human monoclonal antibody, nucleic acid molecule, expression cassette, cell, method for generating a

专利摘要:
human monoclonal antibody, nucleic acid molecule, expression cassette, cell, method for generating an isolated antibody, composition, use of an antibody, and, article of manufacture The present invention relates to anti-masp-2 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the adverse effects of masp-2 dependent complement activation. in one aspect, the invention provides an isolated human monoclonal antibody, or antigen-binding fragment thereof, which binds to masp-2, comprising: (i) a heavy chain variable region comprising the cdr-hi sequences , cdr-h2 and cdr-h3; and (ii) a light chain variable region comprising cdr-li, cdr-12 and cdr-13, wherein the cdr-h3 sequence of the heavy chain variable region comprises an amino acid sequence adjusted with factor b in formation of the c3 convertase from the additional alternative pathway (c3bbb). the alternative pathway c3 convertase is stabilized by binding properdin. properdin prolongs the half-life of the alternative pathway c3 convertase by six to ten times. the addition of c3b to the alternative pathway c3 convertase leads to the formation of the alternative pathway c5 convertase.
公开号:BR112013028429A2
申请号:R112013028429-3
申请日:2012-05-04
公开日:2020-07-21
发明作者:Thomas Dubler；Wayne R. Gombotz；James Brian Parent；Clark E. Tedford；Anita Kavlie；Urs Beat Hagemann；Herald Reiersen；Sergej Kiprijanov
申请人:Omeros Corporation；
IPC主号:

专利说明:

“HUMAN MONOCLONAL ANTIBODY, NUCLEIC ACID MOLECULE, EXPRESSION CASSETTE, CELL, METHOD FOR: GENERATING AN ISOLATED ANTIBODY, COMPOSITION, USE OF A: i ANTIBODY, AND, ARTICLE OF MANUFACTURE”
FIELD OF THE INVENTION The present invention relates to anti-MASP-2 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the adverse effects of MASP-2 dependent complement activation.
CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Interim Application No. 61/482,567 filed May 4, 2011, which is incorporated herein by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING 13 The sequence listing associated with this application is provided in text format rather than a paper copy and is hereby incorporated by reference in the specification. The name of the text file containing the sequence listing is MP 1 0115 PCT SequenceListingasFiled 20120504 ST25. The text file is 158 KB; was created on May 4, 2012; and is being submitted through the EFS-Web with the deposit of the descriptive report.
BACKGROUND The complement system provides an early mechanism of action to initiate, amplify, and coordinate the immune response to microbial infection and other acute insults (MK Liszewski and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by WE Paul, Raven Press, Ltd., New York) in humans and other vertebrates. Although complement activation provides a valuable first-line defense against potential pathogens, complement activities that . promote a protective immune response may also pose a threat to the host (KR Kalli, et al., Springer Semin. Immunopathol." 15: 417-431, 1994; BP Morgan, Eur. J. Clinical Investig. 24: 219- 228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Although indispensable for host defense, activated neutrophils are indiscriminate in their release of destructive enzymes and can cause organ damage. complement can cause the deposition of lytic complement components in neighboring host cells as well as microbial targets, which results in host cell lysis.
The complement system has also been implicated in the pathogenesis of numerous acute and chronic disease states, including: myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), reperfusion injury, septic shock, capillary leakage following thermal burns. , inflammation after cardiopulmonary bypass, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is not the cause but is one of several factors involved in the pathogenesis. Nevertheless, complement activation may be a major pathological mechanism and represents an effective point for clinical management in many of these disease states. The growing recognition of the importance of complement-mediated tissue injury in a variety of disease states emphasizes the need for drugs—effective complement inhibitors. So far, Eculizumab (Soliris&), an antibody against C5, is the only complement-targeting drug that has been approved for human use. To date, C5 is one of several effector molecules located “downstream” in the complement system, and blocking C5 does not inhibit the activation of the complement system. Therefore, an inhibitor of the initiation steps of complement activation would have significant advantages over a "downstream" complement inhibitor.
Í Currently, it is widely accepted that the complement system can be activated through three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually activated by a complex composed of host antibodies bound to a foreign particle (ie, an antigen) and thus requires prior exposure to an antigen for the generation of a specific antibody response. Since activation of the classical pathway depends on a prior adaptive immune response by the host, the classical pathway is part of the acquired immune system. On the contrary, both the lectin and the alternative pathways are independent of adaptive immunity and are part of the innate immune system. Activation of the complement system results in the sequential activation of serine protease zymogens. The first step in activating the classical pathway is the binding of a specific recognition molecule, Clq, to antigen-bound IgG and IgM complexes. Clq is associated with the serine protease proenzymes Clr and Cls as a complex called Cl. In the binding of Cl1q to an immune complex, autoproteolytic cleavage of the Arg-Ile site of Clr is followed by Clr-mediated cleavage and activation of Cls, which thereby acquire the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and, similarly, C2 is cleaved into C2a and C2b. C4b fragments are able to form covalent bonds with adjacent hydroxyl or amino groups and generate C3 convertase (C4b2a) through non-covalent interaction with the C2a fragment of activated C2. C3 — convertase (C4b2a) activates C3 by proteolytic cleavage into subcomponents C3a and C3b that lead to the generation of CS convertase (C4b2a3b), which, by cleavage of C5 leads to formation of membrane attack complex (C5b combined with C6, C7 , C8 and C9, also referred to as “MAC”) which can disrupt cell membranes leading to cell lysis. The activated forms of
C3 and C4 (C3b and C4b) are covalently deposited on target surfaces. foreign, which are recognized by complement receptors on multiple phagocytes.
. Regardless, the first step in activating the complement system via the lectin pathway is also binding of specific recognition molecules, which is followed by activation of associated serine protease proenzymes. However, instead of binding of immune complexes by Clq, the recognition molecules in the lectin pathway comprise a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin and type C lectin CL-11), collectively referred to as lectins. See J. Lu et al., Biochim. Biophys. Acta 1572: 387-400, 2002; Holmskov et al., Annu. Rev. Immunol. 21: 547-578 (2003 ); Teh et al., Immunology 101: 225-232 (2000 )). See also J. Luet et al., Biochim Biophys Acta 1572: 387-400 (2002 ); Holmskov et al, Annu Rev Immunol 21: 547-578 (2003 ); Teh et al. Immunology 101: 225-232 (2000); Hansen S. et al, J. Inmunol 185(10): 6096-6104 (2010 ). Ikeda et al. first demonstrated that, like Cla, MBL can activate the complement system by binding to mannan yeast coated erythrocytes in a C4-dependent manner (Ikeda et al., J. Biol. Chem. 262: 7451-7454, 1987). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxy groups oriented in the equatorial plane of the pyranose ring. Prominent ligands for MBL are thus D-mannose and N-acetyl-D- —glucosamine, while carbohydrates that do not fit this steric requirement have undetectable affinity for MBL (Weis, WL, et al., Nature 360: 127- 134, 1992). The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically in the single-digit millimolar range. MBL achieves tight, specific binding to glycan ligands by avidity, that is, by simultaneous interaction with β residues. multiple monosaccharides located in close proximity to each other (Lee, R.T., et al., Archiv. Biochem. Biophys. 299: 129-136, 1992). MBL recognizes: It is the carbohydrate patterns that usually decorate microorganisms such as bacteria, yeasts, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and last sugars that usually decorate “mature” complex glycoconjugates present in mammalian plasma and cell surface glycoproteins. This binding specificity is thought to promote recognition of “foreign” surfaces and help protect from “self-activation.” However, MBL does not bind with high affinity to high-mannose “precursor” glycan groups on N-linked glycoproteins and sequestered glycolipids in the endoplasmic reticulum and Golgi complex of mammalian cells (Maynard, Y., et al., J. Biol). Chem. 257: 3788-3794, 1982). Therefore, damaged cells are potential targets for activation of the lectin pathway through MBL binding.
Ficolins have a different type of lectin domain than MBL, called a fibrinogen-like domain. Ficolins bind sugar residues in a Cat+-independent manner. In humans, three types of ficolins (L-ficolin, M-ficolin and H-ficolin) have been identified. The two serum Ficolins, L-ficolin and H-ficolin, have in common a specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds N-acetyl-D-galactosamine. The difference in the sugar specificity of L-ficolin, H-ficolin, CL-11, and MBL means that the different lectins can be complementary and target different, albeit overlapping, glycoconjugates. This concept is supported by the recent report that, of the known lectins in the lectin pathway, the only L-ficolin specifically binds to lipoteichoic acid, a cell wall glycoconjugate found in all Gram-positive bacteria (Lynch, N. ] ., et al., J.
Immunol. 172: 1198-1202, (2004)). Collectins (i.e. MBL) and Ficolins. do not carry any significant similarity in amino acid sequence. However, the two groups of proteins have similar domain organizations and, like Clq, come together in oligomeric structures, which maximize the possibility of multi-site binding.
Serum concentrations of MBL are highly variable in healthy populations and this is genetically controlled by polymorphisms/mutations in both the promoter and coding regions of the MBL gene. As an acute phase protein, MBL expression is still upregulated during inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Therefore, the L-phycolin branch of the lectin pathway is potentially comparable to the MBL branch in strength. MBL and Ficolins can also function as opsonins, which allow phagocytes to target surfaces decorated with MBL and Ficolin (see Jack et al., J Leukoc Biol., 77(3): 328-36 (2004), Matsushita and Fujita, Immunobiology , 205(4-5): 490-7 (2002), Aoyagi et al., J Immunol, 174(1): 418-25 (2005). This opsonization requires the interaction of these proteins with phagocytic receptors (Kuhlman, M. , et al., J. Exp. Med. 169: 1733, 1989; Matsushita, M., et al., J. Biol. Chem. 271: 2448-54, 1996), the identity of which has not been established.
Human MBL forms a specific, high-affinity interaction through its collagen-like domain with unique CIr/Cls-like serine proteases, called MBL-associated serine proteases (MASPs). So far, three MASPs have been described. First, a single —"MASP" enzyme was identified and as the enzyme responsible for initiating the complement cascade (i.e., cleavage of C2 and C4) (Matsushita M, and Fujita T., J Exp Med 176(6): 1497 -1502 (1992); Ji, YH, et al., J. Immunol. 150: 571-578, 1993 ). It was subsequently determined that MASP activity was, in fact, a mixture of two proteases: MASP-1 and MASP-2 (Thiel, S., et al., Nature 386: 506-510, 1997). However, the MBL-MASP-2 -complex alone has been shown to be sufficient for β complement activation (Vorup-Jensen, T., et al., J. Imnmunol. 165: 2093-2100, 2000). In addition . i unique MASP-2 cleaved C2 and C4 at high rates ( Ambrus, G., et al., J. Inmunol. 170: 1374-1382, 2003 ). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4b2a. This is a significant difference from the C1 complex of the classical pathway, where the coordinated action of two specific serine proteases (Clr and Cls) leads to the activation of the complement system. In addition, a third novel protease, MASP-3, has been isolated (Dahl, MR, et al., Immunity 15: 127-35, 2001). MASP-1 and MASP-3 are alternatively splice products of the same gene. MASPs share identical domain organizations with those of Clr and Cls, the enzymatic components of the CI complex (Sim, R.B., et al., Biochem. Soc. Trans. 28: 545, 2000). These domains include a sea urchin CIr/CIs/VEGF N-terminal domain/bone morphogenic protein (CUB), an epidermal growth factor-like domain, a second CUB domain, a tandem of complement, and a serine protease domain. As with C1 proteases, activation of MASP-2 occurs through cleavage of an —Arg-Hlle bond adjacent to the serine protease domain, which splits the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain. . Recently, a genetically determined deficiency of MASP-2 has been described (Stengaard-Pedersen, K., et al., New Eng. J. Med. 349: 554-560, 2003). The single nucleotide mutation leads to an Asp-—Glynodomain CUB 1 switch and renders MASP-2 incapable of binding to MBL.
MBL can also be associated with an alternatively split form of MASP-2, known as the 19 kDa MBL-associated protein (MApl19) (Stover, CM, J. Inmunol. 162: 3481-90, 1999) or small MBL-associated protein ( sMAP) Takahashi, M., et al., Int.
Immunol. 11: 859-863, 1999) which lacks the catalytic activity of MASP2. , MAPp19 comprises the first two domains of MASP-2, followed by P : an extra sequence of four unique amino acids.
The MASP-I and . MASP-2 are located on human chromosomes 3 and 1, respectively — (Schwaeble, W., et al., Immunobiology 205: 455-466, (2002)). Several lines of evidence suggest that different MBL-MASP complexes exist and a large fraction of the MASPs in serum are not complexed with MBL (Thiel, S., et al., J.
Immuno 165: 878-887, 2000). Ficolin both H and L bind to all MASPs and activate the lectin "complement" pathway, like MBL (Dahl, M.
R., et al., Immunity 15: 127-35, 2001; Matsushita, M., et al., J.
Immuno 168: 3502-3506, 2002). Both the lectin and classical pathways form a common C3 convertase (C4b2a) and the two pathways converge at this step.
The lectin pathway is widely considered to have a major role in host defense against infection in the naive host.
Strong evidence for the involvement of MBL in host defense results from the analysis of patients with decreased serum levels of functional MBL (Kilpatrick, D.
C., Biochim.
Biophys.
Acta 1572: 401-413, 2002). Such patients demonstrate susceptibility to bacterial and fungal infections—recurrent.
These symptoms are usually evident in youth, during an evident window of vulnerability as maternally derived antibody titers decline, but before a full repertoire of antibody responses develops.
This syndrome often results from mutations at various sites in the collagen portion of MBL, which interfere with the proper formation of MBL oligomers.
However, since MBL can function as a complement-independent opsonin, it is not known to what degree the increased susceptibility to infection is due to impaired complement activation.
Unlike the classical and lectin pathways, no alternative pathway primers were found to fulfill the recognition functions - which Clq and lectins perform in the other two pathways. It is currently widely accepted that the alternative pathway spontaneously undergoes a >i low level of metabolization activation, which can be easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virally infected cells, or damaged tissue) that lack the elements appropriate molecular molecules that keep spontaneous complement activation in check. There are four plasma proteins directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin.
Although there is extensive evidence implicating both classical and alternative complement pathways in the pathogenesis of non-infectious human diseases, the role of the lectin pathway is just beginning to be evaluated. Recent studies provide evidence that activation of the lectin pathway may be responsible for complement activation and related inflammation in ischemia/reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show C3 deposition on exposure to human serum (Collard, CD,, et al., Am. J. Pathol. 156: 1549-1556, 2000) . Furthermore, treatment of human sera with blocking anti-MBL monoclonal antibodies = —inhibited MBL binding and complement activation. These findings were extended to a mouse model of myocardial ischemia-reperfusion in which mice treated with a blocking antibody directed against mouse MBL showed significantly less myocardial damage in coronary artery occlusion than mice treated with a control antibody ( Jordan, J.E, et al., Circulation 104: 1413-1418, 2001 ). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unclear; a recent study suggests that activation of the lectin pathway following oxidative stress may be mediated by binding of MBL to vascular endothelial cytokeratins rather than glycoconjugates (Collard, C.
D., et al., Am. J. Pathol. 159: 1045-1054, 2001). Other studies have . The classical and alternative pathways are implicated in the pathogenesis of ischemia/i-reperfusion injury and the role of the lectin pathway in this disease remains controversial (Riedermann, N.C., et al., Am. J. Pathol. 162: 363-367, 2003).
Ss A recent study has shown that MASP-I (and possibly also MASP-3) is required to convert the alternative pathway activating enzyme Factor D from its zymogen form to its enzymatically active form (See Takahashi M. et al. al., J Exp Med 207(1): 29-37 (2010 )). The physiological importance of this process is emphasized by the 10 — lack of functional activity of the alternative pathway in the plasma of MASP-1/3-deficient mice. Proteolytic generation of C3b from native C3 is required for the alternative pathway to work. Since the alternative pathway C3 convertase (C3bBb) contains C3b as an essential subunit, the question concerning the origin of the first C3b via the alternative pathway has presented an intricate problem and has stimulated considerable research.
C3 belongs to a family of proteins (along with C4 and a-2 macroglobulin) containing a rare post-translational modification known as a thioester bond. The thioester group is composed of a glutamine whose terminal carbonyl group forms a thioester bond—covalently with the sulfhydryl group of a cysteine three amino acids down. This bond is unstable and the electrophilic glutamyl thioester can react with nucleophilic moieties such as hydroxyl or amino groups and thus form a covalent bond with other molecules. The thioester bond is reasonably stable when sequestered within a hydrophobic pocket of (C3 intact. However, proteolytic cleavage from C3 to C3a and C3b results in exposure of the highly reactive thioester bond at C3b and following nucleophilic attack by the thioester moieties). In addition to its well-documented role in covalently binding C3b to complement targets, the C3 thioester is also thought to have a central role in activating the pathway. According to the widely accepted “tick-over theory”, the alternative pathway is initiated by the generation of a fluid-phase convertase, iC3Bb, which is formed from C3 with hydrolyzed thioester (iC3; C3(H5O)) and factor B (Lachmann, PJ et al., Springer Semin. ImmunoPathol. 7: 143-162, 1984) C3(H3zO) as C3b is generated from native C3 by a slow spontaneous hydrolysis of the internal thioester in the protein (Pangburn, MK, et al., J. Exp Med. 154: 856-867, 1981). Through C3(H,O)Bb convertase activity, C3b molecules are deposited on the target surface, thereby initiating the alternative pathway.
Very little is known about alternative pathway activation initiators. Activators are considered to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only common trait for these activators is the presence of carbohydrate, but the complexity and variety of carbohydrate structures has made it difficult to establish the shared molecular determinants that are recognized. It is widely accepted that activation of the alternative pathway is controlled through the fine balance between inhibitor regulatory components of this pathway, such as Factor H, Factor 1, DAF, CRI and properdin, which is the only positive regulator of the alternative pathway (See Schwaeble WJ and Reid KB, Immunol Today 20(1): 17-21 (1999)).
In addition to the apparent dysregulated activation mechanism described above, the alternative pathway may also provide an amplification loop — powerful for the C3 convertase (C4b2a) of the lectin/classical pathway as any C3b generated can participate with factor B in C3 formation. convertase (C3bBb) from the additional alternative pathway. The alternative pathway C3 convertase is stabilized by binding properdin. Properdin prolongs the alternative pathway C3 convertase half-life by six to ten-fold. the addition of
C3b to the alternative pathway C3 convertase leads to the formation of the alternative pathway C5 convertase. All three pathways (i.e. classical, lectin and alternative) were found to converge to C5, which is cleaved to form products with 'multiple pro-inflammatory effects. The converged pathway has been referred to as the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle and vascular tone, as well as vascular permeability. It is also a powerful chemotaxin and activator of both neutrophils and monocytes. C5a-mediated cellular activation can—significantly amplify inflammatory responses by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species. Cleavage of C5 leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic MAC deposition may play an important role in inflammation in addition to its role as a pore-forming lytic complex. In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects.
SUMMARY This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. In one aspect, the invention provides an isolated human monoclonal antibody, or antigen-binding fragment thereof, which binds to human -MASP-2, comprising: (i) a heavy chain variable region: comprising the sequences of CDR-H1, CDR-H2 and CDR-H3; and (ii) a >: light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the heavy chain variable region sequence of CDR-H3 comprises an amino acid sequence shown as SEQ ID NO : 38 or SEQ ID NO: 90, and conservative sequence modifications thereof, wherein the CDR-L3 light chain variable region sequence comprises an amino acid sequence shown as SEQ ID NO: 51 or SEQ ID - NO: 94 , and conservative sequence modifications thereof, and wherein the isolated antibody inhibits MASP-2 dependent complement activation. In another aspect, the present invention provides a human antibody which binds to human MASP-2, wherein the antibody comprises: 1) a) a heavy chain variable region comprising: i) a CDR-H1 heavy chain which comprises amino acid sequence 31 to 35 of SEQID NO: 21; and ii) a CDR-H2 heavy chain comprising amino acid sequence 50 to 65 of SEQ ID NO: 21; and iii) a CDR-H3 heavy chain comprising amino acid sequence 95 to 102 of SEQ ID NO: 21; and b) a light chain variable region comprising: i) a light chain CDR-LI comprising amino acid sequence 24 to 34 of SEQ ID NO: 25 or SEQ ID NO: 27; and ii) a CDR-L2 light chain comprising amino acid sequence 50 to 56 of SEQ ID NO: 25 or SEQ ID NO: 27; and iii) a CDR-L3 light chain comprising amino acid sequence 89 to 97 of SEQ ID NO: 25 or SEQ ID NO: 27; or II) a variant thereof that is otherwise identical to said variable domains, except up to a combined total of 10 amino acid substitutions within said CDR regions of said heavy chain variable region and up to a combined total of 10 amino acid substitutions. amino acid within said CDR regions of said light chain variable region, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation.
- In another aspect, the present invention provides an isolated human monoclonal antibody, or antigen-binding fragment. ; thereof, which binds human MASP-2, wherein the antibody comprises: 1) a) a heavy chain variable region comprising: i) a CDR-HI heavy chain comprising amino acid sequence 31 to 35 of SEQ ID NO: 20; and ii) a CDR-H2 heavy chain comprising amino acid sequence 50 to 65 of SEQ ID NO: 20; and iii) a CDR-H3 heavy chain comprising amino acid sequence 95 to 102 of SEQ ID NO: 18 or SEQID NO: 20; and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising amino acid sequence 24 to 34 of SEQ ID NO: 22 or SEQ ID NO: 24; and ii) a CDR-L2 light chain comprising amino acid sequence 50 to 56 of SEQ ID NO: 22 or SEQ ID NO: 24; and iii) a CDR-L3 light chain comprising amino acid sequence 89 to 97 of SEQ ID NO: 22 or SEQ ID NO: 24; or II) a variant thereof which is otherwise identical to said variable domains, except up to a combined total of 10 amino acid substitutions within said CDR regions of said heavy chain and up to a combined total of 10 amino acid substitutions within said of said -CDR regions of said light chain variable region, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation.
In another aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, which binds to human MASP-2, which comprises a heavy chain variable region comprising any of the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 21.
In another aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, which binds human MASP-2, which comprises a variable region. of the light chain comprising one of the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ - IDNO: 2X.
In another aspect, the present invention provides nucleic acid molecules encoding the amino acid sequences of the anti-MASP-2 antibodies, or fragments thereof, of the present invention, such as those shown in TABLE 2.
In another aspect, the present invention provides a cell "which comprises at least one of the nucleic acid molecules encoding the amino acid sequences of the anti-MASP-2 antibodies, or fragments thereof, of the present invention, such as those shown in TABLE 2.
In another aspect, the invention provides a method for — generating an isolated MASP-2 antibody which comprises culturing cells that comprise at least one of the nucleic acid molecules encoding the amino acid sequences of the anti-MASP-2 antibodies of the present invention under conditions permitting expression of the nucleic acid molecules encoding the anti-MASP-2 antibody and isolating said anti-MASP-2 antibody.
In another aspect, the invention provides a fully isolated human monoclonal antibody or antigen-binding fragment thereof that dissociates from human MASP-2 with a KD of 10 nM or less as determined by surface plasmon resonance and inhibits activation. of C4 on a mannan coated substrate with an ICs 9 of 10 NM or less in 1% serum. In some embodiments, said antibody or antigen-binding fragment thereof specifically recognizes at least part of an epitope recognized by a reference antibody, wherein said reference antibody comprises a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable - region as shown in SEQ ID NO: 24. In another aspect, the present invention provides: compositions comprising the fully human monoclonal anti-MASP-2 antibodies of the invention and an excipient pharmaceutically acceptable.
In another aspect, the present invention provides methods of inhibiting MASP-2 dependent complement activation in a human subject which comprises administering a human monoclonal antibody of the invention in an amount sufficient to inhibit MASP-2 dependent complement activation in the human subject. said human individual.
In another aspect, the present invention provides an article of manufacture comprising a unit dose of human monoclonal MASP-2 antibody of the invention suitable for therapeutic administration to a human subject, wherein the unit dose is in the range of 1 mg to 1000 mg.
DESCRIPTION OF THE DRAWINGS The foregoing aspects and many of the appended advantages of this invention will become more readily appreciated as it becomes — better understood by reference to the detailed description which follows, when considered in conjunction with the accompanying drawings, wherein: A FIGURE IA is a diagram illustrating the genomic structure of human MASP-2; FIGURE 1B is a diagram illustrating the domain structure of the human MASP-2 protein; FIGURE 2 graphically illustrates the results of an ELISA assay performed on selected polyclonal populations from a scFcv phage library panned against various MASP-2 antigens, as described in Example 2; FIGURE 3A and 3B show test results of 45 candidate scFv clones for functional activity in the complement assay. as described in Example 3; FIGURE 4 graphically illustrates the results of an experiment that was performed to compare C3c levels in the three sera 5 (human, rat and NHP) as described in Example 4;
FIGURE 5A is an amino acid sequence alignment of the heavy chain region (residues 1 to 120) of the most active clones reveals two distinct groups belonging to the VH2 and VH6 gene family, respectively, as described in Example 4;
FIGURE 5B is an amino acid sequence alignment of scFv clones 17D20, 17N16, 18L1I6 and 4D9 as described in Example 4;
FIGURE 6 graphically illustrates the inhibitory activities of IgG4 converted precursor clone preparations in a C3b deposition assay using 90% human plasma as described in Example 5;
FIGURE 7A graphically illustrates the results of the ELISA assay on mother clone 17NI6 versus daughter clones titrated on huMASP 2A, as described in Example 6;
FIGURE 7B graphically illustrates the results of the ELISA assay on the mother clone 17D20 versus daughter clones titered on huMASP2A, as described in Example 6;
FIGURE 8 is a protein sequence alignment of the mother clone 17N16 and the daughter clone 17N9 showing that the light chains (starting with SYE) have 17 amino acid residues that differ between the two clones, as described in Example 6;
FIGURE 9 is a protein sequence alignment of the CDR-H3 region of the sequences from Clones 435, 459, and 490 resulting from mutagenesis compared to the parent clone 7D20, as described in
Example 7;
. FIGURE 10A is a protein sequence alignment of the CDR3 region of the mother clone 17D20 with the scrambled chain clone F 17D20md21N11 and the mutagenesis clone CDR-H3 clone 435 shown in Fig.
S—FIGURE 9 combined with the VL of 17D20md21N11 (VH35-VL21INI1]), as described in Example 7;
FIGURE 10B is a protein sequence alignment of the VL and VH regions of the mother clone 17D20 and the daughter clone 17D20md21NI11, as described in Example 7;
FIGURE 11A graphically illustrates the results of the C3b deposition assay performed for the daughter clone isotype variants (MoAbt1-3), derived from the 17N16 mother clone of the human monoclonal anti-MASP-2 antibody, as described in Example 8;
FIGURE 11B graphically illustrates the results of the C3b deposition assay performed for the daughter clone isotype variants (MoAbit4-6), derived from the 17D20 mother clone of the human monoclonal anti-MASP-2 antibody, as described in Example 8;
FIGURES 12A and 12B graphically illustrate the testing of mother clones and MoAbt1-6 in a 95% C3b deposition assay.
— serum, as described in Example 8;
FIGURE 13 graphically illustrates the inhibition of C4b deposition in 95% normal human serum as described in Example 8;
FIGURE 14 graphically illustrates the inhibition of C3b deposition in 95% African green monkey serum as described in
Example8;
FIGURE 15 graphically illustrates the inhibition of the C4 cleavage activity of the pre-assembled MBL-MASP2 complex by MoAbi2-6, as described in Example 8;
FIGURE 16 graphically illustrates the preferential binding of
MoAbt%6 to human MASP2 when compared to Cls, as described in: Example 8; : FIGURE 17 graphically illustrates that the lectin pathway was completely inhibited following intravenous administration of anti-human MoAb*XOMS646 in African Green monkeys, as described in Example 10; FIGURE 18A is a Kaplan-Meier survival plot showing percent survival with time after radiation exposure of 7.0 Gy in control mice and in — mice treated with anti-murine MASP-2 antibody (mAbM11) or anti-human MASP-2 antibody (MALDOMS646) as described in Example 11; FIGURE 18B is a Kaplan-Meier survival plot showing percent survival with time after exposure to 6.5 Gy of radiation in control mice and in mice treated with anti-murine MASP-2 antibody (MAbMI]1) or anti-human MASP-2 antibody (MALDOMS646), as described in Example 11; FIGURE 18C is a Kaplan-Meier survival plot showing percent survival with time after exposure to 8.0 Gy of radiation in control mice and in mice treated with human anti-MASP-2 antibody (mAbOMS646 ), as described in Example 11; FIGURE 19 graphically illustrates the results of surface plasmon resonance (Biacore) analysis on anti-MASP-2 antibody OMS646 (response (binding) units versus time in seconds), which shows that immobilized OMS646 binds to MASP- 2 recombinant with a K-dissociation rate of about 1 to 3 x 10º S" and a K-dissociation rate of about 1.6 to 3 x 10º MS", as described in Example 12;
FIGURE 20 graphically illustrates the results of an ELISA assay to determine the binding affinity of anti-MASP-2 antibody i OMS646 to immobilized human MASP-2, which shows that OMS646 binds to immobilized recombinant human MASP-2 with a KD of — approximately 100 µM, as described in Example 12;
FIGURE 21A graphically illustrates the level of C4 activation on a mannan-coated surface in the presence or absence of anti-MASP-2 antibody (OMS646), which demonstrates that OMS646 inhibits C4 activation on a mannan-coated surface with an ICs , in
— approximately 0.5 nM in 1% human serum, as described in Example 12;
FIGURE 21B graphically illustrates the level of C4 activation on an IgG coated surface in the presence or absence of anti-MASP-2 antibody (OMS646), which shows that OMS646 does not inhibit classical pathway-dependent activation of the C4 component of complement, as described in Example 12;
FIGURE 22A graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions, demonstrating that OMS646 inhibits lectin-mediated MAC deposition with an ICsºK value. approximately 1 nM, as described in Example 12;
FIGURE 22B graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway-specific assay conditions, demonstrating that OMS646
—does not inhibit classical pathway-mediated MAC deposition as described in Example 12;
FIGURE 22C graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions, demonstrating that
OMS646 does not inhibit alternative pathway-mediated MAC deposition, as - described in Example 12; , FIGURE 23A graphically illustrates the deposition level of ; : C3 in the presence or absence of anti-MASP-2 antibody (OMS646) in a — range of concentrations in 90% of human serum under specific conditions of the lectin pathway, demonstrating that OMS646 blocks the deposition of C3 under physiological conditions, as described in Example 12; FIGURE 23B graphically illustrates the level of C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) at a —range of concentrations in 90% of human serum under specific conditions of the lectin pathway, demonstrating that OMS646 blocks deposition of C4 under physiological conditions, as described in Example 12; FIGURE 24A graphically illustrates the level of C4 deposition in the absence or presence of anti-MASP-2 antibody (OMS646) in 90% Cynomolgus monkey serum under specific conditions of the lectin pathway, demonstrating that OMS646 inhibits C4 deposition in the lectin pathway. Cynomolgus monkey Serum lectin pathway in a dose-responsive manner with IC values in the range of 30 to 50 nM as described in Example 12; and FIGURE 24B graphically illustrates the level of —C4 deposition in the absence or presence of anti-MASP-2 antibody (OMS646) in 90% African Green monkey serum under specific conditions of the lectin pathway, demonstrating that OMS646 inhibits the deposition of C4 in the lectin pathway in African Green monkey serum in a dose-responsive manner with ICs9 values in the range of 15 to 30 nM, as described in —Example 2.
DESCRIPTION OF SEQUENCE LISTING SEQ ID NO: 1 human MASP-2 cDNA SEQ ID NO: 2 human MASP-2 protein (with leader) SEQ ID NO: 3 human MASP-2 protein (mature)
SEQ ID NO: 4 rat MASP-2 cDNA - SEQ ID NO: 5 rat MASP-2 protein (with leader) SEQ ID NO: 6 rat MASP-2 protein (mature) z ANTIGENS (in reference) to mature human MASP-2 protein) SEQ ID NO: 7 CUBI domain of human MASP-2 (aa 1-121) SEQ ID NO: 8 CUBI/EGF domains of human MASP-2 (aa 1-166) SEQ ID NO : 9 human MASP-2 CUBI/EGF/CUBII domains (aa 1-277) SEQ ID NO: 10 human MASP-2 EGF domain (aa 122-166) SEQ ID NO: 11 CCPI/CCPII/(SP from Human MASP-2 (aa 278-671) SEQ ID NO: 12 CCPI/CCPII domains of human MASP-2 (aa 278-429) SEQ ID NO: 13 CCPI domain of human MASP-2 (aa 278-347) SEQ ID NO: 14 human MASP-2 CCPII/SP domain (aa348-671) SEQ ID NO: 15 human MASP-2 CCPII domain (aa 348-429) SEQ ID NO: 16 human MASP-2 SP domain (aa 429 -671) SEQ ID NO: 17: serine protease inactivated mutant (aa — 610-625 with mutated Ser 618) ANTI-MASP-2 MONOCLONAL ANTIBODIES VH chains SEQ ID NO: 18 polypeptide 17D20mc pe chain variable region output (VH) SEQ ID NO: 19 DNA encoding 17D20 of the heavy chain (VH) variable region of 35VH21N11VL (OMS646) (no signal peptide). SEQ ID NO: 20 35VH21N11VL heavy chain (VH) variable region 17D20 polypeptide (OMS646): SEQ ID NO: 21 VL heavy chain (VH) heavy chain 17N116mc polypeptide ANTI-MASP-2 MONOCLONAL ANTIBODIES SEQ ID NO: 22 Light chain variable region (VL) 17D20mc polypeptide SEQ ID NO: 23 DNA encoding 21NI1VL light chain (VL) variable region 17D20 (OMS644) (no signal peptide) SEQ ID NO: 24 17D20 polypeptide 21N11VL Light Chain Variable Region (VL) (OMS644) SEQ ID NO: 25 Light Chain Variable Region (VL) 17N16mc Polypeptide SEQ ID NO: 26 DNA Encoding 17N9 Light Chain Variable Region (VL) Region (VL) 17N9 (OMS641) ) (no signal peptide) SEQ ID NO: 27 del7N9 light chain variable region (VL) 17N16 polypeptide (OMS641)
ANTIBODIES HEAVY CHAIN CDRS —"MONOCLONAL ANTI-MASP-2 SEQ ID NOS: 28-31 CDR-H1 SEQ ID NOS: 32-35 CDR-H2 SEQ ID NOS: 36-40 CDR-H3
LIGHT CHAIN CDRS OF ANTI-MASP-2 ANTIBODIES —MONOCLONAL SEQ ID NOS: 41-45 CDR-L1 SEQ ID NOS: 46-50 CDR-L2 SEQ ID NOS: 51-54 CDR-L3 MASP-2 antibody sequences
SEQ ID NO: 55: Full-length ScFv polypeptide. 17D20 parent clone: SEQ ID NO: 56: ScFv full-length 18L16 polypeptide parent clone SEQ ID NO: 57: ScFv full-length 4D9 polypeptide parent clone SEQ ID NO: 58: ScFv full-length 17120 parent clone SEQ ID NO: 59: 17N16 full-length polypeptide from —ScFv clone parent SEQ ID NO: 60: 3F22 full-length polypeptide from ScFv clone parent SEQ ID NO: 61: 9P13 full-length polypeptide from ScFv clone parent SEQ ID NO: 62 : DNA encoding the wild-type IZG4 heavy chain constant region SEQ ID NO: 63: Wild-type IgG4 heavy chain constant region polypeptide SEQ ID NO: 64 DNA encoding the IZG4 heavy chain constant region with 5228P mutant SEQ ID NO: 65: IgG4 heavy chain constant region with 5228P polypeptide mutant SEQ ID NO: 66: full-length polypeptide 17N16m dI7N9 ScFv daughter clone SEQ ID NO: 67: full-length polypeptide 17D20m d21N11 ScFv daughter clone SEQ ID NO: 68: ScFv clone daughter 17D20m d3521INI1 full-length polypeptide SEQ ID NO: 69: DNA encoding wild-type IgG2 heavy chain constant region. SEQ ID NO: 70: Wild-type IgG2 heavy chain constant region polypeptide SEQ ID NO: 71: 17N16m light chain gene sequence — dI7N9 (with signal peptide encoded by nt 1-57)) SEQ ID NO: 72: 17N16m dI7N9 light chain protein sequence (with aa signal peptide 1-19) SEQ ID NO: 73: 17N16m dl7N9 IgG2 heavy chain gene sequence (with signal peptide encoded by nt 1-57 ) SEQ ID NO: 74: 17N16m dI7N9 IgG2 heavy chain protein sequence (with aa 1-19 signal peptide) SEQ ID NO: 75: 17N16m dlI7N9 I2G4 heavy chain gene sequence (with signal peptide encoded by the nt 1-57) SEQ ID NO: 76: IgG417N116m dI7N9 heavy chain protein sequence (with aa signal peptide 1-19) SEQ ID NO: 77: IZ2G4 mutated heavy chain gene sequence 17N16m dl7N9 (with peptide signal encoded by nt 1-57) SEQ ID NO: 78: IZ2G4 17N17m dl7N9 mutated heavy chain protein sequence (with signal peptide aa 1 -19) SEQ ID NO: 79: 17D20 3521N11 light chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO: 80: 17D20 3521N11 light chain protein sequence (with aa 1 signal peptide -19) SEQ ID NO: 81: IgG2 heavy chain gene sequence 17D20 3521N11 (with signal peptide encoded by nt 1-57) SEQ ID NO: 82: IgG2 heavy chain protein sequence 17D20 3521INI11 (with peptide aa 1-19) SEQ ID NO: 83: IgG4 heavy chain gene sequence 17D20 3521N11 (with signal peptide encoded by nt 1-57)
SEQ ID NO: 84: IZG4 17D20 3521N11 heavy chain protein sequence (with aa 1-19 signal peptide) IS SEQ ID NO: 85: IZG4 17D20 3521N11 mutated heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO: 86: Mutated IgG4 heavy chain protein sequence 17D20 3521N11 (with signal peptide as 1-19) SEQ ID NO: 87: DNA encoding full-length polypeptide from daughter clone of ScFv 17N16m dl7N9 (no signal peptide) SEQ ID NO: 88: DNA encoding full-length polypeptide — natural from ScFv 17D20m d21N11 daughter clone (no signal peptide) SEQ ID NO: 89: DNA encoding full-length polypeptide from ScFv daughter clone 17D20m d3521N11 (no signal peptide) SEQ ID NO: 90: CDR-H3 of consensus heavy chain of 17D20m and d3521N11 SEQ ID NO: 91: CDR-L1 of consensus light chain of 17D20m and d3521N11 SEQ ID NO: 92: 17N16m consensus light chain CDR-L1 and d17N9 SEQ ID NO: 93: —17D consensus light chain CDR-L2 20m, d3521INI1,17N16m and d1I7N9 SEQ ID NO: 94: 17N16m and d17N9 consensus light chain CDR-L3
DETAILED DESCRIPTION The present invention provides fully human antibodies—which bind to human MASP2 and inhibit lectin-mediated complement activation while leaving the classical (Clq-dependent) pathway component of the immune system intact. Human anti-MASP-2 antibodies were identified by screening a phage display library as described in Examples 2-9. As described in Examples 10-12, high affinity anti-MASP-2 antibodies were identified with the ability to . inhibit lectin-mediated complement activation, as demonstrated in both in vitro and in vivo assays. The variable heavy and light chain antibody fragments were isolated in either a scFy S-format or a full-size IgG format. Human anti-MASP-2 antibodies are useful for inhibiting cell injury associated with activation of the lectin-mediated complement pathway while leaving the classical (Clq-dependent) pathway component of the immune system intact.
1. DEFINITIONS Unless specifically defined herein, all terms used herein have the same meaning as would be understood by those of ordinary skill in the art of the present invention. The following definitions are provided in order to provide clarity with respect to terms as they are used in the specification and claims to describe the present invention.
As used herein, the term "MASP-2-dependent complement activation" comprises the MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca) leading to the formation of the lectin pathway. lectin C3 convertase C4b2a and the accumulation of the C3 cleavage product, C3b subsequent to C5 convertase C4b2a(C3b)n.
As used herein, the term "alternative pathway" refers to activation of complement that is activated, for example, by zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram negative outer membranes, and erythrocytes from rabbit, as well as very pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which has traditionally been considered to arise from the spontaneous proteolytic generation of C3b from the complement factor C3.
As used herein, the term "lectin pathway" refers to activation. complement that occurs through specific binding of serum and non-serum carbohydrate binding proteins including mannan-binding lectin (MBL), CL-11 and the ficolins (H-ficolin, M-ficolin, or L-ficolin ).
As used herein, the term "classical pathway" refers to complement activation that is activated by an antibody bound to a foreign particle and requires binding of the recognition molecule C11q.
As used herein, the term "MASP-2 inhibitory antibody" refers to any anti-MASP-2 antibody or fragment thereof that binds MASP-2 that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2 activation. of complement. MASP-2 inhibitor antibodies useful in the method of the invention can reduce MASP-2 dependent complement activation by more than 20%, such as more than 30%, or greater than 40%, or greater than 50 %, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%. As used herein, the term "antibody that blocks MASP-2" refers to MASP-2 inhibitory antibodies that reduce MASP-2 dependent complement activation by greater than 90%, such as greater than 95%, or greater than 98% (i.e. resulting in activation of — MASP-2 complement of only 10%, such as only 9%, or only 8%, or only 7%, or only 6%, such as only 5% or less, or just 4%, or just 4%, or just 3%, or just 2%, or just 1%). The terms "antibody" and "immunoglobulin" are used interchangeably herein. These terms are well understood by those in the field, and refer to a protein consisting of one or more polypeptides that specifically binds an antigen. One form of the antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having a light and a heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the . constants are responsible for antibody effector functions. As used herein, the term "antibody" encompasses antibodies and: : antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), or from a hybridoma, phage selection, recombinant expression or transgenic animals (or other methods of producing antibodies or antibody fragments), which specifically bind to MASP-2 polypeptides or portions thereof. The term "antibody" is not intended to be limited with respect to the source of the antibody or the manner in which it is manufactured (eg, by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (eg —Dbispecific antibodies); humanized antibodies; murine antibodies; chimeric monoclonal antibodies, mouse-human, mouse-primate, primate-human; and anti-idiotypic antibodies, and can be any intact molecule or fragment thereof. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab, F(ab'), Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody moiety with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other — modified configuration of the immunoglobulin molecule comprising a binding site of antigen or fragment (antigen binding site) of the required specificity.
As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chain that binds to MASP-2. human. In this regard, an antigen-binding fragment of the antibodies described herein may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence shown herein of antibodies that bind to MASP- two. — An antigen-binding fragment of the MASP-2 specific antibodies described herein is capable of binding to MASP-2. In certain embodiments, an antigen-binding fragment, or an antibody comprising an antigen-binding fragment, mediates inhibition of MASP-2-dependent complement activation.
As used herein the term "anti-MASP-2 monoclonal antibodies" refers to a homogeneous antibody population, wherein the monoclonal antibody is comprised of amino acids that are involved in binding selection of an epitope on MASP-2. Anti-MASP-2 monoclonal antibodies are highly specific for the MASP-2 target antigen. A "monoclonal antibody" refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (both naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific for the target antigen. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-size monoclonal antibodies, but also fragments thereof (such as Fab, Fab, F(ab"), Fv), single-chain (ScFv), variants thereof. thereof, fusion proteins comprising an antigen-binding moiety, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope.
As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not intended to be limited with respect to the source of the antibody or the manner in which it is A manufactured (eg, by hybridoma, phage selection, expression — recombinant, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments etc. described above under the definition of "antibody". Monoclonal antibodies can be obtained using any technique that provides for the production of the antibody molecules by the continuous cell line in culture, such as the hybridoma method described by Kohler, G., et al., Nature 256:495, 1975, or they can be manufactured by recombinant DNA methods (see, for example, US Patent No.
4,816,567 issued to Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352: 624-628, 1991, and Marks, JD, 155 etal, J Mol. Biol . 222: 581-597, 1991. Such antibodies may be of any immunoglobulin class that includes IgG, IgM, IgE, IgA, IgD and any subclass thereof.
Recognized immunoglobulin polypeptides include kappa and lambda light chains and alpha, gamma (IeG1, IgG2, 1IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length immunoglobulin "light chains" (about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH terminus, and a kappa or lambda constant region at the COOH terminus.
Full-length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids) similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
The basic four-chain antibody unit is a - heterotetrameric glycoprotein composed of two identical E light (L) chains and two identical heavy (H) chains.
An IgM antibody consists of 5 of the: 6 basic heterotetrameric units along with an additional polypeptide — called a J chain, and therefore contains 10 antigen-binding sites.
Secreted IgA antibodies can polymerize to form polyvalent assemblies comprising 2 to 5 of the 4 basic chain units along with the J chain.
Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain.
Each of the H and L chains also has regularly spaced intrachain disulfide bridges.
The pairing of a VH and VL together forms a single antigen binding site.
Each H chain has at the N-terminus a variable domain (VH), — followed by three constant domains (CH) for each of the a and 7 chains, and four CH domains (CH) for the un and e isotypes.
Each L chain has at the N-terminus a variable domain (VL) followed by a constant domain (CL) at its other end.
The VL is aligned with the VH and the CL is aligned with the first constant domain of the — heavy chain (CHI). The L-chain of any vertebrate species can be assigned one of two clearly distinct types, called kappa (x) and lambda (2%), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the domain — constant of its heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes.
There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated alpha (a), delta (8), epsilon (e), gamma (7) and mu (u4), respectively.
Classes y and a are further divided into subclasses based on minor differences in CH sequence and function, for example, human classes express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2. “For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, 5 Daniel P.
Stites, Abba 1. Terr and Tristram G.
Parslow (eds); Appleton and Lange, Norwalc, Conn., 1994, page 71 and Chapter 6. The term “variable” refers to the fact that certain segments of the V domain differ extensively in sequence among antibodies.
Domain V mediates antigen binding and defines the specificity of a particular antibody to its particular antigen.
However, the variability is not evenly distributed across the 110 amino acid range of the variable domains.
Instead, the V regions consist of relatively invariant stretches called matrix regions (FRs) of 15 to 30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9 to 12 amino acids in length.
The native heavy and light chain variable domains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops that connect to and in some cases form part of the n-sheet structure. —The hypervariable regions on each chain are held together in immediate intimacy by the FRs and, with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat, et al., Sequences of Proteins of Immunological Interest , 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md (1991)). Domains—constants are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC). As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues - from a "complementarity determining region" or "CDR" (i.e., around about residues 24-34 (L1), 50-56 (L2) and 89-97). (L3) in the light chain variable domain, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain when numbering according to the system Kabat numbering as described in Kabat, ef al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991)); and/or those residues of a "hypervariable loop" (i.e. residues 24-34 (L1), 50-56 (L2) —e89-97(L3) in the light chain variable domain, and 26-32 (H1) , 52-56 (H2) and 95-101 (H3) in the heavy chain variable domain when numbered according to the Chotia numbering system, as described in Chotia and Lesk, J. Mol. Biol. 196: 901917 (1987). )); and/or those residues of a “hypervariable loop”/CDR (e.g. residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56 -65 (H2), and 105-120 (H3) in the VH when numbered according to the IMG DET numbering system as described in Lefranc, JP, et al., Nucleic Acids Res 27: 209-212; Ruiz, M et al., Nucleic Acids Res 28: 219-221 (2000)). As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length anti-MASP-2 antibody, generally including the antigen binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab", F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and bispecific and multispecific antibodies formed from antibody fragments.
Where bispecific antibodies are to be used, these can be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4,
446-449 (1993)), for example chemically prepared or from . hybridomas, or they can be any of the above-mentioned bispecific antibody fragments.
As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Overall, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allow the scFv to form the desired structure for antigen binding. See Pluckthun in The Pharmacology of — Monoclonals Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). “Fv” is the minimal antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy and light chain variable region domain in tight, non-covalent association. From the fold of these two domains emanate six hypervariable loops (three loops each of the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.
As used herein, the term "specific binding" refers to the ability of an antibody to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some embodiments more than about 10 to 100 times or more (e.g., more than about 1000 or 10,000 times ). In certain embodiments, the affinity between a capture agent and the analyte when they are specifically bound to a capture agent/analyte complex is distinguished by a KD (dissociation constant) of E less than about 100 nM, or less than about 50nM, or less than about 25nM, or less than about 10nM, or less than about 5 SnM, or less than about nM
As used herein, the term "variant" anti-MASP-2 antibody refers to a molecule that differs in amino acid sequence from a "precursor" or reference antibody amino acid sequence by virtue of addition, deletion, and/or substitution of one or more amino acid residue(s) in the precursor antibody sequence. In one embodiment, a variant anti-MASP-2 antibody refers to a molecule that contains variable regions that are identical to precursor variable domains, except for a combined total of 1, 2, 3, 4,5,6, 7, 8, 9, or 10 amino acid substitutions within CDR regions of the heavy chain variable region, and/or up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions of amino acid with said CDR regions of the light chain variable region. In some embodiments, the amino acid substitutions are conservative sequence modifications.
As used herein, the term "precursor antibody" refers to an antibody that is encoded by an amino acid sequence used for the preparation of the variant. Preferably, the precursor antibody has a human matrix region and, if present, has human antibody constant region(s). For example, the precursor antibody can be a humanized or fully human antibody.
As used herein, the term "isolated antibody" refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and others.
Ps )»»tt» -FPPFPssenrt ia OE ra 37 proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody. as determined by Lowry's method, and most preferably more than ; i.e. 99% by weight; (2) to a degree sufficient to obtain at least 15 — N-terminal residues or internal amino acid sequence by use of a spinning beaker sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. As used herein, the term "epitope" refers to the portion of an antigen to which a monoclonal antibody specifically binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features as well as specific charge characteristics. More specifically, the term "MASP-2 epitope," as used herein, refers to a portion of the polypeptide corresponding to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by immunoassays. Antigenic epitopes do not necessarily have to be immunogenic. Such epitopes may be linear in nature or may be a discontinuous epitope. Thus, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by a spatial relationship between amino acids of an antigen other than a continuous series of amino acids. As used herein, the term "mannan-binding lectin" & ("MBL") is equivalent to mannan-binding protein ("M BP").
> As used here, the “membrane attack complex”
(“MAC”) refers to a complex of five-terminal complement components (C5-C9) that inserts into and disrupts membranes.
Also alluded to x. such as C5b-9). :i As used herein, "an individual" includes all mammals, S — including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.
As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; O), acid glutamic (Glu; E), glutamine — (Gln;Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; D), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical characteristic of the side chain of the respective amino acids. by "hydrophobic" amino acid is intended Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro.
By "hydrophilic" amino acid is meant Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His.
This grouping of amino acids can be further subclassified as follows.
By "uncharged hydrophiles" amino acid is meant Ser, Thr, Asn or Gln.
By "acidic" amino acid is meant Glu or Asp.
By "basic" amino acid is meant Lys, Arg or His.
As used herein the term "amino acid substitution — conservative" is illustrated by a substitution between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) t-aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and * histidine.
As used herein, an "isolated nucleic acid molecule" is a nucleic acid molecule (e.g., a polynucleotide) that is not integrated into the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism.
A nucleic acid molecule that has been isolated from a particular species is smaller than the entire DNA molecule of a chromosome of that species.
As used herein, a "nucleic acid molecule construct" is a single-stranded or double-stranded nucleic acid molecule that has been modified through human intervention to contain nucleic acid segments combined and juxtaposed in an arrangement not found in nature.
As used herein, an "expression vector" is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Typically, an expression vector comprises a transcriptional promoter, a gene, and a transcriptional terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be —“operably linked to” the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
As used herein, the terms "approximately" or "about" in reference to a number are generally considered to include numbers — falling within a range of 5% in each direction (greater than or less than) of the number. unless otherwise stated or otherwise evident from the context (except where such a number would exceed *100% of a possible value). Where tracks are established, the endpoints . are included within the range unless otherwise stated or otherwise evident from the context.
. As used herein the singular forms "a", "a", and "the", "a" * include plural aspects unless the context clearly dictates otherwise. So, for example, reference to "a cell" includes a single cell, — as well as two or more cells; reference to "an agent" includes an agent as well as two or more agents; reference to "an antibody" includes a plurality of such antibodies and reference to "a matrix region" includes reference to one or more matrix regions and equivalents thereof known to those skilled in the art, and so on.
Each embodiment in this specification shall apply mutatis mutandis to every other embodiment unless expressly stated otherwise.
Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, —electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as customarily performed in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Etan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or other relevant Protocol publications, Current and other equivalent references. Unless specific definitions are provided, nomenclature used in connection with, and laboratory procedures and techniques of, molecular biology, chemistry. analytical, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. : Standard techniques can be used for recombinant technology, biological — molecular, microbiological, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
It is envisaged that any embodiment discussed in this specification may be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. In addition, the "compositions of the invention" can be used to obtain the methods of the invention.
II. Overview Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are the specific recognition molecules that activate the innate complement system and the system includes the lectin initiation pathway and the terminal amplification pathway that amplifies lectin-initiated activation of terminal complement effector molecules. Clq is the specific recognition molecule that activates the acquired complement system, and the system includes the classical initiation pathway and the associated terminal-pathway amplification loop that amplifies Clq-initiated activation of terminal complement effector molecules. We refer to these two major complement activation systems as the lectin-dependent complement system and the Clq-dependent complement system, respectively.
In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. o As described in U.S. Patent No. 7,919,094, Application for
copending U.S. Patent Serial No. 12/905,972 (published as US-2011/0091450), and copending U.S. Patent Application Serial No. F 13/083,441 (published as US2011/0311549), each of which is ; ' assigned to Omeros Corporation, assignee of the present application, and each — of which is hereby incorporated by reference, has been determined through the use of a mouse model MASP 2 -/- that it is possible to inhibit the MASP pathway -2 mediated by the lectin while leaving the classical pathway intact.
With the recognition that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact comes the realization that it would be highly desirable to specifically inhibit only the complement activation system that causes a particular pathology without completely paralyzing the pathways. complement immune defense capabilities.
For example, in disease states where complement activation is mediated predominantly by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system.
This would leave the Clq-dependent complement activation system intact to handle the immune processing of the complex and to aid in the host's defense against infection.
The preferred protein component to target in the development of therapeutic agents to specifically inhibit the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, Factor B, Factor D, and properdin), only MASP- 2 is both unique to the lectin-dependent complement system and required for the system to function.
Lectins (MBL, H-ficolin, M-ficolin, L-ficolin and CL-11) are also unique components in the lectin-dependent complement system.
However, the loss of any of the lectin components R would not necessarily inhibit the activation of the system due to lectin redundancy. It would be necessary to inhibit all five lectins in order to ensure inhibition of the lectin-dependent complement activation system. In addition, since MBL and the Ficolins are also known to have complement-independent opsonic activity, inhibition of lectin function would result in the loss of this beneficial host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity would remain intact if MASP-2 were the target of inhibition. An added benefit of MASP-2 as the therapeutic target for inhibiting the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is among the lowest of any complement protein (= 500 ng/ml); therefore, correspondingly low concentrations of high affinity MASP-2 inhibitors are sufficient to obtain complete inhibition, as demonstrated in the examples herein.
In accordance with the foregoing, as described herein, the present invention provides fully human monoclonal anti-MASP-2 antibodies that bind human MASP-2 with high affinity and are capable of inhibiting the activation of the lectin-mediated complement pathway.
III. MASP-2 INHIBITORY ANTIBODIES In one aspect, the invention provides a fully human monoclonal anti-MASP-2 antibody, or antigen-binding fragment thereof, which specifically binds to human MASP-2 and inhibits or blocks complement activation. MASP-2 dependent. MASP-2 inhibitory antibodies can effectively inhibit or effectively block the MASP-2 dependent complement activation system by inhibiting or blocking the biological function of MASP-2. For example, an inhibitory antibody can effectively inhibit or block protein-to-MASP-2 protein interaction, interfere with the dimerization or assembly of MASP-2, block binding to Ca*+, or interfere with the active site of the serine protease. of MASP-2.
MASP-2 epitopes. The invention provides fully human antibodies that specifically bind human MASP-2. The MASP-2 polypeptide: exhibits a similar molecular structure to MASP-1, MASP-3, and Clr and Cls, the — proteases of the C1 complement system. The cDNA molecule shown in SEQ ID NO: 1 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO: 2) and provides the human MASP-2 polypeptide with a leader sequence (aa 1 at 15) which is cleaved after secretion, resulting in the mature form of human MASP-2 (SEQ ID NO: 3). As shown in FIGURE 1A, the human MASP 2 gene spans twelve exons. Human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, II, KE L. The cDNA molecule shown in SEQ ID NO: 4 encodes mouse MASP-2 (which consists of the amino acid sequence shown in SEQ ID NO: 5) and provides the mouse MASP-2 polypeptide with a leader sequence that is cleaved after secretion, which results in the mature form of mouse MASP-2 (SEQ ID NO : 6).
Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO: 1 and SEQ ID NO: 4 represent unique alleles of human and rat MASP-2 respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4, including those that contain silent mutations and those in which the mutations result in amino acid sequence changes, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Human MASP-2 protein domains (SEQ ID: NO: 3) are shown in FIGURE 1B and Table 1 below and include an N-terminal domain of the sea urchin C1r/Cl1s morphogenic protein/VEGF/bone morphogenic protein (CUBI), a domain equivalent to epidermal growth factor, a second the CUB domain (CUBII), as well as an i tandem of CCP1 and CCP2 complement control protein domains, and — a serine protease domain.
Alternative splicing of the MASP 2 gene results in MApl19. MApl9 is a non-enzymatic protein that contains the N-terminal CUBI-EGF region of MASP-2 with four additional residues (EQSL). Several proteins have been shown to bind to, or interact with, MASP-2 through protein-to-protein interactions.
For example, MASP-2 is known to bind to, and form Ca 2+ -dependent complexes with, the lectin MBL proteins H-ficolin and L-ficolin.
Each MASP-2/lectin complex has been shown to activate complement through MASP-2-dependent cleavage of C4 and C2 proteins (Ikeda, K., et al., J.
Biol. 155 Chem. 262: 7451-7454, 1987; Matsushita, M., et al., J.
Exp.
Med. 176: 1497-2284, 2000; Matsushita, M., et al., J.
Immuno 168: 3502-3506, 2002). Studies have shown that the CUBI-EGF domains of MASP-2 are essential for the association of MASP-2 with MBL (Thielens, N.
M., et al., J.
Immunol. 166: 5068, 2001). The —CUBIEGFCUBII domains have also been shown to mediate the dimerization of MASP-2, which is required for the formation of an active MBL complex (Wallis, R., et al., J.
Biol.
Chem. 275: 30962-30969, 2000). Therefore, MASP-2 inhibitor antibodies can be identified that bind to or interfere with target regions of MASP-2 known to be important for complement activation — dependent on MASP-2. TABLE 1: MASP-2 Polypeptide Domains SEQIDNO:S5 derate MASP-2 protein (nW/lidan— — .
NO: 3 . SEQ ID NO: 11 | Human MASP-2 CCPI/CCPII/SP domains (aa 278-671 of SEQ ID NO: 3 AND SEQID NO: 12 > Human MASP-2 CCPI/CCPII domains (aa 278-429 of SEQ ID NO: 3): SEQID NO: 14 Human MASP-2 CCPII/SP Domains (aa 348-671 of SEQID NO: 3) | SEQ ID NO: 16 Human MASP-2 SP Domain aa 429-671 of SEQ ID NO: 3 SEQ ID NO: 17 | Mutant form inactivated by serine protease [GKDSCRGDAGGALVFL) * Yaa610-625 of SEQ ID NO: 3 with mutated Ser 618.
In one embodiment, the anti-MASP-2 inhibitory antibodies of the invention bind to a portion of the full-length human MASP-2 protein (SEQ ID NO: 3), such as the CUBI, EGF, CUBII, CCPI domains. , CCPII, or SP from MASP-2. In some embodiments, — the anti-MASP-2 inhibitory antibodies of the invention bind to an epitope in the CCP1 domain (SEQ ID NO: 13 (aa 278-347 of SEQ ID NO: 3)). For example, anti-MASP-2 antibodies (eg, OMS646) have been identified that only bind to fragments of MASP-2 that contain the CCP1 domain and inhibit MASP-2-dependent complement activation, — as described in Example 9. Affinity of MASP-2 Inhibitory Antibody Binding Anti-MASP-2 inhibitor antibodies specifically bind to human MASP-2 (shown as SEQ ID NO: 3, encoded by SEQ ID NO: 1), with an affinity of at least ten times greater than — than other antigens in the complement system.
In some embodiments, MASP-2 inhibitor antibodies specifically bind human MASP-2 with at least a 100-fold greater binding affinity than other antigens in the complement system.
In some embodiments, the -MASP-2 inhibitor antibodies specifically bind human MASP-2 with a KD (dissociation constant) of less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 10 nM, * or less than about 5 nM, or less than or equal to about | nM,
or less than or equal to 0.1 nM. The binding affinity of MASP-2 inhibitor antibodies can be determined using a suitable binding assay known in the art, such as an ELISA assay, as described in Examples 3-5 herein. s Potency of MASP-2 Inhibitory Antibodies In one embodiment, a MASP-2 inhibitory antibody is sufficiently potent to inhibit MASP-2 dependent complement activation at an ICso < 30 nM, preferably less than or about of 20 nM, or less than about 10 nM or less than about 5 nM, —or less than or equal to about 3 nM, or less than or equal to about 1 nM when measured at 1% serum.
In one embodiment, a MASP-2 Inhibitory Antibody is potent enough to inhibit MASP-2 dependent complement activation at an IC s 9 < 30 nM, preferably less than or about 20 nM, or less than that about 10 nM or less than about 5 nM, or less than or equal to about 3 nM, or less than or equal to about 1 nM, when measured in 90% serum. Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in one — component of the complement system that occurs as a result of administration of a MASP-2 inhibitory antibody: the inhibition of generation or production of C4a, C3a, C5a and/or C5b-9 (MAC) MASP-2 dependent complement activation system products (measured, for example, as described in Example 2 of US Patent No. 7,919,094) as well as their — catabolic degradation products (e.g. C3desArg), the reduction of C4 cleavage and C4b deposition (measured e.g. as described in Example 5) and their subsequent catabolic degradation products (e.g. C4bc or C4d), or the reduction of C3 cleavage and C3b deposition. (measured, e.g., as described in Example 5), or its subsequent catabolic degradation products (e.g., C3bc, C3d, etc.).
- In some embodiments, the MASP-2 inhibitor antibodies of the invention are capable of inhibiting C3 deposition in whole serum: less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 15%, such as less than 10% from the deposition of control C3.
In some embodiments, the MASP-2 inhibitor antibodies of the invention are capable of inhibiting C4 deposition in whole serum by less than 80%, such as less than 70%, such as less than 60%, such as as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 15%, such as less than 10% C4 deposition of control.
In some embodiments, anti-MASP-2 inhibitor antibodies selectively inhibit complement activation of MASP-2 (i.e., bind to MASP-2 with at least 100-fold or greater affinity than Clr or Cls ), leaving the Clq-dependent complement activation system functionally intact (i.e., at least 80%, or at least 90%, or at least 95%, or at least 98%, or 100% of the — classical pathway activity is retained).
In some embodiments, the object anti-MASP-2 inhibitor antibodies have the following characteristics: (a) high affinity for human MASP-2 (e.g., a KD of 10 nM or less, preferably a KD of 1 nM or less), and (b) inhibits MASP-2-dependent complement activity in 90% of human serum with an IC50 of 30 nM or less, preferably an ICs of 10 nM or less).
As described in Examples 2-12, fully human antibodies were identified that bind with high affinity to MASP-2 and x inhibit lectin-mediated complement activation while leaving the classical (Clq-dependent) pathway component of the immune system intact.
The variable light and heavy chain fragments of the antibodies were sequenced, isolated and analyzed in both an scFv format and a full-size IgG format.
FIGURE 5A is an amino acid sequence alignment of seven anti-MASP-2 scFv clones that were identified as having high affinity binding to MASP-2 and the ability to inhibit MASP-2 dependent activity. FIGURE 5B is an amino acid sequence alignment of four of the mother scFv clones 17D20, 17N16, 18L16 and 4D9, which show the template regions and the CDR regions.
The scFv mother clones 17D20 and 1I7N16 underwent affinity maturation, leading to the generation of daughter clones with higher affinity and increased potency when compared to mother clones, as described in Examples 6 and 7. The amino acid sequences of the variable regions of heavy chain (VH) (aa 1-120) and light chain (VL) variable regions (aa 148-250) of the scFv clones shown in FIGURES 5A and 5B and the resulting daughter clones are provided below in TABLE 2. Replaceable positions of a human anti-MASP-2 antibody inhibitor, as well as the choice of amino acids that can be substituted at these positions, are revealed by aligning the heavy and light chain amino acid sequences of the discussed anti-MASP-2 inhibitor antibodies above, and determining which amino acids occur at these positions of these antibodies.
In an exemplary embodiment, the heavy and light chain amino acid sequences of FIGURES 5A and 5B are aligned, and the identity of the amino acids at each position of the exemplary antibodies is determined.
As illustrated in FIGURES 5A and 5B (which illustrate the amino acids present at each position of the heavy and light chains of exemplary MASP-2 inhibitor antibodies), various substitutable positions, as well as the amino acid residues that can be substituted at these positions, are easily identified.
In another exemplary embodiment, the light chain amino acid sequences of the mother and daughter clones are aligned and the identity of the amino acids at each position of the exemplary antibodies is determined in order to determine the positions. substitutables, as well as the amino acid residues that can be substituted at these positions.
TABLE 2: Representative anti-MASP-2 antibody sequences ae/daughter DOU clone parent — SEQID NO:1I8 SEQ1ID NO:22 EQ ID NO:20 17D20 35VH-5 SEQ ID NO:24 PINHIVL lone daughter Aara Ti) in pes TN0 dl ) changes of I0aaa — fgG2 OMS644) CEI NO le from the precursor VL) EQ ID NO: 20 17D20 35VH- P PINTIVL tone queue — Kumachangedeaain VH rom NO: 24 [gG4 OMS645) A to R) at position 102 of SEQ ID NO : 18. EQ ID NO: 20 : 17D20 35VH-P lIgG4 (PINITIVL tone daughter region A MA) RAT POEIAO 15h SEQ ID NO: 24 WHO linkage646) AND EOIDNOIS 2G4 mutant) Mother SEQIDNO:21 SEQ ID NO: 27 ORISSA TO h SEQ ID NO: 21 IZaa changes from — flgG2 SEQ ID NO: 25 AND OLISSAS NO: SEQID SEQ ID NO: 27 lgG4 (INIG17NO h SEQ ID NO: 21 SEQ ID NO: 27 joint of OMS643 region) 1gG4 mutant) In certain embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to that of any of the heavy chain variable domain sequences shown in TABLE 2. In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g. at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about s1 96% identical, or at least about 97% identical, or at least about z 98% identical, or at least 99% identical) to 17D20 (VH), shown "” as SEQ ID NO: 18. In some embodiments, the antibody; : subject anti-human MASP-2 monoclonal inhibitor has a heavy chain variable domain comprising or consisting of SEQ ID NO: 18. In some embodiments, a subject anti-human MASP-2 monoclonal inhibitor antibody has a variable domain of heavy chain that is substantially identical (e.g. at least about 70%, at least about 75%, at least about 80%, at least about 85%, — at least about 90%, at least about 95%, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical) to 17D20 cd35VH2NI1 (VH), shown as SEQ ID NO: 20. In some forms of In the embodiment, the object = anti-human MASP-2 monoclonal inhibitory antibody has a heavy chain variable v* 15 domain comprising, or consisting of, SEQ ID NO: 20.
In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80% , at least about 85 %, — at least about 90 %, at least about 95 %, or at least about 96 % identical, or at least about 97 % identical, or at least about 98 % identical, or at least 99% identical) to 17N16 (VH), shown as SEQ ID NO: 21. In some embodiments, the subject anti-human MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that comprises, or consists of of SEQ ID NO: 21. In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70 Ss%, at least 75% %, at least about 80 %, at least about 85 %,
at least about 90%, at least about 95%, or at least about: 96% identical, or at least about 97% identical, or at least about *98% identical, or at least 99% identical) , to that of any of the light chain variable domain sequences shown in TABLE 2. In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (for example, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% the same , or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17D20 (VL), shown as SEQ ID NO: 22. In some embodiments, the inhibitory antibody monoclonal anti-human MASP-2 subject has a light chain that - comprises, or consists of SEQ ID NO: 22.
vim 15 In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% the same, or at least about 97% the same, or at least about 98% the same , or at least 99% identical) to 17D20 35VH-21N11VL (VL), shown as SEQ ID NO: 24. In some embodiments, the subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain comprising, or consists of SEQ ID NO: 24. In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70:%, at least at least 75%, at least about 80%, at least about 85%, Y at least about 90%, at least in about 95%, or at least about
96% identical, or at least about 97% identical, or at least about; 98% identical, or at least 99% identical) to 17N16 (VL), shown * as SEQ ID NO: 25. In some embodiments, the subject antibody: anti-human MASP-2 monoclonal inhibitor has a light chain that comprises, or consists of SEQ ID NO: 25.
In some embodiments, a subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80% , at least about 85 %, — at least about 90 %, at least about 95 %, or at least about 96 % identical, or at least about 97 % identical, or at least about 98 % identical, or at least 99% identical) to 17NI6 17N9 (VL), shown as SEQ ID NO: 27. In some embodiments, the subject anti-human MASP-2 monoclonal inhibitory antibody has a light chain comprising, or consisting of, the SEQ ID NO: 27.
In some embodiments, the anti-MASP-2 antibodies of the invention contain a heavy or light chain that is encoded by a nucleotide sequence that hybridizes under high stringency conditions to a nucleotide sequence that encodes a heavy or light chain, such as shown in TABLE 2. High stringency conditions include incubation at 50°C or higher in 0.1 x SSC (15 mM saline/0.15 mM sodium citrate).
In some embodiments, the anti-MASP-2 inhibitory antibodies of the invention have a heavy chain variable region that — comprises one or more CDRs (CDRI, CDR2 and/or CDR3) that are substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97 % identical, or at least about
98% identical, or at least 99% identical), or comprise or . consist of the identical sequence when compared to the amino acid sequence of the CDRs of any of the heavy chain:' variable sequences shown in FIGURES 5A or 5B, or described below in TABLES3A-FeTABLEAA4.
In some embodiments, the anti-MASP-2 inhibitory antibodies of the invention have a light chain variable region that comprises one or more CDRs (CDRI, CDR2, and/or CDR3) that are substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about than 97% identical, or at least about 98% identical, or at least 99% identical), or comprise or. consist of the identical sequence as compared to the -15 amino acid sequence of the CDRs of any of the variable light chain sequences shown in FIGURES 5A or 5B, or described below in TABLES 4A-F and TABLE 5.
Heavy chain variable region TABLE 3A: Heavy chain (aa 1-20) Chain EE E add Me rover Ss e re eo o ser fe rien ste rs TA [sound, [2 |U [D]T jo [95 9 Do | eq qe e 1 je (je js qe "TABLE ;3B: Heavy Chain (aa 21-40) Chain CDR-HI MEe air air to foot SE BETE TRETETITATETITSTATRTPTRE a leseonm | o | Mv 5 [9 [E [8 [é 18 j& JS (É MS |S 15 JWI OR) — snes es e E o qo “sa ONE Ee Es ES ee srs
TABLE 3C: Heavy chain (aa 41-60) o eine Ca A ” heavy - aa — Ja41/42 43/44/45 46/47/48 [49 50/51]/52]53 54 ]55]56]57 ]/58 [59] 60] "OE es a AA Ae . SEQ:18, ; d3521INI1 G AL TE E A | " SEQ:20: an EE Son SEQ:21 eean of 1st je |º (2 1st fe je wie fe exxx ds SEQ:21 TABLE 3D: Heavy Chain (aa 61-80) E Te do o err and Ask heavy lvl. aa — J61762T63] 6465/66/67 [68/69 70/71 /72/73 74/75 /76 77 [7879 [80] 17D20m |YTL [| L|T = TK |[NV SEQ:18, d3s2INIHI | YTE LT KTK |NV SEQ:20) 17N16m "as ve EEE SP" K |N SEQ:21 dI7N9 YjAaA|v YE) “ES PErErr, SEQ:21 a. TABLE 3E: Heavy chain (aa 81-100)
FESNNNNNNNNNNNEEEEAADE heavy aa — 81/82/83 /84/85|86/87]/88 /89 90/91/92 [93/94 /95]96]/97]98]99 [100] SEQ:18 eega | | | [ep ps and vp Te iTikikiea SEQ:20. a 2 eaa ESA FP |T ja xxx|ea SEQ:21 sean e |º 1º [and Jo jr [Ns Iv [Tele eita lx xx ea SEQ:21 TABLE 3F: heavy chain (aa 101-118) heavy jaa — J101 /102 103/1048 [105106 [107 [108] 109 [110111 [112 [113 [14/15] 06 [15 [08 [19 [120 | leeora je [º jo je pn jo qv pe jo je jo fe fe jv jr ly js ds DV SEQ:19 ease A ed Ade o SEQ:20 exoan to | | | and | wo use sr e" SEQ:21 [sto2n [2 | |º [de q rd q je fe de e q vs SEQ:21 ". Shown below are the heavy chain variable region (VH) sequences for the mother clones and daughter clones listed above in TABLE 2 and TABLES 3A-F. . The Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-102 (H3))
are in bold; and the Chotia CDRs (26-32 (H1I), 52-56 (H2) and 95-101 + (H3)) are underlined.
and” Heavy Chain Variable Region (VH) 17D20 (SEQ ID NO: QVTLKESGPVLVKPTETLTLTCTVSGESLSRGKMGVSWIRQPPGKAL EWLAHIFESSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTAT
YYCARIRAGGIDY WGQGTLVTVSS Heavy Chain Variable Region (VH) 17D20 35VH- 21N11VL (SEQ1ID NO: 20) QVTLKESGPVLVKPTETLTLTCTVSGESLSRGKMGVSWIRQPPGKAL EWLAHIFSSDEKSYRTSLKSRLTISKDTSKNQV VLTMTNMDPVDTAT
YYCARIRRGGIDY WGQGTLVTVSS Heavy Chain Variable Region (VH) 17N16 (SEQ ID NO: .21) W QVQLQQOSGPGLVKPSQTLSLTCAISGDSVSSTSAA WNWIRQSPSRGLE WLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTA
VYYCARDPFGVPFDIWGQGTMVTVSS TABLE 4: Heavy Chain CDRs (copy table on pages 43 and 44) — As- and - Consensus 17D20m and d3521N11 — YYCARIRX | FER Clone Reference to Sequenia — ———.. |[SEQIDNO: | |º1D2m = jCpRHi(kaba —[RGKMG À | o |iNÉn —- | CDRHi(kaba) — | Abe SIS ABA Fo .
.
EE sa o O o o
[ Clone Reference [EBR aa Sequenia = ——————— |[SEQIDNO: | |d3s2INI ————[CDRH3(kaba) [YYCARIRR a "A . 17D20m and d352INI1 | CDR-H3 (kabat) YYCARIRX and consensus (where X at position 8 is A (Ala) or R (Arg > z Nor CDR-H3 (chothia)) | AvyyeaAR O QOG Light chain variable region TABLE 5A: Light chain (aa 1-20) Peadeia eve PME ca OR V""r jaa Ja 2 13 14 15 16 |7 8 9 10 [11 [12/13 [14 [145 [146 [17 [18 [19 [20 | |eseoan 1st |" |V [e To [ee js q 15 |v [se Je lo jr [As 11) SEQ:22) [ caca ol Aeee ee e A ae Eh SEQ:24 |people |º | |V |! 7 [e [e e |5 [v [5 [v [a | Jo lo lt ja |& dr SEQ:25)
FESPM SEQ:2 Ç TABLE 5B: Light chain (aa 21-40) and E eee ea light . [yy 121 22/23/2425 ]26]27 128 29130 31 132 [33 [34 135 136 |37 [38 |39 [40] slugs | 5 > 1st e |E je [8 Je JE je ja |x |&|Y |O 19 |E |P 19 | SEQ:22 [eseoan | and the je E is e [9 je [is DX to [+ [8] (and Je |x 1 do | (SEQ:24 |people | oo [8 and [and the fe js je De je fe je | Jo Je [x 1r of | SEQ25, [ese [OIE [E fo | fa e | aoo se and SEQ:27, TABLE 5C: Light chain (aa 41-60) Leadeia had LL TEL OIOCIJOJOCIEJOCCJOE nn CpRa3 jaa — 41/47/ and 19 1% [º |" [ [+ IV 1 [DB |s (e [e (e |s lah Pr lp d*) SEQ:27) ' S — TABLE SD: Light chain (aa 61-80) eadea Teve ER anta ru UT [aa —J61T62T63 64 [65 66 67 68/69 70 |71 [72 |73 74 [75 [76 77 [78 [79 [80] 17D20m |FGN 6 [N ft [AT ft TT G |TA |eseaaa | 1st 9 5 ps js je ps jr fa je jr pe ds de (7 Je da JM [eeoaa | > e > [Se pera e je q fes le jr le ja d8) F SEQ:24
FSTINNNNNNNNNNNANMENMENH SEQ:25 ses or NerENASS” SEQ:27
TABLE SE: Light chain (aa 81-100) Chain CDR-L3 E mao cam o eEcEE ma These [ana DP 1st fe oo geo and ugly a fel je jr e TABLE 5F: Light chain (aa 101-120) oa TETE TETE TETE AND PET or ET TE
MTE ELECTED EEE aa EO [ssoan |º is [| pe nv qe ja ja fe js e fe je je ds je | The light chain variable region (VL) sequences for the mother clones and daughter clones listed above in g S TABELA2e TABLES SA-F are shown below.
The Kabat CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) are in boldface; and the Chotia CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3) are underlined.These regions are the same if numbered by the Kabat or Chotia system.
Light chain variable region (VL) 17D20m (SEQ ID NO: 22) QPVLTQPPSVSVSPGQTASITCSGDKLGDKFAYWYQQKPGHSPVLVI YQDNKRPSGIPGRFSGSNSGNTATLTISGTQAMDEADY YCQAWDSST
AVEGTGTKVTVLA Light Chain Variable Region (VL) 17D20m d3521N11 (SEQ ID NO: 24) QPVLTQPPSLSVSPGQTASITCOSGEKLGDKYAYWYQQKPGQSPVLV . MYQDKQRPSGIPERFSGSNSGNTATLTISGIQAMDEADYYCQAWDS STAVEGGGTKLTVL
Light chain variable region (VL) 17Nl6m (SEQ ID NO: "25) - SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVHWYQQKPGQAPVLV : VYDDSDRPSGIPERFSGSNSGNTATLTVSRVEAGDEADYYCQVWDT
TTDHVVFGGGTKLTVLAAAGSEQKLISE Light chain variable region (VL) 17N116m d17N9 (SEQ ID NO: 27) SYELIQPPSVSVAPGQOTATITCAGDNLGKKRVHWYQQRPGQAPVLVI YDDSDRPSGIPDRFSASNSGNTATLTITRGEAGDEADYYCQVWDIAT
DHVVFGGGTKLTVLAAAGSEQKLISE S — TABLE 6: Light Chain CDRs (Kabat/chotia) [ Reference TER aa Sequene o SEQIDNO: | I7D2Om or EBR GDKLGDKRA DA 17D20m and CDR-L1 GXKLGDKXAYW = d3521IN11 (where X at position 2 is D (Asp) or E consensus (Glu); and where X at position 8 is F (Phe) or Y (T : Lin eDR eNNtGske "A
EN ODR GDNEGRRRE 17N16m and d17N9 CDR-L1 GXNXGXKXVHW 92 consensus (where X at position 2 is N(Asn) or D (Asp); where X at position 4 is I (Ile) or L (Leu); where X at position 6 is S (Ser) or K (Lys); and where X at position 8 is N Asn) or R (Arg Dao ER BRR Na LASS2INDE | CDR-L2 (aas0-60) DRE Ps GR A) Nm ER if the ER sas nn Ng 17D20m, CDR-L2 DXXRPSG 3 d3521INI11, (wherein X at position 2 is N(Asn), K 17N116m, d1I7N9 (Lys) or S (Ser); and where X at consensus position 3 is K (Lys), Q (Gin) or D (Asp aaa eae Eae amena Maraca E near Dam ER ADS SA A [asaintoo — Jepras | awossrae II 17N16m and d17N9 CDR-L3 VWDXXTDHV 94 * consensus (where X in position 4 is T (Thr) or I (Tle); and wherein X at position 5 is T (Thr) or A (Ala)) In one aspect, the invention provides an isolated human monoclonal antibody, or antigen-binding fragment thereof, which binds to human MASP-2, comprising: (1) a heavy chain variable region comprising the sequences of CDR-H1, CDR-H2 and CDR-H3; and (ii) a variable region el da — light chain comprising CDR-LI, CDR-L2 and CDR-L3, wherein the heavy chain variable region sequence CDR-H3 comprises an amino acid sequence shown as SEQ ID NO: 38 or SEQ ID NO: 90, and conservative sequence modifications thereof, wherein the light chain variable region CDR-L3 sequence comprises an amino acid sequence shown as SEQ ID NO: 51 or SEQ ID NO: 94, and conservative sequence modifications thereof , and wherein the antibody alone inhibits MASP-2-dependent complement activation. In one embodiment, the CDR-H2 heavy chain variable region sequence comprises a -15 amino acid sequence shown as SEQ ID NO: 32 or 33, and conservative sequence modifications thereof.
In one embodiment, the CDR-HI heavy chain variable region sequence comprises an amino acid sequence shown as SEQ ID NO: 28 or SEQ ID NO: 29, and conservative modifications thereof.
In one embodiment, the CDR-L2 light chain variable region sequence comprises an amino acid sequence shown as SEQ ID NO: 93 and conservative modifications thereof.
In one embodiment, the CDR-LI light chain variable region sequence comprises an amino acid sequence shown as SEQ ID NO: 91 or SEQ ID NO: 92 and — conservative modifications thereof.
In one embodiment, the heavy chain variable region CDR-HI comprises SEQ ID NO: 28. In one embodiment, the heavy chain variable region CDR-H2 comprises SEQ ID NO: 32. in one embodiment, the heavy chain variable region & CDR-H3 comprises SEQ ID NO: 90,
(as shown in TABLE 4). In one embodiment, the sequence of . The amino acid shown in SEQ ID NO: 90 contains an R (Arg) at position 8.
In one embodiment, the CDR-L1 of the light chain variable region comprises SEQ ID NO: 91 (as shown in TABLE 6). In one embodiment, the amino acid shown in SEQ ID NO: 91 contains an E (Glu) at position 2. In one embodiment, the amino acid sequence shown in SEQ ID NO: 91 contains a Y (Tyr) at position 8.
In one embodiment, the light chain variable region CDR-L2 comprises SEQ ID NO: 93 (as shown in TABLE 6), and wherein the amino acid sequence shown in SEQ ID NO: 93 contains a K (Lys ) at position 2. In one embodiment, the amino acid sequence shown in SEQ ID NO: 93 contains a Q(Gln) at position 3. In one embodiment, the CDR-L3 of the light chain variable region comprises SEQ ID NO: 51.
In one embodiment, said antibody or antigen-binding fragment thereof binds to human MASP-2 with a KD of 10 nM or less. In one embodiment, said antibody or antigen-binding fragment thereof inhibits C4 activation in an in vitro assay in 1% human serum at an IC s 9 of 10 nM or less. In one embodiment, said antibody or antigen-binding fragment thereof inhibits C4 activation in 90% of human serum with an ICs of 30 nM or less. In one embodiment, conservative sequence modifications thereof comprise or consist of a molecule that contains variable regions that are identical to the recited variable domain(s), except for a combined total of 1 , 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid substitutions within CDR regions of the heavy chain variable region, and/or up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uy amino acid substitutions with said light chain variable region CDR regions.
In another aspect, the invention provides an isolated : human antibody, or antigen-binding fragment thereof, which binds : . human MASP-2 wherein the antibody comprises: 1) a) a heavy chain variable region comprising: i) a CDR-HIl heavy chain which — comprises amino acid sequence 31-35 of SEQ ID NO: 21; and ii) a CDR-H2 heavy chain comprising amino acid sequence 50-65 of SEQ ID NO: 21; and iii) a CDR-H3 heavy chain comprising amino acid sequence 95-102 of SEQ ID NO: 21; and b) a light chain variable region comprising: i) a CDR-L1 light chain which — comprises amino acid sequence 24-34 of SEQ ID NO: 25 or SEQ ID NO: 27; and ii) a CDR-L2 light chain comprising amino acid sequence 50-56 of SEQ ID NO: 25 or SEQ ID NO: 27; and ili) a CDR-L3 light chain comprising amino acid sequence 89-97 of SEQ ID NO: 25 or SEQ ID NO: 27; or II) a variant thereof which is otherwise identical to said variable domains, except up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions within the said CDR regions of said heavy chain variable region and up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions within said CDR regions of said variable region of the light chain, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation. In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group consisting of position 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100 or 102 of said heavy chain variable region.
In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group consisting of position 25, 26, 27, 29, 31, 32, '33, 51, 52, 89, 92, 93 , 95, 96 or 97 of said light chain variable region.
In . in one embodiment, the heavy chain of said antibody comprises the
SEQ ID NO: 21. In one embodiment, the light chain of said antibody comprises SEQ ID NO: 25. In one embodiment, the light chain of α. said antibody comprises SEQ ID NO: 27.: In another aspect, the invention provides an isolated human monoclonal antibody that binds human MASP-2, wherein the antibody comprises: 1) a) a heavy chain variable region which comprises: i) a CDR-H1 heavy chain comprising amino acid sequence 31-35 of SEQ ID NO: 20; and ii) a CDRH-2 heavy chain comprising amino acid sequence 50-65 of SEQ ID NO: 20; and —jil) a CDR-H3 heavy chain comprising amino acid sequence 95-102 of SEQ ID NO: 18 or SEQ ID NO: 20; and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising amino acid sequence 24-34 of SEQ ID NO: 22 or SEQ ID NO: 24; and L ii) a CDR-L2 light chain comprising the amino acid sequence of . 15 50-56 of SEQ ID NO: 22 or SEQ ID NO: 24; and iii) a CDR-L3 light chain comprising amino acid sequence 89-97 of SEQ ID NO: 22 or SEQ ID NO: 24; or II) a variant thereof that is otherwise identical to said variable domains, except up to a combined total of 1,2,3,4,5,6, 7, 8, 9, or 10 amino acid substitutions within said CDR regions of said heavy chain variable region and up to a combined total of 1,2,3,4,5, 6,7, 8,9, or 10 amino acid substitutions within said CDR regions of said variable region of light chain, wherein the antibody or variant thereof inhibits MASP-2-dependent complement activation. In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 31, 32, 33, 34,35, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100 or 102 of said heavy chain variable region.
In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96 or 97 of said light chain variable region.
In one embodiment, the heavy chain of said antibody comprises SEQ ID NO: 20, or a variant thereof: which comprises at least 80% identity with SEQ ID NO: 20 (e.g., at least 85% , at least 90%, at least 95% or at least 98% identity to SEQ ID NO: 20). In one embodiment, the heavy chain of said antibody comprises SEQ ID NO: 18, or a variant thereof that comprises at least 80% identity with SEQ ID NO: 18 (e.g., at least 85%, at least at least 90 %, — at least 95 % or at least 98 % identity to SEQ ID NO: 18). In one embodiment, the light chain of said antibody comprises SEQ ID NO: 22, or a variant thereof that comprises at least 80% identity with SEQ ID NO: 22 (e.g., at least 85%, at least ' at least 90%, at least 95% or at least 98% identity to SEQ.15 ID NO:22). In one embodiment, the light chain of said antibody comprises SEQ ID NO: 24, or a variant thereof that comprises at least 80% identity with SEQ ID NO: 24 (e.g., at least 85%, at least least 90%, at least 95% or at least 98% identity to SEQ ID NO: 24). In one embodiment, said antibody binds to an epitope in the CCP1 domain of MASP-2. In one embodiment, said antibody binds human MASP-2 with a KD of 10 nM or less.
In one embodiment, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum at an ICs of 10 nM or less.
In one embodiment, said antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less. ã In one embodiment, said antibody is a fragment. antibody selected from the group consisting of Fv, Fab, Fab, F(ab); and
F(ab”),. In one embodiment, said antibody is a single-chain molecule. In one embodiment, said antibody is an I2G2 s molecule. In one embodiment, said antibody is an IgG1 e molecule. In one embodiment, said antibody is an IgG4 molecule. In one embodiment, said IGG4 molecule comprises an S228P mutation.
In one embodiment, said antibody does not substantially inhibit the classical pathway (i.e., the activity of the classical pathway is at least 80%, or at least 90%, or at least 95%, or at least 95% intact).
In another aspect, the invention provides a fully isolated human monoclonal antibody or antigen-binding fragment thereof that dissociates from human MASP-2 with a KD of 10 nM or less as determined by surface plasmon resonance and inhibits: 15 the activation of C4 on a mannan coated substrate with an IC of 10 nM or less in 1% serum. In some embodiments, said antibody or antigen-binding fragment thereof specifically recognizes at least part of an epitope recognized by a reference antibody that comprises a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable region as shown in SEQ ID NO: 24, such as reference antibody OMS646 (see TABLE 22). In accordance with the foregoing, an antibody or antigen-binding fragment thereof according to certain preferred embodiments of the present application may be one that competes for human MASP-2 binding with any antibody described herein that either (i) specifically binds antigen when (ii) it comprises a VH and/or VL domain disclosed herein, or comprises a CDR-H3 disclosed herein, or is a variant of any of these. Competition between binding members can be easily assayed in vitro, for example using ELISA and/or by labeling a reporter molecule specific to a binding member which can be detected in the presence of other binding member(s). " unlabeled(s), to allow the identification of specific binding members that bind to the same epitope or an overlapping epitope. — Thus, a specific antibody or antigen-binding fragment thereof is presently provided, which comprises a human antibody antigen binding site that competes with an antibody described herein that binds to human MASP-2, such as any one of OMS641 to OMS646 as shown in TABLE 24, for binding to human MASP-2.
MASP-2 Inhibitory Antibodies The human monoclonal antibodies described above can be modified to provide variant antibodies that inhibit MASP-2 dependent complement activation. Variant antibodies can be made by substituting, adding, or deleting at least one α-15 amino acid of a human monoclonal antibody described above. In general, these variant antibodies have the general characteristics of the human antibodies described above and contain at least the CDRs of a human antibody described above, or, in certain embodiments, CDRs that are very similar to the CDRs of a human antibody described above.
In the preferred embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable regions of the precursor antibody. For example, the variant may comprise at least one, for example from about one to about ten, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, — at least 7, at least 8, at least 9 or at least 10 substitutions, and preferably from about two to about six, substitutions in one or more CDR regions of the parent antibody. In one embodiment, said variant comprises an amino acid substitution by one or more. selected positions from the group consisting of positions 31, 32, 33, 34, 35,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100 or 102 of said heavy chain variable region . In one embodiment, said variant comprises an amino acid substitution at: one or more positions selected from the group consisting of positions 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96 or 97 of said light chain variable region.
In some embodiments, the variant antibodies have an amino acid sequence that is otherwise identical to the variable domain of an object antibody shown in TABLE 2, except for up to a combined total of 1, 2, 3, 4, 5, or 6 amino acid substitutions within said CDR regions of said heavy chain variable region and/or up to a combined total of 1, 2, 3, 4, 5 or 6 amino acid substitutions within said CDR regions of said light chain region variable, wherein the antibody or variant thereof inhibits complement-dependent activation - 15 deMASP+ 2.
Ordinarily, the variant will have an amino acid sequence having at least 75% amino acid sequence identity to the precursor antibody heavy or light chain variable domain sequences, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the precursor antibody residues, then align the sequences and introduce gaps, if necessary, to obtain sequence identity. maximum percentage. None of the N-terminal, C-terminal, or internal extensions, deletions, or insertions in the antibody sequence (such as, & for example, signal peptide sequences, linker sequences, or labels,
such as HIS tags) should be interpreted as affecting sequence identity or homology. The variant retains the ability to bind human MASP-2 and preferably has properties that are superior to those of the parent antibody. For example, the variant may have a stronger binding affinity and/or an enhanced ability to inhibit or block MASP-2 dependent complement activation.
To analyze such properties, one must compare a Fab form of the variant with a Fab form of the precursor antibody or a full-size form of the variant with a full-size form of the precursor antibody, for example, since it was discovered that the format of the anti-MASP-2 antibody impacts its activity in the biological activity assays disclosed herein. The variant antibody of particular interest herein is one that demonstrates at least about 10-fold, preferably at least about 20-fold, and most preferably at least about 50-fold, the enhancement in biological activity as compared to the parent antibody. .
Antibodies of the invention may be modified to enhance desirable properties, such as may be desirable to control the serum half-life of the antibody. In general, whole antibody molecules have a very long persistence in serum, whereas fragments (<60-80 —kDa) are filtered very quickly through the kidneys. Consequently, if long-acting MASP-2 antibody is desirable, the MASP-2 antibody is preferably a full-length, full-length IgG antibody (such as IgG2 or IgG4), whereas the shorter-acting of the MASP-2 antibody is preferably a full-length IgG antibody (such as IgG2 or IgG4). MASP-2 is desirable, an antibody fragment may be preferred. As described in Example 5, it was determined that an S228P substitution in the IgG4 hinge region enhances serum stability. Accordingly, in some embodiments, the subject MASP-2 antibody is a full-size IgG4 antibody with an S228P substitution. . single chain antibodies
In one embodiment of the present invention, the MASP-2 inhibitor antibody is a single chain antibody, defined as a genetically engineered molecule that contains the light chain variable region, the heavy chain variable region, linked by a ligand of — polypeptide suitable as a genetically fused single-chain molecule. Such single chain antibodies are also referred to as "single chain Fv" or "scFv" antibody fragments. Overall, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allow the scFv to form the desired structure for —antigen binding. The scFv antibodies that bind to MASP-2 can be oriented with the light variable region amino-terminal to the heavy variable region or carboxyl-terminal to the same. Exemplary scFv antibodies of the invention are shown herein as SEQ ID NOS: 55-]61 and SEQID NOS: 66-68. mos: Methods for Producing Antibodies In many embodiments, nucleic acids encoding an object monoclonal antibody are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded antibody.
In some embodiments, the invention provides a nucleic acid molecule encoding an anti-MASP-2 antibody, or fragment thereof, of the invention, such as an antibody or fragment thereof shown in TABLE 2. In some embodiments the invention provides a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 88 and SEQ ID NO: 89. . In some embodiments, the invention provides a
A cell comprising a nucleic acid molecule encoding an anti-MASP-2 antibody of the invention. f In some embodiments, the invention provides an "expression cassette" comprising a nucleic acid molecule that S — encodes an anti-MASP-2 antibody of the invention.
In some embodiments, the invention provides a method of producing anti-MASP2 antibodies which comprises culturing a cell which comprises a nucleic acid molecule encoding an anti-MASP-2 antibody of the invention.
In accordance with certain related embodiments, a recombinant host cell is provided which comprises one or more constructs as described herein; a nucleic acid encoding any antibody, CDR, VH or VL domain, or antigen-binding fragment thereof; and a method of producing the encoded product, which method comprises expressing the nucleic acid encoding therefor. Expression may be conveniently obtained by culturing recombinant host cells under appropriate conditions which contain the nucleic acid. Following production by expression, an antibody, or antigen-binding fragment thereof, can be isolated and/or purified using any suitable technique, and then used as desired.
For example, any cell suitable for the expression of expression cassettes can be used as a host cell, e.g., yeast, insect, plant, etc. cells. In many embodiments, a mammalian host cell line that does not ordinarily produce antibodies is used, examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7 , ATCC CRL 1651); human embryonic α-renal cells (HEK293, Graham et al., J. Gen Virol. 36:59 (1977)); r baby hamster kidney cells (BHK, ATCC CCL 10); ovary cells of
* 71 Chinese hamster (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77: 4216, (1980); mouse sertoli cells (TMA4, Mather, Biol. Reprod. 23: 243-251 ( 1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells ( MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562 , ATCC CCL 51); TRI cells (Mather et al., Annals NY
academy Sci 383: 44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1). Additional cell lines will become apparent to those of ordinary skill in the art. A wide variety of cell lines is available from the American Type Culture: Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.
= bad Methods of introducing nucleic acids into cells are well known in the art. Suitable methods include electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell that is transformed and the circumstances under which transformation is taking place (ie, in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995. In some embodiments, lipofectamine and calcium-mediated gene transfer technologies are used. .
After the object nucleic acids have been introduced into a cell, the cell is typically incubated, typically at 37°C, sometimes under selection, for an adequate time to allow for antibody expression. In most embodiments, the antibody is typically secreted into the supernatant of the medium in which the cell is grown.
& Ta In mammalian host cells, various viral-based expression systems can be used to express a target antibody. In cases where an adenovirus is used as an expression vector, the sequence encoding the antibody of interest can be linked to an adenovirus transcription/translation control complex, eg, the late promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, E1 or E3 region) will result in a recombinant virus that is viable and capable of — expressing the antibody molecule in infected hosts. (for example, see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). Expression efficiency can be enhanced by the inclusion of appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see A. Bittner et al., Methods in Enzymol. 153: 51-544 (1987)).
- 5 For long-lasting, high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines, which stably express the antibody molecule, can be engendered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selectable marker. Following the introduction of foreign DNA, the engendered cells can be allowed to culture for 1 to 2 days in an enriched medium, and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and — allows cells to stably integrate the plasmid within a chromosome and cultured to form foci which in turn can be cloned and expanded into cell lines. Such engendered A cell lines can be particularly useful in screening and evaluating compounds that interact directly or indirectly with the DNA molecule.
- 73 antibody.
Once an antibody molecule of the invention has been produced, it can be purified by any method known in the art for purifying an immunoglobulin molecule, e.g., by chromatography (e.g., ion exchange, affinity, particularly by affinity for specific antigen after Protein A column chromatography, and sorting by size), centrifugation, differential solubility, or any other standard technique for protein purification. In many embodiments, antibodies are secreted from the cell in the culture medium and harvested from the culture medium. For example, a nucleic acid sequence encoding a signal peptide may be included adjacent to the coding region of the antibody or fragment, for example as provided at nucleotides 1-57 of SEQ ID NO: 71, which encodes the signal peptide as provided at amino acids 1-19 of SEQ ID. NO: 72. Such a signal peptide may be incorporated adjacent to the 5th end of the amino acid sequences presented herein for the object antibodies in order to facilitate the production of the object antibodies.
Pharmaceutical Carriers and Delivery Vehicles In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-2 dependent complement activation which comprise a therapeutically effective amount of an anti-human MASP-2 inhibitory antibody and a pharmaceutically carrier. acceptable.
In general, the human MASP-2 inhibitory antibody compositions of the present invention, combined with any other selected therapeutic agent, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to adversely affect the biological activity of the MASP-2 inhibitor & antibody (and any other therapeutic agents
- 74 combined with this one). Exemplary pharmaceutically acceptable carriers for the polypeptides are described in U.S. Patent.
No. 5,211,657 for Yamada.
Anti-MASP-2 antibodies can be formulated in solid, semi-solid, gel, liquid or gaseous form preparations such as — tablets, capsules, powders, granules, ointments, solutions, deposits, inhalants and injections allowing for oral, parenteral administration. or surgical.
The invention also contemplates the local administration of the compositions through the coating of medical and other devices.
Carriers suitable for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol.
In addition, sterile, fixed oils can be used as: a solvent or suspending medium.
For this purpose, any biocompatible oil can be used including synthetic mono- or diglycerides.
In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The carrier and agent can be composed as a liquid, suspension, polymerizable or non-polymerizable gel, paste or ointment.
The carrier may also comprise a delivery vehicle to sustain (i.e., prolong, delay, or regulate) the release of the agent(s) or to enhance the release, absorption, stability, or pharmacokinetics of the agent(s). therapeutic(s). Such a delivery vehicle may include, by way of non-limiting example, the compounds of microparticles, microspheres, nanospheres or nanoparticles of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.
Suitable hydrogel and micelle delivery systems include the copolymers of PEO: Á PHB: PEO and copolymer/cyclodextrin complexes disclosed in WO h 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in
- 758 U.S. Patent Application Publication No. 2002/0019369 A1. Such hydrogels can be injected locally on the intended side of action, either subcutaneously or intramuscularly to form a sustained release depot.
For intra-articular delivery, the MASP-2 inhibitor antibody can be loaded into the above-described liquid or gel carriers which are injectable, the above-described sustained-release delivery vehicles which are injectable, or a hyaluronic acid or hyaluronic acid derivative. .
For intrathecal (IT) or intracerebro-ventricular (ICV) delivery, appropriately sterile delivery systems (eg, liquids; gels, suspensions, etc.) can be used to administer the present invention.
The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickening agents, flavoring agents (for oral administration) . To obtain high concentrations of anti-MASP-2 antibodies for local release, the antibodies can be formulated as a suspension of particulates or crystals in solution for subsequent injection, such as for intramuscular injection of a depot.
More specifically with respect to anti-MASP-2 antibodies, exemplary formulations may be administered parenterally as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid such as water. , oils, saline, glycerol or ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like may be present in compositions comprising anti-inflammatory antibodies.
- 76 MASP-2. Additional components of pharmaceutical compositions include petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions. Anti-MASP-2 antibodies can also be administered in the form of a depot injection or implant preparation that can be formulated in such a way as to allow a sustained or pulsatile release of the active agents. Pharmaceutical compositions comprising MASP-2 inhibitor antibodies can be administered in a variety of ways depending on whether a site or systemic mode of administration is more appropriate for the condition being treated. Additionally, as described above with respect to reperfusion procedure procedures: extracorporeal, MASP-2 inhibitory antibodies can be administered: 15 via the introduction of the compositions of the present invention to recirculate blood or plasma. In addition, the compositions of the present invention can be delivered by coating or incorporating the compositions into or into an implantable medical device.
SYSTEMIC RELEASE As used herein, the terms “systemic release” and “systemic administration” are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation , sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively result in — dispersion of the released antibody to a local or multiple site of intended therapeutic action. Preferred systemic delivery routes for the present compositions include intravenous, intramuscular, subcutaneous and inhalation. It will be appreciated that the exact route of systemic administration for the selected agents used in the particular compositions herein
The invention will be determined in part to explain the agent's susceptibility to metabolic transformation pathways associated with a given route of administration. MASP-2 inhibitory antibodies and polypeptides can — be delivered to an individual in need thereof by any suitable means. Methods of delivering MASP-2 antibodies and polypeptides include administration via the oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (such as via a formulation fine powder), transdermal, nasal, vaginal, rectal, or sublingual administration, and may be formulated into appropriate dosage forms for each route of administration.
By way of representative example, MASP-2 inhibitory antibodies and peptides can be introduced into a living body by application to a body membrane capable of absorbing the polypeptides, for example, nasal, gastrointestinal and rectal membranes. The polypeptides are typically applied to the absorption membrane together with a permeation enhancer. (See, for example, Lee, VHL, Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, VHL, J. Controlled Delivery 13:213, 1990; Lee, VHL, Ed, PEPTIDE and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, AG, et al., J. Controlled Delivery 13: 241, 1990.) For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant that is similar in structure. to bile salts, and has been used as a permeation enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.) MASP-2 inhibitory antibodies and polypeptides can be introduced in association with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent TF bond of polymers, especially polyethylene glycol (PEG), has been used to protect some proteins from enzymatic hydrolysis in the body and thus prolong their half-life (Fuertges, F., et al., J. Controlled Delivery). 11:139, 1990). Many polymeric systems have been indicated for protein delivery (Bae, YH, et al., J. Controlled Delivery 9: 271, 1989; Hori, R., et al., Pharm. Res. 6: 813, 1989; Yamakawa, 1989; L., et al., J. Pharm. Sci. 79: 505, 1990; Yoshihiro, L., et al., J. Controlled Delivery 10: 195, 1989; Asano, M., et al., J. Controlled Delivery 9: 111, 1989; Rosenblatt, J., et al., J. Controlled Delivery 9: 195, 1989; Makino, K., J. Controlled Delivery 12: 235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78: 117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78: 219, 1989).
Recently, liposomes have been developed with improved serum stability and circulation half-lives (see, for example, U.S. Patent No. 5,741,516, to Webb). In addition, various liposome methods and liposome-like preparations as potential drug carriers have been reviewed (see, for example, US Patent No. 5,567,434 to Szoka; US Patent No. 5,552,157 to Yagi; US Patent No. 5,565,213 to Nakamori; US Patent No. 5,738,868 to Shinkarenko; and US Patent No. 5,795,587 to Gao). For transdermal applications, the “MASP-2” inhibitor antibodies and polypeptides may be combined with other suitable ingredients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration, and cannot degrade the activity of the active ingredients of the composition. Examples of suitable carriers include ointments, creams, gels, or suspensions, with or without purified collagen. MASP-2 inhibitor antibodies and polypeptides may also be impregnated into transdermal patches, patches, and bandages, preferably in liquid or semi-liquid form.
E The compositions of the present invention can be
- 79 systemically administered on a periodic basis at set intervals to maintain a desired level of therapeutic effect. For example, the compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. These factors will include the degree of progress of the condition being treated, the age, sex and weight of the patient, and other clinical factors. The dosage for each individual agent will vary as a function of the MASP-2 inhibitory antibody that is included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained-release delivery vehicle). In addition, the dosage amount can be adjusted to account for the variation in the frequency of administration and the pharmacokinetic behavior of the agent(s) delivered.
LOCAL DELIVERY As used herein, the term “local” encompasses the application of a drug to or around a site of intended localized action, and may include, for example, topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavity, intracranial or intravesicular administration, placement or irrigation. Local administration may be preferred to allow for administration of a lower dose, to avoid systemic side effects, and to more precisely control the release time and concentration of active agents at the site of local release. A — local administration provides a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc. Improved dosing control is also provided through the direct release mode.
e Local release of a MASP-2 inhibitory antibody can
. 80 may be obtained in the context of surgical methods to treat a disease or condition, such as, for example, during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasonic procedures, balloon angioplasty and stent placement. For example, a MASP-2 inhibitor can be administered to a subject in conjunction with a balloon angioplasty procedure. A balloon angioplasty procedure involves inserting a catheter having a deflated balloon into an artery. The deflated balloon is positioned in close proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the vascular wall. As a result, the surface of the balloon is in contact with the vascular endothelial cell layer on the surface of the blood vessel. The MASP-2 inhibitor antibody can be attached to the balloon angioplasty catheter in a manner that allows for the release of the agent at the site of the atherosclerotic plaque. The agent may be attached to the balloon catheter in accordance with standard procedures known in the art. For example, the agent can be stored in a balloon catheter compartment until the balloon is inflated, at which point it is released into the local environment. Alternatively, the agent can be impregnated onto the surface of the balloon such that the balloon communicates with the arterial wall cells as the balloon is inflated. The agent can also be delivered in a punctured balloon catheter such as that disclosed in Flugelman, MY, et al., Circulation 85: 1110-1117, 1992. See also PCT Published Application WO: 95/23161 for an exemplary procedure for attach a therapeutic protein to a balloon angioplasty catheter. Likewise, the MASP-2 inhibitor antibody can be included in a gel or polymeric coating applied to a stent, or it can be incorporated into the stent material, such that the stent elutes the MASP-2 inhibitor antibody after vascular placement.
Treatment Regimens: MASP-2 inhibitory antibody compositions used in the treatment of arthritis and other musculoskeletal disorders can be delivered locally via intra-articular injection. Such compositions may suitably include a sustained release vehicle. As another example of situations in which local delivery may be desired, MASP-2 inhibitory antibody compositions used in the treatment of urogenital conditions may suitably be instilled intravesically or within another urogenital structure.
In prophylactic applications, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk for, a condition associated with MASP-2 dependent complement activation in an amount sufficient to eliminate or reduce the risk of developing symptoms of the disease. condition. In therapeutic applications, pharmaceutical compositions are administered to a subject suspected of, or already suffering from, a condition associated with MASP-2 dependent complement activation in a therapeutically effective amount. 15 sufficient to alleviate, or at least partially reduce, the symptoms of the condition. In both prophylactic and therapeutic regimens, compositions comprising the MASP-2 inhibitor antibodies may be administered in various dosages until a sufficient therapeutic result has been obtained in the subject. Application of the -daMASP-2 inhibitor antibody compositions of the present invention can be accomplished by a single administration of the composition, or a limited sequence of administrations, for the treatment of an acute condition, for example, reperfusion injury or other traumatic injury. Alternatively, the composition may be administered at periodic intervals over an extended period of time for the treatment of chronic conditions, for example, arthritis or psoriasis.
The MASP-2 inhibitory compositions used in the present invention can be released immediately or shortly after an acute Y event that results in activation of the lectin pathway, such as following an E ischemic event and ischemic tissue reperfusion. Examples include myocardial ischemia reperfusion, renal ischemia reperfusion, cerebral ischemia reperfusion, organ transplantation, and finger/extremity reattachment. Other acute examples include septicemia. A MASP-2 inhibitory composition of the present invention can be administered as soon as possible following an acute event that activates the lectin pathway, preferably within twelve hours and more preferably within two to three hours of an activating event, such as via systemic release of the MASP-2 inhibitory composition. The methods and compositions of the present invention can be used to inhibit inflammation and related processes that typically result from medical and surgical diagnostic and therapeutic procedures. To inhibit such processes, the MASP-2 inhibitory composition of the present invention can be applied periprocedurally. As used herein] "periprocedurally" refers to administering the inhibitory composition preprocedurally and/or intraprocedurally and/or postprocedurally, i.e. before the procedure, before and during the procedure, before and after the procedure, before, during and after the procedure, during the procedure, during and after the procedure, or after the procedure. Periprocedural application can be accomplished by local administration of the composition to the surgical or procedural site, such as by continuous or intermittent injection or irrigation of the site, or by systemic administration. Suitable methods for perioperative delivery of MASP-2 inhibitor antibody solutions are disclosed in US Patent Nos.
6,420,432 granted to Demopulos and 6,645,168 granted to Demopulos. Suitable methods for site delivery of chondroprotective compositions including MASP-2 inhibitor antibodies are disclosed in PCT International Patent Application WO 01/07067 A2. Methods and compositions for the targeted systemic delivery of chondroprotective compositions including MASP-2 inhibitor antibodies are disclosed in PCT International Patent Application WO 03/063799 A2.
Dosages MASP-2 inhibitory antibodies can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2 dependent complement activation. A therapeutically effective dose refers to the amount of MASP-2 inhibitor antibody sufficient to result in amelioration of symptoms of the condition.
The toxicity and therapeutic efficacy of MASP-2 inhibitor antibodies can be determined by standard pharmaceutical procedures using experimental animal models, such as the African Green Monkey, as described herein. Using such animal models, the NOAEL (no observed adverse effect level) and MED (the dose is minimally effective) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is expressed as the NOAEL/MED ratio. MASP-2 inhibitory antibodies that exhibit large therapeutic ratios or indices are most preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the MASP-2 inhibitor antibody preferably lies within a range of circulating concentrations that include MED with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used.
For “any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose can be formulated in an animal model to obtain a range of circulating plasma concentrations that includes MED. Quantitative levels of MASP-2 inhibitor antibody in plasma can also be measured, for example, by high performance liquid chromatography.
In addition to toxicity studies, effective dosage can also be estimated based on the amount of MASP-2 protein present in a living subject and the binding affinity of the MASP-2 inhibitor antibody. It has been shown that MASP-2 levels in normal human subjects are present in serum at low levels in the range of 500 ng/ml, and MASP-2 levels in a particular individual can be determined using a quantitative assay for MASP-2 described. in Moller-Kristensen M., et al., J.
Immunol.
Methods 282: 159-167, 2003, hereby incorporated by reference.
In general, the dosage of administered compositions comprising the MASP-2 inhibitor antibodies varies depending on factors such as the age, weight, height, sex, general medical condition, and prior medical history of the individual.
As an illustration, MASP-2 inhibitor antibodies can be administered in the dosage ranges of from about 0.010 to 10.0 mg/kg, preferably from 0.010 to 1.0 mg/kg, more preferably from 0.010 to 0.1 mg/kg of the subject's body weight.
The therapeutic efficacy of MASP-2 inhibitory compositions and methods of the present invention in a given individual, and the appropriate dosages, can be determined in accordance with complement assays well known to those of skill in the art.
The add-on generates numerous specific products.
During the last decade, sensitive and specific assays have been developed and are commercially available for most of these activation products, which include the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays use antibodies that react with new antigens (neoantigens) exposed on the fragment, but not on the native proteins from which they are formed, making these assays very simple and specific. Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the main forms found in the circulation. Unprocessed fragments and C5agesarç are rapidly cleared by binding to cell surface receptors and are therefore present in very low concentrations, whereas C3agesaroe Dão binds to cells and accumulates in plasma. Measurement of C3a provides a sensitive indicator, independent of the —complement activation pathway. Activation of the alternative pathway can be assessed by measuring the Bb fragment. Detection of the fluid phase product of membrane attack pathway activation, sC5b-9, provides evidence that complement is being activated to completion. Because both lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of these two pathways generated the activation products. Inhibition of MASP-2-dependent complement activation is distinguished by at least one of the following changes in a component of the complement system that occur as a result of administration of an anti-MASP-2 antibody in accordance with the present invention: the inhibition of the generation or production of complement activation dependent on the MASP-2 C4b, C3a, C5a and/or C5b-9 (MAC) system products, the reduction of C4 cleavage and C4b deposition, or the reduction of C3 cleavage and deposition of C3b.
Articles of Manufacture In another aspect, the present invention provides an article of manufacture that contains a human MASP-2 inhibitory antibody, or antigen-binding fragment thereof, as described herein in a unit dosage form suitable for therapeutic administration. to one. human subject, such as, for example, a unit dosage in the range of
1mg to 5000mg, such as 1mg to 2000mg, such as 1mg to 1000mg, such as 5mg, 10mg, 50mg, 100mg, 200mg, 500mg, or 1000mg.
In some embodiments, the article of manufacture comprises a container and a label or packaging insert within or associated with the container.
Suitable containers include, for example, bottles, vials, syringe, etc.
Containers can be formed from a variety of materials such as glass or plastic.
The container holds a composition that is effective to treat the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the MASP-2 inhibitory antibody or antigen-binding fragment thereof of the invention.
The label or package insert indicates that the composition is used to treat the particular condition.
The label or packaging insert will further comprise instructions for administering the antibody composition to the patient.
Articles of manufacture and kits comprising combinatorial therapies described herein are also considered.
Therapeutic Uses of Anti-MASP-2 Inhibitory Antibodies In another aspect, the invention provides a method for inhibiting MASP-2 dependent complement activation in a human subject which comprises administering a human monoclonal anti-MASP-2 inhibitory antibody of the invention in an amount sufficient to inhibit MASP-2 dependent complement activation in said human subject.
In accordance with this aspect of the invention, as described in Example 10, the MASP-2 inhibitory antibodies of the present invention are capable of inhibiting the lectin pathway in African Green monkeys following intravenous administration.
As shown in Table 24, Example 8, the antibody already used in this study, OMS646, was found to be more potent in human serum.
As known to those of skill in the art, non-human primates are often used as a model for evaluating therapeutic antibodies. As described in US Patent No. 7,919,094, copending US Patent Application Serial No. 13/083,441, and copending US Patent Application Serial No. 12/905,972 (each of which is granted to Omeros Corporation, the assignee of the present application), each of which is hereby incorporated by reference, MASP-2 dependent complement activation has been implicated as contributing to the pathogenesis of numerous acute and chronic disease states, which include MASP-2 dependent complement, an ischemic reperfusion injury, atherosclerosis, an inflammatory gastrointestinal disorder, a lung condition, an extracorporeal reperfusion procedure, a musculoskeletal condition, a kidney condition, a skin condition, an organ transplant or nervous system tissue, disorder or injury, a blood disorder, a urogenital condition, diabetes, chemotherapy or radiation therapy, malignancy, an endocrine disorder, a clotting, or an eye condition. Therefore, the MASP-2 inhibitor antibodies of the present invention can be used to treat the diseases and conditions given as reference above.
As further described in Example 11, the MASP-2 inhibitor antibodies of the present invention are effective in treating a mammalian subject at risk for, or suffering from the deleterious effects of, acute radiation syndrome, thereby demonstrating therapeutic efficacy in vivo.
The examples that follow merely illustrate the best mode — now considered to practice the invention, but should not be construed as limiting the invention.
EXAMPLE 1 This example describes the recombinant expression and production of full-size recombinant human, rat, and murine MASP-2 protein, MASP-2-derived polypeptides, and catalytically inactivated mutant forms of MASP-2 Expression of MASP-2 human and mouse full-length cDNA sequence: The human MASP-2 full-length cDNA sequence (SEQ ID NO: 1) encoding the human MASP-2 polypeptide with the leader sequence (SEQ ID NO: 2) was subcloned in the pCI-Neo (Promega) mammalian expression vector, which directs eukaryotic expression under the control of the CMV enhancer/promoter region (described in Kaufman R. 5.
et al, Nucleic Acids Research 19: 4485-90, 1991; Kaufman, Methods in Enzymology, 185: 537-66 (1991)). Full-size rat MASP-2 cDNA (SEQ ID NO: 4) encoding rat MASP-2 polypeptide with leader sequence (SEQ ID NO: 5) was subcloned into the pED expression vector.
: MASP-2 expression vectors were then transfected into the adherent DXB1 Chinese hamster ovary cell line using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, implying that the encoded protease is cytotoxic. The mature form of the human MASP-2 protein (SEQ ID NO: 3) and the mature form of the mouse MASP-2 protein (SEQ ID NO: 6) were secreted into the culture medium and isolated as described below.
Expression of catalytically inactive full-size MASP-2: Background Analysis: MASP-2 is activated by autocatalytic cleavage after recognition of the binding of MBL subcomponents, type C lectin CL-11, or ficolins (L-ficolin, H-ficolin or M- Ficolin) collectively alluded to: like lectins, bind to their respective carbohydrate patterns. Ç autocatalytic cleavage that results in MASP-2 activation often
:89 occurs during the isolation procedure of MASP-2 from serum, or during purification following recombinant expression.
In order to obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MASP-2, devised as MASP-2A, was created by — replacing the serine residue that is present in the catalytic triad of the protease domain with an alanine residue in the mature mouse MASP-2 protein (SEQ ID NO: 6 Ser617 to Ala617); or mature human MASP-2 protein (SEQ ID NO: 3 Ser618 to Ala618). In order to generate catalytically inactive human and mouse MASP-2A proteins, site-directed mutagenesis was performed as described in US2007/0172483, hereby incorporated by reference.
PCR products were purified after agarose gel electrophoresis and lane preparation and unique adenosine overlays were generated using a standard tailing procedure.
MASP-2A. The 15th adenosine tail was then cloned into the pPGEM-T easy vector, transformed into E. coli.
The human and mouse MASP-2A were each further subcloned into pED or pCI-Neo mammalian expression vectors and transfected into the Chinese hamster ovary cell line DXB1 as described below.
Construction of Expression Plasmids Containing Polypeptide Regions Derived from Human MASP-2.
The following constructs were produced using the MASP-2 signal peptide (residues 1 to 15 of SEQ ID NO: 2) to secrete various domains of MASP-2. A construct expressing Oo — CUBI domain of human MASP-2 (SEQ ID NO: 7) was made by PCR amplifying the region encoding residues 1 to 121 of MASP-2 (SEQ ID NO: 3) (which corresponds to the N-terminal CUBI1 domain). A construct expressing the CUBI/EGF domain of human MASP-2 C (SEQ ID NO: 8) was made by PCR which amplifies the region encoding
: 90 residues 1 to 166 of MASP-2 (SEQ ID NO: 3) (which corresponds to the N-terminus of the CUBI/EGF domain). A construct expressing the CUBI/EGF/CUBII domain of human MASP-2 (SEQ ID NO: 9) was made by PCR amplification of the region encoding amino acid residues 1 to 277 of MASP-2 (SEQ ID NO: 3) (which corresponds to the N-terminal CUBIEGFCUBII domain). A construct expressing the EGF domain of human MASP-2 (SEQ ID NO: 10) was made by PCR amplification of the region encoding amino acid residues 122-166 of MASP-2 (SEQ ID NO: 3) (which corresponds to the EGF domain). A construct expressing the CCPI/CCPII/SP domains of human MASP-2 (SEQ ID NO: 11) was made by PCR amplification of the region encoding amino acid residues 278-671 of MASP-2 (SEQ ID NO: 3) (which corresponds to the CCPI/CCPII/SP domains). A construct expressing the CCPI/CCPIHI domains of human MASP-2 (SEQ ID NO: 12) was . 15 made by PCR amplification of the region encoding amino acid residues 278-429 of MASP-2 (SEQ ID NO: 3) (which corresponds to the CCPI/CCPII domains). A construct expressing the CCPI domain of MASP-2 (SEQ ID NO: 13) was made by PCR amplification of the region encoding amino acid residues 278-347 of MASP-2 (SEQ ID -NO:3) (which corresponds to to the CCPI domain). A construct expressing the CCPII/SP domains of MASP-2 (SEQ ID NO: 14) was made by PCR amplification of the region encoding amino acid residues 348-671 of MASP-2 (SEQ ID NO: 3) (which corresponds to the CCPII/SP domains). A construct expressing the CCPII domain of MASP-2 (SEQ ID NO: 15) - was made by PCR amplification of the region encoding amino acid residues 348-429 of MASP-2 (SEQ ID NO: 3) (which corresponds to the domain CCPII). A construct expressing the SP domain of MASP-2' (SEQ ID NO: 16) was made by PCR amplification of the region encoding amino acid residues 429-671 of MASP-2 (SEQ ID NO: 3)
: 91 (which corresponds to the SP domain).
The MASP-2 domains mentioned above were amplified by PCR using Ventrg polymerase and pBS-MASP-2 as a standard, according to established PCR methods. The 5th primer sequence of the sense primer introduced a BamHI restriction site (underlined) at the 5th end of the PCR products. The antisense primers for each of the MASP-2 domains were designed to introduce a stop codon followed by an EcoRI site at the end of each PCR product. Once amplified, the DNA fragments were digested with BamHI and EcoRI and cloned into the corresponding sites of the pFastBac11 vector. The resulting constructs were distinguished by restriction mapping and confirmed by dsDNA sequencing.
Recombinant eukaryotic expression of MASP-2 and production of enzymatically inactive mouse and human MASP-2A protein.
: 15 The MASP-2 and MASP-2A expression constructs described above were transfected into DXB1 cells using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that the preparations were not contaminated with other serum proteins. Medium was harvested from confluent cells every second day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium for each of the two species.
MASP-2A protein purification: MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on columns —deMBP-A-agarose. This strategy allowed rapid purification without the use of extraneous labels. MASP-2A (100 to 200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl7) was loaded onto a column of MBP- and agarose affinity (4 ml) pre-equilibrated with 10 ml of loading buffer. Following washing with an additional 10 ml of loading buffer, the protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5, containing 1.25 M NaCl and 10 MM EDTA. Fractions containing MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. Where necessary, MASP-2A was further purified by exchange chromatography. ion on a MonoQ column (HR 5/5).The protein was dialyzed with 50 mM Tris-Cl pH 7.5, containing 50 MM NaCl and loaded onto the column equilibrated in the same buffer. Following washing, MASP- Bound 2A was eluted with a gradient from 0.05 to 1M NaCl in 10 ml.
Results: Yields of 0.25 to 0.5 mg MASP-2A protein were obtained from 200 ml of medium. The molecular mass of 77.5 kDa determined by MALDI-MS is greater than the calculated value of the unmodified polypeptide (73.5 kDa) due to glycosylation. The binding of 'glycans at each of the N-glycosylation sites is responsible for the mass observed. MASP-2A migrates as a single band on SDS-polyacrylamide gels, which demonstrate that it is not proteolytically processed during biosynthesis. The weight average molecular mass determined by equilibrium ultracentrifugation agrees with the calculated value for the glycosylated polypeptide homodimers.
EXAMPLE 2 This example describes the screening method used to identify high affinity fully human anti-MASP-2 scFv antibody that blocks MASP-2 functional activity for progression in affinity maturation.
Background and Rationale: MASP-2 is a complex protein with many functional domains of separation, including: binding site(s) for MBL and Ficolins, a serine protease catalytic site, a binding site for the C2 proteolytic substrate, a binding site for the C4 proteolytic substrate, a site
- 93 of MASP-2 cleavage for MASP-2 zymogen autoactivation, and two Ca binding sites”. The scFV antibody fragments were identified that bind with high affinity to MASP-2, and the identified Fab2 fragments were tested in a functional assay to determine whether S — they were able to block the functional activity of MASP-2.
To block the functional activity of MASP-2, an antibody or scFv or Fab2 antibody fragment must bind and interfere with a structural epitope on MASP-2 that is required for the functional activity of MASP-2. Therefore, many or all of the binding of high-affinity anti-MASP-2 scFvs or Fab2s may not inhibit the functional activity of MASP-2 unless they bind to structural epitopes on MASP-2 that are directly involved in the functional activity of MASP-2. MASP-2.
A functional assay that measures inhibition of C31 convertase formation of the lectin pathway was used to assess "blocking activity". 15. of antic-MASP-2 scFvs. It is known that the primary physiological role of MASP-2 in the lectin pathway is to generate the next functional component of the lectin-mediated complement pathway, namely the C3 convertase of the lectin pathway. The C3 convertase of the lectin pathway is a critical enzyme complex (C4b2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the C3 convertase of the lectin pathway (C4b2a); however, the functional activity of MASP-2 is required in order to generate the two protein components (C4b, C2a) that comprise the C3 convertase of the lectin pathway. Furthermore, all of the separate functional activities of MASP-2 listed above appear to be required in order for MASP-2 to generate the C3 convertase from —viadalectin. For these reasons, a preferred assay for use in evaluating the "blocking activity" of anti-MASP-2 Fab2s and scFvs antibody fragments is believed to be a functional assay that measures inhibition of C3 convertase pathway formation. of the lectin. h The target profile for anti-MASP-2 therapeutic antibodies predicted to produce >90% removal of the lectin pathway in vivo following administration of 1 mg de/kg to a human is an ICs9 < 5 nM in 90% of plasma. The relationship between in vitro pharmacological activity in these assay formats and in vivo pharmacodynamics has been experimentally validated — using anti-rodent MASP-2 antibodies.
Criteria for selection of first-generation MASP-2 blocking antibodies for therapeutic use were as follows: high affinity for MASP-2 and functional values of ICs, down to -25 nM. In addition, candidates were screened for cross-reactivity with primate-non-human serum and with mouse serum.
Methods: Screening of scFv phagemid library against MASP-2 antigen. Human MASP-2A with a 5X His N-terminal tag, and mouse MASP-2A with 6X His N-terminal tags were generated using the reagents described in Example | and purified from the culture supernatants by a nickel affinity chromatograph as previously described ( Chen et al., J. Biol. Chem. 276: 25894-02 (2001 )).
OMS100, an anti-human MASP-2 antibody in Fab2 format, was used as a positive control to bind MASP-2.
Description of Phagomid Library: A phage display library of human light immunoglobulin and heavy chain variable region sequences were — subjected to antigen screening followed by automated antibody screening and selection to identify the high-affinity scFv antibodies to mouse MASP-2 protein and human MASP-2 protein.
Ê Screening Methods: & Overview: Two screening strategies were used to isolate phage from the phagemid library that bind to MASP-2 in a total of three rounds of screening. Both strategies involved sorting in solution and removing MASP-2 bound phage. MASP-2 was immobilized on the magnetic beads via His-tag (using NINTA beads) or via a biotin (using streptavidin beads) on the target.
The first two screening cycles involved an alkaline elution (TEA), and the third screening cycle was first eluted competitively with MBL before a conventional alkaline elution (TEA) step. Negative selection was performed before cycles 2 and 3, and this was against the functional analogues, Cls and Clr, of the classical complement pathway. After screening, the specific enrichment of phages with scFv fragments against MASP-2A was monitored, and it was determined that the screening strategy was successful (data not shown).
The scFv genes from round 3 screen were cloned into a pHOG expression vector, and operated on a small scale filter screen to look for clones specific to MASP-2A, as also described below.
TABLE 7: Phage Screening Methods (biotin/streptavidin) o pm A E ua scho | E E AA amen Peti, Mer Jena. | competition Bam moto EE EA (alkaline) TABLE 8: Phage Screening Methods (HIS/NiNTA)
Screening Reagents: Human MASP-2A WHO antibody 100 (positive control) Goat anti-human IgG (H+L) (Pierce 431412) S NINTA beads (Qiagen XLB13267) Streptavidin Dynabeads” M-280, 10 mg/ml ( LB12321) Normal human serum (LB 13294) Polyclonal rabbit anti-human IgG C3c (LB13137) Goat anti-rabbit IgG, HRP (American Qualex XA102PU) To test for the targeted MASP-2A antigen, an experiment was performed to capture the OMS 100 positive control antibody (200 ng/ml) pre-incubated with biotin-labeled MASP-2A or HIS-labeled MASP-2A antigen (10 µg), with 50 µl NINTA beads in PBS with 4% milk or 200 ul of Estreptavidin beads, -15 respectively.
But bound MASP-2A-OMSI100 antibody was detected with goat anti-human IgG (H+L) HRP (1:5000) and TMB substrate (3,3',5,5'-tetramethylbenzidine). NINTA Beads ELISA Assay 50 µl of NINTA beads were blocked with 1 ml of 4% milk in phosphate-buffered saline (PBS) and incubated on a rotating wheel for | hour at room temperature.
In parallel, 10 µg of MASP-2A and OMS100 antibody (diluted to 200 ng/ml in PBS with 4% milk) were pre-incubated for one hour.
The pearls were then washed three times with | ml of PBS-T using a magnet between each step.
MASP-2A — pre-incubated with OMS 100 antibody was added to the washed beads.
The mixture was incubated on a spinning wheel for 1 hour at room temperature, then washed three times with 1 ml of PBS-T using a magnet as S described above.
The tubes were incubated for 1 hour at room temperature and with goat anti-human IgG (H+L) HRP diluted 1:5000 in PBS with 4%
- 97 of milk. For negative controls, goat anti-human (H+L) HRP IZG (1:5000) was added to washed and blocked Ni-NTA beads in a separate tube.
The samples were incubated on a rotating wheel for 1 hour — at room temperature, then washed three times with 1 ml of PBS-T and once with | x PBS using the magnet as described above. 100 µl of TMB substrate was added and incubated for 3 minutes at room temperature. The tubes were placed on a magnetic shelf for 2 minutes to concentrate the beads, then the TMB solution was transferred to a microtiter plate and the reaction stopped with 100 µl of 2M H2SO4. The absorbance at 450 nm was read on the ELISA beds.
Streptavidin Beads in ELISA Assay This assay was performed as described above for ELISA Assay with NINTA Beads, but instead using 200 µl of '15 Streptavidin Beads per sample, not biotinylated antigens.
Results: His-labeled and biotin-labeled MASP-2A antigen, pre-incubated with the OMS 100 positive control antibody, were each successfully captured with the NINTA beads, or streptavidin beads, respectively.
Screening Three rounds of screening the scFv phage library against HIS-labeled or biotin-labeled MASP-2A were performed as shown in TABLE 7 or TABLE 8, respectively. The third screening cycle was eluted first with MBL, then with TEA (alkaline). To monitor the specific enrichment of scFv fragments displaying phage against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was performed as described below.
: MASP-2A ELISA on polyclonal phage enriched after it's Screening
After three rounds of screening the scFv phage library against human MASP-2 as described above, specific enrichment of the phage with scFv fragments against the target MASP-2A was monitored by performing an ELISA on the phage populations enriched polyclonal antibodies generated by screening against immobilized MASP-2A as described below.
Methods: 5 ng/ml of MASP-2A was immobilized on maxisorp ELISA plates in PBS overnight at 4°C.
Packed phage from all three rounds of sorting were diluted 1:3 in PBS with 4% milk and titrated with 3-fold dilutions.
The negative control was an M13 helper phage. The block was PBS with 4% milk.
Plates were washed 3x in 0.05% (v/v) PBS-Tween between each step.
The primary antibody E was rabbit a-fd (M13 coat protein), 1:5000 in PBS with 4:15% (w/v) treat. The conjugate was 1:10,000 rabbit α-Goat-HRP in PBS with 4% milk (w/v). The substrate was ABTS.
All volumes, except washes and blocks, were 100 ul/reservoir.
All incubations were 1 hour with shaking at room temperature.
Results: Phage ELISA results showed specific enrichment of scFv against MASP-2A for both screening strategies.
See FIGURE 2. As shown in FIGURE 2, the strategy involving magnetic bead capture of NIiNTA provided enrichment of scFv on MASP-2A phage after two rounds of panning, whereas both strategies had good enrichment. in both competitive and TEA elution after the third screening cycle.
The negative control phage was an M13 helper phage, which did not cross-react against: MASP-2A at its lowest dilution.
These results demonstrate that the signal observed is due to the scFv specifically binding to MASP-2A.
J Filter Screening: Colonies of bacteria containing plasmids encoding the scFv fragments from the third round of screening were selected on the crosshatched nitrocellulose membranes and grown — overnight in a non-inducing medium to produce the plaques main. A total of 18,000 colonies were selected and analyzed from the third screening cycle, half from the competitive elution and half from the subsequent TEA elution.
Nitrocellulose membranes with bacterial colonies — were induced with IPTG to express and secrete a soluble scFv protein and were contacted with a secondary nitrocellulose membrane coated with MASP-2A antigen along with a parallel membrane coated with 4% PBS of milk (blocking solution).
: ScFvs that bound to MASP-2A were detected by . 15 of their c-Myc label with Mouse a-cMyc mAb and a-Rabbit Mouse HRP. Hits corresponding to scFv clones that were positive in MASP-2A and negative in Milk-PBS were selected for further expression, and a subsequent ELISA analysis.
Results: Screening of the scFv phagemid library against MASP-2A followed by scFv conversion and a filter screen produced 137 positive clones. Most positive clones came from competitive elution with MBL, using both NINTA and streptavidin strategies. All positive clones were continued with micro expression (200 µl scale) and subsequent extraction. ScFvs were isolated — from the periplasm of the bacteria by incubating the bacterial suspension with sucrose lysis buffer and lysosome for one hour, after which the supernatant was isolated by a centrifugation step. The supernatant containing scFv i secreted into the medium together with the periplasm contents was analyzed by: two assays: ELISA using physically adsorbed MASP-2A, and
. binding using amine-linked MASP-2A to a CMS chip on the Biocore, as described in more detail below.
MASP-2A ELISA on candidate ScFvy clones identified by scFvy screening/conversion and filter screen.
Methods: 4 µg/ml of MASP-2A were immobilized on maxisorp ELISA plates (Nunc) in PBS overnight at 4°C.
The next day, plates were blocked by washing three times with PBS-Tween (0.05%). Crude scFv material (100 µl of medium-periplasm extract) from each of the 137 scFv candidates (generated as described above) was added by reservoir to the plate.
Then, anti-cMyc was added, and in the final step, HRP-conjugated rabbit anti-mouse was applied to detect scFv binding.
The reaction was carried out in 1-step ABTS of peroxidase substrate (Calbiochem). The positive control was OMS100 (an antibody —antic-=MASP-2 in Fab2 format) diluted to 10 µg/ml in PBS-Tween 0.05%. The negative control was plasmid-free XL1-Blue half-periplasm.
Washes of 3 x 200 µl of PBS-Tween 0.05% (v/v) were performed between each step.
The primary antibody was murine a-cMyc, 1:5000 in PBS-Tween0.05% (w/v). The conjugate was α-Goat-rabbit HRP 1:5000 in PBS-Tween 0.05% (w/v) or goat anti-human IgG (H+L, Pierce 31412). The substrate was ABTS, incubated for 15 minutes at room temperature.
All volumes, except washes and blocks, were 100 ul/reservoir. —All incubations were for 1 hour with shaking at room temperature.
Results: 108/137 clones were positive in the ELISA assay (data not shown), of which 45 clones were re-analyzed as described below.
The positive control was OMS100 Fab2 r diluted to 10 µg/ml in PBS-Tween, and this clone was positive.
The negative control was plasmid-free XL 1-Blue half-periplasm, which was negative.
EXAMPLE 3 This example describes the MASP-2 functional screening method used to screen high affinity fully human anti-MASP-2 scFv antibody candidates for the ability to block MASP-2 activity in normal human serum.
Rationale/Rationale The Assay to Measure Inhibition of Lectin C3 Convertase Pathway Formation: A functional assay measuring inhibition of lectin C3 convertase pathway formation was used to assess the “blocking activity” of candidate anti scFv clones. -MASP-2. The lectin C3 convertase pathway is the enzyme complex (C4b2a) that proteolytically cleaves C3 into the two potent pro-inflammatory fragments, anaphylatoxin C3a and opsonic C3b.
The formation of C3 convertase appears to be a key step in the lectin pathway in terms of mediating inflammation.
MASP-2 is not a structural component of the C3 convertase of the lectin pathway (C4b2a); therefore, anti-MASP-2 (or Fab2) antibodies will not directly inhibit pre-existing C3 convertase activity.
However, MASP-2 serine protease activity is required in order to generate the two protein components (C4b, C2a) that comprise the C3 convertase of the lectin pathway.
Therefore, anti-MASP-2 scFv that inhibit the functional activity of MASP-2 (i.e., block anti-MASP-2 scFv) will inhibit de novo formation of the lectin C3 convertase pathway.
C3 contains an unusual and highly reactive thioester group as part of its structure. — Upon cleavage of C3 by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of plastic reservoirs by F via ester or amide bonds, thus facilitating detection of t C3b in the ELISA assay.
Mannan yeast is a known activator of the lectin pathway.
In the following method to measure C3 convertase formation, mannan-coated plastic wells were incubated with diluted human serum to activate the lectin pathway.
The wells were then washed and assayed for C3b immobilized in the wells using standard ELISA methods.
The amount of C3b generated in this assay is a direct reflection of de novo formation of the lectin C3 convertase pathway. Anti-MASP-2 scFv's at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and — consequent generation of C3b.
Methods: The 45 candidate clones identified as described in Example 2 were expressed, purified and diluted to the same * stock concentration, which was further diluted in Ca * and Mg * which contains GVB buffer (4.0 mM barbital , 141 mM NaCl, 1.0 mM MegCl 2 , 2.0 mM CaCl 2 , 0.1% gelatin, pH 7.4) to ensure they all had the same amount of buffer.
The scFv clones were each tested in triplicate at a concentration of 2 µg/ml.
The positive control was OMS100 Fab and was tested at 0.4 ug/ml.
C3c formation was monitored in the presence and absence of scFv/IgG clones.
Mannan was diluted to a concentration of 20 µg/ml (1 µg/well) in 50 mM carbonate buffer (15 mM Na2CO; + 35 MM NaHCO; + 1.5 mM NaN;), pH 9.5 and coated on an ELISA plate overnight at 4°C.
The next day, the mannan coated plates were washed 3X with 200 µl PBS. 100 µl of 1% HSA blocking solution was then added to the wells and incubated for 1 hour at room temperature.
Plates were washed 3X with 200 µl of PBS, and stored on ice with 200 µl of PBS until samples were added. & Normal human serum was diluted to 0.5% in CaMg from
- 103 t GVB buffer, and the scFv clones or OMS 100 Fab2 positive control were added in triplicates at 0.01 ng/ml; 1 µg/ml (OMS100 control only) and 10 µg/ml to this buffer and pre-incubated 45 minutes on ice before the blocked ELISA plate. The reaction was started by incubating for one hour at 37°C and stopped by transferring the plates to an ice bath. C3b deposition was detected with an α-rabbit C3c antibody followed by α-rabbit Goat HRP. The negative control was buffer without antibody (no OMS100 = maximum deposition of C3b), and the positive control was buffer with EDTA (no deposition of C3b). Background was determined by performing the same assay, but in negative mannan reservoirs. The background signal against the mannan-free plates was subtracted from the positive mannan signals. A cutoff criterion was set at half the activity of an irrelevant scFv clone (VZV) and buffer alone.
Results: Based on the cut-off criteria, a total of 13 clones were found to block MASP-2 activity as shown in FIGURES 3A and 3B. All 13 clones produced > 50% of pathway deletion and were selected and sequenced, yielding 10 unique clones, as shown below in TABLE 9. The ten different clones shown in TABLE 9 were found to result in adequate functional activity in the complement. All ten clones were found to have the same light chain subclass, X3, in addition to the three different heavy chain subclasses, VH2, VH3, and VH6. The clone's sequence identity to the germline sequences is also shown in the TABLE 9.
TABLE 9:10 Unique Clones with Functional Anti-MASP-2 Activity Identity of Identity ires] in Je Ie] So EE o Eae POC eee E e e Ee Ba EE Eres feembrr bp ds ba | BRCCEC E fee In fue ess E pm
- 104 E Streptavidin Komp —“NH6 —b63 ——bh3 b3ss | ss eee he esa J16L13/4F2) ESET Streptavidin Comp — fvH2 fon ogss BIBIZ E PO NNTA EA €NA3 bo3l —h3 be | PRI NNTA kom NH hoo — h3 hbs34 | lStreptavidin [TEA/CompvH6 — bo66 ——hbh3 ——b7r8s5 | ma E E Eee | bo fo po be As shown above in TABLE 9, 10 different clones with acceptable functional activity and unique sequences were chosen for further analysis.
As seen in TABLE 9, some of the clones were detected two or three times, based on identical sequences (see — first column of TABLE 9 with clone names). Expression and purification of ten candidate svFc clones The ten candidate clones shown in TABLE 9 were expressed on a liter scale and purified by means of ion exchange in nickel chromatography.
After a sample of each clone has been — run on a chromatography or size exclusion column to assess the content of monomers and dimers.
As shown below in TABLE 10, almost all scFv clones were present in the monomeric form, and this monomeric fraction was isolated for further testing and sorting.
TABLE 10: Monomer Content Analysis Name of eae Monomer Bot ee e oH EP ge 17L20 18L16 Monomeric test fraction for binding and functional activity The clones shown in TABLE 10 were expressed at del 1 scale, purified by metal chromatography and exchange of ions, separated into monomeric fraction by size exclusion chromatography “20 (SEC) and functional assays were repeated to determine ICs values,
E 105 and cross-reactivity.
Functional assay on monomeric fractions: The monomeric fraction of the top ten clones, shown in TABLE 10, was purified and tested for functional ICsy nM in a — dilution series where each received the same concentration of GVB buffer with calcium, magnesium and human serum. The scFv clones were tested at 12 dilutions in triplicate. The positive control was WHO 100 Fab2. C3b deposition was monitored in the presence and absence of antibody. The results are shown below in TABLE 11.
Binding Assay: The binding affinity KD was determined in two different ways for the purified monomeric fractions of the ten candidate scFv clones. MASP-2A was immobilized by amine binding to a CMS chip, or a fixed concentration of scFv (50 nM) was first captured with a high-affinity amine-linked a-cMyc antibody, and then a series of MASP concentration -2A in the solution was passed through the chip. The results are shown below in TABLE 11.
Results: TABLE 11: Summary of functional inhibitory activities (CIs9) and affinity —MASP-2 binding (Kp) for the ten candidate scFv clones tested in the monomeric state Inhibitory Activity in| — Binding affinity for — Human binding affinity Human serum MASP-2 human (immobilized) MASP-2 in solution | Clone name 1C50 (nM KD (nM KD (nM Da to A OEN O | Weighs sn On ae BIS CO gg 5a) BI TARA CORRO A | À a AR ass 8
Description of Results: As shown in TABLE 11, in the functional assay, five of the ten candidate scFv clones provided ICS0 nM values less than the target criterion of 25 nM using 0.5% human serum.
As described below, these clones were also tested in the presence of non-human primate serum and mouse serum to determine functional activity in another species. Regarding binding affinity, in the solution, all binding affinities were in the low or better nM range, whereas in the conventional assay with immobilized MASP-2, only two clones (4D9 and 17D20) had affinities in the nM range. low. The observation of higher affinities in the base assay solution is similar to a result of the fact that the antigen multimerizes when in solution. Furthermore, when the target is immobilized on the chip (via targeted binding) the epitope can be masked, thus reducing the affinities seen in the immobilized assay.
EXAMPLE 4 This example describes the results of testing the ten candidate human anti-MASP-2 scFv clones for cross-reactivity with mouse —“MASP-2” and determining the IC50 values of these scFv clones in a functional assay to determine their ability to inhibit MASP-2 dependent complement activation in human serum, non-human primate serum, and rat serum.
Methods: Cross-reactivity with mouse MASP-2 The ten candidate scFv clones, shown in TABLE 9 of Example 3, were tested for cross-reactivity against mouse MASP-2A in a standard ELISA assay against mouse MASP-2A & adsorbed . Rat MASP-2A was diluted to 4 µg/ml in PBS and coated bh: on a Maxisorp ELISA plate (Nunc) overnight at 4°C.
The next day, the plate was blocked by washing three times in PBS-Tween (0.05%). ScFv clones (100 µl) diluted at 20 µg/ml in PBS-Tween were added to the plate, and titrated again with 3-fold 4-fold dilutions.
Specific MASP-2A svFc clones (reservoirs that contain bound scFv) were detected with anti-cMyc and rabbit anti-mouse HRP secondary antibody.
The reaction was carried out on TMB peroxidase substrate (Pierce). The positive control was WHO 100 Fab2 diluted to 10 µg/ml in PBS-Tween.
All clones tested cross-reacted with mouse MASP-2A, which was expected as the second round of screening was with mouse MASP-2 (data not shown). Functional characterization of ten candidate scFv clones in human serum, non-human primate (NHP) serum and mouse serum Determination of baseline C3c levels in Different Sera First, an experiment was performed to compare the baseline C3b levels base on the three sera (human, rat and NHP) as follows.
Mannan was diluted to 20 µg/ml and coated on an ELISA plate overnight at 4°C.
The next day the reservoirs were blocked with 1% HSA.
Normal human, rat and African green monkey (non-human primate “NHP”) serum was diluted starting at 2% 2-fold dilutions in a buffer of CaMgGVB.
The reaction was started by incubating for one hour at 37°C, and then stopped by transferring the plate to an ice bath.
C3b deposition was detected with a rabbit anti-mouse C3c antibody followed by goat anti-rabbit HRP.
The negative control was an antibody-free buffer (no. OMS 100 results in maximal C3b deposition) and the positive control for inhibition was EDTA buffer (no C3b deposition).
AND FIGURE 4 graphically illustrates baseline C3c levels in the three sera (human, rat and NHP). As shown in FIGURE 4, C3c levels were very different in the different sera tested.
When comparing C3c levels, it appears that 1% human serum provided levels equivalent to 0.2% NHP and 0.375% rat serum.
Based on these results, serum concentrations were normalized so that the scFv results can be directly compared in the three different types of sera.
Functional Assay of ScFv Clones in Different Sera
The purified monomeric fractions of the ten candidate scFv clones were then tested for functional ICso nM in human serum, mouse serum and African green monkey (non-human primate "NHP") serum. The assay was performed as described in Example 3, using 1000 nM purified scFv protein and normal human serum that was diluted to 0.9% in CAMgGVB buffer; African green monkey serum diluted to 0.2% in a buffer of CaMgGVB; or rat serum diluted to 0.375% in CAMgGVB buffer.
All ten scFv clones were tested in a dilution series in which they received the same concentration of GVB buffer with calcium, magnesium and serum.
The scFv clones were tested at twelve dilutions in triplicate.
The positive control was WHO 100 Fab2 at 100 ng/ml or addition of EDTA to the reaction.
The negative control was an irrelevant scFv control or PBS without any scFv.
C3b deposition was monitored in the presence and absence of scFv or Fab2 antibody. The background signal of OMS100 at 100 ng/ml was — subtracted from all signals.
TABLE 12 summarizes the results of the functional assays on all three sera.
: 109 S TABLE: 12: Functional ICs9 activity (nM) of scFv clones in three different types of serum.
Serum Serum Serum Frimeta no | Primate no Serum No Clone name human human human human human mouse Exptl* | Expt2 | expf3 | Expid If Exnot | EXPH 18P15 4D9 BO o lero fe farofa fe 7 17L20 ND —hos3 —Bogr —ambiguous hogo — hi74 hoo7 | 413 Bee actions 65232 6351 6560 76 ND | GDP ND ———hb373 Bsa h347 6383 —BbB664 ND | erI3 bo —joos iz28 —bacs boo 971 ND NG hsa bs —ba7zg how bss7 be78 ND | BF22(18C15) pos —B673 —bi4o br —ha42 h78 ND | Note: * the first human serum dataset (Exp *1) was done on scFv samples that were not concentrated, therefore, clones with low concentration cannot be fully titrated. In the remaining experiments, all clones were concentrated and titrations started at identical concentrations. Summary of results for functional activity in candidate scFv clones in different sera: All ten of the scFv clones showed function in both human and non-human primate (NHP) serum after the serum was normalized with respect to C3b deposition levels . The six most active clones in human serum were: 9P13>17N16>17D20>4D9>3F22>18L16, when ranked from best to worst. In the NHP serum, the clones ranked (best to worst): 17L20>17N16>17D20>9P13>18L16>3F22. Both 17N16 and 17D20 ranked in the top three in both human and NHP serum. 17D20 also showed some activity in mouse serum. Based on these results, the top three scFv clones were determined to be: 18L16, 17D20 and 17N16. These three clones were also analyzed in diluted human serum (1% serum) as shown below FP in TABLE 13.
: 110 h TABLE 13: C3 assay of the three candidate clones: (IC; nM) in diluted serum (1 %) Expo Pano NhoaM IN Expo AMO ba banM Eexpta BonM O É6M brnã “1 average bars Beras as Exp No SAM ksnM Exp AR a RA san FIGURE 5A is an amino acid sequence alignment of full-length scFv clones 17D20, 18L16, 4D9, 17120, 17N16, -3F22e9PI3. The seFv clones comprise a heavy chain variable region (aal-120), a linker region (aal21-145), and a light chain variable region (aa 146-250). As shown in FIGURE 5A, alignment of the heavy chain region (residues 1-120) of the most active clones reveals two different groups that belong to the VH2 and VH6 gene family, — respectively. As shown in FIGURE 5A, the VH region with respect to the VH2 class clones: 17D20, 18L16 and 4D9 has a variability at 20 aa positions in the region of 120 total amino acids (i.e. 83% identity). As also shown in FIGURE 5A, the VH region with respect to the VH6 class clones: 17L20, 17N16, 3F22, and 9P13, has a variability at 18 aa positions in the region of 120 total amino acids (i.e. 85% identity) . FIGURE 5B is a sequence alignment of scFv clones 17D20, 17N16, 18L16 and 4D9. TABLE 14: Sequence of candidate ScFv clones shown in FIGURESSAeSB
DI SEQID NOS é r The ranking priorities were (1) functional power
: 111 human serum and total block; (2) NHP cross-reactivity and (3) sequence diversity. 17D20 and 1I7N16 were selected as the best representatives of each genetic family. 18L16 was selected as the third candidate with appreciable CDR3 sequence diversity.
17N16 and 17D20 were the top two choices due to full functional blocking, with the best functional potencies against humans; appreciable monkey cross-reactivity and different VH gene families. 3F22 and 9P13 were deleted due to VH sequences — nearly identical to 17N16. 18P15, 4J9 and 21B17 were eliminated due to modest power. 17L20 was not continued because it only partially blocked.
18L16 and 4D9 had similar activities and appreciable diversity compared to 17D20. 18L16 was chosen because of greater primate cross-reactivity than 4D9.
Therefore, based on these criteria: three clone mothers follow: 17D20, 17N16 and 18L16 were superior in affinity maturation as described below.
EXAMPLE 5 This Example describes cloning three mother clones 17D20, 17N16 and 18L16 (identified as described in Examples 2 to 4) in wild type IgG4 format, and evaluating the functionality of the three mother clones as full-size IgGs.
Background Analysis: Fully human anti-MASP-2 scFv antibodies with moderate functional potency were identified using phage display as described in Examples 2 to 4. Three such mother clones, 17D20, 17N16 and 18L16 were selected for affinity maturation. To assess the functionality of these mother clones as full-size IgGs, wild-type IgG4 and mutant forms of IgG4 in the S228P hinge region of these antibodies were produced.
The mutant S228P hinge region was included to increase serum stability (see Labrijn A.F. et al., Nature Biotechnology 27: 767 (2009 )). The wild-type IgG4 amino acid sequence is shown as SEQ ID NO: 63, encoded by SEQ ID NO: 62. The IgG4 S228P amino acid sequence is shown as SEQ ID NO: 65, encoded by SEQ ID NO: 64. IZG4 molecules were also cleaved into —F(ab')2 formats with pepsin digestion and fractionated by size exclusion chromatography in order to compare mother clones directly to the OMS 100 control antibody, which is an F (ab)2. Methods: Generate the clones in the full-size format The three mother clones were converted to a wild-type IgG4 format and a mutant IgG4 S228P format.
This was accomplished by PCR isolating the appropriate VH and VL regions from the above-mentioned parent clones and cloning these into the pcDNA3 expression vectors anchoring the appropriate heavy-chain constant region to create fusions in the template to produce the desired antibody.
The three mother clones in the mutant I2gG4 format were then cleaved with pepsin to generate F(ab”) 2 fragments and were purified by fractionation on a size exclusion chromatography column.
Binding Assay Candidate mother clones converted to IgG4 format were transiently transfected into HEK 293 cells and transient transfection supernatants were titered in an ELISA assay.
Clones 6 showed excellent reactivity with physically adsorbed human MASP-2 A, and classified in the following order: 17N16>17D20>18L16 (data
: 113k not shown). The clones were then purified and retested in ELISA and an activity assay as follows.
Human MASP-2 A was coated at 3 µg/ml in PBS on a maxisorp plate, IgG (45 µg/ml) and Fab'2 (30 µg/ml) — were diluted in PBS-Tween to a concentration starting from 300 nM, and again with 3-fold dilutions.
IgGs were detected with HRP-conjugated Goat a-Human IgG (Southern Biotech) and F(ab”) 2 was detected with HRP-conjugated Goat a-Human IgG (Pierce 31412). The reaction was developed with TMB substrate and stopped with 2M H3SO2. The results are shown below in TABLE 15. TABLE 15: Binding affinity for human MASP-2 [ ormatomutateddelgGI(pM) FabrQM EFOM | aa aaa acc Eca —— Ea aaa TT Functional Assay The C3 convertase assay using 1% normal human serum (NHS), as described in Example 4, was used to compare the functional activity of the mother scFv clones and the Id2G4 counterparts full-size at 1% NHS.
Mannan was diluted to a concentration of 20 ng/ml and coated on an ELISA plate overnight at 4°C.
The next day, the wells were blocked with 1% human serum. Human serum was diluted to 1% in a buffer of CaMgGVB and antibodies — were purified; scFv (900 nM), F(ab')2 (300 nM), IgG (300 nM) were added in duplicates in a series of different dilutions to the same amount of buffer, and pre-incubated for 45 minutes on ice before add to blocked ELISA plates.
The reaction was started by incubating at 37°C for one hour and stopped by placing the plate on ice.
C3b deposition was determined with a rabbit-to-mouse C3c antibody followed by a goat-to-rabbit HRP.
The background of 50 nM OMS100 on the positive mannan plates was subtracted from the curves.
; A summary of the results of this analysis is shown below in TABLE 16. TABLE 16: C3 convertase assay using 1% human serum (ICso nM) at IC50 nM) IC50 nM) IICso nM scFv for the bivalent form Note: the two values shown in columns 2-4 of Table 16 refer to the results of the two separate experiments.
The functional potency of the IZG4 mother clones were also compared to the mutant shape of the IZG4 joint (S228P) for each — clone. Numerical IC 50 values for the deposition assay using 1% NHS are shown below in TABLE 17. TABLE 17: Wild-type (IgG4) versus the hinge-type mutant format (S228P) in the human serum C3b deposition assay 1 % (ICso nM) Elore BD Format WT(IEG9) — ———> IgG4 Knuckle Mutant (S228P) | 15X As shown above in TABLE 17, in some cases, unexpected agonist pharmacology was observed for scFvs derived from antagonistic IgGs. The mechanical basis for this observation is not understood.
The activities of IgG4-converted mother clones with inhibitory function at 1% NHS were again evaluated under more stringent assay conditions that more closely mimic physiological conditions.
To estimate antibody activity under physiological conditions, testing of IgG4 mother clone preparations were conducted for their ability to inhibit lectin pathway (LP)-dependent C3b deposition on mannan coated plates under stringent assay conditions using plasma. — minimally diluted human (90 %).
The results of the 90% human plasma C3b deposition assay are shown in FIGURE 6. Since MASP-2 and its substrates are present in the assay mixture at approximately 100-fold greater concentration than in the diluted serum assay using 1
At % normal human serum, a shift to the right of the antagonist dose-response curve is generally expected.
As shown in FIGURE 6, as expected, a shift to the right to lower apparent potentials was observed for OMS 100 and all MASP-2 antibodies tested. — However, surprisingly, no evident reduction in potency was observed for the hinge region (5228P) mutant of 17D20, and potency in this format was comparable to that measured in 1% plasma (see TABLE 17). In the 90% NHS assay format, the functional potency of IgG4 17D20 (S228) was found to be modestly lower than —OMSI00 Fab2, which is, in contrast to the 1% NHS assay results where the OMS100 was 50 to 100-fold more potent than 17D20 IgG4 5228P (data not shown). The wild-type IgG4 form of 17N16 also showed complete inhibition in NHS at 90% but was somewhat less potent in this assay format (ICsy of ISnM) whereas the wild-type IgG4 form of 18L16 was less potent and only partially inhibitory, as shown in FIGURE 6. Based on these findings, the activity of the mother clones converted to IgG4 was further evaluated by examining the deposition of C4b under stringent assay conditions (90% NHS). This assay format — provides a direct measurement of antibody activity in the enzymatic reaction catalyzed by MASP-2. Assay to Measure Inhibition of MASP-2 Dependent C4 Cleavage Background: The serine protease activity of MASP-2 is highly specific and two protein substrates unique to MASP-2 have been identified; C2 and C4. Cleavage of C4 generates C4a and C4b.
Anti-MASP-2 Fab2 can bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for CA; MASP-2 serine protease catalytic site) and by thereby inhibit the
: 116 . functional activity of MASP-2 C4 cleavage.
Mannan yeast is a known activator of the lectin pathway. In the following method to measure the C4 cleavage activity of MASP-2, mannan-coated plastic wells were incubated for 90 —minute4-“°C with 90% human serum to activate the lectin pathway. The wells were then washed and assayed for human C4b immobilized in the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP-2-dependent C4 cleavage activity. Anti-MASP-2 antibodies at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.
Methods: Media binding plates from 96 Costar wells were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1.0 µg/50 µl/well. Each well was washed 3X with 200 µl PBS. The wells were then blocked with 100 µl/well of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing. Each well was washed 3X with 200 µl PBS. Anti-MASP-2 antibody samples were diluted to selected concentrations in Ca and mg containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM mgCl, 2.0 mM CaCl, , 0.1% gelatin, pH 7.4) at 5°C. 90% human serum was added to the above samples at 5°C and 100 µl was transferred to each well. Plates were covered and incubated for 90 min in an ice-water bath to allow complement activation. The reaction was stopped by adding EDTA to the reaction mixture. Each well was washed 5 x 200 µl with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed 2X with 200 µl of PBS. 100 µl/well of a 1:700 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing
It is 117:2.0 mg/ml bovine serum albumin (BSA) and incubated one hour at room temperature with gentle mixing. Each well was washed 5 x 200 µl PBS. 100 µl/well of 0.1 µg/ml peroxidase-conjugated streptavidin (Pierce Chemical 421126) was added to PBS containing -2.0 mg/ml BSA and incubated for one hour at room temperature on a shaker with gentle mixing. Each well was washed 5 x 200 µl with PBS. 100 µl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 16 min. The peroxidase reaction was stopped by the addition of 100 µl/well of 1.0 M H2PO and the ODa5 was measured.
Results: In this format, both IZ2G4 forms of 17D20 inhibited C4b deposition targeted by the Lectin pathway, although ICs values were =3-fold higher compared to the C3b deposition assay.
Interestingly, wild-type 17N16 IgG4 showed good activity in this assay with an IC value, and dose-response profile comparable to the C3b deposition assay. 18L16 was considerably less potent and did not achieve complete inhibition in this format (data not shown).
Discussion: As described in Examples 2-5, fully human anti-MASP-2 scFv antibodies with functional blocking activity were identified using phage display. Three such clones, 17N16, 17D20 and 18L16, were selected for affinity maturation and tested further. To assess the functionality of these mother clones as — full-size IgGs, wild-type IgG4 and IgG4 S228P hinge region mutant forms of these antibodies were produced. As described in this Example, most of the full-size IgGs had improved functional activity when compared to their scFv counterparts when tested in a standard functional assay format with 1% human plasma. To estimate antibody activity under physiological conditions, testing of mother clone IZG4 preparations was conducted under stringent assay conditions using 90% human plasma. Under these conditions, several antibodies showed functional potencies that were S — substantially better than expected based on their performance in standard (1%) plasma functional assays.
EXAMPLE 6 This Example describes the chain shuffling and affinity maturation of the mother clones 17D20, 17N16 and 18L16, and the analysis of the resulting daughter clones.
Methods: To identify antibodies with improved potency, the three parent scFv clones, 17D20, 17N16 and 18L16, identified as described in Examples 2-5, were subjected to light chain scrambling. This process involved the generation of a combinatorial library consisting of the VH from each of the parent clones paired with a library of naive, human lambda light chains (VL) derived from six healthy donors. This library was then screened for scFv clones with improved binding affinity and/or functionality.
9,000 light chain shuffled daughter clones were analyzed per parent clone, for a total of 27,000 clones. Each daughter clone was induced to express and secrete soluble scFv, and screened for the ability to bind human MASP-2A. ScFvs that bound to human MASP-2A were detected via their c-Myc tag. This initial screening resulted in the selection of a total of 119 clones, which included 107 daughter clones from the 17N16 library, 8 daughter clones from the 17D20 library, and 4 daughter clones from the 18L16 library.
The 119 clones were expressed on a small scale, purified on NiNTA columns, and tested for binding affinity in
And an ELISA assay against physically adsorbed human MASP-2A. Results: The results of the ELISA assay on a representative subset of the 119 daughter clones are shown in FIGURES 7A and B. A - FIGURE 7A graphically illustrates the results of the ELISA assay on the mother clone 17N16 versus daughter clones titered on huMASP-2A. FIGURE 7B graphically illustrates the results of the ELISA assay on the mother clone 17D20 versus daughter clones titered on huMASP-2A. As shown in FIGURE 7A, daughter clones 1I7N16m dIl6EI2 and I7N16m dI7N9, derived from the mother clone 17N16 had affinities that were higher than that of the mother clone. Also, as shown in FIGURE 7B, a clone derived from the mother clone 17D20, 17D20m dI8M24, had a higher affinity than the mother clone. These three clones, and three additional clones: 17Nl6m d13L12, 17Nl6m dl16K5, 1I7Nl6m d1G5, and 17D20m —dl1824 that had a low expression level were expressed on the 0.5 L scale, purified on the monomer fraction by size exclusion chromatography and were retested in an ELISA and functional assay. The 18L16 library did not produce any daughter clones with the desired binding affinity.
After purification, the six daughter clones were tested in a complement assay for inhibitory activity. The results are shown in TABLE 18.
TABLE 18: Complement Assay of Mother and Daughter Clones [Cmrdoconedeserv | Tosont | Kpem | Er AA EIA NA o] As shown above in TABLE 18, only one of the clones, 17N16m dI7N9, had affinity and activity in the same range as the mother clone.
FIGURE 8 is an amino acid sequence alignment of the full-length scFv mother clone 17N16 (SEQ ID NO: 59) and the daughter clone I7N16m d17N9 (SEQ ID NO: 66), which shows that the light chains (beginning with SYE) had 17 amino acid residues that differ between the two clones.
Screening of the 17N16 lambda library resulted in several additional candidate daughter clones, of which I7NlI6m d27EI3 was identified in an ELISA and complement assay, and was included in the pool of candidate daughter clones for further analysis.
Assay Daughter Clones in Different Sera Candidate daughter clones were analyzed in different sera as follows.
Mannan was diluted to 20 ng/ml and coated on an ELISA plate overnight at 4°C.
The next day, the wells were blocked with 1% HSA.
African Green monkey serum was diluted to 0.2%, mouse serum was diluted to 0.375% and human serum was diluted to 1% in CaMgGVB buffer.
Purified ScFv from each of the candidate daughter clones was added in duplicates at a series of different concentrations to the same amount of buffer and pre-incubated for 45 minutes on ice prior to addition to the blocked ELISA plate.
The reaction was started by incubating for one hour at 37 °C, and stopped by transferring to the plate in an ice bath.
C3c release was detected with an a-Rabbit-Mouse C3c antibody followed by an a-Goat-Rabbit HRP.
The background of 0.1 µg/ml OMS100 on mannan negative plates was subtracted from these curves.
The results are summarized below in TABLE 19. TABLE 19: ICs.5 values for mother clone 17N16 and daughter clones 17N16m —dlI7N9el7N16md27E13 in different sera.
Note: The two values shown in columns 2-4 of Table 19 refer to the results of two separate experiments.
: Discussion of results: As shown in TABLE 19, the daughter clone 17N116m dI7N9 has higher functional activity than the mother clone. The improved function in mouse serum in addition to the seventeen amino acid sequence differences in the light chain when compared to the parent clone makes this clone a positive candidate. Based on the best data, the 17N16m dI7N9 daughter clone was selected for further analysis.
EXAMPLE 7 This Example describes the generation and analysis of the daughter clone 17D20m d3521NI11, derived from the mother clone 17D20.
Background/Rationale: To improve the affinity of the candidate mother clone 17D20mc, an additional “insertion mutagenesis” was performed on the first three amino acids in the heavy chain CDR3 (CDR-H3). This was a mutagenesis campaign in parallel with the normal light chain scrambling of 17D20mc. Therefore, three different scFv libraries were constructed by PCR where amino acid positions 1, 2 and 3 were randomized to the set of all 20 possible amino acids using degenerate codons. After cloning the libraries, microscale expression was performed and scFv binding was monitored on a MASP-2A coated CMS chip (not shown). BlAcore analysis of microscale expression was performed on the three different libraries on MASP-2A coated chips, randomized at positions 1, 2, or 3, and potentially interesting daughter clones were identified.
It was observed that for amino acid positions 1 and 2 of the CDR-H3, no clone was found to have an improved off-rate compared to the candidate mother clone 17D20m. However, a few candidates with mutations at amino acid position 3 in CDR-H3 demonstrated improved dissociation rates compared to the
& mother clone 17D20m. These clones (435, 459 and 490) were sequenced to identify the mutation. The sequences of two clones derived from “insertional mutagenesis” are compared with 17D20mc (original sequence). Interestingly, all but one (490) sequenced clones harbored an Ala-Arg substitution compared to the parent candidate.
FIGURE 9 is a sequence comparison of the amino acid sequence of the heavy chain region of the mother clone scFv 17D20m (aa 61-119 of SEQ ID NO: 18) and the amino acid sequence of the CRD-H3 region of scFv clones with mutations in the CDR-H3, clone 435 (aa 61-119 of SEQ ID NO: —20, having an R substitution in place of A at position 102 of SEQ ID NO: 18), clone 459 (same sequence as clone f35), and clone 490 (substitution of P in place of A at position 102 of SEQ ID NO: 18).
Analysis of mutant clones 435 and 459 Mutant clones 435 and %59 were expressed at small scale and further tested against candidate mother clone 17D20 in an immobilized MASP-2A titration ELISA (10 µg/ml). The scFvs were serially diluted 5-fold starting from 20 µg/ml and binding was detected using anti-Myc (mouse)/anti-mouse HRP. Slightly improved binding was observed in the ELISA assay for candidate clones 435 and %59 compared to candidate mother clone 17D20 (data not shown).
The improved clone 435 was combined with the best light chain shuffled clone 17D20m d2INIIl. The mutation in the candidate 17D20md35 VH (Ala-Arg) was combined with the candidate 17D20m d21N11 light chain, thus resulting in the clone named VH35-VL21NI11, otherwise alluded to as 3521N11.
FIGURE 10A is an amino acid sequence alignment of the sequence of the CDR3 region of the mother clone 17D20 (aa 61-119 of SEQ ID NO: 18), the same region of the daughter clone 17D20m d21NI11, having the
: same sequence, and same region as mutagenesis clone 435 combined with the VL of 17D20m d21NI1, alluded to as "3521N11" (aa 61-119 of SEQ ID NO: 20). The highlighted regions of the VH sequence comprise CDRHB3, and the mutated target residue region is underlined.
FIGURE 10B is a full-length scFv protein sequence alignment that includes the VL and VH regions of the mother clone 17D20 (SEQ ID NO: 55) and the daughter clone 17D20m d21N11 (SEQ ID NC: 67). The scFv daughter clone 17D20m d3521N11 is shown as SEQ ID NO: 68. Note: Residue X in FIGURE 10B at position 220 was determined to be an "E", as shown in SEQ ID NO: 68.
A titration ELISA assay of the set of scFvs shown in FIGURE 10 was conducted in MASP-2 (10 µg/ml). The results are shown in TABLE 20.
TABLE 20: ELISA on human MASP-2 strokes OO 3 QD ccE5EIIIÊE OO 18 The daughter clone 17D20m d3521N11 was further analyzed for functional activity as described below in Example 8. EXAMPLE 8 This Example describes the conversion and analysis of candidate daughter clones 17N16m dI7N9 and 17D20m d352IN1I1 in IgGA4, IgG4/S228P and elIgG2 formats.
Rationale/Rationale The antibody screening methods described in Examples 2-7 identified two parent clones, 17N16 and 17D20, with adequate functionality. Affinity maturation of these mother clones produced daughter clones that showed approximately 2-fold improvements in potency compared to mother clones in surrogate functional assays at the scFv level. The daughter clones with the best activities are 17Nl6m
: 124K dI7N9 and 17D20m d352INI1. As described in Example 6, in a comparison of the functional activity of the mother clone 17N16 with light chain scrambled daughter clones (scFv format, 1% NHS assay) it was determined that 17N16m dI7N9 is slightly more potent than the mother clone and the best potency. function of all daughter clones tested in this assay. Methods: A comparison of the functional potency of candidate scFv clones was performed in the C3 conversion assay (1% human serum and 90% human serum), and in a C4 conversion assay (90% human serum), performed as described in Example 5. Results are shown below in TABLE 21. TABLE 21: Comparison of functional potency in ICso (nM) of driver daughter clones and their respective mother clones (all in scFv format) C3 assay in 1% of | C3 assay at 90% CI [C4 assay at 90% CI; nM 1Cso nM 1Cso nM pone the Og Og Ong
As shown above in TABLE 21, 1I7Nl6m d17N9 has good activity when assayed in 90% normal human serum (NHS) in the C3 assay and is more potent than than the other daughter clones in this format. Conversion of Candidate Clones to IgGA4, IgG4/S228P and IgG2 Format All these candidate clones were converted to IgG4, IZgG4/S228P and IgG2 format for further analysis. SEQ ID NO: 62: cDNA encoding wild-type IgG4 SEQ ID NO: 63: wild-type IZG4 polypeptide SEQ ID NO: 64 cDNA encoding S228P mutant IZ2G4 SEQ ID NO: 65: S228P mutant IZG4 polypeptide
- 125: SEQ ID NO: 69: cDNA encoding wild-type I2G2 SEQ ID NO: 70: wild-type I2G2 polypeptide TABLE 22: Summary of Candidate Clones: Clone Reference — Daughter Klone Format Tg VA AND FICOMSSA) —> —>— —%7NIGmdi7N9 — ieG2 >> SEQIDNO:2I GNEQIDNO: | FrromMsSa) ——— —%7NnIGKm di7N9 — fieGA——— SEQIDNO:21 SEQIDNO:X | ESCOMSSA45) — —h7D20352INIT lgG4 ————— SEQIDNO:2O SEQIDNOX | Monoclonal antibodies %1-6 were tested for — ability to cross-react with a protein other than human MASP-2 (African Green Monkey (AG)) in a C3 assay to determine whether these antibodies can be used to test for toxicity in an animal model that would be predictive for humans.
Monoclonal antibodies %1-6 were also tested in a C3b deposition assay and a C4 assay in 90% human serum.
The results are shown below in TABLE 23. TABLE 23: Anti-human MASP-2 MoAbs (ICsoy nM) in 90% human serum Ensão — ——— MoAbtl MoAbi2 MoAbisS Moabraá Moabis Moabi6 | [Prumano Bo C3 Assay [Boo Human C4 Assay l Medeameno Monkey C3 Assay op ps pao eo ho o | FIGURE 11A graphically illustrates the results of the C3b deposition assay performed for the daughter clone isotype variants (MoAbf1-3) derived from the 17N16 parent clone of the anti-human MASP-2 monoclonal antibody.
FIGURE 11B graphically illustrates the results of the C3b deposition assay performed for isotype variants of the daughter clone -(MoADbH4-6), derived from the mother clone 17D20 of the human monoclonal anti-MASP-2 antibody.
As shown in TABLE 23 and FIGURES 11A and 11B , anti-human MASP-2 monoclonal antibodies (MoAbf1-6) bind MASP-2 with high affinity, and inhibit the function of C3 and C4 activity in 90% of human serum. It is also observed that anti-human MASP-2 MoAbs cross-react with the non-human MASP-2 protein (African Green monkey), which provides an animal model for toxicity studies that would be predictive for humans.
MoAb*1-6 were further analyzed in 95% human serum, 95% African green serum. The results are summarized below in TABLE 24.
TABLE 24 D of Antibody Binding to Functional Inhibition of | Functional inhibition of | Functional inhibition of hMASP-2 —C3 deposition at 95k, C3 deposition at 95k, C4 deposition at 95) immobilized % human serum % green serum % human serum (mean Kd) | (1Cs5o Mean; African ICo (Average ICso) nM Mean) nM (Average CIso) nM MoabricigGa) — | or roega gesç Moabi2cigGa) — | the | ninth To Pas IMoAbi3 (IeG4 0.323 9.4; 61.0 92 user) imo) — | oox | donkey [using user] FIGURES 12A and 12B graphically illustrate the testing of mother clones and MoAb%1-6 in a C3b deposition assay in 95% normal human serum.
FIGURE 13 graphically illustrates the inhibition of C4b deposition in 95% normal human serum.
FIGURE 14 graphically illustrates inhibition of C3b deposition in 95% African Green monkey serum.
MoAbt1-6 was further tested for the ability to selectively inhibit the lectin pathway by the Mouse C3b inhibition assay, inhibition of pre-assembled MBL-MASP-2 complexes; classical pathway inhibition, and — selectivity over Cls. The results are summarized in TABLE 25.
, TABLE 25: Summary of functional assay results aq Inhibition of ss complexes. , Inhibition of MBL-MASP- 2 Inhibition of the pathway | Corosvity Classic Rat C3b Antibody ID pre-assembled on Cls (ICso nM) 1C59 (nM) ICso (hM to BB nd IMoAbÉ1 (IgG2 to AE sO0O0xX | MoAbr2 (GA not detected ((O200nM)| — not detected =| = >5000x | oAbA3 (IgG 00 tant not detected (()200nM) not detected >5000x the aa O ng o Ab (I8G2 to AO 5000x | Yes, ICs9 = 17nM utant FIGURE 15 graphically illustrates the inhibition of cleavage activity of C4 of the MBL-MASP-2 complex preassembled by MoAb* 2, 3.5€c6 SA FIGURE 16 graphically illustrates preferential binding of MoAbi 6 to human MASP-2 as compared to C1s.
Table 26: Summary of Daughter Clone Sequences in Various Formats: Clone DE O esesion O BEQIDNO | |I7NI6mdI7N9 —Meo light chain gene sequence I7NI6m d17N9 light chain protein sequence I7N16m d17N9 IgG2 heavy chain gene sequence lI72N16ómdI7N9 — —IgG2 heavy chain protein sequence I7NI6m d17N9 IgG4 heavy chain gene sequence ps lI72Nlómdi7N9 — — IgG4 heavy chain protein kequence je I7N16m d1I7N9 IgG4 mutated heavy chain gene sequence |I7%NI7mdi72N9 —— IgG4 mutated heavy chain protein kequence pag 17D20 352IN11 Light chain gene sequence pg 17D20352INI11 — kequeque light chain protein sequence 17D20 352INI1 IgG2 heavy chain gene sequence gi l17D20352IN11 — IgG2 heavy chain protein sequence 17D20352INI1l — IgG4 heavy chain gene sequence 17D20 352IN11 IgG4 heavy chain protein sequence ga = 17D20352IN11 — IgG4 mutated heavy chain gene sequence 17D20 352IN11 IgG4 mutated heavy chain protein sequence TR 17N16m d1I7N9 DNA encoding full-length scFv polypeptide 17D20m d2INI 1 DNA encoding full-length scFv polypeptide jggg Am DNA encoding scFvy polypeptide full-size ego — A EXAMPLE 9 This Example describes the epitope mapping that was — performed for several of the anti-MASP MoAbs -2 human blockers.
Methods: The following recombinant proteins were produced
. 128 h as described in Example 1: MAp 19 human MASP2A human MASP-2 SP human Ss MASP-2 CCP2-SP human MASP-2 CCP1-CCP2-SP human MASP-1/3 CUBI-EGF-CUB2 human MASP-1 CCPI -CCP2-SP The anti-cMASP-2 antibodies OMS100 and MoAb%6 (35VH-21INI1IVL), which have both been shown to bind to human MASP-2 with high affinity and have the ability to block functional complement activity (see Examples 6 -8) were analyzed for epitope binding by spot-spot analysis.
SPOT SPOT ANALYSIS: Serial dilutions of the recombinant MASP-2 polypeptides described above were spotted onto a nitrocellulose membrane. The amount of stained protein ranged from 50 ng to 5 pg, in five-fold steps. In later experiments, the amount of stained protein varied from 50 ng to below 16 pg, again in five-fold steps. Membranes were blocked with 5% skim milk powder in TBS (blocking buffer) after incubation with 1.0 µg/ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca” ). Bound Fab2s were detected using HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL detection kit (Amersham).
— A membrane was incubated with rabbit polyclonal anti-human MASP-2 Ab (described in Stover et al., J Immunol 163: 6848-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2000).
Results:
Y Results are summarized in TABLE 27. TABLE 27: Epitope Mapping A aaa AS PA Trama AND A SP mana human MASP-2 CEP2SP) MASP-2 CCPLICEP2-SP Tama IMASP-1/3 CUB-EGF-CUBI Fman O ) IMASP-1 CCP1-CCP2-SP tiara) (Human MBL/MASP2 O complexes) Results show that MoAb%6 and OMS 100 antibodies are highly specific for MASP-2 and do not bind to MASP-1 or MASP- S —3. None of the antibody fragments bound to Map19 and MASP-2 which do not contain the CCP1 domain of MASP-2, leading to the conclusion that the binding sites span the CCP1 domain. EXAMPLE 10 This Example demonstrates that human anti-MASP-2 MoAbH6 inhibits the lectin pathway in African Green monkeys following intravenous administration.
Methods: MOoAbt6 was administered intravenously to a first group of three African Green monkeys at a dosage of 1 mg/kg and to a second group of three African Green monkeys at a dosage of 3 mg/kg.
Blood samples were obtained 2, 4, 8, 10, 24, 48, 72 and 98 hours after administration and were tested for the presence of lectin pathway activity.
As shown in FIGURE 17, the lectin pathway was completely inhibited following intravenous administration of anti-human MoAbit6.
EXAMPLE 11 This Example demonstrates that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective for treating radiation exposure and/or for treating, ameliorating, or preventing acute radiation syndrome.
Rationale: Exposure to high doses of ionizing radiation causes mortality by two main mechanisms: bone marrow toxicity and — gastrointestinal syndrome. Bone marrow toxicity results in a drop in all hematologic cells, predisposing the organism to death from infection and hemorrhage. Gastrointestinal syndrome is more severe and is triggered by a loss of intestinal barrier function due to disintegration of the epithelial layer of the intestine and a loss of intestinal endocrine function. This leads to septicemia and associated systemic inflammatory response syndrome that can result in death.
The complement lectin pathway is an innate immune mechanism that initiates inflammation in response to tissue injury and exposure to foreign surfaces (ie, bacteria). Blocking this pathway leads to better results in mouse models of ischemic intestinal tissue injury or septic shock. It is hypothesized that the lectin pathway can activate excessive and noxious inflammation in response to radiation-induced tissue injury. Blocking the lectin pathway may thus reduce secondary injury and increase survival following acute radiation exposure.
The purpose of the study performed as described in this Example was to evaluate the effect of blocking the lectin pathway on survival in a mouse model of radiation injury by the administration of murine anti-MASP-2 antibodies.
Study *1 Methods and Materials: Materials. The test articles used in this study were (i) a high affinity murine anti-MASP-2 antibody (mMAbMI11) and (11) a high affinity anti-human MASP-2 antibody (mMAbOMS646) that blocks the protein component MASP-2 of the complement pathway of
: lectin that was produced in the transfected mammalian cells. Dosing concentrations were 1 mg/kg murine anti-MASP-2 antibody (mMmAbMI11), 5 mg/kg human anti-MASP-2 antibody (MAbOMS646), or sterile saline. For each dosing session, an adequate volume of fresh dosing solutions was prepared.
Animals. Young adult male Swiss-Webster mice were obtained from Harlan Laboratories (Houston, TX). Animals were housed in solid bottom cages with Alpha-Dri bedding and provided with certified PMI 5002 Rodent Diet (Animal Specialties, Inc., Hubbard OR) and water ad libitum. The temperature was monitored and the animal restraint room operated with a light cycle of 12 hours light/12 hours dark.
Irradiation. After a 2-week acclimatization at the facility, mice were irradiated with 6.5, 7.0, and 8.0 Gy by whole-body exposure in groups of 10 at a dose rate of 0.78 Gy/mem using a Therapax X-RAD 320 system supplied with a high stability 320 kV x-ray generator, metallized ceramic x-ray tube, variable x-ray beam collimator and filter (Precision X-ray Incorporated, East Haven, CT).
Formulation and Administration of Medication. The appropriate volume of concentrated stock solutions was diluted with ice-cold saline to prepare dosing solutions of 0.2 mg/ml murine anti-MASP-2 antibody (mAbM11) or 0.5 mg/ml anti-MASP antibody. -2 from human (mAbOMS646) according to protocol. Administration of anti-MASP-2 antibody mAbM11 and mAbOMS646 was via IP injection using a 25 gauge needle base over the weight of the animal to deliver 1 mg/kg mAbMI11, 5 mg/kg mAbOMS646, or vehicle solution saline.
Study Planning. Mice were randomly assigned to groups as described in Table 28.
- 132 body and temperature were measured and recorded daily.
Mice in Groups 7, 11 and 13 were sacrificed on day 7 after irradiation and blood collected by cardiac puncture under deep anesthesia.
Animals surviving on day 30 after irradiation were sacrificed in the same way and blood collected.
Plasma was prepared from the blood samples collected according to the protocol and returned to the Sponsor for analysis.
TABLE 28: Study Groups 1 20 6.5 Vehicle 18 hours before irradiation, 2 hours after irradiation, weekly booster
2 20 6.5 ab anti-MASP-2 of | 18 hours before single murine mAbM11 irradiation]
3 20 6.5 ab antic-MASP-2 de | 18 hours before irradiation, 2 murine hours after irradiation, weekly mAbM11 booster
6.5 ab anticMASP-2 2 hours post-irradiation, murine weekly booster mMAbMI11
20 ab antic-MASP-2 of | 18 hours before irradiation, 2 human hours after irradiation, weekly mMAbOMS646 booster
20 7.0 Vehicle 18 hours before irradiation, 2 hours after irradiation, weekly booster
20 7.0 ab anti-MASP-2 of | 18 hours before single mMAbM11 murine irradiation]
20 7.0 ab anti-MASP-2 of | 18 hours before irradiation, 2 murine hours after irradiation, weekly mAbM11 booster
20 7.0 ab antic-MASP-2 2 hours post-irradiation, murine mAbM11 weekly boost
5 7.0 ab anti-MASP-2 of | 2 hours after single mAbM1 murine irradiation]
12 20 7.0 ab anti-MASP-2 of | 18 hours before irradiation, 2 human hours after irradiation, weekly mAbH6 booster
13 MASP-2 ab 18 h before irradiation, 2 h post anti-human irradiation, weekly mMAbOMS646 booster
O o Veialo — | 2h after irradiation only | Statistical analysis.
The Kaplan-survival curves
x 133 y Meier were generated and used to compare median survival time between treatment groups using log-Rank and Wilcoxon methods.
Means with standard deviations, or means with standard error of the mean are reported.
Statistical comparisons were made using a two-tailed unpaired t-test between irradiated control animals and individual treatment groups.
Study Results *1 Kaplan-Meier survival plots for the 6.5, 7.0, and 8.0 Gy exposure groups are provided in FIGURES 18A, 18B, and 18C, respectively, and summarized below in Table 29. Overall, pre-irradiation treatment with murine anti-MASP-2 ab (mMAbM11) increased survival of irradiated mice compared to vehicle-treated irradiated control animals at both the exposure level of 6.5 (20% increase) and 7.0 and 8.0 Gy (30% increase). At the exposure level of 6.5 Gy, post-irradiation treatment with murine anti-MASP-2 ab resulted in a modest increase in survival (15%) compared to vehicle-irradiated control animals.
In comparison, all animals treated at the 7.0 Gy exposure level showed an increase in survival compared to vehicle-treated irradiated control animals.
The greatest change in survival occurred in animals receiving mAbOMS646, with a 45% increase in survival compared to control animals at the 7.0 Gy exposure level, and a 12% increase in survival at the 8.0 Gy exposure level. .0 Gy.
Furthermore, at the 7.0 Gy exposure level, mortalities in the mAbOMS646 treated group first occurred on day 15 post-irradiation compared to day 8 post-irradiation for vehicle-treated irradiated control animals, an increase of 7 days compared to control animals.
The mean time to mortality for mice receiving mAbOMS646 (27.3 + 1.3
: 134 Y days) was significantly increased (p = 0.0087) compared to control animals (20.7 + 2.0 days) at the 7.0 Gy exposure level.
The percent change in body weight compared to pre-irradiation day (day -1) was recorded across the entire study.
A transient weight loss occurred in all irradiated animals, with no evidence of differential change due to treatment with mAbM11 or mALbOMS646 compared to controls (data not shown). At the end of the study, all surviving animals showed an increase in body weight from baseline body weight (day -1). TABLE 29: Survival rates of test animals exposed to radiation Test Group Level of | Survival | Time for Death | First/Last Po exposure (%) Average + SEM, Day) | Death (Day Control Irradiation 27.74 1.5 PANA E am A wings as janean Ja mMAbMI exposure 1 after exposure Control Irradiation exposure 20.7 2.0 8/17 mMAbMI | pre-exposure 23.0 2.3 exposure exposure exposure Control irradiation soe aa *p = 0.0087 by two-tailed unpaired t test between irradiated control and treatment group animals at the same level of irradiation exposure.
Debate Acute radiation syndrome consists of three defined subsyndromes: hematopoietic, gastrointestinal, and cerebrovascular.
The syndrome observed depends on the radiation dose, with hematopoietic effects observed in humans with significant partial or whole-body radiation exposures exceeding 1 Gy.
Hematopoietic syndrome is distinguished by severe depression of bone marrow function leading to pancytopenia with changes in blood counts, blood cells
Only red and white, and platelets occurring concomitantly with damage to the immune system. As nadir occurs, with few neutrophils and platelets present in peripheral blood, neutropenia, fever, complications of septicemia, and uncontrollable bleeding lead to death.
In the present study, administration of mAbHG6 was found to increase survival to whole-body X-ray irradiation in male Swiss-Webster mice irradiated at 7.0 Gy. Notably, at the 7.0 Gy exposure level, 80% of animals receiving mAbOMS646 survived 30 days compared to 35% of vehicle-treated irradiated control animals. Importantly, the first day of death in this treated group did not occur until day 15 post-irradiation, a 7-day increase over those seen in vehicle-treated control irradiated animals. Interestingly, at the lowest X-ray exposure (6.5 Gy), administration of mAbOMS646 did not appear to impact survival or delay in mortality compared to vehicle-treated control irradiated animals. There would be multiple reasons for this difference in response between exposure levels, although verification of any hypothesis may require additional studies, including sample collection at intervals for microbiological culture and hematological parameters. One explanation may simply be that the number of animals assigned to groups may have made it impossible to observe any subtle treatment-related differences. For example, with group sizes of n = 20, the difference in survival between 65% (mMAbOMS646 at 6.5 Gy exposure) and 80% (MAbOMS646 at 7.0 Gy exposure) is 3 animals. On the other hand, the — difference between 35% (vehicle control at 7.0 Gy exposure) and 80% (MAbHG6 at 7.0 Gy exposure) is 9 animals, and provides solid evidence of a difference related to the treatment.
These results demonstrate that anti-MASP-2 antibodies are effective in treating a mammalian subject at risk for, or who
: 136 suffer from the harmful effects of acute radiation syndrome. ; Study 42 Swiss Webster mice (n = 50) were exposed to ionizing radiation (8.0 Gy). 3 The effect of anti-MASP-2 antibody therapy (WHO646 5 mg/kg), administered 18 hours before and 2 hours after radiation exposure, and weekly thereafter, on mortality was evaluated. Results of Study f2: It was determined that administration of anti-MASP-2 antibody OMS646 increased survival in mice exposed to 8.0 Gy, with an adjusted median survival rate of 4 to 6 days when compared to mice that received control of vehicle, and a 12% reduced mortality when compared to mice that received vehicle control (log classification test, p = 0.040). Study t3: Swiss Webster mice (n = 50) were exposed to ionizing radiation (8.0 Gy) in the following groups (1: vehicle) saline control; (II: low) anti-cMASP-2 antibody OMS646 (5 mg/kg) administered 18 hours before irradiation and 2 hours after irradiation; (III: high) OMS646 (10 mg/kg) administered 18 hours before irradiation and 2 hours after irradiation; and (IV: discharged after) OMS646 (10 mg/kg) administered 2 hours after single irradiation.
t3 Study Results: It was determined that administration of anti-MASP-2 antibody pre- and post-irradiation adjusted median survival by 4 to 5 days compared to animals receiving vehicle control. Mortality in the anti-MASP-2 antibody treated mice was reduced by 6 to 12% compared to vehicle control mice. It was further mentioned that no significant harmful treatment effects were
- 137 : observed.
In summary, the results in this Example demonstrate that anti-MASP-2 antibodies of the invention are effective in treating a mammalian subject at risk for, or suffering from the harmful effects of acute radiation syndrome. EXAMPLE 12 This Example describes another characterization of the OMS646 antibody (17D20m d3521IN11), fully human anti-human MASP-2 IgG4 antibody with a hinge region mutation).
Methods: OMS646 (17D20m d3521N11), fully human anti-human MASP-2 IgG4 antibody with a hinge region mutation) was generated as described above in Examples 2-8. The OMS646 antibody was purified from the culture supernatants of a CHO expression cell line stably transfected with expression constructs encoding the heavy and light chains of OMS646. Cells were cultured in PF-CHO medium for 16 to 20 days and cell-free supernatant was collected when cell viability dropped below 50%. OMS646 was purified by Protein A affinity chromatography followed by — ion exchange, concentration and buffer exchange in PBS.
1. OMS646 Binds to Human MASP-2 with High Affinity Surface Plasmon Resonance Analysis (Biocore) of Binding of OMS646 Immobilized to Recombinant Human MASP-2 Methods: OMS646 was immobilized at various densities by amine binding to a flake of CMS and the association and dissociation of recombinant human MASP-2 dissolved at 9 nM, 3 nM, 1 nM or 0.3 nM was recorded with time to determine association (Kiigada) and dissociation (Kaestigated) rate constants: Equilibrium binding constant (KD) was calculated based on experimental Kigada € K turned off EXPperimental values. : Results: FIGURE 19 graphically illustrates the results of surface plasmon resonance analysis (Biocore) on OMS646, which shows that immobilized OMS646 binds to recombinant MASP-2 with a Kes-bound rate of about 1 to 3 x 10° s." and a K ratio of about 1.65a3 x 10ºM's" implying an affinity (Kp of about 92 pM) under these experimental conditions.
Depending on the density of immobilized OMS646 and the concentration of MASP-2 in the solution, experimental KD values in the range of 50 to 250pM were determined.
ELISA Assay of Binding of OM5646 to Immobilized Recombinant Human MASP-2 Methods: An ELISA assay was performed to assess the dose response of OMS646 binding to immobilized recombinant MASP-2.
Recombinant human MASP-2 (50 ng/well) was immobilized on maxisorp ELISA plates (Nunc) in PBS overnight at 4°C.
The next day, the plates were blocked by washing three times with PBS-Tween (0.05%). A series of serial dilutions of OMS646 in blocking buffer (0.001 to 10 nM concentration range) was then added to the MASP-2 coated wells. After a 1 hour incubation to allow binding of OMS646 to the immobilized antigen, the wells were washed to remove unbound OMS646.
Bound OMS646 was detected using HRP-labeled goat anti-human IgG antibody (Qualex; diluted 1:5000 in blocking buffer) followed by — development with TMB peroxidase substrate (Kirkegaard & Perry Laboratories). The peroxidase reaction was stopped by the addition of 100 µl/well of 1.0 M H2PO, and substrate conversion was quantified photometrically at 450 nM using a 96-well plate reader (Spectramax). A single binding site curve fitting algorithm δ (Graphpad) was used to calculate the KD values. Results: FIGURE 20 graphically illustrates the results of the ELISA assay to determine the binding affinity of OMS646 to immobilized human MASP-2. As shown in FIGURE 20, OMS646 was determined to bind to immobilized recombinant human MASP-2 with a KD of 85 + 5 pM, which is consistent with the results obtained in the Biocore analysis, as shown in FIGURE 19. These results demonstrate that the OMS646 has high affinity for human MASP-2, with a KD of approximately 100 10 pM.
2. OMS646 inhibits C4 activation on a mannan coated surface, but not on an immune complex coated surface Methods: C4 activation was measured on a —mannan coated surface or an immune complex coated surface in the presence or absence of OMS646 in the concentration range shown in FIGURES 21A and 21B, respectively, as follows. In the following method for measuring the C4 cleavage activity of MASP-2, mannan-coated plastic reservoirs were — incubated for 60 minutes at 37°C with 1% human serum to activate the lectin pathway. The wells were then washed and assayed for human C4b immobilized in the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP-2 dependent C4 cleavage activity. Anti-MASP-2 antibodies at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage. Methods: Activation of C4 on Mannan Coated Surfaces: In order to determine the effect of OMS646 on the hlectin pathway, Costar Medium Binding plates from 96 wells were coated with mannan by incubating overnight at 5°C with 50 µl of a 40 µg/ml mannan solution diluted in 50 mM carbonate buffer, pH 9.5. Each well was washed 3X with 200 µl PBS.
The wells were then blocked with 100 µl/well of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing.
Each well was washed 3X with 200 µl PBS.
In a separate 96-well plate, serial dilutions of MASP-2 antibody (OMS646) were preincubated with 1% human serum in Ca++emg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl, 2.0 mM CaCl, 0.1 % gelatin, pH 7.4) for 1 hour at 5°C.
These antibody-serum pre-incubation mixtures were subsequently transferred into the corresponding wells of the mannan coated assay plate.
Complement activation was initiated by transferring the assay plate to a 37°C water bath.
Following a 60 minute incubation, the reaction was stopped by adding EDTA to the reaction mixture.
Each well was washed 5 x 200 µl with PBS-Tween (0.05% Tween 20 in PBS), then each well was washed 2X with 200 µl of PBS. 100 µl/well of a 1:100 dilution of Biotin-conjugated chicken anti-20 human C4c (Immunosystem AB, Uppsala, Sweden) was added to PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated one hour at room temperature with gentle mixing.
Each well was washed 5 x 200 µl PBS. 100 µl/well of 0.1 µg/ml peroxidase-conjugated streptavidin (Pierce Chemical —tf21126) was added to PBS containing 2.0 mg/ml BSA and incubated for one hour at room temperature on a shaker with gentle mixing. .
Each well was washed 5 x 200 µl with PBS. 100 µl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes.
The reaction
: 141; of peroxidase was stopped by the addition of 100 ul/well of 1.0 M H3PO, and the OD450 was measured. Activation of C4 on Immune Complex Coated Surfaces In order to measure the effect of OMS646 on the classical pathway, the assay described above was modified to use immune complex coated plates. The assay was performed as detailed for the lectin pathway above, with the difference that the wells were coated with purified sheep IgG used to stimulate C4 activation via the classical pathway. Results: FIGURE 21A graphically illustrates the level of C4 activation on a mannan coated surface in the presence or absence of OMS646. FIGURE 21B graphically illustrates the level of C4 activation on an IgG coated surface in the presence or absence of OMS646.
As shown in FIGURE 21A, OMS646 inhibits C4 activation on the mannan coated surface with an ICs of approximately 0.5 nM in 1% human serum. The ICs value obtained in 10 independent experiments was 0.52 + 0.28 nM (mean + SD). In contrast, as shown in FIGURE 21B, OMS646 does not inhibit C4 activation on an IgG-coated surface. These results demonstrate that OMS646 blocks lectin-dependent activation, but not the classical pathway-dependent C4 component of complement.
3. OMS646 specifically blocks lectin-dependent activation of terminal complement components Methods: The effect of OMS646 on membrane attack complex (MAC) deposition was analyzed using pathway-specific conditions for the lectin pathway, the classical pathway and the alternative way. For this purpose, the Wieslab Comp300 add-on screening kit (Wieslab, Lund, Sweden) was 142' and used following the manufacturer's instructions. Results: FIGURE 22A graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under specific assay conditions of the lectinan pathway. FIGURE 22B graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway specific assay conditions. FIGURE 22C graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions. As shown in FIGURE 22A, OMS646 blocks the lectin pathway-mediated activation of MAC deposition with an :ICso value of approximately 1 nM. However, OMS646 had no effect on MAC deposition generated from activation mediated by the classical pathway (FIGURE 22B) or from activation mediated by the alternative pathway (FIGURE 22C).
4. OMS646 effectively inhibits lectin pathway activation under physiological conditions Methods: Lectin-dependent activation of C3 and C4 was evaluated in 90% human serum in the absence and presence of various concentrations of OMS646 as follows: C4 To assess the effect of OMS646 on lectin-dependent C4 activation, Costar medium binding plates from 96 wells were coated overnight at 5°C with 2 µg mannan (50 µl of a 40 µg/ml solution in 50 mM carbonate buffer, pH 9.5 The plates were then washed three times with 200 µl PBS and blocked with 100 µl/well 1% bovine serum albumin in PBS for one hour at
- 143 room temperature with gentle mixing. In a separate pre-incubation plate, selected concentrations of OMS646 were mixed with 90% human serum and incubated for 1 hour on ice. These antibody-serum pre-incubation mixtures were then transferred into mannan-coated wells of the assay plates on ice. The assay plates were then incubated for 90 minutes in an ice-water bath to allow complement activation. The reaction was stopped by adding EDTA to the reaction mixture. Each well was washed 5 times with 200 µl PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed twice with 200 µl PBS. 100 µl/well of a 1:1000 dilution of Biotin-conjugated chicken anti-human C4c (Immunosystem AB, Uppsala, Sweden) was added in PBS* which contains 2.0 mg/ml bovine serum albumin (BSA) and incubated 1 hour at room temperature with gentle mixing. Each well was washed 5 times with 200 µl PBS. 100 µl/well of 0.1 µg/ml peroxidase-conjugated streptavidin (Pierce Chemical 421126) was added to PBS containing 2.0 mg/ml BSA and incubated for 1 hour at room temperature on a shaker with gentle mixing. Each well was washed five times with 200 µl PBS. 100 µl/well of TMB —peroxidase substrate (Kirdegaard & Perry Laboratories) was added and incubated at room temperature for 16 minutes. The peroxidase reaction was stopped by the addition of 100 ul/well of 1.0 M H2PO, and ODaso was measured.
C3 Activation Assay To assess the effect of OMS646 on lectin-dependent C3 activation, assays were performed in a manner identical to the C4 activation assay described above, except that C3 deposition was assessed as the endpoint. C3 deposition was quantified as follows. At the end of the complement deposition reaction, the plates
- 144: were washed as described above and subsequently incubated for 1 hour with 100 µl/well dilution 1:5000 of rabbit anti-human C3c antibody (Dako) in PBS containing 2.0 mg/ml bovine serum albumin ( BSA). Each plate was washed five times with 200 µl PBS, and then incubated for | hour at room temperature with 100 µl/well of HRP-labeled goat anti-rabbit IgG (American Qualex Antibodies) in PBS containing 2.0 mg/ml BSA. The plates were washed five times with 200 µl of PBS and then 100 µl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by the addition of 100 uL/well of 1.0 M H;PO and the OD450 was measured. The CI values were derived by applying a sigmoidal dose-response curve-fitting algorithm (GraphPad) to the experimental data sets. Results: FIGURE 23A graphically illustrates the level of C3 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under specific conditions of the lectin pathway. FIGURE 23B graphically illustrates the level of —C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under specific conditions of the lectin pathway. As shown in FIGURE 23A, OMS646 blocked C3 deposition in 90% human serum with a CI. =3 + 1.5 nM (n=6). As shown in FIGURE 23B, OMS646 blocked C4 deposition with an ICs9=2.8+1.3 nM(n=6).
These results demonstrate that OMS646 provides potent, effective blockade of lectin pathway activation under physiological conditions, thereby providing support for the use of low therapeutic doses of OMS646. Based on these data, it is expected that OMS646 will block >90%
And the lectin pathway in a patient's circulation at a plasma concentration of 20 nM (31 ug/ml) or less. Based on a typical human plasma volume of approximately 3 L, and the knowledge that all of the administered antibody material is retained in the plasma (Lin YS.etal, JPET 288: 371 (1999), it is expected that a dose of OMS646 as low as 10 mg administered intravenously will be effective in blocking the lectin pathway over an acute period of time (ie, a transient period of time, such as 1 to 3 days). , it may be advantageous to block the lectin pathway for an extended period of time to obtain maximum treatment benefit.Thus, for such chronic conditions, a 100 mg dose of OMS646 may be preferred, which is expected to be effective in blocking the lectin. lectin pathway in an adult human subject for at least a week or longer. Given the slow clearance and long half-life that is commonly observed for antibodies in humans, it is possible that a 100 mg dose of OMS646 could be effective for longer o than a week, such as for 2 weeks, or even 4 weeks. A higher dose of the antibody (i.e., more than 100 mg, such as 200 mg, 500 mg, or greater, such as 700 mg or 1000 mg) is expected to have a longer duration of action (e.g., greater than 2 weeks).
5. OMS646 Blocks Lectin Pathway Activation in Monkeys As described above in Example 10 and shown in FIGURE 17, OMS646 was determined to remove activity from the systemic lectin pathway for a period of time of about 72 hours following — intravenous administration of OMS646 (3 mg/kg) in African Green monkeys, followed by recovery of lectin pathway activity. This Example describes a follow-up study in which lectin-dependent C4 activation was evaluated in 90% African Green monkey serum or 90% Cynomolgus monkey serum in a
: 146
: range of concentrations of OMS646 and in the absence of OMS646, as follows: To assess the effect of OMS646 on lectin-dependent C4 activation in different non-human primate species, Costar binding plates from 96 wells were coated overnight at 5°C with 2 µg mannan (50 µl of a 40 µg/ml solution in 50 mM carbonate buffer, pH 9.5). The plates were then washed three times with 200 µl PBS and blocked with 100 µl/well of 1% bovine serum albumin in PBS for 1 hour at room temperature with gentle mixing.
In a separate pre-incubation plate, selected concentrations of —OMS646 were mixed with 90% serum collected from African Green Monkeys or Cynomolgus Monkeys, and incubated 1 hour on ice.
These antibody-serum pre-incubation mixtures were then transferred into mannan-coated wells of the assay plates on ice. , Assay plates were then incubated for 90 minutes in an ice-water bath to allow complement activation.
The reaction was stopped by adding EDTA to the reaction mixture.
Each well was washed five times with 200 µl PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed twice with 200 µl PBS. 100 µl/well dilution 1:1000 of chicken anti-human C4c conjugated: 20 with Biotin (Immunosystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/ml BSA and incubated one hour at room temperature. with smooth mixing.
Each well was washed five times with 200 µl PBS. 100 µl/well of 0.1 µg/ml peroxidase-conjugated streptavidin (Pierce Chemical 421126) was added to PBS containing 2.0 mg/ml BSA and incubated for one hour at room temperature on a shaker with gentle mixing.
Each well was washed five times with 200 µl PBS. 100 µl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes.
The peroxidase reaction
: 147 was stopped by the addition of 100 µl/well of H2PO, the .0 m and the AODase was measured. ICs9 values were derived by applying a sigmoidal dose-response curve-fitting algorithm (GraphPad) to the experimental data sets. Results: A dose response of lectin pathway inhibition in 90% Cynomolgus monkey serum (FIGURE 24A) and 90% African Green monkey serum (FIGURE 24B) was observed with ICs,o values in the range of 30 nM to 50 nM, and 15 nM to 30 nM, respectively.
In summary, OMS646, a fully human anti-human MASP-2 IgG4 antibody (with a hinge region mutation) was found to have the following advantageous properties: high affinity binding to human MASP-2 (Kp in the range of 50 at 250 pM, with a rate , Kaestigation In the range of 1 to 3 x 10º s' and a rate K., in the range of 1.6 to 3 x 10º 15º M's'; the functional potency in human serum with inhibition of C4 deposition with an ICs of 0.52 + 0.28 nM (n = 10) in 1% human serum, and an IC50 of 3 + 1.5 nM in 90% serum); and monkey cross-reactivity showing inhibition of C4 deposition with an ICs in the range of 15 to 50 nM (90% monkey serum).
As described above, doses as low as 10 mg of OMS646 (which corresponds to 0.15 mg/kg for an average human) are expected to be effective in markedly blocking the lectin pathway in human circulation (e.g., for a period of at least 1 to 3 days), whereas 100 mg doses of WHO646 (which corresponds to 1.5 mg/kg — for an average human) are expected to block the lectin pathway in a patient's circulation for at least a week or longer. Higher doses of WHO646 (e.g. doses greater than 100 mg, such as at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or greater), and preferably subcutaneous (sc) or
- 148' intramuscular (im) administration can be used to further extend the time window of effective lectin pathway removal to two weeks and preferably four weeks.
For example, as shown in the experimental data here, in primates a dose of | mg/kg of OMS646 resulted in inhibition of the lectin pathway for 1 day, and a dose of 3 mg/kg of OMS646 resulted in inhibition of the lectin pathway for about 3 days (72 hours). It is therefore estimated that a higher dosage of 7 to 10 mg/kg would be effective in inhibiting the lectin pathway for a period of time of about 7 days.
As shown here, OMS646 has a 510-fold greater potency against human MASP-2 when compared to monkey MASP-2.
Assuming comparable pharmacokinetics, the expected dose ranges to achieve effective lectin pathway removal in humans are shown in TABLE 30 below. . TABLE 30: Dosage of OMS646 to inhibit the lectin pathway in vivo While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

权利要求:
Claims (23)
[1]
: 1
1. Isolated human monoclonal antibody, or antigen-binding fragment thereof, which binds to human MASP-2, characterized in that it comprises: 1) a) a heavy chain variable region comprising: i ) a heavy chain CDR-HI comprising amino acid sequence 31 to 35 of SEQ ID NO: 20; and ii) a heavy chain CDR-H2 comprising amino acid sequence 50 to 65 of SEQ ID NO: 20; and iii) a heavy chain CDR-H3 comprising amino acid sequence 95 to 102 of SEQ ID NO: 18 or SEQ ID NO: 20; and b) a light chain variable region comprising: i) a light chain CDR-L1 comprising amino acid sequence 24 to 34 of SEQ ID NO: 22 or SEQ ID NO: 24; and ii) a light chain CDR-L2 comprising amino acid sequence 50 to 56 of SEQ ID NO: 22 or SEQ ID NO: 24; and iii) a light chain CDR-L3 comprising amino acid sequence 89 to 97 of SEQ ID NO: 22 or SEQ ID NO: 24; or II) a variant thereof which is otherwise identical to said variable domains, except up to a combined total of 10 amino acid substitutions within said CDR regions of said heavy chain and up to a combined total of 10 amino acid substitutions within said of said CDR regions of said light chain variable region, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation.
[2]
2. An antibody according to claim 1, characterized in that at least one of the following applies: (1) wherein said antibody binds to human MASP-2 with a
KD of 10 nM or less; . (ii) said antibody binds to an epitope in the CCP1 domain of * MASP-2; ] (iii) said antibody inhibits C3b deposition in an in vitro assay in 1% human serum at an IC of 10 nM or less; (iv) said antibody inhibits the deposition of C3b in 90% human serum with an ICs 9 of 30 nM or less; or (v) wherein the antibody does not substantially inhibit the classical pathway.
[3]
3. Antibody according to claim 1, characterized in that the antibody or an antibody fragment selected from the group consisting of Fv, Fab, Fab”, F(ab)», F(ab”), is an antibody chain, an ScFv, a univalent antibody that lacks a hinge region, and a total antibody.
[4]
4. Antibody according to claim 1, characterized in that said antibody is an IgG2 molecule, and an IgG molecule], and an IgG4 molecule.
[5]
5. Antibody according to claim 4, characterized in that the IZG4 molecule comprises an S228P mutation.
[6]
6. The antibody of claim 1, characterized in that said variant is otherwise identical to said heavy chain variable region except up to a combined total of 1, 2, 3, 4, 5 or 6 amino acid substitutions. within said CDR regions of said heavy chain variable region
[7]
7. Antibody according to claim 6, characterized in that said variant comprises an amino acid substitution at one or more positions selected from the group consisting of position 31, 32,33, 34,35, 51,52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97,98, 99, 100 or 102 of said heavy chain variable region.
ê 3
[8]
8. The antibody of claim 1, characterized in that said variant is otherwise identical to said light chain variable region - except up to a combined total of 1, 2, 3, 4, 5, or 6 Amino acid substitutions within said CDR regions of said light chain S-variable region.
[9]
9. Antibody according to claim 8, characterized in that said variant comprises an amino acid substitution at one or more positions selected from the group consisting of position 25, 26, 27, 29, 31, 32, 33, 51 , 52, 89, 92, 93, 95, 96 or 97 of said light-chain variable region.
[10]
10. Antibody according to claim 1, characterized in that said CDR-H3 heavy chain comprising the amino acid sequence from 95 to 102 of SEQ ID NO: 20.
[11]
11. An isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to human MASP-2, characterized in that it comprises a heavy chain variable region comprising any of the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 21.
[12]
12. Isolated monoclonal antibody, or antigen-binding fragment thereof, which binds to human MASP-2, characterized in that it comprises a light chain variable region comprising any of the amino acid sequences shown in SEQ ID NO : 22, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 27.
[13]
13. Nucleic acid molecule, characterized in that —encodes the amino acid sequence of a MASP2 antibody, or a fragment thereof, as shown in claims 1, 11 or 12.
[14]
14. Expression cassette, characterized in that it comprises a nucleic acid molecule that encodes a MASP-2 antibody of the invention as defined in claim 13.
The
[15]
15. Cell, characterized in that it comprises at least one of the nucleic acid molecules that encode an antibody * MASP-2 of the invention according to claim 13 or claim 14. Á
[16]
16. Method for generating an isolated anti-MASP-2 antibody, — characterized in that it comprises culturing the cell as defined in claim 15 under conditions that allow the expression of nucleic acid molecules encoding the anti-MASP-2 antibody and isolating said anti-MASP-2 antibody.
[17]
17. Fully isolated human monoclonal antibody or — antigen-binding fragment thereof, characterized in that it dissociates from human MASP-2 with a KD of 10 nM or less as determined by surface plasmon resonance and inhibits activation of C4 on a mannan coated substrate with an ICs 9 of 10 nM or less in 1% serum.
[18]
18. Isolated human antibody according to claim 17, characterized in that said antibody or antigen-binding fragment thereof specifically recognizes at least part of an epitope recognized by a reference antibody that comprises a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable region as shown in SEQ ID NO: 24.
[19]
19. Composition, characterized in that it comprises the fully human monoclonal anti-MASP-2 antibody, or a fragment thereof, according to claims 1, 11, 12, or 17, and a pharmaceutically acceptable excipient.
[20]
20. Composition according to claim 19, characterized in that the composition is formulated for intra-arterial, intravenous, intracranial, intramuscular, inhalational, nasal or subcutaneous administration.
[21]
21. Use of a MASP-2 antibody as defined in any
[22]
; 5 of claims 1, 11, 12 or 17, characterized in that it is for the manufacture of a medicament to inhibit MASP-2 dependent complement activation in an individual in need thereof. : 22. Article of manufacture, characterized in that it comprises a unit dose of a human monoclonal anti-MASP-2 antibody suitable for therapeutic administration to a human subject, wherein the unit dose is in the range of 1 mg to 1000 mg .
[23]
23. Isolated human monoclonal antibody, or antigen-binding fragment thereof, which binds to human MASP-2, — characterized in that it comprises: (1) a heavy chain variable region comprising the CDR-H1 sequences, CDR-H2 and CDR-H3; and (11) a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the heavy chain variable region CDR-H3 sequence comprises an amino acid sequence shown as SEQ ID NO: 38 or SEQ ID NO: 90, and conservative sequence modifications thereof, wherein the light chain variable region CDR-L3 sequence comprises an amino acid sequence shown as SEQ ID NO: 51 or SEQ ID NO: 94, and modifications sequence — conservative thereof, and wherein the antibody alone inhibits MASP-2-dependent complement activation.

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法律状态:
2020-08-04| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-30| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-12-07| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161482567P| true| 2011-05-04|2011-05-04|

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US61/482567|2011-05-04|

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PCT/US2012/036509|WO2012151481A1|2011-05-04|2012-05-04|Compositions for inhibiting masp-2 dependent complement acitivation|

---