OA19494A - Methods for treating and/or preventing graft-versus-host disease and/or diffuse alveolar hemorrhage and/or veno-occlusive disease associated with hematopoietic stem cell transplant. - Google Patents

Methods for treating and/or preventing graft-versus-host disease and/or diffuse alveolar hemorrhage and/or veno-occlusive disease associated with hematopoietic stem cell transplant. Download PDF

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OA19494A
OA19494A OA1202000072 OA19494A OA 19494 A OA19494 A OA 19494A OA 1202000072 OA1202000072 OA 1202000072 OA 19494 A OA19494 A OA 19494A
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masp
antibody
complément
inhibitory
subject
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OA1202000072
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Gregory A. Demopulos
Hans-Wilhelm Schwaeble
Thomas Dudler
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Omeros Corporation
University Of Leicester
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Abstract

In one aspect, the invention provides methods of inhibiting the effects of MASP-2dependent complement activation in a human subject suffering from graft-versus-host disease and/or diffuse alveolar hemorrhage and/or venoocclusive disease associated with a hematopoietic stem cell transplant. The methods comprise the step of administering, to a subject in need thereof, an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation.

Description

The présent invention is based upon the surprising discovery by the présent inventors that it is possible to inhibit the lectin mediated MASP-2 pathway while leaving 5 the classical pathway intact. The présent invention also describes the use of MASP-2 as a therapeutic target for inhibiting cellular injury associated with lectin-mediated complément pathway activation while leaving the classical (Clq-dependent) pathway component of the immune System intact.
I. DEFINITIONS
Unless specifically defined herein, ail terms used herein hâve the same meaning as would be understood by those of ordinary skill in the art of the présent invention. The following définitions are provided in order to provide clarity with respect to the terms as they are used in the spécification and daims to describe the présent invention.
As used herein, the term “MASP-2-dependent complément activation” comprises 15 MASP-2- dépendent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca++) leading to the formation bf the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n, which has been determined to primarily cause opsonization.
As used herein, the term alternative pathway refers to complément activation that is triggered, for example, by zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram négative outer membranes, and rabbit érythrocytes, as well as from many pure polysaccharides, rabbit érythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which has traditionally been thought to arise from spontaneous proteolytic génération of C3b from complément factor C3.
As used herein, the term lectin pathway refers to complément activation that occurs via the spécifie binding of sérum 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 complément activation that is triggered by antibody bound to a foreign particle and requires binding of the récognition molécule Clq.
As used herein, the term MASP-2 inhibitory agent refers to any agent that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complément activation, including anti-MASP-2 antibodies and MASP-2 binding fragments thereof, natural and synthetic peptides, small molécules, soluble MASP-2 receptors, expression inhibitors and isolated natural inhibitors, and also encompasses peptides that compete with MASP-2 for binding to another récognition molécule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does not encompass antibodies that bind to such other récognition molécules. MASP-2 inhibitory agents useful in the method of the invention may reduce MASP-2-dependent complément activation by greater than 20%, such as greater than 50%, such as greater than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complément activation by greater than 90% (i.e., resulting in MASP-2 complément activation of only 10% or less).
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 sélection, recombinant expression or transgenic animais (or other methods of producing antibodies or antibody fragments”), that specifïcally bind to a target polypeptide, such as, for example, MASP-2, polypeptides or portions thereof. It is not intended that the term “antibody” limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animal, peptide synthesis, etc). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact antibody 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')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molécule that
comprises an antigen-binding site or fragment (epitope récognition site) of the required specificity.
A “monoclonal antibody refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non5 naturally occurring) that are involved in the sélective binding of an epitope. Monoclonal antibodies are highly spécifie for the target antigen. The term monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molécule that comprises an antigenbinding fragment (epitope récognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animais, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the définition of antibody.
As used herein, the term antibody fragment refers to a portion derived from or related to a full-length antibody, such as, for example, an anti-MASP-2 antibody, generally including the antigen binding or variable région thereof. Illustrative examples 20 of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecific antibodies formed from 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 présent in a single 25 polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the and Vl domains, which enables the scFv to form the desired structure for antigen binding.
As used herein, a chimeric antibody is a recombinant protein that contains the variable domains and complementarity-determining régions derived from a non-human 30 species (e.g., rodent) antibody, while the remainder of the antibody molécule is derived from a human antibody.
As used herein, a humanized antibody is a chimeric antibody that comprises a minimal sequence that conforms to spécifie complementarity-determining régions derived from non-human immunoglobulin that is transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody 5 complementarity-determining régions are of non-human origin.
As used herein, the term mannan-binding lectin (MBL) is équivalent to mannan-binding protein (MBP).
As used herein, the membrane attack complex (MAC) refers to a complex of the terminal five complément components (C5b combined with C6, C7, C8 and C-9) that 10 inserts into and disrupts membranes (also referred to as C5b-9).
As used herein, a subject includes ail mammals, including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.
As used herein, the amino acid residues are abbreviated as follows: alanine 15 (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), méthionine (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, the naturally occurring amino acids can be divided into groups based upon the Chemical characteristic of the side chain of the respective amino acids. By hydrophobie amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By hydrophilic amino acid is meant either Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subclassed as 25 follows. By uncharged hydrophilic amino acid is meant either Ser, Thr, Asn or Gin.
By acidic amino acid is meant either Glu or Asp. By basic amino acid is meant either Lys, Arg or His.
As used herein the term conservative amino acid substitution is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, 30 valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
The term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers those oligonucleobases composed of naturally-occurring nucléotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having 5 non-naturally-occurring modifications.
As used herein, an epitope refers to the site on a protein (e.g., a human MASP-2 protein) that is bound by an antibody. Overlapping epitopes include at least one (e.g., two, three, four, five, or six) common amino acid residue(s), including linear and nonlinear epitopes.
As used herein, the terms polypeptide, peptide, and protein are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The MASP-2 protein described herein can contain or be wild-type proteins or can be variants that hâve not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) 15 conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
In some embodiments, the human MASP-2 protein can hâve an amino acid 20 sequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human MASP-2 protein having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, peptide fragments can be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25, 26, 27, 28, 29,30,31,32,33,34, 25 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues in length (e.g., at least 6 contiguous amino acid residues of SEQ ID NO: 5). In some embodiments, an antigenic peptide fragment of a human MASP-2 protein is fewer than 500 (e.g., fewer than 450, 400, 350, 325, 300, 275, 30 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70,
65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length (e.g., fewer than 500 contiguous amino acid residues in any one ofSEQIDNOS: 5).
Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the fulllength of the sequences being compared can be determined by known methods.
II. OverView of the Invention
Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are the spécifie récognition molécules that trigger the innate complément system and the system includes the lectin initiation pathway and the associated terminal pathway amplification loop that amplifies lectin-initiated activation of terminal complément effector molécules. Clq is the spécifie récognition molécule that triggers the acquired complément system and the system includes the classical initiation pathway and associated terminal pathway amplification loop that amplifies Clq-initiated activation of terminal complément effector molécules. We refer to these two major complément activation Systems as the lectin-dependent complément system and the Clq-dependent complément system, respectively.
In addition to its essential rôle in immune defense, the complément system contributes to tissue damage in many clinical conditions. Thus, there is a pressing need to develop therapeutically effective complément inhibitors to prevent these adverse effects. With the récognition that it is possible to inhibit the lectin mediated MASP-2 pathway while leaving the classical pathway intact cornes the realization that it would be highly désirable to specifically inhibit only the complément activation system causing a particular pathology without completely shutting down the immune defense capabilities of complément. For example, in disease States in which complément activation is mediated predominantly by the lectin-dependent complément system, it would be advantageous to specifically inhibit only this System. This would leave the
Clq-dependent complément activation system intact to handle immune complex Processing and to aid in host defense against infection.
The preferred protein component to target in the development of therapeutic agents to specifically inhibit the lectin-dependent complément system is MASP-2. Of ail the known protein components of the lectin-dependent complément 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 complément system and required for the system to function. The lectins (MBL, H-fîcolin, M-ficolin,L-ficolin and CL-11) are also unique components in the lectin-dependent complément system. However, loss of any one of the lectin components would not necessarily inhibit activation of the system due to lectin redundancy. It would be necessary to inhibit ail five lectins in order to guarantee inhibition of the lectin-dependent complément activation system. Furthermore, since MBL and the ficolins are also known to hâve opsonic activity independent of complément, inhibition of lectin function would resuit in the loss of this bénéficiai host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity would remain intact if MASP-2 was the inhibitory target. An added benefit of MASP-2 as the therapeutic target to inhibit the lectin-dependent complément activation System is that the plasma concentration of MASP-2 is among the lowest of any complément protein (~ 500 ng/ml); therefore, correspondingly low concentrations of high-affinity inhibitors of MASP-2 may be sufficient to obtain full inhibition (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).
III. THE ROLE OF MASP-2 IN THROMBOTIC MICROANGIOPATHIES AND THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS
OverView
Thrombotic microangiopathy (TMA) is a pathology characterized by blood clots in small blood vessels (Benz, K.; et al., Curr Opin Nephrol Hypertens 19(3):242-7 (2010)). Stress or injury to the underlying vascular endothélium is believed to be a primary driver. Clinical and laboratory findings of TMA include thrombocytopenia, anémia, purpura, and rénal failure. The classic TMAs are hemolytic urémie syndrome (HUS) and thrombotic thrombocytopénie purpura (TTP). The characteristic underlying pathological feature of TMAs are platelet activation and the formation of microthrombi in the small artérioles and venules. Complément activation initiated, at least in part, by an injury or stress to microvascular endothélium, is also implicated in other TMAs including catastrophic antiphospholipid syndrome (CAPS), systemic Degos disease, and TMAs secondary to cancer, cancer chemotherapy and transplantation.
Direct evidence for a pathological rôle of complément in a host of nephritides is provided by studies of patients with genetic deficiencies in spécifie complément components. A number of reports hâve documented an association of rénal injury with deficiencies of complément regulatory factor H (Ault, B.H., Nephrol. 74:1045-1053, 2000; Levy, M., étal., Kidney Int. 30:949-56, 1986; Pickering, M.C., étal., Nat. Genet. 31:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 due to activation-related consumption of these components. Circulating levels of C5b-9 are also elevated in the sérum of these patients, implying complément activation. Membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic urémie syndrome (HUS) are associated with factor H deficiency or mutations of factor H. Factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998) and factor-H knockout mice (Pickering, M.C., 2002) display MPGN-like symptoms, confirming the importance of factor H in complément régulation. Deficiencies of other complément components are associated with rénal disease, secondary to the development of systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. Sci. 815:261-SI, 1997). Deficiency for Clq, C4 and C2 prédisposé strongly to the development of SLE via mechanisms relating to defective clearance of immune complexes and apoptotic materiaL In many of these SLE patients lupus nephritis occurs, characterized by the déposition of immune complexes throughout the glomerulus.
aHUS
Atypical hemolytic urémie syndrome (aHUS) is part of a group of conditions termed “Thrombotic microangiopathies.” In the atypical form of HUS (aHUS), the disease is associated with defective complément régulation and can be either sporadic or familial. Familial cases of aHUS are associated with mutations in genes coding for complément activation or complément regulatory proteins, including complément factor H, factor I, factor B, membrane cofactor CD46 as well as complément factor H-related protein 1 (CFHR1) and complément factor H-related protein 3 (CFHR3). (Zipfel, P.F., et al., PloS Genetics 3(3):e41 (2007)). The unifying feature of this diverse array of genetic mutations associated with aHUS is a prédisposition to enhanced complément activation on cellular or tissue surfaces. Therefore, one aspect of the présent invention comprises treating a patient suffering with aHUS that is associated with a factor H defiency by administering an effective amount of a MASP-2 inhibitory agent. Another aspect of the présent invention comprises treating a patient suffering with HUS that is associated with a factor I, factor B, membrane cofactor CD46, CFHR1 or CFHR3 deficiency by administering an effective amount of a MASP-2 inhibitory agent.
Significant progress has been made recently toward the understanding of the molecular pathophysiology underlying enhanced complément activation in aHUS caused by the diverse set of mutant complément factors. This mechanism is best understood for factor H mutations. Factor H is an abundant sérum protein comprising 20 short consensus repeat (SCR) domains that acts as a négative regulator of complément activation both in solution as well as on host cell surfaces. It targets the activated form of C3 and, together with factor I and other cofactors, promûtes its inactivation, forestalling further complément activation. To effectively control complément activation on host cell surfaces, factor H needs to interact with host cells, which is mediated by SCR domains 16-20. Ail factor H mutations associated with aHUS described to date are clustered in the C-terminal région encompassing (SCR) domains 16-20. These mutant factor H proteins are fully functional in controlling C3 activation in solution, but are unable to interact with host cell surfaces and consequently cannot control C3 activation on cellular surfaces (Exp Med 204(6):1249-56 (2007)). Thus, certain mutations of factor H are associated with aHUS because the mutant factor H protein fails to interact with host cell surfaces and thus cannot effectively down modulate complément activation on host cell surfaces, including the microvascular endothélium. As a resuit, once initial C3 activation has occurred, subséquent complément activation on microvascular endothélial surfaces proceeds unabated in patients with factor H mutations. This uncontrolled activation of complément ultimately leads to progressive injury to the vascular endothélium, subséquent platelet aggregation and microvascular coagulation, and hemolysis caused by sheer stress of RBC passage through partially occluded microvessels. Thus, aHUS disease manifestations and clinical and laboratory findings are directly linked to a defect in the négative régulation of complément on the surface of the microvascular endothélium.
Analogous to factor H mutation, loss-of-function mutations in the négative complément modulators factor I and membrane cofactor protein (CD46) are also linked to aHUS. The opposite has been observed for factor B the C3 protein in that aHUS was found to be associated with gain-of-function mutations in these proteins (Pediatr Nephrol 25(12):2431-42 (2010)). Thus, a host of converging data implicates complément activation in aHUS pathogenesis. This notion is most convincingly supported by the therapeutic efficacy ofeculizumab, a monoclonal antibody that blocks the terminal complément protein C5 in the treatment of aHUS.
While the central rôle of complément as an effector mechanism in aHUS is widely accepted, the triggers initiating complément activation and the molecular pathways involved are unresolved. Not ail individuals carrying the above described mutations develop aHUS. In fact, familial studies hâve suggested that the penetrance of aHUS is only -50% (Ann Hum Genet 74(1):17-26 (2010)). The natural history of the disease suggests that aHUS most often develops after an initiating event such as an infectious épisode or an injury. Infectious agents are well known to activate the complément System. In the absence of pre-existing adaptive immunity, complément activation by infectious agents may be primarily initiated via the lectin pathway. Thus, lectin pathway activation triggered by an infection may represent the initiating trigger for subséquent pathological amplification of complément activation in aHUS-predisposed individuals, which may ultimately lead to disease progression. Accordingly, another aspect of the présent invention comprises treating a patient suffering with aHUS secondary to an infection by administering an effective amount of a MASP-2 inhibitory agent.
Other forms of injury to host tissue will activate complément via the lectin pathway, in particular injury to the vascular endothélium. Human vascular endothélial cells subject to oxidative stress for example respond by expressing surface moieties that bind lectins and activate the lectin pathway of complément (Am J. Pathol 156(6):1549-56 (2000)). Vascular injury following ischemia/reperfusion also activâtes complément via the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)). Lectin pathway activation in this setting has pathological conséquences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents further host tissue injury and adverse outcomes (Schwaeble PNAS 2011).
Thus, other processes that precipitate aHUS are also known to activate the lectin pathway of complément. It is therefore likely that the lectin pathway may represent the initial complément activating mechanism that is inappropriately amplified in a deregulated fashion in individuals genetically predisposed to aHUS, thus initiating aHUS pathogenesis. By inference, agents that block activation of complément via the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent disease progression or reduce exacerbations in aHUS susceptible individuals.
In further support of this concept, recent studies hâve identified S. pneumonia as an important etiological agent in pédiatrie cases of aHUS. (Nephrology (Carlton), 17:4852 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011)). This particular etiology appears to hâve an unfavorable prognosis, with significant mortality and long-term morbidity. Notably, these cases involved non-enteric infections leading to manifestations of microangiopathy, uremia and hemolysis without evidence of concurrent mutations in complément genes known to prédisposé to aHUS. It is important to note that S. pneumonia is particularly effective at activating complément, and does so predominantly through the lectin pathway. Thus, in cases of non-enteric HUS associated with pneumococcal infection, manifestations of microangiopathy, uremia and hemolysis are expected to be driven predominantly by activation of the lectin pathway, and agents that block the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent progression of aHUS or reduce disease severity in these patients. Accordingly, another aspect of the présent invention comprises treating a patient suffering with non-enteric aHUS that is associated with S. pneumonia infection by administering an effective amount of a MASP-2 inhibitory agent.
In accordance with the foregoing, in some embodiments, in the setting of a subject at risk for developing rénal failure associated with aHUS, a method is provided for decreasing the likelihood of developing aHUS, or of developing rénal failure associated with aHUS, comprising administering an amount of an MASP-2 inhibitory agent for a time period effective to ameliorate or prevent rénal failure in the subject. In some embodiments, the method further comprises the step of determining whether a subject is at risk for developing aHUS prior to the onset of any symptoms associated with aHUS. In other embodiments, the method comprises determining whether a subject is a risk for developing aHUS upon the onset of at least one or more symptoms indicative of aHUS (e.g., the presence of anémia, thrombocytopenia and/or rénal insufficiency) and/or the presence of thrombotic microangiopathy in a biopsy obtained from the subject. The détermination of whether a subject is at risk for developing aHUS comprises determining whether the subject has a genetic prédisposition to developing aHUS, which may be carried out by assessing genetic information (e.g. from a database containing the génotype ofthe subject), or performing at least one genetic screening test on the subject to détermine the presence or absence of a genetic marker associated with aHUS (i.e., determining the presence or absence of a genetic mutation associated with aHUS in the genes encoding complément factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, C3, complément factor H-related protein 1 (CFHR1), or THBD (encoding the anticoagulant protein thrombodulin) or complément factor H-related protein 3 (CFHR3), or complément factor H-related protein 4 (CFHR4)) either via genome sequencing or gene-specific analysis (e.g., PCR analysis), and/or determining whether the subject has a family history of aHUS. Methods of genetic screening for the presence or absence of a genetic mutation associated with aHUS are well established, for example, see Noris M et al. “Atypical Hemolytic-Uremic Syndrome,” 2007 Nov 16 [Updated 2011 Mar 10]. In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews™, Seattle (WA): University of Washington, Seattle.
For example, overall the penetrance of the disease in those with mutations of complément factor H (CFH) is 48%, and the penetrance for mutations in CD46 is 53%, for mutations in CFI is 50%, for mutations in C3 is 56% and for mutations in THBD is 64% (Caprioli J. et al., Blood, 108:1267-79 (2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59 (2010)). As described in Caprioli et al., (2006), supra, a substantial number of individuals with a mutation in complément Factor H (CFH) never develop aHUS, and it is postulated that suboptimal CFH activity in these individuals is sufficient to protect the host from the effects of complément activation in physiological conditions, however, suboptimal CFH activity is not sufficient to prevent C3b from being deposited on vascular endothélial cells when exposure to an agent that activâtes complément produces higher than normal amounts of C3b.
Accordingly, in one embodiment, a method is provided for inhibiting MASP-2dependent complément activation in a subject suffering from, or at risk for developing non-Factor H-dependent atypical hemolytic urémie syndrome, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complément activation. In another embodiment, a method is provided for inhibiting MASP-2-dependent complément activation in a subject at risk for developing Factor H-dependent atypical hemolytic urémie syndrome, comprising periodically monitoring the subject to détermine the presence or absence of anémia, thrombocytopenia or rising créatinine, and treating with a MASP-2 inhibitory agent upon the détermination of the presence of anémia thrombocytopenia, or rising créatinine. In another embodiment, a method is provided for reducing the likelihood that a subject at risk for developing Factor H-dependent aHUS will suffer clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory agent prior to, or during, or after an event known to be associated with triggering aHUS clinical symptoms, for example, drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, an injury, organ or tissue transplant, or pregnancy.
In one embodiment, a method is provided for reducing the likelihood that a subject at risk for developing aHUS will suffer clinical symptoms associated with aHUS, comprising periodically monitoring the subject to détermine the presence or absence of anémia, thrombocytopenia or rising créatinine, and treating with a MASP-2 inhibitory agent upon the détermination of the presence of anémia, thrombocytopenia, or rising créatinine.
In another embodiment, a method is provided for reducing the likelihood that a subject at risk for developing aHUS will suffer clinical symptoms associated with aHUS comprising administering a MASP-2 inhibitory agent prior to, or during, or after an event known to be associated with triggering aHUS clinical symptoms, for example, drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, an injury, organ or tissue transplant, or pregnancy.
In some embodiments, the MASP-2 inhibitory agent is administered for a time period of at least one, two, three, four days, or longer, prior to, during, or after the event associated with triggering aHUS clinical symptoms and may be repeated as determined by a physician until the condition has been resolved or is controlled. In a pre-aHUS setting, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subeutaneous or other parentéral administration.
In some embodiments, in the setting of initial diagnosis of aHUS, or in a subject exhibiting one or more symptoms consistent with a diagnosis of aHUS (e.g., the presence of anémia, thrombocytopenia and/or rénal insufficiency), the subject is treated with an effective amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) as a first line therapy in the absence of plasmapheresis, or in combination with plasmapheresis. As a first line therapy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a first line therapy in the absence of plasmaphersis to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.
In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from aHUS via a cathéter (e.g., intravenously) for a first time period (e.g., at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory agent to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis. In some embodiments, the method further comprises determining the level of at least one complément factor (e.g., C3, C5) in the subject prior to treatment, and optionally during treatmeni, wherein the détermination of a reduced level of at least one complément factor in comparison to a standard value or healthy control subject is indicative of the need for continued treatment with the MASP-2 inhibitory agent.
In some embodiments, the method comprises administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk for developing, aHUS either intravenously, intramuscularly, or preferably, subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every two weeks. The anti-MASP-2 antibody may be administered atone, or in combination with a C5 inhibitor, such as eculizamab.
HUS
Like atypical HUS, the typical form of HUS displays ail the clinical and laboratory findings of a TMA. Typical HUS, however, is often a pédiatrie disease and usually has no familial component or direct association with mutations in complément genes. The etiology of typical HUS is tightly linked to infection with certain intestinal pathogens. The patients typically présent with acute rénal failure, hemoglobinuria, and thrombocytopenia, which typically follows an épisode of bloody diarrhea. The condition is caused by an enteric infection with Shigella dissenteria, Salmonella or shiga toxin-like producing enterohemorrhagic strains ofÆ Coli. such as E.Coli O157:H7. The pathogens are acquired from contaminated food or water supply. HUS is a medical emergency and carries a 5-10% mortality. A significant portion of survivors develop chronic kidney disease (Corrigan and Boineau, Pediatr Rev 22 (11): 365-9 (2011)) and may require kidney transplantation.
The microvascular coagulation in typical HUS occurs predominantly, though not exclusively, in the rénal microvasculature. The underlying pathophysiology is mediated by Shiga toxin (STX). Excreted by enteropathic microbes into the intestinal lumen, STX crosses the intestinal barrier, enters the bloodstream and binds to vascular endothélial cells via the blobotriaosyl ceramide receptor CD77 (Boyd and Lingwood Nephron 51:207 (1989)), which is preferentially expressed on glomerular endothélium and médiates the toxic effect of STX. Once bound to the endothélium, STX induces a sériés of events that damage vascular endothélium, activate leukocytes and cause vWF-dependent thrombus formation (Forsyth et al., Lancet 2: 411^-14 (1989); Zoja et al., Kidney Int. 62: 846-856 (2002); Zanchi et al., J. Immunol. 181:1460-1469 (2008); Morigi et al., Blood 98: 1828— 1835 (2001); Guessou et al., Infect. Immun., 73: 8306—8316 (2005)). These microthrombi obstruct or occlude the artérioles and capillaries of the kidney and other organs. The obstruction of blood flow in artérioles and capillaries by microthrombi increases the shear force applied to RBCs as they squeeze through the narrowed blood vessels. This can resuit in destruction of RBC by shear force and the formation of RBC fragments called schistocytes. The presence of schistocytes is a characteristic finding in HUS. This mechanism is known as microangiopathic hemolysis. In addition, obstruction of blood flow results in ischemia, initiating a complement-mediated inflammatory response that causes additional damage to the affected organ.
The lectin pathway of complément contributes to the pathogenesis of HUS by two principle mechanisms: 1) MASP-2-mediated direct activation of the coagulation cascade caused by endothélial injury, and 2) lectin-mediated subséquent complément activation induced by the ischemia resulting from the initial occlusion of microvascular blood flow.
STX injures microvascular endothélial cells, and injured endothélial cells are known to activate the complément system. As detailed above, complément activation foilowing endothélial cell injury is driven predominantly by the lectin pathway. Human vascular endothélial cells subject to oxidative stress respond by expressing surface moieties that bind lectins and activate the lectin pathway of complément (Collard et al., 10 Am J Pathol. 156(5):1549-56 (2000)). Vascular injury foilowing ischemia reperfusion also activâtes complément via the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)).Lectin pathway activation in this setting has pathological conséquences for the host, and inhibition of the lectin pathway by blockade of MASP-2 prevents further host tissue injury and adverse outcomes (Schwaeble et al., PNAS (2011)). In addition to 15 complément activation, lectin-dependent activation of MASP-2 has been shown to resuit in cleavage of prothrombin to form thrombin and to promote coagulation. Thus, activation of the lectin pathway of complément by injured endothélial cells can directly activate the coagulation system. The lectin pathway of complément, by virtue of MASP2-mediated prothombin activation, therefore is likely the dominant molecular pathway 20 linking the initial endothélial injury by STX to the coagulation and microvascular thrombosis that occurs in HUS. It is therefore expected that lectin pathway inhibitors, including, but not limited to, antibodies that block MASP-2 function, will prevent or mitigate microvascular coagulation, thrombosis and hemolysis in patients suffering from HUS. Indeed, administration of anti-MASP-2 antibody profoundly protects mice in a 25 model of typical HUS. As described in Example 36 and shown in FIGURE 45, ail control mice exposed to STX and LPS developed severe HUS and became moribund or died within 48 hours. On the other hand, as further shown in FIGURE 45, ail mice treated with an anti-MASP-2 antibody and then exposed to STX and LPS survived (Fisher’s exact p<0.01; N=5). Thus, anti-MASP-2 therapy profoundly protects mice in 30 this model of HUS. It is expected that administration of a MASP-2 inhibitory agent, such as a MASP-2 antibody, will be effective in the treatment of HUS patients and provide protection from microvascular coagulation, thrombosis, and hemolysis caused by infection with enteropathic E. coli or other STX-producing pathogens.
While shown here for HUS caused by STX, it is expected that anti-MASP-2 therapy will also be bénéficiai for HUS-like syndromes due to endothélial injury caused by other toxic agents. This includes agents such as mitomycin, ticlopidine, cycplatin, quinine, cyclosporine, bleomycin as well as other chemotherapy drugs and immunosuppresssive drugs. Thus, it is expected that anti-MASP-2 antibody therapy, or other modalities that inhibit MASP-2 activity, will effectively prevent or limit coagulation, thrombus formation, and RBC destruction and prevent rénal failure in HUS and other TMA related diseases (i.e., aHUS and TTP).
Patients suffering from HUS often présent with diarrhea and vomiting, their platelet counts are usually reduced (thrombocytopenia), and RBCs are reduced (anémia). A pre-HUS diarrhea phase typically lasts for about four days, during which subjects at risk for developing HUS typically exhibit one or more of the following symptoms in addition to severe diarrhea: a hematocrit level below 30% with smear evidence of intravascular érythrocyte destruction, thrombocytopenia (platelet count <150 x 103/mm3), and/or the presence of impaired rénal function (sérum créatinine concentration greater than the upper limit of reference range for âge). The presence of oligoanuria (urine output <0.5 mL/kg/h for >1 day) can be used as a measure for progression towards developing HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884-889 (2011)). Testing is typically carried out for the presence of infection with E. coli bacteria (E.coli O157:H7), or Shigella or Salmonella species. In a subject testing positive for infection with enterogenic E. coli (e.g., E. coli 0157:H7), the use of antibiotics is contraindicated because the use of antibiotics may increase the risk of developing HUS through increased STX production (See Wong C. et al., N Engl J. Med 342:1930-1936 (2000). For subjects testing positive for Shigella or Salmonella, antibiotics are typically administered to clear the infection. Other well established first-line therapy for HUS includes volume expansion, dialysis and plasmapheresis.
In accordance with the foregoing, in some embodiments, in the setting of a subject suffering from one or more symptoms associated with a pre-HUS phase and at risk for developing HUS (Le., the subject exhibits one or more of the following: diarrhea, a hematocrit level less than 30% with smear evidence of intravascular érythrocyte destruction, thrombocytopenia (platelet count less than 150 x 103/mm3), and/or the presence of impaired rénal function (sérum créatinine concentration greater than the upper limit of reference range for âge)), a method is provided for decreasing the risk of developing HUS, or of decreasing the likelihood of rénal failure in the subject, comprising administering an amount of an MASP-2 inhibitory agent for a time period effective to ameliorate or prevent impaired rénal function. In some embodiments, the MASP-2 inhibitory agent is administered for a time period of at least one, two, three, four or more days, and may be repeated as determined by a physician until the condition has been resolved or is controlled. In a pre-HUS setting, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, oral, subcutaneous or other parentéral administration.
The treatment of E. coli 0157:H7 infection with bactericidal antibiotics, particularly β-lactams, has been associated with an increased risk of developing HUS (Smith et al., Pediatr Infect Dis J 31(1):37-41 (2012).In some embodiments, in the setting of a subject suffering from symptoms associated with a pre-HUS phase, wherein the subject is known to hâve an infection with enterogenic E. coli for which the use of antibiotics is contra-indicated (e.g., E. coli 0157:H7), a method is provided for decreasing the risk of developing HUS, or of decreasing the likelihood of rénal failure in the subject, comprising administering an amount of a MASP-2 inhibitory agent for a first time period effective to inhibit or prevent the presence of oligoanuria in the subject (e.g., at least one, two, three or four days), wherein the administration of the MASP-2 inhibitory agent during the first time period occurs in the absence of an antibiotic. In some embodiments, the method further comprises administering the MASP-2 inhibitory agent to the subject in combination with an antibiotic for a second time period (such as at least one to two weeks).
In other embodiments, in the setting of a subject suffering from symptoms associated with a pre-HUS phase, wherein the subject is known to hâve an infection with Shigella or Salmonella, a method is provided for decreasing the risk of developing HUS, or of decreasing the likelihood of rénal failure in the subject, comprising administering an amount of a MASP-2 inhibitory agent and for a time period effective to inhibit or prevent the presence of oligoanuria in the subject, wherein the administration of the MASP-2 inhibitory agent is either in the presence or in the absence of a suitable antibiotic.
In some embodiments, in the setting of an initial diagnosis of HUS, or in a subject exhibiting one or more symptoms consistent with a diagnosis of HUS (e.g., the presence of rénal failure, or microangiopathic hemolytic anémia in the absence of low fibrinogen, or thrombocytopenia) the subject is treated with an effective amount of a MASP-2 inhibitory agent (e.g. a anti-MASP-2 antibody) as a first-line therapy in the absence of plasmapheresis, or in combination with plasmapheresis. As a first-line therapy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a first line therapy in the absence of plasmapheresis to avoid the complications of plasmapheresis such as hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.
In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from HUS via a cathéter (e.g., intravenously) for a first time period (e.g., an acute phase lasting at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory agent to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis. In some embodiments, the method further comprises determining the level of at least one complément factor (e.g., C3, C5) in the subject prior to treatment, and optionally during treatment, wherein the détermination of a reduced level of the at least one complément factor in comparison to a standard value or healthy control subject is indicative of the need for treatment, and wherein the détermination of a normal level is indicative of improvement.
In some embodiments, the method comprises administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk for developing, HUS either subcutaneously or intravenously. Treatment is preferably daily, but can be as infrequent as weekly or monthly. Treatment will continue for at least one week and as long as 3 months. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
TTP:
Thrombotic thrombocytopénie purpura (TTP) is a life threatening disorder of the blood-coagulation System, caused by autoimmune or hereditary dysfunctions that activate the coagulation cascade or the complément System (George, JN, N Engl J Med-, 354:1927-35 (2006)). This results in numerous microscopie clots, or thomboses, in small blood vessels throughout the body. Red blood cells are subjected to shear stress which damages their membranes, leading to intravascular hemolysis. The resulting reduced blood flow and endothélial injury results in organ damage, including brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anémia, neurological changes, rénal failure and fever. In the era before plasma exchange, the fatality rate was 90% during acute épisodes. Even with plasma exchange, survival at six months is about 80%.
TTP may arise from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloprotease responsible for cleaving large multimers of von Willebrand factor (vWF) into smaller units. ADAMTS-13 inhibition or deficiency ultimately results in increased coagulation (Tsai, H. J Am Soc Nephrol 14: 1072-1081, (2003)). ADAMTS-13 régulâtes the activity of vWF; in its absence, vWF forms large multimers which are more likely to bind platelets and prédisposés patients to platelet aggregation and thrombosis in the m icro vasculature.
Upshaw-Schulman syndrome (USS, also described as congénital TTP) is a congénital deficiency of ADAMTS13 activity due to ADAMTS13 gene mutations (Schulman et al., Blood, 16(1):943-57, 1960; Upshaw et al., New Engl. J. Med, 298 (24):1350-2, 1978). Numerous mutations in ADAMTS13 hâve been identified in individuals with congénital TTP (Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et aL, Brit. J. Haemat 144:742-754 (2008)). Subjects with USS typically hâve 5-10% of normal ADAMTS13 activity (Kokame et aL, PNAS 99(18):11902-11907, 2002). Although acquired TTP and USS hâve some similarities, USS has some important différences in clinical features. USS usually présents in infancy or childhood and is characterized by severe hyperbilirubinemia with négative Coombs test soon after birth, response to fresh plasma infusion, and frequent relapses (Savasan et al., Blood, 101:4449-4451, 2003). In some cases, patients with this inherited ADAMTS13 deficiency hâve a mild phenotype at birth and only develop symptoms associated with TTP in clinical situations with increased von Willebrand factor levels, such as infection or pregnancy. For example, Fujimura et al.
reported 9 Japanese women from 6 families with genetically confirmed USS who were diagnosed with the disorder during their first pregnancy. Thrombocytopenia occurred during the second to third trimesters in each of their 15 pregnancies, often followed by TTP. Ail of these women were found to be severely déficient in ADAMTS13 activity (Fujimura et aL, Brit. J. Haemat 144:742-754, 2008).
In accordance with the foregoing, in some embodiments, in the setting of a subject with Upshaw-Schulman syndrome (USS) (Le., the subject is known to be déficient in ADAMTS13 activity and/or the subject is known to hâve one or more ADAMTS13 gene mutation(s)), a method is provided for decreasing the likelihood of developing clinical symptoms associated with congénital TTP (e.g., thrombocytopenia, anémia, fever, and/or 15 rénal failure ) comprising administering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2 antibody) for a time period effective to ameliorate or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further comprises the step of determining whether a subject is at risk for developing symptoms associated with congénital TTP prior to the onset of any symptoms associated with TTP, or upon the onset of at least one or more symptoms indicative of TTP (e.g., the presence of anémia, thrombocytopenia and/or rénal insufficiency). The détermination of whether a subject is at risk for developing symptoms associated with congénital TTP (i.e., the subject has USS), comprises determining whether the subject has a mutation in the gene encoding ADAMTS13, and/or determining whether the subject is déficient in
ADAMTS13 activity, and/or determining whether the subject has a family history of USS. Methods of genetic screening for the presence or absence of a genetic mutation associated with USS are well established, for example see Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood, 30 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et aL, Brit. J. Haemat 144:742-754 (2008).
In one embodiment, a method is provided for reducing the likelihood that a subject diagnosed with USS will suffer clinical symptoms associated with TTP comprising periodically monitoring the subject to détermine the presence or absence of anémia, thrombocytopenia or rising créatinine, and treating with a MASP-2 inhibitory agent (e.g., a MASP-2 antibody) upon the détermination of the presence of anémia, thrombocytopenia or rising créatinine, or upon the presence of an event known to be associated with triggering TTP clinical symptoms, for example, drug exposure (e.g., chemotherapy), infection (e.g. bacterial infection), malignancy, injury, transplant, or pregnancy.
In another embodiment, a method is provided for treating a subject with USS and suffering from clinical symptoms associated with TTP comprising administering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2 antibody) for a time period effective to ameliorate or prevent one or more clinical symptoms associated with TTP.
TTP can also develop due to auto-antibodies against ADAMTS-13. In addition, TTP can develop during breast, gastrointestinal tract, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14 (2011)), pregnancy (second trimester or postpartum), George JN., Curr Opin Hematol 10:339-344 (2003)), or is associated with diseases, such as HIV or autoimmune diseases like systemic lupus erythematosis (Hamasaki K, et al., Clin Rheumatol.22:355-8 (2003)). TTP can also be caused by certain drug thérapies, including heparin, Quinine, immunemediated ingrédient, cancer chemotherapeutic agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin and anti-platelet drugs including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16: 353-357 (2011)). Other factors or conditions associated with TTP are toxins such as bee venoms, sepsis, splenic séquestration, transplantation, vasculitis, vascular surgery, and infections like Streptococcus pneumonia and cytomégalovirus (Moake JL., N Engl J Med., 347:589-600 (2002)). TTP due to transient functional ADAMTS-13 deficiency can occur as a conséquence of endothélial cell injury associated with S. pneumonia infection (Pediatr Nephrol., 26:631-5 (2011)).
Plasma exchange is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397 (1991)). Plasma exchange replaces ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in those patients with acquired autoimmune TTP (Tsai, H-M, Hematol Oncol Clin North Am., 21(4): 609-v (2007)). Additional agents such as immunosuppressive drugs are routinely added to therapy (George, JN, N Engl J Med, 354:1927-35 (2006)). However, plasma exchange is not successful for about 20% of patients, relapse occurs in more than a third of patients, and plasmapheresis is costly and technically demanding. Furthermore, many patients are unable to tolerate plasma exchange. Consequently there remains a critical need for additional and better treatments for TTP.
Because TTP is a disorder of the blood coagulation cascade, treatment with antagonists of the complément System may aid in stabilizing and correcting the disease. While pathological activation of the alternative complément pathway is linked to aHUS, the rôle of complément activation in TTP is less clear. The functional deficiency of ADAMTS13 is important for the susceptibility of TTP, however it is not sufficient to cause acute épisodes. Environmental factors and/or other genetic variations may contribute to the manifestation of TTP. For example, genes encoding proteins involved in the régulation of the coagulation cascade, vWF, platelet function, components of the endothélial vessel surface, or the complément System may be implicated in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94: 166-170 (2009)). In particular, complément activation has been shown to play a critical rôle; sérum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC déposition and subséquent neutrophil activation which could be abrogated by complément inactivation (Ruiz-Torres MP, et aL, Thromb Haemost, 93:443-52 (2005)). In addition, it has recently been shown that during acute épisodes of TTP there are increased levels of C4d, C3bBbP, and C3a (M. Réti et aL, J Thromb Haemost. Feb 28.(2012) doi: 10.1111/j. 1538-7836.2012.04674.x. [Epub ahead of print]), consistent with activation of the classical/lectin and alternative pathways. This increased amount of complément activation in acute épisodes may initiate the terminal pathway activation and be responsible for further exacerbation of TTP.
The rôle of ADAMTS-13 and vWF in TTP clearly is responsible for activation and aggregation of platelets and their subséquent rôle in shear stress and déposition in microangiopathies. Activated platelets interact with and trigger both the classical and alternative pathways of complément. Platelet mediated complément activation increases the inflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol, 47:2170-5 (2010)). Platelets may thus serve as targets of classical complément activation in inherited or autoimmune TTP.
As described above, the lectin pathway of complément, by virtue of MASP-2 mediated prothombin activation, is the dominant molecular pathway linking endothélial injury to the coagulation and microvascular thrombosis that occurs in HUS. Similarly, activation of the lectin pathway of complément may directly drive the coagulation System in TTP. Lectin pathway activation may be initiated in response to the initial endothélium injury caused by ADAMTS-13 deficiency in TTP. It is therefore expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will mitigate the microangiopathies associated with microvascular coagulation, thrombosis, and hemolysis in patients suffering from TTP.
Patients suffering from TTP typically présent in the emergency room with one or more of the following: purpura, rénal failure, low platelets, anémia and/or thrombosis, including stroke. The current standard of care for TTP involves intra-catheter delivery (e.g., intravenous or other form of cathéter) of replacement plasmapheresis for a period of two weeks or longer, typically three times a week, but up to daily. If the subject tests positive for the presence of an inhibitor of ADAMTS13 (i.e., an endogenous antibody against ADAMTS13), then the plasmapheresis may be carried out in combination with immunosuppressive therapy (e.g., corticosteroids, rituxan, or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to at least two weeks of plasmapheresis therapy.
In accordance with the foregoing, in one embodiment, in the setting of an initial diagnosis of TTP or in a subject exhibiting one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous System involvement, severe thrombocytopenia (a platelet count of less that or equal to 5000/pL if off aspirin, less than or equal to 20,000/pL if on aspirin), severe cardiac involvement, severe pulmonary involvement, gastro-intestinal infarction or gangrené), a method is provided for treating the subject with an effective amount of a MASP-2 inhibitory agent (e.g., a anti-MASP-2 antibody) as a first line therapy in the absence of plasmapheresis, or in combination with plasmapheresis. As a first-line therapy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a firstline therapy in the absence of plasmapheresis to avoid the potential complications of plasmapheresis, such as hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable. In some embodiments, the MASP-2 inhibitory agent is administered to the subject suffering from TTP in combination (including co-administration) with an immunosuppressive agent (e.g., corticosteroids, rituxan or cyclosporine) and/or in combination with concentrated ADAMTS-13.
In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from TTP via a cathéter (e.g., intravenously) for a first time period (e.g., an acute phase lasting at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory agent to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis. In some embodiments, the method is used to maintain the subject to prevent the subject from suffering one or more symptoms associated with TTP.
In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject that has not responded to at least two weeks of plasmapheresis therapy), by administering an amount of a MASP-2 inhibitor effective to reduce one or more symptoms of TTP. In one embodiment, the MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) is administered to a subject with refractory TTP on a chronic basis, over a time period of at least two weeks or longer via subeutaneous or other parentéral administration. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In some embodiments, the method further comprises determining the level of at least one complément factor (e.g., C3, C5) in the subject prior to treatment, and optionally during treatment, wherein the détermination of a reduced level of the at least one complément factor in comparison to a standard value or healthy control subject is indicative of the need for continued treatment with the MASP-2 inhibitory agent.
In some embodiments, the method comprises administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk for developing, TTP either subcutaneously or intravenously. Treatment is preferably daily, but can be as infrequent as biweekly. Treatment is continuée! until the subject’s platelet count is greater than 150,000/ml for at least two consecutive days. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the foilowing characteristics: said antibody binds human MASP-2 with a Kq of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC5Q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2; wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from TTP by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from TTP at a level of at least 20 percent or greater, (such as at least 30%, at least 40%, at least 50%) more than the inhibitory effect on C5b-9 déposition in sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a TTP patient by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus 5 formation in a subject suffering from TTP comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavychain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID 10 NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDRL1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a lightchain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID 15 NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least
90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 20 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises 25 administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment 30 thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NQ:70.
Degos Disease
Degos disease, also known as malignant atrophie papulosis, is a very rare TMA affecting the endothélium of small vessels of skin, gastrointestinal tract, and CNS. This vasculopathy causes occlusion of venules and artioles, resulting in skin lésions, bowel ischemia, and CNS disorders including strokes, epilepsy and cognitive disorders. In the skin, connective tissue necrosis is due to thrombotic occlusion of the small arteries. However, the cause of Degos disease is unknown. Vasculitis, coagulopathy, or primary dysfunction of the endothélial cells hâve been implicated. Degos disease has a 50% survival of only two to three years. There is no effective treatment for Degos disease although antiplatelet drugs, anticoagulants, and immunosuppressants are utilized to alleviate symptoms.
While the mechanism of Degos disease is unknown, the complément pathway has been implicated. Margo et al., identified prominent C5b-9 deposits in skin, gastrointestinal tract and brain vessels of four terminal patients with Degos disease (Margo et al., Am J Clin Pathol 135(4):599-610, 2011). Experimental treatment with eculizumab was initially effective in the treatment of skin and intestinal lésions, but did not prevent the progression of systemic disease (see Garrett-Bakelman F. et al., “C5b-9 is a potential effector in the pathophysiology of Degos disease; a case report of treatment with eculizumab” (Abstract), Jérusalem: International Society of Hematology; 2010, Poster #156; and Polito J. et al, “Early détection of systemic Degos disease (DD) or malignant atrophie papulosis (MAP) may increase survival” (Abstract), San Antonio, TX: American College of Gastroenterology; 2010, Poster #1205).
Many patients suffering from Degos disease hâve defects of blood coagulation. Thrombotic occlusion of small arteries in the skin is characteristic of the disease. Because the complément pathway is implicated in this disease, as described herein for other TMAs, it is expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will be bénéficiai in treating patients suffering from Degos disease.
Accordingly, in another embodiment, the invention provides methods for treating Degos disease by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from Degos disease or a condition resulting from Degos disease. The MASP-2 inhibitory agent is administered systemically to the subject suffering from Degos disease or a condition resulting from Degos disease, such as by intra-arterial, intravenous, intramuscular, inhalational, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The antiMASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a Kq of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from Degos disease by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from Degos disease at a level of at least 20 percent or greater, (such as at least 30%, at least 40%, at least 50%) more than the inhibitory effect on C5b-9 déposition in sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation 5 in sérum from a Degos disease patient by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the 10 subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from Degos disease comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) 15 a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a lightchain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and 20 ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to
SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding 30 fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment 5 thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
Catastrophic antiphospholipid syndrome (CAPS)
Catastrophic antiphospholipid syndrome (CAPS) is an extreme variant of the antiphospholipid antibody (APLA) syndrome. CAPS is characterized by venous and arterial thrombosis due to pathogenic antibodies. CAPS is a TMA with multiple organ 15 thrombosis, ischemia, and organ failure. Like other TMAs, occlusion of small vessels in various organs is characteristic. There is a high mortality rate in CAPS of about 50% and often it is associated with infection or trauma. Patients hâve antiphospholipid antibodies, generally IgG.
Clinically, CAPS involves at least three organs or tissues with histopathological 20 evidence of small vessel occlusion. Peripheral thrombosis may involve veins and arteries in the CNS, cardiovascular, rénal, or pulmonary Systems. Patients are treated with antibiotics, anticoagulants, corticosteroids, plasma exchange, and intravenous immunoglobulin. Nevertheless, death may resuit from multiple organ failure.
The complément pathway has been implicated in CAPS. For example, studies in 25 animal models indicate that complément inhibition may be an effective means to prevent thrombosis associated with CAPS (Shapira L. et aL, Arthritis Rheum 64(8):2719-23, 2012). Moreover, as further reported by Shapira et al., administration of eculizumab to a subject suffering from CAPS at doses that blocked complément pathway aborted acute progressive thrombotic events and reversed thrombocytopenia (see also Lim W., Curr 30 Opin Hematol 18(5):361-5, 2011). Therefore, as described herein for other TMAs, it is expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will be bénéficiai in treating patients suffering from CAPS.
Accordingly, in another embodiment, the invention provides methods for treating CAPS by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from CAPS or a condition resulting from CAPS. The MASP-2 inhibitory agent is administered systemically to the subject suffering from CAPS or a condition resulting from CAPS, such as by intra-arterial, intravenous, intramuscular, inhalational, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC5Q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from CAPS by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from CAPS at a level of at least 20 percent or greater, (such as at least 30%, at least 40%, at least 50%) more than the inhibitory effect on C5b-9 déposition in sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a CAPS patient by at least 30%, such as at least 40%, such as at least 50%, 5 such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus 10 formation in a subject suffering from CAPS comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavychain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID 15 NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDRL1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a lightchain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID 20 NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least
90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 25 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises 30 administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as 5 set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID
NO:70.
TMA Secondary to Cancer
Systemic malignancies of any type can lead to clinical and pathologie manifestations of TMA (see e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). Cancer-associated TMA is often found in the lungs and appears to be associated with tumor emboli (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor emboli can reduce blood flow and thus lead to a hypo-perfused State in the 15 affected artérioles and venules. The resulting tissue stress and injury is expected to activate the lectin pathway of complément in a localized fashion. The activated lectin pathway in turn can activate the coagulation cascade via MASP-2 dépendent cleavage of prothrombin to thrombin, leading to a pro-thrombotic State characteristic of TMA. MASP-2 inhibition in this setting is expected to reduce the localized activation of thrombin and thereby alleviate the pro-thrombotic State.
Therefore, as described herein for other TMAs, it is expected that lectin pathway inhibitors, including, but not limited to, antibodies that block MASP-2 function, will be bénéficiai in treating patients suffering from TMA secondary to cancer.
Accordingly, in another embodiment, the invention provides methods for treating 25 or preventing TMA secondary to cancer by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from, or at risk for developing, a TMA secondary to cancer. The MASP-2 inhibitory agent is administered systemically to the subject suffering from, or at risk for developing, a TMA secondary to cancer, such 30 as by intra-arterial, intravenous, intramuscular, inhalational, subeutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents.
The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC5Q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2; wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from TMA secondary to cancer by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a patient suffering TMA secondary to cancer by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from TMA secondary to cancer comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
TMA Secondary to Cancer Chemotherapy
Chemotherapy-associated TMA is a condition involving thrombocytopenia, microangiopathic hemolytic anémia, and rénal dysfunction that develops in 2-10% of patients with a history of malignant neoplasms treated with chemotherapeutic agents such as gemcytabin, mitomycin, oxaliplatin and others. Chemotherapy-associated TMA is associated with high mortality poor clinical outcomes (see, e.g., Blake-Haskins et al., Clin Cancer Res 17(18):5858-5866, 2011).
The etiology of chemotherapy-associated TMA is thought to encompass a nonspecific, toxic insult to the microvascular endothélium. A direct injury to endothélial cells has been shown in an animal model of mitomycin-induced TMA (Dlott J. et al., Ther Apher Dial 8:102-11, 2004). Endothélial cell injury through a variety of mechanisms has been shown to activate the lectin pathway of complément. For example, Stahl et al. hâve shown that endothélial cells exposed to oxidative stress activate the lectin pathway of complément both in vitro and in vivo (Collard et al., Am J Pathol. 156(5):1549-56, 2000; La Bonte et al, JImmunol. 15; 188(2):885-91, 2012). In vivo, this process leads to thombosis, and inhibition of the lectin pathway has been shown to prevent thrombosis (La Bonte et al. JImmunol. 15;188(2):885-91, 2012). Futhermôre, as demonstrated in Examples 37-39 herein, in the mouse model of TMA where localized photoexcitation of FITC-Dex was used to induce localized injury to the microvasculature with subséquent development of a TMA response, the présent inventors hâve shown that inhibition of MASP-2 can prevent TMA. Thus, microvascular endothélium injury by chemotherapeutic agents may activate the lectin pathway of complément which then créâtes a localized pro-thrombotic State and promûtes a TMA response. Since activation of the lectin pathway and the création of a pro-thombotic State is MASP-2-dependent, it is expected that MASP-2 inhibitors, including, but not limited to, antibodies that block MASP-2 fonction, will alleviate the TMA response and reduce the risk of cancer chemotherapy-associated TMA.
Accordingly, in another embodiment, the invention provides methods for treating or preventing TMA secondary to chemotherapy by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from, or at risk for developing, a TMA secondary to chemotherapy. The MASP-2 inhibitory agent is administered systemically to a subject that has undergone, is undergoing, or will undergo chemotherapy, such as by intra-arterial, intravenous, intramuscular, inhalational, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the foilowing characteristics: said antibody binds human MASP-2 with a Kq of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2) wherein the antibody is a single-chain moiecule, wherein said antibody is an IgG2 moiecule, wherein said antibody is an IgGl moiecule, wherein said antibody is an IgG4 moiecule, wherein the IgG4 moiecule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from TMA secondary to cancer chemotherapy by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a patient suffering TMA secondary to cancer chemotherapy by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from TMA secondary to cancer chemotherapy comprising administering to the subject a composition comprising an amount of a MASP2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavychain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
TMA Secondary to Transplantation
Transplantation-associated TMA (TA-TMA) is a devastating syndrome that can occur in transplant patients, such as allogeneic hematopoietic stem cell transplant 5 récipients (see e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). The pathogenesis of this condition is poorly understood, but likely involves a confluence of responses that culminate in endothélial cell injury (Laskin B.L. et al., Blood 118(6):1452-62, 2011). As discussed above, endothélial cell injury is a prototypic stimulus for lectin pathway activation and the génération of a pro-thrombotic 10 environment.
Recent data further support the rôle of complément activation via the lectin pathway in the pathogenesis TA-TMA. Laskin et aL, hâve demonstrated that rénal arteriolar C4d déposition was much more common in subjects with histologie TA-TMA (75%) compared with Controls (8%) (Laskin B.L., et al., Transplantation, 27; 96(2):21715 23, 2013). Thus, C4d may be a pathologie marker of TA-TMA, implicating localized complément fixation via the lectin or classical pathway.
Since activation of the lectin pathway and the création of a pro-thombotic State is MASP-2-dependent, it is expected that MASP-2 inhibitors, including, but not limited to, antibodies that block MASP-2 function, will alleviate the TMA response and reduce the 20 risk of transplantation-associated TMA (TA-TMA).
Accordingly, in another embodiment, the invention provides methods for treating or preventing a TMA secondary to transplantation by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from, or at risk for 25 developing a TMA secondary to transplantation. The MASP-2 inhibitory agent is administered systemically to a subject that has undergone, is undergoing, or will undergo a transplant procedure, such as by intra-arterial, intravenous, intramuscular, inhalational, subeutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The anti-MASP-2 antibody may be administered alone, or in 30 combination with a C5 inhibitor, such as eculizamab. In some embodiments, the invention provides methods for treating or preventing a TMA secondary to allogeneic stem cell transplant comprising administering a composition comprising an amount of a
MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject prior to, during or after undergoing an allogeneic stem cell transplant.
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a Kq of 10 nM 5 or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC5q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, 10 wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one 15 embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation 20 in sérum from a subject suffering from TMA secondary to transplant by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation 25 in sérum from a patient suffering TMA secondary to transplant by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the 30 subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from TMA secondary to transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavychain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
IV. THE ROLE OF MASP-2 IN OTHER DISEASES AND CONDITIONS AND
THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS
RENAL CONDITIONS
Activation of the complément System has been implicated in the pathogenesis of a wide variety of rénal diseases; including, mesangioproliferative glomerulonephritis (IgA-nephropathy, Berger's disease) (Endo, M., étal., Clin. Nephrology 55:185-191, 2001), membranous glomerulonephritis (Kerjashki, D., Arch B Cell Pathol. 55:253-71, 1990; Brenchley, P.E., et al., Kidney Int., 41:933-7, 1992; Salant, D.J., et al., Kidney 10 Int. 35:976-84, 1989), membranoproliferative glomerulonephritis (mesangiocapillary glomerulonephritis) (Bartlow, B.G., et al., Kidney Int. 75:294-300, 1979; Meri, S., et al., J. Exp. Med. /75:939-50, 1992), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinémie glomerulonephritis (Ohsawa, L, étal., Clin Immunol. 101:59-66, 2001), lupus nephritis (Gatenby, P.A., 15 Autoimmunity //:61-6, 1991), and Henoch-Schonlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis. 35Ά01-467, 2000). The involvement of complément in rénal disease has been appreciated for several décades but there is still a major discussion on its exact rôle in the onset, the development and the resolution phase of rénal disease. Under normal conditions the contribution of complément is bénéficiai to the host, but 20 inappropriate activation and déposition of complément may contribute to tissue damage.
There is substantial evidence that glomerulonephritis, inflammation of the glomeruli, is often initiated by déposition of immune complexes onto glomerular or tubular structures which then triggers complément activation, inflammation and tissue damage. Kahn and Sinniah demonstrated increased déposition of C5b-9 in tubular 25 basement membranes in biopsies taken from patients with various forms of glomerulonephritis (Kahn, T.N., et al., Histopath. 26:351-6, 1995). In a study of patients with IgA nephrology (Alexopoulos, A., étal., Nephrol. Dial. Transplant 10:1166-1172, 1995), C5b-9 déposition in the tubular epithelial/basement membrane structures correlated with plasma créatinine levels. Another study of membranous nephropathy 30 demonstrated a relationship between clinical outcome and urinary sC5b-9 levels (Kon, S.P., et al., Kidney Int. 48:1953-5%, 1995). Elevated sC5b-9 levels were correlated positively with poor prognosis. Lehto et al., measured elevated levels of CD59, a complément regulatory factor that inhibits the membrane attack complex in plasma membranes, as well as C5b-9 in urine from patients with membranous glomerulonephritis (Lehto, T., et al., Kidney Int. 47:1403-11, 1995). Histopathological analysis of biopsy samples taken from these same patients demonstrated déposition of C3 and C9 proteins in the glomeruli, whereas expression of CD59 in these tissues was diminished compared to that of normal kidney tissue. These various studies suggest that ongoing complement-mediated glomerulonephritis results in urinary excrétion of complément proteins that correlate with the degree of tissue damage and disease prognosis.
Inhibition of complément activation in various animal models of glomerulonephritis has also demonstrated the importance of complément activation in the etiology of the disease. In a model of membranoproliferative glomerulonephritis (MPGN), infusion of anti-Thyl antiserum in C6-deficient rats (that cannot form C5b-9) resulted in 90% less glomerular cellular prolifération, 80% réduction in platelet and macrophage infiltration, diminished collagen type IV synthesis (a marker for mesangial matrix expansion), and 50% less proteinuria than in C6+ normal rats (Brandt, J., et al., Kidney Int. 49:335-343, 1996). These results implicate C5b-9 as a major mediator of tissue damage by complément in this rat anti-thymocyte sérum model. In another model of glomerulonephritis, infusion of graded dosages of rabbit anti-rat glomerular basement membrane produced a dose-dependent influx of polymorphonuclear leukocytes (PMN) that was attenuated by prior treatment with cobra venom factor (to consume complément) (Scandrett, A.L., et aL, Am. J. Physiol. 268:Έ256-Έ265, 1995). Cobra venom factor-treated rats also showed diminished histopathology, decreased long-term proteinuria, and lower créatinine levels than control rats. Employing three models of GN in rats (anti-thymocyte sérum, Con A anti-Con A, and passive Heymann nephritis), Couser et al., demonstrated the potential therapeutic efficacy of approaches to inhibit complément by using the recombinant sCRl protein (Couser, W.G., et al., J. Am. Soc. Nephrol. 5:1888-94, 1995). Rats treated with sCRl showed significantly diminished PMN, platelet and macrophage influx, decreased mesangiolysis, and proteinuria versus control rats. Further evidence for the importance of complément activation in glomerulonephritis has been provided by the use of an anti-C5 MoAb in the NZB/W Fl mouse model. The anti-C5 MoAb inhibits cleavage of C5, thus blocking génération of C5a and C5b-9. Continuons therapy with anti-C5 MoAb for 6 months resulted in significant amelioration of the course of glomerulonephritis. A humanized anti-C5 MoAb monoclonal antibody (5G1.1) that prevents the cleavage of human complément component C5 into its pro-inflammatory components is under development by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential treatment for glomerulonephritis.
Direct evidence for a pathological rôle of complément in rénal injury is provided by studies of patients with genetic deficiencies in spécifie complément components. A number of reports hâve documented an association of rénal disease with deficiencies of complément regulatory factor H (Ault, B.H., Nephrol. 14:1045-1053, 2000; Levy, M., étal., Kidney Int. 30:949-56, 1986; Pickering, M.C., et al., Nat. Genet. 37:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 and in consumption of C5b-9. Both atypical membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic urémie syndrome (HUS) are associated with factor H deficiency. Factor H déficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998) and factor H knockout mice (Pickering, M.C., 2002) display MPGN-like symptoms, confirming the importance of factor H in complément régulation. Deficiencies of other complément components are associated with rénal disease, secondary to the development of systemic lupus erythematosus (SLE) (Walport, M.J., Davies, étal., Ann. N.Y. Acad. Sci. 575:267-81, 1997). Deficiency for Clq, C4 and C2 prédisposé strongly to the development of SLE via mechanisms relating to defective clearance of immune complexes and apoptotic material. In many of these SLE patients lupus nephritis occurs, characterized by the déposition of immune complexes throughout the glomerulus.
Further evidence linking complément activation and rénal disease has been provided by the identification in patients of autoantibodies directed against complément components, some of which hâve been directly related to rénal disease (Trouw, L.A., et al., Mol. Immunol. 35:199-206, 2001). A number of these autoantibodies show such a high degree of corrélation with rénal disease that the term nephritic factor (NeF) was introduced to indicate this activity. In clinical studies, about 50% of the patients positive for nephritic factors developed MPGN (Spitzer, R.E., et al., Clin. Immunol. Immunopathol. 64:177-83, 1992). C3NeF is an autoantibody directed against the alternative pathway C3 convertase (C3bBb) and it stabilizes this convertase, thereby promoting alternative pathway activation (Daha, M.R., et aL, J. Immunol. 116:1-1, 1976).
Likewise, autoantibody with a specificity for the classical pathway C3 convertase (C4b2a), called C4NeF, stabilizes this convertase and thereby promûtes classical pathway activation (Daha, M.R. et al., J. Immunol. 725:2051-2054, 1980; Halbwachs, L., et aL, J. Clin. Invest. 65:1249-56, 1980). Anti-Clq autoantibodies hâve been described to be related to nephritis in SLE patients (Hovath, L., et al., Clin. Exp. Rheumatol. 19:661-12, 2001; Siegert, C., et al., J. Rheumatol. 18:230-34, 1991; Siegert, C., et al., Clin. Exp. Rheumatol. 70:19-23, 1992), and a rise in the titer of these anti-Clq autoantibodies was reported to predict a flare of nephritis (Coremans, LE., et aL, Am. J. Kidney Dis. 26:595-601, 1995). Immune deposits eluted from postmortem kidneys of SLE patients revealed the accumulation of these anti-Clq autoantibodies (Mannick, M., et al., Arthritis Rheumatol. 40:1504-11, 1997). Ail these facts point to a pathological rôle for these autoantibodies. However, not ail patients with anti-Clq autoantibodies develop rénal disease and also some healthy individuals hâve low titer anti-Clq autoantibodies (Siegert, C.E., et al., Clin. Immunol. Immunopathol. 67:204-9, 1993).
In addition to the alternative and classical pathways of complément activation, the lectin pathway may also hâve an important pathological rôle in rénal disease. Elevated levels of MBL, MBL-associated serine protease and complément activation products hâve been detected by immunohistochemical techniques on rénal biopsy material obtained from patients diagnosed with several different rénal diseases, including Henoch-Schonlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000), cryoglobulinémie glomerulonephritis (Ohsawa, L, et al., Clin. Immunol. 101:59-66, 2001) and IgA neuropathy (Endo, M., et aL, Clin.
Nephrology 55:\85-\9\, 2001). Therefore, despite the fact that an association between complément and rénal diseases has been known for several décades, data on how complément exactly influences these rénal diseases is far from complété.
BLOOD DISORDERS
Sepsis is caused by an overwhelming reaction of the patient to invading microorganisms. A major function of the complément System is to orchestrate the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological rôle, complément activation has been shown in numerous studies to hâve a major rôle in the pathogenesis of sepsis (Bone, R.C., Annals. Internai. Med. 775:457-469, 1991). The définition of the clinical manifestations of sepsis is ever evolving. Sepsis is usually defined as the systemic host response to an infection. However, on many occasions, no clinical evidence for infection (e.g., positive bacterial blood cultures) is found in patients with septic symptoms. This discrepancy was first taken into account at a Consensus Conférence in 1992 when the tenu systemic inflammatory response syndrome (SIRS) was established, and for which no definable presence of bacterial infection was required (Bone, R.C., étal., Crit. Care Med. 20:124-126, 1992). There is now general agreement that sepsis and SIRS are accompanied by the inability to regulate the inflammatory response. For the purposes of this brief review, we will consider the clinical définition of sepsis to also include severe sepsis, septic shock, and SIRS.
The prédominant source of infection in septic patients before the late 1980s was Gram-negative bacteria. Lipopolysaccharide (LPS), the main component of the Gram-negative bacterial cell wall, was known to stimulate release of inflammatory mediators from various cell types and induce acute infectious symptoms when injected into animais (Haeney, M.R., étal., Antimicrobial Chemotherapy 41 (Suppl. A):41-6, 1998). Interestingly, the spectrum of responsible microorganisms appears to hâve shifted from predominantly Gram-negative bacteria in the late 1970s and 1980s to predominantly Gram-positive bacteria at présent, for reasons that are currently unclear (Martin, G.S., et al., N. Eng. J. Med. 348:1546-54, 2003).
Many studies hâve shown the importance of complément activation in mediating inflammation and contributing to the features of shock, particularly septic and hémorrhagie shock. Both Gram-negative and Gram-positive organisms commonly precipitate septic shock. LPS is a potent activator of complément, predominantly via the alternative pathway, although classical pathway activation mediated by antibodies also occurs (Fearon, D.T., et al., N. Engl. J. Med. 292:937-400, 1975). The major components of the Gram-positive cell wall are peptidoglycan and lipoteichoic acid, and both components are potent activators of the alternative complément pathway, although in the presence of spécifie antibodies they can also activate the classical complément pathway (Joiner, K.A., et al., Ann. Rev. Immunol. 2:461-2, 1984).
The complément system was initially implicated in the pathogenesis of sepsis when it was noted by researchers that anaphylatoxins C3a and C5a médiate a variety of inflammatory reactions that might also occur during sepsis. These anaphylatoxins evoke vasodilation and an increase in microvascular permeability, events that play a central rôle in septic shock (Schumacher, W.A., étal., Agents Actions 34:345-349, 1991). In addition, the anaphylatoxins induce bronchospasm, histamine release from mast cells, and aggregation of platelets. Moreover, they exert numerous effects on granulocytes, such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, génération of toxic super oxide anion and formation of leukotrienes (Shin, H.S., et al., Science 162:361-363, 1968; Vogt, W., Complément 3:177-86, 1986). These biologie effects are thought to play a rôle in development of complications of sepsis such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, D.E., et aL, Lancet 1:942-949, 1980; Slotman, G.T., étal., Surgery 99:744-50, 1986). Furthermore, elevated levels of the anaphylatoxin C3a is associated with a fatal outcome in sepsis (Hack, C.E., et aL, Am. J. Med. 86:20-26, 1989). In some animal models of shock, certain complement-deficient strains (e.g., C5-deficient ones) are more résistant to the effects of LPS infusions (Hseuh, W., et al., Immunol. 70:309-14, 1990).
Blockade of C5a génération with antibodies during the onset of sepsis in rodents has been shown to greatly improve survival (Czermak, B.J., et aL, Nat. Med. 5:788-792, 1999). Similar findings were made when the C5a receptor (C5aR) was blocked, either with antibodies or with a small molecular inhibitor (Huber-Lang, M.S., et aL, FASEB J. /6:1567-74, 2002; Riedemann, N.C., étal., J. Clin. Invest. 7/0:101-8, 2002). Earlier experimental studies in monkeys hâve suggested that antibody blockade of C5a attenuated E. co/z-induced septic shock and adult respiratory distress syndrome (Hangen, D.H., et aL, J. Surg. Res. 46:195-9, 1989; Stevens, J.H., et aL, J. Clin. Invest. 77:1812-16, 1986). In humans with sepsis, C5a was elevated and associated with significantly reduced survival rates together with multiorgan failure, when compared with that in less severely septic patients and survivors (Nakae, H., et aL, Res. Commun. Chem. Pathol. Pharmacol. <34:189-95, 1994; Nakae, étal., Surg. Today 26:225-29, 1996; Bengtson, A., et aL, Arch. Surg. 123:645-649, 1988). The mechanisms by which C5a exerts its harmful effects during sepsis are yet to be investigated in greater detail, but recent data suggest the génération of C5a during sepsis significantly compromises innate immune functions of blood neutrophils (Huber-Lang, M.S., et al., J. Immunol. 169:3223-31, 2002), their ability to express a respiratory burst, and their ability to generate cytokines (Riedemann, N.C., et aL, Immunity 79:193-202, 2003). In addition, C5a génération during sepsis appears to hâve procoagulant effects (Laudes, LJ., étal., Am. J. Pathol. 760:1867-75, 2002). The complement-modulating protein CI INH has also shown efficacy in animal models of sepsis and ARDS (Dickneite, G., Behring Ins. Mitt. 93:299-305, 1993).
The lectin pathway may also hâve a rôle in pathogenesis of sepsis. MBL has been shown to bind to a range of clinically important microorganisms including both Gram-negative and Gram-positive bacteria, and to activate the lectin pathway (Neth, O., et al., Infect. Immun. 68:688, 2000). Lipoteichoic acid (LTA) is increasingly regarded as the Gram-positive counterpart of LPS. It is a potent immunostimulant that induces cytokine release from mononuclear phagocytes and whole blood (Morath, S., et al., J. Exp. Med. 195Λ635, 2002; Morath, S., étal., Infect. Immun. 70:938, 2002). Recently it was demonstrated that L-ficolin specifically binds to LTA isolated from mimerons Gram-positive bacteria species, including Staphylococcus aureus, and activâtes the lectin pathway (Lynch, N.J., et al., J. Immunol. 772:1198-02, 2004). MBL also has been shown to bind to LTA from Enterococcus spp in which the polyglycerophosphate chain is substituted with glycosyl groups), but not to LTA from nine other species including S. aureus (Polotsky, V.Y., et al., Infect. Immun. 64:380, 1996).
An aspect of the invention thus provides a method for treating sepsis or a condition resulting from sepsis, by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from sepsis or a condition resulting from sepsis including without limitation severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, and systemic inflammatory response syndrome. Related methods are provided for the treatment of other blood disorders, including hémorrhagie shock, hemolytic anémia, autoimmune thrombotic thrombocytopénie purpura (TTP), hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS), or other marrow/blood destructive conditions, by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from such a condition. The MASP-2 inhibitory agent is administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational (particularly in the case of ARDS), subeutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The MASP-2 inhibitory agent composition may be combined with one or more additional therapeutic agents to combat the sequelae of sepsis and/or shock. For advanced sepsis or shock or a distress condition resulting therefrom, the MASP-2 inhibitory composition may suitably be administered in a fast-acting dosage form, such as by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitory agent composition. Repeated administration may be carried out as determined by a physician until the condition has been resolved.
COAGULOPATHIES
Evidence has been developed for the rôle of the complément System in disseminated intravascular coagulation (DIC), such as DIC secondary to significant bodily trauma.
Previous studies hâve shown that C4-/- mice are not protected from rénal reperfusion injury. (Zhou, W., et al, Prédominant rôle for C5b-9 in rénal ischemia/reperfusion injury, J Clin Invest 105:1363-1371 (2000)) In order to investigate whether C4-/- mice may still be able to activate complément via either the classical or the lectin pathway, C3 tum-over in C4-/- plasma was measured in assays spécifie for either the classical, or the lectin pathway activation route. While no C3 cleavage could be observed when triggering activation via the classical, a highly efficient lectin pathwaydependent activation of C3 in C4 déficient sérum was observed (FIGURE 30). It can be seen that C3b déposition on mannan and zymosan is severely compromised in MASP-2-/mice, even under experimental conditions, that according to many previously published papers on alternative pathway activation, should be permissive for ail three pathways. When using the same sera in wells coated with immunoglobulin complexes instead of mannan or zymosan, C3b déposition and Factor B cleavage are seen in MASP-2+/+ mouse sera and MASP-2-/- sera, but not in Clq depleted sera. This indicates that altemate pathway activation is facilitated in MASP-2-/- sera when the initial C3b is provided via classical activity. FIGURE 30C depicts the surprising finding that C3 can efficiently be activated in a lectin pathway-dependent fashion in C4 déficient plasma.
This C4 bypass is abolished by the inhibition of lectin pathway-activation through preincubation of plasma with soluble mannan or mannose.
Aberrant, non-immune, activation of the complément System is potentially hazardous to man and may also play an important rôle in hematological pathway activation, particularly in severe trauma situations wherein both inflammatory and hematological pathways are activated. In normal health, C3 conversion is <5% of the total plasma C3 protein. In rampant infection, including septicaemia and immune complex disease, C3 conversion re-establishes itself at about 30% with complément levels frequently lower than normal, due to increased utilization and changes in pool distribution. Immédiate C3 pathway activation of greater than 30% generally produces obvious clinical evidence of vasodilatation and of fluid loss to the tissues. Above 30% C3 conversion, the initiating mechanisms are predominantly non-immune and the resulting clinical manifestations are harmful to the patient. Complément C5 levels in health and in controlled disease appear much more stable than C3. Significant decreases and or conversion of C5 levels are associated with the patient's response to abnormal polytrauma (e.g., road traffic accidents) and the likely development of shock lung syndromes. Thus, any evidence of either complément C3 activation beyond 30% of the vascular pool or of any C5 involvement, or both, may be considered likely to be a harbinger of a harmful pathological change in the patient.
Both C3 and C5 liberate anaphylatoxins (C3a and C5a) that act on mast cells and basophils releasing vasodilatory Chemicals. They set up chemotactic gradients to guide polymorphonuclear cells (PMN) to the center of immunological disturbances (a bénéficiai response), but here they differ because C5a has a spécifie dumping (aggregating) effect on these phagocytic cells, preventing their random movement away from the reaction site. In normal control of infection, C3 activâtes C5. However, in polytrauma, C5 appears to be widely activated, generating C5a anaphylatoxins systemically. This uncontrolled activity causes polymorphs to clump within the vascular System, and these clumps are then swept into the capillaries of the lungs, which they occlude and generate local damaging effects as a resuit of superoxide libération. While not wishing to be limited by theory, the mechanism is probably important in the pathogenesis of acute respiratory distress syndrome (ARDS), although this view has recently been challenged. The C3a anaphylatoxins in vitro can be shown to be potent platelet aggregators, but their involvement in vivo is less defined and the release of platelet substances and plasmin in wound repair may only secondarily involve complément C3. It is possible that prolonged élévation of C3 activation is necessary to generate DIC.
In addition to cellular and vascular effects of activated complément component outlined above that could explain the link between trauma and DIC, emerging scientific discoveries hâve identified direct molecular links and functional cross-talk between complément and coagulation Systems. Supporting data has been obtained from studies in C3 déficient mice. Because C3 is the shared component for each of the complément pathways, C3 déficient mice are predicted to lack ail complément function. Surprisingly, however, C3 déficient mice are perfectly capable of activating terminal complément components. (Huber-Lang, M., et al., Génération of C5a in the absence of C3: a new complément activation pathway, Nat. Med /2:682-687 (2006)) In depth studies revealed that C3-independent activation of terminal complément components is mediated by thrombin, the rate limiting enzyme of the coagulation cascade. (Huber et al., 2006) The molecular components mediating thrombin activation foilowing initial complément activation remained elusive.
The présent inventors hâve elucidated what is believed to be the molecular basis for cross-talk between complément and clotting cascades and identified MASP-2 as a central control point linking the two Systems. Biochemical studies into the substrate specificity of MASP-2 hâve identified prothrombin as a possible substrate, in addition to the well known C2 and C4 complément proteins. MASP-2 specifically cleaves prothrombin at functionally relevant sites, generating thrombin, the rate limiting enzyme of the coagulation cascade. (Krarup, A., et al., Simultaneous Activation of Complément and Coagulation by MBL-Associated Serine Protease 2, PLoS. ONE. 2:e623 (2007)) MASP-2-generated thrombin is capable of promoting fibrin déposition in a defined reconstituted in vitro system, demonstrating the functional relevance of MASP-2 cleavage. (Krarup et aL, 2007) As discussed in the examples herein below, the inventors hâve further corroborated the physiological significance of this discovery by documenting thrombin activation in normal rodent sérum foilowing lectin pathway activation, and demonstrated that this process is blocked by neutralizing MASP-2 monoclonal antibodies.
MASP-2 may represent a central branch point in the lectin pathway, capable of promoting activation of both complément and coagulation Systems. Because lectin pathway activation is a physiologie response to many types of traumatic injury, the présent inventors believe that concurrent systemic inflammation (mediated by complément components) and disseminated coagulation (mediated via the clotting pathway) can be explained by the capacity of MASP-2 to activate both pathways. These findings clearly suggest a rôle for MASP-2 in DIC génération and therapeutic benefit of
MASP-2 inhibition in treating or preventing DIC. MASP-2 may provide the molecular link between complément and coagulation System, and activation of the lectin pathway as it occurs in settings of trauma can directly initiate activation of the clotting System via the MASP-2-thrombin axis, providing a mechanistic link between trauma and DIC. In 5 accordance with an aspect of the présent invention, inhibition of MASP-2 would inhibit lectin pathway activation and reduce the génération of both anaphylatoxins C3a and C5a. It is believed that prolonged élévation of C3 activation is necessary to generate DIC.
Microcirculatory coagulation (blot clots in capillaries and small blood vessels) occurs in settings such a septic shock. A rôle of the lectin pathway in septic shock is 10 established, as evidenced by the protected phenotype of MASP-2 (-/-) mouse models of sepsis, described in Example 17 and FIGURES 18 and 19. Furthermore, as demonstrated in Example 15 and FIGURES 16A and 16B, MASP-2 (-/-) mice are protected in the localized Schwartzman reaction model of disseminated intravascular coagulation (DIC), a model of localized coagulation in microvessels.
V. MASP-2 INHIBITORY AGENTS
In one aspect, the présent invention provides methods of inhibiting MASP-2-dependent complément activation in a subject suffering from, or at risk for developing a thrombotic microangiopathy. MASP-2 inhibitory agents are administered in 20 an amount effective to inhibit MASP-2-dependent complément activation in a living subject. In the practice of this aspect of the invention, représentative MASP-2 inhibitory agents include: molécules that inhibit the biological activity of MASP-2 (such as small molécule inhibitors, anti-MASP-2 antibodies or blocking peptides which interact with MASP-2 or interfère with a protein-protein interaction), and molécules that decrease the 25 expression of MASP-2 (such as MASP-2 antisense nucleic acid molécules, MASP-2 spécifie RNAi molécules and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complément pathway. The MASP-2 inhibitory agents can be used alone as a primary therapy or in combination with other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.
The inhibition of MASP-2-dependent complément activation is characterized by at least one of the following changes in a component of the complément System that occurs as a resuit of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: the inhibition of the génération or production of MASP-2-dependent complément activation System products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in Example 2), the réduction of complément activation assessed in a hemolytic assay using unsensitized rabbit or guinea pig red blood 5 cells (measured, for example as described in Example 33), the réduction of C4 cleavage and C4b déposition (measured, for example as described in Example 2), or the réduction of C3 cleavage and C3b déposition (measured, for example, as described in Example 2).
According to the présent invention, MASP-2 inhibitory agents are utilized that are effective in inhibiting the MASP-2-dependent complément activation System. MASP-2 10 inhibitory agents useful in the practice of this aspect of the invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molécules, MASP-2 soluble reçeptors and expression inhibitors. MASP-2 inhibitory agents may inhibit the MASP-2-dependent complément activation System by blocking the biological function of MASP-2. For example, an inhibitory agent may 15 effectively block MASP-2 protein-to-protein interactions, interfère with MASP-2 dimerization or assembly, block Ca2+ binding, interfère with the MASP-2 serine protease active site, or may reduce MASP-2 protein expression.
In some embodiments, the MASP-2 inhibitory agents selectively inhibit MASP-2 complément activation, leaving the Clq-dependent complément activation System 20 functionally intact.
In one embodiment, a MASP-2 inhibitory agent useful in the methods of the invention is a spécifie MASP-2 inhibitory agent that specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity of at least ten times greater than to other antigens in the complément System. In another embodiment, a MASP-2 inhibitory agent 25 specifically binds to a polypeptide comprising SEQ ID NO:6 with a binding affinity of at least 100 times greater than to other antigens in the complément System. The binding affinity of the MASP-2 inhibitory agent can be determined using a suitable binding assay.
The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1, MASP-3, and Clr and Cls, the proteases of the Cl complément System. The cDNA 30 molécule set forth in SEQ ID NO:4 encodes a représentative example of MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5) and provides the human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after sécrétion, resulting in the mature form of human MASP-2 (SEQ ID NO:6). As shown in FIGURE 2, the human MASP 2 gene encompasses twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K AND L. An alternative splice results 5 in a 20 kDa protein termed MBL-associated protein 19 (MApl9, also referred to as sMAP) (SEQ ID NO:2), encoded by (SEQ ID NO:1) arising from exons B, C, D and E as shown in FIGURE 2. The cDNA molécule set forth in SEQ ID NO:50 encodes the murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:51) and provides the murine MASP-2 polypeptide with a leader sequence that is cleaved after 10 sécrétion, resulting in the mature form of murine MASP-2 (SEQ ID NO:52). The cDNA molécule set forth in SEQ ID NO:53 encodes the rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:54) and provides the rat MASP-2 polypeptide with a leader sequence that is cleaved after sécrétion, resulting in the mature form of rat MASP-2 (SEQ ID NO:55).
Those skilled in the art will recognize that the sequences disclosed in SEQ ID
NO:4, SEQ ID NO:50 and SEQ ID NO:53 represent single alleles of human, murine and rat MASP-2 respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucléotide sequences shown in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53, including those containing silent mutations and those in 20 which mutations resuit in amino acid sequence changes, are within the scope of the présent invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in FIGURE 1 and 2A and include an N-terminal Clr/Cls/sea urchin Vegf/bone 25 morphogenic protein (CUBI) domain (aa 1-121 of SEQ ID NO:6), an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), as well as a tandem of complément control protein domains and a serine protease domain. Alternative splicing of the MASP 2 gene results in MApl9 shown in FIGURE 1. MApl9 is a nonenzymatic protein containing the N-terminal CUB1-EGF région of MASP-2 with 30 four additional residues (EQSL) derived from exon E as shown in FIGURE 1.
Several proteins hâve 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
Ca2+ dépendent complexes with, the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shown to activate complément through the MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., étal., J. Exp. Med. /76:1497-2284, 2000; Matsushita, M., et aL, J. Immunol. 168:3502-3506, 2002). Studies hâve shown that the CUB 1-EGF domains of MASP-2 are essential for the association of MASP-2 with MBL (Thielens, N.M., étal., J. Immunol. /66:5068, 2001). It has also been shown that the CUB1EGFCUBII domains médiate dimerization of MASP-2, which is required for formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Therefore, MASP-2 inhibitory agents can be identified that bind to or interfère with MASP-2 target régions known to be important for MASP-2-dependent complément activation.
ANTI-MASP-2 ANTIBODIES
In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complément activation system. The anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody producing mammal and may be multispecific, chimeric, humanized, anti-idiotype, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments and single-chain antibodies as further described herein.
Several anti-MASP-2 antibodies hâve been described in the literature, some of which are listed below in TABLE 1. These previously described anti-MASP-2 antibodies can be screened for the ability to inhibit the MASP-2-dependent complément activation system using the assays described herein. For example, anti rat MASP-2 Fab2 antibodies hâve been identified that block MASP-2 dépendent complément activation, as described in more detail in Examples 10 and 11 herein. Once an anti-MASP-2 antibody is identified that functions as a MASP-2 inhibitory agent, it can be used to produce anti-idiotype antibodies and used to identify other MASP-2 binding molécules as further described below.
TABLE l: MASP-2 SPECIFIC ANTIBODIES FROM THE LITERATURE
ANTIGEN ANTIBODY TYPE REFERENCE
Recombinant MASP-2 Rat Polyclonal Peterson, S.V., et al., Mol. Immunol. 37:803-811, 2000
Recombinant human CCP1/2-SP fragment (MoAb 8B5) Rat MoAb (subclass IgGl) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003
Recombinant human MApl9(MoAb 6G12) (cross reacts with MASP-2) Rat MoAb (subclass IgGl) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003
hMASP-2 Mouse MoAb (S/P) Mouse MoAb (N-term) Peterson, S.V., et aL, Mol. Immunol. 35:409, April 1998
hMASP-2 (CCP1-CCP2-SP domain rat MoAb: NimoablOl, produced by hybridoma cell line 03050904 (ECACC) WO 2004/106384
hMASP-2 (full length-his tagged) murine MoAbs: NimoAbl04, produced by hybridoma cell line M0545YM035 (DSMZ) NimoAbl08, produced by hybridoma cell line M0545YM029 (DSMZ) NimoAbl09 produced by hybridoma cell line M0545YM046 (DSMZ) NimoAbl 10 produced by hybridoma cell line M0545YM048 (DSMZ) WO 2004/106384
ANTI-MASP-2 ANTIBODIES WITH REDUCED EFFECTOR FUNCTION
In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies 5 hâve reduced effector function in order to reduce inflammation that may arise from the activation of the classical complément pathway. The ability of IgG molécules to trigger the classical complément pathway has been shown to résidé within the Fc portion of the molécule (Duncan, A.R., étal., Nature 332:Ί38-ΊΑΰ 1988). IgG molécules in which the Fc portion of the molécule has been removed by enzymatic cleavage are devoid of this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be generated as the resuit of lacking the Fc portion of the molécule by having a genetically engineered Fc sequence that minimizes effector function, or being of either the human IgG2 or IgGq isotype.
Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc portion of the IgG heavy chains as described in Example 9 herein and also described in Jolliffe étal., Int'l Rev. Immunol. /0:241-250, 1993, and Rodrigues et aL, J. Immunol. /57:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that hâve a reduced ability to activate complément and/or interact with Fc receptors (Ravetch, J.V., et al., Annu. Rev. Immunol. 9:457-492, 1991; Isaacs, J.D., étal., J. Immunol. 775:3062-3071, 1992; van de Winkel, J.G., étal., Immunol. Today 74:215-221, 1993). Humanized or fully human antibodies spécifie to human MASP-2 comprised of IgG2 or IgG4 isotypes can be produced by one of several methods known to one of ordinary skilled in the art, as described in Vaughan, T.J., et aL, Nature Biotechnical 16:535-539, 1998.
PRODUCTION OF ANTI-MASP-2 ANTIBODIES
Anti-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g., full length MASP-2) or using antigenic MASP-2 epitope-bearing peptides (e.g., a portion of the MASP-2 polypeptide). Immunogenic peptides may be as small as five amino acid residues. For example, the MASP-2 polypeptide including the entire amino acid sequence of SEQ ID NO:6 may be used to induce anti-MASP-2 antibodies useful in the method of the invention. Particular MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI, and CUBIEGF domains, as well as the région encompassing the serine-protease active site, may be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides comprising a portion of at least 6 amino acids of the MASP-2 polypeptide (SEQ ID NO:6) are also useful to induce MASP-2 antibodies. Additional examples of MASP-2 derived antigens useful to induce MASP-2 antibodies are provided below in TABLE 2. The MASP-2 peptides and polypeptides used to raise antibodies may be isolated as natural polypeptides, or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, such as MASP-2A, as further described in Examples 5-7. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using a transgenic mouse strain as described in Examples 8 and 9 and further described 5 below.
Antigens useful for producing anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or with maltose-binding protein. The polypeptide immunogen may be a full-length molécule or a portion thereof. If the polypeptide portion is hapten-like, such 10 portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine sérum albumin (BSA) or tetanus toxoid) for immunization.
TABLE 2: MASP-2 DERIVED ANTIGENS
SEQ ID NO: Amino Acid Sequence
SEQ ID NO:6 Human MASP-2 protein
SEQ ID NO:51 Murine MASP-2 protein
SEQ ID NO:8 CUBI domain of human MASP-2 (aa 1-121 ofSEQ IDNO:6)
SEQ ID NO:9 CUBIEGF domains of human MASP-2 (aa 1-166 ofSEQ ID NO:6)
SEQ ID NO: 10 CUBIEGFCUBII domains of human MASP-2 (aa 1-293 ofSEQ ID NO:6)
SEQ ID NO: 11 EGF domain of human MASP-2 (aa 122-166 ofSEQ ID NO:6)
SEQ ID NO: 12 Serine-Protease domain of human MASP-2 (aa429-671 ofSEQ IDNO:6)
SEQ ID NO: 13 GKDSCRGDAGGALVFL Serine-Protease inactivated mutant form (aa 610-625 ofSEQ ID NO:6 with mutated Ser 618)
SEQ ID NO: 14 TPLGPKWPEPVFGRL Human CUBI peptide
SEQ ID NO: 15: TAPPGYRLRLYFTHFDLEL SHLCE YDF VKLS SG AK VL ATLCGQ Human CUBI peptide
SEQ ID NO: Amino Acid Sequence
SEQ ID NO: 16: TFRSDYSN MBL binding région in human CUBI domain
SEQ ID NO: 17: FYSLGSSLDITFRSDYSNEK PFTGF MBL binding région in human CUBI domain
SEQ ID NO: 18 IDECQVAPG EGF peptide
SEQ ID NO: 19 ANMLCAGLESGGK.DSCRG DSGGALV Peptide from serine-protease active site
POLYCLONAL ANTIBODIES
Polyclonal antibodies against MASP-2 can be prepared by immunizing an animal with MASP-2 polypeptide or an immunogenic portion thereof using methods well known to those of ordinary skill in the art. See, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), page 105, and as further described in Example 6. The immunogenicity of a MASP-2 polypeptide can be increased through the use of an adjuvant, including minerai gels, such as aluminum hydroxide or Freund's adjuvant (complété or incomplète), surface active substances such 10 as lysolecithin, pluronic polyols, polyanions, oil émulsions, keyhole limpet hemocyanin and dinitrophenol. Polyclonal antibodies are typically raised in animais such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep. Altematively, an anti-MASP-2 antibody useful in the présent invention may also be derived from a subhuman primate. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg étal., International Patent Publication No. WO 91/11465, and in Losman, M.J., étal., Int. J. Cancer 46:310, 1990. Sera containing immunologically active antibodies are then produced from the blood of such immunized animais using standard procedures well known in the art.
MONOCLONAL ANTIBODIES
In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly spécifie, being directed against a single MASP-2 epitope. As used herein, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molécules by continuous cell lines in culture, such as the hybridoma method described by Kohler, G., et al., Nature 256Ά95, 1975, or they may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, J.D., et al., J. Mol. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., a BALB/c mouse) with a composition comprising a MASP-2 polypeptide or portion thereof. After a predetermined period of time, splénocytes are removed from the mouse and suspended in a cell culture medium. The splénocytes are then fiised with an immortal cell line to form a hybridoma. The formed hybridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against MASP-2. An example further describing the production of anti-MASP-2 monoclonal antibodies is provided in Example 7. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)
Human monoclonal antibodies may be obtained through the use of transgenic mice that hâve been engineered to produce spécifie human antibodies in response to antigenic challenge. In this technique, éléments of the human immunoglobulin heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous immunoglobulin heavy chain and light chain loci. The transgenic mice can synthesize human antibodies spécifie for human antigens, such as the MASP-2 antigens described herein, and the mice can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B-cells from such animais to suitable myeloma cell lines using conventional Kohler-Milstein technology as further described in Example 7. Transgenic mice with a human immunoglobulin genome are commercially available (e.g., from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L.L., étal., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., Purification of Immunoglobulin G (IgG), in Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).
Once produced, polyclonal, monoclonal or phage-derived antibodies are first tested for spécifie MASP-2 binding. A variety of assays known to those skilled in the art may be utilized to detect antibodies which specifically bind to MASP-2. Exemplary assays include Western blot or immunoprécipitation analysis by standard methods (e.g., as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immuno-sorbent assays, dot blots, inhibition or compétition assays and sandwich assays (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies are identified that specifically bind to MASP-2, the anti-MASP-2 antibodies are tested for the ability to function as a MASP-2 inhibitory agent in one of several assays such as, for example, a lectin-specific C4 cleavage assay (described in Example 2), a C3b déposition assay (described in Example 2) or a C4b déposition assay (described in Example 2).
The affinity of anti-MASP-2 monoclonal antibodies can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 57:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies usefiil for the methods of the invention bind to MASP-2 with a binding affinity of <100 nM, preferably <10 nM and most preferably <2 nM. In some embodiments, a MASP-2 inhibitory monoclonal antibody useful in the methods of the invention is a MASP-2 inhibitory monoclonal antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of
100
SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavychain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
CHIMERIC/HUMANIZED ANTIBODIES
Monoclonal antibodies useful in the method of the invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Patent No. 4,816,567, to Cabilly; and Morrison, S.L., étal., Proc. Nat'l Acad. Sci. USA 57:6851-6855, 1984).
One form of a chimeric antibody useful in the invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring the non-human (e.g., mouse) complementarity determining régions (CDR), from the heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typically, residues of human antibodies are then substituted in the framework régions of the non-human counterparts. Furthermore, humanized antibodies may comprise residues that are not found in the récipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially ail of at least one, and typically two variable domains, in which ail or substantially ail of the hypervariable loops correspond to those of a non-human immunoglobulin and ail or substantially ail of the Fv framework régions are those of a human immunoglobulin sequence. The humanized
101 antibody optionally also will comprise at least a portion of an immunoglobulin constant région (Fc), typically that of a human immunoglobulin. For further details, see Jones, P.T., et al., Nature 321:522-525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.
The humanized antibodies useful in the invention include human monoclonal antibodies including at least a MASP-2 binding CDR3 région. In addition, the Fc portions may be replaced so as to produce IgA or IgM as well as human IgG antibodies. Such humanized antibodies will hâve particular clinical utility because they will specifically recognize human MASP-2 but will not evoke an immune response in humans against the antibody itself. Consequently, they are better suited for in vivo administration in humans, especially when repeated or long-term administration is necessary.
An example of the génération of a humanized anti-MASP-2 antibody from a murine anti-MASP-2 monoclonal antibody is provided herein in Example 6. Techniques for producing humanized monoclonal antibodies are also described, for example, by Jones, P.T., étal., Nature 321:522, 1986; Carter, P., étal., Proc. Nat'l. Acad. Sci. USA 89:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. 12Α3Ί, 1992; Singer, I.I., et aL, J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 1996; and by U.S. Patent No. 5,693,762, to Queen, 1997. In addition, there are commercial entities that will synthesize humanized antibodies from spécifie murine antibody régions, such as Protein Design Labs (Mountain View, CA).
RECOMBINANT ANTIBODIES
Anti-MASP-2 antibodies can also be made using recombinant methods. For example, human antibodies can be made using human immunoglobulin expression libraries (available for example, from Stratagene, Corp., La Jolla, CA) to produce fragments of human antibodies (Vjq, Vp, Fv, Fd, Fab or F(ab')2). These fragments are then used to construct whole human antibodies using techniques similar to those for producing chimeric antibodies.
102
ANTI-IDIOTYPE ANTIBODIES
Once anti-MASP-2 antibodies are identified with the desired inhibitory activity, these_antibodies can be used to generate anti-idiotype antibodies that resemble a portion of MASP-2 using techniques that are well known in the art. See, e.g., Greenspan, N.S., 5 étal., FASEB J. 7Ά3Ί, 1993. For example, antibodies that bind to MASP-2 and competitively inhibit a MASP-2 protein interaction required for complément activation can be used to generate anti-idiotypes that resemble the MBL binding site on MASP-2 protein and therefore bind and neutralize a binding ligand of MASP-2 such as, for example, MBL.
IMMUNOGLOBULIN FRAGMENTS
The MASP-2 inhibitory agents useful in the method of the invention encompass not only intact immunoglobulin molécules but also the well known fragments including Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecific antibodies formed from antibody 15 fragments.
It is well known in the art that only a small portion of an antibody molécule, the paratope, is involved in the binding of the antibody to its epitope (see, e.g., Clark, W.R., The Experimental Foundations of Modem Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc régions of the antibody are effectors of the classical complément 20 pathway, but are not involved in antigen binding. An antibody from which the pFc' région has been enzymatically cleaved, or which has been produced without the pFc' région, is designated an F(ab')2 fragment and retains both of the antigen binding sites of an intact antibody. An isolated F(ab')2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, an antibody from which the 25 Fc région has been enzymatically cleaved, or which has been produced without the Fc région, is designated a Fab fragment, and retains one of the antigen binding sites of an intact antibody molécule.
Antibody fragments can be obtained by proteolytic hydrolysis, such as by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody 30 fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2- This fragment can be further cleaved using a thiol reducing
103 agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that resuit from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, U.S. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., étal., Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959; Edelman, et al., in Methods in Enzymology 7:422, Academie Press, 1967; and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
In some embodiments, the use of antibody fragments lacking the Fc région are preferred to avoid activation of the classical complément pathway which is initiated upon binding Fc to the Fcy receptor. There are several methods by which one can produce a MoAb that avoids Fcy receptor interactions. For example, the Fc région of a monoclonal antibody can be removed chemically using partial digestion by proteolytic enzymes (such as frein digestion), thereby generating, for example, antigen-binding antibody fragments such as Fab or F(ab)2 fragments (Mariani, M., étal., Mol. Immunol. 25:69-71, 1991). Alternatively, the human y4 IgG isotype, which does not bind Fcy receptors, can be used during construction of a humanized antibody as described herein. Antibodies, single chain antibodies and antigen-binding domains that lack the Fc domain can also be engineered using recombinant techniques described herein.
SINGLE-CHAIN ANTIBODY FRAGMENTS
Alternatively, one can create single peptide chain binding molécules spécifie for MASP-2 in which the heavy and light chain Fv régions are connected. The Fv fragments may be connected by a peptide linker to form a single-chain antigen binding protein (scFv). These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the Vjq and Vl domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described for example, by Whitlow, étal., Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird,
104 étal., Science 242:423, 1988; U.S. Patent No. 4,946,778, to Ladner; Pack, P., étal., Bio/Technology 17:1271, 1993.
As an illustrative example, a MASP-2 spécifie scFv can be obtained by exposing lymphocytes to MASP-2 polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for example, through the use of immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage or on bacteria such as E. coïi. These random peptide display libraries can be used to screen for peptides which interact with MASP-2. Techniques for creating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409, to Lardner; U.S. Patent No. 4,946,778, to Ladner; U.S. Patent No. 5,403,484, to Lardner; U.S. Patent No. 5,571,698, to Lardner; and Kay et al., Phage Display of Peptides and Proteins Academie Press, Inc., 1996) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).
Another form of an anti-MASP-2 antibody fragment useful in this aspect of the invention is a peptide coding for a single complementarity-determining région (CDR) that binds to an epitope on a MASP-2 antigen and inhibits MASP-2-dependent complément activation. CDR peptides (minimal récognition units) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable région from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, Genetic Manipulation of Monoclonal Antibodies, in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter étal, (eds.), page 166, Cambridge University Press, 1995; and Ward et al., Genetic Manipulation and Expression of Antibodies, in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).
The MASP-2 antibodies described herein are administered to a subject in need thereof to inhibit MASP-2-dependent complément activation. In some embodiments, the
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MASP-2 inhibitory agent is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.
PEPTIDE INHIBITORS
In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises isolated MASP-2 peptide inhibitors, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit the MASP-2-dependent complément activation system. As used herein, the term isolated MASP-2 peptide inhibitors refers to peptides that inhibit MASP-2 dépendent complément activation by binding to, competing with MASP-2 for binding to another récognition molécule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, and/or directly interacting with MASP-2 to inhibit MASP-2-dependent complément activation that are substantially pure and are essentially free of other substances with which they may be found in nature to an extent practical and appropriate for their intended use.
Peptide inhibitors hâve been used successfully in vivo to interfère with protein-protein interactions and catalytic sites. For example, peptide inhibitors to adhesion molécules structurally related to LFA-1 hâve recently been approved for clinical use in coagulopathies (Ohman, E.M., étal., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) hâve been described that prevent or interfère with integrin-dependent adhesion (Murayama, O., étal., J. Biochem. /20:445-51, 1996). Longer peptides, ranging in length from 25 to 200 amino acid residues, hâve also been used successfully to block integrin-dependent adhesion (Zhang, L., et aL, J. Biol. Chem. 27/(47):29953-57, 1996). In general, longer peptide inhibitors hâve higher affinities and/or slower off-rates than short peptides and may therefore be more potent inhibitors. Cyclic peptide inhibitors hâve also been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory disease (Jackson, D.Y., et aL, J. Med. Chem. 40:3359-68, 1997). One method of producing cyclic peptides involves the synthesis of peptides in which the terminal amino acids of the peptide are cysteines, thereby allowing the peptide to exist in a cyclic form by disulfide bonding between the terminal amino acids, which has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592, to Larson).
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SYNTHETIC MASP-2 PEPTIDE INHIBITORS
MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic the target régions important for MASP-2 function. The inhibitory peptides useful in the practice of the methods of the 5 invention range in size from about 5 amino acids to about 300 amino acids. TABLE 3 provides a list of exemplary inhibitory peptides that may be useful in the practice of this aspect of the présent invention. A candidate MASP-2 inhibitory peptide may be tested for the ability to function as a MASP-2 inhibitory agent in one of several assays including, for example, a lectin spécifie C4 cleavage assay (described in Example 2), and 10 a C3b déposition assay (described in Example 2).
In some embodiments, the MASP-2 inhibitory peptides are derived from MASP-2 polypeptides and are selected from the full length mature MASP-2 protein (SEQ ID NO:6), or from a particular domain of the MASP-2 protein such as, for example, the CUBI domain (SEQ ID NO:8), the CUBIEGF domain (SEQ ID NO:9), the EGF domain 15 (SEQ ID NO:11), and the serine protease domain (SEQ ID NO:12). As previously described, the CUBEGFCUBII régions hâve been shown to be required for dimerization and binding with MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO: 16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identified a human carrying a homozygous 20 mutation at Aspl05 to Glyl05, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New EnglandJ. Med. 349:554-56®, 2003).
In some embodiments, MASP-2 inhibitory peptides are derived from the lectin proteins that bind to MASP-2 and are involved in the lectin complément pathway. Several different lectins hâve been identified that are involved in this pathway, including 25 mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin. (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 776:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 765:3502-3506, 2002). These lectins are présent in sérum as oligomers of homotrimeric subunits, each having N-terminal collagen-like fibers with carbohydrate récognition domains. These different lectins hâve been shown 30 to bind to MASP-2, and the lectin/MASP-2 complex activâtes complément through cleavage of proteins C4 and C2. H-ficolin has an amino-terminal région of 24 amino acids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino
107 acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M., et al., J. Immunol. 765:3502-3506, 2002). H-ficolin binds to GlcNAc and agglutinâtes human érythrocytes coated with LPS derived from 5. typhimurium, S. minnesota and E. coli. H-ficolin has been shown to be associated with MASP-2 and MApl9 and activâtes the 5 lectin pathway. Id. L-ficolin/P35 also binds to GlcNAc and has been shown to be associated with MASP-2 and MApl9 in human sérum and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 764:2281, 2000). Accordingly, MASP-2 inhibitory peptides useful in the présent invention may comprise a région of at least 5 amino acids selected from the MBL protein (SEQ ID NO:21), the 10 H-ficolin protein (Genbank accession number NM_173452), the M-ficolin protein (Genbank accession number 000602) and the L-ficolin protein (Genbank accession number NM_015838).
More specifically, scientists hâve identified the MASP-2 binding site on MBL to be within the 12 Gly-X-Y triplets GKD G RD GTK GEK GEP GQG LRG LQG POG 15 KLG POG NOG PSG SOG PKG QKG DOG KS (SEQ ID NO:26) that lie between the hinge and the neck in the C-terminal portion of the collagen-like domain of MBP (Wallis, R., et al., J. Biol. Chem. 279:14065, 2004). This MASP-2 binding site région is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is présent in ail three lectin proteins comprising the amino acid 20 sequence OGK-X-GP (SEQ ID NO:22) where the letter O represents hydroxyproline and the letter X is a hydrophobie residue (Wallis et al., 2004, supra). Accordingly, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO:22. Peptides derived from MBL that include the amino acid sequence GLR GLQ GPO GKL GPO G (SEQ ID 25 NO:24) hâve been shown to bind MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized that are flanked by two GPO triplets at each end (GPO GPO GLR GLQ GPO GKL GPO GGP OGP O SEQ ID NO:25) to enhance the formation of triple helices as found in the native MBL protein (as further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).
MASP-2 inhibitory peptides may also be derived from human H-ficolin that include the sequence GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO (SEQ ID NO:27) from the consensus MASP-2 binding région in H-ficolin. Also included
108 are peptides derived from human L-ficolin that include the sequence GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO (SEQ ID NO:28) from the consensus MASP-2 binding région in L-ficolin.
MASP-2 inhibitory peptides may also be derived from the C4 cleavage site such 5 as LQRALEILPNRVTIKANRPFLVFI (SEQ ID NO:29) which is the C4 cleavage site linked to the C-terminal portion of antithrombin III (Glover, G.L, et al., Mol. Immunol. 25:1261 (1988)).
TABLE 3: EXEMPLARY MASP-2 INHIBITORY PEPTIDES
SEQ ID NO Source
SEQ ID NO:6 Human MASP-2 protein
SEQ ID NO:8 CUBI domain of MASP-2 (aa 1-121 of SEQ ID NO:6)
SEQ ID NO:9 CUBIEGF domains of MASP-2 (aa 1-166 of SEQ ID NO:6)
SEQ ID NO: 10 CUBIEGFCUBII domains of MASP-2 (aa 1-293 ofSEQ ID NO:6)
SEQ ID NO: 11 EGF domain of MASP-2 (aa 122-166)
SEQ ID NO: 12 Serine-protease domain of MASP-2 (aa 429-671)
SEQ ID NO: 16 MBL binding région in MASP-2
SEQ ID NO:3 Human MAp 19
SEQIDNO:21 Human MBL protein
SEQ ID NO:22 OGK-X-GP, Where O = hydroxyproline and X is a hydrophobie amino acid residue Synthetic peptide Consensus binding site from Human MBL and Human ficolins
SEQ ID NO:23 OGKLG Human MBL core binding site
SEQ ID NO:24 GLR GLQ GPO GKL GPO G Human MBP Triplets 6-10- demonstrated binding to MASP-2
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SEQ ID NO Source
SEQ ID NO:25 GPOGPOGLRGLQGPO GKLGPOGGPOGPO Human MBP Triplets with GPO added to enhance formation of triple helices
SEQ ID NO:26 GKDGRDGTKGEKGEP GQGLRGLQGPOGKLG POGNOGPSGSOGPKG QKGDOGKS Human MBP Triplets 1-17
SEQ ID NO:27 GAOGSOGEKGAOGPQ GPOGPOGKMGPKGEO GDO Human H-Ficolin (Hataka)
SEQ ID NO:28 GCOGLOGAOGDKGE AGTNGKRGERGPOGP OGKAGPOGPNGAOGE 0 Human L-Ficolin P35
SEQ ID NO:29 LQRALEILPNRVTIKA NRPFLVFI Human C4 cleavage site
Note: The letter O represents hydroxyproline. The letter X is a hydrophobie residue.
Peptides derived from the C4 cleavage site as well as other peptides that inhibit the MASP-2 serine protease site can be chemically modified so that they are irréversible 5 protease inhibitors. For example, appropriate modifications may include, but are not necessarily limited to, halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, or appended to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxide or imine-containing groups on the amino or carboxy termini or on functional side chains; or imidate esters on the amino or carboxy 10 termini or on functional side chains. Such modifications would afford the advantage of permanently inhibiting the enzyme by covalent attachment of the peptide. This could resuit in lower effective doses and/or the need for less frequent administration of the peptide inhibitor.
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In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the method of the invention include peptides containing the MASP-2-binding CDR3 région of anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR régions for use in synthesizing the peptides may be determined by methods known in the art. The heavy chain variable région is a peptide that generally ranges from 100 to 150 amino acids in length. The light chain variable région is a peptide that generally ranges from 80 to 130 amino acids in length. The CDR sequences within the heavy and light chain variable régions include only approximately 3-25 amino acid sequences that may be easily sequenced by one of ordinary skill in the art.
Those skilled in the art will recognize that substantially homologous variations of the MASP-2 inhibitory peptides described above will also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides having insertions, délétions, replacements, and/or additional amino acids on the carboxy-terminus or amino-terminus portions of the subject peptides and mixtures thereof. Accordingly, those homologous peptides having MASP-2 inhibitory activity are considered to be useful in the methods of this invention. The peptides described may also include duplicating motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein, and include the exchange of an amino acid for another of like charge, size or hydrophobicity and the like.
MASP-2 inhibitory peptides may be modified to increase solubility and/or to maximize the positive or négative charge in order to more closely resemble the segment in the intact protein. The dérivative may or may not hâve the exact primary amino acid structure of a peptide disclosed herein so long as the dérivative functionally retains the desired property of MASP-2 inhibition. The modifications can include amino acid 25 substitution with one of the commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary désirable characteristics, such as résistance to enzymatic dégradation or with a D-amino acid or substitution with another molécule or compound, such as a carbohydrate, which mimics the natural confirmation and function of the amino acid, amino acids or peptide; amino acid délétion;
amino acid insertion with one of the commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary désirable characteristics, such as résistance to enzymatic dégradation or with a D-amino acid or
111 substitution with another molécule or compound, such as a carbohydrate, which mimics the natural confirmation and function of the amino acid, amino acids or peptide; or substitution with another molécule or compound, such as a carbohydrate or nucleic acid monomer, which mimics the natural conformation, charge distribution and function of the parent peptide. Peptides may also be modified by acétylation or amidation.
The synthesis of dérivative inhibitory peptides can rely on known techniques of peptide biosynthesis, carbohydrate biosynthesis and the like. As a starting point, the artisan may rely on a suitable computer program to détermine the conformation of a peptide of interest. Once the conformation of peptide disclosed herein is known, then the artisan can détermine in a rational design fashion what sort of substitutions can be made at one or more sites to fashion a dérivative that retains the basic conformation and charge distribution of the parent peptide but which may possess characteristics which are not présent or are enhanced over those found in the parent peptide. Once candidate dérivative molécules are identified, the dérivatives can be tested to détermine if they function as MASP-2 inhibitory agents using the assays described herein.
SCREENING FOR MASP-2 INHIBITORY PEPTIDES
One may also use molecular modeling and rational molecular design to generate and screen for peptides that mimic the molecular structures of key binding régions of MASP-2 and inhibit the complément activities of MASP-2. The molecular structures used for modeling include the CDR régions of anti-MASP-2 monoclonal antibodies, as well as the target régions known to be important for MASP-2 function including the région required for dimerization, the région involved in binding to MBL, and the serine protease active site as previously described. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting may be used for the de novo construction of macromolecular structures such as peptides that bind to a particular molécule. See, for example, Shea, K.J., Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sties, TRIP 2(5) 1994.
As an illustrative example, one method of preparing mimics of MASP-2 binding peptides is as follows. Functional monomers of a known MASP-2 binding peptide or the binding région of an anti-MASP-2 antibody that exhibits MASP-2 inhibition (the
112 template) are polymerized. The template is then removed, followed by polymerization of a second class of monomers in the void left by the template, to provide a new molécule that exhibits one or more desired properties that are similar to the template. In addition to preparing peptides in this manner, other MASP-2 binding molécules that are MASP-2 5 inhibitory agents such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroid, lipids and other biologically active materials can also be prepared. This method is useful for designing a wide variety of biological mimics that are more stable than their natural counterparts because they are typically prepared by free radical polymerization of function monomers, resulting in a 10 compound with a nonbiodegradable backbone.
PEPTIDE SYNTHESIS
The MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid-phase synthetic technique initially described by Merrifield, in 15 J. Amer. Chem. Soc. 55:2149-2154, 1963. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Other techniques may be found, for example, in Bodanszky, M., et aL, Peptide Synthesis, second édition, John Wiley & Sons, 1976, as well as in other reference works known to those skilled in the art.
The peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids. The peptide is then purified using chromatographie or 25 electrophoretic techniques, or by means of a carrier protein that can be fused to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding sequence a nucleic acid sequence encoding the carrier protein. The fusion protein-peptide may be isolated using chromatographie, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier 30 protein). The peptide can be cleaved using Chemical methodology or enzymatically, as by, for example, hydrolases.
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The MASP-2 inhibitory peptides that are useful in the method ofthe invention can also be produced in recombinant host cells following conventional techniques. To express a MASP-2 inhibitory peptide encoding sequence, a nucleic acid molécule encoding the peptide must be operably linked to regulatory sequences that control transcriptional 5 expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene, which are suitable for sélection of cells that carry the expression vector.
Nucleic acid molécules that encode a MASP-2 inhibitory peptide can be 10 synthesized with gene machines using protocols such as the phosphoramidite method. If chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and 15 then annealing them. For the production of longer genes, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucléotides in length. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA”, ASM Press, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53:323, 20 1984; and Climie, S., et al., Proc. Nat'l Acad. Sci. USA 87:633, 1990.
SMALL MOLECULE INHIBITORS
In some embodiments, MASP-2 inhibitory agents are small molécule inhibitors including natural and synthetic substances that hâve a low molecular weight, such as for example, peptides, peptidomimetics and nonpeptide inhibitors . (including 25 oligonucleotides and organic compounds). Small molécule inhibitors of MASP-2 can be generated based on the molecular structure of the variable régions of the anti-MASP-2 antibodies.
Small molécule inhibitors may also be designed and generated based on the MASP-2 crystal structure using computational drug design (Kuntz LD., et al., 30 Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., étal., EMBO J. 22:2348-2359, 2003). Using the method described by Kuntz et al., the MASP-2 crystal structure coordinates are used as an input for a computer
114 program such as DOCK, which outputs a list of small moiecule structures that are expected to bind to MASP-2. Use of such computer programs is well known to one of skill in the art. For example, the crystal structure of the HIV-1 protease inhibitor was used to identify unique nonpeptide ligands that are HIV-1 protease inhibitors by evaluating the fit of compounds found in the Cambridge Crystallographic database to the binding site of the enzyme using the program DOCK (Kuntz, I.D., et al., J. Mol. Biol. /67:269-288, 1982; DesJarlais, R.L., et al., PNAS 57:6644-6648, 1990).
The list of small moiecule structures that are identified by a computational method as potential MASP-2 inhibitors are screened using a MASP-2 binding assay such as described in Example 10. The small molécules that are found to bind to MASP-2 are then assayed in a functional assay such as described in Example 2 to détermine if they inhibit MASP-2-dependent complément activation.
MASP-2 SOLUBLE RECEPTORS
Other suitable MASP-2 inhibitory agents are believed to include MASP-2 soluble receptors, which may be produced using techniques known to those of ordinary skill in the art.
EXPRESSION INHIBITORS OF MASP-2
In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is a MASP-2 expression inhibitor capable of inhibiting MASP-2-dependent complément activation. In the practice of this aspect of the invention, représentative MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molécules (such as antisense mRNA, antisense DNA or antisense oligonucleotides), MASP-2 ribozymes and MASP-2 RNAi molécules.
Anti-sense RNA and DNA molécules act to directly block the translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of MASP-2 protein. An antisense nucleic acid moiecule may be constructed in a number of different ways provided that it is capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid moiecule can be constructed by inverting the coding région (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to its normal orientation for transcription to allow for the transcription of its complément.
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The antisense nucleic acid molécule is usually substantially identical to at least a portion of the target gene or genes. The nucleic acid, however, need not be perfectly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter antisense nucleic acid molécule. The minimal percent identity is 5 typically greater than about 65%, but a higher percent identity may exert a more effective repression of expression of the endogenous sequence. Substantially greater percent identity of more than about 80% typically is preferred, though about 95% to absolute identity is typically most preferred.
The antisense nucleic acid molécule need not hâve the same intron or exon pattern 10 as the target gene, and non-coding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments. A DNA sequence of at least about 8 or so nucléotides may be used as the antisense nucleic acid molécule, although a longer sequence is préférable. In the présent invention, a représentative example of a useful inhibitory agent of MASP-2 is an antisense MASP-2 15 nucleic acid molécule which is at least ninety percent identical to the complément of the MASP-2 cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes the MASP-2 protein consisting of the amino acid sequence set forth in SEQ ID NO:5.
The targeting of antisense oligonucleotides to bind MASP-2 mRNA is another 20 mechanism that may be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and the muscarine type 2 acétylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Patent No. 5,739,119, to Cheng, and U.S. Patent No. 5,759,829, to Shewmaker). Furthermore, examples of antisense inhibition hâve been demonstrated 25 with the nuclear protein cyclin, the multiple drug résistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAa receptor and human EGF (see, e.g., U.S. Patent No. 5,801,154, to Baracchini; U.S. Patent No. 5,789,573, to Baker; U.S. Patent No. 5,718,709, to Considine; and U.S. Patent No. 5,610,288, to Reubenstein).
A System has been described that allows one of ordinary skill to détermine which 30 oligonucleotides are useful in the invention, which involves probing for suitable sites in the target mRNA using Rnase H cleavage as an indicator for accessibility of sequences within the transcripts. Scherr, M., et al., Nucleic Acids Res. 26:5079-5085, 1998;
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Lloyd, et aL, Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antisense oligonucleotides that are complementary to certain régions of the MASP-2 transcript is added to cell extracts expressing MASP-2, such as hépatocytes, and hybridized in order to create an RNAseH vulnérable site. This method can be combined with computer-assisted sequence sélection that can predict optimal sequence sélection for antisense compositions based upon their relative ability to form dimers, hairpins, or other secondary structures that would reduce or prohibit spécifie binding to the target mRNA in a host cell. These secondary structure analysis and target site sélection considérations may be performed using the OLIGO primer analysis software (Rychlik, L, 1997) and the
BLASTN 2.0.5 algorithm software (Altschul, S.F., et al., Nucl. Acids Res. 25:3389-3402, 1997). The antisense compounds directed towards the target sequence preferably comprise from about 8 to about 50 nucléotides in length. Antisense oligonucleotides comprising from about 9 to about 35 or so nucléotides are particularly preferred. The inventors contemplate ail oligonucleotide compositions in the range of 9 to 35 nucléotides (i.e., those of9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, or 35 or so bases in length) are highly preferred for the practice of antisense oligonucleotide-based methods of the invention. Highly preferred target régions of the MASP-2 mRNA are those that are at or near the AUG translation initiation codon, and those sequences that are substantially complementary to 5' régions of the mRNA, e.g., between the -10 and +10 régions of the MASP-2 gene nucléotide sequence (SEQ ID NO:4). Exemplary MASP-2 expression inhibitors are provided in TABLE 4.
TABLE 4: EXEMPLARY EXPRESSION INHIBITORS OF MASP-2______
SEQ ID NO:30 (nucléotides 22-680 of SEQ ID NO:4) Nucleic acid sequence of MASP-2 cDNA (SEQ ID NO:4) encoding CUBIEGF
SEQIDNO:31 5'CGGGCACACCATGAGGCTGCTG ACCCTCCTGGGC3 Nucléotides 12-45 of SEQ ID NO:4 including the MASP-2 translation start site (sense)
SEQ ID NO:32 5'GACATTACCTTCCGCTCCGACTC CAACGAGAAG3' Nucléotides 361-396 of SEQ ID NO:4 encoding a région comprising the MASP-2 MBL binding site (sense)
SEQ ID NO:33 5 ' AGCAGCCCTGAATACCCACGGCC GTATCCCAAA3' Nucléotides 610-642 of SEQ ID NO:4 encoding a région comprising the CUBII domain
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As noted above, the term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers those oligonucleobases composed of naturally occurring nucléotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring modifications. These modifications allow one to introduce certain désirable properties that are not offered through naturally occurring oligonucleotides, such as reduced toxic properties, increased stability against nuclease dégradation and enhanced cellular uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense oligonucleotide in which the phosphate substituents are replaced by phosphorothioates. Likewise, one or both ends of the oligonucleotide may be substituted by one or more acridine dérivatives that intercalate between adjacent basepairs within a strand of nucleic acid.
Another alternative to antisense is the use of RNA interférence (RNAi). Double-stranded RNAs (dsRNAs) can provoke gene silencing in mammals in vivo. The natural function of RNAi and co-suppression appears to be protection of the genome against invasion by mobile genetic éléments such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when they become active (see, e.g., Jensen, J., et al., Nat. Genet. 27:209-12, 1999). The double-stranded RNA molécule may be prepared by synthesizing two RNA strands capable of forming a double-stranded RNA molécule, each having a length from about 19 to 25 (e.g., 19-23 nucléotides). For example, a dsRNA molécule useful in the methods of the invention may comprise the RNA corresponding to a sequence and its complément listed in TABLE 4. Preferably, at least one strand of RNA has a 3' overhang from 1-5 nucléotides. The synthesized RNA strands are combined under conditions that form a double-stranded molécule. The RNA sequence may comprise at least an 8 nucléotide portion of SEQ ID NO:4 with a total length of 25 nucléotides or less. The design of siRNA sequences for a given target is within the ordinary skill of one in the art. Commercial services are available that design siRNA sequence and guarantee at least 70% knockdown of expression (Qiagen, Valencia, Calif).
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The dsRNA may be administered as a pharmaceutical composition and carried out by known methods, wherein a nucleic acid is introduced into a desired target cell. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and viral methods. Such methods are taught in
Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.
Ribozymes can also be utilized to decrease the amount and/or biological activity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molécules that can cleave nucleic acid molécules having a sequence that is completely or partially homologous to the sequence of the ribozyme. It is possible to 10 design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a spécifie location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molécules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity 15 upon them, thereby increasing the activity of the antisense constructs.
Ribozymes useful in the practice of the invention typically comprise a hybridizing région of at least about nine nucléotides, which is complementary in nucléotide sequence to at least part of the target MASP-2 mRNA, and a catalytic région that is adapted to cleave the target MASP-2 mRNA (see generally, EPA No. 0 321 201; WO88/04300;
Haseloff, J., et al., Nature 334:585-591, 1988; Fedor, M.J., et al., Proc. Natl. Acad. Sci. USA 57:1668-1672, 1990; Cech, T.R., et al., Ann. Rev. Biochem. 55:599-629, 1986).
Ribozymes can either be targeted directly to cells in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozymes may be used and 25 applied in much the same way as described for antisense polynucleotides.
Anti-sense RNA and DNA, ribozymes and RNAi molécules useful in the methods of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molécules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as for 30 example solid phase phosphoramidite Chemical synthesis. Altematively, RNA molécules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molécule. Such DNA sequences may be incorporated into a wide variety
119 of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Altematively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Various well known modifications of the DNA molécules may be introduced as a means of increasing stability and half-life. Useful modifications include, but are not Iimited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molécule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
VI. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS
DOSING
In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complément activation in a subject suffering from a disease or condition as disclosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. The MASP-2 inhibitory agents can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2-dependent complément activation. A therapeutically effective dose refers to the amount of the MASP-2 inhibitory agent sufficient to resuit in amelioration of symptoms associated with the disease or condition.
Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures employing experimental animal models, such as the murine MASP-2 -/- mouse model expressing the human MASP-2 transgene described in Example 1. Using such animal models, the NOAEL (no observed adverse effect level) and the MED (the minimally effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is expressed as the ratio NOAEL/MED. MASP-2 inhibitory agents that exhibit large therapeutic ratios or indices are most preferred. The data obtained from the 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 inhibitory agent preferably lies within a range of circulating concentrations that include the MED with little or no toxicity. The dosage
120 may vary within this range depending upon the dosage form employed and the route of administration utilized.
For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose may be formulated in an animal 5 model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-2 inhibitory agent in plasma may also be measured, for example, by high performance liquid chromatography.
In addition to toxicity studies, effective dosage may also be estimated based on the amount of MASP-2 protein présent in a living subject and the binding affmity of the 10 MASP-2 inhibitory agent. It has been shown that MASP-2 levels in normal human subjects is présent in sérum in low levels in the range of 500 ng/ml, and MASP-2 levels in a particular subject can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., et al., J. Immunol. Methods 252:159-167, 2003.
Generally, the dosage of administered compositions comprising MASP-2 15 inhibitory agents varies depending on such factors as the subject's âge, weight, height, sex, general medical condition, and previous medical history. As an illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, can be administered in dosage ranges from about 0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject body weight. In some embodiments the composition comprises 20 a combination of anti-MASP-2 antibodies and MASP-2 inhibitory peptides.
Therapeutic efficacy of MASP-2 inhibitory compositions and methods of the présent invention in a given subject, and appropriate dosages, can be determined in accordance with complément assays well known to those of skill in the art. Complément generates numerous spécifie products. During the last decade, sensitive and spécifie 25 assays hâve been developed and are available commercially for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal 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 30 spécifie. 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 major forms found in the circulation.
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Unprocessed fragments and C5adesArg are rapidly cleared by binding to cell surface receptors and are hence présent in very low concentrations, whereas C3a<iesArg does not bind to cells and accumulâtes in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complément activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Détection of the fluid-phase product of membrane attack pathway activation, sC5b-9, provides evidence that complément is being activated to completion. Because both the 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 has generated the activation products.
The inhibition of MASP-2-dependent complément activation is characterized by at least one of the following changes in a component of the complément System that occurs as a resuit of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: the inhibition of the génération or production of MASP-2-dependent complément activation System products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in measured, for example, as described in Example 2, the réduction of C4 cleavage and C4b déposition (measured, for example as described in Example 10), or the réduction of C3 cleavage and C3b déposition (measured, for example, as described in Example 10).
ADDITIONAL AGENTS
The compositions and methods comprising MASP-2 inhibitory agents may optionally comprise one or more additional therapeutic agents, which may augment the activity of the MASP-2 inhibitory agent or that provide related therapeutic fonctions in an additive or synergistic fashion. For example, in the context of treating a subject suffering from TTP, wherein the subject is positive for an inhibitor of ADAM-TS13, one or more MASP-2 inhibitory agents may be administered in combination (including coadministration) with one or more immunosuppressive agents. Suitable immunosuppressive agents include: corticosteroids, rituxan, cyclosporine, and the like. In the context of treating a subject suffering from, or at risk for developing, HUS or aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with a suitable antibiotic. In the context of treating a
122 subject suffering from, or at risk for developing aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with other complément inhibitory agents such as eculizumab (Soliris), TT-30, antibody to factor B, or other agents that inhibit terminal complément components or alternative pathway amplification.
The inclusion and sélection of additional agent(s) will be determined to achieve a desired therapeutic resuit. In some embodiments, the MASP-2 inhibitory agent may be administered in combination with one or more anti-inflammatory and/or analgésie agents. Suitable anti-inflammatory and/or analgésie agents include: serotonin receptor antagonists; serotonin receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; tachykinin receptor antagonists, including neurokinim and neurokinin2 receptor subtype antagonists; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; inhibitors of enzymes active in the synthetic pathway for arachidonic acid métabolites, including phospholipase inhibitors, including PLA2 isoform inhibitors and PLCy isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be either COX-1, COX-2, or nonselective COX-1 and -2 inhibitors), lipooxygenase inhibitors; prostanoid receptor antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists including leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including μ-opioid, δ-opioid, and κ-opioid receptor subtype agonists; purinoceptor agonists and antagonists including P2X receptor antagonists and P2Y receptor agonists; adenosine triphosphate (ATP)-sensitive potassium channel openers; MAP kinase inhibitors; nicotinic acétylcholine inhibitors; and alpha adrenergic receptor agonists (including alpha-1, alpha-2, and nonselective alpha-1 and 2 agonists).
The MASP-2 inhibitory agents of the présent invention may also be administered in combination with one or more other complément inhibitors, such as an inhibitor of C5. To date, Eculizumab (Solaris®), an antibody against C5, is the only complementtargeting drug that has been approved for human use. However some pharmacological agents hâve been shown to block complément in vivo. K76COOH and nafamstat mesilate
123 are two agents that hâve shown some effectiveness in animal models of transplantation (Miyagawa, S., et al., Transplant Proc. 27:483-484, 1992). Low molecular weight heparins hâve also been shown to be effective in regulating complément activity (Edens, R.E., et al., Complément Today, pp. 96-120, Basel: Karger, 1993). It is believed that these small molécule inhibitors may be useful as agents to use in combination with the MASP-2 inhibitory agents of the présent invention.
Other naturally occurring complément inhibitors may be useful in combination with the MASP-2 inhibitory agents of the présent invention. Biological inhibitors of complément include soluble complément factor 1 (sCRl). This is a naturally-occurring inhibitor that can be found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. Recombinant forms hâve been tested for their anti-complement activity in vitro and in vivo. sCRl has been shown to be effective in xenotransplantation, wherein the complément System (both alternative and classical) provides the trigger for a hyperactive rejection syndrome within minutes of perfusing blood through the newly transplanted organ (Platt, J.L., et al., Immunol. Today 77:450-6, 1990; Marino, I.R., étal., Transplant Proc. 1071:6, 1990; Johnstone, P.S., étal., Transplantation 54:573-6, 1992). The use of sCRl protects and extends the survival time of the transplanted organ, implicating the complément pathway in the pathogenesis of organ survival (Leventhal, J.R., étal., Transplantation 55:857-66, 1993; Pruitt, S.K., étal., Transplantation 57:363-70, 1994).
Suitable additional complément inhibitors for use in combination with the compositions of the présent invention also include, by way of example, MoAbs such as an anti-C5 antibody (e.g., eculizumab) being developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, and anti-properdin MoAbs.
PHARMACEUTICAL CARRIERS AND DELIVERY VEHICLES
In general, the MASP-2 inhibitory agent compositions of the présent invention, combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agents combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Patent
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No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides useful in the invention may be formulated into préparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parentéral or surgical 5 administration. The invention also contemplâtes local administration of the compositions by coating medical devices and the like.
Suitable carriers for parentéral 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 10 addition, stérile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the préparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.
The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery Systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0019369 Al. Such hydrogels may be injected locally at the site of intended 25 action, or subcutaneously or intramuscularly to form a sustained release depot.
For intra-articular delivery, the MASP-2 inhibitory agent may be carried in above-described liquid or gel carriers that are injectable, above-described sustained-release delivery vehicles that are injectable, or a hyaluronic acid or hyaluronic acid dérivative.
For oral administration of non-peptidergic agents, the MASP-2 inhibitory agent may be carried in an inert filler or diluent such as sucrose, comstarch, or cellulose.
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For topical administration, the MASP-2 inhibitory agent may be carried in ointment, lotion, cream, gel, drop, suppository, spray, liquid or powder, or in gel or microcapsular delivery Systems via a transdermal patch.
Varions nasal and pulmonary delivery Systems, including aérosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are being developed and may suitably be adapted for delivery of the présent invention in an aérosol, inhalant, or nebulized delivery vehicle, respectively.
For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriately stérile delivery Systems (e.g., liquids; gels, suspensions, etc.) can be used to administer the présent invention.
The compositions of the présent invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, pénétration enhancers, emulsifiers, binders, thickeners, flavouring agents (for oral administration).
PHARMACEUTICAL CARRIERS FOR ANTIBODIES AND PEPTIDES
More specifically with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations can be parenterally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier that can be a stérile liquid such as water, oils, saline, glycerol or éthanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be présent in compositions comprising anti-MASP-2 antibodies and inhibitory peptides. Additional components of pharmaceutical compositions include petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and minerai oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.
The anti-MASP-2 antibodies and inhibitory peptides can also be administered in the form of a depot injection or implant préparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active agents.
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PHARMACEUTICALLY ACCEPTABLE CARRIERS FOR EXPRESSION INHIBITORS
More specifïcally with respect to expression inhibitors useful in the methods of the invention, compositions are provided that comprise an expression inhibitor as 5 described above and a pharmaceutically acceptable carrier or diluent. The composition may further comprise a colloïdal dispersion System.
Pharmaceutical compositions that include expression inhibitors may include, but are not limited to, solutions, émulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not 10 limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. The préparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral 15 buffered saline or saline mixed with non-specific sérum albumin are examples of suitable diluents.
In some embodiments, the compositions may be prepared and formulated as émulsions which are typically heterogeneous Systems of one liquid dispersed in another in the form of droplets (see, Idson, in Pharmaceutical Dosage Forms, Vol. 1, Rieger and 20 Banker (eds.), Marcek Dekker, Inc., N.Y., 1988). Examples of naturally occurring emulsifiers used in émulsion formulations include acacia, beeswax, lanolin, lecithin and phosphatides.
In one embodiment, compositions including nucleic acids can be formulated as microemulsions. A microemulsion, as used herein refers to a System of water, oil, and 25 amphiphile, which is a single optically isotropie and thermodynamically stable liquid solution (see Rosoff in Pharmaceutical Dosage Forms, Vol. 1). The method of the invention may also use liposomes for the transfer and delivery of antisense oligonucleotides to the desired site.
Pharmaceutical compositions and formulations of expression inhibitors for topical 30 administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, as well as aqueous, powder or oily bases and thickeners and the like may be used.
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MODES OF ADMINISTRATION
The pharmaceutical compositions comprising MASP-2 inhibitory agents may be 5 administered in a number of ways depending on whether a local or systemic mode of administration is most appropriate for the condition being treated. Additionally, as described herein above with respect to extracorporeal reperfusion procedures, MASP-2 inhibitory agents can be administered via introduction of the compositions of the présent invention to recirculating blood or plasma. Further, the compositions of the présent 10 invention can be delivered by coating or incorporating the compositions on or into an implantable medical device.
SYSTEMIC DELIVERY
As used herein, the terms systemic delivery and systemic administration are intended to include but are not limited to oral and parentéral routes including 15 intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively resuit in dispersement of the delivered agent to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the présent compositions include intravenous, intramuscular, subcutaneous and inhalational.
It will be appreciated that the exact systemic administration route for selected agents utilized in particular compositions of the présent invention will be determined in part to account for the agents susceptibility to metabolic transformation pathways associated with a given route of administration. For example, peptidergic agents may be most suitably administered by routes other than oral.
MASP-2 inhibitory antibodies and polypeptides can be delivered into a subject in need thereof by any suitable means. Methods of delivery of MASP-2 antibodies and polypeptides include administration by oral, pulmonary, parentéral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (such as via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual routes of 30 administration, and can be formulated in dosage forms appropriate for each route of administration.
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By way of représentative example, MASP-2 inhibitory antibodies and peptides can be introduced into a living body by application to a bodily membrane capable of absorbing the polypeptides, for example the nasal, gastrointestinal and rectal membranes. The polypeptides are typically applied to the absorptive membrane in conjunction with a perméation enhancer. (See, e.g., Lee, V.H.L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 73:213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., et al., J. Controlled Release 73:241, 1990.) For example, STDHF is a synthetic dérivative of fusidic acid, a stéroïdal surfactant that is similar in structure to the bile salts, and has been used as a perméation enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.)
The MASP-2 inhibitory antibodies and polypeptides may be introduced in association with another molécule, such as a lipid, to protect the polypeptides from enzymatic dégradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et aL, J. Controlled Release 77:139, 1990). Many polymer Systems hâve been reported for protein delivery (Bae, Y.H., étal., J. Controlled Release 9:271, 1989; Hori, R., etaL, Pharm. Res. 6:813, 1989; Yamakawa, L, et aL, J. Pharm. Sci. 79:505, 1990; Yoshihiro, L, et aL, J. Controlled Release 10:195, 1989; Asano, M., etaL, J. Controlled Release 9:111, 1989; Rosenblatt, J., etaL, J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 72:235, 1990; Takakura, Y., etaL, J. Pharm. Sci. 75:117, 1989; Takakura, Y., et aL, J. Pharm. Sci. 75:219, 1989).
Recently, liposomes hâve been developed with improved sérum stability and circulation half-times (see, e.g., U.S. Patent No. 5,741,516, to Webb). Furthermore, various methods of liposome and liposome-like préparations as potential drug carriers hâve been reviewed (see, e.g., U.S. Patent No. 5,567,434, to Szoka; U.S. Patent No. 5,552,157, to Yagi; U.S. Patent No. 5,565,213, to Nakamori; U.S. Patent No. 5,738,868, to Shinkarenko; and U.S. Patent No. 5,795,587, to Gao).
For transdermal applications, the MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable ingrédients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingrédients, except that they must be
129 pharmaceutically acceptable for their intended administration, and cannot dégradé the activity of the active ingrédients of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or without purified collagen. The MASP-2 inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.
The compositions of the présent invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. For example, 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 extent of progress of the condition being treated, the patient's âge, sex and weight, and other clinical factors. The dosage for each individual agent will vary as a function of the MASP-2 inhibitory agent 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 quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).
LOCAL DELIVERY
As used herein, the term local encompasses application of a drug in 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 intravesicuiar administration, placement or irrigation. Local administration may be preferred to enable administration of a lower dose, to avoid systemic side effects, and for more accurate control of the timing of delivery and concentration of the active agents at the site of local delivery. Local administration provides a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc. Improved dosage control is also provided by the direct mode of delivery.
Local delivery of a MASP-2 inhibitory agent may be achieved in the context of surgical methods for treating 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
130 can be administered to a subject in conjunction with a balloon angioplasty procedure. A balloon angioplasty procedure involves inserting a cathéter having a deflated balloon into an artery. The deflated balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the vascular wall. As a resuit, 5 the balloon surface is in contact with the layer of vascular endothélial cells on the surface of the blood vessel. The MASP-2 inhibitory agent may be attached to the balloon angioplasty cathéter in a manner that permits release of the agent at the site of the atherosclerotic plaque. The agent may be attached to the balloon cathéter in accordance with standard procedures known in the art. For example, the agent may be stored in a 10 compartment of the balloon cathéter until the balloon is inflated, at which point it is released into the local environment. Alternatively, the agent may be impregnated on the balloon surface, such that it contacts the cells of the arterial wall as the balloon is inflated. The agent may also be delivered in a perforated balloon cathéter such as those disclosed in Flugelman, M.Y., et al., Circulation 55:1110-1117, 1992. See also published PCT 15 Application WO 95/23161 for an exemplary procedure for attaching a therapeutic protein to a balloon angioplasty cathéter. Likewise, the MASP-2 inhibitory agent may be included in a gel or polymeric coating applied to a stent, or may be incorporated into the material of the stent, such that the stent elutes the MASP-2 inhibitory agent after vascular placement.
MASP-2 inhibitory compositions used in the treatment of arthritides and other musculoskeletal disorders may be locally delivered by intra-articular injection. Such compositions may suitably include a sustained release delivery vehicle. As a further example of instances in which local delivery may be desired, MASP-2 inhibitory compositions used in the treatment of urogénital conditions may be suitably instilled intravesically or within another urogénital structure.
COATINGS ON A MEDICAL DEVICE
MASP-2 inhibitory agents such as antibodies and inhibitory peptides may be immobilized onto (or within) a surface of an implantable or attachable medical device.
The modified surface will typically be in contact with living tissue after implantation into an animal body. By implantable or attachable medical device is intended any device that is implanted into, or attached to, tissue of an animal body, during the normal
131 operation of the device (e.g., stents and implantable drug delivery devices). Such implantable or attachable medical devices can be made from, for example, nitrocellulose, diazocellulose, glass, polystyrène, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, stainless Steel, titanium and biodégradable and/or 5 biocompatible polymers. Linkage of the protein to a device can be accomplished by any technique that does not destroy the biological activity of the linked protein, for example by attaching one or both of the N- C-terminal residues of the protein to the device. Attachment may also be made at one or more internai sites in the protein. Multiple attachments (both internai and at the ends of the protein) may also be used. A surface of 10 an implantable or attachable medical device can be modified to include functional groups (e.g., carboxyl, amide, amino, ether, hydroxyl, cyano, nitrido, sulfanamido, acetylinic, epoxide, silanic, anhydric, succinimic, azido) for protein immobilization thereto. Coupling chemistries include, but are not Iimited to, the formation of esters, ethers, amides, azido and sulfanamido dérivatives, cyanate and other linkages to the functional 15 groups available on MASP-2 antibodies or inhibitory peptides. MASP-2 antibodies or inhibitory fragments can also be attached non-covalently by the addition of an affinity tag sequence to the protein, such as GST (D.B. Smith and K.S. Johnson, Gene 67:31, 1988), polyhistidines (E. Hochuli et al., J. Chromatog. 411:71, 1987), or biotin. Such affinity tags may be used for the réversible attachment of the protein to a device.
Proteins can also be covalently attached to the surface of a device body, for example, by covalent activation of the surface of the medical device. By way of ' .^prcscnUŒive example, menoeuuiarptùiwin·) caEDènuacncà towoobvioo oàoÿ üylàqy of the following pairs of reactive groups (one member of the pair being présent on the surface of the device body, and the other member of the pair being présent on the 25 matricellular protein(s)): hydroxyl/carboxylic acid to yield an ester linkage;
hydroxyl/anhydride to yield an ester linkage; hydroxyl/isocyanate to yield a urethane linkage. A surface of a device body that does not possess useful reactive groups can be treated with radio-frequency discharge plasma (RFGD) etching to generate reactive groups in order to allow déposition of matricellular protein(s) (e.g., treatment with 30 oxygen plasma to introduce oxygen-containing groups; treatment with propyl amino plasma to introduce amine groups).
132
MASP-2 inhibitory agents comprising nucleic acid molécules such as antisense, RNAi-or DNA-encoding peptide inhibitors can be embedded in porous matrices attached to a device body. Représentative porous matrices useful for making the surface layer are those prepared from tendon or dermal collagen, as may be obtained from a variety of commercial sources (e.g., Sigma and Collagen Corporation), or collagen matrices prepared as described in U.S. Patent Nos. 4,394,370, to Jefferies, and 4,975,527, to Koezuka. One collagenous material is termed UltraFiber™ and is obtainable from Norian Corp. (Mountain View, California).
Certain polymeric matrices may also be employed if desired, and include acrylic ester polymers and lactic acid polymers, as disclosed, for example, in U.S. Patent Nos. 4,526,909 and 4,563,489, to Urist. Particular examples of useful polymers are those of orthoesters, anhydrides, propylene-cofumarates, or a polymer of one or more α-hydroxy carboxylic acid monomers, (e.g., α-hydroxy acetic acid (glycolic acid) and/or α-hydroxy propionic acid (lactic acid)).
TREATMENT REGIMENS
In prophylactic applications, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, a condition associated with MASP-2-dependent complément activation in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, a condition associated with MASP-2-dependent complément activation in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition. In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. Application of the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of an acute condition, e.g., reperfusion injury or other traumatic injury. Altematively, the composition may be administered at periodic intervals over an extended period of time for treatment of chronic conditions, e.g., arthritides or psoriasis.
133
The methods and compositions of the présent invention may be used to inhibit inflammation and related processes that typically resuit from diagnostic and therapeutic medical and surgical procedures. To inhibit such processes, the MASP-2 inhibitory composition of the présent invention may be applied periprocedurally. As used herein periprocedurally refers to administration of 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 may be carried out by local administration of the composition to the surgical or procédural site, such as by injection or continuous or intermittent irrigation of the site or by systemic administration. Suitable methods for local perioperative delivery of MASP-2 inhibitory agent solutions are disclosed in US Patent Nos. 6,420,432 to Demopulos and 6,645,168 to Demopulos. Suitable methods for local delivery of chondroprotective compositions including MASP-2 inhibitory agent(s) are disclosed in International PCT Patent Application WO 01/07067 A2. Suitable methods and compositions for targeted systemic delivery of chondroprotective compositions including MASP-2 inhibitory agent(s) are disclosed in International PCT Patent Application WO 03/063799 A2.
In one aspect of the invention, the pharmaceutical compositions are administered to a subject suffering from, or at risk for developing a thrombotic microangiopathy (TMA). In one embodiment, the TMA is selected from the group consisting of hemolytic urémie syndrome (HUS), thrombotic thrombocytopénie purpura (TTP) and atypical hemolytic urémie syndrome (aHUS). In one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to an aHUS patient during the acute phase of the disease. In one embodiment, the composition is administered to an aHUS patient during the remission phase (i.e., in a subject that has recovered or partially recovered from an épisode of acute phase aHUS, such remission evidenced, for example, by increased platelet count and/or reduced sérum LDH concentrations, for example as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, hereby incorporated herein by reference). In one embodiment, the subject is suffering from, or at risk for developing a TMA that is (i) a TMA secondary to cancer; (ii) a TMA secondary to chemotherapy; or (iii) a TMA secondary to transplantation (e.g., organ transplantation,
134 such as kidney transplantation or allogeneic hematopoietic stem cell transplantation). In one embodiment, the subject is suffering from, or at risk for developing UpshawSchulman Syndrome (USS). In one embodiment, the subject is suffering from, or at risk for developing Degos disease. In one embodiment, the subject is suffering from, or at 5 risk for developing Catastrophic Antiphospholipid Syndrome (CAPS). In therapeutic applications, the pharmaceutical compositions are administered to a subject suffering from, or at risk for developing a TMA in a therapeutically effective amount sufficient to inhibit thrombus formation, relieve, or at least partially reduce, the symptoms of the condition.
In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of 15 from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pédiatrie patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of TMA. Altematively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of TMA.
In some embodiments, the subject suffering from or at risk for developing a TMA has previously undergone, or is currently undergoing treatment with a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further administering to the subject a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the terminal complément inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complément inhibitor is eculizumab.
135
In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, aHUS in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, aHUS in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition. In one aspect of the invention, prior to administration, the subject may be examined to détermine whether the subject exhibits one or more symptoms of aHUS, including (i) anémia, (ii) thrombocytopenia (iii) rénal insufficiency and (iv) rising créatinine, and the composition of the présent invention is then administered in an effective amount and for a sufficient time period to improve these symptom(s).
In another aspect of the invention, the MASP-2 inhibitory compositions of the présent invention may be used to prophylactically treat a subject that has an elevated risk of developing aHUS and thereby reduce the likelihood that the subject will deliver aHUS. The presence of a genetic marker in the subject known to be associated with aHUS is first determined by performing a genetic screening test on a sample obtained from the subject and identifying the presence of at least one genetic marker associated with aHUS, complément factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, C3, complément factor H-related protein (CFHR1), anticoagulant protein thrombodulin (THBD), complément factor H-related protein 3 (CFHR3) or complément factor Hrelated protein 4 (CFHR4). The subject is then periodically monitored (e.g., monthly, quarterly, twice annually or annually) to détermine the presence or absence of at least one symptom of aHUS, such as anémia, thrombocytopenia, rénal insufficiency and rising créatinine. Upon the détermination of the presence of at least one of these symptoms, the subject can be administered an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dépendent complément activation, in an effective amount and for a sufficient time period to improve said one or more symptoms. In a still further aspect of the présent invention, a subject at increased risk of developing aHUS due to having been screened and determined to hâve one of the genetic markers associated with aHUS may be monitored for the occurrence of an event associated with triggering aHUS clinical symptoms, including drug exposure, infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplant and pregnancy.
136
In a still further aspect of the présent invention, a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dépendent complément activation can be administered to a suffering from or at risk of developing atypical hemolytic urémie syndrome (aHUS) secondary to an infection. For example, a patient suffering from or at risk of developing non-enteric aHUS associated with an S. pneumonia infection may be treated with the compositions of the présent invention.
In a still further aspect of the présent invention, a subject suffering from aHUS may initially be treated with a MASP-2 inhibitory composition of the présent invention that is administered through a cathéter line, such as an intravenous cathéter line or a subeutaneous cathéter line, for a first period of time such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered through regular subeutaneous injections, such as daily, biweekly, weekly, every other week, monthly or bimonthly, injections.
In a still further aspect of the présent invention, a MASP-2 inhibitory composition of the présent invention may be administered to a subject suffering from aHUS in the absence of plasmapheresis (i.e., a subject whose aHUS symptoms hâve not been treated with plasmapheresis and are not treated with plasmapheresis at the time of treatment with the MASP-2 inhibitory composition), to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.
In a still further aspect of the présent invention, a MASP-2 inhibitory composition of the présent invention may be administered to a subject suffering from aHUS coïncident with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment can then be administered the MASP-2 inhibitory composition following or altemating with plasma exchange.
In a still further aspect of the présent invention, a subject suffering from or at risk of developing aHUS and being treated with a MASP-2 inhibitory composition of the présent invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complément factor, wherein the détermination of a reduced level of the at least one complément factor in comparison to a
137 standard value or to a healthy subject is indicative of the need for continued treatment with the composition.
In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pédiatrie patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of aHUS. Altematively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of aHUS.
In some embodiments, the subject suffering from aHUS has previously undergone, or is currently undergoing treatment with a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further administering to the subject a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the terminal complément inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complément inhibitor is eculizumab.
In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, HUS in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, HUS in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.
In another aspect of the présent invention, the likelihood of developing impaired rénal function in a subject at risk for developing HUS can be reduced by administering to
138 the subject a MASP-2 inhibitory composition of the présent invention in an amount effective to inhibit MASP-2 dépendent complément activation. For example, a subject at risk for developing HUS and to be treated with a MASP-2 inhibitory composition of the présent invention may exhibit one or more symptoms associated with HUS, including 5 diarrhea, a hematocrit level of less than 30% with smear evidence of intravascular érythrocyte destruction, thrombocytopenia and rising créatinine levels. As a further example, a subject at risk for developing HUS and to be treated with the MASP-2 inhibitory compositions of the présent invention may be infected with E. coli, shigella or salmonella. Such subjects infected with E. coli, shigella or salmonella may be treated 10 with a MASP-2 inhibitory composition of the présent invention concurrent with antibiotic treatment, or altemately may be treated with a MASP-2 inhibitory composition without concurrent treatment with an antibiotic, particularly for enterogenic E. coli for which antibiotic treatment is contra-indicated. A subject infected with enterogenic E. coli that has been treated with an antibiotic may be at elevated risk of developing HUS, and may 15 be suitably treated with a MASP-2 inhibitory composition of the présent invention to reduce that risk. A subject infected with enterogenic E. coli may be treated for a first period of time with a MASP-2 inhibitory composition of the présent invention in the absence of an antibiotic and then for a second period of time with both a MASP-2 inhibitory composition of the présent invention and an antibiotic.
In a still further aspect of the présent invention, a subject suffering from HUS may initially be treated with a MASP-2 inhibitory composition of the présent invention that is administered through a cathéter line, such as an intravenous cathéter line or a subcutaneous cathéter line, for a first period of time such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered through regular subcutaneous injections, such as daily, biweekly, weekly, every other week, monthly or bimonthly, injections.
In a still further aspect of the présent invention, a MASP-2 inhibitory composition of the présent invention may be administered to a subject suffering from HUS in the 30 absence of plasmapheresis (i.e., a subject whose HUS symptoms hâve not been treated with plasmapheresis and are not treated with plasmapheresis at the time of treatment with the MASP-2 inhibitory composition), to avoid the potential complications of
139 plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.
In a still further aspect of the présent invention, a MASP-2 inhibitory composition 5 of the présent invention may be administered to a subject suffering from HUS coïncident with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment can then be administered the MASP-2 inhibitory composition following or alternating with plasma exchange.
In a still further aspect of the présent invention, a subject suffering from or at risk 10 of developing HUS and being treated with a MASP-2 inhibitory composition of the présent invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complément factor, wherein the détermination of a reduced level of the at least one complément factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment 15 with the composition.
In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be 20 administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pédiatrie patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the présent invention may 25 be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of HUS. Altematively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of HUS.
In some embodiments, the subject suffering from HUS has previously undergone, 30 or is currently undergoing treatment with a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a MASP-2
140 inhibitor and further administering to the subject a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the terminal complément inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complément inhibitor is eculizumab.
In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, TTP in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, TTP in a therapeutically effective amount sufficient to relieve, 10 or at least partially reduce, the symptoms of the condition.
In another aspect of the présent invention, a subject exhibiting one or more of the symptoms of TTP, including central nervous System involvement, thrombocytopenia, severe cardiac involvement, severe pulmonary involvement, gastro-intestinal infarction and gangrené, may be treated with a MASP-2 inhibitory composition of the présent 15 invention. In another aspect of the présent invention, a subject determined to hâve a depressed level of ADAMTS 13 and also testing positive for the presence of an inhibitor of (i.e., an antibody) ADAMTS 13 may be treated with a MASP-2 inhibitory composition of the présent invention. In a still further aspect of the présent invention, a subject testing positive for the presence of an inhibitor of ADAMTS13 may be treated with an 20 immunosupressant (e.g., corticosteroids, rituxan, or cyclosporine) concurrently with treatment with a MASP-2 inhibitory composition of the présent invention. In a still further aspect of the présent invention, a subject determined to hâve a reduced level of ADAMTS13 and testing positive for the presence of an inhibitor of ADAMTS13 may be treated with ADAMTS 13 concurrently with treatment with a MASP-2 inhibitory 25 composition of the présent invention.
In a still further aspect of the présent invention, a subject suffering from TTP may initially be treated with a MASP-2 inhibitory composition of the présent invention that is administered through a cathéter line, such as an intravenous cathéter line or a subcutaneous cathéter line, for a first period of time such as one hour, twelve hours, one 30 day, two days or three days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered through regular subcutaneous
141 injections, such as daily, biweekly, weekly, every other week, monthly or bimonthly, injections.
In a still further aspect of the présent invention, a MASP-2 inhibitory composition of the présent invention may be administered to a subject suffering from HUS in the absence of plasmapheresis (i.e., a subject whose TTP symptoms hâve not been treated with plasmapheresis and are not treated with plasmapheresis at the time of treatment with the MASP-2 inhibitory composition), to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.
In a still further aspect of the présent invention, a MASP-2 inhibitory composition ofthe présent invention may be administered to a subject suffering from TTP coïncident with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment can then be administered the MASP-2 inhibitory composition following or altemating with plasma exchange.
In a still further aspect of the présent invention, a subject suffering from refractory TTP, i.e., symptoms of TTP that hâve not responded adequately to other treatment such as plasmapheresis, may be treated with a MASP-2 inhibitory composition of the présent invention, with or without additionai plasmapheresis.
In a still further aspect of the présent invention, a subject suffering from or at risk of developing TTP and being treated with a MASP-2 inhibitory composition of the présent invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complément factor, wherein the détermination of a reduced level of the at least one complément factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition.
In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0
142 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pédiatrie patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition, or a limited sequence of 5 administrations, for treatment of TTP. Altematively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of TTP.
In some embodiments, the subject suffering from TTP has previously undergone, or is currently undergoing treatment with a terminal complément inhibitor that inhibits 10 cleavage of complément protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further administering to the subject a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the terminal complément inhibitor is a humanized anti-C5 antibody or antigen-binding fragment 15 thereof. In some embodiments, the terminal complément inhibitor is eculizumab.
VI. EXAMPLES
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. Ail literature 20 citations herein are expressly incorporated by reference.
EXAMPLE 1
This example describes the génération of a mouse strain déficient in MASP-2 (MASP-2-/-) but sufficient of MApl9 (MApl9+/+).
Materials and Methods: The targeting vector pKO-NTKV 1901 was designed to disrupt the three exons coding for the C-terminal end of murine MASP-2, including the exon that encodes the serine protease domain, as shown in FIGURE 3. PKO-NTKV 1901 was used to transfect the murine ES cell lineE14.1a (SV129 Ola). Neomycin-resistant and Thymidine Kinase-sensitive clones were selected. 600 ES clones 30 were screened and, of these, four different clones were identified and verified by Southern blot to contain the expected sélective targeting and recombination event as shown in FIGURE 3. Chimeras were generated from these four positive clones by embryo
143 transfer. The chimeras were then backcrossed in the genetic background C57/BL6 to create transgenic males. The transgenic males were crossed with females to generate Fis with 50% of the offspring showing heterozygosity for the disrupted MASP-2 gene. The heterozygous mice were intercrossed to generate homozygous MASP-2 déficient offspring, resulting in heterozygous and wild-type mice in the ration of 1:2:1, respectively.
Results and Phenotype: The resulting homozygous MASP-2-/- déficient mice were found to be viable and fertile and were verified to be MASP-2 déficient by southem blot to confirm the correct targeting event, by Northern blot to confirm the absence of MASP-2 mRNA, and by Western blot to confirm the absence of MASP-2 protein (data not shown). The presence of MApl9 mRNA and the absence of MASP-2 mRNA were further confirmed using time-resolved RT-PCR on a LightCycler machine. The MASP-2-/- mice do continue to express MApl9, MASP-1, and MASP-3 mRNA and protein as expected (data not shown). The presence and abundance of mRNA in the MASP-2-/- mice for Properdin, Factor B, Factor D, C4, C2, and C3 was assessed by LightCycler analysis and found to be identical to that of the wild-type littermate Controls (data not shown). The plasma from homozygous MASP-2-/- mice is totally déficient of lectin-pathway-mediated complément activation as further described in Example 2.
Génération of a MASP-2-/- strain on a pure C57BL6 Background: The MASP-2-/- mice were back-crossed with a pure C57BL6 line for nine générations prior to use of the MASP-2-/- strain as an experimental animal model.
A transgenic mouse strain that is murine MASP-2-/-, MApl9+/+ and that expresses a human MASP-2 transgene (a murine MASP-2 knock-out and a human MASP-2 knock-in) was also generated as follows:
Materials and Methods: A minigene encoding human MASP-2 called mini hMASP-2 (SEQ ID NO:49) as shown in FIGURE 4 was constructed which includes the promoter région of the human MASP 2 gene, including the first 3 exons (exon 1 to exon 3) followed by the cDNA sequence that represents the coding sequence of the following 8 exons, thereby encoding the full-length MASP-2 protein driven by its endogenous promoter. The mini hMASP-2 construct was injected into fertilized eggs of MASP-2-/- in order to replace the déficient murine MASP 2 gene by transgenically expressed human MASP-2.
144
EXAMPLE 2
This example demonstrates that MASP-2 is required for complément activation via the lectin pathway.
Methods and Materials:
Lectin pathway spécifie C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, étal., J. Immunol. Methods 257:107 (2001) that measures lectin pathway activation resulting from lipoteichoic acid (LTA) from 5. aureus, which binds L-ficolin. The assay described by Petersen et al., (2001) was adapted to measure lectin pathway activation via MBL by coating the plate with LPS and mannan or zymosan prior to adding sérum from MASP-2 -I- mice as described below. The assay was also modified to remove the possibility of C4 cleavage due to the classical pathway. This was achieved by using a sample dilution buffer containing 1 M NaCl, which permits high affinity binding of lectin pathway récognition components to their ligands but prevents activation of endogenous C4, thereby excluding the participation of the classical pathway by dissociating the C1 complex. Briefly described, in the modified assay sérum samples (diluted in high sait (1 M NaCl) buffer) are added to ligand-coated plates, followed by the addition of a constant amount of purified C4 in a buffer with a physiological concentration of sait. Bound récognition complexes containing MASP-2 cleave the C4, resulting in C4b déposition.
Assay Methods:
1) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) were coated with 1 pg/ml mannan (M7504 Sigma) or any other ligand (e.g., such as those listed below) diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6).
The foilowing reagents were used in the assay:
a. mannan (1 gg/well mannan (M7504 Sigma) in 100 μΐ coating buffer):
b. zymosan (1 pg/well zymosan (Sigma) in 100 μΐ coating buffer);
c. LTA (lgg/well in 100 μΐ coating buffer or 2 gg/well in 20 μΐ methanol)
d. 1 gg of the H-ficolin spécifie Mab 4H5 in coating buffer
e. PSA from Aerococcus viridans (2 gg/well in 100 μΐ coating buffer)
145
f. 100 μΐ/well of formalin-fixed S. aureus DSM20233 (OD55q=0.5) in coating buffer.
2) The plates were incubated ovemight at 4°C.
3) After ovemight incubation, the residual protein binding sites were 5 saturated by incubated the plates with 0.1% HSA-TBS blocking buffer (0.1% (w/v) HSA in 10 mM Tris-CL, 140 mM NaCl, 1.5 mM NaNs, pH 7.4) for 1-3 hours, then washing the plates 3X with TBS/tween/Ca2+ (TBS with 0.05% Tween 20 and 5 mM CaC12, 1 mM MgC12, pH 7.4).
4) Sérum samples to be tested were diluted in MBL-binding buffer (1 M 10 NaCl) and the diluted samples were added to the plates and incubated ovemight at 4°C. Wells receiving buffer only were used as négative Controls.
5) Following incubation ovemight at 4°C, the plates were washed 3X with TBS/tween/Ca2+. Human C4 (100 μΐ/well of 1 μg/ml diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaC12, 1 mM MgC12, pH 7.4)) was then added to the plates and incubated for 90 minutes at 37°C. The plates were washed again 3X with TBS/tween/Ca2+.
6) C4b déposition was detected with an alkaline phosphatase-conjugated chicken anti-human C4c (diluted 1:1000 in TBS/tween/Ca2+), which was added to the plates and incubated for 90 minutes at room température. The plates were then washed 20 again 3X with TBS/tween/Ca2+.
7) Alkaline phosphatase was detected by adding 100 μΐ of £>-nitrophenyl phosphate substrate solution, incubating at room température for 20 minutes, and reading the OD4Q5 in a microtiter plate reader.
Results: FIGURES 5A-B show the amount of C4b déposition on mannan 25 (FIGURE 5A) and zymosan (FIGURE 5B) in sérum dilutions from MASP-2+/+ (crosses), MASP-2+/- (closed circles) and MASP-2-/- (closed triangles). FIGURE 5C shows the relative C4 convertase activity on plates coated with zymosan (white bars) or mannan (shaded bars) from MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4) relative to wild-type mice (n=5) based on measuring the amount of C4b déposition normalized to 30 wild-type sérum. The error bars represent the standard déviation. As shown in
146
FIGURES 5A-C, plasma from MASP-2-/- mice is totally déficient in lectin-pathway-mediated complément activation on mannan and on zymosan coated plates. These results clearly demonstrate that MASP-2 is an effector component of the lectin pathway.
Recombinant MASP-2 reconstitutes Lectin Pathway-Dependent C4 Activation in sérum from the MASP-2-/- mice
In order to establish that the absence of MASP-2 was the direct cause of the loss of lectin pathway-dependent C4 activation in the MASP-2-/- mice, the effect of adding recombinant MASP-2 protein to sérum samples was examined in the C4 cleavage assay described above. Functionally active murine MASP-2 and catalytically inactive murine MASP-2A (in which the active-site serine residue in the serine protease domain was substituted for the alanine residue) recombinant proteins were produced and purifîed as described below in Example 3. Pooled sérum from 4 MASP-2 -/- mice was pre-incubated with increasing protein concentrations of recombinant murine MASP-2 or inactive recombinant murine MASP-2A and C4 convertase activity was assayed as described above.
Results: As shown in FIGURE 6, the addition of functionally active murine recombinant MASP-2 protein (shown as open triangles) to sérum obtained from the MASP-2 -/- mice restored lectin pathway-dependent C4 activation in a protein concentration dépendent manner, whereas the catalytically inactive murine MASP-2A protein (shown as stars) did not restore C4 activation. The results shown in FIGURE 6 are normalized to the C4 activation observed with pooled wild-type mouse sérum (shown as a dotted line).
EXAMPLE 3
This example describes the recombinant expression and protein production of recombinant full-length human, rat and murine MASP-2, MASP-2 derived polypeptides, and catalytically inactivated mutant forms of MASP-2
Expression of Full-length human, murine and rat MASP-2:
The full length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCI-Neo (Promega), which drives
147 eukaryotic expression under the control of the CMV enhancer/promoter région (described in Kaufman R.J. et al., Nucleic Acids Research 79:4485-90, 1991; Kaufman, Methods in Enzymology, 185:531-66 (1991)). The full length mouse cDNA (SEQ ID NO:50) and rat MASP-2 cDNA (SEQ ID NO:53) were each subcloned into the pED expression vector. The MASP-2 expression vectors were then transfected into the adhèrent Chinese hamster ovary cell line DXB1 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.
In another approach, the minigene construct (SEQ ID NO:49) containing the human cDNA of MASP-2 driven by its endogenous promoter is transiently transfected into Chinese hamster ovary cells (CHO). The human MASP-2 protein is secreted into the culture media and isolated as described below.
Expression of Full-length catalytically inactive MASP-2:
Rationale: MASP-2 is activated by autocatalytic cleavage after the récognition subcomponents MBL or ficolins (either L-fîcolin, H-ficolin or M-ficolin) bind to their respective carbohydrate pattern. Autocatalytic cleavage resulting in activation of MASP-2 often occurs during the isolation procedure of MASP-2 from sérum, or during the purification foilowing recombinant expression. In order to obtain a more stable protein préparation for use as an antigen, a catalytically inactive form of MASP-2, designed as MASP-2A was created by replacing the serine residue that is présent in the catalytic triad of the protease domain with an alanine residue in rat (SEQ ID NO:55 Ser617 to Ala617); in mouse (SEQ ID NO:52 Ser617 to Ala617); or in human (SEQ ID NO:3 Ser618to Ala618).
In order to generate catalytically inactive human and murine MASP-2A proteins, site-directed mutagenesis was carried out using the oligonucleotides shown in TABLE 5. The oligonucleotides in TABLE 5 were designed to anneal to the région of the human and murine cDNA encoding the enzymatically active serine and oligonucleotide contain a mismatch in order to change the serine codon into an alanine codon. For example, PCR oligonucleotides SEQ ID NOS:56-59 were used in combination with human MASP-2 cDNA (SEQ ID NO:4) to amplify the région from the start codon to the enzymatically active serine and from the serine to the stop codon to generate the complété open reading from of the mutated MASP-2A containing the Ser618 to Ala618 mutation. The PCR
148 products were purified after agarose gel electrophoresis and band préparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine tailed MASP-2A was then cloned into the pGEM-T easy vector, transformed into E. coli.
A catalytically inactive rat MASP-2A protein was generated by kinasing and 5 annealing SEQ ID NO:64 and SEQ ID NO:65 by combining these two oligonucleotides in equal molar amounts, heating at 100°C for 2 minutes and slowly cooling to room température. The resulting annealed fragment has Pstl and Xbal compatible ends and was inserted in place of the Pstl-Xbal fragment of the wild-type rat MASP-2 cDNA (SEQ ID NO:53) to generate rat MASP-2A.
5 ’GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO:64)
5' CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO:65)
The human, murine and rat MASP-2A were each further subcloned into either of the mammalian expression vectors pED or pCI-Neo and transfected into the Chinese 15 Elamster ovary cell line DXB1 as described below.
In another approach, a catalytically inactive form of MASP-2 is constructed using the method described in Chen et al., J. Biol. Chem., 276(28):25894-25902, 2001. Briefly, the plasmid containing the full-length human MASP-2 cDNA (described in Thiel et al., Nature 356:506, 1997) is digested with ATzol and EcoRl and the MASP-2 cDNA 20 (described herein as SEQ ID NO:4) is cloned into the corresponding restriction sites of the pFastBacl baculovirus transfer vector (Life Technologies, NY). The MASP-2 serine protease active site at Ser618 is then altered to Ala618 by substituting the double-stranded oligonucleotides encoding the peptide région amino acid 610-625 (SEQ ID NO: 13) with the native région amino acids 610 to 625 to create a MASP-2 fiill 25 length polypeptide with an inactive protease domain. Construction of Expression Plasmids Containing Polypeptide Régions Derived from Human Masp-2.
The following constructs are produced using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO:5) to secrete various domains of MASP-2. A construct expressing the human MASP-2 CUBI domain (SEQ ID NO:8) is made by PCR 30 amplifying the région encoding residues 1-121 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBI domain). A construct expressing the human MASP-2 CUBIEGF domain (SEQ ID NO:9) is made by PCR amplifying the région
149 encoding residues 1-166 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBIEGF domain). A construct expressing the human MASP-2 CUBIEGFCUBII domain (SEQ ID NO: 10) is made by PCR amplifying the région encoding residues 1-293 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBIEGFCUBII domain).
The above mentioned domains are amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, according to established PCR methods. The 5' primer sequence of the sense primer (5'-CGGGATCÇATGAGGCTGCTGACCCTC-3' SEQ ID NO:34) introduces a RumHI restriction site (underlined) at the 5' end of the PCR products. Antisense primers for each of the MASP-2 domains, shown below in
TABLE 5, are designed to introduce a stop codon (boldface) followed by an EcoRI site (underlined) at the end of each PCR product. Once amplified, the DNA fragments are digested with ÆumHI and Eco RI and cloned into the corresponding sites of the pFastBacl vector. The resulting constructs are characterized by restriction mapping and confirmed by dsDNA sequencing.
TABLE 5: MASP-2 PCR PRIMERS
MASP-2 domain 5' PCR Primer 3' PCR Primer
SEQ ID NO:8 CUBI (aa 1-121 ofSEQ ID NO:6) 5'CGGGATCCATGA GGCTGCTGACCCT C-3' (SEQ ID NO:34) 5'GGAATTCCTAGGCTGCAT A (SEQ ID NO:35)
SEQ ID NO:9 CUBIEGF (aa 1-166 of SEQ ID NO:6) 5'CGGGATCCATGA GGCTGCTGACCCT C-3' (SEQ ID NO:34) 5'GGAATTCCTACAGGGCGC T-3' (SEQ ID NO:36)
SEQ ID NO: 10 CUBIEGFCUBII (aa 1-293 ofSEQ ID NO:6) 5'CGGGATCCATGA GGCTGCTGACCCT C-3' (SEQ ID NO:34) 5'GGAATTCCTAGTAGTGGA T 3' (SEQ ID NO:37)
SEQ ID NO:4 human MASP-2 5'ATGAGGCTGCTG ACCCTCCTGGGCC TTC 3' (SEQ ID NO: 56) hMASP-2 forward 5'TTAAAATCACTAATTATG TTCTCGATC 3' (SEQ ID NO: 59) hMASP-2_reverse
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MASP-2 domain 5' PCR Primer 3' PCR Primer
SEQ ID NO:4 human MASP-2 cDNA 5'CAGAGGTGACGC AGGAGGGGCAC 3' (SEQ ID NO: 58) hMASP-2_ala_forwar d 5'GTGCCCCTCCTGCGTCAC CTCTG 3' (SEQ ID NO: 57) hMASP-2_ala_reverse
SEQ ID NO:50 Murine MASP-2 cDNA 5'ATGAGGCTACTC ATCTTCCTGG3' (SEQ ID NO: 60) mMASP-2 forward 5 'TTAGAAATTACTTATTAT GTTCTCAATCC3' (SEQ ID NO: 63) mMASP-2_reverse
SEQ ID NQ:50 Murine MASP-2 cDNA 5'CCCCCCCTGCGT CACCTCTGCAG3' (SEQ ID NO: 62) mMASP-2_ala_forwa rd 5 'CTGC AG AGGTG ACGC AG GGGGGG 3' (SEQ ID NO: 61) mMASP-2_ala_reverse
Recombinant eukaryotic expression of MASP-2 and protein production of enzymatically inactive mouse, rat, and human MASP-2A.
The MASP-2 and MASP-2A expression constructs described above were 5 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 préparations were not contaminated with other sérum proteins. Media 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 three 10 species.
MASP-2A protein purification: The MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns. This strategy enabled rapid purification without the use of extraneous tags. MASP-2A (100-200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, 15 pH 7.5, containing 150 mM NaCl and 25 mM CaCb) was loaded onto an MBP-agarose affmity column (4 ml) pre-equilibrated with 10 ml of loading buffer. Following washing with a further 10 ml of loading buffer, 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 the MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. Where necessary,
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MASP-2A was purified further by ion-exchange chromatography on a MonoQ column (HR 5/5). Protein was dialysed with 50 mM Tris-Cl pH 7.5, containing 50 mM NaCl and loaded onto the column equilibrated in the same buffer. Following washing, bound MASP-2A was eluted with a 0.05-1 M NaCl gradient over 10 ml.
Results: Yields of 0.25-0.5 mg of 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. Attachment of glycans at each of the Y-glycosylation sites accounts for the observed mass. MASP-2A migrâtes as a single band on SDS-polyacrylamide gels, demonstrating that it is not proteolytically processed during biosynthesis. The weight-average molecular mass determined by equilibrium ultracentrifugation is in agreement with the calculated value for homodimers of the glycosylated polypeptide.
PRODUCTION OF RECOMBINANT HUMAN MASP-2 POLYPEPTIDES
Another method for producing recombinant MASP-2 and MASP2A derived polypeptides is described in Thielens, N.M., et al., J. Immunol. 166:5068-5077, 2001. Briefly, the Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from Novagen, Madison, WI) are grown and maintained in Sf900II serum-free medium (Life Technologies) supplemented with 50 lU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). The Trichoplusia ni (High Five) insect cells (provided by Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France) are maintained in TC 100 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 lU/ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses are generated using the Bac-to-Bac System (Life Technologies). The bacmid DNA is purified using the Qiagen midiprep purification System (Qiagen) and is used to transfect Sf9 insect cells using cellfectin in Sf900 II SFM medium (Life Technologies) as described in the manufacturées protocol. Recombinant virus particles are collected 4 days later, titrated by virus plaque assay, and amplified as described by King and Possee, in The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp. 111-114, 1992.
High Five cells (1.75 x 107 cells/175-cm2 tissue culture flask) are infected with the recombinant viruses containing MASP-2 polypeptides at a multiplicity of infection of 2 in
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S1900 II SFM medium at 28°C for 96 h. The supematants are collected by centrifugation and diisopropyl phosphorofluoridate is added to a final concentration of 1 mM.
The MASP-2 polypeptides are secreted in the culture medium. The culture supematants are dialyzed against 50 mM NaCl, 1 mM CaCh, 50 mM triethanolamine 5 hydrochloride, pH 8.1, and loaded at 1.5 ml/min onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm) equilibrated in the same buffer. Elution is conducted by applying al.2 liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing the recombinant MASP-2 polypeptides are identified by Western blot analysis, precipitated by addition of (NH4)2SO4 to 60% (w/v), and left ovemight at4°C. The pellets are resuspended in 145 mM NaCl, 1 mM CaCb, 50 mM triethanolamine hydrochloride, pH 7.4, and applied onto a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas, Montgomeryville, PA) equilibrated in the same buffer. The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration on Microsep microconcentrators (m.w. cut-off= 10,000) (Filtron, Karlstein, Germany).
EXAMPLE 4
This example describes a method of producing polyclonal antibodies against MASP-2 polypeptides.
Materials and Methods:
MASP-2 Antigens: Polyclonal anti-human MASP-2 antiserum is produced by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 (SEQ ID NO:6) isolated from sérum; recombinant human MASP-2 (SEQ ID NO:6), MASP-2A containing the inactive protease domain (SEQ ID NO: 13), as described in Example 3; and recombinant CUBI (SEQ ID NO:8), CUBEGFI (SEQ ID NO:9), and 25 CUBEGFCUBII (SEQ ID NO: 10) expressed as described above in Example 3.
Polyclonal antibodies: Six-week old Rabbits, primed with BCG (bacillus Calmette-Guerin vaccine) are immunized by injecting 100 pg of MASP-2 polypeptide at lOOpg/ml in stérile saline solution. Injections are done every 4 weeks, with antibody titer monitored by ELISA assay as described in Example 5. Culture supematants are 30 collected for antibody purification by protein A affinity chromatography.
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EXAMPLE 5
This example describes a method for producing murine monoclonal antibodies against rat or human MASP-2 polypeptides.
Materials and Methods:
Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, are injected subcutaneously with 100 pg human or rat rMASP-2 or rMASP-2A polypeptides (made as described in Example 3) in complété Freund's adjuvant (Difco Laboratories, Detroit, Mich.) in 200 pl of phosphate buffered saline (PBS) pH 7.4. At two-week intervals the mice are twice injected subcutaneously with 50 μg of human or rat rMASP-2 or 10 rMASP-2A polypeptide in incomplète Freund's adjuvant. On the fourth week the mice are injected with 50 pg of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and are fused 4 days later.
For each fusion, single cell suspensions are prepared from the spleen of an immunized mouse and used for fusion with Sp2/0 myeloma cells. 5x10$ of the Sp2/0 15 and 5xl08 spleen cells are fused in a medium containing 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells are then adjusted to a concentration of 1.5xl05 spleen cells per 200 pl of the suspension in Iscove medium (Gibco, Grand fsland, N.Y.), supplemented with 10% fêtai bovine sérum, 100 units/ml of penicillin, 100 pg/ml of streptomycin, 20 0.1 mM hypoxanthine, 0.4 pM aminopterin and 16 pM thymidine. Two hundred microliters of the cell suspension are added to each well of about twenty 96-well microculture plates. After about ten days culture supematants are withdrawn for screening for reactivity with purified factor MASP-2 in an ELfSA assay.
ELISA Assay: Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) 25 microtest plates are coated by adding 50 pl of purified hMASP-2 at 50 ng/ml or rat rMASP-2 (or rMASP-2A) ovemight at room température. The low concentration of MASP-2 for coating enables the sélection of high-affinity antibodies. After the coating solution is removed by flicking the plate, 200 pl of BLOTTO (non-fat dry milk) in PBS is added to each well for one hour to block the non-specific sites. An hour later, the wells 30 are then washed with a buffer PBST (PBS containing 0.05% Tween 20). Fifty microliters of culture supematants from each fusion well is collected and mixed with 50 pl of
154
BLOTTO and then added to the individual wells of the microtest plates. After one hour of incubation, the wells are washed with PBST. The bound murine antibodies are then detected by reaction with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Fc spécifie) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and diluted at 1:2,000 in BLOTTO. Peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethyl benzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) is added to the wells for color development for 30 minutes. The reaction is terminated by addition of 50 μΐ of 2M H2SO4 per well. The Optical Density at 450 nm of the reaction mixture is read with a BioTek ELISA Reader (BioTek Instruments, Winooski, Vt.).
MASP-2 Binding Assay:
Culture supematants that test positive in the MASP-2 ELISA assay described above can be tested in a binding assay to détermine the binding affinity the MASP-2 inhibitory agents hâve for MASP-2. A similar assay can also be used to détermine if the inhibitory agents bind to other antigens in the complément System.
Polystyrène microtiter plate wells (96-well medium binding plates, Corning Costar, Cambridge, MA) are coated with MASP-2 (20 ng/100 μΐ/well, Advanced Research Technology, San Diego, CA) in phosphate-buffered saline (PBS) pH 7.4 ovemight at 4°C. After aspirating the MASP-2 solution, wells are blocked with PBS containing 1% bovine sérum albumin (BSA; Sigma Chemical) for 2 h at room température. Wells without MASP-2 coating serve as the background Controls. Aliquots of hybridoma supematants or purified anti-MASP-2 MoAbs, at varying concentrations in blocking solution, are added to the wells. Following a 2 h incubation at room température, the wells are extensively rinsed with PBS. MASP-2-bound anti-MASP-2 MoAb is detected by the addition of peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blocking solution, which is allowed to incubate for Ih at room température. The plate is rinsed again thoroughly with PBS, and 100 μΐ of 3,3',5,5'-tetramethyl benzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The reaction of TMB is quenched by the addition of 100 μΐ of IM phosphoric acid, and the plate is read at 450 nm in a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).
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The culture supematants from the positive wells are then tested for the ability to inhibit complément activation in a functional assay such as the C4 cleavage assay as described in Example 2. The cells in positive wells are then cloned by limiting dilution. The MoAbs are tested again for reactivity with hMASP-2 in an ELISA assay as described 5 above. The selected hybridomas are grown in spinner flasks and the spent culture supematant collected for antibody purification by protein A affinity chromatography.
EXAMPLE 6
This example describes the génération and production of humanized murine 10 anti-MASP-2 antibodies and antibody fragments.
A murine anti-MASP-2 monoclonal antibody is generated in Male A/J mice as described in Example 5. The murine antibody is then humanized as described below to reduce its immunogenicity by replacing the murine constant régions with their human counterparts to generate a chimeric IgG and Fab fragment of the antibody, which is usefiil 15 for inhibiting the adverse effects of MASP-2-dependent complément activation in human subjects in accordance with the présent invention.
1. Cloning of anti-MASP-2 variable région genes from murine hybridoma cells. Total RNA is isolated from the hybridoma cells secreting anti-MASP-2 MoAb (obtained as described in Example 7) using RNAzol foilowing the 20 manufacturer's protocol (Biotech, Houston, Tex.). First strand cDNA is synthesized from the total RNA using oligo dT as the primer. PCR is performed using the immunoglobulin constant C region-derived 3' primers and degenerate primer sets derived from the leader peptide or the first framework région of murine Vjq or Vj< genes as the 5' primers. Anchored PCR is carried out as described by Chen and Platsucas (Chen, P.F., Scand. J.
Immunol. 35:539-549, 1992). For cloning the Vk gene, double-stranded cDNA is prepared using a Notl-MAKl primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO:38). Annealed adaptors ADI (5’-GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO:39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO:40) are ligated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are 30 removed by Notl digestion. The digested product is then used as the template in PCR with the ADI oligonucleotide as the 5' primer and MAK2
156 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3' SEQ ID NO:41) as the 3' primer. DNA fragments of approximately 500 bp are cloned into pUC19. Several clones are selected for sequence analysis to verify that the cloned sequence encompasses the expected murine immunoglobulin constant région. The Not 1-MAKI and MAK2 oligonucleotides are derived from the Vjç région and are 182 and 84 bp, respectively, downstream from the First base pair of the C kappa gene. Clones are chosen that include the complété Vjç and leader peptide.
For cloning the V^ gene, double-stranded cDNA is prepared using the Notl MAG1 primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO:42). Annealed adaptors ADI and AD2 are ligated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are removed by Notl digestion. The digested product are used as the template in PCR with the ADI oligonucleotide and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO:43) as primers. DNA fragments of 500 to 600 bp in length are cloned into pUC19. The Notl-MAGl and MAG2 oligonucleotides are derived from the murine Cy.7.1 région, and are 180 and 93 bp, respectively, downstream from the first bp of the murine Cy.7.1 gene. Clones are chosen that encompass the complété Vjq and leader peptide.
2. Construction of Expression Vectors for Chimeric MASP-2 IgG and Fab. The cloned Vjq and Vk genes described above are used as templates in a PCR reaction to add the Kozak consensus sequence to the 5' end and the splice donor to the 3' end of the nucléotide sequence. After the sequences are analyzed to confirm the absence of PCR errors, the V^ and Vjç genes are inserted into expression vector cassettes containing human C.yl and C. kappa respectively, to give pSV2neoVH-huCyl and pSV2neoV-huCy. CsCl gradient-purified plasmid DNAs of the heavy- and light-chain vectors are used to transfect COS cells by electroporation. After 48 hours, the culture supematant is tested by ELISA to confirm the presence of approximately 200 ng/ml of chimeric IgG. The cells are harvested and total RNA is prepared. First strand cDNA is synthesized from the total RNA using oligo dT as the primer. This cDNA is used as the template in PCR to generate the Fd and kappa DNA fragments. For the Fd gene, PCR is carried out using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3'
157 (SEQ ID NO:44) as the 5' primer and a CH1-derived 3' primer (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO:45). The DNA sequence is confirmed to contain the complété Vh and the CH1 domain of human IgGl. After digestion with the proper enzymes, the Fd DNA fragments are inserted at the HindlII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to give pSV2dhfrFd. The pSV2 plasmid is commercially available and consists of DNA segments from various sources: pBR322 DNA (thin line) contains the pBR322 origin of DNA réplication (pBR ori) and the lactamase ampicillin résistance gene (Amp); SV40 DNA, represented by wider hatching and marked, contains the SV40 origin of DNA réplication (SV40 ori), early promoter (5' to the dhfr and neo genes), and polyadenylation signal (3' to the dhfr and neo genes). The SV40-derived polyadenylation signal (pA) is also placed at the 3' end of the Fd gene.
For the kappa gene, PCR is carried out using 5'AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO:46) as the 5' primer and a CR-derived 3' primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO:47). DNA sequence is confirmed to contain the complété Vr and human Cr régions. After digestion with proper restriction enzymes, the kappa DNA fragments are inserted at the HindlII and BamHI restriction sites of the expression vector cassette pSV2neo-TUS to give pSV2neoK. The expression of both Fd and .kappa genes are driven by the HCMV-derived enhancer and promoter éléments. Since the Fd gene does not include the cysteine amino acid residue involved in the inter-chain disulfide bond, this recombinant chimeric Fab contains non-covalently linked heavy- and light-chains. This chimeric Fab is designated as cFab.
To obtain recombinant Fab with an inter-heavy and light chain disulfide bond, the above Fd gene may be extended to include the coding sequence for additional 9 amino acids (EPKSCDKTH SEQ ID NO:48) from the hinge région of human IgGl. The BstEII-BamHI DNA segment encoding 30 amino acids at the 3' end of the Fd gene may be replaced with DNA segments encoding the extended Fd, resulting in pSV2dhfrFd/9aa.
3. Expression and Purification of Chimeric Anti-MASP-2 IgG
To generate cell fines secreting chimeric anti-MASP-2 IgG, NSO cells are transfected with purified plasmid DNAs of pSV2neoVjq-huC.yl and pSV2neoV-huC
158 kappa by electroporation. Transfected cells are selected in the presence of 0.7 mg/ml G418. Cells are grown in a 250 ml spinner flask using serum-containing medium.
Culture supematant of 100 ml spinner culture is loaded on a 10-ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed 5 volumes of PBS. The bound antibody is eluted with 50 mM citrate buffer, pH 3.0. Equal volume of 1 M Hepes, pH 8.0 is added to the fraction containing the purified antibody to adjust the pH to 7.0. Residual salts are removed by buffer exchange with PBS by Millipore membrane ultrafiltration (M.W. cut-off: 3,000). The protein concentration of the purified antibody is determined by the BCA method (Pierce).
4. Expression and purification of chimeric anti-MASP-2 Fab
To generate cell lines secreting chimeric anti-MASP-2 Fab, CHO cells are transfected with purified plasmid DNAs of pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa, by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are amplified in increasing concentrations of 15 methotrexate. Cells are single-cell subcloned by limiting dilution. High-producing single-cell subcloned cell lines are then grown in 100 ml spinner culture using serum-free medium.
Chimeric anti-MASP-2 Fab is purified by affmity chromatography using a mouse anti-idiotypie MoAb to the MASP-2 MoAb. An anti-idiotypic MASP-2 MoAb can be 20 made by immunizing mice with a murine anti-MASP-2 MoAb conjugated with keyhole limpet hemocyanin (KLH) and screening for spécifie MoAb binding that can be competed with human MASP-2. For purification, 100 ml of supematant from spinner cultures of CHO cells producing cFab or cFab/9aa are loaded onto the affmity column coupled with an anti-idiotype MASP-2 MoAb. The column is then washed thoroughly 25 with PBS before the bound Fab is eluted with 50 mM diethylamine, pH 11.5. Residual salts are removed by buffer exchange as described above. The protein concentration of the purified Fab is determined by the BCA method (Pierce).
The ability of the chimeric MASP-2 IgG, cFab, and cFAb/9aa to inhibit MASP-2-dependent complément pathways may be determined by using the inhibitory 30 assays described in Example 2 or Example 7.
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EXAMPLE 7
This example describes an in vitro C4 cleavage assay used as a functional screen to identity MASP-2 inhibitory agents capable of blocking MASP-2-dependent complément activation via L-ficolin/P35, H-ficolin, M-ficolin or mannan.
C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, S.V., étal., J. Immunol. Methods 257:107, 2001, which measures lectin pathway activation resulting from lipoteichoic acid (LTA) from S. aureus which binds L-ficolin.
Reagents: Formalin-fixed £ aureous (DSM20233) is prepared as follows: bacteria is grown ovemight at 37°C in tryptic soy blood medium, washed three times with PBS, then fixed for 1 h at room température in PBS/0.5% formalin, and washed a further three times with PBS, before being resuspended in coating buffer (15 mM Na2Co3, 35 mM NaHCOs, pH 9.6).
Assay: The wells of a Nunc MaxiSorb microtiter plate (Nalgene Nunc International, Rochester, NY) are coated with: 100 μΐ of formalin-fixed S. aureus DSM20233 (00559 = 0.5) in coating buffer with 1 ug of L-ficolin in coating buffer. After overnight incubation, wells are blocked with 0.1% human sérum albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4), then are washed with TBS containing 0.05% Tween 20 and 5 mM CaCl2 (wash buffer). Human sérum samples are diluted in 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl2, 0.05% Triton X-100, 0.1% HSA, pH 7.4, which prevents activation of endogenous C4 and dissociâtes the Cl complex (composed of Clq, Clr and Cl s). MASP-2 inhibitory agents, including anti-MASP-2 MoAbs and inhibitory peptides are added to the sérum samples in varying concentrations. The diluted samples are added to the plate and incubated overnight at 4°C. After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 pg of purified human C4 (obtained as described in Dodds, A.W., Methods EnzymoL 223:46, 1993) in 100 μΐ of 4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4 is added to each well. After 1.5 h at 37°C, the plates are washed again and C4b déposition is detected using alkaline phosphatase-conjugated chicken anti-human C4c (obtained from Immunsystem, Uppsala, Sweden) and measured using the colorimétrie substrate p-nitrophenyl phosphate.
160
C4 Assay on mannan: The assay described above is adapted to measure lectin pathway activation via MBL by coating the plate with LSP and mannan prior to adding sérum mixed with various MASP-2 inhibitory agents.
C4 assay on H-ficolin (Hakata Ag): The assay described above is adapted to 5 measure lectin pathway activation via H-ficolin by coating the plate with LPS and H-ficolin prior to adding sérum mixed with various MASP-2 inhibitory agents.
EXAMPLE 8
The following assay demonstrates the presence of classical pathway activation in 10 wild-type and MASP-2-/-mice.
Methods: Immune complexes were generated in situ by coating microtiter plates (Maxisorb, Nunc, cat. No. 442404, Fisher Scientific) with 0.1% human sérum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 for 1 hours at room température followed by ovemight incubation at 4°C with sheep anti whole sérum antiserum (Scottish Antibody 15 Production Unit, Carluke, Scotland) diluted 1:1000 in TBS/tween/Ca2+. Sérum samples were obtained from wild-type and MASP-2-/- mice and added to the coated plates. Control samples were prepared in which Clq was depleted from wild-type and MASP-2-/- sérum samples. Clq-depleted mouse sérum was prepared using protein-A-coupled Dynabeads (Dynal Biotech, Oslo, Norway) coated with rabbit 20 anti-human Clq IgG (Dako, Glostrup, Denmark), according to the supplier's instructions. The plates were incubated for 90 minutes at 37°C. Bound C3b was detected with a polyclonal anti-human-C3c Antibody (Dako A 062) diluted in TBS/tw/ Ca++ at 1:1000. The secondary antibody is goat anti-rabbit IgG.
Results: FIGURE 7 shows the relative C3b déposition levels on plates coated 25 with IgG in wild-type sérum, MASP-2-/- sérum, Clq-depleted wild-type and Clq-depleted MASP-2-/- sérum. These results demonstrate that the classical pathway is intact in the MASP-2-/- mouse strain.
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EXAMPLE 9
The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of a MASP-2 inhibitory agent under conditions in which the classical pathway is initiated by immune complexes.
Methods: To test the effect of a MASP-2 inhibitory agent on conditions of complément activation where the classical pathway is initiated by immune complexes, triplicate 50 μΐ samples containing 90% NHS are incubated at 37°C in the presence of 10 pg/ml immune complex (IC) or PBS, and parallel triplicate samples (+/-IC) are also included which contain 200 nM anti-properdin monoclonal antibody during the 37°C incubation. After a two hour incubation at 37°C, 13 mM EDTA is added to ail samples to stop further complément activation and the samples are immediately cooled to 5°C. The samples are then stored at -70°C prior to being assayed for complément activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009) following the manufacturées instructions.
EXAMPLE 10
This example describes the identification of high affinity anti-MASP-2 Fab2 antibody fragments that block MASP-2 activity.
Background and rationale: MASP-2 is a complex protein with many separate functional domains, including: binding site(s) for MBL and ficolins, a serine protease catalytic site, a binding site for proteolytic substrate C2, a binding site for proteolytic substrate C4, a MASP-2 cleavage site for autoactivation of MASP-2 zymogen, and two Ca++ binding sites. Fab2 antibody fragments were identified that bind with high affinity to MASP-2, and the identified Fab2 fragments were tested in a functional assay to détermine if they were able to block MASP-2 functional activity.
To block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind and interfère with a structural epitope on MASP-2 that is required for MASP-2 functional activity. Therefore, many or ail of the high affinity binding anti-MASP-2 Fab2s may not inhibit MASP-2 functional activity unless they bind to structural epitopes on MASP-2 that are directly involved in MASP-2 functional activity.
A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of anti-MASP-2 Fab2s. It is
162 known that the primary physiological rôle of MASP-2 in the lectin pathway is to generate the next functional component of the lectin-mediated complément pathway, namely the lectin pathway C3 convertase. The lectin pathway C3 convertase is a critical enzymatic complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, MASP-2 functional activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Furthermore, ail of the separate functional activities of MASP-2. listed above appear to be required in order for MASP-2 to generate the lectin pathway C3 convertase. For these reasons, a preferred assay to use in evaluating the blocking activity of anti-MASP-2 Fab2s is believed to be a functional assay that measures inhibition of lectin pathway C3 convertase formation.
Génération of High Affinity Fab2s: A phage display library of human variable light and heavy chain antibody sequences and automated antibody sélection technology for identifying Fab2s that react with selected ligands of interest was used to create high affinity Fab2s to rat MASP-2 protein (SEQ ID NO:55). A known amount of rat MASP-2 (~1 mg, >85% pure) protein was utilized for antibody screening. Three rounds of amplification were utilized for sélection of the antibodies with the best affinity. Approximately 250 different hits expressing antibody fragments were picked for ELISA screening. High affinity hits were subsequently sequenced to détermine uniqueness of the different antibodies.
Fifty unique anti-MASP-2 antibodies were purified and 250 pg of each purified Fab2 antibody was used for characterization of MASP-2 binding affinity and complément pathway functional testing, as described in more detail below.
Assays used to Evaluate the Inhibitory (blocking) Activity of Anti-MASP-2 Fab2s
1. Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase:
Background: The lectin pathway C3 convertase is the enzymatic complex (C4bC2a) that proteolytically cleaves C3 into the two potent proinflammatory fragments, anaphylatoxin C3a and opsonic C3b. Formation of C3 convertase appears to a key step in the lectin pathway in terms of mediating inflammation. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); therefore anti-MASP-2
163 antibodies (or Fab2) will not directly inhibit activity of preexisting C3 convertase. However, MASP-2 serine protease activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Therefore, anti-MASP-2 Fab2 which inhibit MASP-2 functional activity (i.e., blocking anti-MASP-2 Fab2) will inhibit de novo formation of lectin pathway C3 convertase. 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 on the bottom of the plastic wells via ester or amide linkages, thus facilitating détection of C3b in the ELIS A assay.
Yeast mannan is a known activator of the lectin pathway. In the following method to measure formation of C3 convertase, plastic wells coated with mannan were incubated for 30 min at 37°C with diluted rat sérum to activate the lectin pathway. The wells were then washed and assayed for C3b immobilized onto the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 Fab2s at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and conséquent C3b génération.
Methods:
96-well Costar Medium Binding plates were incubated ovemight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1 ug/50 Tl/well. After ovemight incubation, each well was washed three times with 200 Tl PBS. The wells were then blocked with 100 Tl/well of 1% bovine sérum albumin in PBS and incubated for one hour at room température with gentle mixing. Each well was then washed three times with 200 Tl of PBS. The anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca++ and Mg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCh, 2.0 mM CaCh, 0.1% gelatin, pH 7.4) at 5 C. A 0.5% rat sérum was added to the above samples at 5 C and 100 Tl was transferred to each well. Plates were covered and incubated for 30 minutes in a 37 C waterbath to allow complément activation. The reaction was stopped by transferring the plates from the 37 C waterbath to a container containing an ice-water mix. Each well was washed five times with 200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then washed two times with 200 Tl PBS. A
164
100 Tl/well of 1:10,000 dilution of the primary antibody (rabbit anti-human C3c, DAKO A0062) was added in PBS containing 2.0 mg/ml bovine sérum albumin and incubated 1 hr at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of 1:10,000 dilution of the secondary antibody (peroxidase-conjugated goat 5 anti-rabbit IgG, American Qualex A102PU) was added in PBS containing 2.0 mg/ml bovine sérum albumin and incubated for one hour at room température on a shaker with gentle mixing. Each well was washed five times with 200 Tl with PBS. 100 Tl/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 10 min. The peroxidase reaction was stopped by 10 adding 100 Tl/well of 1.0 Μ H3PO4 and the OD450. was measured.
2. Assay to Measure Inhibition of MASP-2-dependent C4 Cleavage
Background: The serine protease activity of MASP-2 is highly spécifie and only two protein substrates for MASP-2 hâve been identified; C2 and C4. Cleavage of C4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind to structural epitopes on MASP-2 15 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2 serine protease catalytic site) and thereby inhibit the C4 cleavage functional activity of MASP-2.
Yeast mannan is a known activator of the lectin pathway. In the foilowing method to measure the C4 cleavage activity of MASP-2, plastic wells coated with 20 mannan were incubated for 30 minutes at 37 C with diluted rat sérum to activate the lectin pathway. Since the primary antibody used in this ELISA assay only recognizes human C4, the diluted rat sérum was also supplemented with human C4 (1.0 Tg/ml). The wells were then washed and assayed for human C4b immobilized onto the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of 25 MASP-2 dépendent C4 cleavage activity. Anti-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.
Methods: 96-well Costar Medium Binding plates were incubated ovemight at 5 C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1.0 Tg/50 Tl/well. Each well was washed 3X with 200 Tl PBS. The wells were then blocked with 100 Tl/well of 30 1% bovine sérum albumin in PBS and incubated for one hour at room température with gentle mixing. Each well was washed 3X with 200 Tl of PBS. Anti-MASP-2 Fab2
165 samples were diluted to selected concentrations in Ca++ and Mg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCh, 2.0 mM CaCh, 0.1% gelatin, pH 7.4) at 5 C. 1.0 Tg/ml human C4 (Quidel) was also included in these samples. 0.5% rat sérum was added to the above samples at 5 C and 100 Tl was transferred to each well. The plates were covered and incubated for 30 min in a 37 C waterbath to allow complément activation. The reaction was stopped by transferring the plates from the 37 C waterbath to a container containing an ice-water mix. Each well was washed 5 x 200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed with 2X with 200 Tl PBS. 100 Tl/well of 1:700 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/ml bovine sérum albumin (BSA) and incubated one hour at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of 0.1 Tg/ml of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and incubated for one hour at room température on a shaker with gentle mixing. Each well was washed 5 x 200 Tl with PBS. 100 Tl/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 16 min. The peroxidase reaction was stopped by adding 100 Tl/well of LO Μ H3PO4 and the OD450 .was measured.
3. Binding Assay of anti-rat MASP-2 Fab2 to 'Native' rat MASP-2
Background: MASP-2 is usually présent in plasma as a MASP-2 dimer complex that also includes spécifie lectin molécules (mannose-binding protein (MBL) and ficolins). Therefore, if one is interested in studying the binding of anti-MASP-2 Fab2 to the physiologically relevant form of MASP-2, it is important to develop a binding assay in which the interaction between the Fab2 and 'native' MASP-2 in plasma is used, rather than purified recombinant MASP-2. In this binding assay the 'native' MASP-2-MBL complex from 10% rat sérum was first immobilized onto mannan-coated wells. The binding affinity of various anti-MASP-2 Fab2s to the immobilized 'native' MASP-2 was then studied using a standard ELIS A methodology.
Methods: 96-well Costar High Binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1 Tg/50 Tl/well. Each well was washed 3X with 200 Tl PBS. The wells were blocked with 100 Tl/well of 0.5%
166 nonfat dry milk in PBST (PBS with 0.05% Tween 20) and incubated for one hour at room température with gentle mixing. Each well was washed 3X with 200 Tl of TBS/Tween/Ca++ Wash Buffer (Tris-buffered saline, 0.05% Tween 20, containing 5.0 mM CaCl2, pH 7.4. 10% rat sérum in High Sait Binding Buffer (20 mM Tris, 1.0 M 5 NaCl, 10 mM CaCh, 0.05% Triton-X100, 0.1% (w/v) bovine sérum albumin, pH 7.4) was prepared on ice. 100 Tl/well was added and incubated ovemight at 5°C. Wells were washed 3X with 200 Tl of TBS/Tween/Ca^ Wash Buffer. Wells were then washed 2X with 200 Tl PBS. 100 Tl/well of selected concentration of anti-MASP-2 Fab2 diluted in Ca++ and Mg++ containing GVB Buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 10 2.0 mM CaCh, 0.1% gelatin, pH 7.4) was added and incubated for one hour at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of HRP-conjugated goat anti-Fab2 (Biogenesis Cat No 0500-0099) diluted 1:5000 in 2.0 mg/ml bovine sérum albumin in PBS was added and incubated for one hour at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of 15 the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 70 min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 Μ H3PO4 and OD450. was measured.
RESULTS:
Approximately 250 different Fab2s that reacted with high affinity to the rat 20 MASP-2 protein were picked for ELISA screening. These high affinity Fab2s were sequenced to détermine the uniqueness of the different antibodies, and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 ug of each purified Fab2 antibody was used for characterization of MASP-2 binding affinity and complément pathway functional testing. The results of this analysis is shown below in TABLE 6.
TABLE 6: ANTI-MASP-2 FAB2 THAT BLOCK LECTIN PATHWAY
COMPLEMENT ACTIVATION_________________
Fab2 antibody # C3 Convertase (IC50 (nM) Kd C4 Cleavage IC50 (nM)
88 0.32 4.1 ND
41 0.35 ____ 0.30 0.81
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Fab2 antibody # C3 Convertase (IC50 (nM) Kd C4 Cleavage IC50 (nM)
11 0.46 0.86 <2 nM
86 0.53 1.4 ND
81 0.54 2.0 ND
66 0.92 4.5 ND
57 0.95 3.6 <2 nM
40 1.1 7.2 0.68
58 1.3 2.6 ND
60 1.6 3.1 ND
52 1.6 5.8 <2 nM
63 2.0 6.6 ND
49 2.8 8.5 <2 nM
89 3.0 2.5 ND
71 3.0 10.5 ND
87 6.0 2.5 ND
67 10.0 7.7 ND
As shown above in TABLE 6, of the 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocking Fab2 that potently inhibit C3 convertase formation with IC50 equal to or less than 10 nM Fab2s (a 34% positive hit rate). Eight of 5 the seventeen Fab2s identified hâve IC50S in the subnanomolar range. Furthermore, ail seventeen of the MASP-2 blocking Fab2s shown in TABLE 6 gave essentially complété inhibition of C3 convertase formation in the lectin pathway C3 convertase assay. FIGURE 8A graphically illustrâtes the results of the C3 convertase formation assay for Fab2 antibody #11, which is représentative of the other Fab2 antibodies tested, the results of which are shown in TABLE 6. This is an important considération, since it is theoretically possible that a blocking Fab2 may only fractionally inhibit MASP-2 function even when each MASP-2 molécule is bound by the Fab2.
Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat sérum might also activate 15 the classical pathway and generate C3b via the classical pathway C3 convertase.
However, each of the seventeen blocking anti-MASP-2 Fab2s listed in this example
168 potently inhibits C3b génération (>95 %), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.
Binding assays were also performed with ail seventeen of the blocking Fab2s in order to calculate an apparent for each. The results of the binding assays of anti-rat MASP-2 Fab2s to native rat MASP-2 for six of the blocking Fab2s are also shown in TABLE 6. FIGURE 8B graphically illustrâtes the results of a binding assay with the Fab2 antibody #11. Similar binding assays were also carried out for the other Fab2s, the results of which are shown in TABLE 6. In general, the apparent Kjs obtained for binding of each of the six Fab2s to 'native' MASP-2 corresponds reasonably well with the IC5Q for the Fab2 in the C3 convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from an 'inactive' to an 'active' form upon activation of its protease activity (Feinberg et al., EMBO J 22:2348-59 (2003); Gai et al., J. Biol. Chem. 250:33435-44 (2005)). In the normal rat plasma used in the C3 convertase formation assay, MASP-2 is présent primarily in the 'inactive' zymogen conformation. In contrast, in the binding assay, MASP-2 is présent as part of a complex with MBL bound to immobilized mannan; therefore, the MASP-2 would be in the 'active' conformation (Petersen et al., J. Immunol Methods 257:107-16, 2001). Consequently, one would not necessarily expect an exact correspondence between the IC50 and Kj for each of the seventeen blocking Fab2 tested in these two functional assays since in each assay the Fab2 would be binding a different conformational form of MASP-2. Never-the-less, with the exception of Fab2 #88, there appears to be a reasonably close correspondence between the IC50 and apparent Kd for each of the other sixteen Fab2 tested in the two assays (see TABLE 6).
Several of the blocking Fab2s were evaluated for inhibition of MASP-2 mediated cleavage of C4. FIGURE 8C graphically illustrâtes the results of a C4 cleavage assay, showing inhibition with Fab2 #41, with an IC5q=0.81 nM (see TABLE 6). As shown in FIGURE 9, ail of the Fab2s tested were found to inhibit C4 cleavage with ICsqs similar to those obtained in the C3 convertase assay (see TABLE 6).
Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat sérum might also activate the classical pathway and thereby generate C4b by Cls-mediated cleavage of C4.
169
However, several anti-MASP-2 Fab2s hâve been identified which potently inhibit C4b génération (>95 %), thus demonstrating the specificity of this assay for MASP-2 mediated C4 cleavage. C4, like C3, contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C4 by MASP-2 in this assay, the thioester group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic wells via ester or amide linkages, thus facilitating détection of C4b in the ELISA assay.
These studies clearly demonstrate the création of high affinity FAB2s to rat MASP-2 protein that functionally block both C4 and C3 convertase activity, thereby preventing lectin pathway activation.
EXAMPLE 11
This Example describes the epitope mapping for several of the blocking anti-rat MASP-2 Fab2 antibodies that were generated as described in Example 10.
Methods:
As shown in FIGURE 10, the foilowing proteins, ail with N-terminal 6X His tags were expressed in CHO cells using the pED4 vector:
rat MASP-2A, a full length MASP-2 protein, inactivated by altering the serine at the active center to alanine (S613A);
rat MASP-2K, a full-length MASP-2 protein altered to reduce autoactivation (R424K);
CUBI-II, an N-terminal fragment of rat MASP-2 that contains the CUBI, EGF-like and CUBII domains only; and
CUBI/EGF-like, an N-terminal fragment of rat MASP-2 that contains the CUBI and EGF-like domains only.
These proteins were purified from culture supematants by nickel-affmity chromatography, as previously described (Chen et aL, J. Biol. Chem. 276:25894-02 (2001)).
A C-terminal polypeptide (CCPII-SP), containing CCPII and the serine protease domain of rat MASP-2, was expressed in E. coli as a thioredoxin fusion protein using pTrxFus (Invitrogen). Protein was purified from cell lysâtes using Thiobond affinity
170 resin. The thioredoxin fusion partner was expressed from empty pTrxFus as a négative control.
Ail recombinant proteins were dialyzed into TBS buffer and their concentrations determined by measuring the OD at 280 nm.
DOT BLOT ANALYSIS:
Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIGURE 10 (and the thioredoxin polypeptide as a négative control for CCPII-serine protease polypeptide) were spotted onto a nitrocellulose membrane. The amount of protein spotted ranged from 100 ng to 6.4 pg, in five-fold steps. In later experiments, the amount of protein spotted ranged from 50 ng down to 16 pg, again in five-fold steps. Membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer) then incubated with 1.0 pg/ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca2+). Bound Fab2s were detected using HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL détection kit (Amersham). One membrane was incubated with polyclonal rabbit-anti human MASP-2 Ab (described in Stover et al., JImmunol 763:6848-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2,000).
MASP-2 Binding Assay
ELISA plates were coated with 1.0 gg/well of recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0) ovemight at 4°C. Wells were blocked with 1% BSA in TBS, then serial dilutions of the anti-MASP-2 Fab2s were added in TBS containing 5.0 mM Ca2+. The plates were incubated for one hour at RT. After washing three times with TBS/tween/Ca2+, HRP-conjugated anti-human Fab (AbD/Serotec) diluted 1/10,000 in TBS/ Ca2+ was added and the plates incubated for a further one hour at RT. Bound antibody was detected using a TMB peroxidase substrate kit (Biorad).
RESULTS:
Results of the dot blot analysis demonstrating the reactivity of the Fab2s with various MASP-2 polypeptides are provided below in TABLE 7. The numerical values provided in TABLE 7 indicate the amount of spotted protein required to give approximately half-maximal signal strength. As shown, ail of the polypeptides (with the exception of the thioredoxin fusion partner alone) were recognized by the positive control Ab (polyclonal anti-human MASP-2 sera, raised in rabbits).
171
TABLE 7: REACTIVITY WITH VARIOUS RECOMBINANT RAT MASP-2 POLYPEPTIDES ON DOT BLOTS
Fab2 Antibody # MASP-2A CUBI-II CUBI/EGF-like CCPII-SP Thioredoxin
40 0.16 ng NR NR 0.8 ng NR
41 0.16 ng NR NR 0.8 ng NR
11 0.16 ng NR NR 0.8 ng NR
49 0.16 ng NR NR >20 ng NR
52 0.16 ng NR NR 0.8 ng NR
57 0.032 ng NR NR NR NR
58 0.4 ng NR NR 2.0 ng NR
60 0.4 ng 0.4 ng NR NR NR
63 0.4 ng NR NR 2.0 ng NR
66 0.4 ng NR NR 2.0 ng NR
67 0.4 ng NR NR 2.0 ng NR
71 0.4 ng NR NR 2.0 ng NR
81 0.4 ng NR NR 2.0 ng NR
86 0.4 ng NR NR 10 ng NR
87 0.4 ng NR NR 2.0 ng NR
Positive Control <0.032 ng 0.16 ng 0.16 ng <0.032 ng NR
pathway activity was observed over the second and third weeks, with complété lectin pathway restoration in the mice by 17 days post anti-MASP-2 MoAb administration. NR = No reaction. The positive control antibody is polyclonal anti-human MASP-2 sera, raised in rabbits.
Ail of the Fab2s reacted with MASP-2A as well as MASP-2K (data not shown).
The majority of the Fab2s recognized the CCPII-SP polypeptide but not the N-terminal fragments. The two exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and the CUBI-II fragment, but not the CUBI/EGF-like polypeptide or the CCPII-SP polypeptide, suggesting it binds to an epitope in CUBII, or spanning the CUBII and the EGF-like domain. Fab2 # 57 recognizes MASP-2A but not any of the MASP-2 fragments tested, indicating that this Fab2 recognizes an epitope in CCP1. Fab2 #40 and
172 #49 bound only to complété MASP-2A. In the ELISA binding assay shown in FIGURE 11, Fab2 #60 also bound to the CUBI-II polypeptide, albeit with a slightly lower apparent affinity.
These finding demonstrate the identification of unique blocking Fab2s to multiple 5 régions of the MASP-2 protein
EXAMPLE 12
This Example describes the analysis of MASP-2-/- mice in a Murine Rénal Ischemia/Reperfusion Model.
Background/Rationale: Ischemia-Reperfusion (I/R) injury in kidney at body température has relevance in a number of clinical conditions, including hypovolaemic shock, rénal artery occlusion and cross-clamping procedures.
Kidney ischemia-reperfusion (I/R) is an important cause of acute rénal failure, associated with a mortality rate of up to 50% (Levy étal., JAMA 275:1489-94, 1996;
Thadhani et al., N. Engl. J. Med. 534:1448-60, 1996). Post-transplant rénal failure is a common and threatening complication after rénal transplantation (Nicholson et al., Kidney Int. 55:2585-91, 2000). Effective treatment for rénal I/R injury is currently not available and hemodialysis is the only treatment available. The pathophysiology of rénal I/R injury is complicated. Recent studies hâve shown that the lectin pathway of 20 complément activation may hâve an important rôle in the pathogenesis of rénal I/R injury (deVries et al., Am. J. Path. 765:1677-88, 2004).
Methods:
A MASP-2(-/-) mouse was generated as described in Example 1 and backcrossed for at least 10 générations with C57B1/6. Six male MASP-2(-/-) and six wildtype (+/+) 25 mice weighing between 22-25 g were administered an intraperitoneal injection of Hypnovel (6.64 mg/kg; Roche products Ltd. Welwyn Garden City, UK), and subsequently anaesthetized by inhalation of isoflurane (Abbott Laboratories Ltd., Kent, UK). Isoflurane was chosen because it is a mild inhalation anaesthetic with minimal liver toxicity; the concentrations are produced accurately and the animal 30 recovers rapidly, even after prolonged anaesthesia. Hypnovel was administered because it produces a condition of neuroleptanalgesia in the animal and means that less isoflurane needs to be administered. A warm pad was placed beneath the animal in order to
173 maintain a constant body température. Next, a midline abdominal incision was performed and the body cavity held open using a pair of retractors. Connective tissue was cleared above and below the rénal vein and artery of both right and left kidneys, and the rénal pedicle was clamped via the application of microaneurysm clamps for a period 5 of 55 minutes. This period of ischemia was based initially on a previous study performed in this laboratory (Zhou et al., J. Clin. Invest. 105:1363-71 (2000)). In addition, a standard ischémie time of 55 minutes was chosen following ischémie titration and it was found that 55 minutes gave consistent injury that was also réversible, with low mortality, less than 5%. After occlusion, 0.4 ml of warm saline (37°C) was placed in the abdominal 10 cavity and then the abdomen was closed for the period of ischemia. Following removal of the microaneurysm clamps, the kidneys were observed until color change, an indication of blood re-flow to the kidneys. A further 0.4 ml of warm saline was placed in the abdominal cavity and the opening was sutured, whereupon animais were returned to their cages. Tail blood samples were taken at 24 hours after removing the clamps, and at 15 48 hours the mice were sacrificed and an additional blood sample was collected.
Assessment of Rénal Injury: Rénal fonction was assessed at 24 and 48 hours after reperfusion in six male MASP-2(-/-) and six WT (+/+) mice. Blood créatinine measurement was determined by mass spectrometry, which provides a reproducible index of rénal fonction (sensitivity < LOgmol/L). FIGURE 12 graphically illustrâtes the 20 blood urea nitrogen clearance for wildtype C57B1/6 Controls and MASP-2 (-/-) at 24 hours and 48 hours after reperfosion. As shown in FIGURE 12, MASP-2(-/-) mice displayed a significant réduction in the amount of blood urea at 24 and 48 hours, in comparison to wildtype control mice, indicating a protective functional effect from rénal damage in the ischemia reperfusion injury model.
Overall, increased blood urea was seen in both the WT (+/+) and MASP-2 (-/-) mice at 24 and 48 hours following the surgical procedure and ischémie insult. Levels of blood urea in a non-ischemic WT (+/+) surgery animal was separately determined to be 5.8 mmol/L. In addition to the data presented in FIGURE 12, one MASP-2 (-/-) animal showed nearly complété protection from the ischémie insult, with values of 6.8 and 30 9.6 mmol/L at 24 and 48 hours, respectively. This animal was excluded from the group analysis as a potential outlier, wherein no ischémie injury may hâve been présent. Therefore, the final analysis shown in FIGURE 12 included 5 MASP-2(-/-) mice and
174
WT (+/+) mice and a statistically significant réduction in blood urea was seen at 24 and 48 hours in the MASP-2 (-/-) mice (Student t-test p<Q.O5). These findings indicate inhibition of MASP-2 activity would be expected to hâve a protective or therapeutic effect from rénal damage due to ischémie injury.
EXAMPLE 13
This Example describes the results of MASP-2-/- in a Murine Macular Degeneration Model.
Background/Rationale: Age-related macular degeneration (AMD) is the leading cause of blindness after âge 55 in the industrialized world. AMD occurs in two major forms: neovascular (wet) AMD and atrophie (dry) AMD. The neovascular (wet) form accounts for 90% of severe visual loss associated with AMD, even though only -20% of individuals with AMD develop the wet form. Clinical hallmarks of AMD include multiple drusen, géographie atrophy, and choroidal neovascularization (CNV). In December, 2004, the FDA approved Macugen (pegaptanib), a new class of ophthalmic drugs to specifically target and block the effects of vascular endothélial growth factor (VEGF), for treatment of the wet (neovascular) form of AMD (Ng et al., Nat Rev. Drug Discov 5:123-32 (2006)). Although Macugen represents a promising new therapeutic option for a subgroup of AMD patients, there remains a pressing need to develop additional treatments for this complex disease. Multiple, independent lines of investigation implicate a central rôle for complément activation in the pathogenesis of AMD. The pathogenesis of choroidal neovascularization (CNV), the most serious form of AMD, may involve activation of complément pathways.
Over twenty-five years ago, Ryan described a laser-induced injury model of CNV in animais (Ryan, S.J., Tr. Am. Opth. Soc. LXXVIP.101-145, 1979). The model was initially developed using rhésus monkeys, however, the same technology has since been used to develop similar models of CNV in a variety of research animais, including the mouse (Tobeetal., Am. J. Pathol. 753:1641-46, 1998). In this model, laser photocoagulation is used to break Bruch's membrane, an act which results in the formation of CNV-like membranes. The laser-induced model captures many of the important features of the human condition (for a recent review, see Ambati et al., Survey Ophthalmology 45:257-293, 2003). The laser-induced mouse model is now well
175 established, and is used as an experimental basis in a large, and ever increasing, number of research projects. It is generally accepted that the laser-induced model shares enough biological similarity with CNV in humans that preclinical studies of pathogenesis and drug inhibition using this model are relevant to CNV in humans.
Methods:
A MASP-2-/- mouse was generated as described in Example 1 and backcrossed for 10 générations with C57B1/6. The current study compared the results when MASP-2 (-/-) and MASP-2 (+/+) male mice were evaluated in the course of laser-induced CNV, an accelerated model of neovascular AMD focusing on the volume of laser-induced CNV by 10 scanning laser confocal microscopy as a measure of tissue injury and détermination of levels of VEGF, a potent angiogenic factor implicated in CNV, in the retinal pigment epithelium (RPE)/choroids by ELISA after laser injury.
Induction of choroidal neovascularization (CNV): Laser photocoagulation (532 nm, 200 mW, 100 ms, 75pm; Oculight GL, Iridex, Mountain View, CA) was 15 performed on both eyes of each animal on day zéro by a single individual masked to drug group assignment. Laser spots were applied in a standardized fashion around the optic nerve, using a slit lamp delivery system and a coverslip as a contact lens. The morphologie end point of the laser injury was the appearance of a cavitation bubble, a sign thought to correlate with the disruption of Bruch's membrane. The detailed methods 20 and endpoints that were evaluated are as follows.
Fluorescein Angiography: Fluorescein angiography was performed with a caméra and imaging system (TRC 50 IA caméra; ImageNet 2.01 system; Topcon, Paramus , NJ) at 1 week after laser photocoagulation. The photographs were captured with a 20-D lens in contact with the fundus caméra lens after intraperitoneal injection of 25 0.1 ml of 2.5% fluorescein sodium. A retina expert not involved in the laser photocoagulation or angiography evaluated the fluorescein angiograms at a single sitting in masked fashion.
Volume of choroidal neovascularization (CNV): One week after laser injury, eyes were enucleated and fixed with 4% paraformaldéhyde for 30 min at 4°C. Eye cups 30 were obtained by removing anterior segments and were washed three times in PBS, followed by déhydration and rehydration through a methanol sériés. After blocking twice with buffer (PBS containing 1% bovine serumalbumin and 0.5% Triton X-100) for
176 minutes at room température, eye cups were incubated overnight at 4°C with 0.5% FITC-isolectin B4 (Vector laboratories, Burlingame, CA), diluted with PBS containing 0.2% BSA and 0.1% Triton X-100, which binds terminal β-D-galactose residues on the surface of endothélial cells and selectively labels the murine vasculature. After two washings with PBS containing 0.1% Triton X-100, the neurosensory retina was gently detached and severed from the optic nerve. Four relaxing radial incisions were made, and the remaining RPE -choroid-sclera complex was flatmounted in antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories) and cover-slipped.
Flatmounts were examined with a scanning laser confocal microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were visualized by exciting with blue argon wavelength (488 nm) and capturing émission between 515 and 545 nm. A 40X oil-immersion objective was used for ail imaging studies. Horizontal optical sections (1 pm step) were obtained from the surface of the RPE-choroid-sclera complex. The deepest focal plane in which the surrounding choroidal vascular network connecting to the lésion could be identified was judged to be the floor of the lésion. Any vessel in the laser-targeted area and superficial to this reference plane was judged as CNV. Images of each section were digitally stored. The area of CNV-related fluorescence was measured by computerized image analysis with the microscope software (TCS SP; Leica). The summation of whole fluorescent area in each horizontal section was used as an index for the volume of CNV. Imaging was performed by an operator masked to treatment group assignment.
Because the probability of each laser lésion developing CNV is influenced by the group to which it belongs (mouse, eye, and laser spot), the mean lésion volumes were compared using a linear mixed model with a split plot repeated-measures design. The whole plot factor was the genetic group to which the animal belongs, whereas the split plot factor was the eye. Statistical significance was determined at the 0.05 level. Post hoc comparisons of means were constructed with a Bonferroni adjustment for multiple comparisons.
VEGF ELISA. At three days after injury by 12 laser spots, the RPE-choroid complex was sonicated in lysis buffer (20 mM imidazole HCl, 10 mM KC1, 1 mM MgCL2, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM EDTA with protease inhibitor) on ice for 15 min. VEGF protein levels in the supematant
177 were determined by an ELISA kit (R&D Systems, Minneapolis, MN) that recognizes ali splice variants, at 450 to 570 nm (Emax; Molecular Devices, Sunnyvale, CA), and normalized to total protein. Duplicate measurements were performed in a masked fashion by an operator not involved in photocoagulation, imaging, or angiography. VEGF numbers were represented as the mean +/- SEM of at least three independent experiments and compared using the Mann-Whitney U test. The null hypothesis was rejected at P<0.05.
RESULTS:
Assessment of VEGF Levels:
FIGURE 13A graphically illustrâtes the VEGF protein levels in RPE-choroid complex isolated from C57B16 wildtype and MASP-2(-/-) mice at day zéro. As shown in FIGURE 13A, the assessment of VEGF levels indicate a decrease in baseline levels for VEGF in the MASP-2 (-/-) mice versus the C57bl wildtype control mice. FIGURE 13B graphically illustrâtes VEGF protein levels measured at day three following laser induced injury. As shown in FIGURE 13B VEGF levels were significantly increased in the wildtype (+/+) mice three days following laser induced injury, consistent with published studies (Nozaki et al., Proc. Natl. Acad. Sci. USA 103:2328-33 (2006)). However, surprisingly very low levels of VEGF were seen in the MASP-2 (-/-) mice.
Assessment of choroidal neovascularization (CNV):
In addition to the réduction in VEGF levels following laser induced macular degeneration, CNV area was determined before and after laser injury. FIGURE 14 graphically illustrâtes the CNV volume measured in C57bl wildtype mice and MASP-2(-/-) mice at day seven following laser induced injury. As shown in FIGURE 14, the MASP-2 (-/-) mice displayed about a 30% réduction in the CNV area following laser induced damage at day seven in comparison to the wildtype control mice.
These findings indicate a réduction in VEGF and CNV as seen in the MASP (-/-) mice versus the wildtype (+/+) control and that blockade of MASP-2 with an inhibitor would hâve a préventive or therapeutic effect in the treatment of macular degeneration.
EXAMPLE 14
This Example demonstrates that thrombin activation can occur following lectin pathway activation under physiological conditions, and demonstrates the extent of
178
MASP-2 involvement. In normal rat sérum, activation of the lectin pathway leads to thrombin activation (assessed as thrombin déposition) concurrent with complément activation (assessed as C4 déposition). As can be seen in FIGURES 15A and 15B, thrombin activation in this System is inhibited by a MASP-2 blocking antibody (Fab2 5 format), exhibiting an inhibition concentration-response curve (FIGURE 15B) that parallels that for complément activation (FIGURE 15A). These data suggest that activation of the lectin pathway as it occurs in trauma will lead to activation of both complément and coagulation Systems in a process that is entirely dépendent on MASP-2. By inference, MASP2 blocking antibodies may prove efficacious in mitigating cases of 10 excessive systemic coagulation, e.g., disseminated intravascular coagulation, which is one of the hallmarks leading to mortality in major trauma cases.
EXAMPLE 15
This Example provides results generated using a localized Schwartzman reaction 15 model of disseminated intravascular coagulation (DIC) in MASP-2 -/- déficient and
MASP-2 +/+ sufficient mice to evaluate the rôle of lectin pathway in DIC.
Background/Rationale:
As described supra, blockade of MASP-2 inhibits lectin pathway activation and reduces the génération of both anaphylatoxins C3a and C5a. C3a anaphylatoxins can be 20 shown to be potent platelet aggregators in vitro, but their involvement in vivo is less well defined and the release of platelet substances and plasmin in wound repair may only secondarily involve complément C3. In this Example, the rôle of the lectin pathway was analyzed in MASP-2 (-/-) and WT (+/+) mice in order to address whether prolonged élévation of C3 activation is necessary to generate disseminated intravascular 25 coagulation.
Methods:
The MASP-2 (-/-) mice used in this study were generated as described in Example 1 and backcrossed for at least 10 générations with C57B1/6.
The localized Schwartzman reaction model was used in this experiment. The 30 localized Schwartzman reaction (LSR) is a lipopolysaccharide (LPS) -induced response with well-characterized contributions from cellular and humoral éléments of the innate immune System. Dépendent ofthe LSR on complément is well established (Polak, L., et
179 al., Nature 223:738-739 (1969); Fong J.S. et al., J Exp Med 134:642-655 (1971)). In the LSR model, the mice were primed for 4 hours with TNF alpha (500 ng, intrascrotal), then the mice were anaesthetized and prepared for intravital microscopy of the cremaster muscle. Networks of post-capillary venules (15-60 gm diameter) with good blood flow (1-4 mm/s) were selected for observation. Animais were treated with fluorescent antibodies to selectively label neutrophils, or platelets. The network of vessels was sequentially scanned and images of ail vessels were digitally recorded of later analysis. After recording the basal State of the microcirculation, mice received a single intravenous injection of LPS (100 gg), either alone or with the agents Iisted below. The same network of vessels was then scanned every 10 minutes for 1 hour. Spécifie accumulation of fluorophores was identified by subtraction of background fluorescence and enhanced by thresholding the image. The magnitude of reactions was measured from recorded images. The primary measure of Schwartzman reactions was aggregate data.
The studies compared the MASP-2 +/+ sufficient, or wild type, mice exposed to either a known complément pathway depletory agent, cobra venom factor (CVF), or a terminal pathway inhibitor (C5aR antagonist). The results (FIGURE 16A) demonstrate that CVF as well as a C5aR antagonist both prevented the appearance of aggregates in the vasculature. In addition, the MASP-2 -/- déficient mice (FIGURE 16B) also demonstrated complété inhibition of the localized Schwartzman reaction, supporting lectin pathway involvement. These results clearly demonstrate the rôle of MASP-2 in DIC génération and support the use of MASP-2 inhibitors for the treatment and prévention of DIC.
EXAMPLE 16
This Example describes the analysis of MASP-2 (-/-) mice in a Murine Rénal Transplantation Model.
Background/Rationale:
The rôle of MASP-2 in the functional outcome of kidney transplantation was assessed using a mouse model.
Methods:
The functional outcome of kidney transplantation was assessed using a single kidney isograft into uninephrecomized récipient mice, with six WT (+/+) transplant
180 récipients (B6), and six MASP-2 (-/-) transplant récipients. To assess the fonction of the transplanted kidney, the remaining native kidney was removed from the récipient 5 days after transplantation, and rénal fonction was assessed 24 hours later by measurement of blood urea nitrogen (BUN) levels.
Results:
FIGURE 17 graphically illustrâtes the blood urea nitrogen (BUN) levels of the kidney at 6 days post kidney transplant in the WT (+/+) récipients and the MASP-2 (-/-) récipients. As shown in FIGURE 17, strongly elevated BUN levels were observed in the WT (+/+) (B6) transplant récipients (normal BUN levels in mice are < 5 mM), indicating rénal failure. In contrast, MASP-2 (-/-) isograft récipient mice showed substantially lower BUN levels, suggesting improved rénal fonction. It is noted that these results were obtained using grafts from WT (+/+) kidney donors, suggesting that the absence of a functional lectin pathway in the transplant récipient alone is sufficient to achieve a therapeutic benefit.
Taken together, these results indicate that transient inhibition of the lectin pathway via MASP-2 inhibition provides a method of reducing morbidity and delayed graft fonction in rénal transplantation, and that this approach is likely to be useful in other transplant settings.
EXAMPLE 17
This Example demonstrates that MASP-2 (-/-) mice are résistant to septic shock in a Murine Polymicrobial Septic Peritonitis Model.
Backgrou nd/Rationale :
To evaluate the potential effects of MASP-2 (-/-) in infection, the cecal ligation and puncture (CLP) model, a model of polymicrobial septic peritonitis was evaluated. This model is thought to most accurately mimic the course of human septic peritonitis. The cecal ligation and puncture (CLP) model is a model in which the cecum is ligated and punctured by a needle, leading to continuous leakage of the bacteria into the abdominal cavity which reach the blood through the lymph drainage and are then distributed into ail the abdominal organs, leading to multi-organ failure and septic shock (Eskandari et al., J Immunol /45(9):2724-2730 (1992)). The CLP model mimics the course of sepsis observed in patients and induces an early hyper-inflammatory response
181 followed by a pronounced hypo-inflammatory phase. During this phase, the animais are highly sensitive to bacterial challenges (Wichterman et al., J. Surg. Res. 29(2):189-201 (1980)).
Methods:
The mortality of polymicrobial infection using the cecal ligation and puncture (CLP) model was measured in WT (+/+) (n-18) and MASP-2 (-/-) (n= l 6) mice. Briefly described, MASP-2 déficient mice and their wild-type littermates were anaesthetized and the cecum was exteriorized and ligated 30% above the distal end. After that, the cecum was punctured once with a needle of 0.4 mm diameter. The cecum was then replaced into 10 the abdominal cavity and the skin was closed with clamps. The survival of the mice subjected to CLP was monitored over a period of 14 days after CLP. A peritoneal lavage was collected in mice 16 hours post CLP to measure bacterial load. Serial dilutions of the peritoneal lavage were prepared in PBS and inoculated in Mueller Hinton plates with subséquent incubation at 37°C under anaérobie conditions for 24 hours after which 15 bacterial load was determined.
The TNF-alpha cytokine response to the bacterial infection was also measured in the WT (+/+) and MASP-2 (-/-) mice 16 hours after CLP in lungs and spleens via quantitative real time polymerase chain reaction (qRT-PCR). The sérum level of TNFalpha 16 hours after CLP in the WT (+/+) and MASP-2 (-/-) mice was also quantified by 20 sandwich ELISA.
Results:
FIGURE 18 graphically illustrâtes the percentage survival of the CLP treated animais as a function of the days after the CLP procedure. As shown in FIGURE 18, the lectin pathway deficiency in the MASP-2 (-/-) mice does not increase the mortality of 25 mice after polymicrobial infection using the cecal ligation and puncture model as compared to WT (+/+) mice. However, as shown in FIGURE 19, MASP-2 (-/-) mice showed a significantly higher bacterial load (approximately a 1000-fold increase in bacterial numbers) in peritoneal lavage after CLP when compared to their WT (+/+) littermates. These results indicate that MASP-2 (-/-) déficient mice are résistant to septic 30 shock. The reduced bacterial clearance in MASP-2 déficient mice in this model may be due to an impaired C3b mediated phagocytosis, as it was demonstrated that C3 déposition is MASP-2 dépendent.
182
It was determined that the TNF-alpha cytokine response to the bacterial infection was not elevated in the MASP-2 (-/-) mice as compared to the WT (+/+) Controls (data not shown). It was also determined that there was a significantly higher sérum concentration of TNF-alpha in WT (+/+) mice 16 hours after CLP in contrast to MASP-2 5 (-/-) mice, where the sérum level of TNF-alpha remained nearly unaltered. These results suggest that the intense inflammatory response to the septic condition was tempered in MASP-2 (-/-) mice and allowed the animais to survive in the presence of higher bacterial counts.
Taken together, these results demonstrate the potential deleterious effects of lectin 10 pathway complément activation in the case of septicemia and the increased mortality in patients with overwhelming sepsis. These results further demonstrate that MASP-2 deficiency modulâtes the inflammatory immune response and reduces the expression levels of inflammatory mediators during sepsis. Therefore, it is believed that inhibition of MASP-2 (-/-) by administration of inhibitory monoclonal antibodies against MASP-2 15 would be effective to reduce the inflammatory response in a subject suffering from septic shock.
EXAMPLE 18
This Example describes analysis of MASP-2 (-/-) mice in a Murine Intranasal 20 Infectivity Model.
Background/Rationale:
Pseudomonas aeruginosa is a Gram négative opportunistic human bacterial pathogen that causes a wide range of infections, particularly in immune-compromised individuals. It is a major source of acquired nosocomial infections, in particular hospital25 acquired pneumonia. It is also responsible for significant morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa pulmonary infection is characterized by strong neutrophil recruitment and significant lung inflammation resulting in extensive tissue damage (Palanki M.S. et al., J. Med. Chem 57:1546-1559 (2008)).
In this Example, a study was undertaken to détermine whether the removal of the 30 lectin pathway in MASP-2 (-/-) mice increases the susceptibility of the mice to bacterial infections.
183
Methods:
Twenty-two WT (+/+) mice, twenty-two MASP-2 (-/-) mice, and eleven C3 (-/-) mice were challenged with intranasal administration of P. aeruginosa bacterial strain. The mice were monitored over the six days post-infection and Kaplan-Mayer plots were constructed showing percent survival.
Results:
FIGURE 20 is a Kaplan-Mayer plot of the percent survival of WT (+/+), MASP2 (-/-) or C3 (-/-) mice six days post-infection. As shown in FIGURE 20, no différences were observed in the MASP-2 (-/-) mice versus the WT (+/+) mice. However, removal of the classical (Clq) pathway in the C3 (-/-) mice resulted in a severe susceptibility to bacterial infection. These results demonstrate that MASP-2 inhibition does not increase susceptibility to bacterial infection, indicating that it is possible to reduce undesirable inflammatory complications in trauma patients by inhibiting MASP-2 without compromising the patient's ability to fight infections using the classical complément pathway.
EXAMPLE 19
This Example describes the pharmacodynamie analysis of représentative high affinity anti-MASP-2 Fab2 antibodies that were identified as described in Example 10.
Background/Rationale:
As described in Example 10, in order to identify high-affinity antibodies that block the rat lectin pathway, rat MASP-2 protein was utilized to pan a phage display library. This library was designed to provide for high immunological diversity and was constructed using entirely human immunoglobin gene sequences. As described in Example 10, approximately 250 individual phage clones were identified that bound with high affinity to the rat MASP-2 protein by ELISA screening. Sequencing of these clones identified 50 unique MASP-2 antibody encoding phage. Fab2 protein was expressed from these clones, purified and analyzed for MASP-2 binding affinity and lectin complément pathway functional inhibition.
As shown in TABLE 6 of Example 10, 17 anti-MASP-2 Fab2s with functional blocking activity were identified as a resuit of this analysis (a 34% hit rate for blocking antibodies). Functional inhibition of the lectin complément pathway by Fab2s was
184 apparent at the level of C4 déposition, which is a direct measure of C4 cleavage by MASP-2. Importantly, inhibition was equally évident when C3 convertase activity was assessed, demonstrating functional blockade of the lectin complément pathway. The 17 MASP-2 blocking Fab2s identified as described in Example 10 potently inhibit C3 5 convertase formation with IC5Q values equal to or less than 10 nM. Eight of the 17 Fab2s identified hâve IC50 values in the sub-nanomolar range. Furthermore, ail 17 of the MASP-2 blocking Fab2s gave essentially complété inhibition of the C3 convertase formation in the lectin pathway C3 convertase assay, as shown in FIGURES 8A-C, and summarized in TABLE 6 of Example 10. Moreover, each of the 17 blocking anti-MASP10 2 Fab2s shown in TABLE 6 potently inhibit C3b génération (>95%), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.
Rat IgG2c and mouse IgG2a full-length antibody isotype variants were derived from Fab2 #11. This Example describes the in vivo characterization of these isotypes for pharmacodynamie parameters.
Methods:
As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2#ll was identified. Rat IgG2c and mouse IgG2a fulllength antibody isotype variants were derived from Fab2 #11. Both rat IgG2c and mouse IgG2a full length antibody isotypes were characterized in vivo for pharmacodynamie 20 parameters as follows.
In vivo study in mice:
A pharmacodynamie study was carried out in mice to investigate the effect of anti-MASP-2 antibody dosing on the plasma lectin pathway activity in vivo. In this study, C4 déposition was measured ex vivo in a lectin pathway assay at various time 25 points following subeutaneous (sc) and intraperitoneal (ip) administration of 0.3 mg/kg or LO mg/kg of the mouse anti-MASP-2 MoAb (mouse IgG2a full-length antibody isotype derived from Fab2#ll).
FIGURE 21 graphically illustrâtes lectin pathway spécifie C4b déposition, measured ex vivo in undiluted sérum samples taken from mice (n=3 mice/group) at 30 various time points after subeutaneous dosing of either 0.3 mg/kg or LO mg/kg of the mouse anti-MASP-2 MoAb. Sérum samples from mice collected prior to antibody
185 dosing served as négative Controls (100% activity), while sérum supplemented in vitro with 100 nM of the same blocking anti-MASP-2 antibody was used as a positive control (0% activity).
The results shown in FIGURE 21 demonstrate a rapid and complété inhibition of 5 C4b déposition following subcutaneous administration of 1.0 mg/kg dose of mouse antiMASP-2 MoAb. A partial inhibition of C4b déposition was seen following subcutaneous administration of 0.3 mg/kg dose of mouse anti-MASP-2 MoAb.
The time course of lectin pathway recovery was followed for three weeks following a single ip administration of mouse anti-MASP-2 MoAb at 0.6 mg/kg in mice.
As shown in FIGURE 22, a precipitous drop in lectin pathway activity occurred post antibody dosing followed by complété lectin pathway inhibition that lasted for about 7 days after ip administration. Slow restoration of lectin
These results demonstrate that the mouse anti-MASP-2 Moab derived from Fab2 #11 inhibits the lectin pathway of mice in a dose-responsive manner when delivered 15 systemically.
EXAMPLE 20
This Example describes analysis of the mouse anti-MASP-2 Moab derived from Fab2 #11 for efficacy in a mouse model for age-related macular degeneration.
Background/Rationale:
As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2#ll was identified as a functionally active antibody. Full length antibodies of the rat IgG2c and mouse IgG2a isotypes were generated from Fab2 #11. The full length anti-MASP-2 antibody of the mouse IgG2a isotype was 25 characterized for pharmacodynamie parameters as described in Example 19. In this Example, the mouse anti-MASP-2 full-length antibody derived from Fab2 #11 was analyzed in the mouse model of age-related macular degeneration (AMD), described by Bora P.S. et al, JImmunol 774:491-497 (2005).
Methods:
The mouse IgG2a full-length anti-MASP-2 antibody isotype derived from Fab2 #11 as described in Example 19, was tested in the mouse model of age-related macular degeneration (AMD) as described in Example 13 with the following modifications.
186
Administration of mouse-anti-MASP-2 MoAbs
Two different doses (0.3 mg/kg and 1.0 mg/kg) of mouse anti-MASP-2 MoAb along with an isotype control MoAb treatment were injected ip into WT (+/+) mice (n= 8 mice per group) 16 hours prior to CNV induction
Induction of choroidal neovascularization (CNV)
The induction of choroidal neovascularization (CNV) and measurement of the volume of CNV was carried out using laser photocoagulation as described in Example 13.
Results:
FIGURE 23 graphically illustrâtes the CNV area measured at 7 days post laser injury in mice treated with either isotype control MoAb, or mouse anti-MASP-2 MoAb (0.3 mg/kg and 1.0 mg/kg). As shown in FIGURE 23, in the mice pre-treated with 1.0 mg/kg anti-MASP-2 MoAb, a statistically significant (p <0.01) approximately 50% réduction in CNV was observed seven days post-laser treatment. As further shown in FIGURE 23, it was observed that a 0.3 mg/kg dose of anti-MASP-2 MoAb was not efficacious in reducing CNV. It is noted that the 0.3 mg/kg dose of anti-MASP-2 MoAb was shown to hâve a partial and transient inhibition of C4b déposition following subcutaneous administration, as described in Example 19 and shown in FIGURE 21.
The results described in this Example demonstrate that blockade of MASP-2 with an inhibitor, such as anti-MASP-2 MoAb, has a preventative and/or therapeutic effect in the treatment of macular degeneration. It is noted that these results are consistent with the results observed in the study carried out in the MASP-2 (-/-) mice, described in Example 13, in which a 30% réduction in the CNV 7 days post-laser treatment was observed in MASP-2 (-/-) mice in comparison to the wild-type control mice. Moreover, the results in this Example further demonstrate that systemically delivered anti-MASP-2 antibody provides local therapeutic benefit in the eye, thereby highlighting the potential for a systemic route of administration to treat AMD patients. In summary, these results provide evidence supporting the use of MASP-2 MoAb in the treatment of AMD.
EX AMPLE 21
This Example demonstrates that MASP-2 déficient mice are protected from Neisseria meningitidis induced mortality after infection with N. meningitidis and hâve enhanced clearance of bacteraemia as compared to wild type control mice.
187
Rationale: Neisseria meningitidis is a heterotrophic gram-negative diplococcal bacterium known for its rôle in meningitis and other forms of meningococcal disease such as meningococcemia. N. meningitidis is a major cause of morbidity and mortality during childhood. Severe complications include septicaemia, Waterhouse-Friderichsen 5 syndrome, adrenal insufficiency and disseminated intravascular coagulation (DIC). See e.g., Rintala E. et al., Critical Care Medicine 25(7):2373-2378 (2000). In this Example, the rôle of the lectin pathway was analyzed in MASP-2 (-/-) and WT (+/+) mice in order to address whether MASP-2 déficient mice would be susceptible to N. meningitidis induced mortality.
Methods:
MASP-2 knockout mice were generated as described in Example 1 and backcrossed for at least 10 générations with C57B1/6. 10 week old MASP-2 KO mice (n=10) and wild type C57/B6 mice (n=10) were innoculated by intravenous injection with either a dosage of 5x108 cfu/100 μΐ, 2x108 cfu/100 μΐ or 3x107 cfu/100 μΐ of Neisseria 15 meningitidis Serogroup A Z2491 in 400 mg/kg iron dextran. Survival of the mice after infection was monitored over a 72 hour time period. Blood samples were taken from the mice at hourly intervals after infection and analyzed to détermine the sérum level (log cfii/ml) of N meningitidis in order to verify infection and détermine the rate of clearance of the bacteria from the sérum.
Results:
FIGURE 24A graphically illustrâtes the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 5xl08/100 μΐ cfti N. meningitidis. As shown in FIGURE 24A, after infection with the highest dose of 5xl08/100 μΐ cfti N. meningitidis, 100% of the MASP-2 KO mice survived throughout the 72 hour period after 25 infection. In contrast, only 20% of the WT mice were still alive 24 hours after infection. These results demonstrate that MASP-2 déficient mice are protected from N. meningitidis induced mortality.
FIGURE 24B graphically illustrâtes the log cfii/ml of N. meningitidis recovered at different time points in blood samples taken from the MASP-2 KO and WT mice 30 infected with 5x108 cfu/100 μΐ N. meningitidis. As shown in FIGURE 24B, in WT mice the level of N. meningitidis in the blood reached a peak of about 6.5 log cfu/ml at 24
188 hours after infection and dropped to zéro by 48 hours after infection. In contrast, in the MASP-2 KO mice, the level of N. meningitidis reached a peak of about 3.5 log cfii/ml at 6 hours after infection and dropped to zéro by 36 hours after infection.
FIGURE 25A graphically illustrâtes the percent survival of MASP-2 KO and WT 5 mice after infection with 2xl08 cfu/100 μΐ N. meningitidis. As shown in FIGURE 25A, after infection with the dose of 2x108 cfu/100 μΐ N. meningitidis, 100% of the MASP-2 KO mice survived throughout the 72 hour period after infection. In contrast, only 80% of the WT mice were still alive 24 hours after infection. Consistent with the results shown in FIGURE 24A, these results further demonstrate that MASP-2 déficient mice are protected from N. meningitidis induced mortality.
FIGURE 25B graphically illustrâtes the log cfii/ml of N. meningitidis recovered at different time points in blood samples taken from the WT mice infected with 2x108 cfu/100 μΐ N. meningitidis. As shown in FIGURE 25B, the level of N. meningitidis in the blood of WT mice infected with 2xl08 cfu reached a peak of about 4 log cfti/ml at 12 hours after infection and dropped to zéro by 24 hours after infection. FIGURE 25C graphically illustrâtes the log cfu/ml of N. meningitidis recovered at different time points in blood samples taken from the MASP-2 KO mice infected with 2x108 cfti/100 μΐ N. meningitidis. As shown in FIGURE 25C, the level of N. meningitidis in the blood of MASP-2 KO mice infected with 2x108 cfu reached a peak level of about 3.5 log cfu/ml at
2 hours after infection and dropped to zéro at 3 hours after infection. Consistent with the results shown in FIGURE 24B, these results demonstrate that although the MASP-2 KO mice were infected with the same dose of N. meningitidis as the WT mice, the MASP-2 KO mice hâve enhanced clearance of bacteraemia as compared to WT.
The percent survival of MASP-2 KO and WT mice after infection with the lowest 25 dose of 3xl07 cfu/100 μΐ N. meningitidis was 100% at the 72 hour time period (data not shown).
Discussion
These results show that MASP-2 déficient mice are protected from N. meningitidis induced mortality and hâve enhanced clearance of bacteraemia as compared 30 to the WT mice. Therefore, in view of these results, it is expected that therapeutic application of MASP-2 inhibitors, such as MASP-2 MoAb, would be expected to be
189 efficacious to treat, prevent or mitigate the effects of infection with N. meningitidis bacteria (i.e., sepsis and DIC). Further, these results indicate that therapeutic application of MASP-2 inhibitors, such as MASP-2 MoAb would not prédisposé a subject to an increased risk to contract N. meningitidis infections.
EXAMPLE 22
This Example describes the discovery of novel lectin pathway mediated and MASP-2 dépendent C4-bypass activation of complément C3.
Rationale:
The principal therapeutic benefît of utilizing inhibitors of complément activation to limit myocardial ischemia/reperfusion injury (MIRI) was convincingly demonstrated in an experimental rat model of myocardial infarction two décades ago: Recombinant sCRl, a soluble truncated dérivative of the cell surface complément receptor type-1 (CRI), was given intravenously and its effect assessed in a rat in vivo model of MIRI.
Treatment with sCRl reduced infarct volume by more than 40% (Weisman, H.F., et aL, Science 249:146-151 (1990)). The therapeutic potential of this recombinant inhibitor was subsequently demonstrated in a clinical trial showing that the administration of sCRl in patients with MI prevented contractile failure in the post-ischemic heart (Shandelya, S., et aL, Circulation 87:536-546 (1993)). The primary mechanism leading to the activation of 20 complément in ischémie tissue, however, has not been ultimately defined, mainly due to the lack of appropriate experimental models, the limited understanding of the molecular processes that lead to complément activation of oxygen-deprived cells, and the cross-talk and synergisms between the different complément activation pathways.
As a fundamental component of the immune response, the complément System 25 provides protection against invading microorganisms through both antibody-dependent and -independent mechanisms. It orchestrâtes many cellular and humoral interactions within the immune response, including chemotaxis, phagocytosis, cell adhesion, and Bcell différentiation. Three different pathways initiate the complément cascade: the classical pathway, the alternative pathway, and the lectin pathway. The classical pathway 30 récognition subcomponent Clq binds to a variety of targets - most prominently immune complexes - to initiate the step-wise activation of associated serine proteases, Clr and Cl s, providing a major mechanism for pathogen and immune complex clearance
190 foilowing engagement by the adaptive immune system. Binding of Clq to immune complexes converts the Clr zymogen dimer into its active form to cleave and thereby activate Cl s. Cls translates Clq binding into complément activation in two cleavage steps: It first converts C4 into C4a and C4b and then cleaves C4b-bound C2 to form the 5 C3 convertase C4b2a. This complex converts the abundant plasma component C3 into
C3a and C3b. Accumulation of C3b in close proximity of the C4b2a complex shifts the substrate specificity for C3 to C5 to form the C5 convertase C4b2a(C3b)n. The C3 and C5 convertase complexes generated via classical pathway activation are identical to those generated through the lectin pathway activation route. In the alternative pathway, 10 spontaneous low-level hydrolysis of component C3 results in déposition of protein fragments onto cell surfaces, triggering complément activation on foreign cells, while cell-associated regulatory proteins on host tissues avert activation, thus preventing selfdamage. Like the alternative pathway, the lectin pathway may be activated in the absence of immune complexes. Activation is initiated by the binding of a multi-molecular lectin 15 pathway activation complex to Pathogen-Associated Molecular Patterns (PAMPs), mainly carbohydrate structures présent on bacterial, fungal or viral pathogens or aberrant glycosylation patterns on apoptotic, necrotic, malignant or oxygen-deprived cells (Collard, C.D., et al., Am. J. Pathol. 756:1549-1556 (2000); Walport, M.J., N. Engl. J. Med. 344:1058-1066 (2001); Schwaeble, W., et al., Immunobiology 205:455-466 (2002);
and Fujita, T., Nat. Rev. Immunol. 2:346-353 (2002)).
Mannan-binding lectin (MBL) was the First carbohydrate récognition subcomponent shown to form complexes with a group of novel serine proteases, named MBL-associated Serine Proteases (MASPs) and numbered according to the sequence of their discovery (i.e., MASP-1, MASP-2 and MASP-3). In man, lectin pathway activation 25 complexes can be formed with four alternative carbohydrate récognition subcomponents with different carbohydrate binding specificities, i.e., MBL 2, and three different members of the ficolin family, namely L-Ficolin, H-ficolin and M-ficolin and MASPs. Two forms of MBL, MBL A and MBL C, and ficolin-A form lectin activation pathway complexes with MASPs in mouse and rat plasma. We hâve previously cloned and 30 characterised MASP-2 and an additional truncated MASP-2 gene product of 19 kDa, termed MApl9 or sMAP, in human, mouse and rat (Thiel, S., et al., Nature 356:506-510 (1997);. Stover, C.M., et al., J. Immunol. 762:3481-3490 (1999); Takahashi, M., et al.,
191
Int. Immunol. /7:859-863 (1999); and Stover, C.M., et al., J. Immunol. 763:6848-6859 (1999)). MApl9/ sMAP is devoid of protease activity, but may regulate lectin pathway activation by competing for the binding of MASPs to carbohydrate récognition complexes (Iwaki, D. et al., J. Immunol. 777:8626-8632 (2006)).
There is evidence suggesting that of the three MASPs, only MASP-2 is required to translate binding of the lectin pathway récognition complexes into complément activation (Thiel, S., et al. (1997); Vorup-Jensen, T., et al., J. Immunol. 765:2093-2100 (2000); Thiel, S., et al., J. Immunol. 165\^>Ί2>-^Ί (2000); Rossi, V., et al., J. Biol. Chem. 276AQ^A^I (2001)). This conclusion is underlined by the phenotype of a most recently described mouse strain déficient in MASP-1 and MASP-3. Apart from a delay in the onset of lectin pathway mediated complément activation in vitro -MASP-1/3 déficient mice retain lectin pathway functional activity. Reconstitution of MASP-1 and MASP-3 déficient sérum with recombinant MASP-1 overcomes this delay in lectin pathway activation implying that MASP-1 may facilitate MASP-2 activation (Takahashi, M., et al., J. Immunol. 750:6132-6138 (2008)). A most recent study has shown that MASP-1 (and probably also MASP-3) are required to convert the alternative pathway activation enzyme Factor D from its zymogen form into its enzymatically active form (Takahashi, M., et al., J. Exp. Med. 207-.29-3Ί (2010)). The physiological importance of this process is underlined by the absence of alternative pathway functional activity in plasma of MASP-1/3 déficient mice.
The recently generated mouse strains with combined targeted deficiencies of the lectin pathway carbohydrate récognition subcomponents MBL A and MBL C may still initiate lectin pathway activation via the remaining murine lectin pathway récognition subcomponent ficolin A (Takahashi, K., et al., Microbes Infect. 4:773-784 (2002)). The absence of any residual lectin pathway functional activity in MASP-2 déficient mice delivers a conclusive model to study the rôle of this effector arm of innate humoral immunity in health and disease.
The availability of C4 and MASP-2 déficient mouse strains allowed us to define a novel lectin pathway spécifie, but MASP-2 dépendent, C4-bypass activation route of complément C3. The essential contribution of this novel lectin pathway mediated C4bypass activation route towards post-ischemic tissue loss is underlined by the prominent
192 protective phenotype of MASP-2 deficiency in MIRI while C4-deficient mice tested in the same model show no protection.
In this Example, we describe a novel lectin pathway mediated and MASP-2 dépendent C4-bypass activation of complément C3. The physiological relevance of this new activation route is established by the protective phenotype of MASP-2 deficiency in an experimental model of myocardial ischemia/reperfusion injury (MIRI), where C4 déficient animais were not protected.
Methods:
MASP-2 déficient mice show no gross abnormalities. MASP-2 déficient mice were generated as described in Example 1. Both heterozygous (+/‘) and homozygous (*A) MASP-2 déficient mice are healthy and fertile, and show no gross abnormalities. Their life expectancy is similar to that of their WT littermates (>18 months). Prior to studying the phenotype of these mice in experimental models of disease, our MASP-2a line was backcrossed for eleven générations onto a C57BL/6 background. The total absence of MASP-2 mRNA was confirmed by Northern blotting of poly A+ selected liver RNA préparations, while the 1.2kb mRNA encoding MApl9 or sMAP (a truncated alternative splicing product of the MASP2 gene) is abundantly expressed.
qRT-PCR analysis using primer pairs spécifie for either the coding sequence for the serine protease domain of MASP-2 (B chain) or the remainder of the coding sequence for the A-chain showed that no B chain encoding mRNA is détectable in MASP-2 ’Λ mice while the abundance of the disrupted A chain mRNA transcript was significantly increased. Likewise, the abundance of MApl9/sMAP encoding mRNA is increased in MASP-2 +/‘ and MASP-2 -/ mice. Plasma MASP-2 levels, determined by ELISA for 5 animais of each génotype, were 300ng/ml for WT Controls (range 260-330ng/ml), 360ng/ml for heterozygous mice (range 330-395ng/ml) and undetectable inMASP-2 mice. Using qRT-PCR, mRNA expression profiles were established demonstrating that MASP^Tnice express mRNA for MBL A, MBL C, ficolin A, MASP-1, MASP-3, Clq, ClrA, ClsA, Factor B, Factor D, C4, and C3 at an abundance similar to that of their MASP-2 sufficient littermates (data not shown).
Plasma C3 levels of MASP-2'/' (n=8) and MASP-2+/+ (n=7) littermates were measured using a commercially available mouse C3 ELISA kit (Kamiya, Biomédical,
193
Seattle, WA). C3 levels of MASP-2 déficient mice (average 0.84 mg/ml, +/- 0.34) were similar to those of the WT Controls (average 0.92, +/- 0.37).
Results:
MASP-2 is essential for lectin pathway functional activity.
As described in Example 2 and shown in FIGURE 5, the in vitro analyses of MASP-27'plasma showed a total absence of lectin pathway functional activity on activating Mannan- and Zymosan-coated surfaces for the activation of C4. Likewise, neither lectin pathway-dependent C4 nor C3 cleavage was détectable in MASP-2“ plasma on surfaces coated with N-acetyl glucosamine, which binds and triggers activation via MBL A, MBL C and ficolin A (data not shown).
The analyses of sera and plasma of MASP-2-/-mice clearly demonstrated that MASP-2 is essentially required to activate complément via the lectin pathway. The total deficiency of lectin pathway functional activity, however, leaves the other complément activation pathways intact: MASP-2-/-plasma can still activate complément via the classical (FIGURE 26A) and the alternative pathway (FIGURE 26B). In FIGURE 26A and 26B, the symbol * symbol indicates sérum from WT (MASP-2 (+/+)); the symbol · indicates sérum from WT (Clq depleted); the symbol indicates sérum from MASP-2 (-/-); and the symbol Δ indicates sérum from MASP-2 (-/-) (Clq depleted).
FIGURE 26A graphically illustrâtes that MASP-2-/- mice retain a functional classical pathway: C3b déposition was assayed on microtiter plates coated with immune complexes (generated in situ by coating with BSA then adding goat anti-BSA IgG). FIGURE 26B graphically illustrâtes MASP-2 déficient mice retain a functional alternative pathway: C3b déposition was assayed on Zymosan coated microtiter plates under conditions that permit only alternative pathway activation (buffer containing Mg2+ and EGTA). Results shown in FIGURE 26A and FIGURE 26B are means of duplicates and are typical of three independent experiments. Same symbols for plasma sources were used throughout. These results show that a functional alternative pathway is présent in MASP-2 déficient mice, as evidenced in the results shown in FIGURE 26B under experimental conditions designed to directly trigger the alternative pathway, while inactivating both the classical pathway and lectin pathway.
194
The lectin pathway of complément activation critically contributes to inflammatory tissue loss in myocardial ischemia/reperfusion injury (MIRI).
In order to study the contribution of lectin pathway functional activity to MIRI, we compared MASP-2'/'mice and WT littermate Controls in a model of MIRI following 5 transient ligation and reperfusion of the left anterior descending branch of the coronary artery (LAD). The presence or absence of complément C4 has no impact on the degree of ischémie tissue loss in MIRI. We assessed the impact of C4 deficiency on infarct sizes following experimental MIRI. As shown in FIGURE 27A and FIGURE 27B, identical infarct sizes were observed in both C4-deficient mice and their WT littermates. FIGURE
27A graphically illustrâtes MIRI-induced tissue loss following LAD ligation and reperfusion in C4-/- mice (n=6) and matching WT littermate Controls (n=7). FIGURE 27B graphically illustrâtes INF as a function of AAR, clearly demonstrating that C4-/mice are as susceptible to MIRI as their WT Controls (dashed line).
These results demonstrate that C4 déficient mice are not protected from MIRI.
This resuit was unexpected, as it is in conflict with the widely accepted view that the major C4 activation fragment, C4b, is an essential component of the classical and the lectin pathway C3 convertase C4b2a. We therefore assessed whether a residual lectin pathway spécifie activation of complément C3 can be detected in C4-deficient mouse and human plasma.
The lectin pathway can activate complément C3 in absence of C4 via a novel
MASP-2 dépendent C4-bypass activation route.
Encouraged by historical reports indicating the existence of a C4-bypass activation route in C4-deficient guinea pig sérum (May, J.E., and M. Frank, J. Immunol. 777:1671-1677 (1973)), we analyzed whether C4-deficient mice may hâve residual 25 classical or lectin pathway functional activity and monitored activation of C3 under pathway-specific assay conditions that exclude contributions of the alternative pathway.
C3b déposition was assayed on Mannan-coated microtiter plates using re-calcified plasma at plasma concentrations prohibitive for alternative pathway activation (1.25% and below). While no cleavage of C3 was détectable in C4-deficient plasma tested for 30 classical pathway activation (data not shown), a strong residual C3 cleavage activity was observed in C4-deficient mouse plasma when initiating complément activation via the lectin pathway. The lectin pathway dependence is demonstrated by compétitive inhibition
195 of C3 cleavage foilowing preincubation of C4-defïcient plasma dilutions with soluble Mannan (see FIGURE 28A). As shown in FIGURE 28A-D, MASP-2 dépendent activation of C3 was observed in the absence of C4. FIGURE 28A graphically illustrâtes C3b déposition by C4+/+ (crosses) and C4-/- (open circles) mouse plasma. Pre5 incubating the C4-/- plasma with excess (1 gg/ml) fluid-phase Mannan prior to the assay completely inhibits C3 déposition (filled circles). Results are typical of 3 independent experiments. FIGURE 28B graphically illustrâtes the results of an experiment in which wild-type, MASP-2 déficient (open squares) and C4-/-mouse plasma (1%) was mixed with various concentrations of anti-rat MASP-2 mAbMl 1 (abscissa) and C3b déposition assayed on Mannan-coated plates. Results are means (± SD) of 4 assays (duplicates of 2 of each type of plasma). FIGURE 28C graphically illustrâtes the results of an experiment in which Human plasma: pooled NHS (crosses), C4-/- plasma (open circles) and C4-/plasma pre-incubated with 1 gg/ml Mannan (filled circles). Results are représentative of three independent experiments. FIGURE 28D graphically illustrâtes that inhibition of
C3b déposition in C4 sufficient and C4 déficient human plasma (1%) by anti-human MASP-2 mAbH3 (Means ± SD of triplicates). As shown in FIGURE 28B, no lectin pathway-dependent C3 activation was detected in MASP-2-/-plasma assayed in parallel, implying that this C4-bypass activation route of C3 is MASP-2 dépendent.
To further corroborate these findings, we established a sériés of recombinant inhibitory mAbs isolated from phage display antibody libraries by affinity screening against recombinant human and rat MASP-2A (where the serine residue of the active protease domain was replaced by an alanine residue by site-directed mutagenesis to prevent autolytic dégradation of the antigen). Recombinant antibodies against MASP-2 (AbH3 and AbMll) were isolated from Combinatorial Antibody Libraries (Knappik, A., et aL, J. Mol. Biol. 296:51-86 (2000)), using recombinant human and rat MASP-2A as antigens (Chen, C.B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-rat Fab2 fragment that potently inhibited lectin pathway-mediated activation of C4 and C3 in mouse plasma (IC50-1 nM) was converted to a full-length IgG2a antibody. Polyclonal anti-murine MASP-2A antiserum was raised in rats. These tools allowed us to confirm
MASP-2 dependency of this novel lectin pathway spécifie C4-bypass activation route of C3, as further described below.
196
As shown in FIGURE 28B, M211, an inhibitory monoclonal antibody which selectively binds to mouse and rat MASP-2 inhibited the C4-bypass activation of C3 in C4-deficient mouse as well as C3 activation of WT mouse plasma via the lectin pathway in a concentration dépendent fashion with similar IC50 values. AH assays were carried 5 out at high plasma dilutions rendering the alternative pathway activation route dysfunctional (with the highest plasma concentration being 1.25%).
In order to investigate the presence of an analogous lectin pathway spécifie C4bypass activation of C3 in humans, we analyzed the plasma of a donor with an inherited deficiency of both human C4 genes (i.e^, C4A and C4B), resulting in total absence of C4 (Yang, Y., et al., J. Immunol. /73:2803-2814 (2004)). FIGURE 28C shows that this patient’s plasma efficiently activâtes C3 in high plasma dilutions (rendering the alternative activation pathway dysfunctional). The lectin pathway spécifie mode of C3 activation on Mannan-coated plates is demonstrated in murine C4-deficient plasma (FIGURE 28A) and human C4 déficient plasma (FIGURE 28C) by adding excess 15 concentrations of fluid-phase Mannan. The MASP-2 dependence of this activation mechanism of C3 in human C4-deficient plasma was assessed using AbH3, a monoclonal antibody that specifically binds to human MASP-2 and ablates MASP-2 functional activity. As shown in FIGURE 28D, AbH3 inhibited the déposition of C3b (and C3dg) in both C4-sufficient and C4-deficient human plasma with comparable potency.
In order to assess a possible rôle of other complément components in the C4bypass activation of C3, we tested plasma of MASP-l/3-/-and Bf/C2-/-mice alongside MASP-2-/-, C4-/- and Clq-/- plasma (as Controls) under both lectin pathway spécifie and classical pathway spécifie assay conditions. The relative amount of C3 cleavage was plotted against the amount of C3 deposited when using WT plasma.
FIGURE 29A graphically illustrâtes a comparative analysis of C3 convertase activity in plasma from various complément déficient mouse strains tested either under lectin activation pathway or classical activation pathway spécifie assay conditions. Diluted plasma samples (1%) of WT mice (n=6), MASP-2-/-mice (n=4), MASP-1/3-/mice (n=2), C4-/- mice (n=8), C4/MASP-1/3-/- mice (n=8), BPC2-/- (n=2) and Clq-/30 mice (n=2) were tested in parallel. Reconstitution of Bf/C2-/- plasma with 2.5pg/ml recombinant rat C2 (Bf/C2-/- +C2) restored C3b déposition. Results are means (±SD). **p<0.01 (compared to WT plasma). As shown in FIGURE 29A, substantial C3
197 déposition is seen in C4-/- plasma tested under lectin pathway spécifie assay conditions, but not under classical pathway spécifie conditions. Again, no C3 déposition was seen in MASP-2 déficient plasma via the lectin pathway activation route, while the same plasma deposited C3 via the classical pathway. In MASP-1/3-/- plasma, C3 déposition occurred in both lectin and classical pathway spécifie assay conditions. No C3 déposition was seen in plasma with a combined deficiency of C4 and MASP-1/3, either using lectin pathway or classical pathway spécifie conditions. No C3 déposition is détectable in C2/Bf-/- plasma, either via the lectin pathway, or via the classical pathway. Reconstitution of C2/Bf-/- mouse plasma with recombinant C2, however, restored both lectin pathway and classical pathway-mediated C3 cleavage. The assay conditions were validated using Clq-/-plasma.
FIGURE 29B graphically illustrâtes time-resolved kinetics of C3 convertase activity in plasma from various complément déficient mouse strains WT, fB-/-, C4-/-, MASP-1/3-/-, and MASP-2-/-plasma, tested under lectin activation pathway spécifie assay conditions (1% plasma, results are typical of three independent experiments). As shown in FIGURE 29B, while no C3 cleavage was seen in MASP-2-/-plasma, fB-/plasma cleaved C3 with similar kinetics to the WT plasma. A significant delay in the lectin pathway-dependent conversion of C3 to C3b (and C3dg) was seen in C4-/-as well as in MASP-1/3 déficient plasma. This delay of C3 activation in MASP-1/3-/- plasma was recently shown to be MASP-1, rather than MASP-3 dépendent (Takahashi, M., et al., J. Immunol. /50:6132-6138 (2008)).
Discussion:
The results described in this Example strongly suggest that MASP-2 functional activity is essential for the activation of C3 via the lectin pathway both in presence and absence of C4. Furthermore, C2 and MASP-1 are required for this novel lectin pathway spécifie C4-bypass activation route of C3 to work. The comparative analysis of lectin pathway functional activity in MASP-2-/-as well as C4-/- plasma revealed the existence of a previously unrecognized C4-independent, but MASP-2-dependent activation route of complément C3 and showed that C3 can be activated in a lectin pathway-dependent mode in total absence of C4. While the detailed molecular composition and the sequence of activation events of this novel MASP-2 dépendent C3 convertase remains to be elucidated, our results imply that this C4-bypass activation route additionally requires the
198 presence of complément C2 as well as MASP-l. The loss of lectin pathway-mediated C3 cleavage activity in plasma of mice with combined C4 and MASP-l/3 deficiency may be explained by a most recently described rôle of MASP-l to enhance MASP-2 dépendent complément activation through direct cleavage and activation of MASP-2 (Takahashi, 5 M., et al., J. Immunol. 750:6132-6138 (2008)). Likewise, MASP-l may aid MASP-2 functional activity through its ability to cleave C2 (Moller-Kristensen, et al., Int. Immunol. 79:141-149 (2007)). Both activities may explain the reduced rate by which MASP-1/3 déficient plasma cleaves C3 via the lectin activation pathway and why MASP1 may be required to sustain C3 conversion via the C4-bypass activation route.
The inability of C2/fB-/- plasma to activate C3 via the lectin pathway was shown to be C2-dependent as the addition of recombinant rat C2 to C2/fB-/- plasma restored the ability of the reconstituted plasma to activate C3 on Mannan-coated plates.
The finding that C4 deficiency specifically disrupts the classical complément activation pathway while the lectin pathway retains a physiologically critical level of C3 15 convertase activity via a MASP-2 dépendent C4-bypass activation route calls for a reassessment of the rôle of the lectin pathway in various disease models, including experimental S. pneumoniae infection (Brown, J. S., et aL, Proc. Natl. Acad. Sci. U. S. A. 99:16969-16974 (2002); Experimental Allergie Encephalomyelitis (Boos, L.A., et al., Glia 49:158-160 (2005); and models of C3 dépendent murine liver régénération (Clark, 20 A., et aL, Mol. Immunol. 45:3125-3132 (2008)). The latter group demonstrated that C4deficient mice can activate C3 in an alternative pathway independent fashion as in vivo inhibition of the alternative pathway by an antibody-mediated déplétion of factor B functional activity did not effect C3 cleavage-dependent liver régénération in C4-/- mice (Clark, A., et aL (2008)). This lectin pathway mediated C4-bypass activation route of C3 25 may also explain the lack of a protective phenotype of C4 deficiency in our model of MIRI as well as in a previously described model of rénal allograft rejection (Lin, T., et al., Am. J. Pathol. 765:1241-1248 (2006)). In contrast, our recent results hâve independently demonstrated a significant protective phenotype of MASP-2-/-mice in models of rénal transplantation (Farrar, C.A., et aL, Mol. Immunol. 46:2832 (2009)).
In summary, the results of this Example support the view that MASP-2 dépendent
C4-bypass activation of C3 is a physiologically relevant mechanism that may be important under conditions where availability of C4 is limiting C3 activation.
199
EXAMPLE 23
This Example describes activation of C3 by thrombin substrates and C3 déposition on mannan in WT (+/+), MASP-2 (-/-), Fil (-/-), F11/C4 (-/-) and C4 (-/-) mice.
Rationale:
As described in Example 14, it was determined that thrombin activation can occur following lectin pathway activation under physiological conditions, and demonstrates the extent of MASP-2 involvement. C3 plays a central rôle in the activation of complément System. C3 activation is required for both classical and alternative complément activation pathways. An experiment was carried out to détermine whether C3 is activated by thrombin substrates.
Methods:
C3 Activation by thrombin substrates
Activation of C3 was measured in the presence of the following activated forms of thrombin substrates; human FCXIa, human FVIIa, bovine FXa, human FXa, human activated protein C, and human thrombin. C3 was incubated with the various thrombin substrates, then separated under reducing conditions on 10% SDS-polyacrylamide gels. After electrophoretic transfer using cellulose membrane, the membrane was incubated with monoclonal biotin-coupled rat anti-mouse C3, detected with a streptavidin-HRP kit and developed using ECL reagent.
Results:
Activation of C3 involves cleavage of the intact a-chain into the truncated a' chain and soluble C3a (not shown in FIGURE 30). FIGURE 30 shows the results of a Western blot analysis on the activation of human C3 by thrombin substrates, wherein the uncleaved C3 alpha chain, and the activation product a' chain are shown by arrows. As shown in FIGURE 30, incubation of C3 with the activated forms of human clotting factor XI and factor X, as well as activated bovine clotting factor X, can cleave C3 in vitro in the absence of any complément proteases.
C3 déposition on mannan
C3 déposition assays were carried out on sérum samples obtained from WT, MASP-2 (-/-), Fll(-/-), F11 (-/-)/C4(-/-) and C4(-/-). Fil is the gene encoding
200 coagulation factor XL To measure C3 activation, microtiter plates were coated with mannan (1 pg/well), then adding sheep anti-HSA sérum (2 pg/ml) in TBS/tween/Ca2+. Plates were blocked with 0.1% HSA in TBS and washed as above. Plasma samples were diluted in 4 mM barbital, 145 mM NaCl, 2 mM CaCb, 1 mM MgCh, pH 7.4, added to the plates and incubated for 1.5 h at 37°C. After washing, bound C3b was detected using rabbit anti-human C3c (Dako), followed by alkaline phosphatase-conjugated goat antirabbit IgG and pNPP.
Results:
FIGURE 31 shows the results of the C3 déposition assay on sérum samples obtained from WT, MASP-2 (-/-), Fl 1 (-/-), Fl l(-/-)/C4 (-/-) and C4 (-/-). As shown in FIGURE 31, there is a functional lectin pathway even in the complété absence of C4. As further shown in FIGURE 31, this novel lectin pathway dépendent complément activation requires coagulation factor XL
Discussion:
Prior to the results obtained in this experiment, it was believed by those in the art that the lectin pathway of complément required C4 for activity. Hence, data from C4 knockout mice (and C4 déficient humans) were interpreted with the assumption that such organisms were lectin pathway déficient (in addition to classical pathway deficiency). The présent results demonstrate that this notion is false. Thus, conclusions of past studies suggesting that the lectin pathway was not important in certain disease settings based on the phenotype of C4 déficient animais may be false. The data described in this Example also show that in the physiological context of whole sérum the lectin pathway can activate components of the coagulation cascade. Thus, it is demonstrated that there is cross-talk between complément and coagulation involving MASP-2.
EXAMPLE 24
This Example describes methods to assess the effect of an anti-MASP-2 antibody on lysis of red blood cells from blood samples obtained from Paroxysmal nocturnal hemoglobinuria (PNH) patients.
Background/Rationale:
Paroxysmal nocturnal hemoglobinuria (PNH), also referred to as MarchiafavaMicheli syndrome, is an acquired, potentially life-threatening disease of the blood,
201 characterized by complément-induced intravascular hemolytic anémia. The hallmark of PNH is chronic intravascular hemolysis that is a conséquence of unregulated activation of the alternative pathway of complément. Lindorfer, M.A., et al., Blood 115(11) (2010). Anémia in PNH is due to destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine, due to appearance of hemoglobin in the urine, and thrombosis. PNH may develop on its own, referred to as primary PNH or in the context of other bone marrow disorders such as aplastic anémia, referred to as secondary PNH. Treatment for PNH includes blood transfusion for anémia, anticoagulation for thrombosis and the use of the monoclonal antibody eculizumab (Soliris), which protects blood cells against immune destruction by inhibiting the complément system (Hillmen P. et al., N. Engl. J. Med. 350(6):552-9 (2004)). However, a significant portion of PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anémia because the antibody does not block activation of the alternative pathway of complément.
This Example describes methods to assess the effect of an anti-MASP-2 antibody on lysis of red blood cells from blood samples obtained from PNH patients (not treated with Soliris) that are incubated with ABO-matched acidified normal human sérum.
Methods:
Reagents:
Erythrocytes from normal donors and from patients suffering from PNH (not treated with Soliris) are obtained by venipuncture, and prepared as described in Wilcox, L.A., et al., Blood 75:820-829 (1991), hereby incorporated herein by reference. AntiMASP-2 antibodies with functional blocking activity of the lectin pathway may be generated as described in Example 10.
Hemolysis Analysis:
The method for determining the effect of anti-MASP-2 antibodies on the ability to block hemolysis of érythrocytes from PNH patients is carried out using the methods described in Lindorfer, M.A., et al., Blood 75(11):2283-91 (2010) and Wilcox, L.A., et al., Blood 75:820-829 (1991), both référencés hereby incorporated herein by reference. As described in Lindorfer et al., érythrocytes from PNH patient samples are centrifuged, the buffy coat is aspirated and the cells are washed in gelatin veronal buffer (GVB) before each experiment. The érythrocytes are tested for susceptibility to APC-mediated
202 lysis as follows. ABO-matched normal human sera are diluted with GVB containing 0.15 mM CaCl2 and 0.5 mM MgCl2 (GVB+2) and acidified to pH 6.4 (acidified NHS, aNHS) and used to reconstitute the érythrocytes to a hematocrit of 1.6% in 50% aNHS. The mixtures are then incubated at 37°C, and after 1 hour, the érythrocytes are pelleted by 5 centrifugation. The optical density of an aliquot of the recovered supemate is measured at 405 nM and used to calculate the percent lysis. Samples reconstituted in acidified serum-EDTA are processed similarly and used to define background noncomplementmediated lysis (typically less than 3%). Complété lysis (100%) is determined after incubating the érythrocytes in distilled water.
In order to détermine the effect of anti-MASP-2 antibodies on hemolysis of PNH érythrocytes, érythrocytes from PNH patients are incubated in aNHS in the presence of incrémental concentrations of the anti-MASP-2 antibodies, and the presence/amount of hemolysis is subsequently quantified.
In view of the fact that anti-MASP-2 antibodies hâve been shown to block 15 subséquent activation of the alternative complément pathway, it is expected that antiMASP-2 antibodies will be effective in blocking alternative pathway-mediated hemolysis of PNH érythrocytes, and will be useful as a therapeutic to treat patients suffering from PNH.
EXAMPLE 25
This Example describes methods to assess the effect of an anti-MASP-2 blocking antibody on complément activation by cryoglobulins in blood samples obtained from patients suffering from cryoglobulinemia.
Background/Rationale:
Cryoglobulinemia is characterized by the presence of cryoglobulins in the sérum.
Cryoglobulins are single or mixed immunoglobulins (typically IgM antibodies) that undergo réversible aggregation at low températures. Aggregation leads to classical pathway complément activation and inflammation in vascular beds, particularly in the periphery. Clinical présentations of cryoglobulinemia include vasculitis and 30 glomerulonephritis.
Cryoglobulinemia may be classified as follows based on cryoglobulin
203 composition: Type I cryoglobulinemia, or simple cryoglobulinemia, is the resuit of a monoclonal immunoglobulin, usually immunoglobulin M (IgM); Types II and III cryoglobulinemia (mixed cryoglobulinemia) contain rheumatoid factors (RFs), which are usually IgM in complexes with the Fc portion of polyclonal IgG.
Conditions associated with cryoglobulinemia include hepatitis C infection, lymphoproliférative disorders and other autoimmune diseases. Cryoglobulin-containing immune complexes resuit in a clinical syndrome of systemic inflammation, possibly due to their ability to activate complément. While IgG immune complexes normally activate the classical pathway of complément, IgM containing complexes can also activate complément via the lectin pathway (Zhang, M., et al., Mol Immunol 44(1-3):103-110 (2007) and Zhang. M., et al., J. Immunol. 17Ί(Ί)ΆΊ2Ί-3Α (2006)).
Immunohistochemical studies hâve further demonstrated the cryoglobulin immune complexes contain components of the lectin pathway, and biopsies from patients with cryoglobulinémie glomerulonephritis showed immunohistochemical evidence of lectin pathway activation in situ (Ohsawa, I., et al., Clin Immunol /0/(1):59-66 (2001)). These results suggest that the lectin pathway may contribute to inflammation and adverse outcomes in cryoglobulemic diseases.
Methods:
The method for determining the effect of anti-MASP-2 antibodies on the ability to block the adverse effects of Cryoglobulinemia is carried out using the assay for fluid phase C3 conversion as described in Ng Y.C. et al., Arthritis and Rheumatism 31(1):99107 (1988), hereby incorporated herein by reference. As described in Ng et al., in essential mixed cryoglobulinemia (EMC), monoclonal rheumatoid factor (mRF), usually IgM, complexes with polyclonal IgG to form the characteristic cryoprecipitate immune complexes (IC) (type II cryoglobulin). Immunoglobulins and C3 hâve been demonstrated in vessel walls in affected tissues such as skin, nerve and kidney. As described in Ng et al., 125I-labeled mRF is added to sérum (normal human sérum and sérum obtained from patients suffering from cryoglobulinemia), incubated at 37°C, and binding to érythrocytes is measured.
Fluid phase C3 conversion is determined in sérum (normal human sérum and sérum obtained from patients suffering from cryoglobulinemia) in the presence or absence of the following IC: BSA-anti BSA, mRF, mRF plus IgG, or cryoglobulins, in
204 the presence or absence of anti-MASP-2 antibodies. The fixation of C3 and C4 to IC is measured using a coprécipitation assay with F(ab')2 anti-C3 and F(ab')2 anti-C4.
In view of the fact that anti-MASP-2 antibodies hâve been shown to block activation of the lectin pathway it is expected that anti-MASP-2 antibodies will be effective in blocking complément mediated adverse effects associated with cryoglobulinemia, and will be useful as a therapeutic to treat patients suffering from cryoglobulinemia.
EXAMPLE 26
This Example describes methods to assess the effect of an anti-MASP-2 antibody on blood samples obtained from patients with Cold Agglutinin Disease, which manifests as anémia.
Background/Rationale:
Cold Agglutinin Disease (CAD), is a type of autoimmune hemolytic anémia. Cold agglutinins antibodies (usually IgM) are activated by cold températures and bind to and aggregate red blood cells. The cold agglutinin antibodies combine with complément and attack the antigen on the surface of red blood cells. This leads to opsoniation of red blood cells (hemolysis) which triggers their clearance by the réticuloendothélial System. The température at which the agglutination takes place varies from patient to patient.
CAD manifests as anémia. When the rate of destruction of red blood cell destruction exceeds the capacity of the bone marrow to produce an adéquate number of oxygen-carrying cells, then anémia occurs. CAD can be caused by an underlying disease or disorder, referred to as Secondary CAD, such as an infectious disease (mycoplasma pneumonia, mumps, mononucleosis), lymphoproliférative disease (lymphoma, chronic lymphocytic leukemia), or connective tissue disorder. Primary CAD patients are considered to hâve a low grade lymphoproliférative bone marrow disorder. Both primary and secondary CAD are acquired conditions.
Methods:
Reagents:
Erythrocytes from normal donors and from patients suffering from CAD are obtained by venipuncture. Anti-MASP-2 antibodies with functional blocking activity of the lectin pathway may be generated as described in Example 10.
205
The effect of anti-MASP-2 antibodies to block cold aggultinin-mediated activation of the lectin pathway may be determined as follows. Erythrocytes from blood group I positive patients are sensitized with cold aggultinins (i.e., IgM antibodies), in the presence or absence of anti-MASP-2 antibodies. The érythrocytes are then tested for the 5 ability to activate the lectin pathway by measuring C3 binding.
In view of the fact that anti-MASP-2 antibodies hâve been shown to block activation of the lectin pathway, it is expected that anti-MASP-2 antibodies will be effective in blocking complément mediated adverse effects associated with Cold Agglutinin Disease, and will be useful as a therapeutic to treat patients suffering from 10 Cold Agglutinin Disease.
EXAMPLE 27
This Example describes methods to assess the effect of an anti-MASP-2 antibody on lysis of red blood cells in blood samples obtained from mice with atypical hemolytic 15 urémie syndrome (aHUS).
Background/Rationale:
Atypical hemolytic urémie syndrome (aHUS) is characterized by hemolytic anémia, thrombocytopenia, and rénal failure caused by platelet thrombi in the microcirculation of the kidney and other organs. aHUS is associated with defective 20 complément régulation and can be either sporadic or familial. aHUS is associated with mutations in genes coding for complément activation, including complément factor H, membrane cofactor B and factor I, and well as complément factor H-related 1 (CFHR1) and complément factor H-related 3 (CFHR3). Zipfel, P.F., et al., PloS Genetics 3(3):e41 (2007). This Example describes methods to assess the effect of an anti-MASP-2 antibody 25 on lysis of red blood cells from blood samples obtained from aHUS mice.
Methods:
The effect of anti-MASP-2 antibodies to treat aHUS may be determined in a mouse model of this disease in which the endogenouse mouse fH gene has been replaced with a human homologue encoding a mutant form of fH frequently found in aHUS 30 patients. See Pickering M.C. et al., J. Exp. Med. 204(6):1249-1256 (2007), hereby incorporated herein by reference. As described in Pickering et al., such mice develop an aHUS like pathology. In order to assess the effect of an anti-MASP-2 antibody for the
206 treatment of aHUS, anti-MASP-2 antibodies are administered to the mutant aHUS mice and lysis of red blood cells obtained from anti-MASP-2 ab treated and untreated Controls is compared. In view of the fact that anti-MASP-2 antibodies hâve been shown to block activation of the lectin pathway it is expected that anti-MASP-2 antibodies will be 5 effective in blocking lysis of red blood cells in mammalian subjects suffering from aHUS.
EXAMPLE 28
This Example describes methods to assess the effect of an anti-MASP-2 antibody for the treatment of glaucoma.
Rationale/Background:
It has been shown that uncontrolled complément activation contributes to the progression of degenerative injury to retinal ganglion cells (RGCs), their synapses and axons in glaucoma. See Tezel G. et al., Invest Ophthalmol Vis Sci 57:5071-5082 (2010). For example, histopathologie studies of human tissues and in vivo studies using different 15 animal models hâve demonstrated that complément components, including Clq and C3, are synthesized and terminal complément complex is formed in the glaucomatous retina (see Stasi K. et al., Invest Ophthalmol Vis Sci 47:1024-1029 (2006), Kuehn M.H. et al., Exp Eye Res 53:620-628 (2006)). As further described in Kuehn M.H. et al., Experimental Eye Research 57:89-95 (2008), complément synthesis and déposition is 20 induced by retinal ER and the disruption of the complément cascade delays RGC degeneration. In this study, mice carrying a targeted disruption of the complément component C3 were found to exhibit delayed RGC degeneration after transient retinal I/R when compared to normal animais.
Methods:
The method for determining the effect of anti-MASP-2 antibodies on RGC degeneration is carried out in an animal model of retinal I/R as described in Kuehn M.H. et ai., Experimental Eye Research 57:89-95 (2008), hereby incorporated herein by reference. As described in Kuehn et al., retinal ischemia is induced by anesthetizing the animais, then inserting a 30-gauge needle connected to a réservoir containing phosphate 30 buffered saline through the comea into the anterior chamber of the eye. The saline réservoir is then elevated to yield an intraocular pressure of 104 mmHg, sufficient to completely prevent circulation through the retinal vasculature. Elevated intraocular
207 ischemia is confirmed by blanching of the iris and retina and ischemia is maintained for 45 minutes in the left eye only; the right eye serves as a control and does not receive cannulation. Mice are then euthanized either 1 or 3 weeks after the ischémie insult. AntiMASP-2 antibodies are administered to the mice either locally to the eye or systemically to assess the effect of an anti-MASP antibody administered prior to ischémie insult.
Immunohistochemistry of the eyes is carried out using antibodies against Clq and C3 to detect complément déposition. Optic nerve damage can also be assessed using standard électron microscopy methods. Quantitation of surviving retinal RGCs is performed using gamma synuclein labeling.
Results:
As described in Kuehn et al., in normal control mice, transient retinal ischemia results in degenerative changes of the optic nerve and retinal deposits of Clq and C3 détectable by immunohistochemistry. In contrast, C3 déficient mice displayed a marked réduction in axonal degeneration, exhibiting only minor levels of optic nerve damage 1 week after induction. Based on these results, it is expected that similar results would be observed when this assay is carried out in a MASP-2 knockout mouse, and when antiMASP-2 antibodies are administered to a normal mouse prior to ischémie insult.
EXAMPLE 29
This Example demonstrates that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective for the treatment of radiation exposure and/or for the treatment, amelioration or prévention of acute radiation syndrome.
Rationale:
Exposure to high doses of ionizing radiation causes mortality by two main mechanisms: toxicity to the bone marrow and gastrointestinal syndrome. Bone marrow toxicity results in a drop in ail hématologie cells, predisposing the organism to death by infection and hemorrhage. The gastrointestinal syndrome is more severe and is driven by a loss of intestinal barrier function due to disintegration of the gut épithélial layer and a loss of intestinal endocrine function. This leads to sepsis and associated systemic inflammatory response syndrome which can resuit in death.
The lectin pathway of complément is an innate immune mechanism that initiâtes inflammation in response to tissue injury and exposure to foreign surfaces (i.e., bacteria).
208
Blockade of this pathway leads to better outcomes in mouse models of ischémie intestinal tissue injury or septic shock. It is hypothesized that the lectin pathway may trigger excessive and harmful inflammation in response to radiation-induced tissue injury. Blockade of the lectin pathway may thus reduce secondary injury and increase survival 5 following acute radiation exposure.
The objective of the study carried out as described in this Example was to assess the effect of lectin pathway blockade on survival in a mouse model of radiation injury by administering anti-murine MASP-2 antibodies.
Methods and Materials:
Materials. The test articles used in this study were (i) a high affinity anti-murine
MASP-2 antibody (mAbMll) and (ii) a high affmity anti-human MASP-2 antibody (mAbH6) that block the MASP-2 protein component of the lectin complément pathway which were produced in transfected mammalian cells. Dosing concentrations were 1 mg/kg of anti-murine MASP-2 antibody (mAbMll), 5mg/kg of anti-human MASP-2 15 antibody (mAbH6), or stérile saline. For each dosing session, an adéquate volume of fresh dosing solutions were prepared.
Animais. Young adult male Swiss-Webster mice were obtained from Harlan Laboratories (Houston, TX). Animais were housed in solid-bottom cages with Alpha-Dri bedding and provided certified PMI 5002 Rodent Diet (Animal Specialties, Inc., Hubbard 20 OR) and water ad libitum. Température was monitored and the animal holding room operated with a 12 hour light/12 hour dark light cycle.
Irradiation. After a 2-week acclimation in the facility, mice were irradiated at 6.5 and 7.0 Gy by whole-body exposure in groups of 10 at a dose rate of 0.78 Gy/min using a Therapax X-RAD 320 System equipped with a 320-kV high stability X-ray generator, 25 métal ceramic X-ray tube, variable x-ray beam collimator and filter (Précision X-ray Incorporated, East Haven, CT). Dose levels were selected based on prior studies conducted with the same strain of mice indicating the LD50/30 was between 6.5 and 7.0 Gy (data not shown).
Drug Formulation and Administration. The appropriate volume of concentrated 30 stock solutions were diluted with ice cold saline to préparé dosing solutions of 0.2 mg/ml anti-murine MASP-2 antibody (mAbMll) or 0.5 mg/ml anti-human MASP-2 antibody (mAbH6) according to protocol. Administration of anti-MASP-2 antibody mAbMl 1 and
209 mAbH6 was via IP injection using a 25-gauge needle base on animal weight to deliver 1 mg/kg mAbMl 1, 5mg/kg mAbH6, or saline vehicle.
Study Design. Mice were randomly assigned to the groups as described in Table 8. Body weight and température were measured and recorded daily. Mice in 5 Groups 7, 11 and 13 were sacrificed at post-irradiation day 7 and blood collected by cardiac puncture under deep anesthésia. Surviving animais at post-irradiation day 30 were sacrificed in the same manner and blood collected. Plasma was prepared from collected blood samples according to protocol and retumed to Sponsor for analysis.
_____TABLE 8: Study Groups
Group ID N Irradiation Level (Gy) Treatment Dose Schedule
1 20 6.5 Vehicle 18 hr prior to irradiation, 2 hr post irradiation, weekly booster
2 20 6.5 anti-murine MASP-2 ab (mAbMl 1) 18 hr prior to irradiation only
3 20 6.5 anti-murine MASP-2 ab (mAbMl 1) 18 hr prior to irradiation, 2 hr post irradiation, weekly booster
4 20 6.5 anti-murine MASP-2 ab (mAbMl 1) 2 hr post irradiation, weekly booster
5 20 6.5 anti-human MASP-2 ab (mAbH6) 18 hr prior to irradiation, 2 hr post irradiation, weekly booster
6 20 7.0 Vehicle 18 hr prior to irradiation, 2 hr post irradiation, weekly booster
7 5 7.0 Vehicle 2 hr post irradiation only
8 20 7.0 anti-murine MASP-2 ab (mAbMl 1) 18 hr prior to irradiation only
9 20 7.0 anti-murine MASP-2 ab 18 hr prior to irradiation, 2 hr post irradiation, weekly
210
Group ID N Irradiation Level (Gy) Treatment Dose Schedule
(mAbMl 1) booster
10 20 7.0 anti-murine MASP-2 ab (mAbMll) 2 hr post irradiation, weekly booster
11 5 7.0 anti-murine MASP-2 ab (mAbMll) 2 hr post irradiation only
12 20 7.0 anti-human MASP-2 ab (mAbH6) 18 hr prior to irradiation, 2 hr post irradiation, weekly booster
13 5 None None None
Statistical Analysis. Kaplan-Meier survival curves were generated and used to compare mean survival time between treatment groups using log-Rank and Wilcoxon methods. Averages with standard déviations, or means with standard error of the mean 5 are reported. Statistical comparisons were made using a two-tailed unpaired t-test between controlled irradiated animais and individual treatment groups.
Results
Kaplan-Meier survival plots for 7.0 and 6.5 Gy exposure groups are provided in
FIGURES 32A and 32B, respectively, and summarized below in Table 9. Overall, 10 treatment with anti-murine MASP-2 ab (mAbMl 1) pre-irradiation increased the survival of irradiated mice compared to vehicle treated irradiated control animais at both 6.5 (20% increase) and 7.0 Gy (30% increase) exposure levels. At the 6.5 Gy exposure level, postirradiation treatment with anti-murine MASP-2 ab resulted in a modest increase in survival (15%) compared to vehicle control irradiated animais.
In comparison, ail treated animais at the 7.0 Gy exposure level showed an increase in survival compared to vehicle treated irradiated control animais. The greatest change in survival occurred in animais receiving mAbH6, with a 45% increase compared to control animais. Further, at the 7.0 Gy exposure level, mortalities in the mAbH6 treated group first occurred at post-irradiation day 15 compared to post-irradiation day 8 20 for vehicle treated irradiated control animais, an increase of 7 days over control animais.
Mean time to mortality for mice receiving mAbH6 (27.3 ± 1.3 days) was significantly
211 increased (p = 0.0087) compared to control animais (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 throughout the study. A transient weight loss occurred in ail irradiated animais, 5 with no evidence of differential changes due to mAbMl 1 or mAbH6 treatment compared to Controls (data not shown). At study termination, ail surviving animais showed an increase in body weight from starting (day -1) body weight.
TABLE 9: Survival rates of test animais exposed to radiation
Test Group Exposure Level Survival (%) Time to Death (Mean ± SEM, Day) First/Last Death (Day)
Control Irradiation 6.5 Gy 65 % 24.0 ±2.0 9/16
mAbMl 1 preexposure 6.5 Gy 85% 27.7 ± 1.5 13/17
mAbMl 1 pre + post-exposure 6.5 Gy 65 % 24.0 ±2.0 9/15
mAbMl 1 postexposure 6.5 Gy 80% 26.3 ± 1.9 9/13
mAbH6 pre±postexposure 6.5 Gy 65% 24.6 ± 1.9 9/19
Control irraditation 7.0 Gy 35 % 20.7 ±2.0 8/17
mAbMl 1 preexposure 7.0 Gy 65% 23.0 ± 2.3 7/13
mAbMl 1 pre + post-exposure 7.0 Gy 55 % 21.6 ±2.2 7/16
mAbMl 1 postexposure 7.0 Gy 70% 24.3 ±2.1 9/14
mAbH6 pre±postexposure 7.0 Gy 80% 27.3 ± 1.3* 15/20
*p = 0.0087 by two-tailed unpaired t-test between controlled irradiated animais and treatment group at the same irradiation exposure level.
Discussion
Acute radiation syndrome consists of three defined subsyndromes: hematopoietic, gastrointestinal, and cerebrovascular. The syndrome observed dépends on the radiation 15 dose, with the hematopoietic effects observed in humans with significant partial or
212 whole-body radiation exposures exceeding 1 Gy. The hematopoietic syndrome is characterized by severe dépréssion of bone-marrow function leading to pancytopenia with changes in blood counts, red and white blood cells, and platelets occurring concomitant with damage to the immune System. As nadir occurs, with few neutrophils and platelets présent in peripheral blood, neutropenia, fever, complications of sepsis and uncontrollable hemorrhage lead to death.
In the présent study, administration of mAbH6 was found to increase survivability of whole-body x-ray irradiation in Swiss-Webster male mice irradiated at 7.0 Gy. Notably, at the 7.0 Gy exposure level, 80% of the animais receiving mAbH6 survived to 30 days compared to 35% of vehicle treated control irradiated animais. Importantly, the first day of death in this treated group did not occur until post-irradiation day 15, a 7-day increase over that observed in vehicle treated control irradiated animais. Curiously, at the lower X-ray exposure (6.5 Gy), administration of mAbH6 did not appear to impact survivability or delay in mortality compared to vehicle treated control irradiated animais. There could be multiple reasons for this différence in response between exposure levels, although vérification of any hypothesis may require additional studies, including intérim sample collection for microbiological culture and hematological parameters. One explanation may simply be that the number of animais assigned to groups may hâve precluded seeing any subtle treatment-related différences. For example, with groups sizes of n=20, the différence in survival between 65% (mAbH6 at 6.5 Gy exposure) and 80% (mAbH6 at 7.0 Gy exposure) is 3 animais. On the other hand, the différence between 35% (vehicle control at 7.0 Gy exposure) and 80% (mAbH6 at 7.0 Gy exposure) is 9 animais, and provides sound evidence of a treatment-related différence.
These results demonstrate that anti-MASP-2 antibodies are effective in treating a mammalian subject at risk for, or suffering from the detrimental effects of acute radiation syndrome.
EXAMPLE 30
This Example demonstrates that MASP-2 déficient mice are protected from Neisseria meningitidis induced mortality aller infection with either N. meningitidis serogroup A or Neisseria meningitidis serogroup B.
Methods:
213
MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1. 10-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57/BL6 mice (n=10) were inoculated by intraperitoneal (i.p.) injection with a dosage of 2.6 x 107 CFU of Neisseria meningitidis serogroup A Z2491 in a volume of 100 μΐ. The infective dose 5 was administered to mice in conjunction with iron dextran at a final concentration of 400 mg/kg. Survival of the mice after infection was monitored over a 72-hour time period.
In a separate experiment, 10-week-old MASP-2 KO mice (n=10) and wild-type C57/BL6 mice (n=10) were inoculated by i.p. injection with a dosage of 6 x 106 CFU of 10 Neisseria meningitidis serogroup B strain MC58 in a volume of 100 μΐ. The infective dose was administered to mice in conjunction with iron dextran at a final dose of 400 mg/kg. Survival of the mice after infection was monitored over a 72-hour time period. An illness score was also determined for the WT and MASP-2 KO mice during the 72-hour time period after infection, based on the illness scoring parameters described 15 below in TABLE 10, which is based on the scheme of Fransen et aL (2010) with slight modifications.
TABLE 10: Illness Scoring associated with clinical signs in infected mice
Signs Score
Normal 0
Slightly ruffled fur 1
Ruffled fur, slow and sticky eyes 2
Ruffled fur, léthargie and eyes shut 3
Very sick and no movement after stimulation 4
Dead 5
Blood samples were taken from the mice at hourly intervals after infection and analyzed to détermine the sérum level (log cfu/mL) of N. meningitidis in order to verify infection and détermine the rate of clearance of the bacteria from the sérum.
Results:
214
FIGURE 33 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 2.6 x 107 cfti of N. meningitidis serogroup A Z2491. As shown in FIGURE 33, 100% of the MASP-2 KO mice survived throughout the 72-hour period after infection. In contrast, only 80% of the WT mice (p=O.O12) were still alive 24 hours after infection, and only 50% ofthe WT mice were still alive at 72 hours after infection. These results demonstrate that MASP-2deficient mice are protected from N. meningitidis serogroup A Z2491-induced mortality.
FIGURE 34 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 6 x 106 cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 34, 90% ofthe MASP2 KO mice survived throughout the 72-hour period after infection. In contrast, only 20% of the WT mice (p=0.0022) were still alive 24 hours after infection. These results demonstrate that MASP-2-deficient mice are protected from N. meningitidis serogroup B strain MC58-induced mortality.
FIGURE 35 graphically illustrâtes the log cfu/mL of N. meningitidis serogroup B strain MC58 recovered at different time points in blood samples taken from the MASP-2 KO and WT mice after i.p. infection with 6x106 cfu of N. meningitidis serogroup B strain MC58 (n=3 at different time points for both groups of mice). The results are expressed as Means±SEM. As shown in FIGURE 35, in WT mice the level of N. meningitidis in the blood reached a peak of about 6.0 log cfu/mL at 24 hours after infection and dropped to about 4.0 log cfu/mL by 36 hours after infection. In contrast, in the MASP-2 KO mice, the level of N. meningitidis reached a peak of about 4.0 log cfu/mL at 12 hours after infection and dropped to about 1.0 log cfu/mL by 36 hours after infection (the symbol * indicates p<0.05; the symbol ** indicates p=0.0043). These results demonstrate that although the MASP-2 KO mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as the WT mice, the MASP-2 KO mice hâve enhanced clearance of bacteraemia as compared to WT.
FIGURE 36 graphically illustrâtes the average illness score of MASP-2 KO and WT mice at 3, 6, 12 and 24 hours after infection with 6x106 cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 36, the MASP-2-deficient mice showed high résistance to the infection, with much lower illness scores at 6 hours
215 (symbol indicates p=0.0411), 12 hours (symbol ** indicates p=0.0049) and 24 hours (symbol *** indicates p=0.0049) after infection, as compared to WT mice. The results in FIGURE 36 are expressed as means±SEM.
In summary, the results in this Example demonstrate that MASP-2-deficient mice are protected from Neisseria meningitides-ïnduced mortality after infection with either N. meningitidis serogroup A or N. meningitidis serogroup B.
EXAMPLE 31
This Example demonstrates that the administration of anti-MASP-2 antibody after infection with N. meningitidis increases the survival of mice infected with N. meningitidis.
Background/Rationale :
As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2 #11 was identified as a ftinctionally active antibody.
Full-length antibodies of the rat IgG2c and mouse IgG2a isotypes were generated from Fab2 #11. The full-length anti-MASP-2 antibody of the mouse IgG2a isotype was characterized for pharmacodynamie parameters (as described in Example 19).
In this Example, the mouse anti-MASP-2 full-length antibody derived from Fab2 #11 was analyzed in the mouse model of N. meningitidis infection.
Methods:
The mouse IgG2a full-length anti-MASP-2 antibody isotype derived from Fab2 #11, generated as described above, was tested in the mouse model of N. meningitidis infection as follows.
Administration of mouse-anti-MASP-2 Monoclonal antibodies (MoAb) after infection
9-week-old C57/BL6 Charles River mice were treated with inhibitory mouse antiMASP-2 antibody (1.0 mg/kg) (n=12) or control isotype antibody (n=10) at 3 hours after i.p. injection with a high dose (4x106 cfu) of/V. meningitidis serogroup B strain MC58.
Results:
FIGURE 37 is a Kaplan-Meyer plot graphically illustrating the percent survival of mice after administration of an infective dose of 4xl06 cfu of N. meningitidis serogroup B strain MC58, followed by administration 3 hours post-infection of either
216 inhibitory anti-MASP-2 antibody (1.0 mg/kg) or control isotype antibody. As shown in FIGURE 37, 90% of the mice treated with anti-MASP-2 antibody survived throughout the 72-hour period after infection. In contrast, only 50% of the mice treated with isotype control antibody survived throughout the 72-hour period after infection. The Symbol * indicates p=0.0301, as determined by comparison of the two survival curves.
These results demonstrate that administration of anti-MASP-2 antibody is effective to treat and improve survival in subjects infected with N. meningitidis.
As demonstrated herein, the use of anti-MASP-2 antibody in the treatment of a subject infected with N. meningitidis is effective when administered within 3 hours postinfection, and is expected to be effective within 24 hours to 48 hours after infection. Meningococcal disease (either meningococcemia or meningitis) is a medical emergency, and therapy will typically be initiated immediately if meningococcal disease is suspected (Le., before N. meningitidis is positively identified as the etiological agent).
In view of the results in the MASP-2 KO mouse demonstrated in EXAMPLE 30, it is believed that administration of anti-MASP-2 antibody prior to infection with N. meningitidis would also be effective to prevent or ameliorate the severity of infection.
EXAMPLE 32
This Example demonstrates that administration of anti-MASP-2 antibody is effective to treat N. meningitidis infection in human sérum.
Rationale:
Patients with decreased sérum levels of functional MBL display increased susceptibility to récurrent bacterial and fungal infections (Kilpatrick et aL, Biochim Biophys Acta 1572:401-413 (2002)). It is known that N. meningitidis is recognized by MBL, and it has been shown that MBL-deficient sera do not lyse Neisseria.
In view of the results described in Examples 30 and 31, a sériés of experiments were carried out to détermine the efficacy of administration of anti-MASP-2 antibody to treat N. meningitidis infection in complement-deficient and control human sera.
Experiments were carried out in a high concentration of sérum (20%) in order to preserve the complément pathway.
Methods:
217
1. Sérum bactericidal activity in varions complement-deficient human sera and in human sera treated with human anti-MASP-2 antibody
The foilowing complement-deficient human sera and control human sera were used in this experiment:
______TABLE 11: Human sera samples tested (as shown in FIGURE 38)
Sample Sérum type
A Normal human sera (NHS) + human anti-MASP-2 Ab
B NHS + isotype control Ab
C MBL -/- human sérum
D NHS
E Heat-Inactivated (HI) NHS
A recombinant antibody against human MASP-2 was isolated from a Combinatorial Antibody Library (Knappik, A., et al., J. Mol. Biol. 296:51-86 (2000)), using recombinant human MASP-2A as. an antigen (Chen, C.B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-human scFv fragment that potently inhibited lectin pathway-mediated activation of C4 and C3 in human plasma (IC50—20 nM) was identified and converted to a full-length human IgG4 antibody.
N. meningitidis serogroup B-MC58 was incubated with the different sera show in TABLE 11, each at a sérum concentration of 20%, with or without the addition of inhibitory human anti-MASP-2 antibody (3 gg in 100 μΐ total volume) at 37°C with shaking. Samples were taken at the foilowing time points: 0-, 30-, 60- and 90-minute intervals, plated out and then viable counts were determined. Heat-inactivated human sérum was used as a négative control.
Results:
FIGURE 38 graphically illustrâtes the log cfu/mL of viable counts of N. meningitidis serogroup B-MC58 recovered at different time points in the human sera samples shown in TABLE 11. TABLE 12 provides the Student’s t-test results for FIGURE 38.
TABLE 12: Student’s t-test Results for FIGURE 38 (time point 60 minutes)
Mean Diff. (Log) | Significant?P value summary
218
P<0.05?
A vs B -0.3678 Yes ***(0.0002)
A vs C -1.1053 Yes ***(p<0.0001)
A vs D -0.2111 Yes **(0.0012)
C vsD 1.9 Yes ***(p<0.0001)
As shown in FIGURE 38 and TABLE 12, complement-dependent killing of N. meningitidis in human 20% sérum was significantly enhanced by the addition of the human anti-MASP-2 inhibitory antibody.
2. Complement-dependent killing of N. meningitidis in 20% (v/v) mouse sera déficient of MASP-2.
The following complement-deficient mouse sera and control mouse sera were used in this experiment:
TABLE 13: Mouse sera samples tested (as shown in FIGURE 39)
Sample Sérum Type
A WT
B MASP-2 -/-
C MBL A/C -/-
D WT heat-inactivated (HIS)
N. meningitidis serogroup B-MC58 was incubated with different complementdeficient mouse sera, each at a sérum concentration of 20%, at 37°C with shaking.
Samples were taken at the following time points: 0-, 15-, 30-, 60-, 90- and 120-minute intervals, plated out and then viable counts were determined. Heat-inactivated human sérum was used as a négative control.
Results:
FIGURE 39 graphically illustrâtes the log cfu/mL of viable counts of N.
meningitidis serogroup B-MC58 recovered at different time points in the mouse sera samples shown in TABLE 13. As shown in FIGURE 39, the MASP-2 -/- mouse sera hâve a higher level of bactericidal activity for N. meningitidis than WT mouse sera. The symbol ** indicates p=0.0058, the symbol *** indicates p=0.001. TABLE 14 provides the Student's t-test results for FIGURE 39.
219
TABLE 14: Student's t-test Results for FIGURE 39
Comparison Time point Mean Diff. (LOG) Significant? (p<0.05)? P value summary
A vs. B 60 min. 0.39 yes « (0.0058)
A vs. B 90 min. 0.6741 yes *** (0.001)
In summary, the results in this Example demonstrate that MASP-2 -/- sera has a 5 higher level of bactericidal activity for N. meningitidis than WT sera.
EXAMPLE 33
This Example demonstrates the inhibitory effect of MASP-2 deficiency on lysis of 10 red blood cells from blood samples obtained from a mouse model of paroxysmal noctumal hemoglobinuria (PNH).
Background/Rationale :
Paroxysmal noctumal hemoglobinuria (PNH), also referred to as MarchiafavaMicheli syndrome, is an acquired, potentially life-threatening disease of the blood, 15 characterized by complement-induced intravascular hemolytic anémia. The hallmark of
PNH is the chronic complement-mediated intravascular hemolysis that is a conséquence of unregulated activation of the alternative pathway of complément due to the absence of the complément regulators CD55 and CD59 on PNH érythrocytes, with subséquent hemoglobinuria and anémia. Lindorfer, M.A., et aL, Blood 115(11) (2010), Risitano, 20 A.M, Mini-Reviews in Médicinal Chemistry, 11:528-535 (2011). Anémia in PNH is due to destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine, due to appearance of hemoglobin in the urine, back pain, fatigue, shortness of breath and thrombosis. PNH may develop on its own, referred to as primary PNH or in the context of other bone marrow disorders such as aplastic anémia, referred to as secondary PNH.
Treatment for PNH includes blood transfusion for anémia, anticoagulation for thrombosis and the use ofthe monoclonal antibody eculizumab (Soliris®), which protects blood cells against immune destruction by inhibiting the complément System (Hillmen P. et al., N. Engl. J. Med. 350(6):552-9 (2004)). Eculizumab (Soliris®) is a humanized monoclonal
220 antibody that targets the complément component C5, blocking its cleavage by C5 convertases, thereby preventing the production of C5a and the assembly of the MAC. Treatment of PNH patients with eculizumab has resulted in a réduction of intravascular hemolysis, as measured by lactate dehydrogenase (LDH), leading to hemoglobin stabilization and transfusion independence in about half of the patients (Hillmen P, et al., Mini-Reviews in Médicinal Chemistry, vol 11(6) (2011)). While nearly ail patients undergoing therapy with eculizumab achieve normal or almost normal LDH levels (due to control of intravascular hemolysis), only about one third of the patients reach a hemoglobin value above Hgr/dL, and the remaining patients on eculizumab continue to 10 exhibit moderate to severe (z.e.,transfusion-dependent) anémia, in about equal proportions (Risitano A.M. et aL, Blood 113:4094-100 (2009)). As described in Risitano et al., MiniReviews in Médicinal Chemistry 11:528-535 (2011), it was demonstrated that PNH patients on eculizumab contained C3 fragments bound to a substantial portion of their PNH érythrocytes (while untreated patients did not), leading to the conclusion that membrane-bound C3 fragments work as opsonins on PNH érythrocytes, resulting in their entrapment in the réticuloendothélial cells through spécifie C3 receptors and subséquent extravascular hemolysis. Therefore, therapeutic strategies in addition to the use of eculizumab are needed for those patients developing C3 fragment-mediated extravascular hemolysis because they continue to require red cell transfusions.
This Example describes methods to assess the effect of MASP-2- déficient sérum and sérum treated with MASP-2 inhibitory agent on lysis of red blood cells from blood samples obtained from a mouse model of PNH and demonstrates the efficacy of MASP-2 inhibition to treat subjects suffering from PNH, and also supports the use of inhibitors of MASP-2 to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in
PNH subjects undergoing therapy with a C5 inhibitor such as eculizumab.
Methods:
PNH animal model:
Blood samples were obtained from gene-targeted mice with deficiencies of Crry and C3 (Crry/C3-/-) and CD55/CD59-deficient mice. These mice are missing the 30 respective surface complément regulators and their érythrocytes are, therefore, susceptible to spontaneous complément autolysis as are PNH human blood cells.
221
In order to sensitize these érythrocytes even more, these cells were used with and without coating by mannan and then tested for hemolysis in WT C56/BL6 plasma, MBL null plasma, MASP-2 -/- plasma, human NHS, human MBL -/- plasma, and NHS treated with human anti-MASP-2 antibody.
1. Hemolysis assay of Crry/C3 and CD55/CD59 double-deficient murine érythrocytes in MASP-2-deflcient/depleted sera and Controls
Day 1. Préparation of murine RBC (± mannan coating)
Materials included: fresh mouse blood, BBS/Mg2+/Ca2+ (4.4 mM barbituric acid,
1.8 mM sodium barbitone, 145 mM NaCl, pH7.4, 5mM Mg2+, 5mM Ca2+), chromium chloride, CrChôftO (0.5mg/mL in BBS/Mg2+/Ca2+) and mannan, 100 pg/mL in BBS /Mg2+/Ca2+.
Whole blood (2mL) was spun down for 1-2 min at 2000xg in a refrigerated 15 centrifuge at 4°C. The plasma and buffy coat were aspirated off. The sample was then washed three times by re-suspending the RBC pellet in 2 mL ice-cold BBS/gelatin/Mg2+/Ca2+ and repeating centrifugation step. After the third wash, the pellet was re-suspended in 4mL BBS/Mg2+/Ca2+. A 2 mL aliquot of the RBC was set aside as an uncoated control. To the remaining 2 mL, 2 mL CrC13 and 2 mL mannan 20 were added and the sample was incubated with gentle mixing at room température for 5 minutes. The reaction was terminated by adding 7.5mL BBS/gelatin/Mg2+/Ca2+. The sample was spun down as above, re-suspended in 2 mL BBS/gelatin/Mg2+/Ca2+ and washed a further two times as above, then stored at 4°C.
Day 2. Hemolysis assay
Materials included BBS/gelatin/Mg2+/Ca2+ (as above), test sera, 96-well roundbottomed and flat-bottomed plates and a spectrophotometer that reads 96-well plates at 410-414 nm.
The concentration of the RBC was first determined and the cells were adjusted to 109/mL, and stored at this concentration. Before use, the assay buffer was diluted to 30 108/mL, and then lOOul per well was used. Hemolysis was measured at 410-414 nm (allowing for greater sensitivity then 541nm). Dilutions of test sera were prepared in icecold BBS/gelatin/Mg2+/Ca2+. 100μ1 of each sérum dilution was pipetted into round19494
222 bottomed plate (see plate layout). ΙΟΟμΙ of appropriately diluted RBC préparation was added (i.e., 108 /mL) (see plate layout), incubated at 37°C for about 1 hour, and observed for lysis. (The plates may be photographed at this point.) The plate was then spun down at maximum speed for 5 minutes. 1 ΟΟμΙ was aspirated of the fluid-phase, transferred to 5 flat-bottom plates, and the OD was recorded at 410-414 nm. The RBC pellets were retained (these can be subsequently lysed with water to obtain an inverse resuit).
Experiment #1:
Fresh blood was obtained from CD55/CD59 double-deficient mice and blood of Crry/C3 double-deficient mice and érythrocytes were prepared as described in detail in 10 the above protocol. The cells were split and half of the cells were coated with mannan and the other half were left untreated, adjusting the final concentration to Ix 108 per mL, of which 100 μΐ was used in the hemolysis assay, which was carried out as described above.
Results of Experiment #1: The lectin pathway is involved in érythrocyte lysis 15 in the PNH animal model
In an initial experiment, it was determined that non-coated WT mouse érythrocytes were not lysed in any mouse sérum. It was further determined that mannancoated Crry-/- mouse érythrocytes were slowly lysed (more than 3 hours at 37 degrees) in WT mouse sérum, but they were not lysed in MBL null sérum. (Data not shown).
It was determined that mannan-coated Crry-/- mouse érythrocytes were rapidly lysed in human sérum but not in heat-inactivated NHS. Importantly, mannan-coated Crry-/- mouse érythrocytes were lysed in NHS diluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and 1/640 dilutions ail lysed). (Data not shown). In this dilution, the alternative pathway does not work (AP functional activity is significantly reduced below 25 8% sérum concentration).
Conclusions from Experiment #1
Mannan-coated Crry-/- mouse érythrocytes are very well lysed in highly diluted human sérum with MBL but not in that without MBL. The efficient lysis in every sérum concentration tested implies that the alternative pathway is not involved or needed for this 30 lysis. The inability of MBL-deficient mouse sérum and human sérum to lyse the mannan-coated Crry-/- mouse érythrocytes indicates that the classical pathway also has
223 nothing to do with the lysis observed. As lectin pathway récognition molécules are required (i.e., MBL), this lysis is mediated by the lectin pathway.
Experiment #2:
Fresh blood was obtained from the Crry/C3 and CD55/CD59 double-deficient 5 mice and mannan-coated Crry-/- mouse érythrocytes were analyzed in the haemolysis assay as described above in the presence of the foilowing human sérum: MBL null; WT; NHS pretreated with human anti-MASP-2 antibody; and heat-inactivated NHS as a control.
Results of Experiment #2: MASP-2 inhibitors prevent érythrocyte lysis in the 10 PNH animal model
With the Mannan-coated Crry-/- mouse érythrocytes, NHS was incubated in the dilutions diluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and 1/640), human MBL-/serum, NHS pretreated with anti-MASP-2 mAb, and heat-inactivated NHS as a control.
The ELISA microtiter plate was spun down and the non-lysed érythrocytes were 15 collected on the bottom of the round-well plate. The supematant of each well was collected and the amount of hemoglobin released from the lysed érythrocytes was measured by reading the OD415 nm in an ELISA reader.
In the control heat-inactivated NHS (négative control), as expected, no lysis was observed. MBL-/- human sérum lysed mannan-coated mouse érythrocytes at 1/8 and 20 1/16 dilutions. Anti-MASP-2-antibody-pretreated NHS lysed mannan-coated mouse érythrocytes at 1/8 and 1/16 dilutions while WT human sérum lysed mannan-coated mouse érythrocytes down to dilutions of 1/32.
FIGURE 40 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed mouse érythrocytes (Cryy/C3-/-) into the supernatant measured by photometry) 25 of mannan-coated murine érythrocytes by human sérum over a range of sérum concentrations in sérum from heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with anti-MASP-2 antibody, and NHS control.
From the results shown in FIGURE 40, it is demonstrated that MASP-2 inhibition with anti-MASP-2 antibody significantly shifted the CH50 and inhibited complement30 mediated lysis of sensitized érythrocytes with déficient protection from autologous complément activation.
Experiment #3
Φ
224
Fresh blood obtained from the Crry/C3 and CD55/CD59 double-deficient mice in non-coated Crry-/- mouse érythrocytes was analyzed in the hemolysis assay as described above in the presence of the following sérum: MBL WT sera; NHS pretreated with human anti-MASP-2 antibody and heat-inactivated NHS as a control.
Results:
FIGURE 41 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed WT mouse érythrocytes into the supematant measured by photometry) of noncoated murine érythrocytes by human sérum over a range of sérum concentrations in sérum from heat inactivated (HI) NHS, MBL-/-, NHS pretreated with anti-MASP-2 10 antibody, and NHS control. As shown in FIGURE 41, it is demonstrated that inhibiting MASP-2 inhibits complement-mediated lysis of non-sensitized WT mouse érythrocytes.
FIGURE 42 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed mouse érythrocytes (CD55/59 -/-) into the supematant measured by photometry) of non-coated murine érythrocytes by human sérum over a range of sérum concentration 15 in sérum from heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with anti-MASP-2 antibody, and NHS control.
TABLE 12: CH50 va ues expressed as sérum concentrations
Sérum WT CD55/59 -/-
Heat-inactivated NHS No lysis No lysis
MBL AO/XX donor (MBL déficient) 7.2% 2.1%
NHS + anti-MASP-2 antibody 5.4% 1.5%
NHS 3.1% 0.73%
Note: “CH50” is the point at which complément mediated hemolysis reaches 50%.
In summary, the results in this Example demonstrate that inhibiting MASP-2 inhibits complement-mediated lysis of sensitized and non-sensitized érythrocytes with déficient protection from autologous complément activation. Therefore, MASP-2 inhibitors may be used to treat subjects suffering from PNH, and may also be used to
J
225 ameliorate (Le., inhibit, prevent or reduce the severity of) extravascular hemolysis in PNH patients undergoing treatment with a C5 inhibitor such as eculizumab (Soliris®).
EXAMPLE 34
This Example describes a follow on study to the study described above in Example 29, providing further evidence confirming that a MASP-2 inhibitor, such as a MASP-2 antibody, is effective for the treatment of radiation exposure and/or for the treatment, amelioration or prévention of acute radiation syndrome.
Rationale: In the initial study described in Example 29, it was demonstrated that preirradiation treatment with an anti-MASP-2 antibody in mice increased the survival of irradiated mice as compared to vehicle treated irradiated control animais at both 6.5 Gy and 7.0 Gy exposure levels. It was further demonstrated in Example 29 that at the 6.5 Gy 15 exposure level, post-irradiation treatment with anti-MASP-2 antibody resulted in a modest increase in survival as compared to vehicle control irradiated animais. This Example describes a second radiation study that was carried out to confirm the results of the First study.
Methods:
Design of Study A:
Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy). The effect of anti-MASP-2 antibody therapy (mAbH6 5mg/kg), administered 18 hours before and 2 hours after radiation exposure, and weekly thereafter, on mortality was assessed.
Results of Study A:
As shown in FIGURE 43, it was determined that administration of the anti-MASP-2 antibody mAbH6 increased survival in mice exposed to 8.0 Gy, with an adjusted médian survival rate increased from 4 to 6 days as compared to mice that received vehicle control, and a mortality reduced by 12% when compared to mice that received vehicle control (log-rank test, p=0.040).
Design of Study B:
226
Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy) in the following groups (I: vehicie) saline control; (II: low) anti-MASP-2 antibody mAbH6 (5 mg/kg) administered 18 hours before irradiation and 2 hours after irradiation; (III: high) mAbH6 (10 mg/kg) administered 18 hours before irradiation and 2 hours post irradiation; and 5 (IV:high post) mAbH6 (lOmg/kg) administered 2 hours post irradiation only.
Results of Study B:
Administration of anti-MASP-2 antibody pre- and post-irradiation adjusted the mean survival frorn 4 to 5 days in comparison to animais that received vehicie control.
Mortality in the anti-MASP-2 antibody-treated mice was reduced by 6-12% in comparison to vehicie control mice. It is further noted that no significant detrimental treatment effects were observed (data not shown).
In summary, the results shown in this Example are consistent with the results shown in Example 29 and further demonstrate that anti-MASP-2 antibodies are effective in treating a mammalian subject at risk for, or suffering from the detrimental effects of 15 acute radiation syndrome.
EXAMPLE 35
This study investigates the effect of MASP-2-deficiency in a mouse model of LPS (lipopolysaccharide)-induced thrombosis.
Rationale:
Hemolytic urémie syndrome (HUS), which is caused by Shiga toxin-producing E. coli infection, is the leading cause of acute rénal failure in children. In this Example, a Schwartzman model of LPS-induced thrombosis (microvascular coagulation) was carried out in MASP-2-/- (KO) mice to détermine whether MASP-2 inhibition is effective to 25 inhibit or prevent the formation of intravascular thrombi.
Methods:
MASP-2-/- (n=9) and WT (n=10) mice were analyzed in a Schwarztman model of LPS-induced thrombosis (microvascular coagulation). Mice were administered Serratia LPS and thrombus formation was monitored over time. A comparison of the incidence of 30 microthromi and LPS-induced microvascular coagulation was carried out.
Results:
227
Notably, ail MASP-2 -/- mice tested (9/9) did not form intravascular thrombi after Serratia LPS administration. In contrast, microthrombi were detected in 7 of 10 of the WT mice tested in parallel (p=0.0031, Fischer’s exact). As shown in FIGURE 44, the time to onset of microvascular occlusion foilowing LPS infection was measured in MASP-2-/- and WT mice, showing the percentage of WT mice with thrombus formation measured over 60 minutes, with thrombus formation detected as early as about 15 minutes. Up to 80% of the WT mice demonstrated thrombus formation at 60 minutes. In contrast, as shown in FIGURE 44, none of the MASP-2 -/- had thrombus formation at 60 minutes (log rank: p=0.0005).
These results demonstrate that MASP-2 inhibition is protective against the development of intravascular thrombi in an HUS model.
EXAMPLE 36
This Example describes the effect of anti-MASP-2 antibodies in a mouse model of HUS using intraperitoneal co-injection of purified Shiga toxin 2 (STX2) plus LPS.
Background:
A mouse model of HUS was developed using intraperitoneal co-injection of purified Shiga toxin 2 (STX2) plus LPS. Biochemical and microarray analysis of mouse kidneys revealed the STX2 plus LPS challenge to be distinct from the effects of either agent alone. Blood and sérum analysis of these mice showed neutrophilia, thrombocytopenia. red cell hemolysis, and increased sérum créatinine and blood urea nitrogen. In addition, histologie analysis and électron microscopy of mouse kidneys demonstrated glomerular fîbrin déposition, red cell congestion, microthrombi formation, and glomerular ultrastructural changes. It was established that this model ol HUS induces ail clinical symptoms of human HUS pathology in C57BL/6 mice including thrombocytopenia, hemolytic anémia, and rénal failure that defme the human disease. (J. Immunol 187(1):172-80 (201 1))
Methods:
C57BL/6 female mice that weighed between 18 to 20 g were purchased from Charles River Laboratories and divided in to 2 groups (5 mice in each group). One group of mice was pretreated by intraperitoneal (i.p.) injection with the recombinant antiMASP-2 antibody mAbMll (100 gg per mouse; corresponding to a final concentration
228 of 5 mg/kg body weight) diluted in a total volume of 150 μΐ saline. The control group received saline without any antibody. Six hours after i.p injection of anti-MASP-2 antibody mAbMl l, ail mice received a combined i.p. injection of a sublethal dose (3 pg/animal; corresponding to 150 pg/kg body weight) of LPS of Serratia marcescens 5 (L6136; Sigma-Aidrich, St. Louis, MO) and a dose of 4.5 ng/animal (corresponding to
225 ng/kg) of STX2 (two times the LD50 dose) in a total volume of 150 μΐ. Saline injection was used for control.
Survival of the mice was monitored every 6 hours after dosing. Mice were culled as soon as they reached the léthargie stage of HUS pathology. After 36 hours. ail mice 10 were culled and both kidneys were removed for immunohistochemistry and scanning électron microscopy. Blood samples were taken at the end of the experiment by cardiac puncture. Sérum was separated and kept frozen at -80°C for measuring BON and sérum Créatinine levels in both treated and control groups.
Immunohistochemistry
One-third of each mouse kidney was fixed in 4% paraformaldéhyde for 24 h.
processed, and embedded in paraffîn. Three-micron-thick sections were eut and placed onto charged slides for subséquent staining with H & E stain.
Electron Microscopy
The middle section of the kidneys was eut into blocks of approximately 1 to 2 mm3, and fixed overnight at 4°C in 2.5% glutaraldehyde in Ix PBS. The fixed tissue subsequently was processed by the Universtty of Leicester Electron Microscopy Facility
Cryostat sections
The other third of the kidneys was, eut into blocks approximately 1 to 2 mm’ and 25 snap frozen in liquid nitrogen and kept at -80°C for cryostat sections and mRNA analysis.
Results:
FIGURE 45 graphically illustrâtes the percent survival of sali ne-treated control mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in the STX/LPS-induced model over time (hours). Notably, as shown in FIGURE 45, ail of the control mice died 30 by 42 hours. In sharp contrast, 100 % of the anti-MASP-2 antibody-treated mice survived throughout the time course of the experiment. Consistent with the results shown in FIGURE 45, it was observed that ail the untreated mice that either died or had to be
229 culled with signs of severe disease had significant glomerular injuries, while the glomeruli of ail anti-MASP-2-treated mice looked normal (data not shown). These results demonstrate that MASP-2 inhibitors, such as anti-MASP-2 antibodies, may be used to treat subjects suffering from, or at risk for developing a thrombotic microangiopathy (TMA), such as hemolytic urémie syndrome (HUS), atypical HUS (aHUS), or thrombotic thrombocytopénie purpura (TTP).
EXAMPLE 37
This Example describes the effect of MASP-2 deficiency and MASP-2 inhibition in a murine FITC-dextran/light induced endothélial cell injury model of thrombosis.
Background/Rationale: As demonstrated in Examples 35 and 36, MASP-2 deficiency (MASP-2 KO) and MASP-2 inhibition (via administration of an inhibitory MASP-2 antibody) protects mice in a model of typical HUS, wherease ail control mice exposed to STX and LPS developed severe HUS and became moribund or died within 48 hours. For example, as shown in FIGURE 54, ail mice treated with a MASP-2 inhibitory antibody and then exposed to STX and LPS survived (Fisher’s exact p<0.01; N=5). Thus, anti-MASP-2 therapy protects mice in this model of HUS.
The following experiments were carried out to analzye the effect of MASP-2 deficiency and MASP-2 inhibition in a fluorescein isothiocyanate (FITC)-dextraninduced endothélial cell injury model of thrombotic microangiopathy (TMA) in order to demonstrate further the benefit of MASP-2 inhibitors for the treatment of HUS, aHUS, TTP, and TMA’s with other étiologies.
Methods:
Intravital microscopy
Mice were prepared for intravital microscopy as described by Frommhold et al., BMC Immunology 12:56-68, 2011. Briefly, mice were anesthetized with intraperitoneal (i.p.) injection of ketamine (125 mg/kg bodyweight, Ketanest, Pfitzer GmbH, Karlsruhe, Germany) and xylazine (12.5 mg/kg body weight; Rompun, Bayer, Leverkusen, Germany) and placed on a heating pad to maintain body température at 37°C. Intravital microscopy was conducted on an upright microscope (Leica, Wetzlar, Germany) with a saline immersion objective (SW 40/0.75 numerical aperture, Zeiss, Jena, Germany). To ease breathing, mice were intubated using PE 90 tubing (Becton Dickson and Company, Sparks, MD, USA). The left carotid artery was cannuled with PE10 tubing (Becton
230
Dickson and Company, Sparks, MD, USA) for blood sampling and systemic monoclonal antibody (mAb) administration.
Cremaster muscle préparation
The surgical préparation of the cremaster muscle for intravital microscopy was performed as described by Sperandio et al., Blood, 97:3812-3819, 2001. Briefly, the scrotum was opened and the cremaster muscle mobilized. After longitudinal incision and spreading of the muscle over a cover glass, the epididymis and testis were moved and pinned to the side, giving full microscopie access to the cremaster muscle microcirculation. Cremaster muscle venules were recorded via a CCD caméra (CF8/1; Kappa, Gleichen, Germany) on a Panasonic S-VHS recorder. The cremaster muscle was superftised with thermo-controlled (35°C bicarbonate-buffered saline) as previously described by Frommhold et al., BMC Immunology 12:56-68, 20112011.
Light excitation FITC dextran injury model
A controlled, light-dose-dependent vascular injury of the endothélium of cremaster muscle venules and artérioles was induced by light excitation of phototoxic (FITC)-dextran (Cat. No. FD150S, Sigma Aldrich, Poole, U.K.). This procedure initiâtes localized thrombosis. As a phototoxic reagent, 60 gL of a 10% w/v solution of FITCdextran was injected through the left carotid artery access and allowed to spread homogenously throughout the circulating blood for 10 minutes. After selecting a wellperfused venule, halogen light of low to midrange intensity (800-1500) was focused on the vessel of interest to induce FITC-dextran fluorescence and mild to moderate phototoxicity to the endothélial surface in order to stimulate thrombosis in a reproducible, controlled manner. The necessary phototoxic light intensity for the excitation of FITCdextran was generated using a halogen lamp (12V, 100W, Zeiss, Oberkochen, Germany). The phototoxicity resulting from light-induced excitation of the fluorochrome requires a threshold of light intensity and/or duration of illumination and is caused by either direct heating of the endothélial surface or by génération of reactive oxygen radicals as described by Steinbauer et al., Langenbecks Arch Surg 385:290-298, 2000.
The intensity of the light applied to each vessel was measured for adjustment by a wavelength-correcting diode detector for low power measurements (Labmaster LM-2, Cohérent, Auburn, USA). Off-line analysis of video scans was performed by means of a computer assisted microcirculation analyzing System (CAMAS, Dr. Zeintl, Heidelberg)
231 and red blood cell velocity was measured as described by Zeintl et al., Int J Microcirc Clin Exp, 8(3):293-302, 2000.
Application of monoclonal anti-human MASP-2 inhibitory antibody (mAbH6) and vehicle control prior to induction of thrombosis
Using a blinded study design, 9-week-old male C57BL/6 WT littermate mice were given i.p. injections of either the recombinant monoclonal human MASP-2 antibody (mAbH6), an inhibitor of MASP-2 functional activity (given at a final concentration of lOmg/kg body weight), or the same quantity of an isotype control antibody (without MASP-2 inhibitory activity) 16 hours before the phototoxic induction of thrombosis in the cremaster model of intravital microscopy. One hour prior to thrombosis induction, a second dose of either mAbH6 or the control antibody was given. MASP-2 knockout (KO) mice were also evaluated in this model.
mAbH6 (established against recombinant human MASP-2) is a potent inhibitor of human MASP-2 functional activity, which cross-reacts with, binds to and inhibits mouse MASP-2 but with lower affmity due to its species specificity (data not shown). In order to compensate for the lower affmity of mAbH6 to mouse MASP-2, mAbH6 was given at a high concentration (lOmg/kg body weight) to overcome the variation in species specificity, and the lesser affmity for mouse MASP-2, to provide effective blockade of murine MASP-2 functional activity under in vivo conditions.
In this blinded study, the time required for each individual venuole tested (sélection criteria were by comparable diameters and blood flow velocity) to fully occlude was recorded.
The percentage of mice with microvascular occlusion, the time of onset, and the time to occlusion were evaluated over a 60-minute observation period using intravital microscopy video recordings.
Results:
FIGURE 46 graphically illustrâtes, as a function of time after injury induction, the percentage of mice with microvascular occlusion in the FITC/Dextran UV model after treatment with isotype control or human MASP-2 antibody mAbH6 (lOmg/kg) dosed at 16 hours and 1 hour prior to injection of FITC/Dextran. As shown in FIGURE 46, 85% of the wild-type mice receiving the isotype control antibody occluded within 30 minutes or less, whereas only 19% of the wild-type mice pre-treated with the human MASP-2
232 antibody (mAbH6) occluded within the same time period, and the time to occlusion was delayed in the mice that did eventually occlude in the human MASP-2 antibody-treated group. It is further noted that three of the MASP-2 mAbH6 treated mice did not occlude at ail within the 60-minute observation period (i.e., were protected from thrombotic 5 occlusion).
FIGURE 47 graphically illustrâtes the occlusion time in minutes for mice treated with the human MASP-2 antibody (mAbH6) and the isotype control antibody. The data are reported as scatter-dots with mean values (horizontal bars) and standard error bars (vertical bars). This figure shows the occlusion time in the mice where occlusion was 10 observable. Thus, the three MASP-2 antibody-treated mice that did not occlude during the 60 minute observation period were not included in this analysis (there was no control treated mouse that did not occlude). The statistical test used for analysis was the unpaired t test; wherein the Symbol indicates p=0.0129. As shown in FIGURE 47, in the four MASP-2 antibody (mAbH6)-treated mice that occluded, treatment with MASP-2 15 antibody signifîcantly increased the venous occlusion time in the FITC-dextran/lightinduced endothélial cell injury model of thrombosis with low light intensity (800-1500) as compared to the mice treated with the isotype control antibody. The average of the full occlusion time of the isotype control was 19.75 minutes, while the average of the full occlusion time for the MASP-2 antibody treated group was 32.5 minutes.
FIGURE 48 graphically illustrâtes the time until occlusion in minutes for wildtype mice, MASP-2 KO mice, and wild-type mice pre-treated with human MASP-2 antibody (mAbH6) administered i.p. at lOmg/kg 16 hours before, and then administered again i.v.l hour prior to the induction of thrombosis in the FITC-dextran/light-induced endothélial cell injury model of thrombosis with low light intensity (800-1500). Only the 25 animais that occluded were included in FIGURE 48; n=2 for wild-type mice receiving isotype control antibody; n=2 for MASP-2 KO; and n=4 for wild-type mice receiving human MASP-2 antibody (mAbH6). The symbol “*” indicates p<0.01. As shown in FIGURE 48, MASP-2 deficiency and MASP-2 inhibition (mAbH6 at lOmg/kg) increased the venous occlusion time in the FITC-dextran/light-induced endothélial cell injury model 30 of thrombosis with low light intensity (800-1500).
Conclusions:
233
The results in this Example further demonstrate that a MASP-2 inhibitory agent that blocks the lectin pathway (e.g., antibodies that block MASP-2 function), inhibits microvascular coagulation and thrombosis, the hallmarks of multiple microangiopathic disorders, in a mouse model of TMA. Therefore, it is expected that administration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, will be an effective therapy in patients suffering from HUS, aHUS, TTP, or other microangiopathic disorders and provide protection from microvascular coagulation and thrombosis.
EXAMPLE 38
This Example describes a study demonstrating that human MASP-2 inhibitory antibody (mAbH6) has no effect on platelet function in platelet-rich human plasma. Background/Rationale: As described in Example 37, it was demonstrated that MASP-2 inhibition with human MASP-2 inhibitory antibody (mAbH6) increased the venous occlusion time in the FITC-dextran/light-induced endothélial cell injury model of thrombosis. The following experiment was carried out to détermine whether the MASP-2 inhibitory antibody (mAbH6) has an effect on platelet function.
Methods: The effect of human mAbH6 MASP-2 antibody was tested on ADP-induced aggregation of platelets as follows. Human MASP-2 mAbH6 at a concentration of either 1 pg/ml or 0.1 pg/ml was added in a 40 pL solution to 360 pL of freshly prepared platelet-rich human plasma. An isotype control antibody was used as the négative control. After adding the antibodies to the plasma, platelet activation was induced by adding ADP at a final concentration of 2 pM. The assay was started by stirring the solutions with a small magnet in the 1 mL cuvette. Platelet aggregation was measured in a two-channel Chrono-log Platelet Aggregometer Model 700 Whole Blood/Optical LumiAggregometer.
Results:
The percent aggregation in the solutions was measured over a time period of five minutes. The results are shown below in TABLE 13.
TABLE 13: Platelet Aggregation over a time period of five minutes.
Antibody Amplitude Slope
234
(percent aggregation) (percent aggregation over time)
MASP-2 antibody (mAbH6) (1 hg/ml) 46% 59
Isotype control antibody (1 hg/ml) 49% 64
MASP-2 antibody (mAbH6) (0.1 pg/ml) 52% 63
Isotype control antibody (0.1 pg/ml) 46% 59
As shown above in TABLE 13, no significant différence was observed between the aggregation of the ADP-induced platelets treated with the control antibody or the MASP-2 mAbH6 antibody. These results demonstrate that the human MASP-2 antibody 5 (mAbH6) has no effect on platelet function. Therefore, the results described in Example 37 demonstrating that MASP-2 inhibition with human MASP-2 inhibitory antibody (mAbH6) increased the venous occlusion time in the FITC-dextran/light-induced endothélial cell injury model of thrombosis, were not due to an effect of mAbH6 on platelet function. Thus, MASP-2 inhibition prevents thrombosis without directly fO impacting platelet function, revealing a therapeutic mechanism that is distinct from existing anti-thrombotic agents.
EXAMPLE 39
This Example describes the effect of MASP-2 inhibition on thrombus formation and vessel occlusion in a murine model of TMA.
Background/Rationale: The lectin pathway plays a dominant rôle in activating the complément System in settings of endothélial cell stress or injury. This activation is amplified rapidly by the alternative pathway, which is dysregulated in many patients
235 presenting with aHUS. Preventing the activation of MASP-2 and the lectin pathway is thus expected to hait the sequence of enzymatic reactions that lead to the formation of the membrane attack complex, platelet activation, and leukocyte recruitment. This effect limits tissue damage.
In addition, MASP-2 has Factor Xa-like activity and cleaves prothrombin to form thrombin. This MASP-2-driven activation of the coagulation System may imbalance hemostasis and resuit in the pathology of TMA. Thus, inhibition of MASP-2 using a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody that blocks activation of the complément and coagulation Systems is expected to improve outcomes in aHUS and 10 other TMA-related conditions.
As described in Example 37, it was demonstrated that MASP-2 inhibition with human MASP-2 inhibitory antibody (mAbH6) increased the venous occlusion time in the FITCdextran/light-induced endothélial cell injury model of thrombosis. In this model of TMA, mice were sensitized by IV injection of FITC- dextran, followed by localized photo15 activation of the FITC- dextran in the microvasculature of the mouse cremaster muscle (Thorlacius H et al., Eur J Clin. Invest 30(9):804-10, 2000; Agero et al., Toxicon 50(5):698-706, 2007).
The following experiment was carried out to détermine whether the MASP-2 inhibitory antibody (mAbH6) has a dose-response effect on thrombus formation and vessel 20 occlusion in a murine model of TMA.
Methods: Localized thrombosis was induced by photo-activation of fluorescein isothiocyanate-labeled dextran (FITC-dextran) in the microvasculature of the cremaster muscle of C57 Bl/6 mice and intravital microscopy was used to measure onset of thrombus formation and vessel occlusion using methods described in Example 37, with 25 the following modifications. Groups of mice were dosed with mAbH6 (2mg/kg, 10 mg/kg or 20 mg/kg) or isotype control antibody (20 mg/kg) were administered by intravenous (iv) injection one hour before TMA induction. The time to onset of thrombus formation and time to complété vessel occlusion were recorded. Video playback analysis of intravital microscopy images recorded over 30 to 60 minutes was used to evaluate 30 vessel size, blood flow velocity, light intensity, rate of onset of thrombus formation as équivalent of platelet adhesion, time to onset of thrombus formation, rate of total vessel occlusion and time until total vessel occlusion. Statistical analysis was conducted using
236
SigmaPlot vl2.0.
Results:
Initiation of Thrombus Formation
FIGURE 49 is a Kaplan-Meier plot showing the percentage of mice with thrombi as a 5 function of time in FITC-Dextran induced thrombotic microangiopathy in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAbH6 at 2 mg/kg, lOmg/kg or 20 mg/kg) or an isotype control antibody. As shown in FIGURE 49, initiation of thrombus formation was delayed in the mAbH6-treated mice in a dosedependent manner relative to the control-treated mice.
FIGURE 50 graphically illustrâtes the médian time to onset (minutes) of thrombus formation as a function of mAbH6 dose (*p<0.01 compared to control). As shown in FIGURE 50, the médian time to onset of thrombus formation increased with increasing doses of mAbH6 from 6.8 minutes in the control group to 17.7 minutes in the 20 mg/kg mAbH6 treated group (p<0.01). The underlying experimental data and statistical analysis 15 are provided in TABLES 14 and 15.
The time to onset of thrombus formation in individual mice recorded based on évaluation of the videographic recording is detailed below in TABLE 14.
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TABLE 14: Time to Onset of Thrombus Formation After Light Dye-induced Injury
Control Treatment mAbH6 Treatment
Time to Onset (minutes) Control 2 mg/kg 10 mg/kg 20 mg/kg
6.07 5.93 12.75 10.00
1.07 6.95 2.53 10.33
8.00 8.92 14.00 21.00
2.40 11.92 3.05 11.50
8.48 12.75 8.00 19.00
4.00 12.53 8.17 10.37
4.00 15.83 22.65
7.83 11.70 16.37
6.83 50.67 21.75*
15.00 32.25*
15.67
* vessels did not show onset during the indicated observation period
The statistical analysis comparing time to onset of occlusion between control and mAbH6 5 treated animais is shown below in TABLE 15.
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TABLE 15: Time to Onset: data from FITC Dex dose response study
Statistic Control mAbH6 (2 mg/kg) mAbH6 (10 mg/kg) mAbH6 (20 mg/kg)
Number of events/number of animais (%) 11/11 (100%) 6/6 (100%) 9/9 (100%) 8/10 (80.0%)
Médian time (minutes) (95% ci) 6.8 (2.4, 8.5) 10.4 (5.9, 12.8) 11.7 (2.5, 15.8) 17.7 (10.0, 22.7)
Wilcoxon p-value* 0.2364 0.1963 0.0016
Event=Time to onset observed Médian (minutes) and its 95% CI were based on Kaplan-Meier estimate NE=not estimable *p-values were adjusted by Dunnett-Hsu multiple comparison__________________________
Microvascular Occlusion
FIGURE 51 is a Kaplan-Meier plot showing the percentage of mice with microvascular 5 occlusion as a function of time in FITC-Dextran induced thrombotic microangiopathy in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAbH6 at 2 mg/kg, lOmg/kg or 20mg/kg) or an isotype control antibody. As shown in FIGURE 51, complété microvascular occlusion was delayed in the mAbH6 treated groups as compared to the control mice.
FIGURE 52 graphically illustrâtes the médian time to microvascular occlusion as a function of mAbH6 dose (*p<0.05 compared to control). As shown in FIGURE 52, the médian time to complété microvascular occlusion increased from 23.3 minutes in the control group to 38.6 minutes in the 2mg/kg mAbH6 treated group (p<0.05). Doses of 10 mg/kg or 20 mg/kg of mAbH6 performed similarly (médian time for complété 15 microvascular occlusion was 40.3 and 38 minutes, respectively) to the 2 mg/kg mAbH6
239 treated group. The underlying experimental data and statistical analysis are provided in TABLES 16 and 17.
The time to complété vessel occlusion in individual mice recorded based on primary évaluation of the videographic recording is detailed below in TABLE 16.
TABLE 16: Time to Complété Occlusion After Light Dye-Induced Injury
Control Treatment mAbH6 Treatment
Time to Occlusion (minutes) Control 2 mg/kg 10 mg/kg 20 mg/kg
37.50 42.3 30.92 38.00
29.07 21.91 17.53 28.00
27.12 24.4 51.38 40.58
19.38 31.38 36.88 33.00
19.55 61.17* 26.83 39.10
18.00 61.55* 40.28 32.03
16.50 55.83 38.53
23.33 71.93* 21.75*
14.83 98.22* 32.25*
30* 33.17*
61.8*
*vessels did not completely occlude during the indicated observation penod.
The statistical analysis comparing time to complété occlusion between control and mAbEI6 treated animais is shown below in TABLE 17.
240
TABLE 17: Time to Complété Microvascular Occlusion: data from FITC Dex dose response study
Statistic Control mAbH6 (2 mg/kg) mAbH6 (10 mg/kg) mAbH6 (20 mg/kg)
Number of events/number of animais (%) 9/11 (81.8%) 4/6 (66.7%) 7/9 (77.8%) 7/10 (70.0%)
Médian time (minutes) (95% ci) 23.3 (16.5, 37.5) 36.8 (21.9, NE) 40.3 (17.5, NE) 38.0 (28.0, 40.6)
Wilcoxon p-value* 0.0456 0.0285 0.0260
Event=Time to occlusion observed Médian (minutes) and its 95% CI were based on Kaplan-Meier estimate NE=not estimable *p-values were adjusted by Dunnett-Hsu multiple comparison
Summary
As summarized in TABLE 18, the initiation of thrombus formation was delayed in the mAbH6 treated mice in a dose-dependent manner relative to the control-treated mice (médian time to onset 10.4 to 17.7 minutes vs 6.8 minutes). The médian time to complété occlusion was significantly delayed in ail mAbH6-treated groups relative to the controltreated groups (Table 18).
241
TABLE 18: Médian Time to Onset of Thrombus Formation and Complété
Occlusion
Control mAbH6 (2 mg/kg) mAbH6 (10 mg/kg) mAbH6 (20 mg/kg)
Médian# time to onset of thrombus formation (minutes) 6.8 10.4 11.7 17.7*
Médian# time to complété microvascular occlusion (minutes) 23.3 36.8* 40.3* 38.0*
#Median values are based on Kaplan-Meier estimate *p<0.05 compared to control (Wilcoson adjusted by Dunnett-Hsu for multiple 5 comparisons)
These results demonstrate that mAbH6, a human monoclonal antibody that binds to MASP-2 and blocks the lectin pathway of the complément system, reduced microvascular thrombosis in a dose-dependent manner in an experimental mouse model of TMA. Therefore, it is expected that administration of a MASP-2 inhibitory agent, 10 such as a MASP-2 inhibitory antibody, will be an effective therapy in patients suffering from HUS, aHUS, TTP, or other microangiopathic disorders such as other TMAs including catastrophic antiphospholipid syndrome (CAPS), systemic Degos disease, and TMAs secondary to cancer, cancer chemotherapy and transplantation and provide protection from microvascular coagulation and thrombosis.
EXAMPLE 40
242
This Example describes the identification, using phage display, of fully human scFv antibodies that bind to MASP-2 and inhibit lectin-mediated complément activation while leaving the classical (C 1 q-dependent) pathway and the alternative pathway components of the immune system intact.
OverView:
Fully human, high-affmity MASP-2 antibodies were identified by screening a phage display library. The variable light and heavy chain fragments of the antibodies were isolated in both a scFv format and in a full-length IgG format. The human MASP-2 antibodies are useful for inhibiting cellular injury associated with lectin pathwaymediated alternative complément pathway activation while leaving the classical (Clq-dependent) pathway component of the immune system intact. In some embodiments, the subject MASP-2 inhibitory antibodies hâve the foilowing characteristics: (a) high affinity for human MASP-2 (e.g., a KD of 10 nM or less), and (b) inhibit MASP-2-dependent complément activity in 90% human sérum with an IC50 of 30 nM or less.
Methods:
Expression of full-length catalytically inactive MASP-2:
The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4), encoding the human MASP-2 polypeptide with leader sequence (SEQ ID NO:5) was subcloned into the mammalian expression vector pCI-Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter région (described in Kaufman R.J. et al., Nucleic Acids Research /9:4485-90, 1991; Kaufman, Methods in Enzymology, 185:531-66 (1991)).
In order to generate catalytically inactive human MASP-2A protein, site-directed mutagenesis was carried out as described in US2007/0172483, hereby incorporated herein by reference. The PCR products were purified after agarose gel electrophoresis and band préparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine-tailed MASP-2A was then cloned into the pGEM-T easy vector and transformed into E. coli. The human MASP-2A was further subcloned into either of the mammalian expression vectors pED or pCI-Neo.
243
The MASP-2A expression construct described above was 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 préparations were not contaminated with other sérum proteins. Media was harvested from confluent cells every 5 second day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium. The MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns
MASP-2A ELISA on ScFv Candidate Clones identifled by panning/scFv conversion and fllter screening
A phage display library of human immunoglobulin light- and heavy-chain variable région sequences was subjected to antigen panning followed by automated antibody screening and sélection to identify high-affinity scFv antibodies to human MASP-2 protein. Three rounds of panning the scFv phage library against HIS-tagged or biotin-tagged MASP-2A were carried out. The third round of panning was eluted first 15 with MBL and then with TEA (alkaline). To monitor the spécifie enrichment of phages displaying scFv fragments against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was carried out. The scFv genes from panning round 3 were cloned into a pHOG expression vector and run in a small-scale filter screening to look for spécifie clones against MASP-2A.
Bacterial colonies containing plasmids encoding scFv fragments from the third round of panning were picked, gridded onto nitrocellulose membranes and grown ovemight on non-inducing medium to produce master plates. A total of 18,000 colonies were picked and analyzed from the third panning round, half from the compétitive elution and half from the subséquent TEA elution. Panning of the scFv phagemid library against 25 MASP-2A followed by scFv conversion and a filter screen yielded 137 positive clones.
108/137 clones were positive in an ELISA assay for MASP-2 binding (data not shown), of which 45 clones were further analyzed for the ability to block MASP-2 activity in normal human sérum.
Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase 30 A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of the MASP-2 scFv candidate clones. MASP-2 serine protease activity is required in order to generate the two protein
244 components (C4b, C2a) that comprise the lectin pathway C3 convertase. Therefore, a MASP-2 scFv that inhibits MASP-2 functional activity (i.e., a blocking MASP-2 scFv), will inhibit de novo formation of lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C3 by C3 5 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic wells via ester or amide linkages, thus facilitating détection of C3b in the ELISA assay.
Yeast mannan is a known activator of the lectin pathway. In the following method to measure formation of C3 convertase, plastic wells coated with mannan were 10 incubated with diluted human sérum to activate the lectin pathway. The wells were then washed and assayed for C3b immobilized onto the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the de novo formation of lectin pathway C3 convertase. MASP-2 scFv clones at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and conséquent 15 C3b génération.
Methods:
The 45 candidate clones identified as described above were expressed, purified and diluted to the same stock concentration, which was again diluted in Ca++ and Mg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCQ, 2.0 mM CaCQ, 20 0.1% gelatin, pH 7.4) to assure that ail clones had the same amount of buffer. The scFv clones were each tested in triplicate at the concentration of 2 pg/mL. The positive control was OMS 100 Fab2 and was tested at 0.4 pg/mL. C3c formation was monitored in the presence and absence of the scFv/IgG clones.
Mannan was diluted to a concentration of 20 pg/mL (1 pg/well) in 50mM 25 carbonate buffer (15mM Na2CO3 + 35mM NaHCO3 + 1.5 mM NaN3), pH 9.5 and coated on an ELISA plate overnight at 4°C. The next day, the mannan-coated plates were washed 3 times with 200 pl PBS. 100 pl of 1% HSA blocking solution was then added to the wells and incubated for 1 hour at room température. The plates were washed 3 times with 200 pl PBS, and stored on ice with 200 pl PBS until addition of the samples.
Normal human sérum was diluted to 0.5% in CaMgGVB buffer, and scFv clones or the OMS 100 Fab2 positive control were added in triplicates at 0.01 pg/mL; 1 pg/mL
245 (only OMS 100 control) and 10 gg/mL to this buffer and preincubated 45 minutes on ice before addition to the blocked ELISA plate. The reaction was initiated by incubation for one hour at 37°C and was stopped by transferring the plates to an ice bath. C3b déposition was detected with a Rabbit α-Mouse C3c antibody followed by Goat a-Rabbit 5 HRP. The négative control was buffer without antibody (no antibody = maximum C3b déposition), and the positive control was buffer with EDTA (no C3b déposition). The background was determined by carrying out the same assay except that the wells were mannan-free. The background signal against plates without mannan was subtracted from the signais in the mannan-containing wells. A cut-off criterion was set at half of the 10 activity of an irrelevant scFv clone (VZV) and buffer alone.
Results: Based on the cut-off criterion, a total of 13 clones were found to block the activity of MASP-2. Ail 13 clones producing > 50% pathway suppression were selected and sequenced, yielding 10 unique clones. Ail ten clones were found to hâve the same light chain subclass, λ3, but three different heavy chain subclasses: VH2, VH3 and 15 VH6. In the functional assay, five out of the ten candidate scFv clones gave IC50 nM values less than the 25 nM target criteria using 0.5% human sérum.
To identify antibodies with improved potency, the three mother scFv clones, identified as described above, were subjected to light-chain shuffling. This process involved the génération of a combinatorial library consisting of the VH of each of the 20 mother clones paired up with a library of naïve, 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.
TABLE 19: Comparison of functional potency in IC5Q (nM) of the lead daughter 25 clones and their respective mother clones (ail in scFv format)
scFv clone 1% human sérum C3 assay (IC50 nM) 90% human sérum C3 assay (IC50 nM) 90% human sérum C4 assay (IC50 nM)
17D20mc 38 nd nd
17D20m d3521Nll 26 >1000 140
17N16mc 68 nd nd
246
17N16m d!7N9 48 15 230
Presented below are the heavy-chain variable région (VH) sequences for the mother clones and daughter clones shown above in TABLE 19.
The Kabat CDRs (31-35 (Hl), 50-65 (H2) and 95-107 (H3)) are bolded; and the Chothia CDRs (26-32 (Hl), 52-56 (H2) and 95-101 (H3)) are underlined.
17D20 35VH-21N11VL heavy chain variable région (VH) (SEQ ID NO:67, encoded by SEQ ID NO:66)
QVTLKESGPVL VKPTETLTLTCTVSGFSLSRGKMG VS WIRQPPGKALE W LAHIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRRG GIDYWGQGTLVTVSS d!7N9 heavy chain variable région (VH) (SEQ ID NO:68)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWLGRTY YRSKWYNDYAVSVKSRTnNPDTSKNQFSLQLNSVTPEDTAVYYCARDPFGVPF DIWGQGTMVTVSS
Presented below are the light-chain variable région (VL) sequences for the mother clones and daughter clones.
The Kabat CDRs (24-34 (Ll); 50-56 (L2); and 89-97 (L3) are bolded; and the Chothia CDRs (24-34 (Ll); 50-56 (L2) and 89-97 (L3) are underlined. These régions are the same whether numbered by the Kabat or Chothia system.
17D20m d3521Nll light chain variable région (VL) (SEQ ID NQ:70. encoded by SEQ ID NO:69)
QPVT TQPPSI .S VSPGOTASITCSGEKLGDKYAYWYQQKPGQSPVLVMYQ DKQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKL TVL
17N16m d!7N9 light chain variable région (VL) (SEQ ID NO:71)
247
SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYD DSDRPSGTPDRFSASNSGNTATLTITRGEAGDEADYYCQVWDIATDHVVFGGGT KLTVLAAAGSEQKLISE
The MASP-2 antibodies OMS 100 and MoAb_d3521Nl 1VL, (comprising a heavy 5 chain variable région set forth as SEQ ID NO:67 and a light chain variable région set forth as SEQ ID NO:70, also referred to as “OMS646” and “mAbH6”), which hâve both been demonstrated to bind to human MASP-2 with high affinity and hâve the ability to block functional complément activity, were analyzed with regard to epitope binding by dot blot analysis. The results show that OMS646 and OMS 100 antibodies are highly 10 spécifie for MASP-2 and do not bind to MASP-1/3. Neither antibody bound to MApl9 nor to MASP-2 fragments that did not contain the CCP1 domain of MASP-2, leading to the conclusion that the binding sites encompass CCP1.
The MASP-2 antibody OMS646 was determined to avidly bind to recombinant MASP-2 (Kd 60-250pM) with >5000 fold selectivity when compared to Cl s, Clr or 15 MASP-1 (see TABLE 20 below):
TABLE 20: Affinity and Specificity of OMS646 MASP-2 antibody-MASP-2 interaction as assessed by solid phase ELISA studies
Antigen KD (pM)
MASP-1 >500,000
MASP-2 62±23*
MASP-3 >500,000
Purified human C1 r >500,000
Purifîed human C1 s -500,000
*Mean±SD; n=12
QMS646 specifically blocks lectin-dependent activation of terminal complément components
Methods:
The effect of OMS646 on membrane attack complex (MAC) déposition was 25 analyzed using pathway-specific conditions for the lectin pathway, the classical pathway
248 and the alternative pathway. For this purpose, the Wieslab Comp300 complément screening kit (Wieslab, Lund, Sweden) was used following the manufacturer’s instructions.
Results:
FIGURE 53A graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions. FIGURE 53B graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathwayspecific assay conditions. FIGURE 53C graphically illustrâtes the level of MAC 10 déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions.
As shown in FIGURE 5 3A, OMS646 blocks lectin pathway-mediated activation of MAC déposition with an IC50 value of approximately lnM. However, OMS646 had no effect on MAC déposition generated from classical pathway-mediated activation 15 (FIGURE 53B) or from alternative pathway-mediated activation (FIGURE 53C).
Pharmacokinetics and Pharmacodynamies of OMS646 following Intravenous (IV) or Subcutaneous (SC) Administration to Mice
The pharmacokinetics (PK) and pharmacodynamies (PD) of OMS646 were 20 evaluated in a 28 day single dose PK/PD study in mice. The study tested dose levels of 5mg/kg and 15mg/kg of OMS646 administered subcutaneously (SC), as well as a dose level of 5mg/kg OMS646 administered intravenously (IV).
With regard to the PK profile of OMS646, FIGURE 54 graphically illustrâtes the OMS646 concentration (mean of n=3 animals/groups) as a function of time after 25 administration of OMS646 at the indicated dose. As shown in FIGURE 54, at 5mg/kg SC, OMS646 reached the maximal plasma concentration of 5-6 ug/mL approximately 1-2 days after dosing. The bioavailability of OMS646 at 5 mg/kg SC was approximately 60%. As further shown in FIGURE 54, at 15 mg/kg SC, OMS646 reached a maximal plasma concentration of 10-12 ug/mL approximately 1 to 2 days after dosing. For ail 30 groups, the OMS646 was cleared slowly from systemic circulation with a terminal halflife of approximately 8-10 days. The profile of OMS646 is typical for human antibodies in mice.
249
The PD activity of OMS646 is graphically illustrated in FIGURES 55A and 55B. FIGURES 55A and 55B show the PD response (drop in systemic lectin pathway activity) for each mouse in the 5mg/kg IV (FIGURE 55A) and 5mg/kg SC (FIGURE 55B) groups. The dashed line indicates the baseline of the assay (maximal inhibition; naïve mouse sérum spiked in vitro with excess OMS646 prior to assay). As shown in FIGURE 55A, following IV administration of 5mg/kg of OMS646, systemic lectin pathway activity immediately dropped to near undetectable levels, and lectin pathway activity showed only a modest recovery over the 28 day observation period. As shown in FIGURE 55B, in mice dosed with 5mg/kg of OMS646 SC, time-dependent inhibition of lectin pathway activity was observed. Lectin pathway activity dropped to near-undetectable levels within 24 hours of drug administration and remained at low levels for at least 7 days. Lectin pathway activity gradually increased with time, but did not revert to pre-dose levels within the 28 day observation period. The lectin pathway activity versus time profde observed after administration of 15mg/kg SC was similar to the 5 mg/kg SC dose (data not shown), indicating saturation of the PD endpoint. The data further indicated that weekly doses of 5mg/kg of OMS646, administered either IV or SC, is sufficient to achieve continuons suppression of systemic lectin pathway activity in mice.
EXAMPLE 41
This Example demonstrates that a MASP-2 inhibitory antibody (OMS646) inhibits aHUS serum-induced complément C5b-9 déposition on the surface of activated human microvascular endothélial cells (HMEC-1) after exposure to sérum from patients with atypical hemolytic urémie syndrome (aHUS) obtained during the acute phase and the remission phase of the disease.
Background/Rationale: The following study was carried out to analyze aHUS serum-induced complément C5b-9 déposition on the surface of activated HMEC-1 cells after exposure to aHUS patient sérum obtained (1) during the acute phase and (2) during the remission phase of the disease in the presence or absence of OMS646, a MASP-2 antibody that specifically binds to MASP-2 and inhibits lectin pathway activation.
Methods:
Patients: Four patients with aHUS, studied both during the acute phase of the disease and in remission, were selected for this study among those included in the
250
International Registry of HUS/TTP and genotyped by the Laboratory of Immunology and Genetics of Transplantation and Rare Diseases of the Mario Negri Institute. One aHUS patient had a heterozygous p.R1210C complément factor H (CFH) mutation and one had anti-CFH autoantibodies, while no mutation or antibodies to CFH were found in the other two aHUS patients.
Tables 21 and 22 summarize the results of screening for complément gene mutations and anti-CFH autoantibodies in the four aHUS patients analyzed in this study along with clinical and biochemical data measured either during the acute phase or at remission.
TABLE 21 : Clinical Parameters of the four aHUS patients in this study
Case No. Mutation or antiCFH Ab Disease phase Platelets (150- 400*103/μ1) LDH (266-500 IU/1) Hemoglobin (14-18g/dl) s-Creatinine (0.55- 1.25mg/dl)
#1 no mutations, no anti-CFH Ab acute 31,000 1396 12.9 2.37
remission 267,000 n.a. 11.5 3.76
#2 CFH-R1210C acute 46,000 1962 7 5.7
remission 268,000 440 13.4 7.24
#3 anti-CFH Ab acute 40,000 3362 9-5 1.77
remission 271,000 338 8.8 0.84
#4 no mutations, no anti-CFH Ab acute 83,000 1219 7.8 6.8
remission 222,000 495 12.2 13
Note: n.a. = not available
TABLE 22: Complément Parameters of the four aHUS patients in this study
Case No. Mutation of antiCFH Ab Disease phase Sérum C3 (83-180 mg/dl) Plasma SC5b-9 (127-400 ng/ml)
#1 no mutations, no anti-CFH Ab acute 51 69
remission n.a. 117
#2 CFH-R1210C acute 79 421
remission 119 233
#3 anti-CFH Ab acute 58 653
remission 149 591
#4 no mutations, no anti-CFH Ab acute 108 n.a.
remission n.a. n.a.
Experimental Methods: Cells from a human microvascular endothélial cell line (HMEC-1) of dermal origin were plated on glass slides and used when confluent.
Confluent HMEC-1 cells were activated with 10 μΜ ADP (adenosine diphosphate) for 10
251 minutes and then incubated for four hours with sérum from the four aHUS patients described above in Tables 23 and 24 collected either during the acute phase of the disease, or from the same aHUS patients at remission, or from 4 healthy control subjects. The sérum was diluted 1:2 with test medium (HBSS with 0.5% BSA) in the presence or in the absence of a MASP-2 inhibitory antibody, OMS646 (100 pg/mL), generated as described above in Example 40, or in the presence of soluble complément receptor 1 (sCRl) (150 pg/mL), as a positive control of complément inhibition. At the end of the incubation step, the HMEC-1 cells were treated with rabbit anti-human complément C5b9 followed by FITC-conjugated secondary antibody. In each experiment, sérum from one healthy control was tested in parallel with aHUS patient sérum (acute phase and remission). A confocal inverted laser microscope was used for acquisition of the fluorescent staining on the endothélial cell surface. Fifteen fields per sample were acquired and the area occupied by the fluorescent staining was evaluated by automatic edge détection using built-in spécifie functions of the software Image J and expressed as pixel2 per field analyzed. The fields showing the lowest and the highest values were excluded from calculation.
For the statistical analysis (one-way ANOVA followed by Tukey’s test for multiple comparisons) results in pixel2 of the 13 fields considered in each experimental condition for each patient and control were used.
Results:
The results of the complément déposition analysis with the sera from the four aHUS patients are summarized below in Table 23A, and the results with the sera from the four healthy subjects are summarized below in Table 23B.
TABLE 23A: Effect of complément inhibitors on aHUS serum-induced C5b-9 déposition on ADP-activated HMEC-1 cells_______________
aHUS Patient # aHUS acute phase aHUS remission phase
untreated +sCRl +OMS646 untreated ±sCRl ±OMS646
Patient # 1 (no mutation, no antiCFH ab) 5076 ± 562° 551 ± 80* 3312 ±422** 4507 ±533° 598±101§ 1650 ±223§
252
Patient #2 (CFHR1210C) 5103 ±648° 497 ± 67* 2435 ± 394* 3705 ± 570° 420 ±65§ 2151±250§§§
Patient #3 (anti-CFH ab) 3322 ±421° 353 ± 64* 2582 ± 479 6790 ±901° 660±83§ 2077 ±353§
Patient #4 (no mutations, no anti- CFH ab) 4267 ± 488° 205 ± 34* 2369 ± 265** 5032 ± 594° 182±29§ 3290± 552§§
TABLE 23B: Effect of complément inhibitors on sera from four healthy control subjects (not suffering from aHUS) on C5b-9 déposition on ADP-activated HMEC-1 cells
Healthy Control Subject # Untreated +sCRl +OMS646
Control Subject #1 (assayed in parallel with aHUS subject #1) 481 ±66 375 ±43 213 ±57
Control Subject #2 (assayed in parallel with aHUS subject #2) 651 ±61 240 ± 33 490 ± 69
Control Subject #3 (assayed in parallel with aHUS subject #3) 602 ± 83 234 ±35 717 ± 109
Control Subject #4 (assayed in parallel with aHUS subject #4) 370 ± 53 144 ±20 313 ± 36
For Tables 23A and 23B: Data are mean ± SE. °P<0.001 vs control; *P<0.001, **P<0.01 vs aHUS acute phase untreated; §P<0.001, §§P<0.01, §§§P<0.05 vs aHUS remission phase untreated.
253
Figure 56 graphically illustrâtes the inhibitory effect of MASP-2 antibody (OMS646) and sCRl on aHUS serum-induced C5b-9 déposition on ADP-activated HMEC-1 cells. In Figure 56, the data are mean ± SE. °P<0.0001 vs control; *P<0.0001 5 vs aHUS acute phase untreated; ΛΡ<0.0001 vs aHUS acute phase + sCRl; §P<0.0001 vs aHUS remission phase untreated and #P<0.0001 vs aHUS remission phase + sCRl.
As shown in Table 23A, 23B and Figure 56, ADP-stimulated HMEC-1 cells exposed to sérum from aHUS patients (collected either in the acute phase or in remission) for four hours in static conditions showed an intense déposition of C5b-9 on cell surface 10 as detected by confocal microscopy. By measuring the area covered by C5b-9, a significantly higher amount of C5b-9 déposition was observed on cells exposed to sérum from aHUS patients than on cells exposed to sérum from healthy control subjects, irrespective of whether aHUS sérum was collected in the acute phase or during remission. No différence in serum-induced endothélial C5b-9 deposits was observed between acute 15 phase and remission.
As further shown in Table 23A, 23B and Figure 56, addition of the MASP-2 antibody OMS646 to aHUS sérum (either obtained from patients during acute phase or in remission) led to a significant réduction of C5b-9 déposition on endothélial cell surface as compared to untreated aHUS sérum. However, the inhibitory effect of OMS646 on C5b-9 20 déposition was less profound than the effect exerted by the complément pan-inhibitor sCRl. Indeed, a statistically significant différence was observed between aHUS seruminduced C5b-9 deposits in the presence of OMS646 vs. sCRl (Figure 56 and Tables 23A and 23B).
When calculated as a mean of the four aHUS patients, the percentages of réduction 25 of C5b-9 deposits (as compared with C5b-9 deposits induced by the untreated sérum from the same patients taken as 100%) observed in the presence ofthe complément inhibitors were as follows:
Acute Phase:
sCRl (150 pg/ml): 91% réduction in C5b-9 deposits 30 OMS646 (100 pg/ml): 40% réduction in C5b-9 deposits
Remission Phase:
254 sCRl (150 pg/ml): 91% réduction in C5b-9 deposits
OMS646 (100 pg/ml): 54% réduction in C5b-9 deposits
Conclusion: The results described in this Example demonstrate that the lectin 5 pathway of complément is stimulated by activated microvascular endothélial cells and that this stimulation is a significant driver for the exaggerated complément activation response characteristic of aHUS. It is also demonstrated that this stimulation of the lectin pathway and resulting exaggerated complément activation response occurs both during the acute phase and in clinical remission of aHUS. Moreover, this finding does not 10 appear to be limited to any particular complément defect associated with aHUS. As further demonstrated in this Example, sélective inhibition of the lectin pathway with a MASP-2 inhibitory antibody such as OMS646 reduces complément déposition in aHUS patients with diverse étiologies.
EXAMPLE 42
This Example demonstrates that a MASP-2 inhibitory antibody (OMS646) inhibits aHUS serum-induced platelet aggregation and thrombus formation on the surface of activated human microvascular endothélial cells (HMEC-1) after exposure to aHUS patient sérum obtained during (1) the acute phase and (2) the remission phase of aHUS.
Methods:
Patients: Three patients (patients #1, #2 and #4 as described in Tables 21, 22, 23A and 23B in Example 41) with aHUS (one patient had a heterozygous p.R1210C CFH mutation, while no mutation or anti-CFH antibodies were found in the other two patients) were studied both during the acute phase of the disease and in remission. The patients 25 were selected for this study among those included in the International Registry of HUS/TTP and genotyped by the Laboratory of Immunology and Genetics of Transplantation and Rare Diseases of the Mario Negri Institute. Five healthy subjects were also selected as blood donors for perfusion experiments.
Methods: Confluent HMEC-1 cells were activated with 10 μΜ ADP for 10 30 minutes and then were incubated for three hours with sera from three aHUS patients (patients #1,2 and 4 described in Example 41) collected during the acute phase of the disease or from the same patients at remission, or with control sera from healthy subjects.
255
The sérum was diluted 1:2 with test medium (HBSS with 0.5% BSA), in the presence or in the absence of a MASP-2 inhibitory antibody, OMS646 (100 pg/mL), generated as described in Example 40; or with sCRl (150 pg/mL), as a positive control of complément inhibition. For patients #1 and #2 additional wells were incubated with sera (from acute 5 phase and remission) diluted 1:2 with test medium containing 100 pg/mL of irrelevant isotype control antibody or with 20 pg/mL of OMS646 (for the latter, case #1 was tested only in remission and case #2 both during the acute phase and at remission).
At the end of the incubation step, HMEC-1 cells were perfused in a flow chamber with heparinized whole blood (10 UI/mL) obtained from healthy subjects (containing the 10 fluorescent dye mepacrine that labels platelets) at the shear stress encountered in the microcirculation (60 dynes/cm2, three minutes). After three minutes of perfusion, the endothelial-cell monolayers were fixed in acetone. Fifteen images per sample of platelet thrombi on the endothélial cell surface were acquired by confocal inverted laser microscope, and areas occupied by thrombi were evaluated using Image J software. The 15 fields showing the loweest and the highest values were excluded from calculation.
For statistical analysis (one-way ANOVA followed by Tukey’s test for multiple comparisons), results in pixel2 of the 13 fields considered in each experimental condition for each patient and control were used.
Results:
The results of the thrombus formation experiments with the sera from the three aHUS patients are summarized below in Table 24A, and the results with the sera from the five healthy subjects are summarized below in Table 24B.
TABLE 24A: Effect of complément inhibitors on aHUS serum-induced thrombus formation (pixel2±SE) on ADP-activated HMEC-1 Cells
Experimental conditions Disease phase aHUS Case #1 thrombus formation (pixel2±SE) (no mutation, no anti-CFH ab) aHUS Case #2 thrombus formation (pixel2±SE) (CW-R1210C) aHUS Case #4 thrombus formation (pixel2±SE) (no mutations, no anti-CFH ab)
untreated acute 5499 ± 600 22320± 1273° 10291± 1362°
256
remission 6468 ± 1012° 3387 ± 443° 17676± 1106°
+sCRl (150 gg/mL) acute 4311 +676 5539 ± 578* 5336 ±1214***
remission 573 ±316§ 977 ± 102§ 2544 ± 498§
+OMS646 (20 gg/mL) acute not determined 6974 ± 556* not determined
remission 832±150§ 1224±252§ not determined
+OMS646 (100 gg/mL) acute 3705 ± 777 9913 + 984* 2836 ±509*
remission 3321 ±945§§§ 733 ± 102§ 1700±321§
+ irrelevant isotype control antibody ( 100 gg/mL) acute 5995 ± 725 18655± 1699 not determined
remission 10885 + 1380 2711 + 371 not determined
TABLE 24B: Effect of complément inhibitors on sera from five healthy control subjects (not suffering from aHUS) in the thrombus formation (pixel2±SE) assay on ADP-activated HMEC-1 Cells__________________________ ___________
Experimental conditions Control #1 thrombus formation (pixel2±SE) Control #2 thrombus formation (pixel2±SE) Control #3 thrombus formation (pixel2±SE) Control #4 thrombus formation (pixel2±SE) Control #5 thrombus formation (pixel2±SE)
untreated 2880±510 1046± 172 1144 ± 193 735± 124 2811 ±609
±sCRl (150 gg/mL) 5192 ±637 1527± 153 1198± 138 2239 ± 243 2384 ±410
±OMS646 (100 gg/mL) 7637± 888 1036± 175 731 ±203 2000 ±356 7177 ± 1477
257
+irrelevant isotype control antibody ( 100 pg/mL 6325 ± 697 1024 + 235 399 ± 82 45269 not determined
Assayed in parallel with sérum from aHUS subject #1 (acute phase sérum) #1 (remission phase sérum) #2 (acute phase sérum) #2 (remission phase sérum) #5 (acute and remission phase sérum)
For Tables 24A and 24B: Data are mean ± SE. °P<0.001 vs control; *P<0.001, ***P<0.05 vs aHUS acute phase untreated; §P<0.001, §§§P<0.05 vs aHUS remission phase untreated.
Figure 57 graphically illustrâtes the effect of MASP-2 antibody (OMS646) and 5 sCRl on aHUS serum-induced thrombus formation on ADP-activated HMEC-1 cells. In Figure 57, the data shown are mean ± SE. °P<0.0001, ooP<0.01 vs control; *P<0.0001, **P<0.01 vs aHUS acute phase untreated; §P<0.0001 vs aHUS remission phase untreated.
As shown in Table 24A and Figure 57, a marked increase in the area covered by 10 thrombi was observed on HMEC-1 cells treated with aHUS sérum, collected either during acute phase or at remission, in comparison to cells exposed to sérum from healthy control subjects (Table 24B and Figure 57). As shown in Figure 57 and Table 24A, OMS646 (at both 100 pg/ml and 20 pg/ml) showed a partial inhibition of thrombus formation on cells pre-exposed to aHUS sérum taken during the acute phase. The anti-thrombogenic effect 15 was comparable between the two different doses of OMS646 and was not different from the effect of sCRl (Figure 57 and Table 24A). Addition of the irrelevant isotype control antibody had no inhibitory effect on aHUS-serum-induced thrombus formation.
As further shown in Figure 57 and Table 24A, the inhibitory effect of OMS646 was even more évident on aHUS sérum collected during remission phase. Indeed, the 20 addition of OMS646, at both 100 pg/ml and 20 pg/ml doses, to aHUS patient sérum collected at remission resulted in a nearly complété inhibition of thrombus formation, similar to that observed with the addition of sCRl. The irrelevant isotype control antibody showed no significant inhibitory effect.
258
When calculated as a mean of the three aHUS patients, the percentages of réduction of the HMEC-1 surface covered by thrombi deposits (as compared with thrombus area induced by the untreated sera from the same patients taken as 100%) recorded with the complément inhibitors were as follows:
Acute Phase:
sCRl (150 pg/ml): 60% réduction
OMS646 (100 μg/ml): 57% réduction
OMS646 (20 qg/ml): 45% réduction
Remission Phase:
sCRl (150 μg/ml): 85% réduction
OMS646 (100 pg/ml): 79% réduction
OMS646 (20 pg/ml): 89% réduction
Discussion of Results:
The results in this Example demonstrate that a MASP-2 inhibitory antibody, such as OMS646 (generated as described in Example 40), has a strong inhibitory effect on aHUS serum-induced thrombus formation on HMEC-1 cells. Surprisingly, the inhibitory effect of OMS646 on thrombus formation was greater than its effect on C5b-9 deposits induced on HMEC-1 (as described in Example 41). It is also surprising that the addition of OMS646, at both 100 μg/ml and 20 pg/ml doses, to aHUS patient sérum collected at remission resulted in nearly a complété inhibition of thrombus formation. Another surprising finding is the observation that OMS646, in both the acute phase and in remission, was as effective as the positive control sCRl, which is a broad and almost complété inhibitor of the complément System (Weisman H. et al., Science 249:146-151, 1990; Lazar H. et aL, Circulation 100:1438-1442, 1999).
It is noted that the control sérum from healthy subjects also induced a modest thrombus formation on HMEC-1 cells. We did not observe a consistent inhibitory effect on control sérum induced thrombus formation with either OMS646 or with sCRl. While not wishing to be bound by any particular theory, it is believed that the control-induced thrombi do not dépend upon complément, as supported by very low C5b-9 deposits
259 observed on HMEC-1 incubated with control sérum (see Example 41).
Conclusion:
In conclusion, the observed anti-thrombotic effect of a MASP-2 inhibitory antibody, such as OMS646, appears substantially greater than one would hâve expected 5 based on the inhibitory effect of OMS646 on C5b-9 déposition observed in this experimental System (as described in Example 41 and shown in Figure 56). For example, as described in Gastoldi et aL, Immunobiology 217:1129-1222 Abstract 48 (2012) entitled “C5a/C5aR interaction médiates complément activation and thrombosis on endothélial cells in atypical hemolytic urémie syndrome (aHUS),” it was determined that 10 addition of a C5 antibody inhibiting C5b-9 deposits (60% réduction) limited thrombus formation on HMEC-1 to a comparable extent (60% réduction). In contrast, the MASP-2 inhibitory antibody (OMS646 at 100 pg/mL) inhibited C5b-9 deposits with mean values of (acute phase = 40% réduction; remission phase = 54% réduction); and inhibited thrombus formation at a substantially higher percent (acute phase = 57% réduction;
remission phase = 79% réduction). In comparison, OMS646 inhibited complément déposition at a lower percentage than did the positive control complément inhibitor (sCRl at 150 pg/mL, acute phase inhibition of C5b-9 déposition = 91% réduction; remission phase = 91% réduction) yet was equally effective as the sCRl positive control in inhibiting thrombus formation (sCRl at 150 pg/mL, acute phase = 60% réduction;
remission phase = 85% réduction). These results demonstrate that a MASP-2 inhibitory antibody (e.g., OMS646) is surprisingly effective at inhibiting thrombus formation in sérum obtained from aHUS subjects both in the acute phase and remission phase.
In accordance with the foregoing, in one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from, or at risk for 25 developing, a thrombotic microangiopathy (TMA) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In one embodiment, the TMA is selected from the group consisting of hemolytic urémie syndrome (HUS), thrombotic thrombocytopénie purpura (TTP) and atypical hemolytic urémie syndrome (aHUS). In 30 one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to an aHUS patient during the acute phase of the disease. In one embodiment, the composition is administered to an aHUS patient during the remission phase (i.e., in a
260 subject that has recovered or partially recovered from an épisode of acute phase aHUS, such remission evidenced, for example, by increased platelet count and/or reduced sérum LDH concentrations, for example as described in Loirat C et aL, Orphanet Journal of Rare Diseases 6:60, 2011, hereby incorporated herein by reference).
In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the foilowing characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2î wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complément pathways intact).
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from a TMA such as aHUS (acute or remission phase), by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from aHUS at a level of at least 20 percent or greater, (such as at least 30%, at least 40%, at least 50%) more than the inhibitory effect on C5b-9 déposition in sérum.
In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from an aHUS patient in remission phase by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such
261 as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum in an aHUS patient in remission phase at a level of at least 20 percent or greater, (such as at least 30%, at least 40%, at least 50%) more than the inhibitory effect on C5b-9 déposition in sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from a TMA comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavychain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDRL1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a lightchain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In one embodiment, the TMA is selected from the group consisting of atypical hemolytic urémie syndrome (aHUS) (either acute or remission phase), HUS and TTP. In one embodiment, the subject is in acute phase of aHUS. In one embodiment, the subject is in remission phase of aHUS.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises
262 administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70. Compétition between binding members may be assayed easily in vitro, for example using ELISA and/or by tagging a spécifie reporter molécule to one binding member which can be detected in the presence of other untagged binding member(s), to enable identification of spécifie binding members which bind the same epitope or an overlapping epitope. Thus, there is presently provided a spécifie antibody or antigenbinding fragment thereof, comprising a human antibody antigen-binding site, which competes with reference antibody OMS646 for binding to human MASP-2.
EXAMPLE 43
This Example demonstrates that a human MASP-2 inhibitory antibody (OMS646) is able to inhibit TMA patient plasma-mediated induction of apoptosis in primary human microvascular endothélial cells (MVECs) of dermal origin.
Background/Rationale:
The pathophysiology of TMA is known to involve an endothélial cell injury induced by various factors that is followed by occlusions of small vessels (e.g., small artérioles and capillaries) by platelet plugs and/or fibrin thrombi (Hirt-Minkowsk P. et al., Nephron Clin Pract 114:c219-c235, 2010; Goldberg R.J. et al., Am J Kidney Dis 56(6):1168-1174, 2010). It has been shown that MVECs undergo apoptotic injury when exposed in vitro to plasma from patients with TMA-related disorders (see Stefanescu et aL, Blood Vol 112 (2):340-349, 2008; Mitra D. et al., Blood 89:1224-1234, 1997). Apoptotic injury associated with TMAs has been documented in MVEC obtained from tissue biopsies (skin, bone, marrow, spleen, kidney, ileum) of such patients. It has also been shown that apoptotic insults to MVECs reduces the levels of membrane-bound
263 complément regulatory proteins in MVECs (see e.g., Mold & Morris, Immunology 102:359-364, 2001; Christmas et al., Immunology 119:522, 2006).
A positive feedback loop involving terminal complément components is believed to be involved in the pathophysiology of TMAs including atypical hemolytic-uremic 5 syndrome (aHUS), and TMAs associated with catastrophic antiphospholipid syndrome (CAPS), Degos disease, and TMAs secondary to cancer, cancer chemotherapy, autoimmunity and transplantation, each of these conditions are known or thought to be responsive to anti-C5 therapy with the mAb eculizumab (Chapin J. et al., Brit. J. Hematol 157:772-774, 2012; Tsai et al., Br JHaematol 162(4):558-559, 2013); Magro C. M. et al., 10 Journal ofRare Diseases 8:185, 2013).
The following experiment was carried out to analyze the ability of human MASP2 inhibitory antibody (OMS646) to block TMA patient plasma-mediated induction of apoptosis in primary human dermal MVECs in plasma samples obtained from patients suffering from aHUS, ADAMTS13 deficiency-related thrombotic thrombocytopénie 15 purpura (TTP), CAPS and System ic Degos disease, as well as TMAs secondary to cancer, transplantation, autoimmune disease and chemotherapy.
Methods:
An in vitro assay was carried out to analyze the efficacy of a MASP-2 inhibitory antibody (OMS646) to block TMA patient plasma-mediated induction of apoptosis in 20 primary human MVECs of dermal origin as described in Stefanescu R. et al., Blood Vol 112 (2):340-349, 2008, which is hereby incorporated herein by reference. The plasma samples used in this assay were obtained from a collection of healthy control subjects and from individuals with either acute-phase or convalescent thrombotic microangiopathies. The presence of microangiopathy in the TMA patients was assessed by detecting 25 schistocytes on a peripheral blood smear. In addition, TTP was diagnosed as described in Stefanescu R. et al., BloodNol 112 (2):340-349, 2008.
Endothélial Cell (EC) Culture
As described in Stefanescu et al., primary human MVECs of dermal origin were purchased from ScienCell Research Labs (San Diego, CA). MVECs expressed CD34 up 30 through passages 5 and 6 (Blood 89:1224-1234, 1997). The MVECs were maintained in polystyrène flasks coated with 0.1% gelatin in water in ECM1001 medium (ScienCell Research Labs) containing endothélial cell growth supplément, penicillin, streptomycin
264 and 15% fêtai bovine sérum. Ail MVECs were used in passages 2 to 6. Subcultures involved a 5 to 10 minute exposure to 0.25% trypsin-EDTA.
Apoptosis Assay
Représentative primary human MVECs of dermal origin known to be susceptible 5 to TTP/HUS plasma-induced apoptosis were washed with phosphate buffered saline (PBS) and plated in chambers of 12-well plates, coated with 0.1% gelatin in water at 0.15xl06 viable cells/mL. The plated MVEC cells were starved in complété media for 24 hours then exposed to varying concentrations (2% to 20% v/v) of TMA patient plasma samples or healthy donor plasma for 18 hours in the presence or absence of MASP-2 10 mAb OMS646 (150 pg/mL) and the cells were then harvested by trypsinization. Each TMA patient sample was analyzed in duplicate. The degree of plasma-mediated apoptosis was assessed using propidium iodide (PI) staining, with >5 xlO3 cells analyzed in a cytofluorograph and A0 peaks defined by computer software (MCycle Av, Phoenix Flow Systems, San Diego, CA). Enzyme-linked immunosorbent assay (ELISA)-based 15 quantitation of cytoplasmic histone-associated DNA fragments from cell lysate was also performed as per the manufacturer’s directions (Roche Diagnostics, Mannheim, Germany).
Results:
The results of the TMA patient plasma-induced MVEC apoptosis assay in the presence of MASP-2 mAb (OMS646) are shown below in Table 25.
Table 25: TMA patient plasma tested on primary human MVEC of dermal origin in the presence of MASP-2 mAb (OMS646)
Subject # Age/ Sex Clinical Diagnosis (TMA) and other conditions MASP-2 ng/ml Diagnosis based on Cre/LDH C5a sC5b9 ADAM S Activity Diagnosis based on ADAMS activity protection with OMS646
#2 41/f TTP 174 TTP 34.42 772 30% aHUS responder
#3 52/f TTP 150 TTP 48.32 1399 70% aHUS non-
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Subject # Age/ Sex Clinical Diagnosis (TMA) and other conditions MASP-2 ng/ml Diagnosis based on Cre/LDH C5a sC5b9 ADAM S Activity Diagnosis based on ADAMS activity protection with OMS646
responder
#4 20/ m TTP 224 TTP 36.9 1187 <10% TTP responder
#10 60/f TTP 175.4 TTP 49.5 4406 64% aHUS nonresponder
#ll 59/f TTP 144.9 TTP 40.3 1352 <10% TTP non- responder
#13 49/f HUS, Cancer, TTP 142.8 TTP 48.6 3843 86% aHUS non- responder
#42 27/ m TTP 341.5 TTP 100.0 5332 <5% TTP non- responder
#46 25/f TTP, Degos, SLE 225.11 TTP 53.9 3426 ND ND responder
#48 53/f TTP, SLE, nephritis s/p rénal transplan t 788.5 aHUS 31.2 1066 66% aHUS responder
#49 64/f TTP, APLAs, CVA 494.5 35.4 2100 ND ND responder
#51 25/f aHUS, APLAs 313.1 TTP 26.8 1595 23% aHUS responder
#52 56/f aHUS, SLE 333.1 TTP 18.9 1103 97% aHUS non- responder
#53 56/f aHUS 189.9 Remissio 28.69 344 74% aHUS non-
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Subject # Age/ Sex Clinical Diagnosis (TMA) and other conditions MASP-2 ng/ml Diagnosis based on Cre/LDH C5a sC5b9 ADAM S Activity Diagnosis based on ADAMS activity protection with OMS646
remission n TTP responder
Abbreviations used in Table 25:
“APLAs” = antiphospholipid antibodies, associated with Catastrophic antiphospholipid syndrome (CAPS).
“SLE” = systemic lupus erythematosus “CVA” = cerebrovascular accident (stroke)
Consistent with the results reported in Stefanescu R. et al., Blood Vol 112 (2):340349, 2008, significant apoptosis was observed for primary MVECs of dermal origin in the presence of the thirteen TMA patient plasma samples in the absence of MASP-2 10 antibody. Control plasma samples from healthy human subjects were run in parallel and did not induce apoptosis in the MVECs (data not shown). As shown in Table 25, the MASP-2 inhibitory mAb (OMS646) inhibited TMA patient plasma-mediated induction of apoptosis in primary MVECs (“responders” in Table 25) in 6 of the 13 patient plasma samples tested (46%). In particular, it is noted that MASP-2 inhibitory mAb (OMS646) 15 inhibited apoptosis in plasma obtained from patients suffering from aHUS, TTP, Degos disease, SLE, transplant, and APLAs (CAPS). With regard to the seven patient samples tested in this assay in which the MASP-2 mAb did not block apoptosis (“non-responders” in Table 25), it is noted that apoptosis can be induced by several pathways, not ail of which are complément dépendent. For example, as noted in Stefanescu R. et al., Blood 20 Vol 112 (2):340-349, 2008, apoptosis in an EC assay is dépendent on the basal EC activation State which is influenced by plasma factors which may play a rôle in determining the level of insult required to induce apoptosis. As further noted in Stefanescu R. et al., additional factors capable of modulating apoptosis may be présent in the TMA patient plasma, such as cytokines and various components of the complément 25 System. Therefore, due to these complicating factors, it is not surprising that the MASP-2
267 antibody did not show a blocking effect in ail of the plasma samples that exhibited TMAplasma induced apoptosis.
Further in this regard, it is noted that a similar analysis was carried out using TMA-plasma induced apoptosis assay with the anti-C5 antibody eculizumab and very similar results were observed (see Chapin et al., Blood (ASH Annual Meeting Abstracts): Abstract #3342, 120: 2012). Clinical efficacy of eculizumab, a highly successful commercial product, appears greater than the efficacy demonstrated in this model, suggesting that this in vitro model may underestimate the clinical potential of complément inhibitory drugs.
These results demonstrate that a MASP-2 inhibitory antibody such as OMS646 is effective at inhibiting TMA-plasma-induced apoptosis in plasma obtained from patients suffering from a TMA such as aHUS, TTP, Degos disease, SLE, transplant, and APLAs (CAPS). It is known that endothélial damage and apoptosis play a key rôle in the pathology of TMAs such as idiopathic TTP and sporadic HUS (Kim et al., Microvascular Research vol 62(2):83-93, 2001). As described in Dang et al., apoptosis was demonstrated in the splenic red pulp of TTP patients but not in healthy control subjects (Dang et al., Blood 93(4):1264-1270, 1999). Evidence of apoptosis has also been observed in rénal glomerular cells of MVEC origin in an HUS patient (Arends M. J. et al., Hum Pathol 20:89, 1989). Therefore, it is expected that administration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody (e.g., OMS646) will be an effective therapy in patients suffering from a TMA such as aHUS, TTPor other microangiopathic disorder such as other TMAs including CAPS, systemic Degos disease, and a TMA secondary to cancer; a TMA secondary to chemotherapy, or a TMA secondary to transplantation.
In accordance with the foregoing, in one embodiment, the invention provides a method of inhibiting endothélial cell damage and/or endothélial cell apoptosis, and/or thrombus formation in a subject suffering from, or at risk for developing, a thrombotic microangiopathy (TMA) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2dependent complément activation. In one embodiment, the TMA is selected from the group consisting of atypical hemolytic urémie syndrome (aHUS), thrombotic thrombocytopénie purpura (TTP) and hemolytic urémie syndrome (HUS). In one
268 embodiment, the TMA is aHUS. In one embodiment, the composition is administered to an aHUS patient during the acute phase of the disease. In one embodiment, the composition is administered to an aHUS patient during the remission phase (i.e., in a subject that has recovered or partially recovered from an épisode of acute phase aHUS, 5 such remission evidenced, for example, by increased platelet count and/or reduced sérum LDH concentrations, for example as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, hereby incorporated herein by reference).
In one embodiment, the subject is suffering from, or at risk for developing a TMA that is (i) a TMA secondary to cancer; (ii) a TMA secondary to chemotherapy; or (iii) a TMA 10 secondary to transplantation (e.g., organ transplantation, such as kidney transplantation or allogeneic hematopoietic stem cell transplantation). In one embodiment, the subject is suffering from, or at risk for developing Upshaw-Schulman Syndrome (USS). In one embodiment, the subject is suffering from, or at risk for developing Degos disease. In one embodiment, the subject is suffering from, or at risk for developing Catastrophic 15 Antiphospholipid Syndrome (CAPS).
In accordance with any of the disclosed embodiments herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in 20 vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 25 molécule, wherein the IgG4 molécule comprises a S228P mutation, and/or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the 30 classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
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In one embodiment, the MASP-2 inhibitory antibody inhibits plasma induced MVEC apoptosis in sérum from a subject suffering from a TMA such as aHUS (acute or remission phase), hemolytic urémie syndrome (HUS), thrombotic thrombocytopénie purpura (TTP), a TMA secondary to cancer; a TMA secondary to chemotherapy; a TMA 5 secondary to transplantation (e.g., organ transplantation, such as kidney transplantation or allogeneic hematopoietic stem cell transplantation), or in sérum from a subject suffering from Upshaw-Schulman Syndrome (USS), or in sérum from a subject suffering from Degos disease, or in a subject suffering from Catastrophic Antiphospholipid Syndrome (CAPS), wherein the plasma induced MVEC apoptosis is inhibited by at least 5%, such 10 as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% such as at least 85%, such as at least 90%, such as at least 95% up to 99%, as compared to untreated sérum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in sérum from a subject suffering from a TMA (e.g., such as aHUS (acute or 15 remission phase), hemolytic urémie syndrome (HUS), thrombotic thrombocytopénie purpura (TTP), a TMA secondary to cancer; a TMA secondary to chemotherapy; a TMA secondary to transplantation (e.g., organ transplantation, such as kidney transplantation or allogeneic hematopoietic stem cell transplantation), or in sérum from a subject suffering from Upshaw-Schulman Syndrome (USS), or in sérum from a subject suffering from 20 Degos disease, or in a subject suffering from Catastrophic Antiphospholipid Syndrome (CAPS)), at a level of at least 20 percent or greater, (such as at least 30%, at least 40%, at least 50%) more than the inhibitory effect on C5b-9 déposition in sérum.
In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous cathéter or other cathéter delivery method.
In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from a TMA (such as aHUS (acute or remission phase), hemolytic urémie syndrome (HUS), thrombotic thrombocytopénie purpura (TTP), a TMA secondary to cancer; a TMA secondary to chemotherapy; a TMA secondary to transplantation (e.g., organ transplantation, such as kidney transplantation or allogeneic 30 hematopoietic stem cell transplantation), or in sérum from a subject suffering from Upshaw-Schulman Syndrome (USS), or in sérum from a subject suffering from Degos disease, or in a subject suffering from Catastrophic Antiphospholipid Syndrome (CAPS)),
270 comprising administering to the subject a composition comprising an amount of a MASP2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavychain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-102 ofSEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In one embodiment, the subject is suffering from a TMA selected from the group consisting of a TMA secondary to cancer; a TMA secondary to chemotherapy; a TMA secondary to transplantation (e.g., organ transplantation, such as kidney transplantation or allogeneic hematopoietic stem cell transplantation), Upshaw-Schulman Syndrome (USS), Degos disease, and Catastrophic Antiphospholipid Syndrome (CAPS).
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67. In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as
271 set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
EXAMPLE 44
This Example describes the initial results of an ongoing Phase 2 Clinical Trial to Evaluate the Safety and Clinical Efficacy of a fully human monoclonal MASP-2 inhibitory antibody in Adults with Thrombotic Microangiopathies (TMAs).
Background: TMAs are a family of rare, debilitating and life-threatening disorders characterized by excessive thrombi (clots) - aggregations of platelets - in the microcirculation ofthe body’s organs, most commonly the kidney and brain.
Methods:
The first stage of an open-label Phase 2 clinical trial was carried out in subjects with primary atypical hemolytic urémie syndrome (aHUS), plasma therapy-resistant aHUS and thrombotic thrombocytopénie purpura (TTP). This Phase 2 clinical trial has no placebo arm given the life-threatening nature of the disease.
The subjects were âge >18 at screening and were only included in this study if they had a diagnosis of one of the following TMAs:
1) Primary aHUS, diagnosed clinically and having ADAMTS13 activity > 10% in plasma. Patients are eligible with or without a documented complément mutation or anti-CFH antibody. Patients are categorized according to their response to plasma therapy (plasma exchange or plasma infusion):
o Plasma therapy-resistant aHUS patients, for purposes of this study, meet ail ofthe following:
(a) screening platelet count < 150,000/pL despite at least four plasma therapy treatments prior to screening;
(b) evidence of microangiopathic hemolysis (presence of schistocytes, sérum lactate dehydrogenase (LDH) > upper limit of normal (ULN), haptoglobin < LLN); and (c) sérum créatinine > ULN.
o Chronic plasma therapy-responsive aHUS patients (plasma therapysensitive) must require at least once-per-week plasma therapy for four weeks before first dose of OMS646 with sérum créatinine > ULN.
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2) TTP defined as having ail of the following:
o Platelet count < 150,000/pL;
o Evidence of microangiopathic hemolysis (presence of schistocytes, sérum LDH > ULN, or haptoglobin < LLN); and o ADAMTS13 activity < 10% during the current épisode of TTP or historically.
Note: Subjects were excluded from the study if they had eculizumab therapy within three months prior to screening. The criteria for a patient qualifying as plasma therapyresistant were for purposes of determining eligibility in this study. In further aspects of the invention, a plasma therapy-resistant patient to be treated with a MASP-2 inhibitor was previously treated with plasma therapy at least once and after such plasma therapy treatment still had one or more clinical markers of aHUS that were not adequately reduced or eliminated by such plasma therapy treatment.
The monoclonal antibody used in this study, OMS646, is a fully human IgG4 mAb directed against human MASP-2. As demonstrated in Example 40, OMS646 avidly binds to recombinant MASP-2 (apparent equilibrium dissociation constant in the range of 100 pM) and exhibits greater than 5,000-fold selectivity over the homologous proteins Cls, Clr, and MASP-1. In functional assays, OMS646 inhibits the human lectin pathway with nanomolar potency (concentration leading to 50% inhibition [IC50] of approximately 3 nM) but has no significant effect on the classical pathway. OMS646 administered either by intravenous (IV) or subeutaneous (SC) injection to mice, non-human primates, and humans resulted in high plasma concentrations that were associated with suppression of lectin pathway activation in an ex vivo assay. As further described in Example 42, OMS646 treatment reduced C5b-9 déposition and thrombus formation in in vitro models of TMA and thrombus formation in a mouse model of TMA, thus demonstrating that OMS646 has therapeutic utility.
In this study, the OMS646 drug substance was provided at a concentration of 100 mg/mL, which was further diluted for IV administration. The appropriate calculated volume of OMS646 100 mg/mL injection solution was withdrawn from the vial using a
273 syringe for dose préparation. The infusion bag was administered within four hours of préparation.
In Stage 1 of the study, OMS646 was administered to escalating dose cohorts of three subjects per cohort. Each subject in Stage 1 received four weekly doses of OMS646 as shown below in TABLE 26.
TABLE 26: Dosing Schedule For Stage 1
Stage, Cohort Number of
Subjects OMS646 Dose (mg/kg)
1, Cohort 1 3 0.675, weekly x 4
1, Cohort 2 3 2.0, weekly x 4
1, Cohort 3 3 4.0, weekly x 4
Stage 1 Study Design Schematic
Treatment Period
The diluted study drug was infused intravenously over a 30-minute period.
Primary Endpoints
The co-primary endpoints were:
Safety as assessed by AEs, vital signs, ECGs, and clinical laboratory tests
274 • Clinical activity as assessed by change in platelet count
Secondary Endpoints
The secondary endpoints were:
• TMA clinical activity o Sérum LDH o Sérum haptoglobin o Hemoglobin o Sérum créatinine o TMA-related symptoms o Need for plasma therapy (plasma exchange or plasma infusion) o Need for dialysis
Allowed Concomitant Thérapies • Plasma therapy-resistant aHUS - plasma therapy during OMS646 treatment was allowed if the investigator considered it to be medically indicated.
• Chronic plasma therapy-responsive aHUS - investigators were advised that plasma therapy should be continued until there was a sign of improvement in TMA, e.g., increase in platelet count, decrease in LDH, increase in haptoglobin, increase in hemoglobin, decrease in créatinine, at which time the investigator was advised to consider withholding plasma therapy and monitoring TMA parameters to assess whether plasma therapy can be discontinued.
• TTP - plasma therapy was allowed if the investigator considered it to be medically indicated.
• Investigators were advised that rénal dialysis therapy should be managed according to standard of care.
• Eculizumab was not administered during the study.
275
Results:
The first cohort of subjects consisted of three aHUS patients treated with the lowest dose of OMS646. Improvements were observed across TMA disease markers in ail patients in this study cohort. Platelet count, sérum lactate dehydrogenase (LDH) and sérum haptoglobin were measured as markers of disease activity. When compared to baseline levels, platelet counts improved in ail patients. Sérum LDH levels remained normal in one patient, substantially decreased to close to the normal range in another and remained elevated in the third. Haptoglobin improved in two patients, normalizing in one. Créatinine levels in the one patient with independent rénal function also improved.
As designed, three patients were treated in the second, mid-dose cohort of this clinical trial. Two patients hâve plasma therapy-resistant aHUS and one patient has thrombotic thrombocytopénie purpura (TTP). Both patients with aHUS were on rénal dialysis prior to and at the time of study enrollment. In the second or mid-dose cohort, the two patients with plasma therapy-resistant aHUS demonstrated:
• 47% increase in mean platelet count, resulting in both patients having counts in the normal range • 86% decrease in mean schiztocyte count, with schistocytes disappearing in one patient • 71% increase in mean haptoglobin with both patients reaching the normal range during treatment, one slipping slightly below normal at one week following the last dose • 5% decrease in the mean levels of LDH, with levels in both patients remaining slightly elevated above normal range.
The mid-dose-cohort patient with TTP required repeated plasma infusion therapy prior to entering the study. Laboratory parameters did not show consistent improvement, but the patient did not require plasma therapy while on treatment with OMS646, nor, to date, since completion of treatment.
The drug was well tolerated by ail patients throughout the treatment period. Based on the positive data from the second or mid-dose cohort, the third or high-dose cohort was initiated and an aHUS patient has already completed the study treatment
276 period. The data referenced for ail patients include measures to one week following the last dose.
The first patient in the third (high-dose) cohort - a plasma therapy-resistant aHUS patient with additional complicating disorders including hepatitis C, cryoglobulinemia and lymphoma - has also completed treatment with OMS646. Prior to OMS646 treatment, the patient required repeated dialysis. Throughout treatment and following completion of the OMS646 course, to date the patient has remained off dialysis. Hematological and rénal parameters showed:
• 63% improvement in platelet count, retuming to normal levels • 100% decrease in shistocytes • Haptoglobin increased from an undetectable level and normalized • 43% decrease in LDH, resulting in a level just slightly above normal • 24% réduction in créatinine level
As in the first and second cohorts, the drug was well tolerated.
The primary endpoint in this open-label Phase 2 clinical trial is change in platelet count. Platelet counts in ail three aHUS patients in the mid- and high-dose cohorts (two in the mid-dose and one in the high-dose cohort) retumed to normal, with a statistically significant mean increase from baseline of approximately 68,000 platelets/mL (p=0.0055).
In summary, the data obtained so far from this Phase 2 clinical trial show efficacy of OMS646 in patients with primary aHUS, plasma-therapy résistant aHUS and in patients with TTP.
In accordance with the foregoing, in one embodiment, the invention provides a method of treating a subject suffering from plasma therapy-resistant aHUS comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In some embodiments, the method further comprises the step of identifying a subject suffering from plasma therapy-resistant aHUS prior to administering to the subject a composition comprising a MASP-2 inhibitory antibody.
277
In accordance with any of the disclosed embodiments herein, the MASP-2 inhibitory antibody exhibits at least one or more of the foilowing characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in 5 vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected frcm the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain moiecule, wherein said antibody is an IgG2 moiecule, wherein said antibody is an IgGl moiecule, wherein said antibody is an IgG4 10 moiecule, wherein the IgG4 moiecule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an 15 amount effective to improve at least one or more clinical parameters associated with aHUS such as an increase in platelet count, a decrease in LDH, an increase in haptoglobin, an increase in hemoglobin, and/or a decrease in créatinine.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from plasma therapy-resistant aHUS via a cathéter (e.g., 20 intravenously) for a first time period (e.g., at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory antibody to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasma therapy. In some embodiments, the administration in the first and/or 25 second time period occurs in the presence of plasma therapy.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody, to a subject suffering from plasma therapy-resistant aHUS either intravenously, intramuscularly, or preferably, subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every two weeks. The MASP-2 inhibitory 30 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
278
In one embodiment, the method comprises treating a subject suffering from plasma therapy-resistant aHUS comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavy-chain 5 CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-107 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDRL1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light10 chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ
ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding 20 fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment 25 thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
EXAMPLE 45
This Example describes additionai results obtained in the ongoing Phase 2 Clinical trial to evaluate the Safety and Clinical Efficacy of a fully human monoclonal
279
MASP-2 inhibitory antibody in Adults with Thrombotic Microangiopathies (TMAs) described in Example 44.
Background: As described in Example 44, TMAs are a family of rare, debilitating and life-threatening disorders characterized by excessive thrombi (clots) - aggregations of platelets - in the microcirculation of the body’s organs, most commonly the kidney and brain. As described herein, transplantation-associated TMA (TA-TMA) is a devastating syndrome that can occur in transplant patients, such as hematopoietic stem cell transplant (HSCT) récipients. This Example describes the initial results of the Phase 2 Clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in a patient suffering from hematopoietic stem cell transplant-related TMA.
Methods:
As described in Example 44, the Phase 2 TMA trial consists of a three-level doseranging stage, followed by a fixed-dose stage of the MASP-2 inhibitory antibody OMS646. As further described in Example 44, positive results were obtained from aHUS patents and one TTP patient, with consistent and robust improvement in efficacy measures. As in the low-dose cohort, OMS646 was well tolerated by ail patients in the mid- and high-dose cohorts throughout the treatment period. Preclinical toxicity studies hâve been completed and demonstrated no safety concems, allowing chronic dosing in clinical trials.
As described in Example 44, data from the OMS646 Phase 2 TMA clinical trial were obtained from aHUS and TTP patients. Dosing has now been completed for an additional hematopoietic stem cell transplant-related TMA patient in the high-dose cohort using the methods described in Example 44. This is a patient with a history of lymphoma for which he underwent hematopoietic stem cell transplant. His post-transplant course has been complicated by a number of life-threatening disorders, including platelet transfusion-requiring TMA. Despite transfusions, his stem cell transplant-related TMA persisted and he was enrolled in the OMS646 Phase 2 Trial.
Results:
Following the four-week dosing period (high-dose cohort) as described in Example 44, the patient with stem cell transplant-related TMA demonstrated:
280 • Platelet count quadrupled, resulting in a platelet count of more than 100,000;
• Haptoglobin level more than doubled and was normal;
• Plasma lactate dehydrogenase level, a measure of damage within blood vessels, decreased by 35% but was still above normal;
• Shistocyte count remained at only one.
Throughout dosing with OMS646 and since completing OMS646 treatment, the patient has not required any platelet transfusions or plasmapheresis.
In summary, the data obtained so far from this Phase 2 clinical trial (as described in Example 44 and in this Example) show efficacy of OMS646 in patients with primary aHUS, plasma-therapy résistant aHUS, TTP and in a patient with TMA associated with hematopoietic stem cell transplant.
In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from a TMA associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In one embodiment, the subject is suffering from a TMA associated with hematopoietic stem cell transplant that is résistant to treatment with platelet transfusions and/or plasmapheresis. In one embodiment, the method further comprises identifying a human subject suffering from a TMA associated with hematopoietic stem cell transplant that is résistant to treatment with platelet transfusions and/or plasmapheresis prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complément activation.
In accordance with any of the disclosed embodiments herein, the MASP-2 inhibitory antibody exhibits at least one or more of the foilowing characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and
281
F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not 5 substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with TMA associated with hematopoietic stem cell transplant, such as an increase in platelet 10 count (e.g., at least double, at least triple, at least quadruple the platelet count prior to treatment), an increase in haptoglobin, and/or a decrease in lactate dehydrogenase.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from TMA associated with hematopoietic stem cell transplant via a cathéter (e.g., intravenously) for a First time period (e.g., at least one day 15 to a week or two weeks) followed by administering a MASP-2 inhibitory antibody to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasma therapy. In some embodiments, the administration in the first and/or second time period occurs in the presence of plasma 20 therapy.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from TMA associated with hematopoietic stem cell transplant either intravenously, intramuscularly, or preferably, subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every two 25 weeks. The MASP-2 inhibitory antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the method comprises treating a subject suffering from TMA associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen 30 binding fragment thereof, comprising (I) (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65
282 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-107 ofSEQ ID NO:67 and b) a light-chain variable région comprising: i) a lightchain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
EXAMPLE 46
This Example describes additional results obtained in the ongoing Phase 2 Clinical trial to evaluate the Safety and Clinical Efficacy of a fully human monoclonal MASP-2 inhibitory antibody in adults with Thrombotic Microangiopathies (TMAs) described in Examples 44 and 45.
Background: As described in Example 45, transplantation-associated TMA (TATMA) is a devastating syndrome that can occur in transplant patients, such as hematopoietic stem cell transplant (HSCT) récipients. Hematopoietic stem cell transplant-associated TMA (HSCT-TMA) is a life-threatening complication that is
283 triggered by endothélial injury. The kidney is the most commonly affected organ, though HSCT-TMA can be a multi-system disease that also involves the lung, bowel, heart and brain. The occurrence of even mild TMA is associated with long-term rénal impairment. Development of post-allogeneic HSCT-associated TMA differs in frequency based on varying diagnostic criteria and conditioning and graft-versus-host disease prophylaxis regimens, with calcineurin inhibitors being the most frequent drugs implicated (Ho VT et al., Biol Blood Marrow Transplant, 11(8):571-5, 2005). Modification of the immunosuppressive calcineurin inhibitor regimen may resuit in improvement of the TMA in some patients within a few weeks of the réduction or discontinuation of calcineurin inhibitor administration.
TMA is a potentially life-threatening complication of HSCT and is currently managed largely by amelioration of inciting factors including avoidance of immunosuppressive agents (e.g., calcineurin inhibitors) and treatment of ongoing infections, as well as supportive measures such as hemodialysis. Although many HSCTTMA patients respond well to réduction or discontinuation of immunosuppressive agents, there is a subset of patients that hâve persistent HSCT-TMA despite conservative treatment measures (i.e., the TMA did not respond to réduction or discontinuation of immunosuppressives). Patients who do not respond to these conservative treatment measures hâve poor prognosis. Plasma exchange has not shown efficacy and no other therapy is approved.
Example 45 describes the initial positive results of the Phase 2 Clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody (OMS646) in a patient suffering from persistent hematopoietic stem cell transplant-related TMA (HSCT-TMA). This Example describes additional results of the ongoing Phase 2 Clinical trial to evaluate the safety and clinical efficacy of OMS646 in three additional subjects suffering from persistent HSCT-TMA résistant to conservative treatment measures.
Methods:
As described in Example 44, the Phase 2 TMA trial consists of a three-level doseranging stage, followed by a fixed-dose stage of the MASP-2 inhibitory antibody OMS646. OMS646 was well tolerated by ail patients in the low, mid and high-dose
284 cohorts throughout the treatment period. Preclinical toxicity studies hâve been completed and demonstrated no safety concerns, allowing chronic dosing in clinical trials.
The Phase 2 TMA trial includes subjects suffering from persistent HSCTassociated TMA résistant to conservative treatment measures, which is defined, for the purposes of this study, as having ail of the following at least two weeks following réduction or discontinuation of immunosuppression agent (e.g., calcineurin inhibitor treatment) or at least 30 days after transplant:
Thrombocytopenia (Platelet count < 150,000/pL); and
Evidence of microangiopathic hemolytic anémia (presence of schistocytes, sérum lactate dehydrogenase (LDH) > upper limit of normal (ULN), or haptoglobin < lower limit of normal (LLN).
Allowed Concomitant Thérapies for HSCT-associated TMA • Plasma therapy during OMS646 treatment is allowed if the investigator considers it medically indicated. Patients given plasma therapy could receive additional half doses of OMS646.
• Investigators were advised that rénal dialysis therapy should be managed according to standard of care.
• Eculizumab was not administered during the study.
The ongoing TMA study includes subjects suffering from persistent HSCT-TMA that is résistant to standard treatment measures (i.e., persistent TMA at least two weeks after réduction or discontinuation of calcineurin inhibitor treatment).
As described in Example 45, dosing was completed for a patient suffering from persistent HSCT-TMA (patient #1) in the high dose cohort (4 mg/kg OMS646 administered IV once a week for four weeks) using the methods described in Example 44.
Dosing has now been completed for an additional 4 patients suffering from persistent HSCT-TMA, as described below.
Patient #1 (described in Example 44) was treated for four weeks with OMS646 (4mg/kg IV once weekly);
285
Patients #2 and #3 were treated for eight weeks with OMS646 (4mg/kg IV once weekly);
Patients #4 and #5 were treated with OMS646 (4mg/kg IV once weekly) for two and three weeks, respectively. Patients #4 and #5 withdrew from the study after two and three weeks, respectively, did not respond to treatment and deteriorated. It is noted that one of these patients had markedly elevated créatinine at the time of study admission.
Results:
Five patients with persistent HSCT-TMA were enrolled in this study. Ail subjects were adults and received HSCT for hematological malignancies. FIGURES 58, 59 and 60 provide the change from baseline in TMA variables after treatment with MASP-2 inhibitory antibody OMS646. These figures provide data on ail five patients, two of whom discontinued the study after 2-3 weeks, with one subsequently relapsing and the other receiving palliative care. As noted in FIGURES 58, 59 and 60, the last possible treatment given occurred at week 7.
FIGURE 58 graphically illustrâtes the mean change in platelet count from baseline over time (weeks) in subjects suffering from persistent hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) after treatment with MASP-2 inhibitory antibody (OMS646).
FIGURE 59 graphically illustrâtes the mean change in LDH from baseline over time (weeks) in subjects suffering from persistent (HSCT-TMA) after treatment with MASP-2 inhibitory antibody (OMS646).
FIGURE 60 graphically illustrâtes the mean change in haptoglobin from baseline over time (weeks) in subjects suffering from persistent (HSCT-TMA) after treatment with MASP-2 inhibitory antibody (OMS646).
As shown in FIGURES 59 and 60, statistically significant improvement in LDH and haptoglobin were observed during treatment. As shown in FIGURE 58, platelet count improved but did not reach statistical significance in this small number of patients. Of the three patients who completed treatment, one did not show improvement in créatinine but was receiving concomitant nephrotoxic agents. Créatinine improved or remained normal in the other two patients. On extended follow up, one patient
286 experienced graft failure and is awaiting a second transplant. The other two patients remain stable.
Despite potentially confounding effects of médications associated with bone marrow suppression and nephrotoxicity in two of the subjects, bénéficiai treatment effects 5 with OMS646 were observed in three of the subjects suffering from persistent HSCTTMA (one of which is described in Example 45) and further described below. Notably, treatment effects in each of the three responders were initially seen after approximately three weeks of treatment with OMS646.
Patient #1 (treated for 4 weeks as described in Example 45). Briefly summarized, 10 the platelet count quadrupled, resulting in a platelet count of more than 100,000/gL; the haptoglobin level more than doubled and was normal; the plasma lactate dehydrogenase level decreased by 35% but was still above normal; and the shistocyte count remained at only one.
Patient #2 (treated for 8 weeks): the platelet count did not respond to treatment, 15 although the patient also suffered from marrow suppression secondary to concurrent treatment with valganciclovi and later graft failure; the plasma lactate dehydrogenase level decreased from 712 U/L to as low as 256 U/L; the haptoglobin level increased from undetectable levels to as high as 250 mg/dL.
Patient #3 (treated for 8 weeks): the platelet count increased from 13,500/μΕ to 20 133,000/μΕ; the plasma lactate dehydrogenase level decreased from 537 to 225 U/L, the haptoglobin level increased from undetectable levels to as high as 181 mg/dL.
Summary of Results:
For the three patients suffering from persistent HSCT-associated TMA who completed dosing with OMS646, the data demonstrate a strong and consistent efficacy 25 signal. Statistically significant improvements in LDH and haptoglobin were observed during treatment. Platelet counts improved but did not reach statistical significance in this small number of patients. Of the three patients who completed treatment, one did not show an improvement in créatinine but was receiving concomitant nephrotoxic agents. Créatinine improved or remained normal in the other two patients.
Conclusions:
OMS646 improved TMA markers in patients suffering from persistent HSCTTMA who had not responded to conservative treatment measures. The HSCT-TMA
287 patients treated with OMS646 represent some of the most difficult to treat, thereby demonstrating clinical evidence of a therapeutic effect of OMS646 in patients with highrisk persistent HSCT-TMA despite conservative treatment measures.
In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from a TMA associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In one embodiment, the subject is suffering from persistent TMA associated with hematopoietic stem cell transplant that is résistant to conservative treatment measures. In one embodiment, the method further comprises identifying a human subject suffering from persistent TMA associated with hematopoietic stem cell transplant that is résistant to conservative treatment measures prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation.
In accordance with any of the disclosed embodiments herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with TMA associated with hematopoietic stem cell transplant, such as an increase in platelet
288 count (e.g., at least double, at least triple, at least quadruple the platelet count prior to treatment), an increase in haptoglobin, and/or a decrease in lactate dehydrogenase.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from TMA associated with hematopoietic stem cell transplant via a cathéter (e.g., intravenously) for a first time period (e.g., at least one day to a week or two weeks or three weeks or four weeks or longer) followed by administering a MASP-2 inhibitory antibody to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasma therapy. In some embodiments, the administration in the first and/or second time period occurs in the presence of plasma therapy.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from TMA associated with hematopoietic stem cell transplant either intravenously, intramuscularly, or subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every at least every two weeks, or at least once a week, such as twice a week or three times a week. The MASP-2 inhibitory antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the method comprises treating a subject suffering from persistent TMA associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-107 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid
289 sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
In some embodiments, the method comprises administering to a subject suffering from, or at risk for developing TMA associated with HSCT, including a subject suffering from persistent TMA associated with hematopoietic stem cell transplant that is résistant to conservative treatment measures, a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70 in a dosage from 1 mg/kg to 10 mg/kg (i.e., 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg) at least once weekly (such as at least twice weekly or at least three times weekly) for a period of at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks, or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks.
EXAMPLE 47
290
This Example describes a case study demonstrating effective treatment of Graftversus-Host Disease (GVHD) associated microangiopathy with a MASP-2 inhibitory antibody, OMS646.
Background: Graft-versus-host disease is a common complication of 5 hematopoietic stem cell transplant (HSCT). Both acute and chronic forms exist and resuit from donor immune cells recognizing the récipient patient as foreign tissue. This triggers an immune response against the récipient patient. Acute GvHD occurs in up to 50% or more of patients who receive allogeneic transplants. Acute GvHD most commonly targets the skin, gastrointestinal tract, and liver, but can also affect the kidney, eye, lung and 10 blood cells. Chronic GvHD occurs in approximately 40% of patients who receive allogeneic transplants and most commonly affects the skin, liver, eye, gastrointestinal tract and lungs. Both acute and chronic GvHD are related to significant morbidity and mortality.
Methods and Results
A 46-year-old man affected by T-cell acute lymphoblastic leukemia (T-ALL) in second complété remission (CR) underwent a full myeloablative conditioning with a cyclophosphamide and total body irradiation regimen (TBI-Cy) and peripheral blood stem cell (PBSC) allogeneic HSCT transplantation from an unrelated 35-year-old female donor 9/10 HLA compatible (antigenic mismatch at locus A). Graft-versus-host disease 20 (GVHD) prophylaxis was based on antithymocyte globulin (ATG, 5 mg/kg) during conditioning, Cyclosporin A (CSA) and short course of Methotrexate (i.e., Methotrexate on day +1, +3, +6 (dose réduction due to low CD34+)). He achieved a robust hématologie reconstitution (engraftment: WBC >1000/mmc, neutrophils >500/mmc on day +15; platelets >20000/mmc on day +18, 50000/mmc on day +21). Chimerism was 25 observed with full donor (bone marrow and peripheral blood, CD3+ and CD3- fraction) on day +30, +60, +90, +180, +354. Complété remission was confirmed (immunophénotype, molecular MRD) on day +30, +60, +90, +180.
At day +35 post-transplant, he developed gastrointestinal (GI) GVHD (presented with emesis, anorexia, diarrhea, abdominal pain; colonscopy: diffuse érosions; histology: 30 diffuse mucosal ulcérations, lymphogranulocytic inflammation in the lamina propria, gland and crypt distortion, apoptotic bodies), and was diagnosed with acute GvHD, stage 4 (gut), overall grade 4 along with cytomégalovirus (CMV) colitis (determined by
291 positive immunohistochemistry and real-time PCR on gut biopsy; systemic CMV réactivation (2614 UI/mL on peripheral blood). The two conditions were simultaneously treated with méthylprednisolone (1 mg/Kg), foscamet and ganciclovir with good response and steroid tapered to withdrawal.
At day +121 post-transplant he experienced relapse of steroid refractory GI GVHD (presented with emesis, anorexia, diarrhea, abdominal pain; histological findings (stomach and duodénum), and was diagnosed with late onset acute GI GvHD stage 4 (gut), overall grade 4, which was treated, with good response, by continuing CSA and with a sequential administration of pentostatin and mesenchymal stem cells according to a registered clinical study (ClinicalTrials.Gov Identifier NCT02032446).
At day +210 post-transplant the patient presented with weight loss, marked asthenia, generalized muscular atrophy, tetraparesis, reduced deep tendon reflexes, bilateral calf paresthesia, neurogenic bladder and urinary incontinence in absence of minctional (i.e., micturitional) stimulus. Clinical examination and cerebrospinal fluid, neuroradiological and electrophysiological findings described a picture of axonal, sensorimotor and dysautonomic polyneuropathy. Neuroradiology indicated a normal MRI (brain and spine) and cerebrospinal fluid was normal. Relapse of T-ALL, infections, vascular damage, nutritional deficiencies, demyelinating disorder and posterior réversible encephalopathy syndrome (PRES) were excluded. High dose immunoglobulin was administered with no benefit. A concomitant thrombocytopenia (6 x 109/L) with features of macroangiopathic hemolytic anémia (6.7 g/dL) without rénal impairment, reticulocytosis (335 x 109/L), increased LDH (1635 U/L) and undetectable haptoglobin were also documented. A direct antiglobulin test was négative. Schistocytes were observed on peripheral blood smear (2-3/field 50x), as well as absence of anti-B abs (major incompatibility patient/donor), normal ADAMTS13 activity activity, absence of anti-ADAMTS13 Abs, normal coagulation tests and proteinuria with normal créatinine values. He also presented melena with documented colic ulcérations on endoscopie examination; the histopathological study revealed mucosal edema, fibrosis, ectatic small vessels with intraluminal fibrin déposition, glandular atrophy and apoptotic bodies with no microbiological findings. Thus, although there was evidence of persistent GI GVHD, the clinical and histopathological picture was also consistent with a diagnosis of colic transplant-associated thrombotic microangiopathy. Because of the récurrent GI GVHD,
292
CSA was tapered but not withdrawn (trough level 100-150 ng/mL), without obtaining clinical or hematological changes.
At day +263 post-transplant the patient was enrolled into the clinical trial described above in Examples 45 and 46, which involves treatment with OMS646, a MASP-2 inhibitory antibody that inhibits the lectin pathway of complément activation. As shown in FIGURE 61, after the first 2 weekly doses of the drug a clinical and laboratory response was observed with resolution of melena and hemolysis and rise of platelet count. After 4 doses, interestingly, a remarkable neurological improvement was also documented with both clinical and electrophysiological improvement of the sensorimotor déficit. After 8 doses of OMS646, the GvHD response was maintained despite CSA tapering, with no toxicities observed. By day +354 post-transplant, the patient demonsrated further neurological improvement and initial recovery of minctional (i.e., micturitional) déficit. The rapid clinical benefit achieved by this patient suggests that effective inhibition of the complement-mediated endothélial damage, resulting from the administration of OMS646, can be highly effective for the treatment of GVHD associated TMA.
In summary, this Example describes a case report of a HSCT patient posttransplant having co-existing HSCT-TMA and GvHD, which both resolved following treatment with a MASP-2 inhibitory antibody (OMS646). Prior to treatment with OMS646 the patient had a difficult post-transplant course complicated by multiple épisodes of steroid-refractory GvHD, cytomégalovirus infection and HSCT-TMA. After two prior épisodes of GvHD, the patient presented with bloody diarrhea. Intestinal biopsy demonstrated both HSCT-TMA and GvHD. No infections were identified. Notably, the patient also had new onset neurological symptoms of paresthesias, tetraplegia and a neurogenic bladder, which hâve been reported as neurological manifestations of GvHD and TMA. The patient was unable to walk due to the tetraplegia and required blood transfusions at least once daily. Hematological markers demonstrated HSCT-TMA with thrombocytopenia, elevated lactate dehydrogenouse (LDH) and schistocytes. The patient entered the clinical trial and began receiving OMS646. His immunosuppression (cyclosporine) had been decreased 2 weeks earlier and he received only low dose corticosteroids in view of his history of steroid-refractory GvHD. He received no other GvHD treatment. His bloody diarrhea resoleved and his hematological
293 markers improved after 2 OMS646 doses. After 4 OMS646 doses he was able to walk. He completed 8 weeks of OMS646 treatment and is doing well at home. Ail signs and symptoms of HSCT-TMA and GvHD hâve resolved. His neurological symptoms continue to improve. Prior to OMS646 treatment this patient was deteriorating and at 5 high risk for early death. Following treatment with OMS646, improvement of GvHD, HSCT-TMA and the neurological symptoms were observed and the patient is doing well at home.
In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from, or at risk for developing graft-versus10 host-disease (GVHD) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation and thereby treat, or reduce the risk of developing GVHD, or reduce the severity of one or more symptoms associated with GVHD. In one embodiment, the GVHD is active, acute GVHD. In one embodiment, the GVHD is 15 chronic GVHD. In one embodiment, the GVHD is steroid-resistant (i.e., persistant despite steroid treatment). In one embodiment, the GVHD gastrointestinal (GI) GVHD. In one embodiment, the method further comprises the step of determining the presence of GVHD in a subject prior to treatment with the MASP-2 inhibitory antibody.
In one embodiment, the subject is suffering from, or at risk for developing GVHD 20 associated with a hematopoietic stem cell transplant. In one embodiment, the subject is suffering from, or at risk for developing GVHD associated with a TMA associated with hematopoietic stem cell transplant. In one embodiment, the subject is suffering from leukemia, such as T-cell acute lymphoblastic leukemia.
In one embodiment, the subject is suffering from one or more neurological 25 symptoms associated with GvHD and/or TMA, such as, for example, asthenia, paresthesias, tetraplegia, sensorimotor déficit, dysautonomic polyneuropathy, and/or neurogenic bladder and the MASP-2 inhibitory antibody is administered in an amount and for a time period sufficient to ameliorate one or more of the neurological symptoms.
In accordance with any of the disclosed embodiments herein, the MASP-2 30 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in
294 vitro assay in 1% human sérum at an IC50 of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 5 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with GVHD.
In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from GVHD associated with hematopoietic stem cell 15 transplant either intravenously, intramuscularly, or subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every at least every two weeks, or at least once a week, such as twice a week or three times a week. The MASP-2 inhibitory antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
In one embodiment, the method comprises treating a subject suffering from
GVHD associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 25 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 30 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-107 of SEQ ID NO:67 and b) a light-chain variable région comprising:
295
i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NQ:70, or (II) a variant thereof comprising a heavy-chain variable 5 région with at least 90% identity to SEQ ID NO:67 (e.g., at least 9l%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifïcally recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID
NO:70.
In some embodiments, the method comprises administering to a subject suffering from, or at risk for developing GVHD, such as a subject that will undergo, is undergoing, or has undergone HSCT, including a subject suffering from GVHD associated TMA, a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment 25 thereof comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70 in a dosage from 1 mg/kg to 10 mg/kg (i.e., 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg) at least once weekly (such as at least twice weekly or at least three times weekly) 30 for a period of at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks, or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks.
296
EXAMPLE 48
This Example describes a case study demonstrating effective treatment of TMA and diffuse alveolar hemorrhage (DAH) after hematopoietic stem cell transplant with a MASP-2 inhibitory antibody, OMS646.
Background: Hematopoietic stem cell transplantation (HSCT) induces substantial endothélial injury, which contributes to serious post-HSCT complications such as thrombotic microangiopathy (TMA), graft-versus-host disease (GvHD), diffuse alveolar hemorrhage (DAH) and veno-occlusive disease (VOD). (see E. Carreras and M. DiazRicart, Bone Marrow Transplant vol 46:1495-1502, 2011; Akil et al., Biol BloodMarrow Transplant 21:1739-1745, 2015; and Vion et al., Semin Thromb Hemost 41(06):629-643, 2015, each of which is hereby incorporated herein by reference).
Diffuse alveolar hemorrhage (DAH) is a syndrome that can occur in a hematopoietic stem cell transplant (HSCT) receipient. The diagnostic criteria of DAH in the HSCT récipient include lung infiltrâtes, dyspnea and hypoxia (see E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502, 2011, hereby incorporated herein by reference). The suspected pathogenesis of DAH is lung capillary endothélium damages by HSCT conditioning plus engrafted neutrophils and silent infections, allowing the leakage of red blood cells into the pulmonary alveoli (E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502, 2011). Veno-occlusive disease (VOD), also known as sinusoïdal obstructive syndrome (SOS), is another syndrome that can occur in a hematopoietic stem cell transplant (HSCT) patient. The clinical manisfestations of VOD include marked weight gain due to fluid rétention, increased liver size and raised levels of birubin in the blood (see E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502, 2011, hereby incorporated herein by reference).
Methods and Results:
Demographics and Past Medical History
297
The patient was a 14 year-old white female at the time of treatment initation, referred to in this Example as “compassionate use patient #1.” The patient had a history of Diamond-Blackfan anémia and empty sella syndrome.
Patient Outcome
The patient is alive on Day 877.
Transplant Requiring Disease
The patient has a history of Diamond-Blackfan anémia. She had required RBC infusions every 3 weeks since infancy and previously developed iron overload.
Transplant
The patient underwent her stem cell transplant in September 2015. She received a PBSC transplant from a 9/10 matched unrelated donor. Neutrophil engraftment occurred by approximately Day 35. Platelet engraftment had not been achieved by Day 184. She required platelet and RBC infusions for more than 1 year.
Post-transplant Complications
The patient had a complicated post-transplant course. Shortly after discharge she was readmitted on Day 67 for shortness of breath and presumed pneumonia. She was discharged on Day 162. She was readmitted on Day 167 with varicella zoster réactivation treated with acyclovir and polyserositis treated with corticosteroids. She had persistent thrombocytopenia and anémia, elevated LDH, decreased haptoglobin, schistocytes and a négative direct antibody test. A diagnosis of TMA was made. Immunosuppression was changed to MMF and prednisone. She also experienced HHV6 and EBV réactivation. She was discharged on Day 197.
On Day 229 she was readmitted with persistent TMA and severe respiratory distress. She was diagnosed with diffuse alveolar hemorrhage that was treated with CPAP and corticosteroids. On Day 231 she had pleural and pericardial effusions with worsening oxygénation. Her blood pressure increased. She required an ACE inhibitor, calcium channel blocker, beta blocker, diuretic, and nitroglycerin for BP control. She experienced a seizure and clonidine was added. Brain MRI showed iron in the brain consistent with secondary hemochromatosis from her RBC transfusion history. Hemodialysis was
298 initiated on Day 231. On Day 259 she began eculizumab treatment due to continued rénal dysfunction and worsening pulmonary status. Her TMA improved, but she developed acute pulmonary edema and eculizumab was discontinued. Her TMA worsened and she underwent plasma exchange on Days 269, 276, and 278 without improvement. She was treated with defibrotide that was discontinued due to a coagulopathy and no TMA improvement. She again received lower-dose eculizumab starting on approximately Day 320. The eculizumab was stopped again due to pulmonary edema. The TMA persisted. She also developed C. difficile infection. She was discharged in Day 379 receiving prednisone, MMF, voriconazole, cotrimazole, amlodipine, erythropoietin, ursodiol and omeprazole.
She was readmitted on Day 380 with fever, chills, and dyspnea. She had K. pneumoniae pneumonia and E. faecium sepsis. Her MMF was stopped. She was treated with Tazocin, meropenem, and vancomycin. With persistent bacteremia, her central line was changed and her antibiotics were changed to linezolid, daptomycin, and clindamycin.
Her TMA persisted requiring hemodialysis 3 times weekly and daily platelet transfusions. Pulmonary edema diagnosed as diffuse alveolar hemorrhage (treated with corticosteroids without response). On day 404 OMS646 treatment began under a compassionate use protocol. As described herein, OMS646 is a MASP-2 inhibitory antibody that inhibits the lectin pathway of complément activation.
She was treated 3 times weekly with a dose of 4 mg/kg OMS646. Her oxygen was discontinued and her corticosteroid dose was decreased. Her laboratory TMA markers improved. She was discharged on Day 414.
She continued outpatient OMS646 treatment 3 times weekly and on Day 416 her hemodialysis was decreased to once weekly. Her antihypertensive médication was also reduced to amlodipine, clonidine and furosemide only. On Day 423 she could discontinue hemodialysis and on Day 428 her OMS646 was decreased to twice weekly with réductions in her corticosteroid and furosemide doses. Her platelet transfusions were substantially decreased during this period.
She continued to do well until Day 486 when she developed an RSV infection.
Her créatinine and LDH increased, and her platelet count and haptoglobin felL She was diagnosed with récurrent TMA and OMS646 treatment was increased to 3 times weekly. Her TMA resolved with the increased OMS646 treatment.
299
She has remained hemodialysis free and her last platelet transfusions were occurred on about Day 630. She is currently doing well and receiving treatment with OMS646, desftiroxamine, lévothyroxine, sevelamer, Augmentin, levetiracetam, allopurinol, amlodipine, nevibolol, clonidine and erythropoietin. She is receiving 5 phlébotomies for the hemochromatosis.
TMA Episodes
This patient had a diffïcult course with persisting and/or relapsing TMA. Her course is consistent with multi-organ TMA and she had diffuse alveolar hemorrhage 10 (DAH), another endothélial injury syndrome. She initially responded to eculizumab, but did not tolerate the treatment. Prior to initiating OMS646 treatment she was on hemodialysis and requiring daily transfusions. Soon after OMS646 treatment her TMA resolved, her DAH resolved, and she was able to discontinue dialysis. She was later able to substantially reduce and later discontinue platelet and RBC transfusions while 15 maintaining stable or increasing platelet count and hemoglobin values. These results are demonstrated in FIGURES 62A-62E as follows:
FIGURE 62A graphically illustrâtes the level of créatinine over time in the compassionate use patient #1, wherein the vertical line indicates the start of treatment with MASP-2 inhibitory antibody (OMS646).
FIGURE 62B graphically illustrtaes the level of haptoglobin over time in the compassionate use patient #1, wherein the vertical line indicates the start of treatment with MASP-2 inhibitory antibody (OMS646).
FIGURE 62C graphically illustrâtes the level of hemoglobin over time in the compassionate use patient #1, wherein the vertical line indicates the start of treatment 25 with MASP-2 inhibitory antibody (OMS646).
FIGURE 62D graphically illustrâtes the level of LDH over time in the compassionate use patient #1, wherein the vertical line indicates the start of treatment with MASP-2 inhibitory antibody (OMS646).
FIGURE 62E graphically illustrâtes the level of platelets over time in the 30 compassionate use patient #1, wherein the vertical line indicates the start of treatment with MASP-2 inhibitory antibody (OMS646).
300
Overall, this patient’s course demonstrates successful OMS646 treatment of TMA and DAH allowing the patient to discontinue oxygen therapy, hemodialysis, and transfusions.
Summary
In summary, this Example describes an HCST-TMA case report of an adolescent girl (compassionate use patient #1) who did not tolerate eculizumab treatment, but responded well to compassionate use of OMS646 treatment. Soon after OMS646 treatment her TMA resolved, her DAH resolved, and she was able to discontinue dialysis. As described in Example 47, another HSCT-TMA case report was provided describing a patient who had a difficult post-transplant course, including steroid-resistant GvHD and cytomégalovirus infection. He developed TMA that did not respond to conservative measures and had co-existing GvHD with multiple neurological complications and was unable to walk. As described in Example 47, following OMS646 treatment, his TMA and GvHD resolved and his neurological complications improved. He was able to retum to work and his neurological status has continued to improve.
The improvement in overall survival and TMA markers in HSCT receipients combined with resolution of GvHD and resultion of diffuse alveolar hemorrhage in these critically ill patients indicates the rôle the lectin pathway plays in these syndromes and the efficacy for use of a MASP-2 inhibitory antibody OMS646 in treating and/or preventing post-stem cell transplant TMA, graft-versus-host disease and diffuse alveolar hemorrhage.
In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from diffuse alveolar hemorrhage associated with hematopoietic stem cell transplant (i.e., in a HSCT receipient) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In one embodiment, the method further comprises identifying a human subject suffering from diffuse alveolar hemorrhage associated with HSCT prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation.
in accordance with any of the disclosed embodiments herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said
301 antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC5Q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is 5 an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and
F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not 10 substantially inhibit the classical pathway (Le., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with diffuse alveolar hemorrhage in a HSCT récipient. In some embodiments, the method 15 comprises administering a MASP-2 inhibitory antibody to a subject suffering from diffuse alveolar hemorrhage either intravenously, intramuscularly, or subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every at least every two weeks, or at least once a week, such as twice a week or three times a week. The MASP-2 inhibitory antibody may be administered alone, or in combination 20 with a C5 inhibitor, such as eculizamab.
In one embodiment, the method comprises treating a subject suffering from diffuse alveolar hemorrhage associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy chain 25 variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-L1, CDRL2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino 30 acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3
302 comprising the amino acid sequence from 95-107 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
In some embodiments, the method comprises administering to a subject suffering from, or at risk for developing diffuse alveolar hemorrhage associated with HSCT, a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70 in a dosage from 1 mg/kg to 10 mg/kg (i.e., 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg) at least once weekly (such as at least twice weekly or at least three times weekly) for a period of at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks, or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks.
303
In accordance with the foregoing, in another embodiment, the invention provides a method of treating a human subject suffering from veno-occlusive disease (VOD) associated with hematopoietic stem cell transplant (i.e., in a HSCT receipient) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In one embodiment, the method further comprises identifying a human subject suffering from veno-occulsive disease associated with HSCT prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation.
In accordance with any of the disclosed embodiments herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b déposition in an in vitro assay in 1% human sérum at an IC5q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2 wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with veno-occlusive disease in a HSCT récipient. In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from venoocclusive disease either intravenously, intramuscularly, or subcutaneously. Treatment may be chronic and administered daily to monthly, but preferably every at least every two weeks, or at least once a week, such as twice a week or three times a week. The MASP-2 inhibitory antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.
304
In one embodiment, the method comprises treating a subject suffering from venoocclusive disease associated with hematopoietic stem cell transplant comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy chain variable région 5 comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-H1 comprising the amino acid 10 sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-107 ofSEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid 15 sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 ofSEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région with at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light-chain variable région with 20 at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject suffering from veno-occlusive disease associated with HSCT a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, 25 comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment 30 thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as
305 set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
In some embodiments, the method comprises administering to a subject suffering from, or at risk for developing veno-occlusive disease associated with HSCT, a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70 in a dosage from 1 mg/kg to 10 mg/kg (i.e., 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg) at least once weekly (such as at least twice weekly or at least three times weekly) for a period of at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks, or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks.
While illustrative embodiments hâve been illustrated and described, it will be appreciated 15 that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (15)

1. Use of a MASP-2 inhibitory monoclonal antibody, or antigen-binding fragment thereof, in the manufacture of a composition for treating a human subject suffering from, or at risk for developing graft-versus-host disease (GVHD), wherein the composition
5 comprises an amount of the MASP-2 inhibitory antibody effective to inhibit MASP-2dependent complément activation, and wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.
2. The use of Claim 1, wherein the subject has previously undergone, or is currently undergoing, or will undergo a hematopoietic stem cell transplant.
10
3. The use of claim 1, wherein the subject is suffering from acute GVHD.
4. The use of claim 1, wherein the subject is suffering from chronic GVHD.
5. The use of claim 1, wherein the subject is suffering from steroid-resistant GVHD.
6. The use of Claim 1, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof, comprises a heavy chain variable région comprising CDR-H1, 15 CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70.
7. The use of any of claims 1-6, wherein the composition is for administration of said MASP-2 inhibitory antibody in a dosage of from 1 mg/kg to 10 mg/kg at least once 20 weekly.
8. The use of claim 1, wherein the subject is suffering from one or more neurological symptoms associated with graft-versus-host disease.
9. The use of claim 8, wherein the one or more neurological symptoms associated with graft-versus-host-disease or HSCT-TMA is selected from the group consisting 25 of asthenia, paresthesias, tetraplegia, sensorimotor déficit, dysautonomic polyneuropathy, and/or neurogenic bladder.
307
10. Use of a MASP-2 inhibitory monoclonal antibody, or antigen-binding fragment thereof, in the manufacture of a composition for treating a human subject suffering from, or at risk of developing diffuse alveolar hemorrhage, wherein the composition comprises an amount of the MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation, and wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.
11. The use of Claim 10, wherein the subject has previously undergone, or is currently undergoing, or will undergo a hematopoietic stem cell transplant.
12. The use of Claim 10, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof, comprises a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70.
13. Use of a MASP-2 inhibitory monoclonal antibody, or antigen-binding fragment thereof, in the manufacture of a composition for treating a human subject suffering from, or at risk for developing veno-occlusive disease, wherein the composition comprises an amount effective to inhibit MASP-2-dependent complément activation, and wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.
14. The use of Claim 13, wherein the subject has previously undergone, or is currently undergoing, or will undergo a hematopoietic stem cell transplant.
15. The use of Claim 13, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof, comprises a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70.
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