Toward complement inhibition 2.0: Next generation anticomplement agents for paroxysmal nocturnal hemoglobinuria

Therapeutic complement inhibition by eculizumab has revolutionized the treatment of paroxysmal nocturnal hemoglobinuria (PNH) with a major impact on its natural history. Nevertheless, emerging unmet clinical needs may benefit from the development of novel complement inhibitors. Novel strategies of complement inhibition exploit different agents targeting C5, as well as compound intercepting the complement cascade at the level of its key component C3, or even upstream at the level of components involved in complement alternative pathway initiation. Many of these agents are already in their clinical development; preliminary data together with a deep understanding of PNH biology may help to anticipate their possible clinical effect. Novel anti‐C5 agents include monoclonal antibodies (even long‐lasting) as well as other small molecules bioavailable by subcutaneous administration; an anti‐C5 small interfering RNA has been developed too. All these anti‐C5 agents seem to recapitulate safety and efficacy of current eculizumab treatment; their main improvement pertains to better patient's convenience due to longer dosing interval and/or possible subcutaneous self‐administration. The possibility of achieving a deeper C5 inhibition has been shown as well, but its actual clinical meaning remains to be elucidated. Upstream complement inhibitors include the anti‐C3 small peptide compstatin (and its derivatives), and small inhibitors of complement factor D or complement factor B. This class of compounds anticipates a possible efficacy in prevention of C3‐mediated extravascular hemolysis, in addition to inhibition of intravascular hemolysis, eventually leading to improved hematological responses. The availability of all these compounds will result soon in a substantial improvement of PNH management.     

Origin of the classical complement pathway: Lamprey orthologue of mammalian C1q acts as a lectin

The lectin complement pathway in innate immunity is closely related to the classical complement pathway in adaptive immunity, with respect to the structures and functions of their components. Both pathways are initiated by complexes consisting of collagenous proteins and serine proteases of the mannose-binding lectin (MBL)-associated serine protease (MASP)/C1r/C1s family. It has been speculated that the classical pathway emerged after the lectin pathway, and that the activation mechanism of the latter was partially conserved. The classical and lectin pathways can be traced back to at least cartilaginous fish and ascidian (urochordata), respectively. To elucidate the evolution of the complement system, we isolated and characterized a GlcNAc-binding lectin from sera of lamprey (agnathans), the most primitive vertebrate that lacks the classical pathway. Lamprey GlcNAc-binding lectin was an oligomer consisting of 24-kDa subunits. cDNA and phylogenetic analyses revealed that the lamprey GlcNAc-binding lectin is an orthologue of mammalian C1q, a collagenous subcomponent of the first component involved in binding to immunoglobulins in the classical pathway. Lamprey C1q copurified with MASP-A, a serine protease of the MASP/C1r/C1s family, which exhibited proteolytic activity against lamprey C3. Surface plasmon resonance analysis showed that lamprey C1q specifically bound to GlcNAc, but not various other carbohydrates tested. These results suggest that C1q may have emerged as a lectin and may have functioned as an initial recognition molecule of the complement system in innate immunity before the establishment of adaptive immunity such as immunoglobulins in the cartilaginous fish.     

Anti–cyclic citrullinated peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways

Objective

It has been suggested that anti–citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). To exert their pathologic effects, ACPAs must recruit immune effector mechanisms such as activation of the complement system. Mouse models of RA have shown that, surprisingly, arthritogenic antibodies activate the alternative pathway of complement rather than the expected classical pathway. This study was undertaken to investigate whether human anti–cyclic citrullinated peptide (anti‐CCP) antibodies activate the complement system in vitro and, if so, which pathways of complement activation are used.

Methods

We set up novel assays to analyze complement activation by anti‐CCP antibodies, using cyclic citrullinated peptide–coated plates, specific buffers, and normal and complement‐deficient sera as a source of complement.

Results

Anti‐CCP antibodies activated complement in a dose‐dependent manner via the classical pathway of complement, and, surprisingly, via the alternative pathway of complement. The lectin pathway was not activated by anti‐CCP antibodies. Complement activation proceeded in vitro up to the formation of the membrane attack complex, indicating that all activation steps, including the release of C5a, took place.

Conclusion

Our findings indicate that anti‐CCP antibodies activate the complement system in vitro via the classical and alternative pathways but not via the lectin pathway. These findings are relevant for the design of interventions aimed at inhibition of complement‐mediated damage in RA.

     

Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo

Toll-like receptors (TLRs) and complement are 2 components of innate immunity that are critical for first-line host defense and elicitation of adaptive immune responses. Many pathogen-associated molecular patterns activate both TLR and complement, but whether and how these 2 systems, when coactivated in vivo, interact with each other has not been well studied. We demonstrate here a widespread regulation of TLR signaling by complement in vivo. The TLR ligands lipopolysacharride (TLR4), zymosan (TLR2/6), and CpG oligonucleotide (TLR9) caused, in a complement-dependent manner, strikingly elevated plasma interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-1beta, and/or decreased plasma IL-12 levels in mice deficient in the membrane complement inhibitor decay-accelerating factor (DAF). A similar outcome was observed in wild-type mice cotreated with the TLR ligands and cobra venom factor, a potent complement activator. The regulatory effect of complement on TLR-induced cytokine production in vivo was mediated by the anaphylatoxin receptors C5aR and C3aR. Additionally, changes in lipopolysaccharide (LPS)-induced cytokine production in DAF-deficient mice correlated with increased mitogen-activated protein kinase and nuclear factor-kappaB activation in the spleen. These results reveal a strong interaction between complement and TLR signaling in vivo and suggest a novel mechanism by which complement promotes inflammation and modulates adaptive immunity.     

Autoantibody-mediated complement activation on platelets is a common finding in patients with immune thrombocytopenic purpura (ITP)

Background: It is commonly accepted that antibody‐mediated removal of platelets represents a major mechanism of platelet destruction in immune thrombocytopenic purpura (ITP). Although complement activation may participate in platelet clearance, frequency and specificity of complement activation have not yet been studied systematically in ITP. Patients and methods: We examined blood samples from 240 patients with ITP. Samples were assessed for the presence of free and bound platelet autoantibodies by a standard glycoprotein‐specific assay (monoclonal antibody‐specific immobilization of platelet antigens). The ability of all sera to fix complement to a panel of human platelets was investigated in a complement fixation (CF) assay. Fixation of C1q to isolated GP IIb/IIIa was assessed by flow cytometry. Results: Glycoprotein‐specific autoantibodies were detected as platelet‐bound antibodies in 129 (54%) and as additional free antibodies in 26 (11%) and were undetectable in 111 (46%) patients. Assessing these subgroups for CF, 103 (65%), 21 (81%), and 33 (30%) sera gave positive results. If GP IIb/IIIa was absent from the test platelets, 81 (67%) lost their ability to fix complement; if GP Ib/IX was absent, 37 (30%) lost their ability to fix complement. C1q fixation to immunobeads coated with GP IIb/IIIa was observed in 50% of sera containing anti‐GP IIb/IIIa antibodies. Conclusions: In a significant number of patients with chronic ITP, platelet autoantibodies are capable of activating the classical complement pathway. CF is even present in ITP sera without detectable autoantibodies, indicating that current techniques for autoantibody detection may be insufficient. The major targets for complement‐fixing autoantibodies in ITP are GP IIb/IIIa and GP Ib/IX.     

Defining the complement C3 binding site and the antigenic region of Haemonchus contortus GAPDH

Haemonchus contortus is an economically important parasite that survives the host defense system by modulating the immune response. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is secreted by the parasite and the host responds by producing anti‐enzyme antibodies. The enzyme inhibits complement cascade, an arm of the innate immunity, by binding to complement C3. In this study, the C3 binding site and the antigenic region of the enzyme were identified by generating short recombinant fragments and deleting a defined region of the enzyme. Using these proteins in ligand overlay and plate binding assay, the C3 binding region of GAPDH was localized within the 38 residues represented by 77‐114 amino acids whereas one of the antigenic regions was identified in between 77 and 171 amino acids. In addition, deletion of amino acids 77 to 171 from GAPDH (fragment AB) also showed weak immunogenicity but lacked C3 binding activity. Fragment D comprising 95 residues (77‐171), had both the C3 binding activity as well as immunogenicity like the parent enzyme, also stimulated host peripheral blood mononuclear cells in vitro. This truncated GAPDH moiety was stable at refrigerated temperature for at least 12 weeks and appears as a promising new therapeutic tool considering its longer shelf life as compared to the parent protein.     

The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice

The complement system is an important component of the innate immune response to bacterial pathogens, including Streptococcus pneumoniae. The classical complement pathway is activated by antibody-antigen complexes on the bacterial surface and has been considered predominately to be an effector of the adaptive immune response, whereas the alternative and mannose-binding lectin pathways are activated directly by bacterial cell surface components and are considered effectors of the innate immune response. Recently, a role has been suggested for the classical pathway during innate immunity that is activated by natural IgM or components of the acute-phase response bound to bacterial pathogens. However, the functional importance of the classical pathway for innate immunity to S. pneumoniae and other bacterial pathogens, and its relative contribution compared with the alternative and mannose-binding lectin pathways has not been defined. By using strains of mice with genetic deficiencies of complement components and secretory IgM we have investigated the role of each complement pathway and natural IgM for innate immunity to S. pneumoniae. Our results show that the proportion of a population of S. pneumoniae bound by C3 depends mainly on the classical pathway, whereas the intensity of C3 binding depends on the alternative pathway. Furthermore, the classical pathway, partially targeted by the binding of natural IgM to bacteria, is the dominant pathway for activation of the complement system during innate immunity to S. pneumoniae, loss of which results in rapidly progressing septicemia and impaired macrophage activation. These data demonstrate the vital role of the classical pathway for innate immunity to a bacterial pathogen.     

The local production of complement in the pathogenesis of renal inflammation

The deposition of complement components is a feature of many immune mediated human glomerular diseases. Experimental models provide evidence that complement activation within the glomerulus has a pathogenic role in immune complex and antibody mediated glomerulonephritis. It was thought that the complement components deposited within the kidney were derived from the systemic circulating pool. However, recent work has shown that the kidney is able to produce many of the components of the complement cascade. In vitro work has shown that cells of glomerular and tubular origin can produce complement, as can inflammatory cells present within the kidney during inflammation. Analysis of human biopsy material has shown that expression of complement genes is increased during inflammatory renal disease implicating local complement synthesis as a potential source of complement components. At present no direct evidence for a pathogenic role of local complement synthesis exists. In this review we consider the current experimental evidence which suggests that local production of complement may be contributing to renal injury in a variety of diseases.     

The role of complement inhibitors beyond controlling inflammation

The complement system is an arm of innate immunity that aids in the removal of pathogens and dying cells. Due to its harmful, pro‐inflammatory potential, complement is controlled by several soluble and membrane‐bound inhibitors. This family of complement regulators has been recently extended by the discovery of several new members, and it is becoming apparent that these proteins harbour additional functions. In this review, the current state of knowledge of the physiological functions of four complement regulators will be described: cartilage oligomeric matrix protein (COMP), CUB and sushi multiple domains 1 (CSMD1), sushi domain‐containing protein 4 (SUSD4) and CD59. Complement activation is involved in both the development of and defence against cancer. COMP expression is pro‐oncogenic, whereas CSMD1 and SUSD4 act as tumour suppressors. These effects may be related in part to the complex influence of complement on cancer but also depend on unrelated functions such as the protection of cells from endoplasmic reticulum stress conveyed by intracellular COMP. CD59 is the main inhibitor of the membrane attack complex, and its deficiency leads to complement attack on erythrocytes and severe haemolytic anaemia, which is now amenable to treatment with an inhibitor of C5 cleavage. Unexpectedly, the intracellular pool of CD59 is crucial for insulin secretion from pancreatic β‐cells. This finding is one of several relating to the intracellular functions of complement proteins, which until recently were only considered to be present in the extracellular space. Understanding the alternative functions of complement inhibitors may unravel unexpected links between complement and other physiological systems, but is also important for better design of therapeutic complement inhibition.     

Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration

We introduce a human retinal pigmented epithelial (RPE) cell-culture model that mimics several key aspects of early stage age-related macular degeneration (AMD). These include accumulation of sub-RPE deposits that contain molecular constituents of human drusen, and activation of complement leading to formation of deposit-associated terminal complement complexes. Abundant sub-RPE deposits that are rich in apolipoprotein E (APOE), a prominent drusen constituent, are formed by RPE cells grown on porous supports. Exposure to human serum results in selective, deposit-associated accumulation of additional known drusen components, including vitronectin, clusterin, and serum amyloid P, thus suggesting that specific protein-protein interactions contribute to the accretion of plasma proteins during drusen formation. Serum exposure also leads to complement activation, as evidenced by the generation of C5b-9 immunoreactive terminal complement complexes in association with APOE-containing deposits. Ultrastructural analyses reveal two morphologically distinct forms of deposits: One consisting of membrane-bounded multivesicular material, and the other of nonmembrane-bounded particle conglomerates. Collectively, these results suggest that drusen formation involves the accumulation of sub-RPE material rich in APOE, a prominent biosynthetic product of the RPE, which interacts with a select group of drusen-associated plasma proteins. Activation of the complement cascade appears to be mediated via the classical pathway by the binding of C1q to ligands in APOE-rich deposits, triggering direct activation of complement by C1q, deposition of terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular aspects of AMD pathogenesis, and the testing of new therapeutic agents for its treatment.     

Activation of the alternative complement pathway by Torulopsis glabrata

Torulopsis glabrata is a yeast-like fungus with increasing importance as an opportunistic human pathogen. The role of serum complement in resistance to this infection was studied by chemotactic and opsonic assays. The results indicate that T. glabrata activates complement via the alternative pathway. This may be an important mechanism in host immunity to this fungus.     

Expression of complement components and inhibitors on platelet microparticles

Platelet microparticles (PMP) are released from activated platelets and play an important role in hemostasis, thrombosis and inflammation. Since platelets were recently found to demonstrate an intrinsic capacity for activating both classical and alternative pathways of the complement system, the present study extended these observations to PMP. PMP were generated by treating platelets with 10 microM A23187 (37 degrees C, 5 min). PMP were identified by flow cytometry, based on size, Annexin V binding, and expression of P-selectin and GPIIb (CD41). PMP expressed gC1qR/p33, a multifunctional cellular protein that was recently described to activate the classical complement cascade. PMP also expressed the classical pathway and contact system regulator, C1 inhibitor (C1-INH), as well as CD55 and CD59. Despite C1-INH expression, PMP supported classical pathway C4 activation in the presence of purified C1 and C4. Moreover, statistically significant deposition of C3b and C5b-9 was detected on PMP exposed to plasma, concurrently with expression of CD55 and CD59. These data provide the first evidence for the ability of PMP to support in situ complement activation. Complement activation contributes to a variety of vascular and inflammatory disease states including atherosclerosis and ischemia/reperfusion injury.     

Complement in immune thrombocytopenia (ITP): The role of complement in refractory ITP

Immune thrombocytopenia (ITP) is a disorder characterized by low platelets due to increased clearance and decreased platelet production. While ITP has been characterized as an acquired disorder of the adaptive immune system, the resulting platelet autoantibodies provide ancillary links to the innate immune system via antibody interaction with the complement system. Most autoantibodies in patients with ITP are of the IgG1 subclass, which can be potent activators of the classical complement pathway. Antibody-coated platelets can initiate complement activation via the classical pathway leading to both direct platelet destruction and enhanced clearance of C3b-coated platelets by complement receptors. Similar autoantibody interactions with bone marrow megakaryocytes can also result in complement injury and ineffective thrombopoiesis. The development of novel therapeutic complement inhibitors has revived interest in the role of complement in autoantibody-mediated disorders, such as ITP. A recent early-phase clinical trial of a classical complement pathway inhibitor has demonstrated efficacy in a subset of ITP patients refractory to conventional immune modulation. In this review, we will analyse the role of complement in refractory ITP.     

Expression of complement components and inhibitors on platelet microparticles

Platelet microparticles (PMP) are released from activated platelets and play an important role in hemostasis, thrombosis and inflammation. Since platelets were recently found to demonstrate an intrinsic capacity for activating both classical and alternative pathways of the complement system, the present study extended these observations to PMP. PMP were generated by treating platelets with 10 µM A23187 (37°C, 5 min). PMP were identified by flow cytometry, based on size, Annexin V binding, and expression of P-selectin and GPIIb (CD41). PMP expressed gC1qR/p33, a multifunctional cellular protein that was recently described to activate the classical complement cascade. PMP also expressed the classical pathway and contact system regulator, C1 inhibitor (C1-INH), as well as CD55 and CD59. Despite C1-INH expression, PMP supported classical pathway C4 activation in the presence of purified C1 and C4. Moreover, statistically significant deposition of C3b and C5b-9 was detected on PMP exposed to plasma, concurrently with expression of CD55 and CD59. These data provide the first evidence for the ability of PMP to support in situ complement activation. Complement activation contributes to a variety of vascular and inflammatory disease states including atherosclerosis and ischemia/reperfusion injury.     

Molecules Great and Small: The Complement System

The complement cascade, traditionally considered an effector arm of innate immunity required for host defense against pathogens, is now recognized as a crucial pathogenic mediator of various kidney diseases. Complement components produced by the liver and circulating in the plasma undergo activation through the classical and/or mannose-binding lectin pathways to mediate anti-HLA antibody-initiated kidney transplant rejection and autoantibody-initiated GN, the latter including membranous glomerulopathy, antiglomerular basement membrane disease, and lupus nephritis. Inherited and/or acquired abnormalities of complement regulators, which requisitely limit restraint on alternative pathway complement activation, contribute to the pathogenesis of the C3 nephropathies and atypical hemolytic uremic syndrome. Increasing evidence links complement produced by endothelial cells and/or tubular cells to the pathogenesis of kidney ischemia-reperfusion injury and progressive kidney fibrosis. Data emerging since the mid-2000s additionally show that immune cells, including T cells and antigen-presenting cells, produce alternative pathway complement components during cognate interactions. The subsequent local complement activation yields production of the anaphylatoxins C3a and C5a, which bind to their respective receptors (C3aR and C5aR) on both partners to augment effector T-cell proliferation and survival, while simultaneously inhibiting regulatory T-cell induction and function. This immune cell–derived complement enhances pathogenic alloreactive T-cell immunity that results in transplant rejection and likely contributes to the pathogenesis of other T cell–mediated kidney diseases. C5a/C5aR ligations on neutrophils have additionally been shown to contribute to vascular inflammation in models of ANCA-mediated renal vasculitis. New translational immunology efforts along with the development of pharmacologic agents that block human complement components and receptors now permit testing of the intriguing concept that targeting complement in patients with an assortment of kidney diseases has the potential to abrogate disease progression and improve patient health.     

Usefulness of detection of complement activation products in evaluating SLE activity

Complement activation products, such as C1rs-C1inh, specific for the activation of the classical pathway, C3b(Bb)P, specific for the activation of the alternative pathway and SC5b-9, specific for common terminal pathway of the complement cascade, were measured in healthy donors and in patients with clinically active and inactive systemic lupus erythematosus (SLE). Plasma levels of C3b(Bb)P and SC5b-9 were moderately, those of C1rs-C1inhibitor (C1rs-C1inh) were markedly elevated in patients with clinically inactive SLE, compared with healthy controls. The difference between active and inactive stages of the disease was best reflected by C3b(Bb)P plasma concentration (P<0.001), which also showed the highest correlation with the SLEDAI (Rs=0.41 P<0.001) and which was the most useful in distinguishing active and inactive sample pairs as well. The difference between SC5b-9 levels in the active and inactive stages was also significant (P=0.007), while that of C1rs-C1inh did not differ significantly (P=0.136). The correlation of the SLEDAI with SC5b-9 was 0.3 (P=0.015), while with C1rs-C1inh it was 0.21 (P=0. 089). These findings suggest that the measurement of complement activation products, especially that of the alternative pathway, are sensitive markers of the activity of SLE and can be used for clinical purposes.     

Susceptibility to mycobacterial infections due to interferon-gamma and interleukin-12 pathway defects.

A case of interferon-gamma (IFN-gamma)/interleukin-12 (IL-12) pathway defect is presented. Pathophysiology, clinical characteristics, diagnostic test, and case management are reviewed. Clinical Pearls and Pitfalls include: (1) A high probability of a defect in the IFN-gamma/IL-12 cascade exists in patients with disseminated or recurrent infection due to poorly pathogenic mycobacteria or systemic infections caused by non-typhi Salmonella species that are persistent and recurrent despite antibiotic therapy. (2) Although less frequent, patients with impaired IFN-gamma/IL-12 mediated immunity are more susceptible to cytomegalovirus, human herpesvirus 8, herpes simplex virus, Listeria monocytogenes, and Histoplasma capsulatum.     

Germline variation in complement genes and event-free survival in follicular and diffuse large B-cell lymphoma

The complement pathway plays a central role in innate immunity, and also functions as a regulator of the overall immune response. We evaluated whether polymorphisms in complement genes are associated with event‐free survival (EFS) in follicular lymphoma (FL) and diffuse large B‐cell (DLBCL) lymphoma. We genotyped 167 single nucleotide polymorphisms (SNPs) from 30 complement pathway genes in a prospective cohort study of newly diagnosed FL (N = 107) and DLBCL (N = 82) patients enrolled at the Mayo Clinic from 2002 to 2005. Cox regression was used to estimate hazard ratios (HRs) for individual SNPs with EFS, adjusting for FLIPI or IPI and treatment. For gene‐level analyses, we used a principal components based gene‐level test. In gene‐level analyses for FL EFS, CFH (P = 0.009), CD55 (P = 0.006), CFHR5 (P = 0.01), C9 (P = 0.02), CFHR1 (P = 0.03), and CD46 (P = 0.03) were significant at P < 0.05, and these genes remained noteworthy after accounting for multiple testing (q < 0.15). SNPs in CFH, CFHR1, and CFHR5 showed stronger associations among patients receiving any rituximab, while SNPs from CD55 and CD46 showed stronger associations among patients who were observed. For DLBCL, only CLU (P = 0.001) and C7 (P = 0.03) were associated with EFS, but did not remain noteworthy after accounting for multiple testing (q>0.15). Genes from the regulators of complement activation (CFH, CD55, CFHR1, CFHR5, CD46) at 1q32–q32.1, along with C9, were associated with FL EFS after adjusting for clinical variables, and if replicated, these findings add further support for the role of host innate immunity in FL prognosis. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.     

JAK kinase targeting in hematologic malignancies: a sinuous pathway from identification of genetic alterations towards clinical indications

Constitutive JAK-STAT pathway activation occurs in most myeloproliferative neoplasms as well as in a significant proportion of other hematologic malignancies, and is frequently a marker of poor prognosis. The underlying molecular alterations are heterogeneous as they include activating mutations in distinct components (cytokine receptor, JAK, STAT), overexpression (cytokine receptor, JAK) or rare JAK2 fusion proteins. In some cases, concomitant loss of negative regulators contributes to pathogenesis by further boosting the activation of the cascade. Exploiting the signaling bottleneck provided by the limited number of JAK kinases is an attractive therapeutic strategy for hematologic neoplasms driven by constitutive JAK-STAT pathway activation. However, given the conserved nature of the kinase domain among family members and the interrelated roles of JAK kinases in many physiological processes, including hematopoiesis and immunity, broad usage of JAK inhibitors in hematology is challenged by their narrow therapeutic window. Novel therapies are, therefore, needed. The development of more selective inhibitors is a questionable strategy as such inhibitors might abrogate the beneficial contribution of alleviating the cancer-related pro-inflammatory microenvironment and raise selective pressure to a threshold that allows the emergence of malignant subclones harboring drug-resistant mutations. In contrast, synergistic combinations of JAK inhibitors with drugs targeting cascades that work in concert with JAK-STAT pathway appear to be promising therapeutic alternatives to JAK inhibitors as monotherapies.     

Severe granulocytic dysplasia with vacuolar rosettes in a myelodysplastic syndrome with low‐copy KMT2A gain

Cold agglutinin disease (CAD) is an uncommon autoimmune haemolytic anaemia in which a well‐defined, clonal low‐grade lymphoproliferative disorder of the bone marrow results in erythrocyte destruction mediated by the classical complement pathway. The pathogenesis, clinical features and diagnostic criteria are reviewed. Although anaemia is mild in some patients, approximately one‐third of untreated patients have a haemoglobin level of ≤80 g/l, and about 50% have been considered transfusion dependent for shorter or longer periods. Therapy has improved greatly during the last 15 years. Mild disease can be managed by avoidance of cold and adequate precautions in specific situations, without drug therapy. Corticosteroids should not be used to treat CAD. Patients requiring pharmacological therapy should be considered for prospective trials. Outside clinical studies, the rituximab‐bendamustine combination or rituximab monotherapy is recommended in the first line, depending on individual patient characteristics. Second‐line options are rituximab‐fludarabine in fit patients or, although less strongly documented, a bortezomib‐based regimen. Therapies targeting the classical complement pathway are promising, and the complement C1s inhibitor, BIVV009, has shown favourable results in preliminary studies.