R

CRt

JJa

b

a

ARRAA

KCIATAC

1i

sem(11tccp1ad

N

h0

Molecular Immunology 67 (2015) 117–130

Contents lists available at ScienceDirect

Molecular Immunology

j ourna l ho me pa ge: www.elsev ier .com/ locate /mol imm

eview

omplement in therapy and diseaseegulating the complement system with antibody-basedherapeutics�

oost P.M. Melisa,1, Kristin Strumanea,1, Sigrid R. Ruulsa, Frank J. Beurskensa,anine Schuurmana, Paul W.H.I. Parrena,b,∗

Genmab, Utrecht, The NetherlandsDepartment of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

r t i c l e i n f o

rticle history:eceived 16 December 2014eceived in revised form 26 January 2015ccepted 27 January 2015vailable online 17 February 2015

a b s t r a c t

Complement is recognized as a key player in a wide range of normal as well as disease-related immune,developmental and homeostatic processes. Knowledge of complement components, structures, interac-tions, and cross-talk with other biological systems continues to grow and this leads to novel treatments forcancer, infectious, autoimmune- or age-related diseases as well as for preventing transplantation rejec-tion. Antibodies are superbly suited to be developed into therapeutics with appropriate complement

eywords:omplement

mmunologyntibodyherapyntibody engineering

stimulatory or inhibitory activity. Here we review the design, development and future of antibody-baseddrugs that enhance or dampen the complement system.

© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

ancer

. The complement cascade: a fine interplay of molecularnteractions regulates functional outcome

The complement system is an evolutionarily well-conservedystem of which the current complexity in jawed vertebratesmerged 600 million years ago, while some components of theost primitive complement system date 1.6 billion years back

Nonaka and Kimura, 2006). Complement was identified in the late800s as a heat-labile, bactericidal component in serum (Bordet,895; Ehrlich, 1899). This activity of innate immunity was fur-her unraveled in the following century. One by one, essentialomponents were identified, purified, and assigned a place in theascade from pathogen recognition to response amplification andathogen inactivation (Fig. 1A) (Hadding and Mueller-Eberhard,

969; Mueller-Eberhard and Biro, 1963; Nilsson, 1967; Nilssonnd Mueller-Eberhard, 1965; Pillemer and Ecker, 1941). Nowa-ays, three activation pathways of complement, initiated through

� This article belongs to Special Issue on Therapeutic Antibodies.∗ Corresponding author at: Genmab, Yalelaan 60, 3584 CM Utrecht, Theetherlands. Tel.: +31 30 212 3108.

E-mail address: P.Parren@genmab.com (P.W.H.I. Parren).1 These authors contributed equally to this paper.

ttp://dx.doi.org/10.1016/j.molimm.2015.01.028161-5890/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article u

distinct ligand–receptor interactions, are well-defined: the clas-sical pathway, the lectin pathway, and the alternative pathway(Fig. 1A). The pathways converge at an amplification stage charac-terized by the formation of C3 and C5 convertases, and cooperateclosely to form opsonins, anaphylatoxins, chemoattractants, andmembrane attack complexes (MACs). Complement is heavily regu-lated by both soluble and cell-surface expressed proteins, by whichit is constantly activated and quenched to maintain a delicatebalance. However, it can be amplified rapidly when an immedi-ate immune reaction against a pathogenic challenge is required.Approximately 50 soluble and cell surface-attached componentsare currently known to comprise the major proteolytic compo-nents, cofactors, and regulators of the entire complement cascade(Gros et al., 2008; Holers, 2014; Kemper et al., 2014; Meyer et al.,2014; Ricklin et al., 2010) (Fig. 1B).

Antibodies can deploy the complement cascade as a potenteffector mechanism. IgG or IgM antibodies activate the classi-cal pathway of complement through binding of C1q via their Fcregion. C1q consists of six collagen-like arms, each containing anN-terminal triple helix and a C-terminal immunoglobulin-binding

globular head domain (Reid and Porter, 1976) with an overall shapeof a bunch of tulips (Fig. 2). The affinity of a single C1q globularhead for immunoglobulin Fc is low (Feinstein, 1986; Hughes-Jonesand Gardner, 1979), such that physiological binding and activation

nder the CC BY license (http://creativecommons.org/licenses/by/4.0/).

118 J.P.M. Melis et al. / Molecular Immunology 67 (2015) 117–130

Fig. 1. A simplified (A) and more detailed (B) overview of the complement system. Activation can be achieved via the classical pathway, the lectin pathway, or the alternativepathway. Initiation of the classical pathway occurs when C1 (C1q in complex with the serine proteases C1r and C1s) interacts with the Fc region of IgG or IgM antibodiesattached to antigenic surfaces. In the lectin pathway, mannose-binding lectin (MBL) and ficolins assemble with MBL-associated serine proteases (MASPs). The alternativepathway is induced by C3 hydrolysis, either spontaneously at low rate or enhanced by interaction of C3 with pathogen’s cell surfaces. All three pathways lead to the formationof C3 and C5 convertases, which rapidly amplify the complement response. The outcome of complement activation is three-pronged: (1) opsonization of the target surfaceby C3b, (2) a boost in inflammation through the generation of anaphylatoxins C3a and C5a and subsequent recruitment of effector cells and (3) formation of the terminalmembrane attack complex (MAC), which is responsible for target cell lysis. In addition to the processes described above, several complement regulatory proteins (indicatedin orange) are able to inhibit complement by inactivation of C3b and C3 convertases, or by preventing successful formation of the MAC.

J.P.M. Melis et al. / Molecular Immunology 67 (2015) 117–130 119

Fig. 2. Schematic representation of human IgG (top right), human C1q (top left), and illustration of IgG hexamerization after antigen binding on cells, generating an optimaldocking site for C1q (bottom). The C1q binding site in human IgG maps to residues D270, K322, P329 and P331 in the CH2 domain, highlighted in light green in the IgG cartoon.C1q binding is further determined by features in other regions of IgG, such as flexibility of the hinge region, variations in N-glycosylation at position N297 and the CH2–CH3

i multia hexam

rU

epc(wtsailbsChte

mkoB2hIIbTd

sacihWP

nterface that mediates Fc:Fc interactions in antibody hexamers. C1 consists of the

nd C1s. C1q has six globular heads that can each bind a single IgG molecule in the

equires a multivalent, high avidity interaction (Burton et al., 1980;daka et al., 1986; Utsumi et al., 1985).

IgM exists naturally in covalently pentameric and hexam-ric forms (Cattaneo and Neuberger, 1987). We propose thatentameric and hexameric IgM should be seen as two distinct sub-lasses (i.e. IgMp and IgMh). IgMp, representing the vast majority95%) of IgM, is organized as a pentamer associated with a J-chain,hich enables its transepithelial transport via the poly-Ig recep-

or. IgMp therefore is adapted for providing protection at mucosalurfaces. IgMh is organized in a hexamer, which gives it a superiorbility to interact with C1q and activate complement, as shown byts greater complement activating potential (approximately one-og compared to IgMp) (Collins et al., 2002). For IgMp, the C1qinding sites are sequestered, but become available by a star-to-taple conformational change upon antigen binding (Burton, 1986;zajkowsky and Shao, 2009; Feinstein, 1986). IgMh, on the otherand, appears to be a planar molecule, in which potential perturba-ions required for regulation of C1q binding are less clear (Arnoldt al., 2005; Muller et al., 2013).

For IgG antibodies, high avidity C1q binding requires multi-erization (Burton, 1986; Diebolder et al., 2014). It has been long

nown that multiple closely spaced Fc regions increase the strengthf binding of C1q for IgG dramatically (Borsos and Rapp, 1965;urton, 1985; Hughes-Jones and Gardner, 1978; Kishore et al.,004). Recently, we demonstrated that high avidity binding of C1 touman IgG occurs through antibody hexamers that form after the

gG molecules bind antigen on a cell surface (Diebolder et al., 2014).gG hexamers are assembled via specific non-covalent interactionsetween the Fc segments of neighboring IgG molecules (Fig. 2).he hexamerized antibody complex is thought to form the optimalocking structure for C1 binding and complement activation.

A number of additional factors influence activation of the clas-ical complement pathway (Fig. 2). The epitope recognized by thentibody is a critical factor in determining its ability to engage theomplement system. Indeed antigen size, density, geometry, flu-

dity, as well as IgG orientation imposed by epitope positioning,ave a strong impact (Bindon et al., 1988; Cragg et al., 2003; deeers et al., 2011; Hughes-Jones et al., 1985; Parce et al., 1983;

awluczkowycz et al., 2009; Teeling et al., 2004, 2006; Xia et al.,

meric pattern recognition molecule C1q and a heterotetramer of the proteases C1reric ring structure.

1993b). These observations can be explained by structural prereq-uisites affecting Fc movement and Fc:Fc positioning required forefficient hexamer formation (Diebolder et al., 2014). Furthermore,placement of the hexameric platform relative to the cell surfaceappears of importance. Thus, even though antibodies of the IgG3isotype demonstrate stronger C1q binding than IgG1 molecules, thelatter may induce cell killing more potently, potentially becauseit situates the C1q binding site more proximal to the cell surface(Bindon et al., 1988; Bruggemann et al., 1987; Diebolder et al.,2014). The length and flexibility of the hinge region as well as theheterogeneity of glycans in the CH2 domain are also factors thatinfluence C1q binding and complement activation (Coloma et al.,1997; Dangl et al., 1988; Raju, 2008; Tan et al., 1990). Finally, IgG-mediated complement activation can be dampened by the presenceof specific antibodies of other isotypes, such as human IgG4 or IgA,which are both impaired in their interaction with C1q (Davies et al.,2014; Dechant and Valerius, 2001; Kerr, 1990; Labrijn et al., 2008;van der Neut Kolfschoten et al., 2007; Woof and Kerr, 2006). Suchdampening may result from competition for antigen binding and/orfrom steric interference of antigen-bound IgG4 or IgA with com-plement factors. Potentially, the binding of complement-impairedisotypes in the close vicinity of IgG1 molecules interferes with Fc:Fccontact formation thereby inhibiting hexamerization, C1q bindingand complement activation.

2. The complement system off balance

An imbalance in complement, either by insufficient or excessivecomplement activity, can have important pathological conse-quences (Botto et al., 2009; Grumach and Kirschfink, 2014;Mayilyan, 2012; Ricklin and Lambris, 2013a, 2013b; Skattum et al.,2011). Antibody-based treatments can be employed to restore thebalance in the complement network in order to achieve thera-peutic effects (Fig. 3). Complement inhibition can be beneficial inpathologies where the system is hyperactivated (e.g. sepsis, trans-

plant rejection, ischemia and reperfusion (I/R) injury) or where itis chronically activated and attacks or damages healthy tissues (e.g.autoimmune disease). It is envisioned that such inhibition can beachieved by targeting key components of the complement cascade

120 J.P.M. Melis et al. / Molecular Immunology 67 (2015) 117–130

Table 1Complement-modulating antibodies in development. (A) Antibodies and Fc fusion proteins that target and regulate complement factors directly. Thomson Reuters Cortellisdatabase was interrogated for complement-targeting antibody-based therapies, together with www.clinicaltrials.gov, www.fda.gov and company websites (as of October30, 2014). (B) Antibodies that have been reported to (partly) rely on complement activation as part of their mechanism of action are indicated. Key references are shown.

(A)

Product Company Target Indication Development

Eculizumab Alexion Pharmaceuticals C5 Atypical Hemolytic uremic syndrome (aHUS); Paroxysmal nocturnal hemoglobinuria LaunchedAntibody-associated vasculitis; Antiphospholipid syndrome; Budd Chiari syndrome;Chronic hemolysis; Cold agglutinin disease; Dry age related macular degeneration;Glomerulonephritis; Heart transplant rejection; Kidney transplant rejection; Kidneytransplantation; Myasthenia gravis

Clinical

Neuromyelitis optica; Neuropathy; STEC-HUS; Dermatomyositis; Nephritis;Pemphigus; Psoriasis; Rheumatoid arthritis; Systemic lupus erythematosus

Discontinued

Mubodina Adienne Pharma & Biotech C5 Hemolytic uremic syndrome; Membrano-proliferative glomerulonephritis Launched

Ergidina Adienne Pharma & Biotech C5 Ischemia; Reperfusion injury LaunchedInflammatory disease Discontinued

LFG-316 Novartis; MorphoSys C5 Age-related macular degeneration; Uveitis ClinicalLampalizumab Genentech Factor

DAge-related macular degeneration Clinical

IFX-1, CaCP-29 InflaRx C5a Severe sepsis; Septic shock ClinicalTNT-009 True North Therapeutics C1s Cold agglutinin disease; Neurodegenerative disease; Renal disease PreclinicalBikaciomab NovelMed Therapeutics Factor

BAge-related macular degeneration Preclinical

B

Product Company Target Indication Development Reference

Rituximab Biogen Idec; Genentech; Hoffmann-LaRoche; Chugai Pharmaceuticals;Zenyaku Kogyo; AryoGen

CD20 B-cell lymphoma; Chroniclymphocytic leukemia;Microscopic polyangiitis;Non-Hodgkin lymphoma;Rheumatoid arthritis; Wegenergranulomatosis

Launched Anderson et al. (1997); Glennieet al. (2007); Lindorfer et al.(2014)

Central nervous system tumor;Colorectal tumor; Diffuse largeB-cell lymphoma; Factor VIIIdeficiency; Follicle centerlymphoma; Graft versus hostdisease; Hodgkins disease; Kidneytransplant rejection; Lupusnephritis; Mantle cell lymphoma;Nephrotic syndrome; Orbitalinflammatory disease; Polyarteritisnodosa; Polymyositis; Sarcoidosis;Scleritis; Thrombocytopenicpurpura; Vasculitis

Clinical

Ofatumumab Genmab; GSK CD20 Chronic lymphocytic leukemia Launched Beurskens et al. (2012);Lindorfer et al. (2014); Teelinget al. (2004)

Diffuse large B-cell lymphoma;Follicular lymphoma; Multiplesclerosis; Pemphigus vulgaris;Waldenstrom’s macroglobulinemia

Clinical

Diffuse large B-cell lymphoma;Follicle center lymphoma; Mantlecell lymphoma; Non-Hodgkinlymphoma

Clinical

Alemtuzumab Genzyme Corp; Sanofi CD52 Multiple sclerosisMycosis fungoides; Sezarysyndrome

Clinical Xia et al. (1993a, 1993b); Zentet al. (2008)

Chronic lymphocytic leukemia;Peripheral T-cell lymphoma

Discontinued

Daratumumab Genmab; Janssen Biotech, Inc CD38 Multiple myeloma Clinical de Weers et al. (2011)Ocaratuzumab Mentrik Biotech LLC CD20 Follicle center lymphoma;

Autoimmune disease; Chroniclymphocytic leukemia;Non-Hodgkin lymphoma;Rheumatoid arthritis

Clinical Cheney et al. (2014)

Veltuzumab Immunomedics Inc CD20 Chronic lymphocytic leukemia;Immune thrombocytopenicpurpura; Non-Hodgkin lymphoma;Systemic lupus erythematosus

Clinical Goldenberg et al. (2010)

J.P.M. Melis et al. / Molecular Immunology 67 (2015) 117–130 121

Fig. 3. Modulation of complement for therapeutic opportunities in several disorders. Orange boxes indicate possible therapeutic options for complement inhibition; greenb role. I

mt

bacbopwim

2

2

pegodumectpts(asuWm2

rb2sjo2w

oxes illustrate pathologies where complement stimulation could play a beneficial

ediating the initiation, amplification, and/or termination steps ofhe system (Table 1A).

Antibody-mediated activation of complement, in contrast, cane a valuable approach in the treatment of infectious diseasesnd cancer. Here, it is anticipated that antibody-based therapyan mediate complement-dependent lysis via Fab-mediated inhi-ition of specific negative complement regulatory proteins (CRPs),r via Fc-induced complement activation (Table 1B) to eliminateathogens and infected or malignant cells, respectively. Below,e will first discuss therapeutic approaches for complement

nhibition, followed by strategies to induce and augment antibody-ediated complement activation.

.1. Antibody-mediated complement inhibition

.1.1. Autoimmune and inflammatory diseasesIncreased complement activation plays an important role in the

athogenesis of several autoimmune and auto-inflammatory dis-ases (Ballanti et al., 2013; Chen et al., 2010). This can result fromain of function mutations in complement activation componentsr dysregulation of CRPs leading to chronic inflammation and tissueamage (Wagner and Frank, 2010). For example, atypical hemolyticremic syndrome (aHUS) can be caused by C3 gain of functionutations or CD46 deficiency (Richards et al., 2003; Stuhlinger

t al., 1974). In paroxysmal nocturnal hemoglobinuria (PNH), tworitical CRPs (CD55 and CD59) are absent because of somatic muta-ion of the PIGA gene, leading to a defect in the synthesis of thehosphatidylinositol tails which are required for their attachmento the membrane. The deficiency, amongst others, leads to sen-itization of red blood cells to complement-induced cytotoxicityBessler et al., 1994; Takeda et al., 1993). The therapeutic anti-C5ntibody eculizumab, that inhibits terminal pathway initiation, hasignificant clinical benefit for both PNH and aHUS and is widelysed to treat these disorders (Hillmen et al., 2013; Keating, 2013;ong et al., 2013). Eculizumab is also in clinical development forany other complement-related indications (Barilla-Labarca et al.,

013) (Table 1).Treatment with eculizumab did not show much efficacy in

heumatoid arthritis (RA), an autoimmune disease characterizedy chronic inflammation of synovial joints (Barilla-Labarca et al.,013). However, selective delivery of an anti-C5 antibody fused to aynovial homing peptide to locally inhibit complement at inflamed

oints showed therapeutic potential in prevention or treatmentf arthritis in a preclinical animal model for RA (Durigutto et al.,013; Macor et al., 2012). Besides anaphylaxis (via C3a and C5a),hich is the prominent pathway responsible for tissue damage in

/R = ischemia/reperfusion.

arthritis, also opsonization, MAC formation, and subsequent lysisis suggested to play a role in RA pathogenesis (Banda et al., 2012;Okroj et al., 2007; Romero et al., 2013). Therefore, these featuresof complement activation are candidate targets for complementinhibition in RA.

Systemic lupus erythematosus (SLE) is another autoimmunedisease associated with numerous immune-based abnormalitiesincluding, paradoxically, both complement deficiency and comple-ment activation. Deficiencies within the early classical pathwaycomponents (C1q, C4 and C2) predispose for development ofSLE, whereas complement also takes part in the auto-antibodyinflammatory reaction in the disease leading to tissue and organdamage (Bryan and Wu, 2014; Truedsson et al., 2007). Reducingcomplement-related inflammatory responses with antibody ther-apeutics, potentially via the simultaneous inhibition at multiplelevels could, therefore, be incorporated in future treatment regi-mens for SLE, RA and other autoimmune disorders.

2.1.2. SepsisSepsis is characterized by hyperactivation of the complement

cascade in a systemic response to severe infection or inflamma-tion. It may be possible to constrain such an exaggerated responsewith a therapeutic antibody-based approach. C3a and especiallyC5a would represent favorable targets in this context, as high lev-els of these anaphylotoxins are thought to contribute to acute tissueinjury (Guo and Ward, 2005; Silasi-Mansat et al., 2010). The anti-C5a antibody IFX-1 for example is currently in clinical developmentfor sepsis, although it could be challenging to effectively target thismolecule due to its rapid turnover. In addition, it will be impor-tant to use C5a inhibiting antibodies that do not cross-react withwhole C5 in order to avoid its depletion resulting in reduced MACformation and cell lysis, thereby potentially increasing the suscep-tibility to infection (Ward et al., 2012). Alternatively, the effects ofC5a could be neutralized by blocking its receptor C5aR. A proof-of-concept for this was provided with antibodies targeting C5aR oranother C5a receptor, C5L2, in treating experimental sepsis in mice(Rittirsch et al., 2008). Upstream factors of C5 could also representtherapeutic targets in sepsis, with C1, C3 or MASP2 antibodies ofparticular interest.

Simultaneously targeting of complement and Toll-like recep-tors, that play a role in innate defense against bacteria, was shownto be effective for Staphylococcus aureus-induced inflammation ina human whole blood model and polymicrobial sepsis in mice

(Huber-Lang et al., 2014; Skjeflo et al., 2014). These observationssuggest that dampening the immune response with antibody mix-tures or bispecific antibodies in a multi-targeting approach mayhave therapeutic potential.

1 r Imm

2

dtpVltaA2aa(22plw(cAte2ivrtf

rctsdtWlebce

2

iiaocHAihidpoi

(acpa

22 J.P.M. Melis et al. / Molecula

.1.3. Age-related degenerative diseasesComplement has been associated with several age-related

egenerative diseases. The most advanced antibody therapeutic inhis area is focused on age-related macular degeneration (AMD), therimary cause of blindness in the Western world (Bora et al., 2015).ision loss in this condition results from a gradual deterioration of

ight-sensing cells due to chronic low-grade complement activa-ion resulting in inflammation. Since the initial demonstration ofn association between complement Factor H polymorphisms andMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al.,005; Klein et al., 2005), additional associations between AMDnd other complement component polymorphisms have becomepparent (C2, C3, C7, Factor B, Factor D, Factor I, CFHR1/CFHR3)Ansari et al., 2013; Bergeron-Sawitzke et al., 2009; Dinu et al.,007; Fagerness et al., 2009; Francis et al., 2009; Stanton et al.,011; Yates et al., 2007). This provides opportunities for thera-eutic approaches, such as shown for the anti-Factor D antibody

ampalizumab, which appeared to be safe and effective for patientsith geographic atrophy, the late stage of the dry form of AMD

Weber et al., 2014). Since inhibiting C5 has shown to dampen theomplement response in other pathologies, C5 is also a target forMD. However, systemic eculizumab treatment did not decrease

he progression of late stage AMD and clinical development ofculizumab appears to be halted in this indication (Weber et al.,014). Other complement inhibiting antibodies that are currently

n clinical development for AMD are administered as direct intra-itreal injections to achieve adequate drug levels in the retina or theetinal pigment epithelium (Yehoshua et al., 2014). The outcome ofhese trials will indicate whether local complement inhibition is aeasible therapeutic approach in late stage AMD.

Other correlations between complement and aging have beeneported. C3 levels have been associated with the development ofhronic and age-related diabetes (Engstrom et al., 2005). Also, inhe normal aging mouse and human brain, C1q levels are increasedignificantly (Stephan et al., 2013). Interestingly, it was recentlyescribed that C1q may activate Wnt signaling via a direct interac-ion with Frizzled and subsequent C1s-dependent cleavage of the

nt coreceptors LRP5/6. Together, these observations appear toink C1q with the age-related decline in tissue regeneration (Naitot al., 2012). Finally, in Alzheimer’s disease, complement activationy C1q in amyloid plaques has been suggested to significantlyontribute to the pathology (Bonifati and Kishore, 2007; Yasojimat al., 1999)

.1.4. Transplant rejection and ischemia/reperfusion (I/R) injuryImmune responses are generally heavily activated follow-

ng allo-transplantation. The suppression of adaptive and innatemmune responses, including complement inhibition, is therefore

prerequisite for successful graft survival. Hyperacute rejectionf transplanted solid organs can occur within minutes, due toomplement activation by pre-existing antibodies against donorLA molecules or blood group antigens expressed on the graft.lthough the implementation of blood group and histocompat-

bility screening almost completely prevents the occurrence ofyperacute rejection, the application of efficacious complement-

nhibiting antibodies could have value for transplantation withonor tissue across ABO- and HLA-barriers as well as in xenotrans-lantion. Indeed, clinical studies with C5 antibody eculizumab arengoing to determine its efficacy and safety in ABO-blood group-ncompatible transplantation.

Antibodies and complement activation can contribute to acutedays to weeks after transplantation) and chronic (months to years

fter transplantation) rejection in patients with pre-existing non-omplement fixing HLA antibodies (Otten et al., 2012) and inatients that generate complement-binding donor-specific HLAntibodies de novo after transplantation (Loupy et al., 2013; Safavi

unology 67 (2015) 117–130

et al., 2014). C4d deposition in diagnostic biopsies from organtransplants has been adopted as a marker for antibody-mediatedrejection and is central in clinical decision making (Racusen et al.,2003). Both clinical and subclinical antibody-mediated rejectionhave been recognized as major causes of allograft loss (Amico et al.,2009), which is usually unresponsive to conventional anti-rejectiontherapy, such as suppression of the T-cell-dependent antibodyresponse, removal of donor reactive antibodies, blocking residualallo-antibodies, and depletion of naive and memory B-cells. Con-vincing, but still limited, evidence has shown that eculizumab isefficient in preventing both acute and chronic antibody-mediatedrejection and clinical studies further extending these results areongoing (Legendre et al., 2013).

Graft-versus-host disease (GvHD) is at the other side of thespectrum of clinical complications of allo-transplantation and ismediated by donor T-cells that attack host cells, leading to epithelialtissue injury in skin, intestine, and liver (Shlomchik, 2007). Total-body irradiation, a conditioning regimen to permit hematopoieticcell transplantations, contributes to GvHD by activating host den-dritic cells. Mouse models of bone marrow transplantation showedthat GvHD involved up-regulation and activation of complementcomponents, including C3a and C5a, by the recipient dendriticcells, and expression of CD55, C3aR, and C5aR on the donor T-cells (Kwan et al., 2012; Ma et al., 2012). Recent findings indicatedthat immune cell-derived complement also impacts human T-cellimmunity and GvHD, creating the possibility of targeting comple-ment for GvHD treatment (Cravedi et al., 2013; Ma et al., 2014).Complement is involved in I/R injury, a condition which also appliesto transplantation. Low oxygen and insufficient nutrient levelsduring the restriction in blood supply creates inflammatory con-ditions, which together with oxidative stress during reperfusionof the (transplant) organ leads to tissue damage. Complementplays an important role in such ischemic tissue infarction byanaphylatoxin-mediated immune responses (Peng et al., 2012) andthrough MAC-directed lysis of parenchymal cells (Zhou et al., 2000).It is believed that both the classical and the lectin pathways are partof the injurious processes upon I/R (Castellano et al., 2010; Gorsuchet al., 2012; Sacks and Zhou, 2012). MASP2-deficient mice were pro-tected from gastrointestinal and myocardium I/R injury comparedto wild type mice (Schwaeble et al., 2011) and due to encour-aging results, a C5 minibody fused with an RGD-motif (Ergidina,Table 1A), has been approved for treatment of I/R injury.

2.2. Antibody-mediated complement stimulation

2.2.1. Infectious diseasesThe best-known function of complement is the role in protec-

tion against microbial infections. The importance of complement incombatting infectious disease is supported by the observation thatmany pathogens have evolved mechanisms to evade complement-dependent elimination (see Supplemental Table 1 for a compre-hensive overview). Various bacterial or viral pathogens inhibitcomplement by the acquisition or mimicking of membrane-boundCRPs that interfere with the complement cascade. Successful elim-ination of infectious agents by pathogen-specific antibodies couldtherefore entail the inhibition of the pathogen’s evasive CRPs. As anexample, hijacking CD59 from the host’s cell membrane appears tobe a mechanism of HIV-1 to avoid complement-mediated viroly-sis (Saifuddin et al., 1997, 1995). Indeed, anti-HIV-1 gp120/gp160polyclonal antibody treatment combined with anti-CD59 antibodyor with the CD59-inhibiting recombinant form of the bacterial toxinintermedilysin domain 4 (rILYd4), sensitized HIV-1 to virolysis (Hu

et al., 2010; Lan et al., 2014). The role of complement in protectionagainst HIV-1 infection, however, has been debated as complementactivation did not contribute to antibody-mediated protectionagainst HIV-1 infection in a macaque model (Hessell et al., 2007).

r Immu

i2

itciitre(ts2bitsotewmoRbep

2

nbultiaacCci

tesifBeSmpt(BCk(

stD

J.P.M. Melis et al. / Molecula

Supplemental Table 1 related to this article can be found,n the online version, at http://dx.doi.org/10.1016/j.molimm.015.01.028.

The development of antibody therapy for the treatment ofnfectious disease has met with little success when compared tohe development of therapies in inflammatory disease and can-er. Currently only palivizumab is widely used in this aria, whichs employed as an RSV prophylaxis. The recent progress in thedentification of antibodies that react with highly conserved epi-opes on diverse variants of a single virus or common epitopes onelated viruses, such as for HIV-1, hepatitis C and influenza (Burtont al., 2012; Corti and Lanzavecchia, 2013) or paramyxovirusesCorti et al., 2013), bears promise for the development of antibodyherapy of infectious diseases. Similar strategies are being pur-ued for treatment of bacterial infections (Garcia-Rodriguez et al.,007; Theilacker et al., 2012). Potentially, such antibodies coulde combined with strategies to improve their complement activat-

ng capabilities, such as described in the next section. In addition,he combination of broadly cross-strain reactive antibodies withtrategies to limit complement evasion mechanisms is potentiallyf interest. In this respect, there is an unexpected paucity in clinicalrials with therapeutic antibodies against microbial complementvasion molecules (e.g. as those listed in Supplemental Table 1),hich can in part be explained by the fact that multiple comple-ent escape mechanisms may be employed making development

f therapies targeting single pathways difficult (Foster, 2005;ooijakkers and van Strijp, 2007; van Wamel et al., 2006). Anti-ody combinations against various evasion molecules to increasefficacy and limit escape and resistance should therefore beursued.

.2.2. CancerThe overexpression of certain molecules or expression of

eo-antigens in cancer cells provides potential targets for attacky the immune system. However, in progressive disease the nat-ral immune response apparently lacks specificity or potency to

imit disease spreading. For many cancer indications, therapeu-ic monoclonal antibodies are currently available that redirect themmune system to attack and suppress malignant cells. There isccumulating evidence that complement activation contributes tochieve this (Table 1B). Nevertheless, controversy on the role ofomplement activation in cancer therapy remains. We will useD20 antibodies to exemplify the role of complement activation inancer treatment and give our view on the place for complementn the cancer immunotherapy field.

Rituximab and ofatumumab are both licensed CD20 antibodieshat have been shown to employ complement activation for thelimination of malignant B-cells. Ofatumumab has demonstrateduperior complement activation and CDC compared to rituximabn a range of in vitro and in vivo studies (see Lindorfer et al., 2014or a comprehensive review) (Baig et al., 2014; Barth et al., 2012;eum et al., 2008; Beurskens et al., 2012; Bologna et al., 2013; Lit al., 2009; Middleton et al., 2015; Pawluczkowycz et al., 2009;ebejova et al., 2014; Teeling et al., 2004). Remarkably, ofatu-umab allows for much lower CD20 expression levels and the

resence of much higher levels of (negative) complement regula-ory proteins in complement-mediated cell killing than rituximabBarth et al., 2012; Teeling et al., 2004; van Meerten et al., 2010).ased on these latter findings, antibody constructs targeting bothD20 and CD55 or CD59 were developed, which showed increasedilling in pre-clinical studies compared to targeting CD20 aloneMacor et al., 2014, 2007; Ziller et al., 2005).

Rituximab and ofatumumab are both type I CD20 antibodies thattrongly cluster CD20 into lipid rafts, a feature that is linked toheir ability to potently engage complement (Cragg et al., 2003;eans et al., 2002; Teeling et al., 2004). The differences in the

nology 67 (2015) 117–130 123

strength of complement-mediated cell lysis between the two CD20antibodies have been studied intensively and can be explained bytwo key molecular differences. Firstly, clustering facilitates inter-molecular antibody Fc:Fc contacts that mediate the assembly ofIgG hexamers required for optimal C1 binding and complementactivation (Diebolder et al., 2014). Ofatumumab-opsonized cellsbind C1q with much higher avidity than rituximab-opsonized cells(Pawluczkowycz et al., 2009), which suggests that ofatumumab Fctails assemble more readily into hexameric platforms (with highC1q avidity) than the Fcs in rituximab. This increased clusteringpotentially also explains the relatively slow functional off-rate ofofatumumab (i.e. its long retention of CDC capacity after targetcell opsonization) (Teeling et al., 2004). We speculate that theofatumumab epitope on CD20, as well as the antibody’s bindingorientation, offer a more favorable structural arrangement for hex-amer formation compared to rituximab. Secondly, ofatumumabrecognizes an epitope that includes the small loop of CD20 andconsequently binds more closely to the cell surface than rituximab(Du et al., 2009; Niederfellner et al., 2011; Teeling et al., 2006).Activated C4 and C3 only exist for a very short period until theyare hydrolyzed, during which time they may diffuse away andform a covalent bond either with a cell surface protein or the anti-body itself. The potential for cell surface-opsonization with C4 andC3 and subsequent complement proteins therefore is thought todepend on the distance from the cell membrane at which C1 acti-vation occurs (Sim et al., 1981; Teeling et al., 2004; Xia et al., 1993a,1993b). We believe that a potent ability of ofatumumab to facili-tate hexamerization of its Fc segments at close proximity to the cellmembrane leads to an unusually effective complement depositionon the target cell surface. This notion is supported by the observa-tion that a much larger fraction of C4b locates to the surface afterofatumumab-induced complement activation compared to that forrituximab (Beurskens et al., 2012).

The in vivo contribution of complement activation to the mech-anism of action of CD20 antibodies in cancer therapy is still heavilydebated. In part, this can be explained by the absence of clear-cutcorrelations between complement polymorphisms and clinicalresponses after CD20 antibody treatment. In contrast, such correla-tions convinced the field of the importance of Fc receptor-mediatedeffector functions (e.g. ADCC) a long time ago (Cartron et al., 2002;Dall’Ozzo et al., 2004), even though correlations were observedin some indications and not in others (Boross and Leusen, 2012;Dornan et al., 2010; Farag et al., 2004). A correlation between a sin-gle nucleotide polymorphism (SNP) in the C1qA gene (rs172378,also described as C1qA[276]) with prolonged response rates to rit-uximab therapy of follicular lymphoma and diffuse large B-celllymphoma has been described (Jin et al., 2012; Racila et al., 2008).However, as the SNP does not lead to a change in the C1q aminoacid composition, the mechanism is poorly understood and thisobservation therefore does not provide an insight into the con-tribution of complement in CD20 antibody therapy (Derer et al.,2014). The importance of complement in tumor cell killing thusneeds to be drawn from in vitro, in vivo and ex vivo models. Arole for complement in therapy is supported by the observationsthat complement depletion by CVF markedly reduced cell-killingefficacy of rituximab (Cragg and Glennie, 2004) and that in vivodepletion of NK cells and neutrophils does not affect therapeu-tic activity of rituximab in xenograft models (Boross et al., 2011;Di Gaetano et al., 2003; Golay et al., 2006). Here, it is impor-tant to note that both rituximab and ofatumumab utilize multipleeffector mechanisms, including CDC, ADCC (antibody-dependentcellular cytotoxicity), ADCP (antibody-dependent cellular phago-

cytosis), and CDCC (complement-dependent cellular cytotoxicity).Interestingly, it has been observed that in absence of ADCC andADCP, in knock-out mice lacking activating Fc receptors, therapyby CD20 antibodies is inhibited (Clynes et al., 2000), but that in

124 J.P.M. Melis et al. / Molecular Immunology 67 (2015) 117–130

Fig. 4. Interplay between complement activation and cell-mediated effector mechanisms. Local complement activation results in complement-dependent-cytotoxicity(CDC) of target cells, plus leads to production of opsonins (C3b, iC3b and C4b) and anaphylatoxin C5a. These mediators enable cross-talk between the complement andcellular immune system, which results in combined and synergistic effects. Opsonization of target cells allows complement-dependent-cellular-cytotoxicity (CDCC) viacomplement receptors (CR) binding of macrophages and NK cells. C5a acts as a chemoattractant for the recruitment of inflammatory cells and up-regulates expressiono c�RIIba

asccF(mtoiA2fti2cmchttat

f the activating Fc� receptors while it down-regulates the inhibitory receptor Fntibody-dependent-cellular-phagocytosis (ADCP).

bsence of complement activation, in C1q knock-out mice, theame is achieved (Di Gaetano et al., 2003). Similarly, whereasomplement activation alone was effective against a low tumorell burden, larger tumor loads required the presence of activec receptors, complement, as well as the complement receptor 3Boross et al., 2011). In our view, this emphasizes that comple-

ent and cell-mediated functions should not be seen in isolation;hese mechanisms contain important links by which they aid eachther (Fig. 4). Indeed, complement activation has been shown toncrease the elimination of mAb-opsonized cells via ADCC andDCP (Boross et al., 2011; de Haij et al., 2010; Teeling et al.,004, 2006). C5a, furthermore, acts as a potent chemoattractantor the recruitment of inflammatory effector cells and, in addi-ion, up-regulates expression of the activating Fc� receptors, whilet down-regulates the inhibitory receptor Fc�RIIb (Godau et al.,004; Karsten and Kohl, 2012; Shushakova et al., 2002). Finally,omplement opsonization of target cells increased their killing byacrophages via ADCC. Such complement-enhanced cell-mediated

ytotoxicity may be crucial for effective tumor cell depletion atigh tumor burden (Boross et al., 2011). Not only for CD20 mAb

reatment, but for antibody therapy of cancer in general, weherefore envisage an intricate interplay between complementnd cell-mediated effectors, in which local complement activa-ion not only leads to cell killing, but also attracts and activates

. This results in enhanced antibody-dependent-cellular-cytotoxicity (ADCC) and

effector cells leading to additional and synergistic killing activity(Fig. 4).

A further complicating factor is that the killing mechanismsemployed by therapeutic agents, such as CD20 antibodies, that relyon innate effector mechanisms may be depleted. Indeed, rituximabinfusion promotes rapid complement depletion and acute CD20loss in chronic lymphocytic leukemia (CLL) patients. Sera of treatedpatients therefore have a reduced capacity to C3b-opsonize and killCD20-positive cells, unless supplemented with normal serum orcomponent C2 (Kennedy et al., 2004). In ofatumumab-treated CLLpatients it was observed that, following ofatumumab infusion andinitial depletion, CLL cells coated with C3d and CD20 antibody couldbe observed in the blood, suggesting that the combined effects ofcomplement and Fc-mediated effector mechanisms became insuf-ficient for further B-cell depletion soon after dosing. The observedexhaustion was transient as depletion of CLL cells was again appar-ent at the next doses (Beurskens et al., 2012). In vitro studiesindicated that exhaustion and suboptimal killing were associatedwith high antibody concentrations, suggesting that it is importantto identify optimal antibody dosing regimens aimed at maximizing

killing while minimizing effector reservoir exhaustion (Baig et al.,2014; Beurskens et al., 2012; Zent et al., 2014).

In summary, convincing evidence exists for a critical role of com-plement in the in vivo mechanism of action of complement-active

r Immu

ace

3b

araa

tCd

3

orpaaartpa

apppeid2bar(Tdpme(Mt2

aaceattl

3

e

J.P.M. Melis et al. / Molecula

ntibodies. Herein, complement activation, in addition to its directytotoxic effects, attracts and activates effector cells to induce syn-rgistic tumor cell killing.

. Improving antibody therapies – restoring or shifting thealance

Complement represents an important target or effector mech-nism for a range of therapeutic approaches, as is argued in thiseview. In this section, we will discuss antibody format and ther-py innovations to further optimize, strengthen and develop thispproach.

We envisage three main strategies by which antibody-basedherapies that target complement activation could be improved: (i)ombination therapies, (ii) Antibody engineering, and (iii) Therapyesign.

.1. Combination therapies

Targeting multiple proteins by the use of antibody cocktailsr bispecific antibodies is a promising strategy to dampen orecruit complement and to counter immune evasion molecules onathogens and on cancer cells. In its crudest form, polyclonals exists antibody preparations derived from immune sera generated innimals. Despite their drawbacks, such non-human polyclonalsre still in use as anti-venoms to treat live-threatening conditionsesulting from snake bites. The interaction between various animaloxins and the human complement system and its inhibition mayrovide leads for potential therapeutic intervention (Tambourgind van den Berg, 2014).

The development of combination therapies using recombinantntibody technologies has recently been coming of age. Thera-ies based on the combination of two existing antibodies haverogressed up to approval for human use e.g. trastuzumab andertuzumab (Baselga et al., 2012; Blumenthal et al., 2013; Swaint al., 2013) and technologies to generate antibody cocktails exist-ng of recombinant oligoclonal or polyclonal antibodies are beingeveloped (Andersen et al., 2007; Logtenberg, 2007; Nielsen et al.,010; Wang et al., 2013). Bispecific IgG antibodies that com-ine two distinct antibody binding sites, offering the ability toddress two distinct targets with a single antibody, can now beoutinely generated and manufactured using recent innovationsGramer et al., 2013; Kontermann, 2012; Labrijn et al., 2013).he approach can not only be used to target epitopes on twoistinct proteins, but also to target two epitopes on a singlerotein. The potential of the latter is supported by studies onultiple cell surface antigens that demonstrated increased or syn-

rgistic CDC by certain non-cross blocking antibody combinationsDechant et al., 2008; Fogler et al., 1988; Klitgaard et al., 2013;

acor et al., 2006; Spiridon et al., 2002) and was demonstratedo increase complement activation of CD20 antibodies (Li et al.,008).

Combination therapies, by dual or multispecific targetingpproaches, may allow the combined inhibition of complementctivation pathways to treat complex pathologies, such as dis-ussed for sepsis or RA. Alternatively, these approaches may bemployed to maximize the effects of complement activation, suchs aimed for in therapies for infectious disease or cancer. Combina-ion therapies of complement activating antibodies with antibodieshat inhibit the activity of host or pathogen CRPs could be particu-arly rewarding.

.2. Antibody engineering

Molecular engineering of functional sites represents a well-stablished approach to inhibit or enhance complement activation

nology 67 (2015) 117–130 125

of therapeutic antibodies. Mutations in the C1q binding site or thehinge region can be used to selectively abrogate complement acti-vating capabilities (Duncan and Winter, 1988; Hezareh et al., 2001;Xu et al., 2000). Antibodies in which both complement-mediatedand cellular effector functions were knocked out have a.o. beeninvestigated for the treatment of diabetes (Herold et al., 2005; Xuet al., 2000).

The structure of the hinge region affects antibody flexibilitywhich is correlated with the antibody’s capacity to activate comple-ment (Oi et al., 1984). Hinge modifications to enhance C1q bindingand complement activation therefore have been investigated. Therequirements of the hinge region for complement activation washowever minimal (Brekke et al., 1995). Varying the hinge lengthby itself was not sufficient to regulate complement activity in IgGmolecules (Dangl et al., 1988; Tan et al., 1990) and amino acid mod-ifications in IgG hinge regions only resulted in minor improvementsin C1q binding and CDC (Dall’Acqua et al., 2006). The opportunitiesfor enhancement of complement activation by hinge modificationtherefore appear to be modest.

A second approach to enhance complement activation is byincreasing the affinity of the C1q binding site on IgG. Mutationsin or near the C1q binding site can indeed result in enhanced C1qaffinity and increased CDC (Idusogie et al., 2001; Moore et al., 2010).Also IgG1/IgG3 chimeric antibodies show stronger C1q binding andincreased CDC capacity (Kellner et al., 2014; Natsume et al., 2008,2009). A potential drawback of strengthening the binding of C1q tomonomeric IgG is that C1q may bind the therapeutic IgG in solu-tion independent of target binding, leading to non-specific effectsand/or C1q may bind to very small immune complexes leading tosuboptimal complement stimulation.

Glycoengineering provides a third approach to modify com-plement activation by IgG antibodies. Fc glycosylation linkedto the asparagine residue at amino acid position 297 is crit-ical for the binding of IgG Fc to C1q and the induction ofCDC and mutating this site strongly impacts C1q interaction(Nimmerjahn and Ravetch, 2008; Wright and Morrison, 1997).N-glycans in the Fc region vary in terminal residues, in whichterminal galactose, N-acetylglucosamine, and mannose residuesmay affect C1q binding and CDC activity. The opportunitieshowever are limited, as maximal terminal galactosylation ofglycans (G2) was reported to increase CDC of the CD20 anti-body rituximab up to a maximum of only 2-fold compared tothe non-galactosylated (G0) antibody (Ferrara et al., 2006; Raju,2008).

Recently, we described a completely novel approach to mod-ulate complement activation by IgG antibodies. This approachbuilds on our insight into the molecular mechanism of complementactivation by IgG. We found that IgG antibodies form hexam-eric structures after they bind antigen on the cell surface. Thesehexamers are characterized by extensive intermolecular Fc:Fc con-tacts that can be manipulated to decrease or enhance complementactivating capabilities (Diebolder et al., 2014). A number of Fc:Fcinterface mutations that enhance hexamer formation, C1q bind-ing, and complement activation were identified. The versatilityof the approach is suggested by the observation that a mutationof glutamic acid into arginine at position 345 can enhance com-plement activation of all four IgG isotypes of an antibody againstCD38 (Diebolder et al., 2014). The approach appears particularlyattractive to specifically increase the assembly of the functionalIgG antibody unit that activates complement. IgG hexamerizationcould therefore be conceptually applicable in the engineering oftherapeutic antibodies with enhanced activity and creates opportu-

nities for potentiating existing or novel monoclonal antibody drugsfor which effector functions are essential mechanisms. Applicationof this novel technology to drug development is currently beingpursued.

1 r Imm

3

moariiadeaCmmr2Lbapccica

4

famabnooio

C

a

A

c

R

A

A

A

A

26 J.P.M. Melis et al. / Molecula

.3. Therapy design

A third strategy to maximize the effects of complement engage-ent by therapeutic antibodies is to further improve the design

f therapeutic regimens. It has been argued that exaggeratedntibody-mediated complement activation could lead to tempo-ary exhaustion of critical complement components and therebynterfere with the efficacy of the treatment. Preclinical studiesndeed suggested that a killing optimum is achieved at intermedi-te antibody concentrations, whereas high antibody concentrationsepleted complement, leading to suboptimal cell lysis (Beurskenst al., 2012). Therapy design affects in vivo characteristics, suchs antibody pharmacokinetics, tissue penetration, opsonization,DCC, ADCC, ADCP and trogocytosis. Different therapeutic regi-ens, such as more frequent injections with lower antibody doses,ay prevent exhaustion and trogocytosis and thereby allow for

ecovery of effector activity between administrations (Baig et al.,014; Beurskens et al., 2012; Lindorfer et al., 2012; Taylor andindorfer, 2014; Zent et al., 2014). Therapies should thereforee carefully designed to take the interplay between complementctivation, cell-mediated effector functions, and various otherarameters into account. The inclusion of biomarkers to measureomplement titers and complement activation products appearsritical in this respect. Therapy design represents a highly interest-ng yet complex field in which further preclinical experiments, inombination with innovative and state-of-the-art clinical studies,re required to increase insight and establish general rules.

. Conclusions

Modulation of complement activation represents a power-ul strategy with a strong potential for therapeutic antibodypproaches in a wide variety of diseases. Novel antibody for-ats, including bispecific antibodies, antibody combinations and

ntibodies engineered for optimized complement activating capa-ilities, provide extensive opportunities for the development ofew and improved treatments. To achieve the full potential of thesepportunities, it will be critical for pre-clinical and clinical devel-pment to collaborate closely to bring together antibody biology,mmunology, pharmacology and drug development knowledge inrder to optimally tweak clinical trial designs.

onflict of interest

All authors are Genmab employees and own Genmab warrantsnd/or stock.

cknowledgements

We thank René Toes, Henny Otten and Suzan Rooijakkers forritically reviewing the manuscript.

eferences

mico, P., Honger, G., Mayr, M., Steiger, J., Hopfer, H., Schaub, S., 2009. Clinical rele-vance of pretransplant donor-specific HLA antibodies detected by single-antigenflow-beads. Transplantation 87, 1681–1688.

ndersen, P.S., Haahr-Hansen, M., Coljee, V.W., Hinnerfeldt, F.R., Varming, K., Bre-genholt, S., Haurum, J.S., 2007. Extensive restrictions in the VH sequence usageof the human antibody response against the Rhesus D antigen. Mol. Immunol.44, 412–422.

nderson, D.R., Grillo-Lopez, A., Varns, C., Chambers, K.S., Hanna, N., 1997. Targetedanti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin’s B-cell lymphoma. Biochem. Soc. Trans.

25, 705–708.

nsari, M., McKeigue, P.M., Skerka, C., Hayward, C., Rudan, I., Vitart, V., Polasek, O.,Armbrecht, A.M., Yates, J.R., Vatavuk, Z., Bencic, G., Kolcic, I., Oostra, B.A., VanDuijn, C.M., Campbell, S., Stanton, C.M., Huffman, J., Shu, X., Khan, J.C., Shahid,H., Harding, S.P., Bishop, P.N., Deary, I.J., Moore, A.T., Dhillon, B., Rudan, P., Zipfel,

unology 67 (2015) 117–130

P.F., Sim, R.B., Hastie, N.D., Campbell, H., Wright, A.F., 2013. Genetic influenceson plasma CFH and CFHR1 concentrations and their role in susceptibility toage-related macular degeneration. Hum. Mol. Genet. 22, 4857–4869.

Arnold, J.N., Wormald, M.R., Suter, D.M., Radcliffe, C.M., Harvey, D.J., Dwek, R.A.,Rudd, P.M., Sim, R.B., 2005. Human serum IgM glycosylation: identificationof glycoforms that can bind to mannan-binding lectin. J. Biol. Chem. 280,29080–29087.

Baig, N.A., Taylor, R.P., Lindorfer, M.A., Church, A.K., LaPlant, B.R., Pettinger,A.M., Shanafelt, T.D., Nowakowski, G.S., Zent, C.S., 2014. Induced resistanceto ofatumumab-mediated cell clearance mechanisms, including complement-dependent cytotoxicity, in chronic lymphocytic leukemia. J. Immunol. 192,1620–1629.

Ballanti, E., Perricone, C., Greco, E., Ballanti, M., Di Muzio, G., Chimenti, M.S.,Perricone, R., 2013. Complement and autoimmunity. Immunol. Res. 56,477–491.

Banda, N.K., Hyatt, S., Antonioli, A.H., White, J.T., Glogowska, M., Takahashi, K.,Merkel, T.J., Stahl, G.L., Mueller-Ortiz, S., Wetsel, R., Arend, W.P., Holers,V.M., 2012. Role of C3a receptors, C5a receptors, and complement protein C6deficiency in collagen antibody-induced arthritis in mice. J. Immunol. 188,1469–1478.

Barilla-Labarca, M.L., Toder, K., Furie, R., 2013. Targeting the complement system insystemic lupus erythematosus and other diseases. Clin. Immunol. 148, 313–321.

Barth, M.J., Hernandez-Ilizaliturri, F.J., Mavis, C., Tsai, P.C., Gibbs, J.F., Deeb, G.,Czuczman, M.S., 2012. Ofatumumab demonstrates activity against rituximab-sensitive and -resistant cell lines, lymphoma xenografts and primary tumourcells from patients with B-cell lymphoma. Br. J. Haematol. 156, 490–498.

Baselga, J., Cortes, J., Kim, S.B., Im, S.A., Hegg, R., Im, Y.H., Roman, L., Pedrini, J.L.,Pienkowski, T., Knott, A., Clark, E., Benyunes, M.C., Ross, G., Swain, S.M., 2012.Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N.Engl. J. Med. 366, 109–119.

Bergeron-Sawitzke, J., Gold, B., Olsh, A., Schlotterbeck, S., Lemon, K., Visvanathan,K., Allikmets, R., Dean, M., 2009. Multilocus analysis of age-related maculardegeneration. Eur. J. Hum. Genet. 17, 1190–1199.

Bessler, M., Mason, P.J., Hillmen, P., Miyata, T., Yamada, N., Takeda, J., Luzzatto, L.,Kinoshita, T., 1994. Paroxysmal nocturnal haemoglobinuria (PNH) is caused bysomatic mutations in the PIG-A gene. EMBO J. 13, 110–117.

Beum, P.V., Lindorfer, M.A., Beurskens, F., Stukenberg, P.T., Lokhorst, H.M.,Pawluczkowycz, A.W., Parren, P.W., van de Winkel, J.G., Taylor, R.P., 2008. Com-plement activation on B lymphocytes opsonized with rituximab or ofatumumabproduces substantial changes in membrane structure preceding cell lysis. J.Immunol. 181, 822–832.

Beurskens, F.J., Lindorfer, M.A., Farooqui, M., Beum, P.V., Engelberts, P., Mackus, W.J.,Parren, P.W., Wiestner, A., Taylor, R.P., 2012. Exhaustion of cytotoxic effector sys-tems may limit monoclonal antibody-based immunotherapy in cancer patients.J. Immunol. 188, 3532–3541.

Bindon, C.I., Hale, G., Bruggemann, M., Waldmann, H., 1988. Human monoclonal IgGisotypes differ in complement activating function at the level of C4 as well asC1q. J. Exp. Med. 168, 127–142.

Blumenthal, G.M., Scher, N.S., Cortazar, P., Chattopadhyay, S., Tang, S., Song, P., Liu, Q.,Ringgold, K., Pilaro, A.M., Tilley, A., King, K.E., Graham, L., Rellahan, B.L., Wein-berg, W.C., Chi, B., Thomas, C., Hughes, P., Ibrahim, A., Justice, R., Pazdur, R.,2013. First FDA approval of dual anti-HER2 regimen: pertuzumab in combina-tion with trastuzumab and docetaxel for HER2-positive metastatic breast cancer.Clin. Cancer Res. 19, 4911–4916.

Bologna, L., Gotti, E., Da Roit, F., Intermesoli, T., Rambaldi, A., Introna, M., Golay, J.,2013. Ofatumumab is more efficient than rituximab in lysing B chronic lympho-cytic leukemia cells in whole blood and in combination with chemotherapy. J.Immunol. 190, 231–239.

Bonifati, D.M., Kishore, U., 2007. Role of complement in neurodegeneration andneuroinflammation. Mol. Immunol. 44, 999–1010.

Bora, N.S., Matta, B., Lyzogubov, V.V., Bora, P.S., 2015. Relationship between thecomplement system, risk factors and prediction models in age-related maculardegeneration. Mol. Immunol. 63, 176–183.

Bordet, J., 1895. Les leukocytes et les proprietes actives du serum chez les vaccines.Ann. Inst. Pasteur, 462–506.

Boross, P., Jansen, J.H., de Haij, S., Beurskens, F.J., van der Poel, C.E., Bevaart, L., Ned-erend, M., Golay, J., van de Winkel, J.G., Parren, P.W., Leusen, J.H., 2011. Thein vivo mechanism of action of CD20 monoclonal antibodies depends on localtumor burden. Haematologica 96, 1822–1830.

Boross, P., Leusen, J.H., 2012. Mechanisms of action of CD20 antibodies. Am. J. CancerRes. 2, 676–690.

Borsos, T., Rapp, H.J., 1965. Complement fixation on cell surfaces by 19S and 7Santibodies. Science 150, 505–506.

Botto, M., Kirschfink, M., Macor, P., Pickering, M.C., Wurzner, R., Tedesco, F., 2009.Complement in human diseases: lessons from complement deficiencies. Mol.Immunol. 46, 2774–2783.

Brekke, O.H., Michaelsen, T.E., Sandlie, I., 1995. The structural requirements for com-plement activation by IgG: does it hinge on the hinge? Immunol. Today 16,85–90.

Bruggemann, M., Williams, G.T., Bindon, C.I., Clark, M.R., Walker, M.R., Jefferis, R.,Waldmann, H., Neuberger, M.S., 1987. Comparison of the effector functions of

human immunoglobulins using a matched set of chimeric antibodies. J. Exp.Med. 166, 1351–1361.

Bryan, A.R., Wu, E.Y., 2014. Complement deficiencies in systemic lupus erythemato-sus. Curr. Allergy Asthma Rep. 14, 448.

Burton, D.R., 1985. Immunoglobulin G: functional sites. Mol. Immunol. 22, 161–206.

r Immu

B

B

B

C

C

C

C

C

C

C

C

C

C

C

C

C

C

D

D

D

D

d

d

D

D

D

D

D

J.P.M. Melis et al. / Molecula

urton, D.R., 1986. Is IgM-like dislocation a common feature of antibody function?Immunol. Today 7, 165–167.

urton, D.R., Boyd, J., Brampton, A.D., Easterbrook-Smith, S.B., Emanuel, E.J., Novotny,J., Rademacher, T.W., van Schravendijk, M.R., Sternberg, M.J., Dwek, R.A., 1980.The Clq receptor site on immunoglobulin G. Nature 288, 338–344.

urton, D.R., Poignard, P., Stanfield, R.L., Wilson, I.A., 2012. Broadly neutralizing anti-bodies present new prospects to counter highly antigenically diverse viruses.Science 337, 183–186.

artron, G., Dacheux, L., Salles, G., Solal-Celigny, P., Bardos, P., Colombat, P., Watier,H., 2002. Therapeutic activity of humanized anti-CD20 monoclonal antibody andpolymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood 99, 754–758.

astellano, G., Melchiorre, R., Loverre, A., Ditonno, P., Montinaro, V., Rossini, M.,Divella, C., Battaglia, M., Lucarelli, G., Annunziata, G., Palazzo, S., Selvaggi, F.P.,Staffieri, F., Crovace, A., Daha, M.R., Mannesse, M., van Wetering, S., Paolo Schena,F., Grandaliano, G., 2010. Therapeutic targeting of classical and lectin pathwaysof complement protects from ischemia–reperfusion-induced renal damage. Am.J. Pathol. 176, 1648–1659.

attaneo, A., Neuberger, M.S., 1987. Polymeric immunoglobulin M is secreted bytransfectants of non-lymphoid cells in the absence of immunoglobulin J chain.EMBO J. 6, 2753–2758.

hen, M., Daha, M.R., Kallenberg, C.G., 2010. The complement system in systemicautoimmune disease. J. Autoimmun. 34, J276–J286.

heney, C.M., Stephens, D.M., Mo, X., Rafiq, S., Butchar, J., Flynn, J.M., Jones, J.A.,Maddocks, K., O’Reilly, A., Ramachandran, A., Tridandapani, S., Muthusamy,N., Byrd, J.C., 2014. Ocaratuzumab, an Fc-engineered antibody demonstratesenhanced antibody-dependent cell-mediated cytotoxicity in chronic lympho-cytic leukemia. MAbs 6, 749–755.

lynes, R.A., Towers, T.L., Presta, L.G., Ravetch, J.V., 2000. Inhibitory Fc receptorsmodulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443–446.

ollins, C., Tsui, F.W., Shulman, M.J., 2002. Differential activation of human andguinea pig complement by pentameric and hexameric IgM. Eur. J. Immunol.32, 1802–1810.

oloma, M.J., Trinh, K.R., Wims, L.A., Morrison, S.L., 1997. The hinge as a spacer con-tributes to covalent assembly and is required for function of IgG. J. Immunol.158, 733–740.

orti, D., Bianchi, S., Vanzetta, F., Minola, A., Perez, L., Agatic, G., Guarino, B., Silacci,C., Marcandalli, J., Marsland, B.J., Piralla, A., Percivalle, E., Sallusto, F., Baldanti,F., Lanzavecchia, A., 2013. Cross-neutralization of four paramyxoviruses by ahuman monoclonal antibody. Nature 501, 439–443.

orti, D., Lanzavecchia, A., 2013. Broadly neutralizing antiviral antibodies. Annu. Rev.Immunol. 31, 705–742.

ragg, M.S., Glennie, M.J., 2004. Antibody specificity controls in vivo effector mech-anisms of anti-CD20 reagents. Blood 103, 2738–2743.

ragg, M.S., Morgan, S.M., Chan, H.T., Morgan, B.P., Filatov, A.V., Johnson, P.W.,French, R.R., Glennie, M.J., 2003. Complement-mediated lysis by anti-CD20 mAbcorrelates with segregation into lipid rafts. Blood 101, 1045–1052.

ravedi, P., Leventhal, J., Lakhani, P., Ward, S.C., Donovan, M.J., Heeger, P.S., 2013.Immune cell-derived C3a and C5a costimulate human T cell alloimmunity. Am.J. Transplant. 13, 2530–2539.

zajkowsky, D.M., Shao, Z., 2009. The human IgM pentamer is a mushroom-shaped molecule with a flexural bias. Proc. Natl. Acad. Sci. U. S. A. 106,14960–14965.

all’Acqua, W.F., Cook, K.E., Damschroder, M.M., Woods, R.M., Wu, H., 2006. Modu-lation of the effector functions of a human IgG1 through engineering of its hingeregion. J. Immunol. 177, 1129–1138.

all’Ozzo, S., Tartas, S., Paintaud, G., Cartron, G., Colombat, P., Bardos, P., Watier,H., Thibault, G., 2004. Rituximab-dependent cytotoxicity by natural killer cells:influence of FCGR3A polymorphism on the concentration-effect relationship.Cancer Res. 64, 4664–4669.

angl, J.L., Wensel, T.G., Morrison, S.L., Stryer, L., Herzenberg, L.A., Oi, V.T., 1988. Seg-mental flexibility and complement fixation of genetically engineered chimerichuman, rabbit and mouse antibodies. EMBO J. 7, 1989–1994.

avies, A.M., Jefferis, R., Sutton, B.J., 2014. Crystal structure of deglycosylated humanIgG4-Fc. Mol. Immunol. 62, 46–53.

e Haij, S., Jansen, J.H., Boross, P., Beurskens, F.J., Bakema, J.E., Bos, D.L., Martens,A., Verbeek, J.S., Parren, P.W., van de Winkel, J.G., Leusen, J.H., 2010. In vivocytotoxicity of type I CD20 antibodies critically depends on Fc receptor ITAMsignaling. Cancer Res. 70, 3209–3217.

e Weers, M., Tai, Y.T., van der Veer, M.S., Bakker, J.M., Vink, T., Jacobs, D.C., Oomen,L.A., Peipp, M., Valerius, T., Slootstra, J.W., Mutis, T., Bleeker, W.K., Anderson,K.C., Lokhorst, H.M., van de Winkel, J.G., Parren, P.W., 2011. Daratumumab, anovel therapeutic human CD38 monoclonal antibody, induces killing of multiplemyeloma and other hematological tumors. J. Immunol. 186, 1840–1848.

eans, J.P., Li, H., Polyak, M.J., 2002. CD20-mediated apoptosis: signalling throughlipid rafts. Immunology 107, 176–182.

echant, M., Valerius, T., 2001. IgA antibodies for cancer therapy. Crit. Rev. Oncol.Hematol. 39, 69–77.

echant, M., Weisner, W., Berger, S., Peipp, M., Beyer, T., Schneider-Merck, T., Lam-merts van Bueren, J.J., Bleeker, W.K., Parren, P.W., van de Winkel, J.G., Valerius,T., 2008. Complement-dependent tumor cell lysis triggered by combinations ofepidermal growth factor receptor antibodies. Cancer Res. 68, 4998–5003.

erer, S., Beurskens, F.J., Rosner, T., Peipp, M., Valerius, T., 2014. Complement inantibody-based tumor therapy. Crit. Rev. Immunol. 34, 199–214.

i Gaetano, N., Cittera, E., Nota, R., Vecchi, A., Grieco, V., Scanziani, E., Botto, M.,Introna, M., Golay, J., 2003. Complement activation determines the therapeuticactivity of rituximab in vivo. J. Immunol. 171, 1581–1587.

nology 67 (2015) 117–130 127

Diebolder, C.A., Beurskens, F.J., de Jong, R.N., Koning, R.I., Strumane, K., Lindorfer,M.A., Voorhorst, M., Ugurlar, D., Rosati, S., Heck, A.J., van de Winkel, J.G., Wilson,I.A., Koster, A.J., Taylor, R.P., Saphire, E.O., Burton, D.R., Schuurman, J., Gros, P.,Parren, P.W., 2014. Complement is activated by IgG hexamers assembled at thecell surface. Science 343, 1260–1263.

Dinu, V., Miller, P.L., Zhao, H., 2007. Evidence for association between multiple com-plement pathway genes and AMD. Genet. Epidemiol. 31, 224–237.

Dornan, D., Spleiss, O., Yeh, R.F., Duchateau-Nguyen, G., Dufour, A., Zhi, J., Robak,T., Moiseev, S.I., Dmoszynska, A., Solal-Celigny, P., Warzocha, K., Loscertales, J.,Catalano, J., Afanasiev, B.V., Larratt, L., Rossiev, V.A., Bence-Bruckler, I., Geisler,C.H., Montillo, M., Wenger, M.K., Weisser, M., 2010. Effect of FCGR2A and FCGR3Avariants on CLL outcome. Blood 116, 4212–4222.

Du, J., Yang, H., Guo, Y., Ding, J., 2009. Structure of the Fab fragment of therapeuticantibody Ofatumumab provides insights into the recognition mechanism withCD20. Mol. Immunol. 46, 2419–2423.

Duncan, A.R., Winter, G., 1988. The binding site for C1q on IgG. Nature 332, 738–740.Durigutto, P., Macor, P., Ziller, F., De Maso, L., Fischetti, F., Marzari, R., Sblattero,

D., Tedesco, F., 2013. Prevention of arthritis by locally synthesized recombinantantibody neutralizing complement component C5. PLOS ONE 8, e58696.

Edwards, A.O., Ritter, R. 3rd, Abel, K.J., Manning, A., Panhuysen, C., Farrer, L.A., 2005.Complement factor H polymorphism and age-related macular degeneration.Science 308, 421–424.

Ehrlich, P.M.J., 1899. Zur Theorie der Lysenwirkung Berlin Klin. Wchsr. 6.Engstrom, G., Hedblad, B., Eriksson, K.F., Janzon, L., Lindgarde, F., 2005. Complement

C3 is a risk factor for the development of diabetes: a population-based cohortstudy. Diabetes 54, 570–575.

Fagerness, J.A., Maller, J.B., Neale, B.M., Reynolds, R.C., Daly, M.J., Seddon, J.M., 2009.Variation near complement factor I is associated with risk of advanced AMD.Eur. J. Hum. Genet. 17, 100–104.

Farag, S.S., Flinn, I.W., Modali, R., Lehman, T.A., Young, D., Byrd, J.C., 2004. Fc gammaRIIIa and Fc gamma RIIa polymorphisms do not predict response to rituximabin B-cell chronic lymphocytic leukemia. Blood 103, 1472–1474.

Feinstein, A., 1986. Immunoglobulin flexibility in complement activation. Immunol.Today 7, 169–174.

Ferrara, C., Brunker, P., Suter, T., Moser, S., Puntener, U., Umana, P., 2006. Modulationof therapeutic antibody effector functions by glycosylation engineering: influ-ence of Golgi enzyme localization domain and co-expression of heterologousbeta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II.Biotechnol. Bioeng. 93, 851–861.

Fogler, W.E., Klinger, M.R., Abraham, K.G., Gottlinger, H.G., Riethmuller, G., Daddona,P.E., 1988. Enhanced cytotoxicity against colon carcinoma by combinations ofnoncompeting monoclonal antibodies to the 17-1A antigen. Cancer Res. 48,6303–6308.

Foster, T.J., 2005. Immune evasion by staphylococci. Nat. Rev. Microbiol. 3, 948–958.Francis, P.J., Hamon, S.C., Ott, J., Weleber, R.G., Klein, M.L., 2009. Polymorphisms in

C2, CFB and C3 are associated with progression to advanced age related maculardegeneration associated with visual loss. J. Med. Genet. 46, 300–307.

Garcia-Rodriguez, C., Levy, R., Arndt, J.W., Forsyth, C.M., Razai, A., Lou, J., Geren, I.,Stevens, R.C., Marks, J.D., 2007. Molecular evolution of antibody cross-reactivityfor two subtypes of type A botulinum neurotoxin. Nat. Biotechnol. 25, 107–116.

Glennie, M.J., French, R.R., Cragg, M.S., Taylor, R.P., 2007. Mechanisms of killing byanti-CD20 monoclonal antibodies. Mol. Immunol. 44, 3823–3837.

Godau, J., Heller, T., Hawlisch, H., Trappe, M., Howells, E., Best, J., Zwirner, J., Ver-beek, J.S., Hogarth, P.M., Gerard, C., Van Rooijen, N., Klos, A., Gessner, J.E., Kohl,J., 2004. C5a initiates the inflammatory cascade in immune complex peritonitis.J. Immunol. 173, 3437–3445.

Golay, J., Cittera, E., Di Gaetano, N., Manganini, M., Mosca, M., Nebuloni, M., vanRooijen, N., Vago, L., Introna, M., 2006. The role of complement in the therapeuticactivity of rituximab in a murine B lymphoma model homing in lymph nodes.Haematologica 91, 176–183.

Goldenberg, D.M., Morschhauser, F., Wegener, W.A., 2010. Veltuzumab (humanizedanti-CD20 monoclonal antibody): characterization, current clinical results, andfuture prospects. Leuk. Lymphoma 51, 747–755.

Gorsuch, W.B., Chrysanthou, E., Schwaeble, W.J., Stahl, G.L., 2012. The complementsystem in ischemia-reperfusion injuries. Immunobiology 217, 1026–1033.

Gramer, M.J., van den Bremer, E.T., van Kampen, M.D., Kundu, A., Kopfmann, P.,Etter, E., Stinehelfer, D., Long, J., Lannom, T., Noordergraaf, E.H., Gerritsen, J.,Labrijn, A.F., Schuurman, J., van Berkel, P.H., Parren, P.W., 2013. Production ofstable bispecific IgG1 by controlled Fab-arm exchange: scalability from benchto large-scale manufacturing by application of standard approaches. MAbs 5,962–973.

Gros, P., Milder, F.J., Janssen, B.J., 2008. Complement driven by conformationalchanges. Nat. Rev. Immunol. 8, 48–58.

Grumach, A.S., Kirschfink, M., 2014. Are complement deficiencies really rare?Overview on prevalence, clinical importance and modern diagnostic approach.Mol. Immunol. 61, 110–117.

Guo, R.F., Ward, P.A., 2005. Role of C5a in inflammatory responses. Annu. Rev.Immunol. 23, 821–852.

Hadding, U., Mueller-Eberhard, H.J., 1969. The ninth component of humancomplement: isolation, description and mode of action. Immunology 16,719–735.

Hageman, G.S., Anderson, D.H., Johnson, L.V., Hancox, L.S., Taiber, A.J., Hardisty,L.I., Hageman, J.L., Stockman, H.A., Borchardt, J.D., Gehrs, K.M., Smith, R.J., Sil-vestri, G., Russell, S.R., Klaver, C.C., Barbazetto, I., Chang, S., Yannuzzi, L.A., Barile,G.R., Merriam, J.C., Smith, R.T., Olsh, A.K., Bergeron, J., Zernant, J., Merriam, J.E.,Gold, B., Dean, M., Allikmets, R., 2005. A common haplotype in the complement

1 r Imm

H

H

H

H

H

H

H

H

H

H

H

I

J

K

K

K

K

K

K

K

K

K

K

K

L

L

28 J.P.M. Melis et al. / Molecula

regulatory gene factor H (HF1/CFH) predisposes individuals to age-related mac-ular degeneration. Proc. Natl. Acad. Sci. U. S. A. 102, 7227–7232.

aines, J.L., Hauser, M.A., Schmidt, S., Scott, W.K., Olson, L.M., Gallins, P., Spencer,K.L., Kwan, S.Y., Noureddine, M., Gilbert, J.R., Schnetz-Boutaud, N., Agarwal, A.,Postel, E.A., Pericak-Vance, M.A., 2005. Complement factor H variant increasesthe risk of age-related macular degeneration. Science 308, 419–421.

erold, K.C., Gitelman, S.E., Masharani, U., Hagopian, W., Bisikirska, B., Donaldson,D., Rother, K., Diamond, B., Harlan, D.M., Bluestone, J.A., 2005. A single course ofanti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improvementin C-peptide responses and clinical parameters for at least 2 years after onset oftype 1 diabetes. Diabetes 54, 1763–1769.

essell, A.J., Hangartner, L., Hunter, M., Havenith, C.E., Beurskens, F.J., Bakker, J.M.,Lanigan, C.M., Landucci, G., Forthal, D.N., Parren, P.W., Marx, P.A., Burton, D.R.,2007. Fc receptor but not complement binding is important in antibody protec-tion against HIV. Nature 449, 101–104.

ezareh, M., Hessell, A.J., Jensen, R.C., van de Winkel, J.G., Parren, P.W., 2001. Effec-tor function activities of a panel of mutants of a broadly neutralizing antibodyagainst human immunodeficiency virus type 1. J. Virol. 75, 12161–12168.

illmen, P., Muus, P., Roth, A., Elebute, M.O., Risitano, A.M., Schrezenmeier, H., Szer, J.,Browne, P., Maciejewski, J.P., Schubert, J., Urbano-Ispizua, A., de Castro, C., Socie,G., Brodsky, R.A., 2013. Long-term safety and efficacy of sustained eculizumabtreatment in patients with paroxysmal nocturnal haemoglobinuria. Br. J. Haema-tol. 162, 62–73.

olers, V.M., 2014. Complement and its receptors: new insights into human disease.Annu. Rev. Immunol. 32, 433–459.

u, W., Yu, Q., Hu, N., Byrd, D., Amet, T., Shikuma, C., Shiramizu, B., Halperin, J.A.,Qin, X., 2010. A high-affinity inhibitor of human CD59 enhances complement-mediated virolysis of HIV-1: implications for treatment of HIV-1/AIDS. J.Immunol. 184, 359–368.

uber-Lang, M., Barratt-Due, A., Pischke, S.E., Sandanger, O., Nilsson, P.H., Nunn,M.A., Denk, S., Gaus, W., Espevik, T., Mollnes, T.E., 2014. Double blockade of CD14and complement C5 abolishes the cytokine storm and improves morbidity andsurvival in polymicrobial sepsis in mice. J. Immunol. 192, 5324–5331.

ughes-Jones, N.C., Gardner, B., 1978. The reaction between the complement sub-component C1q, IgG complexes and polyionic molecules. Immunology 34,459–463.

ughes-Jones, N.C., Gardner, B., 1979. Reaction between the isolated globular sub-units of the complement component C1q and IgG-complexes. Mol. Immunol. 16,697–701.

ughes-Jones, N.C., Gorick, B.D., Howard, J.C., Feinstein, A., 1985. Antibody densityon rat red cells determines the rate of activation of the complement componentC1. Eur. J. Immunol. 15, 976–980.

dusogie, E.E., Wong, P.Y., Presta, L.G., Gazzano-Santoro, H., Totpal, K., Ultsch, M.,Mulkerrin, M.G., 2001. Engineered antibodies with increased activity to recruitcomplement. J. Immunol. 166, 2571–2575.

in, X., Ding, H., Ding, N., Fu, Z., Song, Y., Zhu, J., 2012. Homozygous A polymorphism ofthe complement C1qA276 correlates with prolonged overall survival in patientswith diffuse large B cell lymphoma treated with R-CHOP. J. Hematol. Oncol. 5,51.

arsten, C.M., Kohl, J., 2012. The immunoglobulin, IgG Fc receptor and complementtriangle in autoimmune diseases. Immunobiology 217, 1067–1079.

eating, G.M., 2013. Eculizumab: a review of its use in atypical haemolytic uraemicsyndrome. Drugs 73, 2053–2066.

ellner, C., Derer, S., Valerius, T., Peipp, M., 2014. Boosting ADCC and CDC activityby Fc engineering and evaluation of antibody effector functions. Methods 65,105–113.

emper, C., Pangburn, M.K., Fishelson, Z., 2014. Complement nomenclature 2014.Mol. Immunol. 61, 56–58.

ennedy, A.D., Beum, P.V., Solga, M.D., DiLillo, D.J., Lindorfer, M.A., Hess, C.E., Dens-more, J.J., Williams, M.E., Taylor, R.P., 2004. Rituximab infusion promotes rapidcomplement depletion and acute CD20 loss in chronic lymphocytic leukemia. J.Immunol. 172, 3280–3288.

err, M.A., 1990. The structure and function of human IgA. Biochem. J 271,285–296.

ishore, U., Ghai, R., Greenhough, T.J., Shrive, A.K., Bonifati, D.M., Gadjeva, M.G.,Waters, P., Kojouharova, M.S., Chakraborty, T., Agrawal, A., 2004. Structuraland functional anatomy of the globular domain of complement protein C1q.Immunol. Lett. 95, 113–128.

lein, R.J., Zeiss, C., Chew, E.Y., Tsai, J.Y., Sackler, R.S., Haynes, C., Henning, A.K., San-Giovanni, J.P., Mane, S.M., Mayne, S.T., Bracken, M.B., Ferris, F.L., Ott, J., Barnstable,C., Hoh, J., 2005. Complement factor H polymorphism in age-related maculardegeneration. Science 308, 385–389.

litgaard, J.L., Koefoed, K., Geisler, C., Gadeberg, O.V., Frank, D.A., Petersen, J.,Jurlander, J., Pedersen, M.W., 2013. Combination of two anti-CD5 mono-clonal antibodies synergistically induces complement-dependent cytotoxicityof chronic lymphocytic leukaemia cells. Br. J. Haematol. 163, 182–193.

ontermann, R.E., 2012. Dual targeting strategies with bispecific antibodies. MAbs4, 182–197.

wan, W.H., Hashimoto, D., Paz-Artal, E., Ostrow, K., Greter, M., Raedler, H., Medof,M.E., Merad, M., Heeger, P.S., 2012. Antigen-presenting cell-derived complementmodulates graft-versus-host disease. J. Clin. Invest. 122, 2234–2238.

abrijn, A.F., Aalberse, R.C., Schuurman, J., 2008. When binding is enough: nonacti-vating antibody formats. Curr. Opin. Immunol. 20, 479–485.

abrijn, A.F., Meesters, J.I., de Goeij, B.E., van den Bremer, E.T., Neijssen, J., van Kam-pen, M.D., Strumane, K., Verploegen, S., Kundu, A., Gramer, M.J., van Berkel, P.H.,van de Winkel, J.G., Schuurman, J., Parren, P.W., 2013. Efficient generation of

unology 67 (2015) 117–130

stable bispecific IgG1 by controlled Fab-arm exchange. Proc. Natl. Acad. Sci. U.S. A. 110, 5145–5150.

Lan, J., Yang, K., Byrd, D., Hu, N., Amet, T., Shepherd, N., Desai, M., Gao, J., Gupta,S., Sun, Y., Yu, Q., 2014. Provirus activation plus CD59 blockage triggersantibody-dependent complement-mediated lysis of latently HIV-1-infectedcells. J. Immunol. 193, 3577–3589.

Legendre, C., Sberro-Soussan, R., Zuber, J., Rabant, M., Loupy, A., Timsit, M.O.,Anglicheau, D., 2013. Eculizumab in renal transplantation. Transplant. Rev.(Orlando) 27, 90–92.

Li, B., Shi, S., Qian, W., Zhao, L., Zhang, D., Hou, S., Zheng, L., Dai, J., Zhao, J., Wang,H., Guo, Y., 2008. Development of novel tetravalent anti-CD20 antibodies withpotent antitumor activity. Cancer Res. 68, 2400–2408.

Li, B., Zhao, L., Guo, H., Wang, C., Zhang, X., Wu, L., Chen, L., Tong, Q., Qian, W., Wang,H., Guo, Y., 2009. Characterization of a rituximab variant with potent antitumoractivity against rituximab-resistant B-cell lymphoma. Blood 114, 5007–5015.

Lindorfer, M., Bakker, J., Parren, P., Taylor, R., 2014. In: Dübel, S., Reichert, J. (Eds.), Ofa-tumumab: A Next-Generation Human Therapeutic CD20 Antibody with PotentComplement-Dependent Cytotoxicity. Wiley-Blackwell, pp. 1733–1774.

Lindorfer, M.A., Wiestner, A., Zent, C.S., Taylor, R.P., 2012. Monoclonal antibody(mAb)-based cancer therapy: Is it time to reevaluate dosing strategies? Oncoim-munology 1, 959–961.

Logtenberg, T., 2007. Antibody cocktails: next-generation biopharmaceuticals withimproved potency. Trends Biotechnol. 25, 390–394.

Loupy, A., Lefaucheur, C., Vernerey, D., Prugger, C., Duong van Huyen, J.P., Mooney,N., Suberbielle, C., Fremeaux-Bacchi, V., Mejean, A., Desgrandchamps, F.,Anglicheau, D., Nochy, D., Charron, D., Empana, J.P., Delahousse, M., Legendre,C., Glotz, D., Hill, G.S., Zeevi, A., Jouven, X., 2013. Complement-binding anti-HLAantibodies and kidney-allograft survival. N. Engl. J. Med. 369, 1215–1226.

Ma, Q., Li, D., Carreno, R., Patenia, R., Tsai, K.Y., Xydes-Smith, M., Alousi, A.M.,Champlin, R.E., Sale, G.E., Afshar-Kharghan, V., 2014. Complement componentC3 mediates Th1/Th17 polarization in human T-cell activation and cutaneousGVHD. Bone Marrow Transplant. 49, 972–976.

Ma, Q., Li, D., Nurieva, R., Patenia, R., Bassett, R., Cao, W., Alekseev, A.M., He,H., Molldrem, J.J., Kroll, M.H., Champlin, R.E., Sale, G.E., Afshar-Kharghan, V.,2012. Reduced graft-versus-host disease in C3-deficient mice is associated withdecreased donor Th1/Th17 differentiation. Biol. Blood Marrow Transplant. 18,1174–1181.

Macor, P., Durigutto, P., De Maso, L., Garrovo, C., Biffi, S., Cortini, A., Fischetti, F.,Sblattero, D., Pitzalis, C., Marzari, R., Tedesco, F., 2012. Treatment of experimentalarthritis by targeting synovial endothelium with a neutralizing recombinantantibody to C5. Arthritis Rheum. 64, 2559–2567.

Macor, P., Mezzanzanica, D., Cossetti, C., Alberti, P., Figini, M., Canevari, S., Tedesco,F., 2006. Complement activated by chimeric anti-folate receptor antibodiesis an efficient effector system to control ovarian carcinoma. Cancer Res. 66,3876–3883.

Macor, P., Secco, E., Mezzaroba, N., Zorzet, S., Durigutto, P., Gaiotto, T., De Maso,L., Biffi, S., Garrovo, C., Capolla, S., Tripodo, C., Gattei, V., Marzari, R., Tedesco,F., Sblattero, D., 2014. Bispecific antibodies targeting tumor-associated antigensand neutralizing complement regulators increase the efficacy of antibody-basedimmunotherapy in mice. Leukemia.

Macor, P., Tripodo, C., Zorzet, S., Piovan, E., Bossi, F., Marzari, R., Amadori, A., Tedesco,F., 2007. In vivo targeting of human neutralizing antibodies against CD55 andCD59 to lymphoma cells increases the antitumor activity of rituximab. CancerRes. 67, 10556–10563.

Mayilyan, K.R., 2012. Complement genetics, deficiencies, and disease associations.Protein Cell 3, 487–496.

Meyer, S., Leusen, J.H., Boross, P., 2014. Regulation of complement and modulationof its activity in monoclonal antibody therapy of cancer. MAbs, 6.

Middleton, O., Cosimo, E., Dobbin, E., McCaig, A.M., Clarke, C., Brant, A.M., Leach,M.T., Michie, A.M., Wheadon, H., 2015. Complement deficiencies limit CD20monoclonal antibody treatment efficacy in CLL. Leukemia 29, 107–114.

Moore, G.L., Chen, H., Karki, S., Lazar, G.A., 2010. Engineered Fc variant antibodieswith enhanced ability to recruit complement and mediate effector functions.MAbs 2, 181–189.

Mueller-Eberhard, H.J., Biro, C.E., 1963. Isolation and description of the fourth com-ponent of human complement. J. Exp. Med. 118, 447–466.

Muller, R., Grawert, M.A., Kern, T., Madl, T., Peschek, J., Sattler, M., Groll, M., Buchner,J., 2013. High-resolution structures of the IgM Fc domains reveal principles ofits hexamer formation. Proc. Natl. Acad. Sci. U. S. A. 110, 10183–10188.

Naito, A.T., Sumida, T., Nomura, S., Liu, M.L., Higo, T., Nakagawa, A., Okada, K.,Sakai, T., Hashimoto, A., Hara, Y., Shimizu, I., Zhu, W., Toko, H., Katada, A.,Akazawa, H., Oka, T., Lee, J.K., Minamino, T., Nagai, T., Walsh, K., Kikuchi, A.,Matsumoto, M., Botto, M., Shiojima, I., Komuro, I., 2012. Complement C1q acti-vates canonical Wnt signaling and promotes aging-related phenotypes. Cell 149,1298–1313.

Natsume, A., In, M., Takamura, H., Nakagawa, T., Shimizu, Y., Kitajima, K., Waki-tani, M., Ohta, S., Satoh, M., Shitara, K., Niwa, R., 2008. Engineered antibodiesof IgG1/IgG3 mixed isotype with enhanced cytotoxic activities. Cancer Res. 68,3863–3872.

Natsume, A., Shimizu-Yokoyama, Y., Satoh, M., Shitara, K., Niwa, R., 2009. Engineeredanti-CD20 antibodies with enhanced complement-activating capacity mediate

potent anti-lymphoma activity. Cancer Sci. 100, 2411–2418.

Niederfellner, G., Lammens, A., Mundigl, O., Georges, G.J., Schaefer, W., Schwaiger,M., Franke, A., Wiechmann, K., Jenewein, S., Slootstra, J.W., Timmerman, P.,Brannstrom, A., Lindstrom, F., Mossner, E., Umana, P., Hopfner, K.P., Klein, C.,2011. Epitope characterization and crystal structure of GA101 provide insights

r Immu

N

N

N

N

N

O

O

O

P

P

P

P

R

R

R

R

R

R

R

R

R

R

R

S

S

S

S

J.P.M. Melis et al. / Molecula

into the molecular basis for type I/II distinction of CD20 antibodies. Blood 118,358–367.

ielsen, L.S., Baer, A., Muller, C., Gregersen, K., Monster, N.T., Rasmussen, S.K., Weil-guny, D., Tolstrup, A.B., 2010. Single-batch production of recombinant humanpolyclonal antibodies. Mol. Biotechnol. 45, 257–266.

ilsson, U., 1967. Separation and partial purification of the sixth, seventh and eighthcomponents of human haemolytic complement. Acta Pathol. Microbiol. Scand.70, 469–480.

ilsson, U.R., Mueller-Eberhard, H.J., 1965. Isolation of beta IF-globulin from humanserum and its characterization as the fifth component of complement. J. Exp.Med. 122, 277–298.

immerjahn, F., Ravetch, J.V., 2008. Fcgamma receptors as regulators of immuneresponses. Nat. Rev. Immunol. 8, 34–47.

onaka, M., Kimura, A., 2006. Genomic view of the evolution of the complementsystem. Immunogenetics 58, 701–713.

i, V.T., Vuong, T.M., Hardy, R., Reidler, J., Dangle, J., Herzenberg, L.A., Stryer, L., 1984.Correlation between segmental flexibility and effector function of antibodies.Nature 307, 136–140.

kroj, M., Heinegard, D., Holmdahl, R., Blom, A.M., 2007. Rheumatoid arthritis andthe complement system. Ann. Med. 39, 517–530.

tten, H.G., Verhaar, M.C., Borst, H.P., Hene, R.J., van Zuilen, A.D., 2012. Pretransplantdonor-specific HLA class-I and -II antibodies are associated with an increasedrisk for kidney graft failure. Am. J. Transplant. 12, 1618–1623.

arce, J.W., Kelley, D., Heinzelmann, K., 1983. Measurement of antibody-dependentbinding, proteolysis, and turnover of C1s on liposomal antigens localizes thefluidity-dependent step in C1 activation. Biochim. Biophys. Acta 736, 92–98.

awluczkowycz, A.W., Beurskens, F.J., Beum, P.V., Lindorfer, M.A., van de Winkel,J.G., Parren, P.W., Taylor, R.P., 2009. Binding of submaximal C1q promotescomplement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDCare induced by OFA than by RTX. J. Immunol. 183, 749–758.

eng, Q., Li, K., Smyth, L.A., Xing, G., Wang, N., Meader, L., Lu, B., Sacks, S.H., Zhou,W., 2012. C3a and C5a promote renal ischemia-reperfusion injury. J. Am. Soc.Nephrol. 23, 1474–1485.

illemer, L., Ecker, E.E., 1941. The terminology of the components of complement.Science 94, 437.

acila, E., Link, B.K., Weng, W.K., Witzig, T.E., Ansell, S., Maurer, M.J., Huang, J., Dahle,C., Halwani, A., Levy, R., Weiner, G.J., 2008. A polymorphism in the comple-ment component C1qA correlates with prolonged response following rituximabtherapy of follicular lymphoma. Clin. Cancer Res. 14, 6697–6703.

acusen, L.C., Colvin, R.B., Solez, K., Mihatsch, M.J., Halloran, P.F., Campbell, P.M.,Cecka, M.J., Cosyns, J.P., Demetris, A.J., Fishbein, M.C., Fogo, A., Furness, P., Gibson,I.W., Glotz, D., Hayry, P., Hunsickern, L., Kashgarian, M., Kerman, R., Magil, A.J.,Montgomery, R., Morozumi, K., Nickeleit, V., Randhawa, P., Regele, H., Seron,D., Seshan, S., Sund, S., Trpkov, K., 2003. Antibody-mediated rejection criteria– an addition to the Banff 97 classification of renal allograft rejection. Am. J.Transplant. 3, 708–714.

aju, T.S., 2008. Terminal sugars of Fc glycans influence antibody effector functionsof IgGs. Curr. Opin. Immunol. 20, 471–478.

eid, K.B., Porter, R.R., 1976. Subunit composition and structure of subcomponentC1q of the first component of human complement. Biochem. J 155, 19–23.

ichards, A., Kemp, E.J., Liszewski, M.K., Goodship, J.A., Lampe, A.K., Decorte, R.,Muslumanoglu, M.H., Kavukcu, S., Filler, G., Pirson, Y., Wen, L.S., Atkinson, J.P.,Goodship, T.H., 2003. Mutations in human complement regulator, membranecofactor protein (CD46), predispose to development of familial hemolytic ure-mic syndrome. Proc. Natl. Acad. Sci. U. S. A. 100, 12966–12971.

icklin, D., Hajishengallis, G., Yang, K., Lambris, J.D., 2010. Complement: a key systemfor immune surveillance and homeostasis. Nat. Immunol. 11, 785–797.

icklin, D., Lambris, J.D., 2013a. Complement in immune and inflammatory disor-ders: pathophysiological mechanisms. J. Immunol. 190, 3831–3838.

icklin, D., Lambris, J.D., 2013b. Complement in immune and inflammatory disor-ders: therapeutic interventions. J. Immunol. 190, 3839–3847.

ittirsch, D., Flierl, M.A., Nadeau, B.A., Day, D.E., Huber-Lang, M., Mackay, C.R.,Zetoune, F.S., Gerard, N.P., Cianflone, K., Kohl, J., Gerard, C., Sarma, J.V., Ward,P.A., 2008. Functional roles for C5a receptors in sepsis. Nat. Med. 14, 551–557.

omero, V., Fert-Bober, J., Nigrovic, P.A., Darrah, E., Haque, U.J., Lee, D.M., van Eyk, J.,Rosen, A., Andrade, F., 2013. Immune-mediated pore-forming pathways inducecellular hypercitrullination and generate citrullinated autoantigens in rheuma-toid arthritis. Sci. Transl. Med. 5, 209ra150.

ooijakkers, S.H., van Strijp, J.A., 2007. Bacterial complement evasion. Mol. Immunol.44, 23–32.

acks, S.H., Zhou, W., 2012. The role of complement in the early immune responseto transplantation. Nat. Rev. Immunol. 12, 431–442.

afavi, S., Robinson, D.R., Soresi, S., Carby, M., Smith, J.D., 2014. De novo donor HLA-specific antibodies predict development of bronchiolitis obliterans syndromeafter lung transplantation. J. Heart Lung Transplant. 33, 1273–1281.

aifuddin, M., Hedayati, T., Atkinson, J.P., Holguin, M.H., Parker, C.J., Spear, G.T.,1997. Human immunodeficiency virus type 1 incorporates both glycosylphosphatidylinositol-anchored CD55 and CD59 and integral membrane CD46at levels that protect from complement-mediated destruction. J. Gen. Virol. 78(Pt 8), 1907–1911.

aifuddin, M., Parker, C.J., Peeples, M.E., Gorny, M.K., Zolla-Pazner, S., Ghassemi,M., Rooney, I.A., Atkinson, J.P., Spear, G.T., 1995. Role of virion-associatedglycosylphosphatidylinositol-linked proteins CD55 and CD59 in complementresistance of cell line-derived and primary isolates of HIV-1. J. Exp. Med. 182,501–509.

nology 67 (2015) 117–130 129

Schwaeble, W.J., Lynch, N.J., Clark, J.E., Marber, M., Samani, N.J., Ali, Y.M., Dudler, T.,Parent, B., Lhotta, K., Wallis, R., Farrar, C.A., Sacks, S., Lee, H., Zhang, M., Iwaki, D.,Takahashi, M., Fujita, T., Tedford, C.E., Stover, C.M., 2011. Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardialand gastrointestinal ischemia/reperfusion injury. Proc. Natl. Acad. Sci. U. S. A.108, 7523–7528.

Sebejova, L., Borsky, M., Jaskova, Z., Potesil, D., Navrkalova, V., Malcikova, J., Sramek,M., Doubek, M., Loja, T., Pospisilova, S., Mayer, J., Trbusek, M., 2014. Distinctin vitro sensitivity of p53-mutated and ATM-mutated chronic lymphocyticleukemia cells to ofatumumab and rituximab. Exp. Hematol. 42, 867–874.e1.

Shlomchik, W.D., 2007. Graft-versus-host disease. Nat. Rev. Immunol. 7, 340–352.Shushakova, N., Skokowa, J., Schulman, J., Baumann, U., Zwirner, J., Schmidt, R.E.,

Gessner, J.E., 2002. C5a anaphylatoxin is a major regulator of activating versusinhibitory FcgammaRs in immune complex-induced lung disease. J. Clin. Invest.110, 1823–1830.

Silasi-Mansat, R., Zhu, H., Popescu, N.I., Peer, G., Sfyroera, G., Magotti, P., Ivanciu, L.,Lupu, C., Mollnes, T.E., Taylor, F.B., Kinasewitz, G., Lambris, J.D., Lupu, F., 2010.Complement inhibition decreases the procoagulant response and confers organprotection in a baboon model of Escherichia coli sepsis. Blood 116, 1002–1010.

Sim, R.B., Twose, T.M., Paterson, D.S., Sim, E., 1981. The covalent-binding reaction ofcomplement component C3. Biochem. J 193, 115–127.

Skattum, L., van Deuren, M., van der Poll, T., Truedsson, L., 2011. Complement defi-ciency states and associated infections. Mol. Immunol. 48, 1643–1655.

Skjeflo, E.W., Christiansen, D., Espevik, T., Nielsen, E.W., Mollnes, T.E., 2014.Combined inhibition of complement and CD14 efficiently attenuated the inflam-matory response induced by Staphylococcus aureus in a human whole bloodmodel. J. Immunol. 192, 2857–2864.

Spiridon, C.I., Ghetie, M.A., Uhr, J., Marches, R., Li, J.L., Shen, G.L., Vitetta, E.S.,2002. Targeting multiple Her-2 epitopes with monoclonal antibodies results inimproved antigrowth activity of a human breast cancer cell line in vitro and invivo. Clin. Cancer Res. 8, 1720–1730.

Stanton, C.M., Yates, J.R., den Hollander, A.I., Seddon, J.M., Swaroop, A., Stambolian,D., Fauser, S., Hoyng, C., Yu, Y., Atsuhiro, K., Branham, K., Othman, M., Chen,W., Kortvely, E., Chalmers, K., Hayward, C., Moore, A.T., Dhillon, B., Ueffing, M.,Wright, A.F., 2011. Complement factor D in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 52, 8828–8834.

Stephan, A.H., Madison, D.V., Mateos, J.M., Fraser, D.A., Lovelett, E.A., Coutellier, L.,Kim, L., Tsai, H.H., Huang, E.J., Rowitch, D.H., Berns, D.S., Tenner, A.J., Shamloo, M.,Barres, B.A., 2013. A dramatic increase of C1q protein in the CNS during normalaging. J. Neurosci. 33, 13460–13474.

Stuhlinger, W., Kourilsky, O., Kanfer, A., Sraer, J.D., 1974. Letter: Haemolytic-uraemicsyndrome: evidence for intravascular C3 activation. Lancet 2, 788–789.

Swain, S.M., Kim, S.B., Cortes, J., Ro, J., Semiglazov, V., Campone, M., Ciruelos, E., Fer-rero, J.M., Schneeweiss, A., Knott, A., Clark, E., Ross, G., Benyunes, M.C., Baselga,J., 2013. Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastaticbreast cancer (CLEOPATRA study): overall survival results from a randomised,double-blind, placebo-controlled, phase 3 study. Lancet Oncol. 14, 461–471.

Takeda, J., Miyata, T., Kawagoe, K., Iida, Y., Endo, Y., Fujita, T., Takahashi, M., Kitani, T.,Kinoshita, T., 1993. Deficiency of the GPI anchor caused by a somatic mutationof the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 73, 703–711.

Tambourgi, D.V., van den Berg, C.W., 2014. Animal venoms/toxins and the comple-ment system. Mol. Immunol. 61, 153–162.

Tan, L.K., Shopes, R.J., Oi, V.T., Morrison, S.L., 1990. Influence of the hinge regionon complement activation, C1q binding, and segmental flexibility in chimerichuman immunoglobulins. Proc. Natl. Acad. Sci. U. S. A. 87, 162–166.

Taylor, R.P., Lindorfer, M.A., 2014. Analyses of CD20 monoclonal antibody-mediatedtumor cell killing mechanisms: rational design of dosing strategies. Mol.Pharmacol. 86, 485–491.

Teeling, J.L., French, R.R., Cragg, M.S., van den Brakel, J., Pluyter, M., Huang, H.,Chan, C., Parren, P.W., Hack, C.E., Dechant, M., Valerius, T., van de Winkel, J.G.,Glennie, M.J., 2004. Characterization of new human CD20 monoclonal antibod-ies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 104,1793–1800.

Teeling, J.L., Mackus, W.J., Wiegman, L.J., van den Brakel, J.H., Beers, S.A., French, R.R.,van Meerten, T., Ebeling, S., Vink, T., Slootstra, J.W., Parren, P.W., Glennie, M.J.,van de Winkel, J.G., 2006. The biological activity of human CD20 monoclonalantibodies is linked to unique epitopes on CD20. J. Immunol. 177, 362–371.

Theilacker, C., Kropec, A., Hammer, F., Sava, I., Wobser, D., Sakinc, T., Codee, J.D.,Hogendorf, W.F., van der Marel, G.A., Huebner, J., 2012. Protection againstStaphylococcus aureus by antibody to the polyglycerolphosphate backbone ofheterologous lipoteichoic acid. J. Infect. Dis. 205, 1076–1085.

Truedsson, L., Bengtsson, A.A., Sturfelt, G., 2007. Complement deficiencies and sys-temic lupus erythematosus. Autoimmunity 40, 560–566.

Udaka, K., Okada, M., Utsumi, S., 1986. Co-operation between the pair of C gamma2 domains in Clq-binding by rabbit IgG. Mol. Immunol. 23, 1103–1110.

Utsumi, S., Okada, M., Udaka, K., Amano, T., 1985. Preparation and biologic charac-terization of fragments containing dimeric and monomeric C gamma 2 domainof rabbit IgG. Mol. Immunol. 22, 811–819.

van der Neut Kolfschoten, M., Schuurman, J., Losen, M., Bleeker, W.K., Martinez-Martinez, P., Vermeulen, E., den Bleker, T.H., Wiegman, L., Vink, T., Aarden, L.A.,De Baets, M.H., van de Winkel, J.G., Aalberse, R.C., Parren, P.W., 2007. Anti-

inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.Science 317, 1554–1557.

van Meerten, T., Rozemuller, H., Hol, S., Moerer, P., Zwart, M., Hagenbeek, A., Mackus,W.J., Parren, P.W., van de Winkel, J.G., Ebeling, S.B., Martens, A.C., 2010. HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small

1 r Imm

v

W

W

W

W

W

W

W

X

X

X

Invest. 105, 1363–1371.

30 J.P.M. Melis et al. / Molecula

loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cellsthat resist rituximab-mediated lysis. Haematologica 95, 2063–2071.

an Wamel, W.J., Rooijakkers, S.H., Ruyken, M., van Kessel, K.P., van Strijp, J.A.,2006. The innate immune modulators staphylococcal complement inhibitorand chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages. J. Bacteriol. 188, 1310–1315.

agner, E., Frank, M.M., 2010. Therapeutic potential of complement modulation.Nat. Rev. Drug Discov. 9, 43–56.

ang, X.Z., Coljee, V.W., Maynard, J.A., 2013. Back to the future: recombinant poly-clonal antibody therapeutics. Curr. Opin. Chem. Eng. 2, 405–415.

ard, P.A., Guo, R.F., Riedemann, N.C., 2012. Manipulation of the complement sys-tem for benefit in sepsis. Crit. Care Res. Pract. 2012, 427607.

eber, B.H., Charbel Issa, P., Pauly, D., Herrmann, P., Grassmann, F., Holz, F.G., 2014.The role of the complement system in age-related macular degeneration. Dtsch.Arztebl. Int. 111, 133–138.

ong, E.K., Goodship, T.H., Kavanagh, D., 2013. Complement therapy in atypicalhaemolytic uraemic syndrome (aHUS). Mol. Immunol. 56, 199–212.

oof, J.M., Kerr, M.A., 2006. The function of immunoglobulin A in immunity. J. Pathol.208, 270–282.

right, A., Morrison, S.L., 1997. Effect of glycosylation on antibody function: impli-cations for genetic engineering. Trends Biotechnol. 15, 26–32.

ia, M.Q., Hale, G., Lifely, M.R., Ferguson, M.A., Campbell, D., Packman, L., Waldmann,H., 1993a. Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complementlysis. Biochem. J 293 (Pt 3), 633–640.

ia, M.Q., Hale, G., Waldmann, H., 1993b. Efficient complement-mediated lysisof cells containing the CAMPATH-1 (CDw52) antigen. Mol. Immunol. 30,

1089–1096.

u, D., Alegre, M.L., Varga, S.S., Rothermel, A.L., Collins, A.M., Pulito, V.L., Hanna, L.S.,Dolan, K.P., Parren, P.W., Bluestone, J.A., Jolliffe, L.K., Zivin, R.A., 2000. In vitrocharacterization of five humanized OKT3 effector function variant antibodies.Cell. Immunol. 200, 16–26.

unology 67 (2015) 117–130

Yasojima, K., Schwab, C., McGeer, E.G., McGeer, P.L., 1999. Up-regulated productionand activation of the complement system in Alzheimer’s disease brain. Am. J.Pathol. 154, 927–936.

Yates, J.R., Sepp, T., Matharu, B.K., Khan, J.C., Thurlby, D.A., Shahid, H., Clayton, D.G.,Hayward, C., Morgan, J., Wright, A.F., Armbrecht, A.M., Dhillon, B., Deary, I.J.,Redmond, E., Bird, A.C., Moore, A.T., 2007. Complement C3 variant and the riskof age-related macular degeneration. N. Engl. J. Med. 357, 553–561.

Yehoshua, Z., de Amorim Garcia Filho, C.A., Nunes, R.P., Gregori, G., Penha, F.M.,Moshfeghi, A.A., Zhang, K., Sadda, S., Feuer, W., Rosenfeld, P.J., 2014. Sys-temic complement inhibition with eculizumab for geographic atrophy inage-related macular degeneration: the COMPLETE study. Ophthalmology 121,693–701.

Zent, C.S., Secreto, C.R., LaPlant, B.R., Bone, N.D., Call, T.G., Shanafelt, T.D., Jelinek, D.F.,Tschumper, R.C., Kay, N.E., 2008. Direct and complement dependent cytotoxicityin CLL cells from patients with high-risk early-intermediate stage chronic lym-phocytic leukemia (CLL) treated with alemtuzumab and rituximab. Leuk. Res.32, 1849–1856.

Zent, C.S., Taylor, R.P., Lindorfer, M.A., Beum, P.V., LaPlant, B., Wu, W., Call, T.G.,Bowen, D.A., Conte, M.J., Frederick, L.A., Link, B.K., Blackwell, S.E., Veeramani, S.,Baig, N.A., Viswanatha, D.S., Weiner, G.J., Witzig, T.E., 2014. Chemoimmunother-apy for relapsed/refractory and progressive 17p13-deleted chronic lymphocyticleukemia (CLL) combining pentostatin, alemtuzumab, and low-dose rituximabis effective and tolerable and limits loss of CD20 expression by circulating CLLcells. Am. J. Hematol. 89, 757–765.

Zhou, W., Farrar, C.A., Abe, K., Pratt, J.R., Marsh, J.E., Wang, Y., Stahl, G.L., Sacks, S.H.,2000. Predominant role for C5b-9 in renal ischemia/reperfusion injury. J. Clin.

Ziller, F., Macor, P., Bulla, R., Sblattero, D., Marzari, R., Tedesco, F., 2005. Controllingcomplement resistance in cancer by using human monoclonal antibodies thatneutralize complement-regulatory proteins CD55 and CD59. Eur. J. Immunol.35, 2175–2183.