The dimerization of glucagon-like peptide-2 MIMETIBODY™ is linked to leucine-17 in the glucagon-like peptide-2 region.

Glucagon-like peptide-2 (GLP-2) is a member of the glucagon multigene family that is produced by intestinal enteroendocrine cells in response to food intake. GLP-2 stimulates growth of the intestinal epithelium, enhances its barrier functions, and increases nutrient uptake. Therefore, a GLP-2 agonist may be efficacious in human diseases characterized by malabsorption or injury to the gastrointestinal epithelium. MIMETIBODY™ refers to a proprietary scaffold developed to extend the half-life of rapidly cleared peptides. It consists of a peptide linked to a scaffold that contains sequence elements from a human immunoglobulin G including those that allow recycling through the FcRn. The GLP-2 sequence was engineered into the MIMETIBODY™ scaffold. The primary state of both GLP-2 and the GLP-2 MIMETIBODY™ in DPBS was a noncovalently associated dimer indicative of self-interaction. The increased heterogeneity and the decreased lot-to-lot reproducibility caused by the self-interaction of therapeutic proteins are a challenge to drug development. A similar protein, GLP-1 MIMETIBODY™, contains the related GLP-1 peptide and does not form a dimer under similar conditions. Therefore, to minimize or abrogate dimerization, several variants were made by substituting GLP-2 amino acids with the corresponding amino acids from GLP-1. Molecular weight and secondary structure analyses reveal that substituting leucine for glutamine at position 17 (L17Q) reduces dimerization and α-helix content yet retains bioactivity.

Synthesis by native chemical ligation and crystal structure of human CCL2.

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) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full-length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP-1 cells, as expected. CCL2 was crystallized and the structure was determined by X-ray diffraction at 1.9-A resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9-Thr10-Cys11, which form an intersubunit antiparallel beta-sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety-catch linker and NCL.

Human anti-IgG1 hinge autoantibodies reconstitute the effector functions of proteolytically inactivated IgGs.

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.

Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches.

Chimeric 101F (ch101F) is a mouse-human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422-438 spanning antigenic site IV, V, VI was analysed. Residues 423-436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

Synthesis and biological characterization of human monocyte chemoattractant protein 1 (MCP-1) and its analogs.

Novel analogs of human monocyte chemoattractant protein 1 (MCP-1) were designed, synthesized and characterized to be used as tools to generate monoclonal antibodies as potential human therapeutics. MCP-1 and three analogs were synthesized by step-wise Fmoc solid phase synthesis. After oxidation to form the two-disulfide bonds, affinity chromatography using an immobilized mouse anti-human MCP-1 monoclonal antibody (mAb) was utilized for a simple and highly effective purification procedure for the proteins. The final products were extensively characterized and compared with recombinant human MCP-1 (rhMCP-1). All proteins showed identical binding with mouse anti-human MCP-1 mAbs as measured by surface plasmon resonance. Synthetic MCP-1 and the analogs were comparable to recombinant MCP-1 in competition radio-ligand binding to CCR2 receptors on THP-1 cells, and MCP-1-induced, calcium mobilization and chemotaxis assays.

Synthetic, site-specific biotinylated analogs of human MCP-1.

Human monocyte chemoattractant protein 1 (MCP-1, CCL2) is a 8.6-kDa protein that has been implicated in a number of diseases including atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease and cancer. As part of a program to identify antibodies against MCP-1, we synthesized site-specific, biotinylated human MCP-1 analogs to be used for panning of an antibody phage display library. In contrast to material obtained from random biotinylation, the site-specific biotinylated analogs were homogeneous and retained full activity.

Pharmacodynamic enhancement of the anti-platelet antibody fab abciximab by site-specific pegylation.

Monoclonal antibodies have been firmly established as human therapeutics. Their high affinity and specificity for target antigens minimize adverse reactions and their molecular size results in extended circulation times relative to small molecule pharmaceuticals. The ability to customize the pharmacokinetics in a rational manner can enhance the potential for these and other classes of biologicals. We have systematically studied the effect of site-specific pegylation of the Fab' fragment of the anti-GPIIb/IIIa, alphavbeta3 antibody c7E3. Regardless of the molecular weight of the PEG molecules, the intrinsic affinity of the resulting constructs remained unchanged. However, in functional assays measuring inhibition of platelet aggregation, the calculated IC50 values of the conjugates decreased with increasing molecular weight of the conjugated PEG. It was determined that the molecular size of the conjugates affects antigen accessibility and whereas high levels of binding to antigen molecules on cells with high antigen density can be demonstrated with the Fab fragment, comparable levels are not achievable with large molecular weight conjugates. In spite of the inability of the larger PEG constructs to achieve saturation binding, functional inhibition of platelet aggregation consistent with saturation binding was demonstrated and the increased molecular size of the conjugates led to predictably prolonged inhibition of platelet aggregation.