Inhibition of complement C1s improves severe hemolytic anemia in cold agglutinin disease: a first-in-human trial.

Cold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase >2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement-mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.

C1s Inhibition by BIVV009 (Sutimlimab) Prevents Complement-Enhanced Activation of Autoimmune Human B Cells In Vitro.

The classical pathway of complement (CP) can mediate C3 opsonization of Ags responsible for the costimulation and activation of cognate B lymphocytes. In this manner, the complement system acts as a bridge between the innate and adaptive immune systems critical for establishing a humoral response. However, aberrant complement activation is often observed in autoimmune diseases in which C3 deposition on self-antigens may serve to activate self-reactive B cell clones. In this study, we use BIVV009 (Sutimlimab), a clinical stage, humanized mAb that specifically inhibits the CP-specific serine protease C1s to evaluate the impact of upstream CP antagonism on activation and proliferation of normal and autoimmune human B cells. We report that BIVV009 significantly inhibited complement-mediated activation and proliferation of primary human B cells. Strikingly, CP antagonism suppressed human Ig-induced activation of B cells derived from patients with rheumatoid arthritis. These results suggest that clinical use of CP inhibitors in autoimmune patients may not only block complement-mediated tissue damage, but may also prevent the long-term activation of autoimmune B cells and the production of autoantibodies that contribute to the underlying pathologic condition of these diseases.

Recruitment of Human C1 Esterase Inhibitor Controls Complement Activation on Blood Stage Plasmodium falciparum Merozoites.

The complement system is a front-line defense system that opsonizes and lyses invading pathogens. To survive, microbes exposed to serum must evade the complement response. To achieve this, many pathogens recruit soluble human complement regulators to their surfaces and hijack their regulatory function for protection from complement activation. C1 esterase inhibitor (C1-INH) is a soluble regulator of complement activation that negatively regulates the classical and lectin pathways of complement to protect human tissue from aberrant activation. In this article, we show that Plasmodium falciparum merozoites, the invasive form of blood stage malaria parasites, actively recruit C1-INH to their surfaces when exposed to human serum. We identified PfMSP3.1, a member of the merozoite surface protein 3 family of merozoite surface proteins, as the direct interaction partner. When bound to the merozoite surface, C1-INH retains its ability to complex with and inhibit C1s, MASP1, and MASP2, the activating proteases of the complement cascade. P. falciparum merozoites that lack PfMSP3.1 showed a marked reduction in C1-INH recruitment and increased C3b deposition on their surfaces. However, these ΔPfMSP3.1 merozoites exhibit enhanced invasion of RBCs in the presence of active complement. This study characterizes an immune-evasion strategy used by malaria parasites and highlights the complex relationship between merozoites and the complement system.

Anti-C1s monoclonal antibody BIVV009 in late antibody-mediated kidney allograft rejection-results from a first-in-patient phase 1 trial.

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of 4 weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During 7 weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of 8 C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.

Effect of the Anti-C1s Humanized Antibody TNT009 and Its Parental Mouse Variant TNT003 on HLA Antibody-Induced Complement Activation-A Preclinical In Vitro Study.

The classic pathway (CP) of complement is believed to significantly contribute to alloantibody-mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti-C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody-triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen-coated flow beads or HLA-mismatched aortic endothelial cells and splenic lymphocytes. Anti-C1s antibodies profoundly inhibited C3 activation at concentrations >20 µg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti-C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody-triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement-mediated tissue injury in sensitized transplant recipients.

TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2.

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro. In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro.

Pathogenesis and laboratory diagnosis of hereditary angioedema.

Hereditary angioedema (HAE) was first described in the 19th century. Over the past 50 years, many details of the pathophysiology and molecular biology of HAE have been elucidated. Two types of HAE, type I and type II, result from mutations in the gene for the broad-spectrum protease inhibitor C1 inhibitor (C1INH). Type I HAE is characterized by low antigenic and functional C1INH levels and type II HAE has normal antigenic but low functional C1INH levels. Type III HAE, by contrast, has normal antigenic and functional C1INH levels. In some families, type III HAE has been linked to mutations in Hageman factor. C1INH is the primary inhibitor of the complement proteases C1r and C1s as well as the contact system proteases activated Hageman factor (coagulation factor XIIa and XIIf) and plasma kallikrein. It is also an inhibitor of plasmin and coagulation factor XIa. The primary mediator of swelling in HAE has now been unequivocally shown to be bradykinin, generated from activation of the plasma contact system. The knowledge gained concerning the underlying mechanisms of the different types of HAE allow the clinician to approach the laboratory diagnosis with confidence and provides opportunities for novel therapeutic strategies.

Genetic analysis of complement C1s deficiency associated with systemic lupus erythematosus highlights alternative splicing of normal C1s gene.

Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of C1r has been observed to occur concomitantly with deficiency in C1s and 9 out of 15 reported cases presented systemic lupus erythematosus (SLE). Here, we describe a family in which all four children are deficient in C1s but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children's sera. C1s was undetectable, while in the parents' sera it was lower than in the normal controls. The levels of C1r observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of C1s synthesis was observed in the patients' fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the C1s cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of C1s mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3' splice site within intron 1 which increases the size of exon 2 by 87 nucleotides.

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes.

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

Update on C1 Esterase Inhibitor in Human Solid Organ Transplantation.

Complement plays important roles in both ischemia-reperfusion injury (IRI) and antibody-mediated rejection (AMR) of solid organ allografts. One approach to possibly improve outcomes after transplantation is the use of C1 inhibitor (C1-INH), which blocks the first step in both the classical and lectin pathways of complement activation and also inhibits the contact, coagulation, and kinin systems. C1-INH can also directly block leukocyte-endothelial cell adhesion. C1-INH contrasts with eculizumab and other distal inhibitors, which do not affect C4b or C3b deposition or noncomplement pathways. Authors of reports on trials in kidney transplant recipients have suggested that C1-INH treatment may reduce IRI and delayed graft function, based on decreased requirements for dialysis in the first month after transplantation. This effect was particularly marked with grafts with Kidney Disease Profile Index ≥ 85. Other clinical studies and models suggest that C1-INH may decrease sensitization and donor-specific antibody production and might improve outcomes in AMR, including in patients who are refractory to other modalities. However, the studies have been small and often only single-center. This article reviews clinical data and ongoing trials with C1-INH in transplant recipients, compares the results with those of other complement inhibitors, and summarizes potentially productive directions for future research.

Human astrovirus coat protein: a novel C1 inhibitor.

C1 is a multimolecular complex that initiates the classical pathway of complement. It is composed of the pattern recognition component C1q and the serine proteases C1r and C1s. Activation of C1 elicits a series of potent effector mechanisms directed at limiting infection by invading pathogens as well as participating in other biological functions such as immune tolerance. While many molecules in addition to antibody have been demonstrated to activate C1, only a handful of C1 inhibitors have been described. Disregulated control of complement activation is associated with numerous autoimmune and inflammatory disease processes, thus tight regulation of C1 activation is highly desirable. We have recently discovered a novel inhibitor of C1, the coat protein of the human astroviruses, a family of enteric pathogens that infect young children. The astrovirus coat protein binds to the A-chain of C1q and inhibits spontaneous as well as antibody-mediated activation of the C1 complex resulting in suppression of classical pathway activation and complement-mediated terminal effector functions. This is the first description of a non-enveloped icosahedral virus inhibiting complement activation and the first description of a viral inhibitor of C1. The known inhibitors of C1 are reviewed and then discussed in the context of this novel viral C1 inhibitor. Additionally, the properties of this compound are elucidated highlighting its potential as an anti-complement therapeutic for the many diseases associated with inappropriate complement activation.

A Randomized, First-in-Human, Healthy Volunteer Trial of sutimlimab, a Humanized Antibody for the Specific Inhibition of the Classical Complement Pathway.

Aberrant activation of the classical complement pathway is the common underlying pathophysiology of orphan diseases such as bullous pemphigoid, antibody-mediated rejection of organ transplants, cold agglutinin disease, and warm autoimmune hemolytic anemia. Therapeutic options for these complement-mediated disorders are limited and sutimlimab, a humanized monoclonal antibody directed against complement factor C1s, may be potentially useful for inhibition of the classical complement pathway. A phase I, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of sutimlimab or placebo was conducted in 64 volunteers to evaluate safety, tolerability, pharmacokinetic, and pharmacodynamic profiles. Single and multiple infusions of sutimlimab were well tolerated without any safety concerns. sutimlimab exhibited a steep concentration-effect relationship with a Hill coefficient of 2.4, and an IC90 of 15.5 µg/mL. This study establishes the foundation for using sutimlimab as a highly selective inhibitor of the classical complement pathway in different diseases.

Blockade of HLA Antibody-Triggered Classical Complement Activation in Sera From Subjects Dosed With the Anti-C1s Monoclonal Antibody TNT009-Results from a Randomized First-in-Human Phase 1 Trial.

BACKGROUND: Complement may play a key role in antibody-mediated rejection. A promising therapeutic approach may be classical pathway (CP) inhibition at the level of early component C1. METHODS: In this first-in-human, double-blind, randomized placebo-controlled phase 1 trial, we evaluated the safety and complement inhibitory effect of TNT009, a humanized monoclonal anti-C1s antibody. Sixty-four adult healthy volunteers received either single (n = 48; 7 consecutive cohorts, 0.3-100 mg/kg) or 4 weekly infusions (n = 16; 2 consecutive cohorts, 30 and 60 mg/kg per infusion) of TNT009 or placebo. To assess the effect of treatment on complement activity, sera from dosed subjects were analyzed in a CP activation assay evaluating C3d deposition on HLA-coated microbeads spiked with alloantibodies. RESULTS: Single doses of TNT009 at 3 to 100 mg/kg uniformly and profoundly inhibited HLA antibody-mediated C3d deposition (≥86% after 60 minutes), whereby the duration of CP inhibition (2-14 days) was dose-dependent. Four weekly doses persistently blocked complement for 5 to 6 weeks. Ex vivo serum CP activity was profoundly inhibited when TNT009 concentrations exceeded 20 µg/mL. Infusions were well tolerated without serious or severe adverse events. CONCLUSIONS: Treatment with TNT009 was safe and potently inhibited CP activity. Future studies in patients are required to assess the potential of TNT009 for preventing or treating antibody-mediated rejection.

Purification and characterization of recombinant hamster tissue complement C1s.

Hamster complement C1s cDNA was inserted into expression plasmid BCMGSNeo, and transfected to SEA7 cells, A31 mouse fibroblasts transformed by polyoma virus. The transfectant secreted a large amount of recombinant C1s that was activated in the serum free culture medium and hydrolyzed acetyl-Gly-L-Lys-naphthyl ester (AGLNE). C1s was purified to a homogeneity from the culture medium of the transfectant by DEAE-Sephadex, Dymatrex orange A and size-exclusion HPLC. Purified hamster C1s consumed human complement in hemolytic assay and hydrolyzed gelatin in enzymography. To investigate the enzymic action of C1s at molecular levels, several antibodies were prepared against hamster C1s. One peptide (amino-acid residues 379-391) and two peptides (amino-acid residues 478-496 and 560-583) corresponding to the heavy and the light chain, respectively, were synthesized. The amino-acid sequences of these regions is not conserved between hamster and human C1s. Antibodies against these peptides were raised in rabbits. The anti-peptide antibodies bound specifically to hamster serum and recombinant C1s but not to human C1s. They inhibited the esterase activity of recombinant C1s to varying degrees depending on each antibody's binding site.

Immune complex independent activation of complement, C1s secreted from hamster embryo malignant fibroblasts, Nil2C2 in serum free culture medium.

Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of C1s.

Acceleration of site-to-site transfer of C1- by a monoclonal antibody to C1-s.

A monoclonal antibody to human C1-s (a subcomponent of C1-), M365 blocked the complement-mediated lysis of C1(-)-coupled IgM-sensitized sheep erythrocytes (EAIgMC1). However, M365, via its binding to C1-s, did not inhibit C2 activation and only partially inhibited C4 activation, both attributable to the proteolytic action of C1s. Therefore, the inhibition of lysis of EAIgMC1 is not due to the direct effect of antibody binding to C1-s in the C1- molecule. M365, when added to the pre-formed EAIgMC1, deprived the whole C1 molecule from the cells. It was also found that M365-bound C1 could not bind to EAIgM. These phenomena were induced only when M365, one of seven monoclonal antibodies to C1-s, was employed and when IgM antibody-sensitized E was used. The observed C1- liberation and C1 binding inhibition by M365 were found to be less effective when the C4b bearing cells, EAIgMC4, were used. But the addition of M365-bound C1- to EAIgMC4 promoted the formation of the C3 convertase, C4b2a, which caused an incremental lysis of the C4b bearing cells. The results are interpreted to mean that C1-, once complexed with M365, still remains active and acquires the ability to transfer from one C1 binding site on IgM to another to activate the convertases. C4b must be a prerequisite on the cells to induce the effective C1- transfer and resulting increase of C3 convertase sites. We hypothesize that the M365-C1-s association alters the conformation of C1q and thereby leads to the dissociation of whole C1- from IgM. The M365-C1- complex, therefore, can move from site to another.

C1-inhibitor prevents PEG fractionation-induced, EDTA-resistant activation of mouse complement.

Fractionation of mouse serum by precipitation with a critical amount of polyethylene glycol 6000 (PEG; 11% w/v) results in a classical and alternative pathway-independent activation of the terminal complement route. The activation can take place after the separation of an activating principle together with the terminal route components from a natural regulator. The isolation and identification of the regulatory component preventing this activation in serum, is subject of this paper. The regulator was purified by fractionated PEG-precipitation (15-25%), followed by heparin-Sepharose affinity, Mono Q anion-exchange, and Superose 12 gel filtration chromatography. The regulator appeared to be a single-chain protein with a Mr of 96 k. A protein with similar activity purified from human serum had a Mr of 104 k and was functionally and antigenically indistinguishable from C1-INH. The mouse 96 k protein inhibited C1-esterase activity indicating that this protein is indeed C1-INH. Mouse C1-INH regulates the PEG fractionation-induced bypass activation of complement, but does not interfere with the assembly or the lytic activity of membrane attack complexes. alpha 2-Macroglobulin appeared also to be capable of inhibiting the PEG-precipitation-induced activation process, but with lower efficiency.

Complement classical pathway expression by human skeletal myoblasts in vitro.

Human myoblasts express immunological properties in vitro and we have previously reported that they produce Complement (C) components of the alternative pathway. Myoblasts activate the classical pathway but are fully protected against C attack by the expression of major C regulators. In order to fully understand the relationship between myoblasts and C, we here report the biosynthesis of C components of the classical pathway by skeletal muscle cells. Human myoblasts in vitro produced C1q, C1r, C1s, C2 and C4 constitutively and all syntheses were upregulated after stimulation with IFN-gamma. We suggest that human myoblasts may constitute a local source of C and therefore C could be implicated in inflammatory or physiopathological processes developed in skeletal muscle.

A rapid and efficient method for the purification of the complement subcomponents C1r and C1s in zymogen form using fast protein chromatography.

The purification of the subcomponents C1r and C1s of the first component of complement involves multiple steps and is time-consuming. This accounts for the frequently observed partial activation of the subcomponents. In this report we propose a simplified procedure of purification using a batch method and fast protein chromatography avoiding a shift of pH. The method provides C1r and C1s in a yield of 35 and 60% respectively. In addition, this study provides a simple and sensitive test to assess functional purity of C1r and C1s with respect to the other C1 subcomponents.