Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity by antibodies present in the cerebrospinal fluid of patients with multiple sclerosis.

We have previously shown that B cells and Abs reactive with GAPDH and antitriosephosphate isomerase (TPI) are present in lesions and cerebrospinal fluid (CSF) in multiple sclerosis (MS). In the current study, we studied the effect of anti-GAPDH and anti-TPI CSF IgG on the glycolytic enzyme activity of GAPDH and TPI after exposure to intrathecal IgG from 10 patients with MS and 34 patients with other neurologic diseases. The degree of inhibition of GAPDH activity by CSF anti-GAPDH IgG in the seven MS samples tested varied from 13 to 98%, which seemed to correlate with the percentage of anti-GAPDH IgG in the CSF IgG (1-45%). Inhibition of GAPDH activity (18 and 23%) by CSF IgG was seen in two of the 34 patients with other neurologic diseases, corresponding to the low percentage of CSF anti-GAPDH IgG (1 and 8%). In addition, depletion of anti-GAPDH IgG from CSF IgG, using immobilized GAPDH, removed the inhibitory effect of the IgG on GAPDH. No inhibition of GAPDH activity was seen with CSF samples not containing anti-GAPDH IgG. No inhibition of TPI activity was seen with any purified CSF IgG sample. These findings demonstrate an increased percentage of anti-GAPDH Abs in the CSF of patients with MS that can inhibit GAPDH glycolytic enzyme activity and may contribute to neuroaxonal degeneration.

Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography.

The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.

Engineering of human complement component C3 for catalytic inhibition of complement.

As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies.

Functional analysis of Cobra Venom Factor/human C3 chimeras transiently expressed in mammalian cells.

The complement activating venom component Cobra Venom Factor (CVF), a functional and structural homologue of the human complement component C3, forms a stable CVF-dependent C3 convertase complex, which, in contrast to C3-dependent convertase effects continuous activation of the complement and, thereby, decomplementation. In order to elucidate the mechanism underlying the enhanced activity of CVF compared to human C3, we generated two CVF/C3 chimeras and established different affinity-based assay systems for functional analysis of these constructs. To allow for convenient expression and subsequent functional characterisation, the CVF/C3 chimeras as well as CVF and C3 were transiently expressed in mammalian cells. Problems due to the low concentration of the recombinant proteins in the supernatants of transient expressions were circumvented by fusion to peptide tags enabling their efficient immobilisation onto suitable surfaces and subsequent characterisation. In an alternative approach monoclonal antibody fragments generated from a semisynthetic phage display scFv library were employed for concentrating the recombinant proteins by immunoprecipitation. Utilising both approaches all transiently expressed proteins could be characterised for their complement consumption activity. The data obtained with the CVF/C3 chimeras demonstrate that the increased stability of the CVFBb complex is independent of the domains in CVF corresponding to binding sites of factor B and H and the cleavage sites of factor I in the human C3 molecule.

Characterization and screening of IgG binding to the neonatal Fc receptor.

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties.

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.

HuCAL PLATINUM, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems.

This article describes the design of HuCAL (human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production. Gene optimization has been used to improve expression of the antibody master genes in the library context. As a result, full-length IgGs derived from the library show both significant improvements in expression levels and less undesirable glycosylation sites when compared to the previous HuCAL GOLD library. Additionally, in-depth analysis of sequences from public databases revealed that diversity of CDR-H3 is a function of loop length. Based upon this analysis, the relatively uniform diversification strategy used in the CDR-H3s of the previous HuCAL libraries was changed to a length-dependent design, which replicates the natural amino acid distribution of CDR-H3 in the human repertoire. In a side-by-side comparison of HuCAL GOLD and HuCAL PLATINUM, the new library concept led to isolation of about fourfold more unique sequences and to a higher number of high-affinity antibodies. In the majority of HuCAL PLATINUM projects, 100-300 antibodies each having different CDR-H3s are obtained against each antigen. This increased diversity pool has been shown to significantly benefit functional antibody profiling and screening for superior biophysical properties.

B lymphocyte autoimmunity in rheumatoid synovitis is independent of ectopic lymphoid neogenesis.

B lymphocyte autoimmunity plays a crucial role in the pathogenesis of rheumatoid arthritis. The local production of autoantibodies and the presence of ectopic lymphoid neogenesis in the rheumatoid synovium suggest that these dedicated microenvironments resembling canonical lymphoid follicles may regulate the initiation and maturation of B cell autoimmunity. In this study, we assessed experimentally the relevance of ectopic lymphoid neogenesis for B cell autoimmunity by a detailed structural, molecular, and serological analysis of seropositive and seronegative human synovitis. We demonstrate that synovial lymphoid neogenesis is a reversible process associated with inflammation which is neither restricted to nor preferentially associated with autoantibody positive rheumatic conditions. Despite the abundant expression of key chemokines and cytokines required for full differentiation toward germinal center reactions, synovial lymphoid neogenesis in rheumatoid arthritis only occasionally progresses toward fully differentiated follicles. In agreement with that observation, we could not detect Ag-driven clonal expansion and affinity maturation of B lymphocytes. Furthermore, ectopic lymphoid neogenesis is not directly associated with local production of anti-citrullinated protein Abs and rheumatoid factor in the rheumatoid joint. Therefore, we conclude that synovial lymphoid neogenesis is not a major determinant of these rheumatoid arthritis-specific autoantibody responses.

Triosephosphate isomerase- and glyceraldehyde-3-phosphate dehydrogenase-reactive autoantibodies in the cerebrospinal fluid of patients with multiple sclerosis.

Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B cells in the cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) bind to axons in MS brains. To study the axonal Ags involved in MS, we identified the glycolytic enzymes, triosephosphate isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B cells in the CSF and in lesions from MS patients. Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), clinically isolated syndrome (CIS) suggestive of MS (40%), other inflammatory neurological diseases (OIND; 29%), and other noninflammatory neurological diseases (ONIND; 31%). Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%). The coexistence of both autoantibodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND. Two scFv-Abs generated from the CSF and from lesions of a MS brain showed immunoreactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an autoimmune response to neurons and axons in MS.

Complement inactivation by recombinant human C3 derivatives.

From the implications of the complement system in a large number of diseases, an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. In this study we report the design of a novel class of enzymes of human origin that obliterate functional complement by a noninhibitory, catalytic mechanism. Combining the framework of human C3 and the enzymatic mechanism of cobra venom factor, a nontoxic snake venom protein, we established molecules capable of forming stable C3 convertase complexes. Although the half-life of naturally occurring C3 convertase complexes ranges between 1 and 2 min, these complexes exhibit a half-life of up to several hours. Because the overall identity to human C3 could be extended to >90%, the novel C3 derivatives can be assumed to exhibit low immunogenicity and, therefore, represent promising candidates for therapeutic reduction of complement activity in vivo.