George A. Heavner

Structural basis for the dual recognition of IL-12 and IL-23 by ustekinumab.

Interleukin (IL)-12 and IL-23 are heterodimeric proinflammatory cytokines that share a common p40 subunit, paired with p35 and p19 subunits, respectively. They represent an attractive class of therapeutic targets for the treatment of psoriasis and other immune-mediated diseases. Ustekinumab is a fully human monoclonal antibody (mAb) that binds specifically to IL-12/IL-23p40 and neutralizes human IL-12 and IL-23 bioactivity. The crystal structure of ustekinumab Fab (antigen binding fragment of mAb), in complex with human IL-12, has been determined by X-ray crystallography at 3.0 Å resolution. Ustekinumab Fab binds the D1 domain of the p40 subunit in a 1:1 ratio in the crystal, consistent with a 2 cytokines:1 mAb stoichiometry, as measured by isothermal titration calorimetry. The structure indicates that ustekinumab binds to the same epitope on p40 in both IL-12 and IL-23 with identical interactions. Mutational analyses confirm that several residues identified in the IL-12/IL-23p40 epitope provide important molecular binding interactions with ustekinumab. The electrostatic complementarity between the mAb antigen binding site and the p40 D1 domain epitope appears to play a key role in antibody/antigen recognition specificity. Interestingly, this structure also reveals significant structural differences in the p35 subunit and p35/p40 interface, compared with the published crystal structure of human IL-12, suggesting unusual and potentially functionally relevant structural flexibility of p35, as well as p40/p35 recognition. Collectively, these data describe unique observations about IL-12p35 and ustekinumab interactions with p40 that account for its dual binding and neutralization of IL-12 and IL-23.

Biologically active analogs of thymopentin with enhanced enzymatic stability

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32-36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro2-TP5-NH2 and Aib2-TP5-NH2 retained activity and were shown to exhibit a high degree of stability when incubated in human serum.

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, αVβ3 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.

Structural requirements for the biological activity of thymopentin analogs

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity.

Proton NMR investigation of Ln3+ complexes of thymopoietin32–36

The pentapeptide Arg-Lys-Asp-Val-Tyr (TP5) is a biologically active fragment of thymopoietin, the thymic hormone that induces selective T-cell differentiation. The formation of lanthanide(III) complexes of TP5 is demonstrated through the observation of Tb3+ fluorescence enhancement. The equilibria, stoichiometry and solution conformation of the La3+, Pr3+ and Yb3+ complexes of TP5 have been investigated using NMR spectroscopy. In addition, the dissociation constants of two methyl ester analogs of TP5 have been studied. Evidence is presented supporting an interaction between the arginine guanidino N epsilon H and the aspartate carboxylate of TP5. Binding of Ln3+ appears to be accompanied by a disruption (or weakening) of this interaction and a concomitant increase in the 180 degrees rotamer population for the aspartate carboxylate group. The observed trends in the magnitudes of the dissociation constants and the rotamer populations appear to suggest that, although a significant amount of monodentate complexes may also exist, the metal ion binds predominantly to both carboxylates in a bidentate fashion.

Cooperativity of thymopoietin 32–36 (the active site) and thymopoietin 38–45 in receptor binding

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.

Nuclear magnetic resonance analysis of Gd3+-induced perturbations in thymopoietin32–36: A study of amide and aromatic proton resonances

Abstract The Gd 3+ -induced perturbations in the NMR spectra of a cell differentiating peptide fragment, Argue5f8Lysue5f8Aspue5f8Value5f8Tyr (TP5), have been examined. This pentapeptide fragment retains the selective T-cell differentiating activity of its parent polypeptide thymic hormone, thymopoietin. The observed relaxation enhancements induced by Gd 3+ have been analyzed to determine the relative and absolute amide and aromatic proton-Gd 3+ distances. The data are compatible with a bidentate model, in which both the aspartyl and tyrosyl carboxylates bind the metal ion simultaneously in a chelate fashion, being the dominant conformer. From these studies a picture of the conformation of Ln 3+ complexes of TP5 begins to emerge.

Peptide analogs of thymopentin distinguish distinct thymopoietin receptor specificities on two human T cell lines

Thymopoietin, a polypeptide hormone of the thymus, and the synthetic pentapeptide thymopentin, corresponding to thymopoietin32-36, both induced elevations of intracellular cyclic GMP in two human T cell lines, CEM and MOLT-4. In contrast, the closely related polypeptide thysplenin, which differs from thymopoietin at position 34, induced intracellular cyclic GMP elevation in MOLT-4 but not in CEM. We synthesized a series of penta- and tetrapeptide analogs of amino acids 32-36 of human thymopoietin and thysplenin, and now show that distinct patterns of activity can be obtained in these small peptides, with selectivity for cyclic GMP elevation in MOLT-4 alone or CEM alone. This suggests that the thymopoietin receptors (TPR) on these two human T cell lines are distinguishable by their differing ligand specificities, and we have termed them alpha TPR and beta TPR for CEM and MOLT-4 receptors, respectively.

Probing the solution structure of tumor necrosis factor-α homotrimer and heterotrimer after complex perturbation using electrospray ionization mass spectrometry†

There are a number of proteins whose active forms are non‐covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild‐type (wt) proteins. We have used tumor necrosis factor‐α (TNF‐α) to establish that it is possible to use mass spectrometry to monitor the non‐covalent solution structure of therapeutically relevant proteins and correlate the results with binding data. Mass spectrometry is shown to be able to directly monitor the state of the solution complexes to within 5u2009Da errors mass accuracy of theoretical mass at 50u2009kDa, as well as to resolve homocomplex from heterocomplex. Furthermore, it was determined that perturbation of the TNF‐α complex, at or below pH 4.0, results in monomers that cannot reform into the multimeric complex, and the resulting protein solution can no longer bind to an anti‐TNF‐α antibody. Dissociation and re‐association of the trimer was possible with the use of dimethyl sulfoxide at pH 5.5 and allowed for the resulting detection of both homotrimer and heterotrimer in solution with no impact on antibody binding. This work demonstrates that mass spectrometric techniques offer a means to monitor native solution interactions of non‐covalent complexes and to differentiate multiple complexes from each other in solution. This method has applicability in the biopharmaceutical arena for monitoring engineering non‐covalent drug complexes for the purpose of altering biological activity. Copyright