Structure-Guided Design of an Anti-dengue Antibody Directed to a Non-immunodominant Epitope.

Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.

Characterization of an Antibody Recognizing the Conserved Inner Core of Pseudomonas aeruginosa Lipopolysaccharides.

Bacterial infections are a growing public health threat with carbapenem-resistant Pseudomonas aeruginosa being classified as a Priority 1 critical threat by the World Health Organization. Antibody-based therapeutics can serve as an alternative and in some cases supplement antibiotics for the treatment of bacterial infections. The glycans covering the bacterial cell surface have been proposed as intriguing targets for binding by antibodies; however, antibodies that can engage with high affinity and specificity with glycans are much less common compared to antibodies that engage with protein antigens. In this study, we sought to characterize an antibody that targets a conserved glycan epitope on the surface of Pseudomonas. First, we characterized the breadth of binding of VSX, demonstrating that the VSX is specific to Pseudomonas but can bind across multiple serotypes of the organism. Next, we provide insight into how VSX engages with its target epitope, using a combination of biolayer interferometry and nuclear magnetic resonance, and verify our results using site-directed mutagenesis experiments. We demonstrate that the antibody, with limited somatic hypermutation of the complementarity-determining regions (CDRs) and with a characteristic set of arginines within the CDRs, specifically targets the conserved inner core of Pseudomonas lipopolysaccharides. Our results provide important additional context to antibody-glycan contacts and provide insight useful for the construction of vaccines and therapeutics against Pseudomonas aeruginosa, an important human pathogen.

A Proliferation Inducing Ligand (APRIL) targeted antibody is a safe and effective treatment of murine IgA nephropathy.

IgA nephropathy (IgAN) is the most prevalent primary chronic glomerular disease for which no safe disease-specific therapies currently exist. IgAN is an autoimmune disease involving the production of autoantigenic, aberrantly O-glycosylated IgA1 and ensuing deposition of nephritogenic immune complexes in the kidney. A Proliferation Inducing Ligand (APRIL) has emerged as a key B-cell-modulating factor in this pathogenesis. Using a mouse anti-APRIL monoclonal antibody (4540), we confirm both the pathogenic role of APRIL in IgAN and the therapeutic efficacy of antibody-directed neutralization of APRIL in the grouped mouse ddY disease model. Treatment with 4540 directly translated to a reduction in relevant pathogenic mechanisms including suppressed serum IgA levels, reduced circulating immune complexes, significantly lower kidney deposits of IgA, IgG and C3, and suppression of proteinuria compared to mice receiving vehicle or isotype control antibodies. Furthermore, we translated these findings to the pharmacological characterization of VIS649, a highly potent, humanized IgG2κ antibody targeting and neutralizing human APRIL through unique epitope engagement, leading to inhibition of APRIL-mediated B-cell activities. VIS649 treatment of non-human primates showed dose-dependent reduction of serum IgA levels of up to 70%. A reduction of IgA+, IgM+, and IgG+ B cells was noted in the gut-associated mucosa of VIS649-treated animals. Population-based modeling predicted a favorable therapeutic dosing profile for subcutaneous administration of VIS649 in the clinical setting. Thus, our data highlight the potential therapeutic benefit of VIS649 for the treatment of IgAN.

Structural prediction of antibody-APRIL complexes by computational docking constrained by antigen saturation mutagenesis library data.

IgA nephropathy (IgAN) is the most prevalent cause of primary glomerular disease worldwide, and the cytokine A PRoliferation-Inducing Ligand (APRIL) is emerging as a key player in IgAN pathogenesis and disease progression. For a panel of anti-human APRIL antibodies with known antagonistic activity, we sought to define their structural mode of engagement to understand molecular mechanisms of action and aid rational antibody engineering. Reliable computational prediction of antibody-antigen complexes remains challenging, and experimental methods such as X-ray co-crystallography and cryoEM have considerable technical, resource, and throughput barriers. To overcome these limitations, we implemented an integrated and accessible experimental-computational workflow to more accurately predict structures of antibody-APRIL complexes. Specifically, a yeast surface display library encoding site-saturation mutagenized surface positions of APRIL was screened against a panel of anti-APRIL antibodies to rapidly obtain a comprehensive biochemical profile of mutational impact on binding for each antibody. The experimentally derived mutational profile data were used as quantitative constraints in a computational docking workflow optimized for antibodies, resulting in robust structural models of antibody-antigen complexes. The model results were confirmed by solving the cocrystal structure of one antibody-APRIL complex, which revealed strong agreement with our model. The models were used to rationally select and engineer one antibody for cross-species APRIL binding, which quite often aids further testing in relevant animal models. Collectively, we demonstrate a rapid, integrated computational-experimental approach to robustly predict antibody-antigen structures information, which can aid rational antibody engineering and provide insights into molecular mechanisms of action.

Crystal structures of ß-1,4-galactosyltransferase 7 enzyme reveal conformational changes and substrate binding.

The ß-1,4-galactosyltransferase 7 (ß4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human ß4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type ß4GalT7 and D211N ß4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N ß4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an SN2 type catalytic mechanism for the ß4GalT7 enzyme.

Binding of N-acetylglucosamine (GlcNAc) ß1-6-branched oligosaccharide acceptors to ß4-galactosyltransferase I reveals a new ligand binding mode.

N-acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The ß1-4-galactosyltransferase-I (ß4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the ß4Gal-T1 molecule. Here we report seven crystal structures of ß4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a ß1-6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of ß4Gal-T1 with I-antigen analog pentasaccharide, the ß1-6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the ß4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the ß4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that ß4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.

Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-acetylglucosamine (GlcNAc) residues on live cell surface.

On the basis of the crystal structure of bovine ß4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289L-ß4Gal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn(2+) concentrations required to selectively modify GlcNAc residues on the surface of live cells, we have engineered a Mg(2+)-dependent enzyme. Previously, we found that the mutation of the metal-binding residue Met-344 to His-344 in bovine ß4Gal-T1 enzyme altered its metal-ion specificity in such a way that the M344H-ß4Gal-T1 enzyme exhibits better catalytic activity with Mg(2+) than with Mn(2+). Here, we find that, when these two mutations are combined, the double mutant, Y289L-M344H-ß4Gal-T1, transfers GalNAc and its analogue sugars to the acceptor GlcNAc in the presence of Mg(2+). Using this mutant enzyme, we have detected free GlcNAc residues on the surface glycans of live HeLa cells and platelets. The specific transfer of a synthetic sugar with a chemical handle to the terminal GlcNAc residues on the surface of live cells provides a novel tool for selective modification, detection, and isolation of GlcNAc-ending glycans present on the cellular surface.

Investigations on ß1,4-galactosyltransferase I using 6-sulfo-GlcNAc as an acceptor sugar substrate.

6-sulfate modified N-acetylglucosamine (6-sulfo-GlcNAc) is often found as part of many biologically important carbohydrate epitopes such as 6-sulfo-Le(X). In these epitopes, the 6-sulfo-GlcNAc moiety is extended by a galactose sugar in a ß1-4 linkage. The ß4GalT1 enzyme transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) in the presence of manganese. Here we report that the ß4GalT1 enzyme transfers Gal to the 6-sulfo-GlcNAc and 4-methylumbelliferyl-6-sulfo-N-acetyl-ß-D-glucosaminide (6-sulfo-ßGlcNAc-MU) acceptor substrates, although with very low efficiency. To understand the effect that the 6-sulfate group on the GlcNAc acceptor has on the catalytic activity of the ß4GalT1 molecule, we have determined the crystal structure of the catalytic domain of bovine ß4GalT1 mutant enzyme M344H-ß4GalT1 complex with the 6-sulfo-GlcNAc molecule. In the crystal structure, the 6-sulfo-GlcNAc is bound to the protein in a way that is similar to the GlcNAc molecule. However, the 6-sulfate group engages in additional interactions with the hydrophobic region, residues 276-285, of the protein molecule, and this group is found wedged between the aromatic side chains of Phe-280 and Trp314 residues. Since the side chain of the Trp314 residue undergoes conformational changes during the catalytic cycle of the enzyme, molecular interaction between Trp314 and the 6-sulfate group might hinder this conformational change. Therefore, the lack of a favorable binding environment, together with hindrance to the conformational changes, might be responsible for the poor catalytic activity.

The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2.

In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-ß1,4-galactosyltransferase I (Y289L-ß4Gal-T1) and the wild-type polypeptide-α-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-ß4Gal-T1 mutant enzyme, the wild-type human ß1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-ß4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar.

Crystal structure of the catalytic domain of Drosophila beta1,4-Galactosyltransferase-7.

The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

Galectin-1 as a fusion partner for the production of soluble and folded human beta-1,4-galactosyltransferase-T7 in E. coli.

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, beta-1,4-galactosyltransferase-7 (beta4Gal-T7), in E. coli. The enzyme beta4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, beta4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-beta4Gal-T7 fusion protein, the unique protease cleavage site allows the protein beta4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded beta4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.

Substrate-induced conformational changes in glycosyltransferases.

Oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans are synthesized by glycosyltransferases by the transfer of specific glycosyl moieties from activated sugar-nucleotide donors to specific acceptors. Structural studies on several of these enzymes have shown that one or two flexible loops at the substrate-binding site of the enzymes undergo a marked conformational change from an open to a closed conformation on binding the donor substrate. This conformational change, in which the loop acts as a lid covering the bound donor substrate, creates an acceptor-binding site. After the glycosyl unit is transferred from the donor to the acceptor, the saccharide product is ejected and the loop reverts to its native conformation, thereby releasing the remaining nucleotide moiety. The specificity of the sugar donor is determined by a few residues in the sugar-nucleotide-binding pocket of the enzyme that are conserved among the family members from different species.

VIS832, a novel CD138-targeting monoclonal antibody, potently induces killing of human multiple myeloma and further synergizes with IMiDs or bortezomib in vitro and in vivo.

Therapeutically targeting CD138, a define multiple myeloma (MM) antigen, is not yet approved for patients. We here developed and determined the preclinical efficacy of VIS832, a novel therapeutic monoclonal antibody (MoAb) with differentiated CD138 target binding to BB4 that is anti-CD138 MoAb scaffold for indatuximab ravtansine (BT062). VIS832 demonstrated enhanced CD138-binding avidity and significantly improved potency to kill MM cell lines and autologous patient MM cells regardless of resistance to current standard-of-care therapies, via robust antibody-dependent cellular cytotoxicity and phagocytosis mediated by NK and macrophage effector cells, respectively. Specifically, CD38-targeting daratumumab-resistant MM cells were highly susceptible to VIS832 which, unlike daratumumab, spares NK cells. Superior maximal cytolysis of VIS832 vs. daratumumab corresponded to higher CD138 vs. CD38 levels in MM cells. Furthermore, VIS832 acted synergistically with lenalidomide or bortezomib to deplete MM cells. Importantly, VIS832 at a sub-optimal dose inhibited disseminated MM1S tumors in vivo as monotherapy (P < 0.0001), and rapidly eradicated myeloma burden in all mice concomitantly receiving bortezomib, with 100% host survival. Taken together, these data strongly support clinical development of VIS832, alone and in combination, for the therapeutic treatment of MM in relapsed and refractory patients while pointing to its potential therapeutic use earlier in disease intervention.

Iterative cluster-NMA: A tool for generating conformational transitions in proteins.

Computational models provide insight into the structure-function relationship in proteins. These approaches, especially those based on normal mode analysis, can identify the accessible motion space around a given equilibrium structure. The large magnitude, collective motions identified by these methods are often well aligned with the general direction of the expected conformational transitions. However, these motions cannot realistically be extrapolated beyond the local neighborhood of the starting conformation. In this article, the iterative cluster-NMA (icNMA) method is presented for traversing the energy landscape from a starting conformation to a desired goal conformation. This is accomplished by allowing the evolving geometry of the intermediate structures to define the local accessible motion space, and thus produce an appropriate displacement. Following the derivation of the icNMA method, a set of sample simulations are performed to probe the robustness of the model. A detailed analysis of beta1,4-galactosyltransferase-T1 is also given, to highlight many of the capabilities of icNMA. Remarkably, during the transition, a helix is seen to be extended by an additional turn, emphasizing a new unknown role for secondary structures to absorb slack during transitions. The transition pathway for adenylate kinase, which has been frequently studied in the literature, is also discussed.

Bioconjugation and detection of lactosamine moiety using alpha1,3-galactosyltransferase mutants that transfer C2-modified galactose with a chemical handle.

Studies on wild-type and mutant glycosyltransferases have shown that they can transfer modified sugars with a versatile chemical handle, such as keto or azido group, that can be used for conjugation chemistry and detection of glycan residues on glycoconjugates. To detect the most prevalent glycan epitope, N-acetyllactosamine (LacNAc (Galbeta1-4GalNAcbeta)), we have mutated a bovine alpha1,3-galactosyltransferse (alpha3Gal-T)() enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives. The alpha3Gal-T enzyme belongs to the alpha3Gal/GalNAc-T family that includes human blood group A and B glycosyltransferases, which transfer GalNAc and Gal, respectively, to the Gal moiety of the trisaccharide Fucalpha1-2Galbeta1-4GlcNAc. On the basis of the sequence and structure comparison of these enzymes, we have carried out rational mutation studies on the sugar donor-binding residues in bovine alpha3Gal-T at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant alpha3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants (280)SGG(282) and (280)AGG(282) with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of asialofetuin, thus enabling the detection of LacNAc moiety of glycoproteins and glycolipids by a chemiluminescence method.

Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection.

The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using beta1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant beta1,4-galactosyltransferase (beta1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect enzyme linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods.

Multiple site-specific in vitro labeling of single-chain antibody.

For multiple site-specific conjugations of bioactive molecules to a single-chain antibody (scFv) molecule, we have constructed a human anti HER2 receptor, scFv, with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the acceptor substrate for human polypeptide-alpha-Nu-acetylgalactosaminyltransferase II (h-ppGalNAc-T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). The recombinant scFv fusion proteins are expressed in E. coli as inclusion bodies and in vitro refolded and glycosylated with h-ppGalNAc-T2. Upon protease cleavage, the MALDI-TOF spectra of the glycosylated C-terminal fusion polypeptides showed that the glycosylated scFv fusion protein with a single Thr residue is fully glycosylated with a single 2-keto-Gal, whereas the glycosylated scFv fusion protein with 3 and 17 Thr residues is found as an equal mixture of 2-3 and 5-8 2-keto-Gal glycosylated fusion proteins, respectively. These fusion scFv proteins with the modified galactose are then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bind specifically to a human breast cancer cell line (SK-BR-3) that overexpresses the HER2 receptor, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen.

Role of a single amino acid in the evolution of glycans of invertebrates and vertebrates.

Structures of glycoconjugate N-glycans and glycolipids of invertebrates show significant differences from those of vertebrates. These differences are due largely to the vertebrate beta1,4-galactosyltransferase-1 (beta4Gal-T1), which is found as a beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAc-T1) in invertebrates. Mutation of Tyr285 to Ile or Leu in human beta4Gal-T1 converts the enzyme into an equally efficient beta4GalNAc-T1. A comparison of all the human beta4Gal-T1 ortholog enzymes shows that this Tyr285 residue in human beta4Gal-T1 is conserved either as Tyr or Phe in all vertebrate enzymes, while in all invertebrate enzymes it is conserved as an Ile or Leu. We find that mutation of the corresponding Ile residue to Tyr in Drosophila beta4GalNAc-T1 converts the enzyme to a beta4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold. Furthermore, we find that, similar to the vertebrate/mammalian beta4Gal-T1 enzymes, the wild-type Drosophila beta4GalNAc-T1 enzyme binds to a mammary gland-specific protein, alpha-lactalbumin (alpha-LA). Thus, it would seem that, during the evolution of vertebrates from invertebrates over 500 million years ago, beta4Gal-T1 appeared as a result of the single amino acid substitution of Tyr or Phe for Leu or Ile in the invertebrate beta4GalNAc-T1. Subsequently, the pre-existing alpha-LA-binding site was utilized during mammalian evolution to synthesize lactose in the mammary gland during lactation.

Applications of glycosyltransferases in the site-specific conjugation of biomolecules and the development of a targeted drug delivery system and contrast agents for MRI.

BACKGROUND: The delivery of drugs to the proposed site of action is a challenging task. Tissue and cell-specific guiding molecules are being used to carry a cargo of therapeutic molecules. The cargo molecules need to be conjugated in a site-specific manner to the therapeutic molecules such that the bioefficacy of these molecules is not compromised. METHODS: Using wild-type and mutant glycosyltransferases, the sugar moiety with a unique chemical handle is incorporated at a specific site in the cargo or therapeutic molecules, making it possible to conjugate these molecules through the chemical handle present on the modified glycan. RESULTS/CONCLUSIONS: The modified glycan residues introduced at specific sites on the cargo molecule make it possible to conjugate fluorophores for ELISA-based assays, radionuclides for imaging and immunotherapy applications, lipids for the assembly of immunoliposomes, cytotoxic drugs, cytokines, or toxins for antibody-based cancer therapy and the development of a targeted drug delivery system.

Extending human IgG half-life using structure-guided design.

Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.