Multivalent Antibody-Recruiting Macromolecules: Linking Increased Binding Affinity with Enhanced Innate Immune Killing.

Antibody-recruiting molecules (ARMs) are a novel class of immunotherapeutics. They are capable of introducing antibodies onto disease-relevant targets such as cancer cells, bacterial cells or viruses. This can induce antibody-mediated immune responses such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent phagocytosis (ADCP), which can kill the pathogen. In contrast to the classic ARMs, multivalent ARMs could offer the advantage of increasing the efficiency of antibody recruitment and subsequent innate immune killing. Such compounds consist of multiple target-binding termini (TBT) and/or antibody-binding termini (ABT). Those multivalent interactions are able to convert low binding affinities into increased binding avidities. This minireview summarizes the current status of multivalent ARMs and gives insight into possible benefits, hurdles still to be overcome and future perspectives.

Tricellulin is regulated via interleukin-13-receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis.

In the two inflammatory bowel diseases, ulcerative colitis (UC) and Crohn's disease (CD), altered expression of tight junction (TJ) proteins leads to an impaired epithelial barrier including increased uptake of luminal antigens supporting the inflammation. Here, we focused on regulation of tricellulin (Tric), a protein of the tricellular TJ essential for the barrier against macromolecules, and hypothesized a role in paracellular antigen uptake. We report that Tric is downregulated in UC, but not in CD, and that its reduction increases the passage of macromolecules. Using a novel visualization method, passage sites were identified at TJ regions usually sealed by Tric. We show that interleukin-13 (IL-13), beyond its known effect on claudin-2, downregulates Tric expression. These two effects of IL-13 are regulated by different signaling pathways: The IL-13 receptor α1 upregulates claudin-2, whereas IL-13 receptor α2 downregulates Tric. We suggest to target the α2 receptor in future developments of therapeutical IL-13-based biologicals.

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks ­ each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

Synthesis and immunological effects of heroin vaccines.

Three haptens have been synthesized with linkers for attachment to carrier macromolecules at either the piperidino-nitrogen or via an introduced 3-amino group. Two of the haptens, with a 2-oxopropyl functionality at either C6, or at both the C3 and C6 positions on the 4,5-epoxymorphinan framework, as well as the third hapten (DiAmHap) with diamido moieties at both the C3 and C6 positions, should be much more stable in solution, or in vivo in a vaccine, than a hapten with an ester in one of those positions, as found in many heroin-based haptens. A "classical" opioid synthetic scheme enabled the formation of a 3-amino-4,5-epoxymorphinan which could not be obtained using palladium chemistry. Our vaccines are aimed at the reduction of the abuse of heroin and, as well, at the reduction of the effects of its predominant metabolites, 6-acetylmorphine and morphine. One of the haptens, DiAmHap, has given interesting results in a heroin vaccine and is clearly more suited for the purpose than the other two haptens.

Peptide-conjugation induced conformational changes in human IgG1 observed by optimized negative-staining and individual-particle electron tomography.

Peptides show much promise as potent and selective drug candidates. Fusing peptides to a scaffold monoclonal antibody produces a conjugated antibody which has the advantages of peptide activity yet also has the pharmacokinetics determined by the scaffold antibody. However, the conjugated antibody often has poor binding affinity to antigens that may be related to unknown structural changes. The study of the conformational change is difficult by conventional techniques because structural fluctuation under equilibrium results in multiple structures co-existing. Here, we employed our two recently developed electron microscopy (EM) techniques: optimized negative-staining (OpNS) EM and individual-particle electron tomography (IPET). Two-dimensional (2D) image analyses and three-dimensional (3D) maps have shown that the domains of antibodies present an elongated peptide-conjugated conformational change, suggesting that our EM techniques may be novel tools to monitor the structural conformation changes in heterogeneous and dynamic macromolecules, such as drug delivery vehicles after pharmacological synthesis and development.

Human α-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets.

Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human α-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.

Recognition of membrane-bound fusion-peptide/MPER complexes by the HIV-1 neutralizing 2F5 antibody: implications for anti-2F5 immunogenicity.

The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen.

Antibody targeted siRNA delivery.

It is very clear that RNA interference (RNAi) is a potent and versatile tool for gene silencing. One of the hurdles to making siRNA/miRNA a human therapeutic includes effective in vivo delivery and being able to deliver drugs to target cells only. The commercial success of in vivo applications of RNAi hinges on the development of new delivery methods. Our strategy involves the use of antibody-based delivery agents to target and deliver siRNA into specific cell types. We have developed antibody-based agents for directed delivery into cultured cells and animal disease models. Using antibodies against various cell surface receptors, modified siRNAs are attached to antibody complexes using RNA carrier proteins. The complex can then be intravenously administered to in vivo models and taken up by specific cells via receptor-mediated endocytosis. The labile structure of the linking agents enables release of siRNA molecules post internalization. Using this targeting strategy, we have developed a method that allows any commercially available or recombinant antibody to be conjugated to siRNA for delivery purposes.

Supramolecular complexing of membane Siglec CD22 mediated by a polyvalent heterobifunctional ligand that templates on IgM.

We report the synthesis and in vitro evaluation of a multivalent homing device, a polymer which contains preordered pendant groups with dual specificity, a trisaccharide moiety, which is specific for the siglec CD22, and an antibody specific hapten, nitrophenol. The device efficiently attracts antihapten IgM to the surface of human lymphoma B cells as well as to CD22-conjugated magnetic beads by mediating the formation of a ternary complex on the surface of the target.

LL-37 complexation with glycosaminoglycans in cystic fibrosis lungs inhibits antimicrobial activity, which can be restored by hypertonic saline.

There is an abundance of antimicrobial peptides in cystic fibrosis (CF) lungs. Despite this, individuals with CF are susceptible to microbial colonization and infection. In this study, we investigated the antimicrobial response within the CF lung, focusing on the human cathelicidin LL-37. We demonstrate the presence of the LL-37 precursor, human cathelicidin precursor protein designated 18-kDa cationic antimicrobial protein, in the CF lung along with evidence that it is processed to active LL-37 by proteinase-3. We demonstrate that despite supranormal levels of LL-37, the lung fluid from CF patients exhibits no demonstrable antimicrobial activity. Furthermore Pseudomonas killing by physiological concentrations of exogenous LL-37 is inhibited by CF bronchoalveolar lavage (BAL) fluid due to proteolytic degradation of LL-37 by neutrophil elastase and cathepsin D. The endogenous LL-37 in CF BAL fluid is protected from this proteolysis by interactions with glycosaminoglycans, but while this protects LL-37 from proteolysis it results in inactivation of LL-37 antimicrobial activity. By digesting glycosaminoglycans in CF BAL fluid, endogenous LL-37 is liberated and the antimicrobial properties of CF BAL fluid restored. High sodium concentrations also liberate LL-37 in CF BAL fluid in vitro. This is also seen in vivo in CF sputum where LL-37 is complexed to glycosaminoglycans but is liberated following nebulized hypertonic saline resulting in increased antimicrobial effect. These data suggest glycosaminoglycan-LL-37 complexes to be potential therapeutic targets. Factors that disrupt glycosaminoglycan-LL-37 aggregates promote the antimicrobial effects of LL-37 with the caveat that concomitant administration of antiproteases may be needed to protect the now liberated LL-37 from proteolytic cleavage.

Immunomodulatory effects of beta-glucans on porcine alveolar macrophages and bone marrow haematopoietic cell-derived dendritic cells.

The immunopharmacological activities of beta-glucans with a backbone of beta-1,3/beta-1,6-linkages associated with anti-tumor, anti-viral, bacterial and fungal infections have been well documented. Dectin-1, a specific pattern recognition receptor for beta-1,3/beta-1,6-glucans, is expressed mainly on phagocytes, especially macrophages and dendritic cells (DCs). In this study, the encoding nucleotide for the carbohydrate-recognition domain (CRD) of porcine dectin-1 was sequenced for the first time, and the immunomodulatory functions of a synthetic particulate beta-glucan (p-beta-glucan) were examined. Results showed that p-beta-glucan significantly enhanced cell activity and phagocytosis in porcine alveolar macrophages (AMs), immature DCs (imDCs) and mature DCs (mDCs), in a similar way to zymosan. Zymosan enhanced dectin-1/TLR2/TLR4 expression and TNF-alpha/IL-10 production in all of three types of cell, whereas p-beta-glucan increased dectin-1/TLR4 and TNF-alpha/IL-12 production in AMs but inhibited IL-10 in mDCs. These results indicate that the complex collaborating interactions between dectin-1 and TLRs in the recognition of beta-1,3/beta-1,6-glucans with different structural features may direct different cellular responses.

HLA-specific B cells: II. Application to transplantation.

BACKGROUND: Differences in the antibody response to allogeneic transplantation exist between groups defined by race or gender. These differences may reflect differences in immune competency and/or exposure to alloantigens. We have investigated the frequencies and phenotypes of HLA-specific B cells to address those possibilities. METHODS: HLA-specific B cells were identified by staining with HLA tetramers (tet) as described previously and the distribution of CD27 and CD38 among those cells were measured in groups defined by various parameters. Possible correlation between frequencies of HLA-specific B cells and production of HLA-specific antibody after transplantation was also investigated. RESULTS: We found no correlation between the frequencies of CD27+tet+ (33%-44% vs. 34%-36%) or CD38+tet+ (57%-65% vs. 59%-66%) B cells and a previous mismatch for the HLA antigen of the tetramer. However, there was an increase in CD38+tet+ B cells among patients making antibody to the tetramer antigen (67%-72% vs. 53%-56%). Blacks had lower frequencies of CD27+ B cells than did whites (11.8% vs. 28.9%, P=0.003), but had greater increases of these cells among tet+ cells than did whites. There was a higher frequency of tet+ B cells among patients who developed "new" antibody to the HLA antigen (3.9%-8.6%) of the tetramer after transplantation than among those who did not (1.1%-3.7%). CONCLUSIONS: The phenotype of HLA-specific B cells reflects current or historic sensitization to HLA and may reflect inherent differences between groups defined by race and/or gender. The frequencies of HLA-specific B cells may predict patients at risk for production of donor-specific antibody after transplantation.

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2.

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A(426-434)) or a self-peptide (Tyr(369-377)). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A(426-434) pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr(369-377) pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag(77-85)) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%+/-3.72% vs 7.01%+/-0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%+/-0.33% vs 0.05%+/-0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%+/-2.58% vs 6.87%+/-1.01%, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%+/-0.3% vs 0.06%+/-0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.

Coupling of C3bi to IgG inhibits the tyrosine phosphorylation signaling cascade downstream Syk and reduces cytokine induction in monocytes.

The effect of coupling C3bi to immunoglobulin G (IgG) immune complexes (IC) on their ability to produce protein tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and the Akt/protein kinase B (PKB) routes was assessed in human monocytes. Cross-linking Fc receptors for IgG activated the protein tyrosine kinase Syk, phospholipases Cgamma1 and Cgamma2, the MAPK cascade, and the Akt/PKB route. Linkage of C3bi to the gamma-chain of IgG produced a decrease of the protein bands displaying tyrosine phosphorylation, whereas the MAPK cascades and the Akt/PKB route remained almost unaffected. Zymosan particles, which because of their beta-glucan content mimic the effect of fungi, produced a limited increase of tyrosine-phosphorylated protein bands, whereas treatment of zymosan under conditions adequate for C3bi coating increased its ability to induce protein tyrosine phosphorylation. Noteworthy, this was also observed under conditions where other components of serum might be bound by zymosan particles, for instance, serum IgG, thereby suggesting their potential involvement in Syk activation. The induction of cytokines showed a changing pattern consistent with the changes observed in the signaling pathways. IC induced monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 (CCL2), interleukin (IL)-1beta, and eotaxin-2/CCL24, which were not observed with C3bi-coated IC. Zymosan induced the expression of tumor necrosis factor alpha (TNF-alpha), TNF-beta, IL-10, IL-6, and MCP-2/CCL8, whereas the cytokine signature of C3bi-coated zymosan also included interferon-inducible protein 10/CXC chemokine ligand 10, platelet-derived growth factor-BB, and I-309/CCL1. Taken together, these findings indicate that C3bi targets the phagocytic cargo, and engagement or diversion of the Syk route determines the phagocyte response.

Immunogenicity of heme complexes of peptides designed to mimic the heme environment of myoglobin and hemoglobin.

In the preceding paper (Protein J. 25, pages 37-49, 2005), we reported the preparation and oxygen-binding properties of peptides that form stable complexes with heme mimic. The design of the peptides was based on the natural environment of the heme group in myoglobin (Mb) and in the alpha- and beta-subunits of human adult hemoglobin (Hb). In the present work, the heme-peptides were each administered into mice, either as emulsions in adjuvant (both for injections and boosters) or intravenously as solutions in phosphate-buffered saline. Antibody (Ab) responses, monitored up to 14 weeks after the first administration, showed that when the heme-peptides were injected with adjuvant they stimulated Ab responses against the immunizing peptide, which in most cases bound to the correlate protein (Mb or Hb). However these heme-peptides were non-immunogenic when administered in PBS intravenously. It is concluded that heme-peptides:(a) would not trigger an adverse immune response if used for transfusion purposes.

Immunological detection of DNA-protein complexes induced by chromate.

A select group of non-histone proteins becomes complexed to DNA after Chinese hamster ovary (CHO) cells are treated with potassium chromate. The most abundant complexed protein has a mol. wt of approximately 45 kd and is thought to be actin. An antiserum to the chromate-induced DNA-protein complexes (DPCs) was prepared to facilitate the study of these complexes. Rather than detecting the predominant silver-stained proteins of DPCs described in an earlier study, this antiserum reacts primarily with an acidic 95-kd protein (p95) that does not silver stain. The antiserum can be used routinely to assay for the induction of p95-DNA complexes produced by chromate and perhaps by other carcinogens. Immunofluorescent staining of CHO cells and immunoblotting of cell fractions show the reactive antigens are within the cell nucleus. Blotting experiments with the antiserum indicate the chromate-induced p95-DNA complex dissociates in the presence of 2-mercaptoethanol, suggesting similarity to the DPCs formed by carcinogenic platinum compounds. A reduced species of chromium (probably Cr3+) may form DPCs by binding to the nitrogen, oxygen or sulfur atoms of proteins and DNA. These results illustrate the usefulness of immunological detection methods to study specific DNA-protein interactions induced by carcinogens. The possible relevance of DPCs in the carcinogenic process is discussed.