Stage-specific expression of mucosal addressin cell adhesion molecule-1 during embryogenesis in rats

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is essential for lymphocyte trafficking to gut-associated lymphoid tissues and is implicated in inflammatory disorders in the gut and pancreatic islets. In this study, we examined the functional role of MAdCAM-1 during rat ontogeny using newly generated specific mAb. As previously observed in mice and humans, MAdCAM-1 was preferentially expressed in high endothelial venules (HEV) in gut-associated lymphoid tissues and venules of lamina propria in adult rats. Lymphocyte rolling and adhesion on HEV in Peyer's patches (PP) were completely abrogated with neutralizing anti-MAdCAM-1 mAb, in agreement with the notion that MAdCAM-1 is the principal HEV ligand for lymphocyte rolling and adhesion in adult PP. In the developing gastrointestinal tract, MAdCAM-1 was widely expressed in the venules of the lamina propria of fetal rats. In addition, MAdCAM-1 was also expressed in follicular dendritic cells in the neonatal PP. Interestingly, MAdCAM-1 expression was found also in nonmucosal tissues during ontogeny. MAdCAM-1 was transiently expressed in blood vascular endothelial cells in the fetal skin and neonatal thymus. Notably, MAdCAM-1-positive blood vessels were localized mainly in the cortico-medullary junction in the neonatal thymus and about 10-20% of thymocytes, most of which were either CD4, CD8 double positive or single positive specifically reacted with soluble MAdCAM-1 via integrin alpha4beta7. After birth, MAdCAM-1 expression in thymus blood vessels disappeared and concomitantly, the soluble MAdCAM-1-reactive thymocytes were rapidly down-regulated. Our results suggest that MAdCAM-1 functions as a vascular addressin in not only mucosal, but also nonmucosal lymphoid tissues during ontogeny.     

Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site.

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer's patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than alpha4beta7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.     

Prevention of a chronic progressive form of experimental autoimmune encephalomyelitis by an antibody against mucosal addressin cell adhesion molecule-1, given early in the course of disease progression

A role for alpha4 and beta7 integrins in mediating leucocyte entry into the central nervous system in the multiple sclerosis (MS)-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated. However, the individual contributions of their respective ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin expressed on the blood-brain barrier has not been determined. In the present paper, it is shown that an antibody directed against MAdCAM-1, the preferential ligand for alpha4beta7, effectively prevented the development of a progressive, non-remitting, form of EAE, actively induced by injection of myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) autoantigen. Combinational treatment with both anti-MAdCAM-1, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) (ligand for integrin lymphocyte function-associated antigen (LFA)-1) mAbs led to more rapid remission than that obtained with anti-MAdCAM-1 antibody alone. However, neither MAdCAM-1 monotherapy, nor combinational antibody blockade was preventative when administered late in the course of disease progression. In conclusion, MAdCAM-1 plays a major contributory role in the progression of chronic EAE and is a potential therapeutic target for the treatment of MS. Critically, antivascular addressin therapy must be given early in the course of disease prior to the establishment of irreversible damage if it is to be effective, as a single treatment modality.     

Mutational analysis of MAdCAM-1/α4β7 interactions reveals significant binding determinants in both the first and second immunoglobulin domains

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin, MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin α4β7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin α4β7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/α4β7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.     

Mutational analysis of MAdCAM-1/alpha4beta7 interactions reveals significant binding determinants in both the first and second immunuglobulin domains

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.     

Extension of neurites on axons is impaired by antibodies against specific neural cell surface glycoproteins

We have developed an in vitro assay which measures the ability of growth cones to extend on an axonal substrate. Neurite lengths were compared in the presence or absence of monovalent antibodies against specific neural cell surface glycoproteins. Fab fragments of antibodies against the neural cell adhesion molecule, NCAM, have an insignificant effect on the lengths of neurites elongating on either an axonal substrate or a laminin substrate. Fab fragments of polyclonal antibodies against two new neural cell surface antigens, defined by mAb G4 and mAb F11, decrease the lengths of neurites elongating on an axonal substrate, but have no effect on the lengths of neurites elongating on a laminin substrate. G4 antigen is related to mouse L1, while F11 antigen appears to be distinct from all known neural cell surface glycoproteins. Our results suggest that the G4 and F11 antigens help to promote the extension of growth cones on axons.     

Chronic allergy to dietary ovalbumin induces lymphocyte migration to rat small intestinal mucosa that is inhibited by MAdCAM-1

Few models have described a chronic food allergy with morphological changes in the intestinal mucosa. Here we established an ovalbumin (OVA)-induced, cell-mediated, allergic rat model and examined lymphocyte migration in the gut. Brown Norway rats were intraperitoneally sensitized to OVA and then given 10 mg OVA/day by gastric intubation for 6 wk. Lymphocyte subsets and adhesion molecules were examined immunohistochemically, and the migration of T lymphocytes to microvessels of Peyer's patches and villus mucosa was observed by using an intravital microscope. Serum OVA-specific IgG and IgE levels were increased in animals repeatedly exposed to OVA. Significant villus atrophy and increased crypt depth was accompanied by increased infiltration of T lymphocytes in the small intestinal mucosa of the group given OVA. Expression of rat mast cell protease II and of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was also increased in these groups. The administration of anti-MAdCAM-1 antibody significantly attenuated the OVA-induced changes in the mucosal architecture and in CD4 T lymphocyte infiltration. Intravital observation demonstrated that in rats with a chronic allergy, T lymphocytes significantly accumulated in villus microvessels as well as in Peyer's patches via a MAdCAM-1-dependent process. Our model of chronic food allergy revealed that lymphocyte migration was increased with MAdCAM-1 upregulation.     

A Novel Monoclonal Antibody to Human Laminin α5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521

Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.     

Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.     

Targeting the gut vascular endothelium induces gut effector CD8 T cell responses via cross-presentation by dendritic cells

Systemic delivery of Ag usually induces poor mucosal immunity. To improve the CD8 T cell response at mucosal sites, we targeted the Ag to MAdCAM-1, a mucosal addressin cell adhesion molecule expressed mainly by high endothelial venules (HEV) in mesenteric lymph nodes (MLN) and Peyer's patches of gut-associated lymphoid tissue. When chemical conjugates of anti-MAdCAM-1 Ab and model Ag OVA were injected i.v., a greatly enhanced proliferative response of Ag-specific OT-I CD8 T cells was detected in MLN. This was preceded by prolonged accumulation, up to 2 wk, of the anti-MAdCAM OVA conjugate on HEV of Peyer's patches and MLN. In contrast, nontargeted OVA conjugate was very inefficient in inducing OT-I CD8 T cell proliferation in MLN and required at least 20-fold more Ag to induce a comparable response. In addition, MAdCAM targeting elicits an endogenous OVA-specific CD8 T cell response, evident by IFN-gamma production and target killing. Induced response offers protection against an OVA-expressing B cell lymphoma. We propose that the augmentation of gut CD8 T cell responses by MAdCAM targeting is due to both accumulation of Ag in the HEV and conversion of a soluble Ag to a cell-associated one, allowing cross-presentation by DCs.     

Exploration of delta-subunit interactions in beef heart mitochondrial F1-ATPase by monoclonal antibodies

Three monoclonal antibodies (mAbs) recognizing distinct epitopes on the delta-subunit of beef heart mitochondrial F1-ATPase were studied for their reactivity towards the delta-subunit both in isolated F1 and in the F0-F1 complex of submitochondrial particles. Two of the antibodies termed mAb delta 195 and mAb delta 239 had free access to delta in F1 and the F0-F1 complex. Partial hindrance was observed for the third antibody mAb delta 22. By a double antibinding assay, it was found that the binding sites for mAb delta 195 and mAb delta 239 were close to each other and possibly overlapping. Mapping studies conducted with the isolated delta-subunit showed that mAb delta 195 and mAb delta 239 interacted with the N-terminal portion of delta extending from Ala-1 to Met-16, whereas mAb delta 22 interacted with the fragment spanning Ser-17-Glu-68. It was concluded that the Ala-1-Met-16 segment of the delta-subunit in F1 and the F0-F1 complex is freely accessible from the outside, whereas the Ser-17-Glu-68 segment of delta is partially hidden, possibly as a result of interactions with other subunits.     

Membrane glycoproteins involved in neurite fasciculation

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.     

Genomic organization, chromosomal mapping, and analysis of the 5’ promoter region of the human MAdCAM-1 gene

 MAdCAM-1, the endothelial addressin cell adhesion molecule-1, interacts preferentially with the leukocyte β7 integrin LPAM-1 (α4β7), but also with L-selectin, and with VLA-4 (α4β1) on myeloid cells, and serves to direct leukocytes into mucosal and inflamed tissues. Overlapping cosmid and phage λ genomic clones were isolated, revealing that the human MAdCAM-1 gene contains five exons where the signal peptide, two Ig domains, and mucin domain are each encoded by separate exons. The transmembrane domain, cytoplasmic domain, and 3′ untranslated region are encoded together on exon 5. The mucin domain contains eight repeats in total that are subject to alternative splicing. Despite the absence of a human counterpart of the third IgA-homologous domain and lack of sequence conservation of the mucin domain, the genomic organizations of the human and mouse MAdCAM-1 genes are similar. An alternatively spliced MAdCAM-1 variant was identified that lacks exon 4 encoding the mucin domain, and may mediate leukocyte adhesion to LPAM-1 without adhesion to the alternate receptor, L-selectin. The MAdCAM-1 gene was located at p13.3 on chromosome 19, in close proximity to the ICAM-1 and ICAM-3 genes (p13.2-p13.3). PMA-inducible promotor activity was contained in a 700 base pair 5’ flanking fragment conserved with the mouse MAdCAM-1 gene including tandem NF-kB sites, and an Sp1 site; and in addition multiple potential AP2, Adh1 (ETF), PEA3, and Sp1 sites. In summary, the data establish that the previously reported human MAdCAM-1 cDNA does indeed encode the human homologue of mouse MAdCAM-1, despite gross dissimilarities in the MAdCAM-1 C-terminal structures. Received: 5 December 1996 / Revised: 2 January 1997     

Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (betaG-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-betaG-sPEG conjugate showed that mAb 1E8 (IgG1) bound betaG and betaG-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the betaG activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared betaG-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared betaG-sPEG more completely and rapidly than 1E8, reducing the serum concentration of betaG-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-betaG-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of betaG-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-betaG-PEG conjugates for glucuronide prodrug therapy of cancer.     

Activation of beta1 integrins induces cell-cell adhesion

Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.     

Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

ABSTRACT

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen–deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs.

Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-β-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen–deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance–solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.     

Potent neutralization of botulinum neurotoxin/b by synergistic action of antibodies recognizing protein and ganglioside receptor binding domain

Background

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.

Methods and Findings

We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.

Conclusions

The combination of two mAbs recognizing different receptors'' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.     

Inhibitory monoclonal antibody targeting ADAM17 expressed on cancer cells

ADAM17 is upregulated in many cancers and in turn activates signaling pathways, including EGFR/ErbB, as well as those underlying resistance to targeted anti-EGFR therapies. Due to its central role in oncogenic pathways and drug resistance mechanisms, specific and efficacious monoclonal antibodies against ADAM17 could be useful for a broad patient population with solid tumors. Hence, we describe here an inhibitory anti-ADAM17 monoclonal antibody, named D8P1C1, that preferentially recognizes ADAM17 on cancer cells. D8P1C1 inhibits the catalytic activity of ADAM17 in a fluorescence-based peptide cleavage assay, as well as the proliferation of a range of cancer cell lines, including breast, ovarian, glioma, colon and the lung adenocarcinoma. In mouse models of triple-negative breast cancer and ovarian cancer, treatment with the mAb results in 78% and 45% tumor growth inhibition, respectively. Negative staining electron microscopy analysis of the ADAM17 ectodomain in complex with D8P1C1 reveals that the mAb binds the ADAM17 protease domain, consistent with its ability to inhibit the ADAM17 catalytic activity. Collectively, our results demonstrate the therapeutic potential of the D8P1C1 mAb to treat solid tumors.     

Proposed mechanism of off-target toxicity for antibody–drug conjugates driven by mannose receptor uptake

Antibody–drug conjugates (ADCs) are developed with the goal of increasing compound therapeutic index by specific and targeted delivery of a toxic payload to the site of action while considerably reducing damage to normal tissues. Yet, off-target hepatic toxicities have been reported for several ADC. Locations of these off-target toxicities coincide with the reported locations of cell surface mannose receptor (MR). The relative proportion of agalactosylated glycans on the Fc domain (G0F vs. G1F and G2F components) in monoclonal antibody (mAb)–based biotherapeutics is closer to some disease state IgG rather than to a normal serum-derived immunoglobulin. The lack of the terminal galactose on a G0F glycan creates an opportunity for the mAb to interact with soluble and cell surface MRs. MR is a known multi-domain lectin that specifically binds and internalizes glycoproteins and immune complexes with relatively high G0F content and has been found on the surface of various cell types, including immune cells of myeloid lineage, endothelial cells, and hepatic and splenic sinusoids. In this review paper it is proposed that the mechanism of the off-target toxicities for ADC biotherapeutics is at least in part driven by the carbohydrates, specifically agalactosylated glycans, such as G0F, their interactions with MR and resulting glycan-derived cellular uptake of ADCs. Several case studies are reviewed presenting corroborating information.