Transgenic expression of IL-10 in T cells facilitates development of experimental myasthenia gravis.

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8(+)-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8(+)-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-gamma, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.

Construction and binding kinetics of a soluble granulocyte-macrophage colony-stimulating factor receptor alpha-chain-Fc fusion protein.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha-chain (GM-CSFRalpha), which specifically binds GM-CSF, and a beta-chain (betac), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha) and a soluble form (sGM-CSFRalpha). We designed an sGM-CSFRalpha-Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha. The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFRalpha-Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions. The sGM-CSFRalpha-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF. The sGM-CSFRalpha-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFRalpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFRalpha-Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined. This revealed a rapid koff of 2.43 x 10(-2)/s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha-Fc in solution. The dissociation rate constant (koff) for the sGM-CSFRalpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha-Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity.

Bioabsorbable polyphosphazene matrices as systems for calcitonin controlled release.

To provide a suitable delivery system for the calcitonin controlled release 80 mg bioabsorbable polyphosphazene matrices were obtained with entrapped 50 or 250 micrograms calcitonin. The in vitro behaviour demonstrated a release burst for about 24 hours, followed by a period of slow release of the peptide lasting for weeks. Matrices containing 250 micrograms calcitonin were implanted under general anaesthesia in osteoporotic female rats, while a group of animals (control group) received unloaded matrices. After thirty days a second batch of matrices was implanted in both groups to prolong the period of treatment until two months. The explanted matrices were histologically evaluated together with the surrounding tissues, and the dosage of the residual calcitonin was also performed. Results demonstrated the good biocompatibility of the system and the complete release of the calcitonin from the matrices 30 days after implantation. The therapeutic effect, after sixty days of treatment was confirmed by the better densitometric values observed in the femoral bone of treated animals than in controls.

Rational design, analysis, and potential utility of GM-CSF antagonists.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine involved in many immune and inflammatory processes and is believed to act in the early stages of immune responses. GM-CSF stimulates antigen-presenting cells, enhancing antigen presentation and inducing macrophage tumoricidal activity. GM-CSF binds to specific cellular receptors that are potential targets for pharmacological design. Rational design of small-molecule mimics is an important approach to pharmacophore design. One of the strategies in the development of small-molecular mimics of larger polypeptyde ligands is analysis of alternative ligands that bind the same site as does the native ligand. Molecular studies of GM-CSF-receptor interactions have led to the development of interaction site analogs and the development of an "anti-anti-GM-CSF" recombinant antibody (rAb) analog of a site on GM-CSF important for biological activity and receptor binding. This rAb and a peptide derived from the rAb first complementarity determining region (CDR) sequence bind to a monoclonal anti-GM-CSF antibody that mimics the GM-CSFR alpha chain, compete with GM-CSF for binding to GM-CSF receptor alpha chain (GM-CSFR alpha), and are specific biological antagonists. Molecular modeling of the rAb suggests structural similarity with a site previously implicated in GM-CSF binding to the GM-CSFR alpha. Two cyclic peptides, 1785 and 1786, also were developed on the basis of structural analysis of the GM-CSF region mimicked by anti-anti-GM-CSF recombinant antibody (rAb) 23.2. These peptides were designed to mimic structurally the positions of specific residues on the B and C helicies of human GM-CSF implicated in receptor binding and bioactivity. Both 1785 and 1786 were recognized specifically by polyclonal anti-GM-CSF antibody. 1786 also competitively inhibited binding of GM-CSF to the GM-CSF receptor and demonstrated antagonist bioactivity, as shown by its reversal of GM-CSF's ability to inhibit apoptosis of the GM-CSF-dependent cell line MO7E. These studies support the role of residues on the GM-CSF B and C helicies in receptor binding and bioactivity and suggest strategies for mimicking binding sites on four-helix bundle proteins with cyclic peptides.

Rational design of granulocyte-macrophage colony-stimulating factor antagonist peptides.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the four-helix bundle family of cytokines/growth factors which exhibit several activities. It is a hematopoietic growth factor, a cytokine involved in inflammatory and immune processes, an adjunct for cancer therapy, and an anti-tumor immunomodulator. Studies of interactions between GM-CSF and its receptor and identification of small peptides presenting binding capacity to the receptor are important goals for the development of GM-CSF analogs. Here we describe the study of two cyclic peptides, 1785 and 1786, developed based on structural analysis of the GM-CSF region mimicked by anti-anti-GM-CSF recombinant antibody 23.2. These peptides were designed to structurally mimic the positions of specific residues on the B and C helices of human GM-CSF implicated in receptor binding and bioactivity. Both 1785 and 1786 were specifically recognized by polyclonal anti-GM-CSF antibody (stronger for 1786 than 1785). 1786 also competitively inhibited binding of GM-CSF to the GM-CSF receptor on HL-60 cells and demonstrated antagonist bioactivity, as shown by its reversal of GM-CSF's ability to inhibit apoptosis of the GM-CSF-dependent cell line MO7E. These studies support the role of residues on the GM-CSF B and C helices in receptor binding and bioactivity and suggest strategies for mimicking binding sites on four-helix bundle proteins with cyclic peptides.

Recombinant antibodies in bioactive peptide design.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody. Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDABL vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones isolated. One clone (23.2) which inhibited 126.213 binding to GM-CSF was sequenced revealing a murine kappa light chain of subgroup III. Comparison of the 23.2 sequence with the human GM-CSF sequence revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.

A branched monomethoxypoly(ethylene glycol) for protein modification.

Procedures are described for linking monomethoxypoly(ethylene glycol) (mPEG) to both epsilon and alpha amino groups of lysine. The lysine carboxyl group can then be activated as a succinimidyl ester to obtain a new mPEG derivative (mPEG2-COOSu) with improved properties for biotechnical applications. This branched reagent showed in some cases a lower reactivity toward protein amino groups than the linear mPEG from which it was derived. A comparison of mPEG- and mPEG2-modified enzymes (ribonuclease, catalase, asparaginase, trypsin) was carried out for activity, pH and temperature stability, Km and Kcat values, and protection to proteolytic digestion. Most of the adducts from mPEG and mPEG2 modification presented similar activity and stability toward temperature change and pH change, although in a few cases mPEG2 modification was found to increase temperature stability and to widen the range of pH stability of the adducts. On the other hand, all of the enzymes modified with the branched polymer presented greater stability to proteolytic digestion relative to those modified with the linear mPEG. A further advantage of this branched mPEG lies in the possibility of a precise evaluation of the number of polymer molecules bound to the proteins; upon acid hydrolysis, each molecule of mPEG2 releases a molecule of lysine which can be detected by amino acid analysis. Finally, dimerization of mPEG by coupling to lysine provides a needed route to monofunctional PEGs of high molecular weight.

Development of GM-CSF antagonist peptides.

Granulocyte/macrophage colony stimulating factor (GM-CSF) is both a hematopoietic growth factor and a cytokine implicated in inflammatory disease. The development of GM-CSF antagonist peptides corresponding to the GM-CSF native sequence should allow their modification into higher affinity analogs, but this is hampered by the low affinity of linear peptides. To adequately evaluate such low affinity peptides, the use of several independent assays should allow specific versus nonspecific inhibitors to be distinguished. In this study, inhibition of GM-CSF-dependent cell growth, inhibition of GM-CSF binding and immunologic cross-reactivity between GM-CSF-derived peptides and native protein by neutralizing antibodies have been used to evaluate peptide analogs with potential bioactivity. The GM-CSF sequence was divided into 6 peptides ranging in size from 15-24 amino acids. Antisera were raised to these peptides in mice and assayed for immunologic cross-reactivity. 4/6 anti-peptide antisera bound GM-CSF on ELISA and 3/6 on immunoprecipitation. Antisera to two of the peptides (corresponding to residues 17-31 and 96-112) inhibited GM-CSF-dependent cellular proliferation in two cell lines, with one peptide derived from residues 17-31 demonstrating inhibition of GM-CSF binding and direct biological inhibitory activity. A peptide that did not elicit native GM-CSF reactive antibodies, corresponding to residues 54-78, was recognized by two neutralizing monoclonal antibodies. It exhibited inhibition of GM-CSF binding and direct biological antagonist activity. These studies implicate two sites in mediating GM-CSF biological activity, and indicate that biological antagonists can be developed based on these sites.

Granulocyte-macrophage colony-stimulating factor mimicry and receptor interactions.

Development of small molecular mimics of larger polypeptide ligands is an important approach to pharmacophore design. One strategy for the development of such mimics is analysis of alternative ligands that bind to the same site as the native ligand. These allow examination of the structural and chemical constraints for binding within the setting of diverse backbone geometries. The use of antireceptor antibodies as alternative ligands has allowed the development of biologically active peptides in several ligand-receptor systems. This technology has been applied to the study of interactions between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR). GM-CSF is one of a family of signal-transducing cytokines and growth factors characterized by a four-helix bundle core structure. The GM-CSFR is comprised of an alpha-chain (GM-CSFR alpha) specific for GM-CSF, and a beta-chain (beta c) shared with the interleukin-3 and interleukin-5 receptors. At least two sites on GM-CSF have been implicated in the GM-CSF-GM-CSFR alpha/beta c ternary complex. In studies summarized here, synthetic peptide analogs of GM-CSF sequences were designed and used to map neutralizing epitopes. One neutralizing epitope corresponded to the A helix of GM-CSF, and a synthetic analog displayed biological activity as a GM-CSF antagonist in vitro, suggesting interaction with the GM-CSFR alpha/beta c complex. A second peptide comprising the B and C helices was recognized by monoclonal neutralizing antibodies and similarly displayed antagonist activity. Recombinant antibody (rAb) technology was also employed. An expression library of rAbs from mice immunized with neutralizing anti-GM-CSF antibodies was developed and screened with a neutralizing anti-GM-CSF monoclonal antibody. One clone which displayed receptor binding activity exhibited structural similarity with epitopes on GM-CSF previously implicated as interaction sites with the neutralizing monoclonal antibody. A synthetic peptide analog of the rAb inhibited GM-CSF bioactivity. Critical contact residues were predicted on the basis of structural similarity of the rAb peptide and GM-CSF. These studies indicate the feasibility of using rAbs in bioactive peptide design, providing lead compounds and information regarding contact residues for drug design.

Preparation and properties of monomethoxy poly(ethylene glycol) doxorubicin conjugates linked by an amino acid or a peptide as spacer.

Polymeric doxorubicin prodrugs were prepared linking monomethoxy poly(ethylene glycol), 5000 D molecular weight, to the doxorubicin amino group, using an amino acid or a peptide as a spacer arm. As spacers glycine, L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine were used. The conjugates showed enhanced stability to alkaline degradation compared to the free doxorubicin. Towards Ehrlich solid tumor in mice the glycin spaced derivative was devoid of activity, whereas the phenylalanine and tryptophan derivatives were 20% and 16% active and the tripeptide one 50% active with respect to free doxorubicin. On the other hand the derivatization was accompanied by a great decrease of toxicity in mice with respect to the free drug. Doxorubicin was not released from conjugates by chymotrypsin incubation or in plasma.

Accurate evaluation method of the polymer content in monomethoxy(polyethylene glycol) modified proteins based on amino acid analysis.

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.