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.

The complement system consists of about 31 proteins, acting within a cascade-like reaction sequence, serving as control proteins or as cellular receptors. Some of the proteins are enzymes (C1r, C1s, C2, factor B, factor D), some cofactors, some inhibitors, and other are composed of membrane-integrated proteins.

Complement proteins are made mainly by the liver and circulate in the blood and extracellular fluid. A majority of the protein componetns of the system are syntehsized by liver hepatocytes; however, blood monocytes, tissue macrophages and the epithelial cells lining the gastrointestinal and genitourinary tracts can also synthesize some complement proteins. 

Both in vivo and in vitro studies have confirmed that cytokine mediators of the acute phase response, for example, the interleukins (particularly interlukin y), TNF and dexamethasone can increase the hepatic synthesis of complement proteins 2-5 fold in cultured hepatocytes. (Figueroa, Clinical Microbiology Reviews, July 1991, p. 359-395)

Most complement proteins are inactive until they are triggered by an infection. Most the circulating proteins are innnocuous and as such are regarded as “pro-enzymes”. However, upon initiation there occurs a sequential proteolysis and ensuing activation of these proteins. Activation of a complement protein changes its structure, revealing an “active” site then catalyzes, and usually cleaves, a portion of the next protein in the sequence, ultimately producing a multiple subunit protein complex that lyses the invading cell. 

They were originally identified by their ability to “complement” the action of antibodies but some components of complement are also pattern recognition receptors that can be activated directly by pathogen associated immunostimulants. Complement can be activated by any of three pathways, either the antibody-dependent classical pathway, the alternative pathway or the lectin pathway. On activation, these pathways result in the formation of unstable protease complexes, the C3-convertases. The classical pathway C3-convertase, C4b2a, and the AP C3-convertase, C3bBb, are both able to cleave the alpha chain of C3 generating C3b. C3b has the potential to bind covalently to biological surfaces which leads to opsonization for phagocytosis by polymorphonuclear cells and macrophages. When additional C3b is available, the C3-convertases can function as C5-convertases, cleaving C5 and initiating the assembly of the TCC, or the membrane attack complex (MAC), which mediates cellular lysis by insertion of pore-forming protein complexes into targeted cell membranes. (Kaleko, WO2008/106644).

Companies: Editas  Excision Bio Therapeutics Prime Medicine. Pairwise.  Tome Biosciences. EditCo

See also chimeric antigen receptors (CARs) under Cancer treatment

Programmable nucleases such as CRISPR-Cas9 make double strand DNA breaks (DSBs) that can disrupt genes by inducing mixtures of insertions and deletions (indels) at target sites. DSBs, however, are associated with undersired outcomes including complex mixtures of products, translocations and p53 activaiton. Moreover, the vast majority of pathogenic alleles arise form specific insertions, deletions or base substitutions tha require more precie editing technologies to correct. Homology-directed repair (HDR) stimulateed by DSBs has been used to insall precise DNA changes. HDR, however, relies on exogenous donor DNA repair templates, typically generates an excess of indels form end-joining repair of DSBs, and is inefficient in most therapeutically relevant cell types (T cells and some types of stem cells being important exceptions). (liu, Nature 2019, 576(7785): 149-157).

CRISPR-Cas componetns readily access the gehnome of bacteria as prokaryotes lack a nucleus. However, unmodified CRISPR-Cas components do not readily enter the nucleus of eukaryotic cells, wehre genomic DNA is located, which greatly limits the efficiency of DNA editing. A single nuclear localization signal (NLS) is typically sufficient to facilitate efficient nuclear entry of most prtoeins. However, multiple NLSs are necessary to drive Cas variants to the nucleus in eukaryotic cells, including Sterptococcus pyogenes (Sp) Cas9 and Neisseria meningitidis (Nm) Cas9, as well as base editors, prime editors, Cas12a, and nuclease dead Cas9. This may be because Cas9 is sequenstered in the cytoplams of mammalian cells, inn part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants. (Zylka, “Exploring the cytoplasmic retention of CRISPR-Cas9 in eukaryotic cells: the role of nuclear localizaiton signals and ribosomal interactions” CRISPR Journal, volume 00, number 00, 2025.)

Classes of CRISPR Nucleases:

In brief, CRISPR based gene editing reagents are generally used as two-component systems: a nuclease protein is complexed with a single-guid RNA (sgRNA) designed to target a specific site in the genome. Much work has gone into engineering both nucleases and guides to optimize function and stabilitiy across an array of cell types. The CRISPR associated nucleases most commonly used for double stranded DNA targeting belong to class II and can be put into two groups, type II and type V, that differ in the position of their protospacer-adjacent motif (PAM) vis-a-vis the spacer region (3′ vs. 5′) and the DNA ends that result from the clevage reaction (blunt vs. overhnag). (Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)

Type II Nucleases:

The type II CRISPR-Cas system includes three components: (1) a crRNA molecule, which is called a “guide sequence” and “targeter-FNA”, (2) a tracr RNA, also known as an activtor-RNA and (3) a protein called Cas9. To alter a DNA molecule, the ssytem must acheive three intereactions: (1) crRNA binding by specific base pairing to a specific sequence in the dNA of interest (target DNA), (2) crFNA bing by specific base pairing at naother sequence to a tracer RNA, and (3) tracr RNA interacting with a Cas9 protein, which then cuts the target DNA at the specific site.

A CRISPR-Cas9 system is a combination of protein and ribonucleic acid (RNA) that can alter the genetic sequence of an organisms. In their natural environment, CRISPR-Cas systems protect bacterai agaisnt infection by viruses. Te system is now being developed as a powerful tool to modify specific deoxyribonucleic acid (DNA) in the genomes of other organisms, from plants to animals. With CRISP, scientists can create mouse models of human diseases much quicker, study indivdual genes much faster and easily change multiple genes in cells at once to study their interactions.

Although Streptococcus pyrogenes Cas9 is a highly active gene editing enzyme, its use is complicated by a low editing specificity. Furthermore, Cas9 is derived from a Streptococcus bacterium, a very commonly pathogenic genus. As a result, between one third and half of people have a preexisting immune response to the Cas9 enzyme and thus are less than optimal candidates for a Cas9 based gene editing therapy. (Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)

Base Editors:

Base editors generate targeted base conversions without requiring DSBs. Cystosine base editors and adenine base editors for example comine deaminases with CRISPR systems to produce C:G-to T:A and A:T to G:C base transcritions, respectively. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

Prime Editors (Prime editing):

Prime editing relies on Cas9 and a reverse transcriptase.

Prime editors are powerful tools for installing base substitutions and precise DNA insertions and deletions. They are composed of two components: an engineered Cas9 nickase (H840A) reverse transcriptase (RT) fusion protein and a prime editing guide RNA (pegRNA). The pegRNA contains an RT template (RTT) encoding the desired edits and a primer binding site (PBS) for hybridization of the 3′ end of the nicked DNA strand to initiate reverse transcription. After reverse transcription, the RT template is reverse transcribed, forming a 3′ DNA flap followed by a 5′ DNA flap, and this enables the desired edit to be integrated into the target site. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

liu, (Nature 2019, 576(7785): 149-157) discloses a search-and-replace genome editing technology called “prime editing” that mediates targeted insertions, deletions, all 12 possible base to base conversions and combinations thereof in human cells without requiring DSBs or donor DNA templates. Prime editiors (PEs) use a reverse transcriptase (RT) fused to an RNA programmable nickage and a prime editing guide RNA (pegRNA) to direclty copy genetic informaiton from an extension on the pegRNA into the target genomic locus. The DNA nicked at the target site to expose a 3′-hydroxyl group can be used to prime the reverse transcription of an edit-encoding extension of the engineered guide RNA (prime editing guid RNA, or “pegRNA) directly into the target site. These initial steps result in a branched intermedaite with two redundant single-stranded DNA falps: a 5′ flap that contains the uneditind DNA sequence and a 3′ flap that contains the edited sequence opied form the pegRNA. %’ flaps are hte preferred substrate for structure-specific endonucleases such as FEN1 which excises 5′ flaps engenerated during lagging-strand DNA syntehsis and long-patch base excision repair. Alternativley, the reduncant uneidted DNA may be removed by 5′ exonucleases such as EXO1. 5′ flap excision and 3′ flap ligation can drive the incorproation of the edited DNA strand, creating heteroduplex DNA containing one edited strand and one unedited strand. DNA repair to resolved the hteroduplex by copying the informaiton in the edited strand to the complementary strand will permanently install the edit.

DNA-Dependent Polymerase (DDP) Editing (DPE):

DNA-dependent polymerase (DDP) editing presents adnvatages over prime editing by allowing researchers to cale materials up for clinical use. DPE benefits fro the highly processive and accurate synthesis abilities of DNA polyemrases. The approach avoids a range of problems associated with prime editing: fewer erros, less reliance on a cell cycle due to higher dNTP affinity, and easier template synthesis.

–Click Editing:

An even newer form of genome eidint that relies on a DDP, a nickase Cas9 (nCas9) and a histidine-hyrophogic histidine (HUH) endonuclease to alter the geneome allows for mass screening of DNA templates and futher refines DDNA polymerase editing (DPE). Click editing adds HUH edonucleases to DPE. HUH endonucleases are smalle (10-40 kDa) proteins common across all domains of life and which are often found playing a role in replication. HUH enzymes are guided by 8-40 nucleotide ssDNA regoniction sequences. Click editors eploy HUH endonculeases for teplate recruitment to teh target site, which covalently and sequence specifically bind ssDNA. Click editing invovles 5 components: nCas9, a single guid RNA (sgRNA), an HUH endonuclease, a DDP and a click DNA (clkDNA). clkDNA guides the DDP and contains the edit of itnerest, which an sgRNA directs nCas9. In this way, clkDNA mimics the role of DPETs. Click editing follows a methodical set of steps to alter the geome. Frist, the click editor binds and nicks the genome at the target site. The, HUH tethers or “clicks” -clkDNA to the target site. The primer binding sequence of the clkDNA subsequently binds to the flap created by the nickase, allowing clkDNA to serve as a tplate for polymerization. After the primer has synthesized the new sequence, the click editor dissociates and leaes the edited flap behind. Finally the new sequence is integrated into the gebome. Using a DNA based template has a range of benefits over prime editing, which uses a long pegRNA to direct and initate its reverse trasncriptase appraoch. Click editing extends these benefits by removing RNA form teh template to facilitate easier synthesis. (Seren hough, “Polymerase editing ‘clicks’ together  in trailbalzing study’ Tides Global, Oct 25, 2024.)

Applications:

See FDA Approved Cellular Therapies

Gene editing has been applied to cell therapy with many types of primary immune cells –especially T cells –via electroporation of Cas9 ribonucleoprotein particles (RNPs). Such engineering has been used to knockout the T cell receptor (TCR), checkpoint inhibitors, and ot knock in chimeric antigen receptors (CARs), among many examples. Gene editing systems (type II and type V) have also been used to edit B cells, NK cells, indicued pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs). Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)

HIV Gene Editing:

Manucuso et al. (“CRISPR based gene editing of SIV proviral DNA in ART treated non-human primates; Nature Communications 2020) discloses creating an adeno-associated virus mediated plasmid DNA vector that allows for simultaneous expression of Cas9 endonuclease and multiple guide RNA. The Cas9 endonucleas and gRNAs specifically recognize LTR and Gag region of the HIV-like simian immunodeficiency virus genome. Excision of large segments of the integrated proviral DNA spanning from cleavage site mitgates the chance of the replication -competent virus’s emergence.

Based on these promising results, Excision Bio Therapeutics has started clinical trials to evaluate the construct, termed EBT-101 as a potential cure of HIV infection.

Colesterol lowering/PCSK9:

Musunuro (“In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates”  Nature, 2021) demonstrates durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment.

Deafness (Hearing Loss):

–Autosomal dominant deafness-50 (DFNA50) is a form of progressive hearing loss which starts with mild symptoms before getting significantly worse later in life. The condition is caused by a variant in the microRNA gene MIR96. A new CRISPR-Cas9 based treatment for FFNA50 has been desmontrated in an artile published in Science Translational Medicin (Zheng-Yi). The study showed that hearing loss was restored in mice after targeting and disrupting the DFNA50 causing version of MIR96. Chen’s team used an adeno-associated virsu (AAV) system to deliver Cas9 and a single guid RNA (sgRNA) specifically to teh cochlea in the inner ear, the region responsbiel for hearing. AAV has become a popualr choice for delivery becasue it is less likely to interate into the target cell. Moreover, different serotypes of AAV can be used to target specific parts of the body. On the other hand, the small cargo size of AAV prohibits the delivery of a large gene like traditional Cas9. To get around this limitation, the team used a specific kind of Cas9 derived form Staphylococcus aureus bacteria. This ortholog is smaller than the typical streptococcus pyogenes Cas9-making it an idea match for AAV delivery.

Sickle Cell Disease and beta-thalassemia:

–Casgevy (CRISPR Therapeutics & Vertex Pharmaceuticals): is a one time treatment for people age 12 and over for sickle-cell diesease as well as beta-thalassemia. Vertex recieved the FDA first approval for a CRIP based gene edited therapy, Casgevy for sickle cell disease. The following month, the FDA approved Casgevy to treat tranfusion dependent beta-thalassemia.

Beam Therapeutics also expects to conduct Phase I/II trails to asess its lead candicate BEAM-101 in severe sickle cell diseas.

Duchenne Mascular Dystrophy:

–ELEVIDYS (Sarepta Therapeutics): is a prescription gene therapy used to treat ambulatory children aged 4 through 5 years old with Duchenne muscular dystrophy (DMD) who have a confirmed mutation in the dystrophin gene. ELEVIDYS was approved under accelerated approval.

Animal Genetics:

Genus, a British animal genetics company with research facilites in Wisconsin and Tennessee, has developed a new generation of CISPR edited pigds that are resistant to procine reproductive and respiratory syndrome (PRRS) virus, a disease that has had a widespread impact on porcine populations around the world for decades. Several genes are invovled in viral infection, including CD163, which encoes the entry receptor for teh virus. In pigs, this particular protein is expressed on teh surface of macrophage and monocytes and mediates inflammation among other functions. A single modified CD163 allele was introduced into four geentically diverse, elete porcine lines. Genus scientifits injected CRISPR-Cas9 editing reagents into the genomes of pig zygotes. Their goal was to make a preice deletion in CD163 that removed a single expon encoding the domain that directly interacts with the virus. Importantly, the edit did not impact CD163’s function in the new population. The work produced healthy pigs that resisted PRRS virus infections. The company is now seeking regulatory approval in the US which is the only country which regulated intentional genetic alterations including single base pair deletions as an animal drug and reqires a new animal drug approval for commercialization. This regulatory path is very hard for small companies. Until the pigs are approved, they are considered unsalable.

Gene Editing in Plants:

Classical genome editing in plants involves a sgRnA guided Cas dsDNA nuclease with subsequent induction of the non-homologous end-joing (NHEJ) DNA repair pathway, frequently resulting in loss of function (LOF) mutaitons. Modulation or gain of function may be obtained through the use of sgRNA wity subsequent induction of the base excision repair pathway or by prime editor RNA (pegRNA) guided prime editors with subsequent induction of the NHEJ or the homology directed repair pathway, depending on cell type. Petersen “Strategies and Protocols for optimized genome editing in potato” CRISPR Journal, volume 00, number 00, 2024).

Diagnostics:

–Zika Detection:

Collins (“Rapid, low-cost detection of Zika virus using programmable biomolecular components” Cell 165, 1255-1266, 2016) demonstrate the rapid development of a diagnostic workflow for sequence-specific detection of Zika virus that can be employed in low-resource settings. Discriminatio of different viral strains at single-base resolution was achieved using a CRISPR-based tool. By simply boiling (95C) virsu samples for 2 min, sufficient quantities of RNA for amplication and detection were generated. NASBA is an extremely sensitive and has a proven track record in the field-based diagnostic applications. The amplificaiton process betgins with reverse transcription of a target RNA that is mediated by a sequence-specific reverse primer to create an RNA/DNA duplex. RNase H then degrades the RNA template, allowing a forward primer containing the T7 promoter to bind and initiate elongation of the complementary. strand, generating a double-stranded DNA product. T6-mediated transcription of the DNA template then creates copies of the target RNA sequence. Importantly, each new target RNA can be detected by a towhold switch sensors and also serve as starting material for futher amplication cycles. NASBA requires an initial heating step (65C), followed by isothermal amplication at 41 C. NASBA was performed on trigger RNA corresponding to Zika genomic regions for sensors 27B and 32B. Toehold switch sensors are programmable syntehtic riboregulators that control the translation of a gene via the binding of a trans-acting trigger RNA. The switches contain a hairpin structure that blocks gene translation in cis by sequestration of the ribosome binding stie (RBS) and start condon. Upon a switch binding to a complementary trigger RNA, sequenstraiton of the RBS and start condon is relieved, activating gene translation. To allow for colrimetric detection of trigger RNA sequencs, the sensors can be desigend to regulate translation of the enzyme LacA, which mediates a color change by converting a yellow substrate to a purpose product. Toehold switch sensors for sequence based detectin of Zika virus were generated using a modified version of a developed in silico design algorithm. The modifed algorithm screened the genome of the Zika strain prevalent in the American (Genbank: KU312312) for regions compatible with RNA amplication and toehold switch activation. The slected Zika genome regions were then computationally filtered to eliminate potential homology to the human transcriptome and to a panel of related virsues, incuding Dengue and Chikungunya. A total of 24 unique regions of the Zika genome compatible with downstream sensing efforts were identified. The presence of a train-sepcific PAm leads to the production of either truncated or full lenght trigger RNA, which differentially activate a towhold switch. The probability that a non-biased single nucleotide polymorphism (SNP) between two strains can be discriminated by CRISPR/Cas9 is 48%.

A species identified as a mAb monomer with an additional light chain covalently associated through a disulfide bond formed between a L chain cystein and an engineered cystein in either the H or L chains is known. Genetech researchers also characterized mAb species containing variants with one and two additional light chains present at 0.2% when produced in CHO cells. They determined that the relative quantity of this species was related to the redox environment of the cell culture, which implicates disulfide bonding as the mode of binding for tehe additional light chains. In addition, a report characterizaing a mAb size variant using SE-HPLC coupled with multi-angle light scattering (MALS), matrix-assisted laser desportion/ionization-time of light (MALDI-TOF) mass spectrometry, capillary gel elctrophoresis and N-terminal sequences showed a mAb with incorporation of a third light chain; however the extra L chain was associated through non-covalent interactions. A cell based potency assay showed the reduced aiblity of the extra L chain species to neutralzie the target antigen. Wollacot (mAbs, 5(6): 925-935 (2013)

Jensen (US 2016/0347833) discloses an antibody pre-monomer impurity that includes two heavy chains (HC) and three light chains (LC) of which one is non-covalently attached (LC2HC2: LC). Based on the biophysical, spectroscopic and function characterists of LC2HC2:LC, the molecular structure is an antibody where an addition light chain has taken up the position normally occupied by a HC. The additional LC is bound via non-covalent itneractions to another LC, which in turn is covalently bound to HC. The C-terminal cysteine in the non-covalently attached LC is capped by forming a disulfide bond with either glutathione or cysteine. 

Challenges Posed in removing Extra Light chains 

Due to the similarities to the monomer species, the separation of H2L3 antibodeis generates a challenge during downstream purificaiton of monoclonal H2L2 cysteine-modified antibodies. (Liu US 2019/0112359). 

Purification Strategies for Intermediate HMW species:

CEX:

Liu (US 2019/0112359) discloses a purificaiton strategy to seperate triple light chain (H2L3) antibodies from double light chain (H2L2) antibodies using CEX. using optimzied POROS SC strong CEX, the amount of H2L3 antibody can be reduced from 11% to less than 1%. In one embodiment, an antibody composition is applied to the CEX so that H2L3 antibodies and H2L2 antibodies bind and eluting with a pH of about 3.8 to about 5 and collecting an H2L2 composition eluted form the resin. In some embodiments, no more than 0.5 of the antibodies in the H2L2 composition are H2L3 antibodies. 

HIC

Jensen (US 2016/0347833) disclosing a method for removing an antibody pre-monomer that includes two H chains and 3 L chains (LC2HC2:LC) which includes the step of purifying the monomeric antibodies using hydrophobic interaction chromatography (HIC). Antibody products after the method include anti-IL21 comprising at most 1% antibody pre-monomer aggregates. Elution from the HIC was done by decreasing the ammonium sulphage concetnration, such as by using alinear gradient of ammonium sulphage to 0 mol/kg. In one embodiment, the monomeric antibody componets elutes as the first antibody componentIn such examples, the antibody solution of interest may be obtained by collecting the fractions eluting prior to the fractions including the majority of pre-monomers and antibody dimers. Pre-monomer mayb be excluded form the antibody composition by deselecting subsequent eluate fractions. 

Wollacot (mAbs, 5(6): 925-935 (2013) disclsoes a robust purificaiton method to reduce an intermedaite HMW species using hydrophobic interaction chromatography (HIC) from about 3% to less than 0.5% with good step recovery. 

SEC:

Size-exclusion chromatogrpahy (SEC) is the most commonly used method to separate and quantify mAbs size variants. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. Elctron spray ionization -time of light mass spectrometry (ESI-TOF MS) microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and mAb-A with two extral light chains (2H4L). Teh C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cystein or another light chain. the Peak 1 fraction also shows two major peaks with calculated molecular weights of 175 kDa and 191 kDa which are the approximate sizes of 2H3L and 2H4L. (Liu, “Chracterization of monoclonal antibody size variants containing extra light chain” mAbs 5:1, 102-111, 2013). 

Companies:  ALV.  Brookhaven Instruments   Malvern. Wyatt  

Light scattering is one of the few absolute methods available for the determination of molecular mass and structure and is applicable over a broader range of Mw than any other methods. When size-exclusion chromtography (SEC) is used with on-line multi-angle laser light scattering (MALLS) detection, the weight, number and z-average values for obth mass and size may be obtained for most samples. Included in such measurements is the ability to caclulate both differential and accumulative distributions of the Mw and mean square radii. (Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004). 

Liquid chromatography and in particular high performance size exclusive chromatography, HPSEC, is a useful tool for the characterization of polymers. Typically, samples are prepared and injected into a chromatgoraphy where they are pumped thorugh columns that seaprate the molecules based on their hydrodynamic size, smaller molecuels tend to remain longer in the interstices of the columns and thus elute at later times than large molecules. Historically, the chromatograph with its separating columns and concentraiton sensitive detector was claibrated by using nearly monodisperse polymeric standards spanning a broad range of molecular weight, MW. The MWs present in the unkown sample were thus derived form a measurement of the time required for each separated fraction of sample to pass through the chromatograph relative to the corresponding times for the narrow calibration standards. With the advent of in-line light scattering detectors, the need to calibrate was no longer required, since a light scattering detector combined with a concentraiton detector permitted the determiantion of MWs and sizes, and their distributions, on an absoltue basis. (Shortt US Patent No: 5,528366). 

Dynamic Light scattering (DLS):

products: 

Wyatt Technology DyanPro Plate Readr: can perform high-throughput screening with dyanmic light scattering (HTS-DLS), It contains 96, 384 or 1536 well plates and performs temeprature scans of all samples simultaneously for a temperature range of 4-85C. A biomolecule’s staility is not an entirely intrinsci property, as it is influenced by the concetnraiton at which the protein is formulated and buffer composition. Protein stability must be measrued as a function of specific ion type, ionic strengh, pH and excipient profile for an optimal and successful formulation. (Wyatt Technology “The diffusion interaction parameter (Kd) as an indicator of colloidal and thermal stability” 

High throughput light scattering applications are routinely applied in the pharmaceutical industry including dynamic light scattering (DLS) and multi-angle light scattering (MALS). DLS is based on the measurement of intensity fluctuations of scatterid light due to Brownian motion. Autocorrelation curves can then be calculated form the intesity flucutations and fitted to dervie the translational diffusion coefficient D. In the prsence of interactions the moelcuels diffuse according to an effective diffusion coefficient D whcih deviates form the diffusion coefficient of the moelcuels D0. This effective diffusion coefficient D exhibits a linear relationship as a function of mAb concetnration, D=D1(1+KDc), with the slope KD equal to the diffusion itneraction parameter. A positive slope indicates repulsive intermoelcuels interactions, whereas a negative slope indicates attractive intermoelcular interaction.  (Lorenzen Chapter 14, “Mesauring self-assocaition of antibody lead candidates with dynamic light scattering” in “Therapeutic Antibodies Methods and Protocols, Methods in Molecular Biology, springer Protocols, pp. 1-347, 2022)

Various studies suggest that undesriable solution behaviors such as elevated viscosity are caused by short live transient “clusters” that result form attractive prtoein-protein interactions (PPI), The protein interaction parameter (Kd) calculated form low-protein-concetnraiton dyynamic light scattering (DLS) mreasurements is often used to relate PPI to solution viscosity at high protein concetnration. For example, the potein interaction parameter (Kd) calculated from low protein concenration DLS measruements were related to solution viscosity at high protein concentration. These empirical correlations were used to conclude that negative KD values, and therefore attractive PPI, at least qualitatively, predict the large increases in viscosity observed at high mAb concetnration. (Woldeyes, “Molecular-scale understanding of protein interactions and solution viscosities” dissertation, 2018). 

DLS is based on the fluctuations of the scatterd light intensity. It is robust and amendable to high throughput methodeoloy (about 100 samples/day per instrucement). Colloidal stability can be measrued by measuring the concetnration depednence of the prtoein collective diffusion coefficeint via DLS with protein-protein interactions being parameterized via the interaction parameter KD and comparing diffusion coefficient values measrued usying Taylor disperson analysis. (Sikka, sirat “Studying Protein-Protein interactions using dynamic light scatteringa nd Taylor Disperson Analysis” Dissertation. 

A negative KD value (protein-protien interation parameter obtainable from DLS) can be used to udnerstand the effects of NaCL and ArgHCL on protein-protein interactions. A negative KD value indicates an attractive prtein-protein interaction, while a positive KD value indicates a repulsive protein-protein interaction. (Alsabih “Towards an improved predictor for the colloidal stability of unfolded prtoeins through probing aggregation behaviour in solutions containing chemical denaturants” Thesis. 

With UV absorbance (multi-angle light scattering (MALS) +UV):

Ion Exchange:

Bailey (US Patent Application 16/579,220, published as US 2020/0018771 and US 13/850,664, published as US 10481164; see also US 17/495,963 published as US 2022/0026442) disclose a method for determing a stop collection point during IEX which includes monitoring the eluate with a laser light scattering detector and a UV absorbance detector and calculating a fraction LS/UV ratio for each fraction until he peak max LS/UV is determined, calculating a normalized LS/UV ratio for all subsequent fractions, wherein an increase in the normalized LS/UV ratio indicates an increase in the amount of impurities in the eluate pool and terminating the eltuion when the normalized LS/UV ratio reaches a predetermined value. In one embodiment, the normalized LS/UV ratio is calculated by dividing the fraction LS/UV ratio by the peak max LS/UV ratio. When a purificaiton process is operated in bind and elute or gradeint elution mode, the protein product is eluted first and aggregated species are present in the tail of the elution peak. Real time monitoring of the change in the ratio allows for a real time determiantion of the stop collection point based on the properties of the desired product at the time of elution. A monomeric protein peak, for instance, will have a constant ratio of LS/UV. Any deviation in that ratio signifies the product stream is contaminated with non-monomer impurities. The ration of the light scattering (LS) to absorbance concentration (for example, UV) is determiend real time. In one embodiment, eluate from a protein A column was diverted using a slipstream port to a AKTA UV-900 by pump and then passed to a Heleous II (Wyatt Technology) light scattering detector. The signals were processed in real time using a software program that cacluated the LS/UV ration of the elution preaks, “fraction LS/UV ratio” using the signals form the light scattering detector and UV absorbance. The purest fraction was the peak max. The stop collection point for the elution was set at a predetermiend normalized LS/UV ratio in the elution pool. An increase in high molecular eight cotaminants in the elution was reflected in an increase in the normalized LS/UV ratio. 

With Size-exclusion (LS +SEC):

The SEC-MALLS technique does not rely on relative Mw standrds for column calibration and yields absolute Mw estimates direclty from the angular dependence of scatterd light intesity as a funciton of concentration, as formulated by light scattering theory. Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004).

Harman “Charcterization and analysis of thermal denaturation of antibodies by size exlusion high-performance liquid chromatography with quadruple detection” (Analytical Biochemistry 325 (2004) 227-239) discloses that SEX coupled with online light scattering and UV visible spectroscopy provides a very powerful tool for studying protein size, shape and aggregation. 

Kalonia (US 2007/0178013) discloses appatuses and methods for simltaneous measurment of protein concentraiton and scatered light intensity.