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.