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Gary L. Gilliland

Gary L. Gilliland
Janssen Research & Development, LLC, Spring House, United States · Biologics Research, BCoE

Ph.D.

About

253
Publications
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Publications

Publications (253)
Article
This study uses differential scanning calorimetry, X-ray crystallography and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5 °C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-st...
Article
Full-text available
Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indicat...
Article
The cover image, by Alexey Teplyakov et al., is based on the Short Communication Crystal structure of B‐cell co‐receptor CD19 in complex with antibody B43 reveals an unexpected fold, DOI: 10.1002/prot.25485.
Article
Full-text available
In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subtypes, many strategies are pursued to simulate the human system in the test animal. However, t...
Article
CD19 is a transmembrane protein expressed on malignant B cells, but not in other lineages or other tissues, which makes it an attractive target for monoclonal antibody-mediated immunotherapy. Anti-CD19 antibody B43 was utilized in a bispecific T-cell engager (BiTE) blinatumomab, that demonstrated potency for the treatment of relapsed acute lymphobl...
Article
Full-text available
Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the re...
Article
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The homeostatic chemokine CCL17, also known as thymus and activation regulated chemokine (TARC), has been associated with various diseases such as asthma, idiopathic pulmonary fibrosis, atopic dermatitis and ulcerative colitis. Neutralization of CCL17 by antibody treatment ameliorates the impact of disease by blocking influx of T cells. Monoclonal...
Article
Full-text available
The Protein Data Bank (PDB) is the global archive for structural information on macromolecules, and a popular resource for researchers, teachers and students, amassing more than one million unique users each year. Crystallographic structure models in the PDB (more than 100,000 entries) are optimized against the crystal diffraction data and geometri...
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The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A bin...
Article
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Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2...
Article
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Backgroundβ-Amyloid (Aβ) peptide is believed to play a pivotal role in the development of Alzheimer’s disease. Passive immunization with anti-Aβ monoclonal antibodies may facilitate the clearance of Aβ in the brain and may thus prevent the downstream pathology. Antibodies targeting the immunodominant N-terminal epitope of Aβ and capable of binding...
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Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signalin...
Preprint
Crystallographic structure models in the Protein Data Bank (PDB) are optimized against the crystal diffraction data and geometrical restraints. This process of crystallographic refinement typically ignored hydrogen bond (H-bond) distances as a source of information. However, H-bond restraints can improve structures, especially at low resolution whe...
Article
Full-text available
CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been...
Article
Full-text available
Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibo...
Article
CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding af...
Article
Designed ankyrin repeat proteins (DARPin(®)) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9...
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Full-text available
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the...
Article
Centyrins are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with increased tissue penetration and simplified manufacturing. To identify optimal positions for chemical conjugation within the Centyrin scaffold, the crystal structure of an anti-EGFR Centyrin was solved and a library consisting of all...
Article
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The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characte...
Article
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To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of four different kappa light chains paired with four different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in...
Article
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Microtubule-associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF-tau). AT8 is a PHF-tau specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports des...
Article
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The crystal structure of DARPin 44C12V5 that neutralizes IL-4 signaling has been determined alone and bound to human IL-4. A significant conformational change occurs in the IL-4 upon DARPin binding. The DARPin binds to the face of IL-4 formed by the A and C α-helices. The structure of the DARPin remains virtually unchanged. The conformational chang...
Article
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tencons are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with the biophysical features of smaller molecules and hold high promise as biotherapeutic agents. We present the crystal structures and antigen binding data of several a...
Article
Three Fab structures used as targets in the Antibody Modeling Assessment presented a challenge for modeling CDR-L3 due to deviations from the most typical canonical structure. In all three antibodies CDR-L3 has 8 residues, which is one residue shorter than usual, and has a conformation that is rarely observed in crystal structures. We analyzed the...
Article
Full-text available
Despite sequence diversity, five out of six hypervariable loops in antibodies assume a limited number of conformations called canonical structures. Their correct identification is essential for successful prediction of antibody structure. This in turn requires regular updates of the classification of canonical structures to match the expanding expe...
Article
To assess the state of the art in antibody structure modeling, a blinded study was conducted. Eleven unpublished Fab crystal structures were used as a benchmark to compare Fv models generated by 7 structure prediction methodologies. In the first round, each participant submitted 3 non-ranked complete Fv models for each target. In the second round,...
Article
The functional role of human anti-hinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclon...
Article
To assess the state of the art in antibody 3D modeling, eleven unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as benchmark to compare Fv models generated by seven structure prediction methodologies. The participants included: Accerlys Inc, Chemi...
Article
Full-text available
The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Ap...
Article
The crystal structures of six different fibronectin type III consensus derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C-termin...
Article
The crystal structure of an N-terminal β-strand-swapped consensus derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydr...
Book
With a focus on practical applications of biophysical techniques, this book links fundamental biophysics to the process of biopharmaceutical development. • Helps formulation and analytical scientists in pharma and biotech better understand and use biophysical methods • Chapters organized according to the sequential nature of the drug development...
Chapter
This chapter focuses primarily on antibodies, but the principles and methods outlined and described in the chapter, with few exceptions, have general applicability to all classes of biotherapeutics. It describes experimental methods for directly and indirectly assessing biologic solubility and stability. Useful computational methods for predicting...
Article
Full-text available
The 24(th) Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modelin...
Article
Full-text available
The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progre...
Article
The analyses of two human IgG2 Fc structures, determined in different crystal forms, and the comparison with IgG1 Fc structures reveals molecular features that are involved in accommodating and stabilizing structural conformations. In the IgG2 Fc structures relative positions of the CH2 domains with respect to the CH3 domains vary significantly fro...
Article
Full-text available
Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-...
Article
Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epito...
Article
Human CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), is a member of the β chemokine family whose actions are mediated through the G-protein-coupled receptor CCR2. Binding of CCL2 to its receptor CCR2 triggers calcium mobilization and chemotaxis. CCL2 is implicated in the pathogenesis of certain inflammatory...
Article
The use of consensus design to produce stable proteins has been applied to numerous structures and classes of proteins. Here, we describe the engineering of novel FN3 domains from two different proteins, namely human fibronectin and human tenascin-C, as potential alternative scaffold biotherapeutics. The resulting FN3 domains were found to be robus...
Article
Antibodies make up the largest, growing segment of protein therapeutics in the pharmaceutical and biotechnology industries. The development or engineering of therapeutic antibodies is based to a large extent on our knowledge of antibody structure and requires sophisticated methods that continue to evolve. In this chapter, after a review of what is...
Article
Full-text available
In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s...
Article
A blinded study to assess the state of the art in three-dimensional structure modeling of the variable region (Fv) of antibodies was conducted. Nine unpublished high-resolution x-ray Fab crystal structures covering a wide range of antigen-binding site conformations were used as benchmark to compare Fv models generated by four structure prediction m...
Article
C836 is a neutralizing monoclonal antibody to human interleukin IL-13 generated by mouse immunization. The crystal structure of the C836 Fab was determined at 2.5 Å resolution and compared with the IL-13-bound form determined previously. This comparison indicates an induced-fit mechanism of antigen recognition through rigid-body rotation of the VL...
Article
The mechanism of action of therapeutic antibodies can be elucidated from the three-dimensional crystal structures of their complexes with antigens, but crystallization remains the primary bottleneck to structure determination. Methods that resulted in the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting,...
Article
Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β-amyloid (Aβ) plaques. Aggregation of the Aβ(42) peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti-Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used fo...
Article
The human antibody repertoire is dominated by a few combinations of germline sequences, with the genes 3-23 for the heavy chain and B3 for the light chain being among the most frequently used. Despite this fact, there was no experimental structure of the antibody composed of 3-23 and B3. The crystal structure of the Fab fragment of the synthetic an...
Article
Antibodies recognize antigens through six hypervariable loops, five of which have a limited set of conformations known as canonical structures. For κ light chains, the majority of CDR-L3 [the third hypervariable loop of the light chain variable domain (V(L))] adopts the type 1 canonical structure (CS1), with a cis-proline at position 95. Here, we p...
Article
Full-text available
Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface h...
Article
A chimeric antibody was constructed from two unrelated antibodies by combining their heavy and light chains. The "double chimera" consists of the mouse variable regions of different specificity (IL-13 and EMMPRIN) and the constant regions of different origin (mouse and human). The Fab fragment of this chimeric antibody was expressed in mammalian ce...
Article
Full-text available
The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-1...
Article
Full-text available
CNTO4088 is a monoclonal antibody to human IL-23. The X-ray structure of the Fab fragment revealed an unusual noncanonical conformation of CDR L1. Most antibodies with the kappa light chain exhibit a canonical structure for CDR L1 in which residue 29 anchors the CDR loop to the framework. Analysis of the residues believed to define the conformation...
Article
Humanization of a potent neutralizing mouse anti-human IL-13 antibody (m836) using a method called human framework adaptation (HFA) is reported. HFA consists of two steps: human framework selection (HFS) and specificity-determining residue optimization (SDRO). The HFS step involved generation of a library of m836 antigen binding sites combined with...
Article
The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP-1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester-peptide segment was synthesized using the sulfonamide safety-catch linker and 9-fluorenylmethoxycarbonyl (Fmoc) S...
Article
Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasi...
Article
CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at...
Article
The enzyme 2-hydroxychromene-2-carboxylic acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion (Keq = 1.5) of HCCA and trans-o-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The dimeric protein binds one molecule of GSH very tightly (Kd approximately 5 nM) and a second...