Patent Grant
9540626
This invention relates to the field of the innate immune system and complement activation. In particular, the invention provides regulators of complement activation, particularly via the alternative pathway, comprising a complement regulatory region connected by a flexible linker to a multifunctional binding region that enables binding to C3b activation/inactivation products and/or oxidation end products, as well as to polyanionic surface markers on host cells.
Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety. Full citations for publications not cited fully within the specification are set forth at the end of the specification.
The efficacy of the complement-mediated immune response relies on a delicate balance between activation and regulation. Although continuous generation of the strong opsonin complement fragment C3b by the alternative pathway (AP) allows rapid reaction to foreign or abnormal cells, its indiscriminate deposition may potentially cause host tissue damage. Host cells are therefore protected by proteins of the regulator of complement activation (RCA) family, which either impair the generation of new C3b by accelerating the decay of the C3 convertases or act as cofactor for factor 1 (F1) in degrading existing C3b. In addition to cell surface-bound RCA proteins such as decay accelerating factor (DAF (A000571); also called CD55), membrane cofactor protein (MCP (A000568); also called CD46) and complement receptor 1 (CR1; also called CD35), the soluble and highly abundant regulator factor H (FH) offers an additional layer of protection, as it controls the steady-state alternative pathway activation in circulation and on surfaces to which it specifically binds.
Factor H, the second most abundant complement protein in plasma, is the primary regulator of the AP. It is a large (155 kDa), soluble glycoprotein that circulates in human plasma. It has an elongated structure consisting of 20 homologous short consensus repeats (SCR, also referred to as complement control protein (CCP) domains or modules), each comprising approximately 60 residues that are common to all RCA proteins (Schmidt C Q et al., 2008, Clin Exp Immunol 151: 14-24), held together by four conserved cysteine residues. Whereas the complement regulatory functions are concentrated to the N-terminus (SCR 1-4) of factor H, two distinct regions (SCR 6-8, SCR 19-20) define the recognition of self-surfaces via binding to polyanion patches (e.g., glycosaminoglycans; GAG) on host cells. Factor H regulates the AP by inhibiting the formation of the AP C3 convertase and accelerating its dissociation, or by acting as cofactor for the degradation of C3b by factor I (Wu, J et al., 2009, Nat Immunol 10:728-733; Schmidt, C et al., 2008, J Immunol 181:2610-2619; Schmidt, C et al., 2008, Clin Exp Immunol 151:14-24; Pickering, M C et al., 2008, Clin Exp Immunol 151:210-230; Jozsi, M, & P F Zipfel, 2008, Trends Immunol 29:380-387; Ross, G D et al., 1982, J Immunol 129:2051-2060).
Though a soluble protein, FH may be recruited to host membranes by recognizing and binding self-components, such as glycosaminoglycans, and thereby may prevent the opsonization of host tissue with low surface expression of RCA. The importance of FH in maintaining a well-balanced immune response is reflected by the increasing number of diseases found to have strong association with mutations and polymorphisms in the gene encoding FH, as found in age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS) and membrano-proliferative glomerulonephritis type II (MPGN-II) (Meri, S, 2007, Ann NY Acad Sci 1109: 93-105; de Cordoba, S R & de Jorge, E G, 2008, Clin Exp Immunol 151: 1-13). Because of their vital function in immune modulation, therapeutic targeting of FH and other RCA proteins has been considered important for the treatment of diseases associated with abnormal or loss of complement control.
In accordance with a current model of FH engagement of C3b on a self-surface, concurrent recognition of C3b-opsonins and host-surface markers enable FH to control complement activation effectively on self, but not on foreign surfaces. This specificity depends on the simultaneous binding of FH19-20 to the thioester domain (TED or C3d domain) of C3b, and to polyanions. Proteolytic cleavage within the complement C1r/C1s, Uegf, Bmp1 (CUB) domain of C3b inactivates C3b and yields iC3b (inactive C3b). With proteolysis of CUB, C3b loses one interaction patch for the N-terminal FH domains (Wu J et al., 2009 Nat Immunol 10: 728-733; Schmidt C Q et al., 2008, J Immunol 181: 2610-2619), but iC3b still contains TED, the interaction partner of C-terminal domains 19-20 of FH. Even so, FH no longer binds readily to iC3b under physiological conditions, irrespective of iC3b being positioned on a cell surface (Ross G D et al., 1983, J Exp Med 158: 334-352) or directly on a biosensor (Alcorlo M et al., 2011, Proc Natl Acad Sci USA 108: 13236-13240). In contrast, FH19-20 binds indiscriminately well to the TED of C3b, C3dg and C3d (Morgan H P et al., 2011, Nat Struct Mol Biol 18, 463-470).
A role of FH in abatement of oxidative stress was established recently. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathological processes, including AMD. FH has been identified as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice (Weismann D et al., 2011, Nature 478: 76-81). SCRs 7 and 20 were demonstrated to mediate the binding of FH to MDA (Id.).
The AP has been shown to play a particularly important role in preclinical disease models and by studies of human diseases, where mutations or dysfunctional polymorphisms that promote activation of the AP are highly associated with diseases such as aHUS, dense deposit disease, AMD and paroxysomal nocturnal hemoglobinuria (PNH) (see Holers, V M, 2008, Immunol Rev 223: 300-316). Diseases in which a primary pathogenic role for the alternative pathway has been shown or postulated in or from preclinical studies in animals include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), several autoimmune and autoinflammatory kidney diseases, autoimmune myocarditis, multiple sclerosis, traumatic brain and spinal cord injury, intestinal and renal ischemia-reperfusion (IR) injury, spontaneous and recurrent pregnancy loss, anti-phospholipid syndrome (APS), asthma, anti-nuclear cytoplasmic antigen-associated pauci-immune vasculitis (Wegener's syndrome), non-lupus autoimmune skin diseases such as pemphigus, bullous pemphigoid, and epidermolysis bullosa, post-traumatic shock and atherosclerosis (Holers, 2008, supra).
A milestone in complement intervention strategies was marked by the approval of the humanized monoclonal antibody Eculizumab for the treatment of the orphan diseases PNH and aHUS. Both diseases are characterized by malfunctioning of complement regulators and result in insufficient control of the AP. Eculizumab binds C5 and inhibits its activation into C5a, a potent anaphylatoxin, and C5b, the initiator of the terminal pathway, and consequently inhibits inflammatory signaling and cell lysis by MAC. However, use of Eculizumab can be disadvantageous; for instance, its use increases susceptibility to infections. Additionally, Eculizumab treatment costs are extremely high and an appreciable proportion of PNH patients do not respond to Eculizumab treatment. These disadvantages have boosted the preclinical development of the chimeric fusion protein TT30, which intervenes earlier in the complement cascade at C3-level, is AP-specific and targets to sites of C3-inactivation products predominantly found on host surfaces (Fridkis-Hareli M, et al., 2011, Blood 118: 4705-4713).
From the foregoing discussion, it is clear that improved regulators of the AP of complement activation are needed. The present invention satisfies that need.
One aspect of the present invention features a complement regulator comprising a complement regulating region that includes a plurality of complement control proteins (CCPs), linked by a flexible linker to a multifunctional binding region that enables binding of the complement regulator to one or more C3b activation/inactivation products, one or more oxidation end products and/or one or more polyanionic surface markers on host cells. The complement regulator at least regulates AP-mediated complement activation. In one embodiment, the C3b activation/inactivation products to which the complement regulators bind contain thioester domains (TEDs). These include C3b, iC3b, C3dg and C3d. The polyanionic surface markers on host cells to which the complement regulators bind include N-linked glycosyl units with sialic acid end-groups or glycosaminoglycans (GAGs) selected from heparin, heparan sulfate, chondroitin sulfate, dermantan sulfate, keratan sulfate and hyaluronan. The oxidation end products to which the complement regulators bind include malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), carboxyethylpyrrole (CEP), oxidized phosphatidylserine (OxPS), oxidized cardiolipin (OxCL) and phosphocholine (PC).
In one embodiment, the CCPs of the complement regulatory region and/or the multifunctional binding region are derived from factor H. In particular, the CCPs of the complement regulatory region are derived from SCRs 1-4 of factor H. In one embodiment, the multifunctional binding region is derived from SCRs 19 and 20 of factor H. In certain embodiments, the linker that links the complement regulatory region to the multifunctional binding region is of the same length and flexibility as a poly-Gly peptide at least 10, 11 or 12 residues in length. In particular, the linker is a poly-Gly peptide at least 12 residues in length.
In one embodiment, the aforementioned complement regulator comprises, from N- to C-terminus, SCRs 1-4 of factor H, a poly-Gly linker at least 12 residues in length, and SCRs 19-20 of factor H. The factor H is human factor H in certain embodiments. In particular the factor H comprises a polypeptide that is functionally equivalent to SEQ ID NO:2. That polypeptide typically comprises a sequence that is more than 90% identical to that of SEQ ID NO:2. An exemplary embodiment of the complement regulator comprises SEQ ID NO:2.
Another aspect of the invention features a polypeptide comprising, from N- to C-terminus, SCRs 1-4 of factor H, a poly-Gly linker at least 12 residues in length, and SCRs 19-20 of factor H. In one embodiment, the factor H is human factor H. In a particular embodiment, the polypeptide comprises SEQ ID NO:2.
Another aspect of the invention features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and a complement regulator complement regulating region that includes a plurality of complement control proteins (CCPs), linked by a flexible linker to a multifunctional binding region that enables binding of the complement regulator to one or more C3b activation/inactivation products, one or more oxidation end products and/or one or more polyanionic surface markers on host cells. The complement regulator in the pharmaceutical composition regulates AP-mediated complement activation.
Another aspect of the invention features a method of regulating complement activation comprising contacting a medium in which regulation of complement activation is desired with a complement regulator comprising a complement regulating region that includes a plurality of complement control proteins (CCPs), linked by a flexible linker to a multifunctional binding region that enables binding of the complement regulator to one or more C3b activation/inactivation products, one or more oxidation end products and/or one or more polyanionic surface markers on host cells, wherein the contacting results in regulation of complement activation in the medium. In one embodiment, the method comprises regulation of AP-mediated complement activation. In one embodiment, the medium includes cells or tissues of an organism. The cells or tissues can be cultured cells or tissues. Alternatively, the cells or tissues are disposed within a living organism. In one embodiment, the medium includes a biomaterial. The biomaterial can be included in an extracorporeal shunt system for cells or tissues of a living organism.
Other features and advantages of the invention will be understood by reference to the drawings, detailed description and examples that follow.
Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, protein chemistry and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.
Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel et al., 2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.
The nomenclature used herein and the laboratory procedures used in analytical chemistry and organic syntheses described below are those well known and commonly employed in the art. Standard techniques or modifications thereof, are used for chemical syntheses and chemical analyses.
As used herein, each of the following terms has the meaning associated with it in this section.
A “complement regulator” as used herein is an agent that possesses the type of complement regulating activity found in the family of mammalian proteins known as the regulator of complement activation (RCA) family, also referred to herein as “RCA proteins”, which regulate complement activity through the AP. RCA proteins impair the generation of new C3b by accelerating the decay of the C3 convertases or act as cofactor for factor 1 (F1) in degrading existing C3b. RCA proteins include cell surface-bound proteins such as decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1), as well as the soluble factor H (FH), which controls the steady-state alternative pathway activation in circulation and on surfaces to which it specifically binds.
The terms “complement control protein (CCP) domain or module”, “complement control protein (CCP), “short consensus repeat” (SCR) and “sushi domain” are used interchangeably herein to describe domains found in all RCAs that contribute to their ability to regulate complement activation in the blood or on host cell surfaces to which they specifically bind. CCPs typically are composed of about 60 amino acids, with four cysteine residues disulfide bonded in a 1-3 2-4 arrangement and a hydrophobic core built around an almost invariant tryptophan residue.
The terms “host” and “self” are used interchangeably herein to describe cells or tissues belonging to a particular organism, as compared to foreign cells, e.g., of invading microorganisms, or abnormal cells, which the immune system is designed to recognize as “other” or “non-self.”
A “subject”, “individual” or “patient” refers to an animal of any species. In various embodiments, the animal is a mammal. In one embodiment, the mammal is a human. In another embodiment, the mammal is a non-human animal.
“Treating” refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. “Treating” can also refer to reducing or eliminating a condition of a part of the body, such as a cell, tissue or bodily fluid, e.g., blood.
“Preventing” refers to the partial or complete prevention of the disease or condition in an individual or in a population, or in a part of the body, such as a cell, tissue or bodily fluid (e.g., blood). The term “prevention” does not establish a requirement for complete prevention of a disease or condition in the entirety of the treated population of individuals or cells, tissues or fluids of individuals.
The term “treat or prevent” is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.
A “prophylactic” treatment is a treatment administered to a subject (or sample) that does not exhibit signs of a disease or condition, or exhibits only early signs of the disease or condition, for the purpose of decreasing the risk of developing pathology associated with the disease or condition. This term may be used interchangeably with the term “preventing,” again with the understanding that such prophylactic treatment or “prevention” does not establish a requirement for complete prevention of a disease in the entirety of the treated population of individuals or tissues, cells or bodily fluids.
As used herein, a “therapeutically effective amount” or simply an “effective amount” is the amount of a composition sufficient to provide a beneficial effect to the individual to whom the composition is administered, or who is otherwise treated using a method involving the composition.
The term “extracorporeal treatment” as used herein refers generally to treatment or manipulation of cells, tissues or bodily fluids that have been removed from an individual and are thereafter returned to the same individual or to another individual. Examples of extracorporeal treatments include, but are not limited to, extracorporeal shunting of blood during surgical procedures, for example, hemodialysis, and cell or tissue transplantation, to name a few.
The term “biomaterials” as used herein refers to components of equipment, devices or articles that come into contact with, biological substances such as cells, tissues or biological fluids, such as those being subjected to the extracorporeal treatment, or tissues surrounding an implanted device or tissue, such as stents, tubes, artificial tissues or other implants.
Dosages expressed herein are in units per kilogram of body weight (e.g., μg/kg or mg/kg) unless expressed otherwise.
Ranges are used herein in shorthand, to avoid having to list and describe each and every value within the range. Any appropriate value within the range is intended to be included in the present invention, as is the lower terminus and upper terminus, independent of each other.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some embodiments±5%, in some embodiments±1%, and in some embodiments±0.1% from the specified value, as such variations are appropriate to practice the disclosed methods or to make and used the disclosed compounds, compositions or articles of manufacture.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Thus, for example, a container comprising one tab may have two or three tabs. Additionally, the term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of” Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting of.”
The present invention springs in part from the inventors' rational design and development of a recombinant protein therapeutic (mini-FH) by joining the four N-terminal and two C-terminal domains of FH, from a total number of 20, through an optimized linker in such a way that FH functionality is preserved. Importantly, the design attributes a novel function to mini-FH in that mini-FH shows a strong preference over FH in binding complement inactivation products. Thus mini-FH targets to and protects host cells via its simultaneous recognition of (i) complement activation/inactivation products and (ii) polyanionic host surface markers. When probed in an AP-mediated disease assay on patient-derived cells, mini-FH conferred efficient complement regulation at an IC50 of approximately 0.02 μM.
To construct an exemplary targeted AP-specific regulator, the inventors analyzed available structure-function data on the major AP-regulator FH, reconciled apparently contradictory findings with a hypothesis on how FH works, tested this hypothesis experimentally and employed it for the rational design of the novel protein-therapeutic mini-FH.
The inventors surmised that a compact conformation of FH (Oppermann M et al., 2006, Clin Exp Immunol 144: 342-352; Schmidt C Q et al., 2010, J Mol Biol 395: 105-122; Aslam M & Perkins S J, 2001, J Mol Biol 309: 1117-1138) prevents unhindered engagement of FH C-terminal modules with TED on inactivated C3b (cf.
Compositions:
One aspect of the present invention thus features a complement regulator that is uniquely targeted to the AP. The complement regulator comprises a complement regulating region that includes at least 3, preferably 4, complement control protein domains (CCPs), linked by a flexible linker to a multifunctional binding region that enables binding of the complement regulator to one or more C3b activation/inactivation products and/or one or more oxidation end products, as well as to polyanionic surface markers on host cells.
Because CCPs are found in all RCAs, they can be derived from any RCA. Suitable RCAs from which CCPs may be derived include, but are not limited to Factor H, decay accelerating factor (DAF, CD55), complement receptor 1 (CR1, CD35), membrane cofactor protein (MCP, CD46), C4 binding protein (C4BP) or Smallpox inhibitor of complement enzymes (SPICE). In one embodiment, the CCPs are derived from FH. Any FH can serve as the source of those CCPs. One embodiment utilizes human FH as the source of CCPs. In one embodiment, the CCPs are derived from SCRs 1-4 of FH, such as human FH.
The multifunctional binding region of the complement regulator binds to one or more C3 activation/inactivation products, to polyanionic surface markers on host cells and optionally advanced oxidation end products. Such C3 activation/inactivation products include but are not limited to thioester domain (TED)-containing C3 fragments, namely C3b, iC3b, C3dg and C3d. The polyanionic surface markers on host cells include but are not limited to N-linked glycosylation with sialic acid end-groups and diverse glycosaminoglycans (GAGs) like heparin, heparan sulfate, chondroitin sulfate, dermantan sulfate, keratan sulfate and hyaluronan. The oxidation end products include but are not limited to malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), carboxyethylpyrrole (CEP), oxidized phosphatidylserine (OxPS), oxidized cardiolipin (OxCL) and phosphocholine (PC).
In one embodiment, the multifunctional binding region is derived from SCRs (CCPs) 19 and/or 20 of FH; preferably both SCRs 19 and 20 are utilized. In other embodiments, SCRs 6-8 of FH, and/or SCR 13 of FH, can be utilized as an alternative or supplement to SCRs 19 and/or 20. Additionally, domains in other proteins that bind glycosaminoglycans (GAGs) may also be suitable for polyanion targeting, particularly in combination with other domains. Examples of such proteins include hepatocyte growth factor, which acts upon endothelial cells in a manner similar to FH, and antithrombin, which also binds heparin.
The complement regulatory region is linked to the multifunctional binding region by a flexible linker designed to provide appropriate spacing between the two regions to enable each region to function for its intended purpose, either on its own or cooperatively. The linker may be peptidic, non-peptidic or partially peptidic. In certain embodiments, the linker is of substantially the same length and flexibility as a poly-Gly peptide at least 9 residues in length, or at least 10 residues in length, or at least 11 residues in length, or at least 12 residues in length, or at least 13 residues in length, or at least 14 residues in length, or at least 15 residues in length, or at least 16 residues in length, or at least 17 residues in length, or at least 18 residues in length. In certain embodiments, the linker is of substantially the same length and flexibility as a poly-Gly peptide up to 18 residues in length. An exemplary embodiment utilizes a poly-Gly peptide at least 12 residues in length. Other amino acid residues, or analogs of such residues, may be included in the linker. For instance, addition of one or more Ala residues into the linker may introduce beneficial features while also maintaining the relative size and flexibility of the linker Substitution of a single Gly residue in the exemplary linker with another residue is not likely to substantially alter the features of the linker. If additional substitutions are made, in may be beneficial to increase the overall linker length to maintain proper flexibility (e.g., a 17 residue linker with 15 Gly residues and two other residues).
The two regions can be linked together in either order; i.e., the complement regulatory region can be on the amino-terminal side of the linker and the multifunctional binding region can be on the carboxy-terminal side of the linker, or the arrangement can be reversed such that the multifunctional binding region is on the N-terminal side of the linker and the complement regulatory region is on the C-terminal side of the linker. In one embodiment, the complement regulator comprises, from N- to C-terminus, SCRs 1-4 of FH, a poly-Gly linker at least 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18 residues in length, and SCRs 19-20 of FH. In a particular embodiment, the functional elements are derived from human FH, including but not limited to the human FH represented by SEQ ID NO:1 herein. FH domains 19-20 may exhibit surface recognition functions in species other than human, particularly in higher mammals. There is significant sequence conservation for Factor H across the species, especially for the regulatory domains 1-4 and the recognition domain 19-20 (see, e.g., Schmidt C Q et al., 2010, supra (supplement). Thus, examples of suitable FH molecules include but are not limited to those found under GenBank Accession Nos. AAI42700 (human), ABB02180 (human), CAA68704 (human), CAI19672 (human), NP_000177 (human), PO8603 (human), NP_034018 (mouse), PO6909 (mouse), CAC67513 (rat), NP_569093 (rat), AAI05259 (cow), NP_001029108 (cow), CAC81999 (pig) and NP_999446 (pig), to name a few.
In an exemplary embodiment, the complement regulator is a polypeptide referred to herein as “mini-FH,” and comprises the amino acid sequence represented by SEQ ID NO:2 or a functional equivalent. By “functional equivalent,” it is meant that the precise sequence can vary, but only to the extent that each functional element still performs the recited function, e.g., complement regulation, multifunctional binding and flexible linking. In certain embodiments, functional equivalents comprise an amino acid sequence at least 80% identical to that of SEQ ID NO:2, excluding the linker. In other embodiments, functional equivalents comprise an amino acid sequence at least 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to that of SEQ ID NO:2, excluding the linker.
The exemplary complement regulator, mini-FH, displays several improved properties as compared with FH and with other agents known to regulate AP-mediated complement activation. For instance, the experimental results set forth in Example 1 demonstrate that mini-FH shows a strong preference over FH in binding complement inactivation products. Thus, mini-FH targets to and protects host cells via its simultaneous recognition of complement activation/inactivation products and polyanionic host surface markers. When probed in an AP-mediated disease assay on PNH patient-derived erythrocytes, mini-FH conferred efficient complement regulation at an IC50 of approximately 0.02 μM. In that assay, 0.06 to 0.1 μM of mini-FH stopped AP-mediated lysis completely. By comparison, addition of FH to a concentration of 1 μM was needed to stop lysis completely.
Using a similar assay, Ristano et al. (2010, ASH Annual Meeting Abstracts, Blood 116: Abstract 637), reported an IC50 of the chimeric peptide TT30 of 30 μg/ml, or 0.48 μM based on an estimated molecular weight of 63 kDa which corresponds to the 9 CCPs comprising TT30. Thus, mini-FH was over ten-fold more active than TT30 in an in vitro protection assay of patient-derived PNH erythrocytes.
The complement regulators of the present invention can be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods. For example, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 431A peptide synthesizer (Applied Biosystems, Foster City, Calif.), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art. During the course of peptide synthesis, branched chain amino and carboxyl groups may be protected/deprotected as needed, using commonly-known protecting groups.
Alternatively, certain types of complement regulators can be produced by expression in a suitable prokaryotic or eukaryotic system. For example, a DNA construct can be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae or Pichia pastoris), or into a baculovirus vector for expression in an insect cell or a viral vector for expression in a mammalian cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. In an exemplary embodiment, a FH-based complement regulator is produced by expression in Pichia pastoris, and thereafter purified in accordance with known methods, as summarized in Example 1.
Polypeptides produced by gene expression in a recombinant procaryotic or eucaryotic system can be purified according to methods known in the art. A combination of gene expression and synthetic methods may also be utilized to produce the complement regulators. For example, a polypeptide can be produced by gene expression and thereafter subjected to one or more post-translational synthetic processes.
Another aspect of the invention features a pharmaceutical composition comprising the complement regulator described above and a pharmaceutically acceptable carrier. Such a pharmaceutical composition may include the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
The formulations of the pharmaceutical compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-does unit.
As used herein, the term “pharmaceutically-acceptable carrier” means a chemical composition with which a complement regulator may be combined and which, following the combination, can be used to administer the complement regulator to an individual.
As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg and 100 mg/kg body weight as a single bolus, or in a repeated regimen, or a combination thereof as readily determined by the skilled artisan. In certain embodiments, the dosage comprises at least 0.1 mg/kg, or at least 0.2 mg/kg, or at least 0.3 mg/kg, or at least 0.4 mg/kg, or at least 0.5 mg/kg, or at least 0.6 mg/kg, or at least 0.7 mg/kg, or at least 0.8 mg/kg, or at least 0.9 mg/kg, or at least 1 mg/kg, or at least 2 mg/kg, or at least 3 mg/kg, or at least 4 mg/kg, or at least 5 mg/kg, or at least 6 mg/kg, or at least 7 mg/kg, or at least 8 mg/kg, or at least 9 mg/kg, or at least 10 mg/kg, or at least 15 mg/kg, or at least 20 mg/kg, or at least 25 mg/kg, or at least 30 mg/kg, or at least 35 mg/kg, or at least 40 mg/kg, or at least 45 mg/kg, or at least 50 mg/kg, or at least 55 mg/kg, or at least 60 mg/kg, or at least 65 mg/kg, or at least 70 m/kg, or at least 75 mg/kg, or at least 80 mg/kg, or at least 85 mg/kg, or at least 90 mg/kg, or at least 95 mg/kg, or at least 100 mg/kg, on a daily basis or on another suitable periodic regimen. In a particular embodiment, the dosage is between about 0.5 mg/kg and about 20 mg/kg, or between about 1 mg/kg and about 10 mg/kg, or between about 2 mg/kg and about 6 mg/kg, based on preclinical studies of TT30 (Fridkis-Hareli et al., 2011, supra) and taking into account the comparatively greater efficacy of mini-FH in an in vitro assay.
In one embodiment, the invention envisions administration of a dose that results in a serum concentration of the complement regulator between about 0.01 μM and 10 μM in an individual. In certain embodiments, the combined dose and regimen will result in a serum concentration, or an average serum concentration over time, of the complement regulator of at least about 0.01 μM, or at least about 0.02 μM, or at least about 0.03 μM, or at least about 0.04 μM, or at least about 0.05 μM, or at least about 0.06 μMI, or at least about 0.07 μM, or at least about 0.08 μM, or at least about 0.09 μM, or at least about 0.1 μM, 0.11 μM, or at least about 0.12 μM, or at least about 0.13 μM, or at least about 0.14 μM, or at least about 0.15 μM, or at least about 0.16 μM, or at least about 0.17 μM, or at least about 0.18 μM, or at least about 0.19 μM, or at least about 0.2 μM, or at least about 0.3 μM, or at least about 0.4 μM, or at least about 0.5 μM, or at least about 0.6 μM, or at least about 0.7 μM, or at least about 0.8 μM, or at least about 0.9 μM, or at least about 1 μM or at least about 1.5 μM, or at least about 2 μM, or at least about 2.5 μM, or at least about 3 μM, or at least about 3.5 μM, or at least about 4 μM, or at least about 4.5 μM, or at least about 5 μM, or at least about 5.5 μM, or at least about 6 μM, or at least about 6.5 μM, or at least about 7 μM, or at least about 7.5 μM, or at least about 8 μM, or at least about 8.5 μM, or at least about 9 μM, or at least about 9.5 μM, or at least about 10 μM. In certain embodiments, the combined dose and regimen will result in a serum concentration, or an average serum concentration over time, of the complement regulator of up to about 0.1 μM, 0.11 μM, or up to about 0.12 μM, or up to about 0.13 μM, or up to about 0.14 μM, or up to about 0.15 μM, or up to about 0.16 μM, or up to about 0.17 μM, or up to about 0.18 μM, or up to about 0.19 μM, or up to about 0.2 μM, or up to about 0.3 μM, or up to about 0.4 μM, or up to about 0.5 μM, or up to about 0.6 μM, or up to about 0.7 μM, or up to about 0.8 μM, or up to about 0.9 μM, or up to about 1 μM or up to about 1.5 μM, or up to about 2 μM, or up to about 2.5 μM, or up to about 3 μM, or up to about 3.5 μM, or up to about 4 μM, or up to about 4.5 μM, or up to about 5 μM, or up to about 5.5 μM, or up to about 6 μM, or up to about 6.5 μM, or up to about 7 μM, or up to about 7.5 μM, or up to about 8 μM, or up to about 8.5 μM, or up to about 9 μM, or up to about 9.5 μM, or up to about 10 μM. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of patient and type of disease state being treated, the age of the patient and the route of administration, such dosage is readily determinable by the person of skill in the art.
The pharmaceutical composition can be administered to a patient as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the patient, as described above.
Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, parenteral, ophthalmic (including intravitreal), suppository, aerosol, topical or other similar formulations. Such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer a complement inhibitor according to the methods of the invention.
As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution can be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, in microbubbles for ultrasound-released delivery or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents including replacement pulmonary surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
Methods:
Another aspect of the invention features methods of regulating complement activation. In particular embodiments, practice of the methods results in modulation of complement activation via the AP. In general, the methods comprise contacting a medium in which regulation of complement activation is desired with a complement regulator of the present invention, wherein the contacting results in regulation of complement activation in the medium. The medium can be any medium in which regulation of complement activation is desired. In certain embodiments, the medium includes cells or tissues of an organism, including (1) cultured cells or tissues, (2) cells or tissues within the body of a subject or patient, and (3) cells or tissues that have been removed from the body of one subject and will be replaced into the body of the same patient (e.g., extracorporeal shunting of blood or autologous transplantation) or transferred to another patient. In connection with the latter embodiment, the medium may further comprise a biomaterial, such as tubing, filters or membranes, that contact the cells or tissues during extracorporeal shunting. Alternatively, the medium may comprise biomaterials that are implanted into a subject.
In certain embodiments, the methods of regulating complement activation apply to living patients or subjects and comprise part or all of a method of treating the patient for a pathological condition associated with complement activation, particularly AP-mediated complement activation. Many such pathological conditions are known in the art (see, e.g., Holers, 2008, supra) and include, but are not limited to, as atypical hemolytic uremic syndrome (aHUS), dense deposit disease, age-related macular degeneration (AMD), paroxysomal nocturnal hemoglobinuria (PNH), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), several autoimmune and autoinflammatory kidney diseases, autoimmune myocarditis, multiple sclerosis, traumatic brain and spinal cord injury, intestinal and renal ischemia-reperfusion (IR) injury, spontaneous and recurrent pregnancy loss, anti-phospholipid syndrome (APS), asthma, anti-nuclear cytoplasmic antigen-associated pauci-immune vasculitis (Wegener's syndrome), non-lupus autoimmune skin diseases such as pemphigus, bullous pemphigoid, and epidermolysis bullosa, post-traumatic shock and atherosclerosis. In particular embodiments, the pathological condition has been associated with mutations and polymorphisms in the gene encoding FH, including but not limited to: AMD, aHUS and membrano-proliferative glomerulonephritis type II (MPGN-II, also referred to as dense deposit disease (DDD)). In other embodiments, the complement regulators of the present invention are suitable for use as a substitute for Eculizumab or TT30 in treatment of diseases for which those agents are currently prescribed, or for which they are being developed in pre-clinical and clinical studies. Those diseases include, but are not limited to, aHUS, PNH and AMD.
The treatment methods typically comprise (1) identifying a subject with a disease or condition treatable by regulation of complement activation as described hereinabove, and (2) administering to the subject an effective amount of a complement regulator of the invention using a treatment regimen and duration appropriate for the condition being treated. Development of appropriate dosages and treatment regimens will vary depending upon any number of factors, including but not limited to, the type of patient and type of disease state being treated, the age of the patient and the route of administration. The skilled artisan is familiar with the design of dosage regimens that take such variables into account. As one example, a dosage regimen similar to that of Eculizumab may be developed for treatment of PNH or aHUS with a complement regulator of the invention, taking into account the difference in potency between the two substances. Eculizumab typically is administered by intravenous infusion for treatment of PNH or aHUS according to the following schedule: 600 mg weekly for the first 4 weeks, 900 mg for the fifth dose 1 week later, then 900 mg every 2 weeks thereafter (Alexion Pharmaceuticals, Inc. Package Insert version dated “Last Revised 28 Sep. 2011”). As another example, a dosage regimen based on preclinical and clinical results developed for similar agents may be utilized. For example, a pharmacokinetic study of the chimeric protein TT30 in cynomolgus monkeys revealed that an intravenous or subcutaneous bolus of 15-60 mg/kg resulted in inhibition of complement AP activity for several to many hours (Fridkis-Hareli et al., 2011, supra). The skilled artisan can readily develop a dosage and regimen for the complement regulators of the invention based on this type of information.
The following example is provided to describe the invention in greater detail. It is intended to illustrate, not to limit, the invention.
This example describes the synthesis of mini-FH and demonstration of its ability to regulate complement activation through the AP.
Surface plasmon Resonance.
All sensorgrams were recorded on a Biacore 2000 instrument (GE Healthcare Corp., Piscataway, N.J.), processed in Scrubber (v2.0c; BioLogic Pty, Australia), and are shown as duplicates of dummy surface-subtracted response curves.
C3-Fragment Binding.
Equal amounts of C3b (˜4000 RUs) (from Complement Technology, Tyler, Tex., USA) were immobilized onto 3 flow-cells (fc) of a CM5 chip (Biacore) by initial amine-coupling of a small amount of C3b onto fc 2-4 and further deposition of the vast majority of C3b molecules via its reactive thioester through an on-chip assembly of the AP-convertase (C3bBb) and subsequent exposure to and proteolytic activation of C3 (24). The surface was washed overnight with a buffer flow of 10 ul/min to allow stabilization of the physiologically prepared C3b-surface. Test-injections of 1 μM FH or 10 μFH19-20 yielded similar response units (RUs) for all three C3b surfaces (on fc 2-4: 600, 680 and 690 RUs for FH and 240, 230 and 230 RUs for FH19-20, respectively) and proved very similar loadings of C3b molecules. C3b molecules on fc3 and fc4 were processed with FI in the presence of the cofactors FH or soluble complement receptor 1 (sCR1) to yield iC3b or C3dg (the latter as described in Morgan et al., 2011, supra), respectively. A dummy surface (fc1) was prepared by two consecutive cycles of surface activation with standard amine coupling reagents and quenching by ethanolamine.
Successful processing to iC3b and C3dg was demonstrated with injections of sCR1 and FH15-18. As expected, FH15-18 did not associate with either of the deposited C3-opsonins (Morgan et al., 2011, supra), while sCR1 at 0.33 μM showed a strong binding to the C3b surface (925 RUs), a moderate to weak response to the iC3b surface (245 RUs) and negligible binding to the C3dg surface (15 RUs) consistent with previous findings. Due to the physiological deposition of C3b and subsequent trimming with FI, both, the iC3b and the C3dg, surfaces likely carry miniscule amounts of residual C3b molecules, which were not readily accessible by FI. However, the predicted responses of the positive and negative control, sCR1 and FH15-18, show that the physiological processing worked close to completion. Deposition, proteolytic processing and all interactions studies were performed in HBS-P+buffer (i.e. 10 mM HEPES-buffered 150 mM saline (pH7.4), 0.005% (v/v) surfactant p20, 1 mM MgCl2) at 25° C.
Decay Acceleration Assay.
Similarly as described above C3b was immobilized onto a CM5-chip by initially fixing some C3b via amine coupling (2740RUs) and subsequent physiological deposition of the majority of C3b molecules (5090RUs) through the reactive thioester of C3b in the presence of the AP-convertase. An extensive wash with buffer was performed as above. A mix of 100 nM Factor D and 500 nM Factor B in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% (vol/vol) Tween-20 and 1 mM MgCl2, pH 7.4) was flowed for 3 min at 10 μl/min over the immobilized C3b-surface to build the AP-convertase C3bBb on the chip. Following an undisturbed decay of 1 min, the analytes, all at 100 nM, were injected for 3.5 min. To regenerate the surface, residual convertases were decayed by consecutive injections of 2 μM FH1-4 and 1 M NaCl. For comparative visualization of the decay acceleration response, SPR signals of the analytes obtained for C3b in the absence of the convertase were subtracted from the respective once obtained in presence of the convertase. Scaling up to a maximum of 5% was performed on some response curves to compensate for the small drift in signal due to the physiological immobilization procedure in order to facilitate an excellent overlay of sensorgrams at the time point of analyte injection.
Computational Modeling.
The complex structure of C3b:mini-factor H was constructed by superposing the C3d domain (or TED) of the C3b:FH1-4 (2WII, Wu et al., 2009, supra) and C3d:FH19-20 (3OXU, Morgan et al., 2011, supra) complex structures. The root mean square distance between two superposed C3d domains was 0.285 Å. The linker between CCP4 and CCP19 was modeled by the dope_loopmodel module of MODELLER 9v8 (25). The energy of dope_loopmodel, which is the energy we used to evaluate the model of mini-FH, with different linker length, bound to C3b, is composed by several terms, including bond length, bond angle, torsion, improper torsion, dihedral, 6-12 Lennard-Jones potential, DOPE potential and GBSA solvent potential. For each linker length, 100 conformations were sampled. The top 80 models of each linker length were selected to evaluate the effect of different length on energy and conformation.
Heparin Chromatography.
A 5 ml HiTrap heparin column equilibrated in PBS was used. Elution was performed by applying a linear gradient from PBS to PBS substituted with 0.5 M NaCl in 5 column volumes.
Cofactor Activity.
A fluid phase, time-course cofactor assay was performed in PBS, similar to Schmidt et al., 2011, supra. A mastermix of Factor I, C3b (both from Complement Technology) and respective cofactor (added last) was kept on ice and aliquoted into 20 μl aliquots prior to incubation at 37° C. for increasing amounts of time (as specified in
Classical Pathway ELISA.
Nunc maxisorb 96-well plates were coated with 1% ovalbumin (Sigma) in PBS, pH 7.4, 50μ/well for 2 h at room temperature (RT) or overnight (O/N) at 4° C. Washing twice with PBST (0.05% Tween 20), 200 μl/well was followed by blocking with 1% BSA (bovine serum albumin) in PBS, pH 7.4, 200 μl/well, for 1 h at RT. Then rabbit anti-ovalbumin antibody at a dilution of 1:1000 in 1% BSA/PBS, was bound for 1 h at RT (50 μl/well). After another washing step, as above, serial dilutions of analytes in PBS were added to the 96 well plate. To 10 μl of analyte, 20 μl of PBS++ (=PBS containing 1 mM MgCl2 and 0.9 mM CaCl2) was added into each well of the ELISA plate, followed by 30 μl of a 1:40 serum dilution in PBS++. The mix was incubated at RT for 15 min prior to another washing step as above and the subsequent exposure to goat anti-human C3 (HRP-conjugate from MP Biomedicals, LLC) at a 1:1000 dilution in 1% BSA/PBS, 50 μl/well for 30 min at RT. After washing three times with PBST, detection was achieved by adding 50 μl/well of a freshly mixed solution containing 0.1 M sodium citrate at pH 4.3, 5 mg ABTS (Roche) and 0.03% H2O2. Absorbance was measured at 405 nm. EDTA at a final concentration of 10 mM was used as negative control.
Alternative Pathway ELISA.
Nunc maxisorb 96 well plates were coated with 40 μg/ml LPS (lipopolysaccharide from Sigma), in PBS, pH 7.4, 50 μl/well for 2 h at room temperature (RT) or overnight (O/N) at 4° C. Washing, blocking, addition of analytes was performed as above. To 30 μl of analyte in PBS, 30 μl of a solution containing 50% serum and 10 mM MgEGTA in PBS was added. The mix was incubated for 1 h at 37° C. Negative control, washing and detection were performed as mentioned above.
Induction of PNH-Phenotype in Normal Erythrocytes and Protection/Lysis Assay.
Erythrocytes from a healthy donor were washed and treated (Ezzell J L et al., 1991, Blood 77: 2764-2773) in order to induce PNH phenotype (sensitized erythrocytes). A protection assay of those erythrocytes was performed similarly, but with a few modifications, as published (Ezzell et al., 1991, supra). In brief, erythrocytes were treated with an 8% solution of the sulfhydryl reagent 2-aminoethylisothiouronium-bromide (AET) and then washed with PBSE (=PBS containing 5 mM EDTA) and PBS. The concentration of red blood cells (RBC) in PBS was adjusted so that the absorbance at 405 nm of 100 μl of a dilution of 10 μl erythrocyte stock in 190 μl water yielded a value between 1.5 and 2.0. To such a stock an anti-DAF antibody (Clone: BRIC216, Isotype: IgG1, from SeroTech) was added to a final concentration of 6.7 μg/ml and the mix was incubated for 30 min on ice. After washing with PBS, the treated erythrocytes were resuspended with PBS-Mg (PBS containing 1 mM MgCl2 at pH 6.4) to the initial volume (to yield the same concentration of RBCs) and subjected to the protection/lysis assay. When desialylated erythrocytes were used, a RBC suspension in PBS-Mg was split in half. Both halves were incubated at 37° C. on a rotating disk, but only one had been substituted with 36U of Neuraminidase (New England Biolabs) per 100 μl of RBC-stock.
Fresh human serum was acidified with 0.2 M HCl to pH 6.4 and substituted with MgCl2 and EGTA to yield final concentrations of 2.5 mM and 8 mM, respectively. In a round bottom 96 well plate, 60 μl of this serum-mix were added to 10 μl PBS at pH 6.4 containing 1 mM MgCl2 and 20 μl of analyte in PBS. The plate was shaken and incubated on ice for 5 min prior to pipetting 10 μl of sensitized, or sensitized and desialylated, RBC into each well. The final serum concentration in this reaction mix was 52%. The 96-well plate was incubate on a shaking platform for 30 min at 37° C. and thereafter the reaction was immediately quenched by addition of 100 μl ice-cold PBS containing 5 mM EDTA. Remaining, i.e. non-lysed, cells were separated by centrifugation at 1000 g for 3 min and 100 μl of the supernatant was transferred into a fresh 96-well plate to measure the absorbance at 405 nm.
Results:
Design Objectives.
To gain AP-specific regulatory function without blocking CP and LP, the new regulator was designed based on functional domains of FH, but was determined to be more potent than FH. The complement regulatory functions residing in FH CCPs 1-4 and the polyanion host surface recognition feature, which maps to FH CCPs 19-20, were included. With the incorporation of C-terminal CCPs 19-20, the recently described FH-function of localizing to and protection of sites of oxidative damage was also incorporated (Weismann D et al., 2011, Nature 478: 76-81). To gain extra potency in complement regulation over FH, the CCPs 1-4 and 19-20 were connected with a sufficient degree of flexibility so that the ability of FH19-20 to bind indiscriminately to all C3-opsonins would be unrestricted to allow efficient targeting to sites of complement inactivation, as opposed to a compact FH molecule which shows substantially reduced ability to bind iC3b. Finally, despite the flexibility needed for targeting of C3b-inactivation products, direct linking of CCPs 1-4 and 19-20 additionally requires that the new regulator matches the affinity of FH for its major target C3b. Accordingly, the new regulator was designed to occupy both FH-binding sites on C3b simultaneously to cooperate upon C3b-engagment.
Twelve Glycines Link the Four N- and Two C-Terminal Domains of FH without Imposing Restraints on C3b-Binding.
To design an unstructured linker with low immunogenic potential and high solubility any hydrophobic, bulky and charged amino acids were avoided (Schellenberger V, et al., 2009, Nat Biotechnol 27: 1186-1190) and glycine was chosen to insure a high degree of flexibility. Superimposition of the crystal structure complexes FH1-4:C3b and FH19-20:C3d (
Computational modeling was employed to optimize the number of linking glycine residues to provide enough space and flexibility as to allow the covalently linked CCPs 1-4 and 19-20 to bind simultaneously to C3b. In silico constructions of such fusion proteins were submitted to energy calculations. Two stages of energy change in relation to the number of linking glycines were found (
Engineered Protein Mini-FH Obtained Recombinantly at High Purity.
Mini-FH was expressed in Pichia pastoris and purified using conservative chromatography methods, as detailed by Schmidt et al. (Schmidt C Q et al., 2011, Protein Expr Purif 76: 254-263). Briefly, codon-optimized coding DNA was cloned it into the Pst1-Xba1 restriction sites of vector pPICZalphaB (Invitrogen) and transformed into P. pastoris. The culture was expanded in a Bioflow 310 fermenter (New Brunswick Scientific) without deglycosylation and purified from the organism using SP-sepharose size exclusion and ion exchange chromatography.
Denaturing gel analysis of the purified protein under both reducing and non-reducing conditions showed a clean preparation in the absence of significant cleavage bands. The migration pattern was consistent with a 43.3 kDa protein that contained multiple (12 in total) disulfide bridges (
Binding Activities of Mini-FH and FH are Nearly Identical for C3b and Heparin, but Differ for the C3b-Inactivation Products iC3b and C3dg.
The analytes FH1-4, FH19-20, mini-FH and FH were tested for their ability to adhere to heparin, a model substance for polyanionic host surface markers, under physiological conditions in phosphate-buffered saline (PBS). FH1-4 did not bind to the model substance while FH and FH19-20 did bind to it. Mini-FH eluted slightly later than FH and just prior to FH19-20 from the heparin affinity column, which is consistent with the notion of all three proteins being able to recognize polyanionic host surfaces.
Surface plasmon resonance (SPR) was used to analyze the binding behavior between FH and mini-FH and the key functional FH sites, i.e. FH1-4 and FH19-20, with physiologically deposited C3-opsonins C3b, iC3b and C3dg (all at equal molar loading density) in physiological buffer. Binding of FH1-4 (
Mini-FH Retains Known FH Functionality but is Substantially More Active in Controlling AP-Activation.
When tested for their ability to accelerate the decay of AP-convertases C3bBb, assembled on a Biacore CM5 chip, FH19-20 failed to exhibit such activity, as reported before (Wu et al., 2009, supra). All other proteins assayed showed decay accelerating activity (DAA,
Next, FH and mini-FH were probed for their ability to act as a cofactor for factor I mediated proteolytic inactivation of fluid phase C3b into iC3b. Both analytes exhibited cofactor-activity; FH facilitates a somewhat faster consecutive cleavage of the first two scissile bonds (between residues 1303-1304 and 1320-1321) of the α-chain of C3b when compared to mini-FH (
To determine the complement regulatory activity in an ELISA assay, LPS was used as activating agent. Addition of mini-FH to a final concentration of 0.04 μM inhibited AP-activation by 50% (
To probe the pathway specificity all analytes were also submitted to a CP-specific complement activation ELISA. Analytes were added to a 1:80 serum dilution in PBS++ (phosphate-buffered saline containing 5 mM MgCl2 and 0.9 mM CaCl2. At the concentration range tested, only the compstatin analog CP30 achieved inhibition of CP-activation. At a concentration of 0.13 μM, CP30 inhibited half of the CP-activation in this assay. Towards higher concentrations (6-8 μM) mild attenuation of CP-activation was also noticeable for mini-FH and to a lesser extent also for FH.
Mini-FH has Higher Protective Activity than FH in PNH Hemolytic Assays.
Next we evaluated AP-regulation in a physiological setting on a cell surfaces in a hemolytic assay, which mimics the PNH disease condition.
PNH phenotype was induced in healthy erythrocytes prior to exposing them to acidified serum that had been substituted with various analytes (according to Ezzell et al., 1991, supra). Brisk activation of the AP in acidified serum renders PNH erythrocytes, in contrast to healthy erythrocytes, susceptible to lysis by complement. Addition of the controls lin-scr-CP30 and FH12-13, which are void of any complement regulatory-function, produced the same levels of lysis as addition of PBS alone (
To analyze the contribution of polyanionic surface markers in conferring complement protection to RBCs of PNH-phenotype, the negatively charged sialic acid, which is abundant on erythrocyte surfaces, was removed enzymatically with neuraminidase (ND). Only analytes that contained the host-surface recognition domain CCP 20 of FH showed a differential protection behavior (
The dose-dependent protection of vulnerable PNH-induced erythrocytes was confirmed when the same analytes were tested on erythrocytes derived from PNH-patients (
The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.
This is a U.S. national filing, pursuant to 35 U.S.C. §371, of International Application No. PCT/US2013/032350, filed Mar. 15, 2013, which claims benefit of U.S. Provisional Application No. 61/612,512, filed Mar. 19, 2012, the entire contents of each of which are incorporated by reference herein.
This invention was made with government support under Grant Nos. GM62134 and AI068730 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2013/032350 | 3/15/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2013/142362 | 9/26/2013 | WO | A |
| Number | Date | Country |
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| WO 9823638 | Jul 1998 | WO |
| WO 2007056227 | Oct 2007 | WO |
| WO 2010034015 | Jul 2010 | WO |
| WO 2011107591 | Sep 2011 | WO |
| WO 2011113641 | Sep 2011 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20150110766 A1 | Apr 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61612512 | Mar 2012 | US |
