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 5 Da errors mass accuracy of theoretical mass at 50 kDa, 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.

Discovery and mechanism of ustekinumab: a human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders.

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, "humanized" and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor ß1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.

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.

The oxidation of methionine-54 of epoetinum alfa does not affect molecular structure or stability, but does decrease biological activity.

Erythropoietin therapy is used to treat severe anemia in renal failure and chemotherapy patients. One of these therapies based on recombinant human erythropoietin is marketed under the trade name of EPREX and utilizes epoetinum alfa as the active pharmaceutical ingredient. The effect of oxidation of methionine-54 on the structure and stability of the erythropoietin molecule has not been directly tested. We have observed partial and full chemical oxidation of methionine-54 to methionine-54 sulfoxide, accomplished using tert-Butylhydroperoxide and hydrogen peroxide, respectively. A blue shift in the fluorescence center of spectral mass wavelength was observed as a linear response to the level of methionine sulfoxide in the epoetinum alfa molecule, presumably arising from a local change in the environment near tryptophan-51, as supported by potassium iodide quenching studies. Circular dichroism studies demonstrated no change in the folded structure of the molecule with methionine oxidation. The thermal unfolding profiles of partial and completely oxidized epoetinum alfa overlap, with a T(m) of 49.5 degrees C across all levels of methionine sulfoxide content. When the protein was tested for activity, a decrease in biological activity was observed, correlating with methionine sulfoxide levels. An allosteric effect between Met54, Trp51, and residues involved in receptor binding is proposed. These results indicate that methionine oxidation has no effect on the folded structure and global thermodynamic stability of the recombinant human erythropoietin molecule. Oxidation can affect potency, but only at levels significantly in excess of those seen in EPREX.

Recognition and identification of UV-absorbing leachables in EPREX pre-filled syringes: an unexpected occurrence at a formulation-component interface.

During the period of 1998 to 2002, there was an increase in the incidence of antibody-positive pure red cell aplasia (PRCA) in patients receiving subcutaneous administration of EPREX (epoetinum alfa). As part of the investigation of this event, the aqueous formulation containing polysorbate 80, introduced in 1998, facilitated the leaching of small-molecule, aromatic compounds from the uncoated rubber syringe stoppers. The leachables were identified using Liquid Chromatography-Mass Spectroscopy, Electrospray Ionisation-MS/MS, Dithiothreitol reduction, and Hydrogen/Deuterium exchange. The major leachable was identified as a dialkylphenol disulfide, and the majority of the remaining peaks were identified as structural variants containing different numbers of sulfur atoms in the sulfide bridge. In this report, we describe the strategies and experimental designs that were used to overcome the analytical challenges and that led to successful structural identification of the leachables in EPREX pre-filled syringes with uncoated syringe stoppers.

Engineering human IL-18 with increased bioactivity and bioavailability.

Cytokines in plasmid form can act as potent adjuvants when co-administered with DNA vaccines, resulting in an enhanced immune response to the DNA-encoded antigen. This is true of interleukin-18 (IL-18), which has been shown to serve as an adjuvant in conjunction with certain DNA vaccines. To determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce interferon gamma (IFNgamma) expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFNgamma response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant.

An in vivo model to assess factors that may stimulate the generation of an immune reaction to erythropoietin.

The incidence of pure red cell aplasia (PRCA) in patients with chronic kidney disease associated with the subcutaneous (s.c.) administration of epoetin alfa (EPREX) began to increase in 1998. As part of an intensive investigation into the reasons for this increase, in vivo models were developed to assess the ability of potential causative factors to stimulate an immune response to recombinant human erythropoietin (rHuEPO). It was difficult to generate anti-EPO antibodies in mice. In animals injected with rHuEPO alone, anti-EPO antibodies were either absent or present at very low levels. The addition of an adjuvant to the immunization protocol was able to increase both the frequency of occurrence and titer of the immune response and resulted in the generation of anti-EPO antibodies that, in most cases, recognized both human and mouse EPO. Some mice exhibited a reduction in hematocrit, suggesting neutralization of endogenous EPO by anti-EPO antibodies. To evaluate the primary lead identified in the technical investigation, leachates from the uncoated syringe stoppers of EPREX syringes, a surrogate antigen (chicken egg albumin, OVA) was used to avoid possible interferences that could arise from the use of an endogenous protein like EPO. These leachates yielded a positive, concentration-dependent antibody response in the OVA animal model, demonstrating their adjuvant properties and providing support for the hypothesis generated through the technical investigation that leachates were capable of enhancing the immune response to rHuEPO.

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.

Interaction of polysorbate 80 with erythropoietin: a case study in protein-surfactant interactions.

PURPOSE: The cause of antibody positive pure red cell aplasia associated with the subcutaneous administration of EPREX to patients with chronic kidney failure has been determined to be due to the leaching of weakly adjuvant compounds from the uncoated rubber stoppers that were formerly used in prefilled syringes. Other researchers have suggested that polysorbate 80 micelles containing erythropoietin may be a causative factor. The purpose of this work was to repeat previously published studies in a more controlled manner and to define the precise nature of the interactions between polysorbate 80 and erythropoietin. METHODS: The contents of EPREX prefilled syringes and laboratory-prepared, well-characterized formulations of EPREX were analyzed by size exclusion chromatography. Fractions were analyzed for the presence of erythropoietin by ELISA. EPREX formulations prepared with increasing amounts of polysorbate 80 were analyzed by light scattering. RESULTS: Well-controlled chromatographic studies showed that when EPREX formulations containing no aggregate were analyzed by high-performance liquid chromatography, erythropoietin monomer could not be detected under the polysorbate 80 peak. Dimer and oligomers of erythropoietin coeluted under the polysorbate 80 peak as the molecular weights overlapped on the size exclusion chromatogram. Solution light scattering indicated that polysorbate 80 associates with erythropoietin in a defined stoichiometric ratio of 1:12. CONCLUSIONS: Based on controlled studies, previous results suggesting that EPREX contains micelle-associated erythropoietin were incorrect. As with other surfactants and proteins, polysorbate 80 associates with erythropoietin in a defined stoichiometric ratio.

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.

A monoclonal antibody and an enzyme immunoassay for human Ala-IL-8(77).

Interleukin-8 (IL-8) plays a central role in neutrophil chemotaxis and exerts a wide range of effects on various cells, ranging from tumor angiogenesis to impairment of neuronal signaling. Two main forms of IL-8 exist, one containing 77 amino acids (Ala-IL-8(77)) and a second containing 72 amino acids (Ser-IL-8(72)), which comprise more than 90% of IL-8 protein in cell cultures. IL-8(77) was reported to be produced predominantly by endothelial cells and is known as "endothelial" IL-8. IL-8(72) predominates in monocyte cultures and is known as "leukocyte" IL-8. While both forms have equal chemotactic activity in vivo, recent data suggest that their biological activities might be different. Here we describe the generation of a mouse monoclonal antibody (mAb) specific for IL-8(77) and the development of a corresponding immunoassay. Various immunization protocols were investigated. Immunization with conjugates of a peptide from the N-terminus of IL-8(77) (NTP(77)) resulted in the production of an IgG1 mAb (N11) that recognizes human IL-8(77) and neutralizes its chemotactic activity. A sensitive ELISA specific for IL-8(77) was developed using N11 for capture and a biotinylated mAb to IL-8(72) for detection. Using this immunoassay it was shown that the only form of IL-8 secreted in cell culture was IL-8(77) and that the IL-8(72) present was the result of proteolysis of IL-8(77). IL-8(77) was detected in plasma and cerebrospinal fluid (CSF) from patients with sepsis and meningitis.