Structural basis for high selectivity of anti-CCL2 neutralizing antibody CNTO 888.

Human CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), is a member of the ß 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 diseases and cancer. CNTO 888, a neutralizing human anti-CCL2 antibody, was derived by antibody phage display. The antibody binds human CCL2 with high affinity (K(D)=22 pM) and inhibits CCL2 binding to its receptor. The crystal structure of the CNTO 888 Fab alone and in complex with the monomeric form of CCL2 (P8A variant) was determined at 2.6 Å and 2.8 Å resolution, respectively. CNTO 888 recognizes a conformational epitope encompassing residues 18-24 and 45-51 that overlaps the mapped receptor binding site. The epitope of CNTO 888 does not overlap with the dimerization site of CCL2, and thus its inhibitory activity is not expected to result from interference with the oligomeric state of CCL2. Comparison of the X-ray-determined epitopes of CNTO 888 and another CCL2-neutralizing antibody, 11K2, provides insight into the molecular basis of antibody selectivity and functional inhibition.

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.

Distinctive circular dichroism signature for 14-helix-bundle formation by beta-peptides.

We identify a distinctive circular dichroism (CD) signature for self-assembled 14-helical beta-peptides. Our data show that self-assembly leads to a mimimum at 205 nm, which is distinct from the well-known minimum at 214 nm for a monomeric 14-helix. The onset of assembly is indicated by [theta]205/[theta]214>0.7. Our results will facilitate rapid screening for self-assembling beta-peptides and raise the possibility that far-UV CD will be useful for detecting higher-order structure for other well-folded oligoamide backbones.