See also complement inhibitor targetting under “Pharmacology” and “Drug delivery” See also Human Complement Regulatory Proteins (CRegs) 

See also disease mechanisms of viruses and bacteria for how pathogens use complement regulatory proteins to evade the immune system

Mechanisms of Complement Regulation

The complement system is regulated via a number of interrelated mechanisms. There are two broad mechanisms for inhibition of the complement system; inhibitors of the complement activation pathway (inibitors of C3 convertase formation) and inhibitors of the terminal complement pathway (inhibit MAC formation).

(1) Inactivation of Complement Enzymes: In the first mechanism, CRegs function by inactivating enzymes, such as the C3 and C5 convertases, which are formed during C activation and which are responsible for cleavage of C3 and C5. The first mechanism is generally reversible, facilitating the dissociation of the C3 convertases (i.e., C3b from Bb and C4b from C2a). The dissociation may also involve reversible binding of the antagonist proteins to C3b or C4b components, thus preventing their reassociation.

(2) Interference with MAC: In the second mechanism, CRegs function by interfering with MAC formation. This second mechanism, which is an irreversible inactivation process, results from proteolytic cleavage of the C3 convertase components C3b or C4b by the serine protease factor I. 

Both general regulatory mechanisms, the facilitation of dissociation of C3b and C4b and the inactivation of C3b and C4b through cleavage by factor I, also apply to the inhibition of the alternative pathway C5 convertase (C3bBbC3b) and the classical pathway C5 convertase (C4bC2aC3b).

The proteins encoded by a region of the genome which designated the regulators of complement activation (RCA) gene cluster are involved in both mechanisms.

Various assays/methods can be used to mesasure activities of complement pathway molecules and activaiton of the complement system. (see US Patent No. 6,087,120 and Newell, J Lab Cin Med, 100:437-44, 1982). The two most commonly used techniques are hemolytic assays and immunological assays.

Definitions/General Principals

(1) EGTA blocks the classical C pathway because it chelates Ca++ and thus inactivates C1 (Platts-Mills, J. Immunology, 113(1), 1974).

(2) Amplifcation loops of the AP: The AP pathway involves an amplifcaiton loop utilizing C3b producec by the CP and lectin pathways. Some molecules of C3b generated by the CP C3 convertase are funneled into the AP. Surface bound C3b binds Factor B to yield C3bB, which becomes a substrate for Factor D of the AP. Factor D is a serine esterase that cleaves the Ba fragment, leaving C3bBb bound to the surface of the target cell. C3bBb is stablized by proeprdin, forming C3bBbP, which acts as the AP C3 convertase. This C3 convertase participates in an amplifcaiton loop to cleave many C3 molecules, resulting in the deposition of C3b on the target cell. Some of these C3b bind back to C3bBb to form C3bBb3b, the AP C5 convertase which cleaves C5 into C5a and C5b. C5b binds to the surface of the cell to initiate the formation of MAC. (Fung, 2006/0140939).

Antibody sensitized sRBCs only activate the CP. They do not, by themselves, activate the AP. However, in the presence of sufficient NHS (e.g., 7-10%) activated CP will utilize the amplication loop of the AP. (Bansal, US13/646,286).

(3) Calcium/magnesium: The CP is a calcium/magnesium dependent cascade whereas the AP is a magnesium dependent cascade. With the lectinpathway, Ca++ dependent binding of MBL to a mannan coated surface triggers activaiton of C3. C2 is a single chain plasma protein that is specific for the CP and LP. Membrane bound C4b expresses a binding site which, in the presence of Mg++, binds the proenzyme C2 near its amino termus and present it for cleavage by C1s (for the CP) or MASP-2 (for the LP) to yeild a 30 kD amino terminal fragment, C2b, and 70 kD carboxy terminal fragment, C2a.  (Fung WO01/70818).

Hemolytic Assays/techniques

Hemolytic Assays measure the functional capacity of the entire pathway.

Erythrocyte Lysis Assays: 

Erythrocyte lysis assays are based on the formation of a terminal complement complex on the surface of rabbit red blood cells (rRBC). As a reuslt of the formation of this complex, the rRBCs are lysed. The progressive decrease in light scatter at 700 nm is a direct measure of erythrocyte lysis.  

Assays for the Alternative Pathway (AP)

Rabbit erythrocyte hemolysis:

It is well established that rabbit erythrocytes specifically activate the complement AP, with a resulting lysis of the cells by the C5b-9 complex (Bansal, US 2005/0107319)

Alternative Pathway Isolated from the Classical Pathway: 

It is accepted that AP activation is dependent on Mg++ but not Ca++ (Platts-Mills, J. Immunology, 113(1), 1974). AP activation in Mg++ ions without calcium ions guarantees only the AP activaiton (Bansal, 13/646286). 

—-Rabbits RBCs in 10% NHS/Mg2+:EGTA: Introducing rabbit Erythrocytes (rRBC) into 10% human serum (with Mg2+/EGTA) represent the introduction of a foreign cell surface which initiates the AP cascade. AP activation in Mg++ ions without calcium ions (EGTA is used to chelate the calcium ions to prevent CP activation)guarantees only AP activation. Rabbit RCs can be used to activate the AP in 10% NHS in the presence of Mg2+ in the absence of Ca2+ and in the presence of sufficient NHS concentration . Because the CP requires the presence of Ca2+, the CP will not be active under these conditions. 

Example (anti-properdin): To determine the effects of a MoAb to propderin on alternative pathway (AP) function, increasing amounts of the antibody are added to human serum that is incubated with rabbit erythrocytes in buffer containin EGTA and Mg++. The cells cause AP activation which results in C5b-9 formation and consequent lysis of the cells. The extent of erythrocyte lysis can be monitored by examining the amount of light scattered by intact red blood cells (Polhill, 1978). Human serum + rabbit erythrocytes in buffer containing EGTA and Mg++. These cells cause AP activation, which results in C5b-9 formation and consequent lysis of the cells. (Bansal, US 13/646286)

—-Rabbit erythrocytes + 7.5% NHS + GVB-Mg+++-EGTA: Song (US2010/0263061) disclose incubation of rabbit erythrocytes with 7.5% NHS in GVB with or withoug anti-P antibodies or EDTA. Song shows properdin mAbs which dose dependently inhibited complement mediated lysis of the rabbit red blood cells. Anti-P antibodies and EDTA were used as positive controls for inhibition (EDTA blocks CP complement).

Assays for the Classical Pathway (CP)

The classical CP is typically triggered by immune complexes, for example, an antibody boudn to a foreign particle and thus requires prior exposure to that particle for the generation of specific antibody. (Gupta-Bansal (US 6,333,034). The CP is a calcium/magnesium dependent cascade. C1, the first enzyme complex in the cascade, is a pentamolecular complex consisting of C1q, 2 C1r molcules and 2 C1s molecules. This complex binds to an antigen-antibody complext through the C1q domain to initiate the cascase. (Fung, US 2005/1096394)

CH50 Assay:

The complement system is a group of proteins that wehn activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Inidividual ocmmplement components can be quantified; however this does not provide any informaiton as to the activity of the pathway. The CH50 is a screening assay for the activaiton of the CP and it is sensitie to the reduciton, absence and/or inactivity of any component of the pathway. The CH50 tests the funciotnal capability of serum complement components of the CP to lyse sheep red blood cells pre-coated with rabbit anti-sheep red blood cell antibody (haemolysis). When antibody-coated SRBC are incubated with test serum, the classical pathway of coplement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zerio; if one or mroe components are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the sueprnatant at 540 nm. The amount of complement acitvity is determiend by examining teh capacity of various dilutions of test serum to lyse antibody coated SRBC. (Costabile “Measuring the 50% haemolytic complement (CH50) activity of serum” J Visualized experiments, 2010). 

Classical Pathway isolated from alternative pathway: 

—-Antibody Sensitized Sheep cells in 1% NHS/Ca2+/Mg2+: To measure the functional capacity of the classical pathway, sheep red blood cells coated with hemolysin (rabbit IgG to sheep red blood cells) are used as target cells (sensitized cells). These ag-Ab complexes activate the classical pathway and result in lysis of the target cells when the components are functional and present in adequate concentration. To determine functional capacity of the alternative pathway, rabbit red blood cells are used as the target cell (see US Patent No. 6,087,120). The antibody sensitized sheep cells are used as an activator in 1% normal human serum in the presence of Ca2+/Mg2+. The calcium ionsare required for activation of the CP for the initial trigger of the C1q/C1r/s complexes. CP will not occur in the basence of the calcium ions. Mg2+ is required for AP activation. However, in 1% normal human serum containing Ca2+/Mg2+, only the CP proceeds to completion. Without the requisite levels of NHS which is 10%, the AP pathway will not have a significant presence.  (Bansal, US 13/646286) 

Classical Pathway activation of the Alternative Pathway Via way of the Amplication Loop of the AP

—-Antibody sensitized sheep erythrocytes + 7.5 NHS + GVB-Mg++: Antibody sensitized sheep erythrocytes were incubated with 7.5 NHS in GVB-Mg++ buffer with or without anti-P antibodies or EDTA. Antibody sensitized sheep erythrocytes are another well established assay for the classical pathway complement activation.  Cells completely lysed by hypotonic shock were aslo sued as a control (100% lysis). The degree of lysis was determined by hemoglobin release using a spectrophotometer. Polyclonal anti-P antibody had no effect on human complement mediated lysis of the antibody sensitized sheep erythrocytes. In contrast, EDTA inhibited human complement mediated lysis and was used as a positive control. (Song, 2010/0263061).  Antibody sensitized sRBCs only activate CP. They do not, by themselves, activate AP. However, in the presence of sufficient NHS, activated CP will utilize the amplifcation loop of the AP. (Bansal, US 13/646,286).

—-Antibody sensitized sheep cells in in 10% NHS/Ca2+/Mg2+: For CP and AP action, antibody sensitized sheep red blood cells are used as an activator in 10% ca2+/Mg2+ in human human serum. The difference between the assay above for the CP only is that NHS is 10%. The Ca2+/Mg2+ provides the level of Mg2+ required for AP activation and allows for both CP and AP to be active. Antibody sensitized sRBCs only activate the CP. They do not by themselves activate the AP. However, in the presence of sufficient NHS, activated CP will utilize the amplification loop of the AP. Under these conditions, the C3b produced vial the CP can feed into the AP causing amplificaiton of the AP loop. In other words, the AP has been activated by the CP. (Bansal, US 13/646286)

Assays for the Lectin pathway

 Assays for the lectin pathway include measuring the functional activity of lectin-MASP complext through activation of added exogenous C4 or measuring endogenous activation of C4 after blocking of CP and AP using a high ionic strenght dilution buffer (1M NaCl) Palarasah (US2010/0196927).

Polyanethol sulphonate (PAS) + activation of lectin pathway:

Palarasah (US2010/0196927) discloses a method of determining functional deficiencies in the lectin pathway by diluting a sample with polyanethol sulphonate (PAS) which blocks the CP and AP, activating the lectin pathway and then determining activation of one or more complement factors C3, C4 of one or mroe components of the C5-C9 complex.

Immunologic Assays/techniques: 

Immunologic assays use antibodies agaisnt the different epitopes of the various complement components (e.g., C3, C4, and C5) to detect split product of the complement components (e.g., C3a, C4a, C5a and the C5b-9 terminal complex).

Alternative Pathway: To accomplish this assay one immobilizes LPS onto microtiter wells to activate the AP in diluted serum samples. Can then measure AP components like C5b-9 (MAC).

Particular Complement Components

—-C3b:

Example: LPS + 10% NHS/Mg++: LPS is a specific activator of the AP.  (Bansal, US 13/646286) AP activation generates C3 and C3b as a result of C3 cleavage by the C3 convertase of the AP. AP is acivated in NHS by LPS under conditions that allow activation of the AP. This assay was used to demonstrate whether an anti-properdin antibody would inhibit the foramtion and deposition of C3b which initiates the start of AP. As a way of mechanims, activated and deposited C3b provides high affinity binding to properdin. Properdin-C3b complexes bind factor B and the complex is cleaved by factor D to generate PC3bBb, an AP C3 convertase. As the AP proceeds, C5b-9 complexes are formed and deposited. Polystyrene microtiter plate wells were coated with LPS. Normal human serum (NHS) at 10% in AP buffer was mixed with varying concentrations of an anti-properdin antibody and C3b was detected with rabbit anti-human C3c, properdin was detected with goat anti-human P, Bb was detected with goat anti-human factor Bb and C5b-9 was detected with HRPO-conjugated neo-anti-human C5b-9. The presence of C3b, p and Bb and MAC together was indicative of AP C3 convertase formation, and the antibody was shown to inhibit C3b formation. (Bansal, US8,664,362 and 13/583879; 14/195, 458). 

—-C5 protein and fragments: Methods for determining whether an antibody can block the generation or activity of the C5a and/or C5b active fragments of C5 or binding to complement component C4b or C3b are known in the art (US6355245). 

—-C5a: Methods for measuring C5a activity include chemotaxis assays, RIAs, or ELISas (Ward and Zvaifler (1971) J Clin Invest 50(3): 606-16). 

Classical Pathway:

–CH50 EIA:  The binding of the C1q component of C1 to immune complexes triggers the CP which results in a cascade of enzymatic and non-enzymatic reactions culminating in the formation of terminal complement complexes (TCC). Th CH50 EIA measures the hemolytic complement (CG50) in human serum and allowed detection of a deficiency of one or more complement components C1-C9. It is a tradtional method for measuring the CP. In this lytic assay, antibody sensitized sheep erythroytes (EA) are used to activate the CP and various dilutions of the test serum are used to determine the amount required to give 50% lysis. The assay uses a mAb to a unique neoantigen to caputre the TCC analytic.

Lectin Pathway: Mannan can be as a ligand and coated on plates and then serum is added. Detection of MBL, C4 and C3 can then be performed with mAbs against these components. As both the LP and the CP are calcium dependent and lead to activation of C4, one challenge in devising an assay to assess the functional characterization of the LP is distinguishing its activation from activation of the CP. Using manna, for example, to activate the LP is also likely to activate the CP via anti-annan Ab which is present in huan serum.

Roos (Mol Immunol. 2003; 39: 655-68) describe a lectin pathway assay which uses a mAb to C1q to inhibit CP (blocks CP but allows lectin activation as by mannan to proceed normally) and a mAb to factor D to inhibit AP.

Petersen (J Immunol Methods, 2001, 257: 107-16) escribe blocking CP and AP using a high ionic strenght dilution buffer (1M NaCL) and then measuring endogenous activation of C4. 

Palarasah (US 12/676283) disclose an in vitro method for determining functional deficiencies in the lectin pathway by diluting a sample with a polyanion, preferably a sulphated polyanion and even more preferably polyanethol sulphonate (PAS), which is an inhibitor of the AP and CP, but not the lectin pathway, then activating the lectin pathway and then determining the activation of one or more of the complement factors C3, C4 or one or more of the components of the C5-C9 complex such as with an antibody, wherein a lower level compared to a normal reference level is indicative of a functional deficiency in the lectin pathway. 

C3a and C5a (below) are anaphylatoxins which can trigger mast cell degranulation, which releases histamine and other mediators of inflammation, resulting in smooth muscle contraction, increased vascular permeability, leukocyte activaiton, and other inflammatory phenomena including cellular proliferation resulting in hypercellularity. C5a also functions as a chemotactice peptide that serves to attract pro-inflammatory granulocytes to the site of complement activation.

C5a and C3a are about 10kDa anaphylatoxins able to ligate the C5a receptor (C5aR) and the C3a receptor (C3aR) which are G-protein coupled receptors (GPCRs) generally expressed on APCs and reported under some conditions to be detectable on T cells (US 12/920293).

C3 and its Components (C3a, C3b) and Receptors

C3: Both the AP and CL pathways converge at the point where complement component C3 is cleaved by an active protease (which is different in each pathway) to yeild C3a and C3b. C3 is thus reguard as the central protein in the complement reaction sequence since it is essential to both the AP and CI pathways. In particular, proteolytic activation of C3 by classical (C4b,2a) or alternative (C3b,Bb) pathway C3 convertase leads to cleavage of C3 into an anaphylotoxic peptide C3a and an opsonic fragment C3b. Covalent attachment of metastable C3b to target cells underoging complement attack results in generation of C5a and formation of C5b-9 membrane attack complex. (Lambris, US6,319,897B1).

–Inhibitors of C3:

—-Compstatin and its analogs:

Compstatin is a small molecule weight disulfide bonded cyclic peptide having the sequence Ile-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr (Lambris, US2011/0142837)

C3 inhibitors such as compstain or their analogs are known. Compstatin inhibits the cleavage of C3 to C3a and C3b by C3 convertase. Compstatin has been tested in a series of in vitro, in vivo, ex vivo experiments and has been demonstrated to inhibit complement activaiton in human serum and prolong the lifteim of a porcine to human xenograft perfused with human blood.  (US6,319,897, WO/1999/013899, WO/2004/026328; (Lambris (US13/059482)). 

US 6,319,897 to Lambris describes the use of a phage-displayed combinatorial random peptide library to identify a 27 residue peptide that binds to C3 and inhibits complement activation. This peptide was then truncated to a 13 residue cyclic segment that maintained complete activity. Compstatin inhibits the cleavage of C3 to C3a and C3b by C3 convertase. Compstain comprising the sequence ICVVQDWGHHRCT-NH2 where Cys2 and Cys12 form a disulfide bridge.

Compstatin analogues are made by altering compstatin’s amino acid composition are known. Replacement of Val4, His9, His10 and Arg11 results in minimal change in the functional activity of compstatin whereas replacing Gly8 dramatically reduces the activity of the peptide by more than 100-fold. (Marikis, Protein Science, 1998, 7, p. 623, 2nd column, 1st ¶).

Lambris (WO2004/026328) discloses analogs of Compsatin which have improved complement inhibting activity.

C3a: During complement activation, complement protein C3 is proteolytically cleaved, resulting in a large fragment (C3b) and the smaller 77 residue peptide, C3a. C3a is knwon to regulate vasodilation increasing the permeability of small blood vessels, induce contraction of smooth muslces, induce oxidative burst, regulate cytokine release, and stimulate chemotaxis.

C3aR: The receptor for C3a is a seven transmembrane G protein coupled receptor protein that shares 40% homology with the C5a Receptor.

C3b:  C3b originates from classical pathway activation and/or from natural spontaneous hydrolysis of C3. C3b is generated by the cleavage of C3, which is carried out by classical and/or alternative C3 convertases and results in the generation of both C3a and C3b. In addition to its role in C3 and C5 convertases, C3b also functions as an opsonin through its interaction with complement receptors present on the surfaces of antigen-presenting cells. The opsonic function of C3b is generally considered to be one of the most important anti-infective functions of the coplement system. Patients with genetic lesions that block C3b function are prone to infection by a broad variety of pathogenic organisms, while patients with lesions later in the complement cascade sequence, ie.., patients with lesions that block C5 functions, are found to be more prone only to Neisseria infection, and then only somewhat more prone.

In order not to impair the opsonization functions associated with C3b, if one wants to inhibit C5 with an antibody, one would need to design the antibody such that the antibody that binds C5 does not substantially also interfere with the cleavage of complement component C3 in a body fluid into C3a and C3b.

Resulting C3b binds to the surface of an activating substance. In the presence of magnesium, Factor B binds to the C3b which is bound to the activating suface. Factor Dthen cleaves B, releasing the a fragment and forming C3bBb. Properdin stabilizes the C3bBb complex and protects it from decay. C3bBbP is the alternative pathway convertase which can cleave many C3 molecules. Cleavage of C3 results in the formation of C3bBb3b, the C5 convertase. This enzyme is also stablized by P to form C3bBb3bP. C5 convertase can cleave many molecules of C5 into C5a and C5b.

C3b is also an anaphylatoxin and has multiple functions. As opsonin, it binds to bacteria, viruses and other cells and tags them for removal form the circulation. Patients with genetic lesions that block C3b function are prone to infection by a broad variety of pathogenic organisms, while patients with lesions later in the complement cascade (e.g., lesions that block C5 functions) are more prone only to Neisseria infection.

C3b can form a complex with other components unique to each pathway to form classical or alternative C5 convertase, which cleaves C5 into C5a and C5b. C3b is regulated by the serum protease Factor I, which acts on C3b to produce iC3b. While still functional as opsonin, iC3b cannot form an active C5 convertase. To take a look at the interation of C3b with other compoments more specifically, C3b forms a complex with C4b,C2a to produce C4b,2a,3b (classical C5 convertase) which cleaves C5 into C5a (another anaphylatoxin) and C5b. Alternative C5 convertase is C3b,Bb,C3b and performs the same function. C5b combines with C6 yeilding C5b,6, and this complex combines with C7 to form the ternary complex C5b,6,7 which binds C8 at the surface of a cell membrane. Upon binding of C9, the complex membrane attack complex (MAC) is formed (C5b-9) which mediates the lysis of roeign cells, microriganisms and viruses.

C5, its Components, Receptors and Inhibitors

C5: is a 190 kDa beta globulin found in normal serum. The pro-C5 precursor, a 1676 amino acid residue precursor protein, is cleaved after amino acid 655 and 659, to yeild the beta chain as an amino terminal fragment (amino acid reisudes +1 to 655) and the alpha chain as a carboxyl terminal fragment (amino acid reisudes 660 to 1658), with 4 amino acids deleted between the two. The CP or AP C5 convertase activates mature C5 by cleaving the alpha chain between residues 733 and 734, which results in the liberation of C5a fragment (amino acids 660 to 733). The remaining portion of mature C5 is fragment C5b, which contains the residues 734 to 1650 of the alpha chain disulfide bonded to the beta chain. 

–Inhibitors of C5:

—-Eculizumab (Trade name Soliris by Alexion Pharmaceuticals) is a humanixed anti-human C5 monoclonal antibody (Alexion Pharmaceutical, Inc) with a human IgG2/IgG4 hybrid constant region, so as to reduce the potential to elicity proinflammatory responses. (US 2017/0073399). Soliris is a formulationof eculizumab which is a recombinant humanized monoclonal IgG2/4 kappa antibody produced by murine myeloma cell culture and purified by stadnard bioprocess technology. Eculizumab contains human constant regions form human IgG2 sequence and human IgG4 sequences and murine heavy chain variable regions. Eculizumab is composed of two 448 amino acid heavy cahins and two 214 amino acid light cahins and has a MW of about 148 kDa. (US 2020/0057046), 

–—Pexelizumab: (alexion pharmaceuticals), an scFv fragment of Eculizumab has the same activity.

–—BNJ441: (also known as ALXN1210) is a humanized monocloanl antibody that is structurally related to eulizumab. BNJ441 selectiely binds to human complent protein C5, inhibiting its cleavage to C5a and C5b during complement activation (US 2020/0057046). 

—-ARC1905 (Archemix), an anti-C5 aptamer, binds to and inhibits cleavage of C5, inhibiting the generation of C5b and C5a. C5a can also be reduced through the use of a C3 inhibitor. (Lambris (US13/059482).

C5a: is an anaphylatoxin which is cleaved from the alpha chain of C5 by either alternative or classical B5 convertase as an amino terminal fragment comprising the first 74 amino acids of the alpha chain (i.e., amino acid resiudes 660-733). C5 is thus a 74 amino acid peptide and is a complement component generated early in the terminal phase of the complement cascade by the proteolytic cleavage (by C5 convertase) of the complement component plasma protein C5. C5a mediates inflammation and is a chemotactic attractant for induction of neutrophilic release of antimicrobila proteases and oxygen radicals. C5a is readily metabolized by serum and cell-surface carboxypeptidases that remove the C-teminal arginine to form C5a des Arg, reducing potency to only 3-10% for promoting neutrophil chemotactice activity.

C5a also enhances blood thrombogenicity, mainly through the upregulation of Tissue Factor (TF) and PAI-1 epxression on various cell types (Markiewski, TRENDS in Immunology 28(4), 2007).

–Inhibitors of C5a formation: C5a formation may be accomplished in a number of ways such as by preventing binding to its receptor C5aR through a number of C5aR inhibitors (see below) or by antibodies such as TNX-558 (Tanox) that neutralize C5a. Formation of C5a may also be inhibited by inhibiting the cleavage of C5 by C5-convertase through the use of antibody like Eculizumab that is an anti-C5 antibody (see above) which prevents its cleavage into C5a and C5b. 

C5b: combines with C6, C7, and C8 to form the C5b-8 complex at the surface of the target cell. Upon binding of several C9 molecules, the membrane attack complex(MAC, C5b-9, erminal complement complex-TCC) is formed. When sufficient numbers of MACs insert into target cell membranes the opening they create mediate rapid osmatic lysis of the target cells.

C5aR: The C5a receptor is present on neutrophils and monocytes. Its activation by C5a causes the directed migration of these cells toward the source of C5a generation. (Figueroa, Clinical Microbiology Reviews, July 1991, p. 359-395)

The first human C5aR was clone in 1991 and the second, C5L2, was identified in 2000. Both C5aR and C5L2 have been placed in the GPCR family AB. 

–C5aR Inhibitors: A number of C5aR inhibitors are known in the art including small cyclic hexapeptides, antibodies that bind to C5aR such as Neutrazumab (Lambris (US13/059482). 

Pathways Leading up to C3b and C5a

Formation of the C3 convertases: Although triggered differently, the complement pathways culminate in the formation of the C3 convertases (C3bBb and C4bC2a) and C5 convertases (C3bBbC3b and C4bC2aC3b), which involves cleavage of C2 and C4 (classical and lectin pathways) or the serine proteases factor B and factor D (althernative pathway). This then results in the generation of the main effector molecules of the complement system: the opsonins C3b and C4b, the analphylatoxins C3a and C5a, and the membrane-attack complex (MAC; membrane-bound C5b-C6-C7-C8-C9, denoted as C5b-C9).

Conversion C3 to C3a and C3b: In both the classical and altenrative pathways, the critical step in the activation of complement is the proteolytic conversion of C3 to the fragments C3b and C3a. The third complement component, C3, is known to have an important role in all of the pathways of complement activation. Proteolytic activation of C3 by classical (C4b, 2a) or alternative (C3b, Bb) pathway C3 convertase leads to cleavage of C3 into an anaphylotoxic peptide C3a and an opsonic fragment C3b. Covalent attachment of metastable C3b to target cells undergoing complement attack results in generation of C5a and and formation of C4b-9 membrane attack complex. However, the tissue injury that results form complement activation is directly mediated by the membrane attack complex, C5b-C9, and indirectly by the generation of anaphylotoxic peptides C3a and C5a. These peptides induce damage through their effect on neutrophils and mast cells. Upon stimulation with C5a, neutrophils produce a serine elastase that causes tissue injury. C5a also triggers the generation of toxic oxygen-derived free radicals from neutorphils, and both C3a and C5a stimulate rapid and enhanced production of leukotrienes form Il-3-primed basophils.

Conversion of C5 to C5a and C5b: Each of the three complement pathway produces C5a and C5b. C5b assembles with C6, C7, C8 an dC9 serum proteins to forom the membrane attack complex.

Inhibitors of C5A

Synthetic peptides dervied from C5a are known antagonists of C5a. However, the antagonistic activity is highly dependent upon the structure of the peptides. For example, whereas a 14 amino acid residue peptide from the C5a C-terminal region can function as an antagonist of C5a, 5-8 amino acid residue peptide analogues of the same region have been reported to be agonists rather than antagonists (Kaneko, Immunology, 1995, 86, 149-154).

Several antagonists for C5a receptor (CD88) have been described, including peptides, a non-peptidic compound, C5a mutants and anti-C5R-antibodies. All of these compounds are potent CD88 antagonists in vitro; however, only the C5a mutatns C5aRAM and jun/fos-A8, the cyclic peptide AcPhe[AcF-(pdChaWR), and a nonpeptidc antagonist have been proven useful in vivo (Otto, J. Biological Chemistry, 279, 1, 2004, 142-151).

Rother (WO2011137395) discloses humanized anti-C5a antibodies such as BNJ364, BNJ366, BNJ369, BNJ378, BNJ381 and BNJ383. The BNJ383 antibody depelted plasma C5a/C5a desarg levels to below detectable limits in cynomolgus monkey at 10 ug/mL. The anti-C5a antibodies are useful for treating or preventing RA and other complement assocaited disorders such as aHUS. Unlike C5, C5a is present in blood at much lower concetnrations and is often restricted to specific areas of local complement activation sucha s the lungs in asthma patietns, the joints of RA patietns and the drusen in the eys of patients with AMD. Thus, the anti-C5a antibodies can be adminsitered locally to sites of complement activation at much lower dose and/or less frequently than for example an anti-C5 antibody. as by subcutaneous, intramscular and intrapulmonary delivery. A lower concentraiton of antigen C5a versus C5 also faors a longer half-life of the anti-C5a antibody. 

Overview

The Lectin pathway is initiated by mannan-binding lectin (MBL) which is a protein that binds specifically to mannose residues in bacterial cell walls. It is a phylogenetically ancient mechanism which was first shown to be activated by MBL when it interacts with repeating mannose residues on the surface of pathogens. Mannose is a monosaccharide isomer of glucose that is an integral part of the cell surface structure of potentially pathogenic bacteria. These sugars are usually absent from mammalian cells and thus provide a unique target for activaiton of the immune system. MBL is an oligomeric serum lectin that is associated with several homologus, modular lectin specific serine proteases (MASPs). The bining of MBL within this complex to microbial mannose residues activates MASP-1 and MASP-2. MASP-2 protease can hydrolyze circulating C4 and C2 proteins to produce the C4b2a complex (C3 convertase) as seen in the classical pathway. Unlike MASP-2, MASP-1 hydroyzes C3 directly. Two additional MASPs ahve been observed, an alternative spliced variant of MASP-1 called MASP-3 and a truncated form of MASP-2 called sMASP. (Pugsley, Cardiovascular Toxicology, 2003, pp. 43-69)

The binding of MBP to ther terminal mannose group I residue on the microbial cell surface is calcium dependent and serves the unique biological funciton of recognizing exogenous CHO moieties on cells. Thus, lectin binds these mannose or CHO ligands by the formation of a series of hydrogen and calcium coordinated bonds with the 2-OH groups of the terminal mannose residues. MBP binding then triggers an immune response that is mechanistically simlar to the classical pathway since C3 and C5 convertase are generated. (Pugsley, Cardiovascular Toxicology, 2003, pp. 43-69). 

Activation

Activation of the LP occurs through recognition of PAMPs by either MBL or ficolins in association with MBL-associated serine proteases (MASPs). MBL is a plasma protein belonging to a family of proteins known as the collectins. Collectins are oligomers of polypeptide chains containing a C type lectin carbohydrate recognition domain (CRD) attached to a collage like region. The overall structure of MBL resembles that of C1q. The PAMPs recognized by MBL comprise various simple and complex carbohydrate motifs. MBL does not selectively bind only mannose or its multimers as the name implies but rather in general recognizes sugars with 3- and 4-OH groups placed in the equatorial plae of the sugar ring structure. A group of proteins known as ficolins can also activate the LP. Ficolins are structurally similar to MBL and other collectins but instead of C-type lectin domains they possess fibrinogen like domain for PAMP recognition. Three ficolins are found in humans: H-ficolin, L-ficolin, and M-ficolin, while the mouse has only two. (Degn, Immunobiology 212, 2007, 301-311). Binding of MBL or serum ficolins indcues conformational changes in these complexes, leading to autoactivation of the MASPs, which in turn activate a downstream reaction cascade. 

With a prevalence of 5-10% in the Caucasian population, MBL deficiency is the most common imunodeficiency.  Functional MBL deficiency is explained largely by three single point mutations in codons 52, 54 and 57 of exon 1 in the MBL2 gene. 

Lectin Pathway 

Once MBL is bound to its target, MBL-associated proteins 1, 2, and 3 (MASP-1, MASP-2, MASP-3 and sMAP) are activated, resulting in the cleavage of C4 and C2, which is then followed by the assembly of the remainder of the complement pathway. MASPs are serine proteases. MASP-2 has been shown to be entirely associated with MBL, whereas MASP-1 cirulcates in both bound and in unbound forms. The overall structures of MASPs resemble C1r and C1s of the CP and mimic their activities. Upon bind of MBL to carbohydrate structures on teh surface of microbes, the proenzymes of MASPs are cleaved resulting in the active form. The activated MASPs are then  able to exert their proteolytic activites against complement components. MASP-2 activates C4 and C2, and form the C3 convertase (C4bC2a), an effect similar to the effect of CP C1s. 

The lectin pathway is particularly important in the recognition and clearance of pathogens, as deficiencies are strongly associated with recurrent infections and earlier death in patients with cystic fibrosis and recurrent pulmonary infection. MBL deficiency is the most common inherited immunodeficiency (MBL concentration <100 ng/ml) with a prevalence of 5-7% in the general population. It is alrgely explained by three single point mutations in codons 52, 54 and 57 of exon 1 of the MBL gene. 

See also Fc receptors under signal transduction

The classical pathway is is a major effector of the humoral branch of the human immune response. the CP is typically triggered by immune complexes such as an antibody bound to a foreign particle, and thus requires prior exposure to that particle for the generation of specific antibody. 

The classical pathway is a calcium/magnesium dependent cascade (Fung, 2002/0081293). 

Activation of the Classical Pathway

Activation of the classical pathway occurs primarily during immunologic recognition of antigen by specific antibody that exposes a C1 binding site on the Fc portion of the antibody. (Figueroa, Clinical Microbiology Reviews, July 1991, p. 359-395)

There are four plasma proteins involved in the initial steps of the CP: C1, C2, C4 and C3.

C1: Activation of the CP is initiated by binding of C1, the first component in the complement cascase, to an antigen-antibody complex and the subsequent activation of the antibody bound C1. C1 can also be activated by certain pathogens including HIV. C1 is a complex of threee subcomponents, C1q, C1r and C1s, the latter two being homologous single chain zymogens of 85 kD each. The first proteins (i.e., C1r, C1s, C4, C2 and C3 in the complement cascade exist as inactive precursor molecules which when converted to active proteases by protolytic reactions participate in cleavage of the next protein in the sequence. Thus, the activation of C1r and C1s results in a complex protease that can cleave C4 and C2. Hourcade (US5,869,615). 

The interaction of C1 with Fc regions of IgG or IgM in immune complexes activates a C1 protease that can cleave plasma protein C4, resulting in the c4a and C4b fragments. With respect to binding of C1q to antigen-antibody complexes,  IgG or IgM isotypes bind antigen, resulting in increased affinity of the Fc domains for the first component of complement C1q. Binding of antibody to antigen exposes a site on the antibody which is a binding site for C1. Since IgM is a pentamer whereas IgG is a monomer, IgM is a better activator since there are more regions for the complement to bind. C4b can bind another plasma protein, C2. The resulting species, C4b2, is cleaved by the C1 protease to from the classical pathway C3 convertase, C4b2a. Addition of the C3 cleavage product, C3b, to the C3 convertase leads to the formation of the CP C5 convertase, C4b2a3b.

–C1q: 

The first step in the complement cascade is the binding of C1q to the antibody. Therea are six heads on C1q, connected by collagen-like stems to a central stalk and the isolated heads bind to the Fc portion of antibody rather weakly. Binding of antibody to multiple epitopes on an antigenic surface, aggregates the antibody and this facilitates the binding of several C1q heads, leading to an enhanced affinity. Within the Fc portion of the antibody, C1q binds to the CH2 domain. The interation is sensitive to ionic strengh, and appears to be highly conserved throughout evolution as C1q reacts with IgG form different speies. (Winter, The binding stie for C1q on IgG” Nature, 332(21), 1988). 

C2: has 39% sequence identity to Factor B, and is a 102 kD zyogen composed of three SCRs followed by a vWF repeat and a serine protease domain. In the Mg2+ dependent formation of the convertase C4bC2a, a weakly associated C4bC2 allows the clevage of the C2 subunit by activated C1, yielding two C2 fragmetns, C2a and C2b. The C2b fragment, derived form the amino terminus, dissociates while the remaining C2a fragment associates more tightly with C4b, to for the proteolytically active classical pathway C3 convertase, C4bC2a. Hourcade (US5,869,615)

C3: is cleavged to produce the important fragment C3b, an opsonin that binds covalently to nearby cells and macromolecules. When these reactions occur on a cell surface, they can direct the assembly of complemnt lytic components in the membrane. Hourcade (US5,869,615)

Classical pathway C3 convertase cleaves C3 into C3b which can work independent of the alternative pathway with full amplification of the classical pathway in 1% normal human serum in the presence of Ca2+/Mg2+ ions. Classical pathway C5 convertase can cleave C5 to generate C5a and C5b. The C5b molecule then inserts into the lipid bilayer of the cell to initiate the formation of C5b-9 or sC5b-9. Bansal (US 13/646286).

C5b: initiates the assembly of the membrane attack complex (MAC) by a series of protein-protein interactions that involve the components C6 to C9. Attachment of this C5b6789 complex to the membrane leads to formation of a channel that traverses the membrane. Hourcade (US5,869,615)

Classical Pathway Initatied Amplifcation of the Alternative Pathway

The classical pathway, like the AP, generates C3b. This C3b can feed into the AP which can couple to target pahtogens and serve as a substrate for the alternative pathway (Song, US2010/0263061).

Overview

The AP is responsible for 80-95% of total complement activity. The AP is a complement activation pathway which is triggered by artificial surfaces such as LPS from Gram negative outer membranes and rabbit erythrocytes, zymosan from fungal and yeast cell walls, as well as from many pure polysaccharides, viruses, bacterial, animal tumor cells, parasites and damaged cells. The pathway is initiated mainly by cell surface constituents that are foreign to the host like gram negative and positive bacteria. The alternative pathway generates bound C5b, the same product that the classical pathway generates but it does so without the need for antigen-antibody complexes for initiation. Because no antibody is required, it is a component of the innate immune system. This pathway involves 4 serum proteins like factor B and D. The alternative pathway is also an ancient immune mechanism that is primarily activated onto the surface of pathogens by a process called “tickover”. This formation of C3(H2O) allows for the binding of plasma protein Factor B, which in turn allows Factor D to cleave Factor B into Ba and Bb.Tickover is facilitated by the presence of surfaces that lack complement regulatory proteins and which support the binding of activated C3. It occurs through the spontaneous cleavage of a thioester bond in C3 to form C3i or C3(H2O). Tickover is facilitated by the presence of surfaces that support the binding of activated C3 and/or have neutral or positive charge characteristics (e.g., bacterial cell surfaces). Potential therapeutic targets specific to the alternative pathway include factor B, factor D, and properdin.

C3b Binding to foreign surface: AP recognition occurs in the presence of C3b and an activating substance such as bacterial lipoprotein, surfaces of certain parasites, yeasts, viruses and other foreign body surfaces, such as biomaterials. C3b originates from classical pathway activation and/or from natural spontaneous hydrolysis of C3. The resulting C3b binds to the surface of the activating substance. C3b does not usually bind to mammalian cells because they contain high levels of naturally occurring n- and o-acyl derivative of the deoxy-amino sugar nueraminic (or sialic) acid on the cell membrane. Prokaryotes and parasites lack this cell surface constituent and are, therefore, highly susceptible to C3b binding. (Bansal, US 13/583879).

Binding of Factor B to C3b: Binding of factor B to immobilized C3b is dependent on Mg2+ (Hourcade, J. Biological Chemistry, 270(34), 19716-19722, 1995). Initial binding of B to C3b is believed to be effected through the cooperative binding of two low affinity sites; one of them is located on Ba and the other, which is Mg2+ dependent, on the Bb region of factor B. (Ueda, J. Immunology, 138(4), 1143-1149 1987). In the presence of magnesium, Factor B binds to the C3b which is bound to the activating surface. Once bound, the Factor B protein is susceptible to immediate hydrolysis by a sserine protease called Factor D. Factor D cleaves B, releasing the Ba fragment and forming C3bBb. Properdin stabilizes the C3bBb complex and protects it from decay. C3bBbP enzyme complex exhibits C3 convertase activity (in a manner similar to C4b2a in the classical pathway) and converts C3 to C3b, launching a positive feedback arrangement that significantly amplifies the genesis of C3b molecules. Deposition of these C3b molecules on the foreign cell surface enhances nuetrophil opsonizaiton of the cell. The released C3a analphylatoxin molecules mediate a localized, inflammatory, chemotactic response that recruits neutrophils to the area. The autocatalytic activity of C3bBb convertase results in the produciton of high levels of C3b that can attach to this convertase to produce a C3bB3b complex that demonstrates C5 convertase activity. Circulating C5 is bound by the C3b component and enzymatically hydrolyzed by the activated Bb serine protease into the products C5a and C5b. The the hydrolysis of C5 and the commencement of the formation of the membrane attack complex. (Pugsley, Cardiovascular Toxicology (2003), pp. 43-69). To say another way, cleavage of C3 results in the formation of C3bBb3b, the C5 convertase. This enzyme is also stabilized by P to form C3bBb3bP. C5 convertase can cleave many molecules of C5 into C5a and C5b. If the PC3bB complex can not form, then the MAC complex (C5b-9) will also not form (Bansal, US 13/583879).

To review, conversion of C3 to C3b produces a product that can combine with factor B, giving C3bB.  These complexes are acted upon by factor D to generate C3bBb, which is a C3 convertase capable of cleaving more C3 to C3b, leading to more C3bBb and even more C3 conversion. Under certain circumstances, the C3bBb complex is stabilized by association with the positive regulator properdin (P) by association of C3b and Bb. The C3 convertases can associate with an additional C3b subunit to form the C5 convertase, C3bBbC3b, which is active in the production of the C5-C9 MAC. C3a is an anaphylatoxin that attracts mast cells to the site of challenge, resulting in local release of histamine, vasodilation and other inflammatory effects. The nascent C3b has an ability to bind to surfaces around its site of generation and functions as a ligand for C3 receptors mediating, for example, phagocytosis. The rate limiting step of activation of the alternative pathway in humans is the enzymatic action of factor D on the cleavage of factor B to form the alternative pathway C3 convertase.

Components of the AP

Factor B: 

Human factor B is required for the initiation and propagation of the complement alternative pathway (AP). It also participates in the amplificaiton of the complementCP. Alone, factor B is a zymogen with little known biochemical activity, but in the context of the AP covertases, the factor B serine protease is activated in a process that first involves the association with C3b and subsequently the cleavage of factor B into two fragments, Ba and Bb. Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995). 

Complement Protein B is a single polypeptide chain serum glycoprotein with about a 90k MS. It carries the catalytic center of the AP C3 convertase and represents a novel type of serine protease characterized by an unusual structure in the NH2 terminal region of its catalytic fragment Bb, when compared to otehr serine proteases. Assembly of the bimolecular, C3bBb, C3 convertase proceeds in two well defined steps. First, B binds stoichiometrically to C3b in a reaction requiring Mg2+ or Ni2+ ions. Second, complement protein D catalyzes the cleavage of a single arginyllysyl peptide bond of B, resulting in the relase of fragment Ba and the formation of the C3bBb protease. Ueda (J. Immunology) 138(4), 1143-1149, 1987)

Factor B is a tightly regulated, highly specific serine protease. It is the zymogen of the alternative pathway C3/C5 convertase, and is activated when it is split by factor D into two fragments, Ba and Bb, after it has formed a complex with C3b.The interaction between factor B and surface-bound C3b triggers a conformational change in factor B that ultimately creates the C3 convertase (PC3bBb) of the alternative complement pathway. The activation of the AP hinges on a Mg ion-enhanced interaction between factor B and C3b. Upon binding, factor B is rendered susceptible to proteolytic cleavage by factor D, forming fragments Ba and Bb. Bb, in association with C3b, comprises the AP C3 convertase. This complex has serine protease activity and functions to cleave native C3 in C3a and C3b.tic cleavage by factor D, forming Ba and Bb. Bb, in association with C3b comprises the APC3 convertase. This complex has serine protease activity and functions to cleave native C3 into C3a and C3b. 

Binding of factor B to immobilized C3b is depndent on Mg2+. Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995).

More specifically, Facto B is a 90 kDa protein consisting of threee domains: a 3 module complement control protein (CCP1, CCP2 and CCP3), a von Willebrand factor A domain, and a C terminal serine protease domain that adopts a default inactive  (zymogen) conformation. The interaction between factor B and surface bound C3b triggers a conformational change in factor B that ultimately creates the C3 convertase (PC3bBb)of the AP. The activation of the AP hinges on a Magensium ion enchanced interaction between factor B and C3b. Upon binding, facotr B is rendered susceptible to proteolytic cleavage by factor D, forming fragments Ba and Bb. Bb, in association with C3b, comprises the AP C3 convertase which has serine protease activity and functions to cleave native C3 into C3a and C3b (Bansal, US 12/675, 220). 

To state yet another way, the first step in the assembly of the AP C3 convertase is the association of factor B with C3b. In this context, factor B can be cleaved by factor D, resulting in Ba and Bb, a process that requires a divalent cation. Ba then dissociates from the complex while Bb remains bound to C3b. C3bBb can be partially stabilized by assocaition with properdin. C3bBb and C3bBbP are active enzymes that cleave C3 at a single point, generating more C3b and ultimately more convertases. AP C5 convertase activity occurs thorugh the association of C3 convertase and additional C3b. In all cases the dissociation of Bb from the convertases is inevitable, irreversible, and followed by inactivation of proteolytic funciton. Hourcade (J. Biological Chemistry, 270(34), 1995, pp. 19716-19722. 

–Ba fragment: Ba, the NH2 terminal fragment of Factor B, is composed mainly of three tandem short consensus repeats, globular domains found in other complement proteins. It dissociates from the convertase during assembly, leaving the active C3 convertase, C3bBb. The Ba fragment has affinity for C3b and some monoclonal antibodies directed against Ba block factor B-C3b interations. Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995).

Factor D: is a highly specific serine protease essential for activation of the AP. (Fung US2002/0081293). Factor D’s only known natural substrate is factor B bound to C3b. It is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor activation by a serpin for its control. It is a highly specific serine protease and cleaves factor B bound to C3b, generating the C3bBb enzyme which is the alternative pathway C3 convertase. 

Properdin:  is a serum glycoprotein which stabilizes the labile C3 convertase (C3bBb) of the AP of the complement system. Properdin binding to the complex C3bB promotes factor D induced cleavage of factor B. Properdin is central to deposition of the activated complement fragment C3b on the surfaces of pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. Individuals with properdin frequently develop meningococcal disease. In the absence of properdin, the C3bB complex cannot be formed and cleaved with factor D. Only oligomeric forms of properdin are considered active. Oligomeric properdin binds C3b and initiates the AP activation. Human properdin is a 469 amino acid protein that includes a signal peptide (amiono  acids 1-28), and six, non-identical thrombospondin type 1 repeates (TSR) of about 60 amiono acids each, as follows: amino acids 80-134 (TSR), amino acids 139-191 (TSR2), amino acids 196-255 (TSR3), amino acids 260-313 (TSR4), amino acids 318-377 (TSR5), and amino acids 382-462 (TSR6). Properdin is formed by oligomerization of a rod-like monomer into cyclic dimers, trimers, and tegramers.

The amino acid sequences of mammalian properdin as well as human properdin are known. Properdin has an unusual structure formed by oligomerisation of a rod like monomer into cyclic dimers, trimers and teramers.  The monomer contains a N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids called “thrombospondin type 1 repeats” or TSR modules. Polyclonal antibodies raised against each TSR have been found to be module specific. Perdikoulis, Biochimica et Biophysica Acta 1548 2001) pp. 265-277. Human properdin is a 469 amino acid protein that includes a signal peptide (amino acids 1-28), 6 non-identical thrombospondin type 1 repeats (TSR) of about 60 amino acids each as follows: amino acics 80-134 (TSR1), amino acids 139-191 (TSR2), amino acids 196-255 (TSR3), amino acids 260-313 (TSR4), amino acids 318-377 (TSR5) and amino acids 3829-462 (TSR6). Bansal US13/849092) discloses that monoclona antibodies that specifically bind to an epitope of the N terminal region consisting of amino acids 71-110 of human properdin which can inhibit properdin function. 

Properdin is composed of multiple identical protein subunits, with each subunit carrying a separate ligand-binding site. Previous reprots suggest that properdin function depends on multiple interactions between its subunits with its ligands. Hourcade (J Biological Chemistry, 281(4), pp. 2128-2132). The amino acid sequences of mammalina properdin are known (GenBank database under Accession No. AAA36489). Human properdin is a 469 amino acid protein that includes a signal peptide (amino acids 1-28), and six-non-identical thrombospondin type 1 repeats (TSR) of about 60 amino acids each. All six TSRs of properdin have different function with TSR5 being involved in properdin function. The N-terminal region of properdin including the first half of the TSR1 are important for properdin functions and monoclonal antibodies which specifcally bind to an peitope of the N-terminal region can inhibit properdin function (Bansal (12/920,997, now US 8,435,512; see also US 13/849092). 

Amplification Loops: 

The AP has a critical role in amplifying the complement response independent of the initiating pathway and in exacerbating inflammatory pathologies. When a C3b molecule is deposited on a activating surface, either by the CP and LP C3 covertases or as a result of bystander effects or tick over, the subsequent binding of the serine proteases factor B (FB) and factor D (FD) leads to the assembly of the AP C3 convertase (C3bBb complex) that cleaves C3 into C3a and additional C3b, which can participiate in the formation of new convertases. In many settings, this amplificaiton loop is the major source of opsonization and feeds all effect arms of the complement system, including inflammatory and adaptive signalling, phagocytosis and the formation of C3 covertases with subsequent MAC assembly. (Lambris, “The renaissance of complement therapeutics” (2018))

The AP involves amplification loops utilizing C3b produced by the CP and LP. Some molecules of C3b generated by the CP C3 convertase are funneled into the AP. Surface bound C3b binds Factor B to yield C3bB, which becomes a substrate for Factor D. Factor D is a serine esterase that cleaves the Ba fragment, leaving C3bBb bound to the surface of the target cell. C3bBb is stabilized by properdin (P), forming the complex C3bBbP, which acts as the AP C3 convertase. This C3 convertase participates in an amplification loop (the AP amplification loop”) to cleave many C3 molecules, resulting in the deposition of C3b molecules on the target cell. In other words, the C3b formed by the AP pathway makes AP C3 convertase which in turn cleaves C3 and generates even more C3b, which feeds back into the loop. This self-perpetuating cycle of reactions generates large amounts of C3b. Some of these C3b molecules bind back to C3bBb to form C3bBbC3b, the AP C5 convertase. C5 convertase cleaves C5 into C5a and C5b. C5b binds to the surface of the cell to initiate the formation of the membrane attack complex.

See also therapeutic antibodies against specific complement components in the “antibodies” section. 

See also CR2 targetting of complement inhibitors for treatment of disease under “pharmacology”  

See also specific diseases throughout ypatent for the role which complement plays in these diseases. 

The complement system has been implicated in many autoimmune diseases such a rheumatoid arthritis, systemic lupus erythemaosus, renal diseases, myocarditis, multiple sclerosis, Type I diabetes mellitus and asthma. Individuals with inherited deficiency of any of the complement molecules prior to C5 are vulnerable to both pyogenic organisms and to autoimmune disorders. In contrast, deficiency of any of the molecules after C5 remarkably has little phenotype except for an increased risk of infection by encapsulated organisms, namely, Haemophilus influenza, Neisseria meningitides and N gonorrhea.

Inherited deficiencies have been recognized in humans for nearly every complement component. Deficiencies of components of the same pathway cause similar clinical problems. Classical pathway component deficiencies (C1, C4, C2) commonly cause infections by a variety of pyrogenic organisms and immune complex diseases, as does defieincy of C3. Alternative pathway component deficiences (P, D) often results in Neisserial infections. (US 2003/0198636). 

Unpredictability in the Art: 

The activation of the complement system may cause substantial injury when activated inappropriately and that while complement activaiton is probably not the primary etiology of many diseases such as those which affect the cardiovascular, the damage to tissues in certain conditions is clearly complement mediated (p. 59, 1st #s1-2). Key constituents of the formation of the AP is the association of factor B with C3b, and adminsitration of antibodies against components of the AP such as C5 are therapeutic approaches for blocking AP pathways and thus complement actiation. Makrides (“therapeutic inhibition of the complement system” Pharmacological Reviews, 60(1), 1998).

Still, succcessful marketing of complement targeted drugs has proved to be more difficult than initially expected, and many strategies have been discontinued. In light of these complications, most of the major pharmaceutical companies seem to have abandoned their initial efforts to develop drugs that target complement. Despite numerous attempts to inhibit or modulate complement therapeutically, the success rate has been disappointly low. The multifaceted nature of both the cascade and its disease involvement may be one central problem. Many questions about the exact disease related mechanisms of complement, both at the molecular and clinical level are still unresolved, and this lack fo clarity complicates specific targetting. Inhibition of a single pathway may be insuffcient for many diseases. (Ricklin “Complement-targeted therapeutics” Nat Biotechnol. 2007, 25(11); 1265-1276). 

One of skill in the art would recognize that a particular complement inhibitor which may be effective for one type of complement mediated disorder may not be effective in a different complement mediator disorder and that treatment of any one particular disorder will depend upon not only the particular complement inhibitor being administered but also the complement component and organ being targeted. For example, C3a protects neurons against glutamate-induced excitoxicity and induces the production of nerve grwoth factor and anti-inflammatory hormones, while targeted expression in the brain proteins against endotoxic short, indicating that “complemet therapeutics may be highly disease specific in practice (Stahel, Expert Rev. Clin. Immunol. 2(3), p. 451, 2nd column, lines 4-16, (2006).

The development of mice deficient in elements of the complement system are stimulating investigators to study the role of different complement components in systemic injury. Such experiments suggest that not all components of the complement system may enhance injury but some may in fact be beneficial. (Yang, “the role of complement C3 in intracerebral hemorrhage-induced brain injury” J. Cerebral Blood Flow & Metabolism (2006), 26, 1490-1495).

Role in Specific Diseases 

Ischemia/Reperfusion Injury (IRI):  Activation of the complement cascade represents an important event during ischemia/reperfuion injury (IRI). Fondevilla, (Liver Transpl. 2008, 14(8): 1133-1141).

–Hepatic IRI: 

Fondevilla (Liver Transpl. 2008, 14(8): 1133-1141) discloses that terminal products of the complement system are essential in the mechanism of hepatic IRI. Using a clinically relevant liver cold ischemia model, they show that local MAC inhibition attenuates IRI cascade in orthotopic liver transplantation recipients. 

–Intestinal IRI:

Huang (J. Immunology, 2008, 181: 8068-8076) discloses that the alternative pathway of complement plays a role in intestine ischemia/eperfusion injury. 

Pulmonary diseases:

Francois (US14/052545 and US8,580,735) teaches administration of compstatin analogs for treatment of respiratory diseases.

Levin (WO94/17822) discloses pulmonary administration of a complement inhibitory protein by inhalation for the therapeutic treatment of diseases involving complement. Deseases include bronchoconstriction and inhibitors are directed to tComplement receptors.

Wang (WO2004022096) discloses method of treating asthma which a complement inhbitiory such as an antibody.