binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRPalpha as a probe show that neither human CD47 nor SIRPalpha requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRPalpha-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRPalpha. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRPalpha to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.