Synthesis of LJP 993, a multivalent conjugate of the N-terminal domain of beta2GPI and suppression of an anti-beta2GPI immune response.

LJP 993, a tetravalent conjugate of the amino-terminal domain (domain 1) of beta2GPI, was synthesized, and studies were carried out to explore the ability of LJP 993 to bind anti-beta2GPI antibodies and to function as a B cell toleragen. Domain 1 was expressed in Pichia pastoris, and the N-terminus was site-specifically modified by a transamination reaction converting the N-terminal glycine to a glyoxyl group. A tetravalent platform was synthesized with linkers that terminate in aminooxy groups. This was accomplished by preparing an ethylene glycol-based heterobifunctional linker that contains both a Boc-protected aminooxy group and a free primary amine. The linker was used to modify a tetravalent platform molecule by reacting the amino groups on the linker with 4-nitrophenyl carbonate esters on the platform to provide a linker-modified platform, and the Boc protecting groups were removed to provide a tetravalent aminooxy platform. Glyoxylated domain 1 was attached to the platform to provide LJP 993 by formation of oxime bonds. The protein domains of LJP 993 retain activity as evidenced by the ability of LJP 993 to bind to anti-beta2GPI antibodies. Dissociation constants (Kd) for domain 1 and LJP 993 bound to immobilized affinity-purified anti-beta2GPI antibodies from autoimmune thrombosis patients were determined using surface plasmon resonance. An immunized mouse model was developed to test the ability of LJP 993 to act as a toleragen. A thiol containing domain 1 analogue was expressed in insect cells using the baculovirus expression system, and it was used to prepare an immunogenic conjugate of domain 1 and maleimide-derivatized keyhole limpet hemocyanin (KLH). Mice were immunized with the KLH conjugate, and spleen cells were harvested from the immunized mice. The cells were incubated with various concentrations of LJP 993 and transferred to mice whose immune systems had been compromised by irradiation. The hosts were then boosted with the KLH-domain 1 conjugate, and after 7 days their antibody levels were measured. Host mice receiving cells that were treated with LJP 993 produced significantly lower amounts of anti-domain 1 antibodies than controls which received untreated cells, indicative of B cell tolerance.

Pre-treatment affinity for LJP 394 influences pharmacodynamic response in lupus patients.

Five prospective clinical studies in lupus patients have shown that LJP 394 can reduce circulating anti-dsDNA antibody levels without causing generalized immunosuppression. The compound is currently being evaluated in a phase III clinical trial for the prevention of renal flares in patients with high-affinity antibodies to LJP 394 and a history of lupus nephritis. The current study analyzed the affinity of patient IgG for LJP 394 prior to and following 4 months of treatment with LJP 394 to determine if pretreatment affinity influenced pharmacodynamic response. Patient serum samples from a multicenter, double-blind, placebo-controlled trial were evaluated prior to and following 4 months of weekly, biweekly or monthly treatment with placebo (n = 9) or weekly treatment with 10 mg LJP 394 (n = 6) or 50 mg LJP 394 (n = 4). After treatment there was a dose-dependent reduction in affinity in the 10 mg/week and 50 mg/week groups (P < 0.05 and P < 0.01, respectively), whereas the placebo group was unchanged. This study demonstrates that weekly treatment with LJP 394 produces a dose-dependent reduction in titer-weighted average affinity. These results suggest it may be possible to use an affinity assay to define prospectively patients that are most likely to exhibit the desired pharmacodynamic response to LJP 394.

Beta2-glycoprotein I-dependent anticardiolipin antibodies preferentially bind the amino terminal domain of beta2-glycoprotein I.

Many of the autoantibodies in antiphospholipid syndrome (APS) are directed against beta2-glycoprotein I (beta2-GPI). Recent studies from our laboratories have indicated that the immunodominant binding epitope(s) for high titer, affinity purified antibodies from 11 APS patients are localized to the amino terminal domain (domain 1) of beta2-GPI. The present study employed surface plasmon resonance to localize the immunodominant domain in serum samples from a large cohort of patients with GPL values ranging from 21 to 230 units (n = 106 patients). Eighty-eight percent of patients showed > or = threefold selectivity for beta2-GPI containing domain 1 relative to the domain deletion mutant that lacked domain 1. The domain 1 binding activity in patient serum was abolished by removing the IgG fraction from the serum and the binding activity could be fully reconstituted with the IgG fraction. Thus, analysis of serum samples from a large cohort of APS patients indicates that the immunodominant binding epitope(s) for anti-beta2 antibodies are localized to the amino terminal domain of beta2-GPI.

Identification of a peptide that protects the human acetylcholine receptor against antigenic modulation.

mAb 192 is a rat monoclonal antibody with very high affinity for the major immunogenic region (MIR) of the human muscle acetylcholine receptor (AChR). An epitope mimic of this antibody was selected from a phage display peptide library screened with mAb 192. The peptide-presenting phage has been shown to specifically bind to solid phase mAb 192 with an equilibrium dissociation constant (K(d)) of 8.45x10(-9) M, as directly measured with surface plasmon resonance. This value represents the avidity of the interaction between selected phage and mAb 192. A synthetic version of this peptide QPSPYNGWRMEI, referred to as MG15, binds to its selecting antibody and blocks the interaction of mAb 192 with human AChR. Peptide MG15 was able to protect acetylcholine receptors on human RD cells from antibody-mediated down-modulation. The negative charge of glutamic acid plays a important role in antibody binding. Replacement of the glutamic acid with an alanine completely abolishes the inhibitory activity.

Antibody affinities and relative titers in polyclonal populations: surface plasmon resonance analysis of anti-DNA antibodies.

This paper presents the equations and methodology for the measurement and interpretation of apparent dissociation constants for polyclonal populations of antibodies, where antigen is kept trace relative to antibody concentration. Surface plasmon resonance is used to determine K(d)s for the binding of anti-DNA antibodies to trace amounts of DNA antigen on a chip. Since the approach taken relies on equilibrium measurements, kinetic mass transport artifacts are avoided. The apparent K(d) is a weighted average of all the K(d)s for the clonally related subpopulations within the polyclonal pool, where each weighting factor is the relative titer (fractional presence) of the subpopulation. Titration curves appear as if there is one monoclonal population with that titer-weighted-average K(d). Implications of changes in the antibody affinity distribution within the population are discussed. The equations described herein provide a better physical understanding of the apparent K(d) that is obtained when a heterogeneous population of receptors is titrated against a trace ligand.

Application of fluorescence polarization to the steady-state enzyme kinetic analysis of calpain II.

This paper presents the application of fluorescence polarization to the determination of dissociation constants for competitive inhibitors that bind to enzymes. This steady-state enzyme kinetic study measures the inhibition of the conversion of a fluorescently tagged substrate to a lower molecular weight fluorescent product by calpain II. It relies on the measurement of a parameter proportional to velocity, which is sufficient for this type of analysis. The strengths and limitations of the method are discussed. Inhibition constants for filamin and spectrin determined by this method are 125 nM and 13 nM respectively.

RS-102221: a novel high affinity and selective, 5-HT2C receptor antagonist.

The 5-HT2C receptor is one of three closely related receptor subtypes in the 5-HT2 receptor family. 5-HT2A and 5-HT2B selective antagonists have been described. However, no 5-HT2C selective antagonists have yet been disclosed. As part of an effort to further explore the function of 5-HT2C receptors, we have developed a selective 5-HT2C receptor antagonist, RS-102221 (a benzenesulfonamide of 8-[5-(5-amino-2,4-dimethoxyphenyl) 5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione). This compound exhibited nanomolar affinity for human (pKi = 8.4) and rat (pKi = 8.5) 5-HT2C receptors. The compound also demonstrated nearly 100-fold selectivity for the 5-HT2C receptor as compared to the 5-HT2A and 5-HT2B receptors. RS-102221 acted as an antagonist in a cell-based microphysiometry functional assay (pA2 = 8.1) and had no detectable intrinsic efficacy. Consistent with its action as a 5-HT2C receptor antagonist, daily dosing with RS-102221 (2 mg/kg intraperitoneal) increased food-intake and weight-gain in rats. Surprisingly, RS-102221 failed to reverse the hypolocomotion induced by the 5-HT2 receptor agonist 1-(3-chlorophenyl)piperazine (m-CPP). It is concluded that RS-102221 is the first selective, high affinity 5-HT2C receptor antagonist to be described.

Evaluation of human astrocytoma and glioblastoma cell lines for nerve growth factor release.

Nerve growth factor (NGF) prevents degeneration of cholinergic neurons in the central nervous system (CNS), and has potential as a therapeutic treatment for Alzheimer's disease. The inability of NGF to cross the blood-brain barrier has prompted pharmacological approaches investigating peripherally administered compounds that stimulate release of endogenous NGF. This study describes the NGF-releasing properties of six human astrocytoma and glioblastoma cell lines (SW 1088, SW 1783 and CRL 1718 astrocytomas, and U-138, U-373, and T98G glioblastomas). Using a highly specific two-site ELISA for human NGF, basal NGF release could be detected in all cell lines, with the lowest level in the T98G line (approximately 80 pg NGF/ml). Cell lines tested with a variety of compounds for 24 h in serum-free media demonstrated stimulation of NGF release by distinct mechanisms. NGF levels were markedly elevated (up to 8-fold above vehicle-treated cells) when stimulated with the cytokine interleukin-1 beta (IL-1 beta). Phorbol ester stimulated NGF release 4-fold. Clenbuterol, 4-methyl catechol, and propentofylline had little activity, while 6-(4-hydroxybutyl)-2,3,5,-trimethyl-1,4,benzoquinone (TMQ), dexamethasone and 1,25-dihydroxyvitamin D3 elevated NGF levels 3-fold. These data indicate differences in the ability of human astrocytoma and glioblastoma cells to release NGF when stimulated with mechanistically distinct compounds.

Recombinant human ciliary neurotrophic factor stimulates the metabolic activity of SH-SY5Y cells as measured by a cytosensor microphysiometer.

Information on the transmembrane signaling events and subsequent biochemical processes initiated by ciliary neurotrophic factor (CNTF) receptor activation in neurons is lacking. SH-SY5Y cells, a human neuroblastoma cell line expressing CNTF receptors, were used to study metabolic changes associated with functional ligand-receptor interactions. Real-time measurements quantifying the rate of extracellular acidification by SH-SY5Y cells (a measure of metabolic activity) were made using a silicon-based cytosensor. Application of recombinant human CNTF (rhCNTF) to resting SH-SY5Y cells increased their acidification rate in a concentration and time-dependent manner with an apparent EC50 of 60 ng/ml. Pretreatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) prevented the CNTF, but not an NGF-stimulated increase in acidification rate. Collectively, these results demonstrate that: (1) SH-SY5Y cells express functional CNTF receptors; and (2) the initial signal transduction mechanism activated by the CNTF receptor in SH-SY5Y cells is distinct from that activated by the NGF receptor; however, both may ultimately stimulate the same downstream biochemical messengers to increase cellular metabolism.