Raymond W. Sweet

Antibody modeling assessment.

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 Nine unpublished high‐resolution x‐ray Fab crystal structures covering a wide range of antigen‐binding site conformations were used as benchmark to compare Fv models generated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 methodologies. The methodologies included two homology modeling strategies independently developed by CCG (Chemical Computer Group) and Accerlys Inc, and two fully automated antibody modeling servers: PIGS (Prediction of ImmunoGlobulin Structure), based on the canonical structure model, and Rosetta Antibody Modeling, based on homology modeling and Rosetta structure prediction methodology. The benchmark structure sequences were submitted to Accelrys and CCG and a set of models for each of the nine antibody structures were generated. PIGS and Rosetta models were obtained using the default parameters of the servers. In most cases, we found good agreement between the models and x‐ray structures. The average rmsd (root mean square deviation) values calculated over the backbone atoms between the models and structures were fairly consistent, around 1.2 Å. Average rmsd values of the framework and hypervariable loops with canonical structures (L1, L2, L3, H1, and H2) were close to 1.0 Å. H3 prediction yielded rmsd values around 3.0 Å for most of the models. Quality assessment of the models and the relative strengths and weaknesses of the methods are discussed. We hope this initiative will serve as a model of scientific partnership and look forward to future antibody modeling assessments. Proteins 2011;

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V(L) and three V(H) domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V(H) by keeping V(L) in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V(H) diversity was sufficient for isolating Fabs to most antigens, whereas variability in V(L) was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V(L) diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.

Promoting crystallization of antibody-antigen complexes via microseed matrix screening

The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate.

Structure and specificity of an antibody targeting a proteolytically cleaved IgG hinge

The functional role of human antihinge (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 monoclonal reagents. 2095‐2 is a rabbit monoclonal antibody targeting the IdeS‐cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095‐2 and its complex with a hinge analog peptide. The antibody is selective for the C‐terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095‐2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656–1667.

Structure of the EMMPRIN N-terminal domain 1: dimerization via beta-strand swapping.

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 invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which hasmorexa0» a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last {beta}-strand (strand G). {beta}-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell-cell adhesion by cadherins. 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains and amyloidogenesis. In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state. It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations. Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis-trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell-cell adhesion.«xa0less

Measurement of protein interaction bioenergetics: Application to structural variants of anti-sCD4 antibody

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies

Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity. A phage display Fab library was selected on recombinant F antigen in the presence of three representative antibodies recovered in the unblocked and subsequent single-blocked panning procedures. Restriction endonuclease fingerprinting of 13 F+ clones revealed seven unique Fabs. DNA sequence analysis of five of these clones revealed five new light chains in combination with different heavy chains, three of which were very similar or identical to Fabs previously isolated from this library. The blocking antibodies did not compete with the new Fabs, demonstrating effective masking of their binding sites in the panning procedure. Conversely, these Fabs did show variable inhibition of two of the blocking antibodies suggesting a close proximity or interdependence of their epitopes. One of the antibodies did inhibit virus infection, albeit with modest potency. These results demonstrate that epitope-blocked panning is a self-progressing approach to retrieving diverse antibodies from phage libraries.

Crystallization of a challenging antigen-antibody complex: TLR3 ECD with three noncompeting Fabs.

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, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 with the three Fab fragments combined with microseed matrix screening and additive screening. Fine purification entailed the application of a very shallow gradient in anion-exchange chromatography, resulting in the resolution of two separate complex peaks which had different crystallizabilities. Subsequent structure determination defined the epitopes of the respective antibodies and revealed a mechanistic hypothesis that is currently under investigation. The results also showed that cocrystallization with multiple noncompeting Fab fragments can be a viable path when an antigen complex with a single Fab proves to be recalcitrant to crystallization.

CD4 mAb induced apoptosis of peripheral T cells: multiparameter subpopulation analysis by flow cytometry using Attractors™

Studies describing the induction of apoptosis for CD4 mAbs do not delineate between epitope-dependent and Fc-driven epitope cross-linking induced cell death. Keliximab and clenoliximab are two CD4 mAbs that differ only in their heavy chain isotypes, being an IgG1 and a modified IgG4, respectively. These antibodies suppress CD4 T cell responses in vitro and in vivo and have been in human clinical trials for the treatment of RA and asthma. Here we compared the apoptotic activity of these mAbs to differentiate between the contributions of epitope-dependent vs. Fc-driven epitope cross-linking induced cell death in vitro as a link to differential CD4 cell depletion in vivo. We developed a simple flow cytometry procedure that measures apoptosis within intact and compromised subpopulations of PBMCs within a few hours of culture. Attractors software was used to quantitate the percentage of apoptotic CD4 T cells, which generate reactive oxygen species (ROS), express external phosphatidyl serine (PS) and cleaved fluorescein diacetate (FDA), within the intact and compromised lymphocyte populations. Treatment of freshly isolated PBMCs with keliximab resulted in the appearance of characteristic apoptotic condensed CD4 T cells that contained reactive oxygen species, were annexin V positive and had intact esterase activity. Apoptosis was evident within 3 h and continued throughout the 72-h culture period. In contrast, clenoliximab alone did not induce apoptosis. The use of multiparameter flow cytometry and Attractors to analyze subpopulations based on scatter properties and biochemical processes during apoptosis provides a sensitive assay in which to quantitate and characterize the induction of cell death. Depletion of CD4 T cells in vivo by keliximab may reflect, in part, antibody-mediated apoptosis of these cells that is dependent on Fcgamma receptors.