Brolucizumab: Evolution through Preclinical and Clinical Studies and the Implications for the Management of Neovascular Age-Related Macular Degeneration.

Improving or maintaining visual acuity is the main goal for the treatment of neovascular age-related macular degeneration (nAMD). Current nAMD standard of care dictates frequent intravitreal (IVT) anti-vascular endothelial growth factor (VEGF) injections, which places a substantial burden on patients, caregivers, and physicians. Brolucizumab, a newly developed anti-VEGF molecule for nAMD treatment, has demonstrated longer durability and improvement in visual and anatomic outcomes in clinical studies in a q12-week regimen, indicating its potential to reduce treatment burden as an important therapeutic tool in nAMD management. This review focuses on the development of brolucizumab and the preclinical and clinical studies evaluating its efficacy, tolerability, and safety. Brolucizumab (also known as "RTH258" and "ESBA1008") is a humanized, single-chain variable fragment (scFv) antibody with a molecular mass of approximately 26 kDa that inhibits VEGF-A. Preclinical studies show that brolucizumab readily penetrates the retina to reach the retinal pigment epithelium (RPE)/choroid with minimal subsequent systemic exposure. The safety, tolerability, and efficacy of a single IVT brolucizumab administration in patients with treatment-naïve nAMD were first demonstrated in the SEE Phase 1/2 study. The OSPREY Phase 2 study showed brolucizumab to be as efficacious as aflibercept in a q8-week regimen with regard to best-corrected visual acuity (BCVA) and brolucizumab achieving greater fluid resolution. Brolucizumab-treated patients in the OSPREY study were subsequently challenged with a q12-week dosing interval, and the outcomes provided key information for the study design and end points of the Phase 3 studies. In the HAWK and HARRIER Phase 3 studies, after 3 monthly loading injections, brolucizumab treatment regimen (q12-week or q8-week) was guided by individual disease activity assessment using functional and anatomic parameters (central subfield thickness [CST], intraretinal fluid [IRF], or subretinal fluid [SRF]) versus aflibercept (q8-week). Fewer brolucizumab 6-mg treated eyes had disease activity versus aflibercept, and anatomic outcome results at weeks 16 and 48 demonstrate brolucizumab as a potent drying agent. Moreover, of patients treated with 6 mg brolucizumab, 55.6% and 51.0% maintained a q12-week dosing interval immediately after the loading phase until week 48 in HAWK and HARRIER, respectively. These Phase 3 studies demonstrated that the brolucizumab q12-week regimen maintains efficacy and safety while reducing treatment burden associated with regular IVT injections for patients with nAMD.

Development of an Interleukin-1ß Vaccine in Patients with Type 2 Diabetes.

Interleukin-1ß (IL-1ß) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1ß activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1ß. The vaccine hIL1bQb consisting of full-length, recombinant IL-1ß coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1ß-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1ß-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1ß and represents a new treatment option for IL-1ß-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).

Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease.

Monoclonal antibodies (mAbs) inhibiting cytokines have recently emerged as new drug modalities for the treatment of chronic inflammatory diseases. Interleukin-17 (IL-17) is a T-cell-derived central mediator of autoimmunity. Immunization with Qß-IL-17, a virus-like particle based vaccine, has been shown to produce autoantibodies in mice and was effective in ameliorating disease symptoms in animal models of autoimmunity. To characterize autoantibodies induced by vaccination at the molecular level, we generated mouse mAbs specific for IL-17 and compared them to germline Ig sequences. The variable regions of a selected hypermutated high-affinity anti-IL-17 antibody differed in only three amino acid residues compared to the likely germline progenitor. An antibody, which was backmutated to germline, maintained a surprisingly high affinity (0.5 nM). The ability of the parental hypermutated antibody and the derived germline antibody to block inflammation was subsequently tested in murine models of multiple sclerosis (experimental autoimmune encephalomyelitis), arthritis (collagen-induced arthritis), and psoriasis (imiquimod-induced skin inflammation). Both antibodies were able to delay disease onset and significantly reduced disease severity. Thus, the mouse genome unexpectedly encodes for antibodies with the ability to functionally neutralize IL-17 in vivo.

Virus-like particles induce robust human T-helper cell responses.

Among synthetic vaccines, virus-like particles (VLPs) are used for their ability to induce strong humoral responses. Very little is reported on VLP-based-vaccine-induced CD4(+) T-cell responses, despite the requirement of helper T cells for antibody isotype switching. Further knowledge on helper T cells is also needed for optimization of CD8(+) T-cell vaccination. Here, we analysed human CD4(+) T-cell responses to vaccination with MelQbG10, which is a Qß-VLP covalently linked to a long peptide derived from the melanoma self-antigen Melan-A. In all analysed patients, we found strong antibody responses of mainly IgG1 and IgG3 isotypes, and concomitant Th1-biased CD4(+) T-cell responses specific for Qß. Although less strong, comparable B- and CD4(+) T-cell responses were also found specific for the Melan-A cargo peptide. Further optimization is required to shift the response more towards the cargo peptide. Nevertheless, the data demonstrate the high potential of VLPs for inducing humoral and cellular immune responses by mounting powerful CD4(+) T-cell help.

Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody.

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (>99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge-charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation-π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine-carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine-carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine-linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.

Immunization against angiotensins for the treatment of hypertension.

Current vaccination approaches against hypertension target angiotensin I and angiotensin II, key components of the renin-angiotensin system. The effectiveness and long-term safety of blockade of the renin-angiotensin system with antihypertensive small-molecule drugs is well documented. Phase I/II testing of the angiotensin I vaccine PMD3117 demonstrated safety and immunogenicity in humans. While angiotensin I-specific antibodies were induced blood pressure was not lowered, presumably due to insufficient antibody levels. A second vaccine, which targets angiotensin II, has been clinically tested. Administration of CYT006-AngQb to subjects with mild to moderate hypertension was safe and well tolerated. After three administrations of 300 microg of the vaccine, ambulatory blood pressure was significantly reduced compared to placebo. The vaccine was particularly effective early in the morning as systolic and diastolic blood pressure were lowered by -25 mm Hg and -13 mm Hg, respectively. Further studies are required to show long-term safety and to assess how robust and long-lived the blood pressure reduction is. It will also be important to ascertain whether the strong reduction of blood pressure in the early morning, when most cardiovascular events occur, might result in long-term benefits over current therapies.

Isolation of human monoclonal antibodies by mammalian cell display.

Due to their low immunogenicity in patients, humanized or fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infections, and immune disorders. Here, we describe a technology allowing for the rapid isolation of fully human mAbs. In contrast to previously described methods, B cells specific for an antigen of interest are directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant, antigen-specific single-chain Fv (scFv) libraries are generated from this pool of B cells and screened by mammalian cell surface display by using a Sindbis virus expression system. This method allows isolating antigen-specific antibodies by a single round of FACS. The variable regions (VRs) of the heavy chains (HCs) and light chains (LCs) are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this manner, several hypermutated high-affinity antibodies binding the Qbeta virus like particle (VLP), a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high expression levels in cell culture. The human nicotine-specific mAbs were validated preclinically in a mouse model. Thus, the technology presented here allows for rapid isolation of high-affinity, fully human antibodies with therapeutic potential from human volunteers.

A vaccine against nicotine for smoking cessation: a randomized controlled trial.

BACKGROUND: Tobacco dependence is the leading cause of preventable death and disabilities worldwide and nicotine is the main substance responsible for the addiction to tobacco. A vaccine against nicotine was tested in a 6-month randomized, double blind phase II smoking cessation study in 341 smokers with a subsequent 6-month follow-up period. METHODOLOGY/PRINCIPAL FINDINGS: 229 subjects were randomized to receive five intramuscular injections of the nicotine vaccine and 112 to receive placebo at monthly intervals. All subjects received individual behavioral smoking cessation counseling. The vaccine was safe, generally well tolerated and highly immunogenic, inducing a 100% antibody responder rate after the first injection. Point prevalence of abstinence at month 2 showed a statistically significant difference between subjects treated with Nicotine-Qbeta (47.2%) and placebo (35.1%) (P = 0.036), but continuous abstinence between months 2 and 6 was not significantly different. However, in subgroup analysis of the per-protocol population, the third of subjects with highest antibody levels showed higher continuous abstinence from month 2 until month 6 (56.6%) than placebo treated participants (31.3%) (OR 2.9; P = 0.004) while medium and low antibody levels did not increase abstinence rates. After 12 month, the difference in continuous abstinence rate between subjects on placebo and those with high antibody response was maintained (difference 20.2%, P = 0.012). CONCLUSIONS: Whereas Nicotine-Qbeta did not significantly increase continuous abstinence rates in the intention-to-treat population, subgroup analyses of the per-protocol population suggest that such a vaccination against nicotine can significantly increase continuous abstinence rates in smokers when sufficiently high antibody levels are achieved. Immunotherapy might open a new avenue to the treatment of nicotine addiction. TRIAL REGISTRATION: Swiss Medical Registry 2003DR2327; ClinicalTrials.gov NCT00369616.

Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study.

BACKGROUND: Hypertension can be controlled adequately with existing drugs such as angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Nevertheless, treatment success is often restricted by patients not adhering to treatment. Immunisation against angiotensin II could solve this problem. We investigated the safety and efficacy of CYT006-AngQb-a vaccine based on a virus-like particle-that targets angiotensin II to reduce ambulatory blood pressure. METHODS: In this multicentre, double-blind, randomised, placebo-controlled phase IIa trial, 72 patients with mild-to-moderate hypertension were randomly assigned with a computer-generated randomisation list to receive subcutaneous injections of either 100 mug CYT006-AngQb (n=24), 300 mug CYT006-AngQb (24), or placebo (24), at weeks 0, 4, and 12. 24-h ambulatory blood pressure was measured before treatment and at week 14. The primary outcomes were safety and tolerability. Analyses were done by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00500786. FINDINGS: Two patients in the 100 mug group, three in the 300 mug group, and none in the placebo group discontinued study treatment. All patients were included in safety analyses; efficacy analyses did not include the five dropouts, for whom no data were available at week 14. Five serious adverse events were reported (two in the 100 mug group, two in the 300 mug group, and one in the placebo group); none were deemed to be treatment related. Most side-effects were mild, transient reactions at the injection site. Mild, transient influenza-like symptoms were seen in three patients in the 100 mug group, seven in the 300 mug group, and none in the placebo group. In the 300 mug group, there was a reduction from baseline in mean ambulatory daytime blood pressure at week 14 by -9.0/-4.0 mm Hg compared with placebo (p=0.015 for systolic and 0.064 for diastolic). The 300 mug dose reduced the early morning blood-pressure surge compared with placebo (change at 0800 h -25/-13 mm Hg; p<0.0001 for systolic, p=0.0035 for diastolic). INTERPRETATION: Immunisation with CYT006-AngQb was associated with no serious adverse events; most observed adverse events were consistent with local or systemic responses similar to those seen with other vaccines. The 300 mug dose reduced blood pressure in patients with mild-to-moderate hypertension during the daytime, especially in the early morning. FUNDING: Cytos Biotechnology AG.

Vaccination against nicotine: an emerging therapy for tobacco dependence.

Tobacco dependence is an addiction characterised by compulsive drug-seeking and drug-taking behavior and intensive craving in the absence of tobacco. Nicotine is the major addictive component of tobacco and acts on the reward system in the brain. Together with strong conditional reinforcements, unaided smoking cessation attempts are notoriously unsuccessful and even the most recently introduced pharmacotherapy, varenicline, only achieves a 23% continuous abstinence rate after 1 year. Vaccination against nicotine represents a promising novel concept for treating nicotine addiction. Antibodies against nicotine inhibit the passage of nicotine to brain and thus inhibit its addiction-reinforcing activities. There are three nicotine vaccines that are in clinical development. The first proof-of-concept study in smoking cessation with the vaccine NicQb (Cytos Biotechnology), a nicotine vaccine based on virus-like particles, demonstrated that continuous abstinence rates can be significantly increased by vaccination; however, as expected from the mode of action, a sufficient antibody level had to be achieved. Antibody level dependence of abstinence was also observed with the nicotine vaccine NicVAX (Nabi Biopharmaceuticals). Vaccination against nicotine has the potential of becoming an important therapy against tobacco dependence.

TLR9 signaling in B cells determines class switch recombination to IgG2a.

Although IgG2a is the most potent Ab isotype in the host response to viral and bacterial infections, the regulation of class switch recombination to IgG2a in vivo is not yet well understood. Recognition of pathogen-associated molecular patterns by dendritic cells expressing TLRs, like TLR7, recognizing ssRNA, or TLR9, recognizing DNA rich in nonmethylated CG motifs (CpG), favors induction of Th1 responses. It is generally assumed that these Th1 responses are responsible for the TLR-mediated induction of IgG2a. Using virus-like particles loaded with CpGs, we show here that TLR9 ligands can directly stimulate B cells to undergo isotype switching to IgG2a. Unexpectedly, TLR9 expression in non-B cells did not affect isotype switching in the Ab response against virus-like particles. Thus, TLR9 can regulate isotype switching to IgG2a directly by interacting with B cells rather than indirectly by inducing Th1 responses.

A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity.

BACKGROUND: Despite the availability of efficacious drugs, the success of treating hypertension is limited by patients' inconsistent drug intake. Immunization against angiotensin II may offer a valuable alternative to conventional drugs for the treatment of hypertension, because vaccines induce relatively long-lasting effects and do not require daily dosing. Here we describe the preclinical development and the phase I clinical trial testing of a virus-like particle (VLP)-based antihypertensive vaccine. METHODS AND RESULTS: An angiotensin II-derived peptide was conjugated to the VLP Qbeta (AngQb). AngQb was highly immunogenic in mice and rats. To test for efficacy, spontaneously hypertensive rats (SHR) were immunized with 400 microg AngQb or VLP alone. Group mean systolic blood pressure (SBP) was reduced by up to 21 mmHg (159 +/- 2 versus 180 +/- 5 mmHg, P < 0.001), and total angiotensin II levels (antibody-bound and free) were increased ninefold (85 +/- 20 versus 9 +/- 1 pmol/l, P = 0.002) compared with VLP controls. SHR treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril (1 mg/kg per day by mouth) reached an SBP of 155 +/- 2 mmHg. Twelve healthy volunteers of a placebo-controlled randomized phase I trial were injected once with 100 microg AngQb. Angiotensin II-specific antibodies were raised in all subjects (100% responder rate) and AngQb was well tolerated. CONCLUSIONS: AngQb reduces blood pressure in SHR to levels obtained with an ACE inhibitor, and is immunogenic and well tolerated in humans. Therefore, vaccination against angiotensin II has the potential to become a useful antihypertensive treatment providing long-lasting effects and improving patient compliance.

Therapeutic vaccines for nicotine dependence.

Smoking remains the leading cause of preventable death worldwide. Despite the development of a number of drugs for smoking cessation, overall efficacy of these substances is limited and the majority of smokers fail to quit smoking long-term. These drugs aim to help people quit by either replacing nicotine with an alternative source of nicotine (eg, patches or gums) or by reducing the withdrawal symptoms. A vaccine against nicotine has a different strategy: by inducing sufficient nicotine-specific antibodies, it is possible to sequester the drug in the blood and prevent it from entering the brain. In this way, the addictive properties of cigarettes are eliminated and smokers attempting to quit may be able to smoke one or two cigarettes without becoming hooked again. Essentially, a vaccine against nicotine targets the progression from lapses to full relapse rather than withdrawal symptoms. Recent research with vaccines against nicotine has clearly demonstrated in animals that antibodies can interfere with the addictive properties of nicotine in different settings. The first phase II clinical trial has confirmed the validity of the concept and shown that a vaccine against nicotine can be efficacious for smoking cessation in humans provided anti-nicotine antibody levels are sufficiently high.

Protection against osteoporosis by active immunization with TRANCE/RANKL displayed on virus-like particles.

TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-kappaB ligand (RANKL), is the key molecule responsible for the bone loss observed in osteoporosis. Passive administration of osteoprotegerin, the soluble decoy receptor of TRANCE/RANKL, is efficient in blocking disease progression, but may not find widespread clinical use due to patient compliance problems and the expected high costs. In this study, we describe an efficient, safe, and potentially cost-effective active immunization strategy against TRANCE/RANKL. We show in mice that immunization with TRANCE/RANKL covalently linked to virus-like particles can overcome the natural tolerance of the immune system toward self proteins and produce high levels of specific Abs without the addition of any adjuvant. Serum Abs of immunized mice neutralized TRANCE/RANKL activity in vitro and were highly active in preventing bone loss in a mouse model of osteoporosis. Active immunization against TRANCE/RANKL was essentially reversible and did not produce any measurable immunosuppressive side effects, underscoring its potential as a new therapeutic approach to the treatment of human bone-degenerative disorders.

A therapeutic vaccine for nicotine dependence: preclinical efficacy, and Phase I safety and immunogenicity.

Nicotine is the principal addictive component in tobacco, and following uptake acts in the central nervous system. The smoking-cessation efforts of most smokers fail because a single slip often delivers sufficient nicotine to the brain to reinstate the drug-seeking behaviour. Blocking nicotine from entering the brain by induction of specific antibodies may be an effective means to prevent such relapses. The hapten nicotine was coupled to virus-like particles (VLP) formed by the coat protein of the bacteriophage Qb. In preclinical experiments, this Nicotine-Qb VLP (NicQb) vaccine induced strong antibody responses. After intravenous nicotine challenge, vaccinated mice exhibited strongly reduced nicotine levels in the brain compared with control mice. In a phase I study, 32 healthy non-smokers were immunized with NicQb. The vaccine was safe and well-tolerated. All volunteers who received NicQb showed nicotine-specific IgM antibodies at day 7 and nicotine-specific IgG antibodies at day 14. Antibody levels could be boosted by a second injection or the addition of Alum as an adjuvant and the antibodies had a high affinity for nicotine. These data suggest that antibodies induced by NicQb may prevent relapses by sequestering nicotine in the blood of immunized smokers.

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity.

We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein.

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.

Structural characterization of TSC-36/Flik: analysis of two charge isoforms.

Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties.

Characterization of a pseudoachondroplasia-associated mutation (His587-->Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP).

We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.

Characterization of SMOC-2, a modular extracellular calcium-binding protein.

We have isolated the novel gene SMOC-2, which encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain found only in the homologous SMOC-1. Phylogenetic analysis of the calcium-binding domain sequences showed that SMOC-1 and -2 form a separate group within the BM-40 family. The human and mouse SMOC-2 sequences are coded for by genes consisting of 13 exons located on chromosomes 6 and 17, respectively. Analysis of recombinantly expressed protein showed that SMOC-2 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots and reverse transcription PCR revealed a widespread expression in many tissues.