Modulation of complement activity

Abstract

The present invention relates to modulation of complement activity, in particular, the present invention relates to providing modulators of complement activity. Also provided are methods of utilizing such modulators as therapy.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201580031341.8 (2020100206781), application date June 12, 2015, and invention name “Regulation of Complement Activity”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 011,368, filed on June 12, 2014, entitled MODULATION OF COMPLEMENT ACTIVITY, U.S. Provisional Patent Application No. 62 / 077,460, filed on November 10, 2014, entitled MODULATION OF COMPLEMENT ACTIVITY, and U.S. Provisional Patent Application No. 62 / 108,772, filed on January 28, 2015, entitled MODULATION OF COMPLEMENT ACTIVITY, the contents of each of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format, and the sequence listing is hereby incorporated by reference in its entirety. The ASCII copy created on June 11, 2015 is named 20111005PCT_SL.txt and is 130,110 bytes in size. Field of the Invention

[0006] The present invention relates to compounds, including polypeptides, which are useful as inhibitors and / or antagonists of complement activity. Methods of using the inhibitors as therapy are also provided. Background of the Invention

[0007] The immune response of vertebrates consists of adaptive and innate immune components. The adaptive immune response is selective for specific pathogens and responds slowly, while components of the innate immune response recognize a wide range of pathogens and respond rapidly upon infection. One such component of the innate immune response is the complement system.

[0008] The complement system includes about 20 circulating proteins synthesized mainly by the liver. The components of this specific immune response were first referred to as "complement" because they were observed to supplement antibody responses in bacterial destruction. Before activation in response to infection, these proteins remain in an inactive form. Activation occurs by means of a pathway of proteolytic cleavage that is triggered by pathogen recognition and causes pathogen destruction. Three such pathways are known in the complement system and are referred to as the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is activated when IgG or IgM molecules are bound to the surface of the pathogen. The lectin pathway is triggered by the lectin protein that binds mannans that recognizes sugar residues in the bacterial cell wall. The alternative pathway remains active at a low level in the absence of any specific stimulant. Although all three pathways are different about initiation events, all three pathways converge due to the cleavage of complement component C3. C3 is cleaved into two products called C3a and C3b. In these two products, C3b becomes covalently attached to the pathogen surface, and C3a acts as a diffusible signal to promote inflammation and recruit circulating immune cells. Surface-bound C3b forms complexes with other components to initiate a cascade of reactions in other subsequent components of the complement system. Due to the requirement for surface attachment, complement activity remains localized and damage to non-target cells is minimized.

[0009] Pathogen-associated C3b promotes pathogen destruction in two ways. In one approach, C3b is directly recognized by phagocytes and leads to ingestion of the pathogen. In the second approach, pathogen-associated C3b triggers the formation of the membrane attack complex (MAC). In the first step, C3b is complexed with other complement components to form the C5-convertase complex. Depending on the initial complement activation pathway, the components of this complex can be different. The C5-convertase formed by the classical complement pathway contains C4b and C2a in addition to C3b. When formed by the alternative pathway, the C5-convertase contains two subunits of C3b and a Bb component.

[0010] Complement component C5 is cleaved into C5a and C5b by either C5-convertase complex. C5a, much like C3a, diffuses into the circulation and promotes inflammation, acting as a chemoattractant for inflammatory cells. C5b remains attached to the cell surface, where it triggers the formation of MAC via interaction with C6, C7, C8 and C9. MAC is a hydrophilic pore across the membrane and promotes the free flow of fluid in and out of the cell, thereby destroying the cell.

[0011] An important component of all immunological activities is the ability of the immune system to distinguish between autologous cells and non-autologous cells. Pathology occurs when the immune system cannot make this distinction. With respect to the complement system, vertebrate cells express proteins that protect them from the effects of the complement cascade. This ensures that the targets of the complement system are confined to pathogenic cells. Many complement-related disorders and diseases are associated with abnormal destruction of autologous cells caused by the complement cascade. In one example, a subject suffering from paroxysmal nocturnal hemoglobinuria (PNH) cannot synthesize functional versions of complement regulatory proteins CD55 and CD59 on hematopoietic stem cells. This leads to complement-mediated hemolysis and various downstream complications. Other complement-related disorders and diseases include, but are not limited to, autoimmune diseases and disorders, neurological diseases and disorders, blood diseases and disorders, and infectious diseases and disorders. Experimental evidence shows that many complement-related disorders are alleviated via the inhibition of complement activity. Therefore, there is a need to develop compounds that can selectively block complement-mediated cell destruction. Summary of the invention

[0012] The present invention provides compounds including polypeptides (e.g., mimetic peptides, cyclic polypeptides and cyclic mimetic peptides), small molecules, antibodies, antibody fragments and aptamers, and methods for using the compounds for complement regulation. According to the present invention, the polypeptide may have the sequence shown in SEQ ID NO 1-201 or 211. The polypeptide may be linear or cyclic and may include a cyclic ring formed by a bridging moiety between two amino acids. The bridging moiety may include features selected from the group consisting of a disulfide bond, an amide bond (lactam), a thioether bond, an aromatic ring, an unsaturated aliphatic hydrocarbon chain, a saturated aliphatic hydrocarbon chain, a triazole ring or a combination thereof. In addition, the cyclic ring may vary in length and may be 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, or more.

[0013] The bridging moiety may comprise a disulfide group between two cysteine ​​residues or an aromatic ring generated by reaction with poly(bromomethyl)benzene, and such poly(bromomethyl)benzene may be selected from the group consisting of 1,2-bis(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene and 1,4-bis(bromomethyl)benzene.

[0014] The bridging portion may also include an aromatic ring generated by reacting with a compound selected from the group consisting of 2,6-bis(bromomethyl)pyridine, (E)-1,4-dibromobut-2-ene, and 1,2-bis(bromomethyl)-4-alkylbenzene.

[0015] The bridging moiety may also comprise an amide bond produced by reaction of the side chains of the following amino acid pairs: lysine and glutamate, lysine and aspartate, ornithine and glutamate, ornithine and aspartate, homolysine and glutamate, homolysine and aspartate, other combinations of amino acids, unnatural amino acids, or non-amino acid residues including primary amines and carboxylic acids.

[0016] According to the present invention, polypeptides are useful because they can exhibit complement C5 inhibitory activity. In some cases, such activity can be at a half-maximal inhibitory concentration (IC) of about 50 nM to about 200 nM or less than 50 nM. 50 ).

[0017] In some embodiments, the present invention provides polypeptides having tryptophan or one or more tryptophan analogs selected from the group consisting of 5-fluorotryptophan, 5-methyl-O-tryptophan, 1-methyltryptophan, D-tryptophan and 7-azatryptophan present in the amino acid sequence. Such compounds particularly include those of SEQ ID NO: 3, 9, 11, 19, 45, 46, 50 and 59.

[0018] In some embodiments, the polypeptides of the invention can bind to a region on C5, wherein the region is distal to the C5a-C5b cleavage site. In some cases, the polypeptides of the invention can inhibit the cleavage of C5 into C5a and C5b cleavage products.

[0019] In some cases, a polypeptide of the invention may be part of an antibody.

[0020] The polypeptides of the present invention may be included in a composition. Such compositions may include one or more acceptable carriers or excipients. Such polypeptides may be further bound to another non-polypeptide moiety such as, for example, a water-soluble polymer. These polymers may be hydrophilic or hydrophobic. In some embodiments, the hydrophilic polymer is selected from the group consisting of polyalkylene oxide homopolymers, polypropylene glycol, polyoxyethylated polyols, and copolymers thereof. The hydrophilic polymer may also include polyethylene glycol (PEG). The composition may further include a polypeptide bound to an albumin binding polypeptide selected from the group consisting of SEQ ID NOs: 202-204. In some cases, the composition may include a polypeptide bound to a cell penetrating polypeptide selected from the group consisting of SEQ ID NOs: 205-210. The composition may further include a polypeptide bound to a lipid moiety. The lipid moiety may include, but is not limited to, fatty acids, phospholipids, and sterols. In some embodiments, the lipid moiety may be directly bound to the polypeptide of the present invention or linked to a polypeptide-PEG conjugate. Some compositions may include a pharmaceutically acceptable excipient.

[0021] In some embodiments, the invention provides methods of inhibiting C5 cleavage in a cell system, the methods comprising using a composition according to the invention such as those described above. Some such methods may reduce C5a levels. Other methods may reduce membrane attack complex (MAC) formation.

[0022] Some methods of the invention can result in a molecule having an IC of about 50 nM to about 200 nM. 50 In some cases, the methods of the invention may include an IC of less than 50 nM. 50 Inhibition of C5 cleavage.

[0023] Some methods of the present invention can be applied to cell systems selected from in vitro systems, in vivo systems and ex vivo systems. Some in vivo systems may include subjects, such as human subjects. In some cases, polypeptides and polypeptide compositions can be used to treat or prevent complement-related diseases, disorders and / or conditions in subjects. Administration to subjects may be selected from the group consisting of buccal, intranasal, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal and intravitreal. In some cases, the composition can be used to treat paroxysmal nocturnal hemoglobinuria and / or atypical hemolytic uremic syndrome. In some cases, the composition of the present invention can be used to treat one or more of inflammatory indications, trauma, injury, autoimmune diseases, vascular indications, neurological indications, renal-related indications and eye diseases.

[0024] In some embodiments, the present invention provides a method of inhibiting thrombin-induced complement activation in a cell system, the method comprising contacting such a system with a polypeptide of the present invention. Further provided is a method of treating hemolysis in a subject, the method comprising administering a polypeptide as described herein. According to some such methods, the hemolysis treated is caused by thrombin-induced complement activation.

[0025] In certain embodiments, a kit is provided for diagnosing, prognosing, preventing or treating a disease, illness and / or condition in a mammal including a human. Such kit will comprise one or more polypeptides or polypeptide compositions of the present invention, and optionally, reagents and / or instructions for use. In such kit, the polypeptide may comprise a detectable label or be bound to a detectable label to form the ability to detect a complex. According to one embodiment, the label comprises a BODIPY-TMR label. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The foregoing and other objects, features and advantages will be apparent from the following description of specific embodiments of the present invention as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The accompanying drawings are not necessarily drawn to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present invention.

[0027] Figure 1 Graph showing the results of an enzyme immunoassay (EIA) for detecting C5a in supernatants from a human red blood cell (RBC) hemolysis assay as concentrations of the inhibitors R3002 (SEQ ID NO: 3) and R3008 (SEQ ID NO: 9) increase. The level of C5a correlates with complement activity and is therefore an indicator of the ability of the tested compound to inhibit complement activity. Supernatants from the hemolysis assay were diluted 1:50 and assayed for C5a levels. C5a levels decreased in human hemolysis assay supernatant samples as levels of either assayed inhibitor increased. R3002 (SEQ ID NO: 3) had an IC of 5.4 nM. 50 , while R3008 (SEQ ID NO: 9) had an IC of 54.5 nM 50 As used herein, the term “IC 50 ” refers to the half-maximal inhibitory concentration, a value used to indicate the amount of inhibitor required to reduce a given reaction or process by half.

[0028] Figure 2 Graph showing the results of an EIA for detecting membrane attack complex (MAC) in supernatants from human RBC hemolysis assays as concentrations of R3008 (SEQ ID NO: 9) increase. The level of MAC correlates with complement activity and is therefore an indicator of the ability of R3008 (SEQ ID NO: 9) to inhibit complement activity. Supernatants from hemolysis assays were diluted 1:5 and MAC levels were determined. MAC levels increased with increasing concentrations of R3008 (SEQ ID NO: 9) with an IC of 33 nM. 50 Levels of the assayed inhibitor were increased in the hemolysis assay supernatant samples and decreased in the hemolysis assay supernatant samples.

[0029] Figure 3is a graph showing competitive fluorescence polarization (FP) data for test articles R3003 (SEQ ID NO: 4), R3011 (SEQ ID NO: 31), R3014 (SEQ ID NO: 55), R3023 (SEQ ID NO: 104), R3043 (SEQ ID NO: 50) and R3050 (SEQ ID NO: 23). FP allows the measurement of binding events in homogeneous solutions. Competitive binding assays were performed as follows: a 25 nM solution of compound R3076 (SEQ ID NO: 40) with a fluorescent tag was combined with increasing amounts of the test article and the change in FP (in millipolarization units; mP) was measured. Reduced mP levels are associated with successful competition of C5 by the test article. The average (+ / - standard deviation) of two independent experiments performed in triplicate is shown. Among the tested articles, R3003 (SEQ ID NO: 4) was the most potent, while the control polypeptide R3023 (SEQ ID NO: 104) showed no activity at the highest concentration tested.

[0030] Figure 4 Results from a study in cynomolgus monkeys are depicted. Changes in plasma concentrations of R3152 (SEQ ID NO: 153) (circles) following a single 3 mg / kg IV dose in cynomolgus monkeys are shown. Changes in hemolytic activity (squares) at the same time points are also shown.

[0031] Figure 5 Depicted are the results of monitoring compounds in plasma following intravenous (IV; squares) or subcutaneous (SC; circles) administration of 2 mg / kg R3152 (SEQ ID NO: 153) in male Sprague-Dawley rats. Monitoring included determination of combined plasma concentrations of R3152 (SEQ ID NO: 153) and its equivalent C-terminal deamidated metabolite R3201 (SEQ ID NO: 211).

[0032] Figure 6The pharmacokinetics of the compounds of the present invention in rats are shown. Left panel: Male Sprague-Dawley rats (n=3) were injected intravenously with a single 2 mg / kg dose. Blood samples were collected at the indicated time points, processed into plasma, and analyzed by LC-MS for the indicated compounds. Black circles: R3176 (SEQ ID NO: 177) (non-lipidated compound); open circles: R3183 (SEQ ID NO: 184) (C16 lipidated compound). Right panel: Male Sprague-Dawley rats (n=3) were injected subcutaneously with a single 15 mg / kg dose. Blood samples were collected at the indicated time points, processed into plasma, and analyzed by LC-MS for the indicated compounds. Black circles: R3176 (SEQ ID NO: 177) (non-lipidated compound); open circles: R3183 (SEQ ID NO: 184) (C16 lipidated compound).

[0033] Figure 7 is present R3183 (SEQ ID NO: 184) (C16 lipidated compound) or with Similar graphs showing the inhibitory effect of anti-C5 monoclonal antibodies on hemolysis via the thrombin-inducible complement pathway. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention relates to the discovery of novel C5 modulating compounds. Such compounds may include, but are not limited to, polypeptides (e.g., cyclic polypeptides, mimetic peptides and cyclic mimetic peptides, small molecules, antibodies, antibody fragments and aptamers. In some cases, the C5 modulating compound is a polypeptide such as a cyclic polypeptide, mimetic peptide and cyclic mimetic peptide, which can be used for the diagnosis and / or treatment of diseases in which inhibition of complement activation is desired. In some embodiments, the polypeptides of the invention specifically bind to complement component C5. In other embodiments, the polypeptides of the invention reduce complement-mediated cytolysis by preventing the cleavage of C5 into C5a and C5b fragments.

[0035] As used herein, "mimetic" refers to a molecule that exhibits some properties or characteristics of another molecule. "Mimetic peptide" or "polypeptide mimetic" is a mimetic in which the molecule contains structural elements not found in natural polypeptides (i.e., polypeptides consisting of only 20 protein amino acids). In a preferred embodiment, a mimetic peptide can replay or simulate one or more biological effects of a natural peptide. A mimetic peptide can differ from a natural polypeptide in many ways, including but not limited to changes in backbone structure and the presence of amino acids that do not exist in nature. In some cases, a mimetic peptide may include amino acids with the following: side chains not found in the known 20 protein amino acids, non-polypeptide-based bridging moieties used to achieve cyclization between the ends or internal parts of the molecule, amide bond hydrogen moieties replaced by methyl (N-methylation) or other alkyl groups, peptide bonds replaced by chemical groups or chemical bonds that resist chemical or enzymatic treatment, N-terminal and C-terminal modifications, and combined with non-peptide extensions (such as polyethylene glycol, lipids, carbohydrates, nucleosides, nucleotides, nucleoside bases, various small molecules, or phosphate or sulfate radicals).

[0036] Some polypeptides of the present invention may be cyclic. Cyclic polypeptides include any polypeptide having one or more cyclic features such as rings, bridging moieties and / or internal connections as part of their structure. As used herein, the term "bridging moiety" refers to one or more components of a bridge formed between two adjacent or non-adjacent amino acids, non-natural amino acids or non-amino acids in a polypeptide. The bridging moiety may have any size or composition. In some embodiments, the bridging moiety may include one or more chemical bonds between two adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues or combinations thereof. In some embodiments, such chemical bonds may be between one or more functional groups on adjacent or non-adjacent amino acids, non-natural amino acids, non-amino acid residues or combinations thereof. The bridging moiety may include one or more features, including but not limited to amide bonds (lactams), disulfide bonds, thioether bonds, aromatic rings, triazole rings and hydrocarbon chains. In some embodiments, the bridging moiety includes an amide bond between an amine functional group and a carboxylic acid functional group, each of which is present in an amino acid, non-natural amino acid or non-amino acid residue side chain. In some embodiments, the amine or carboxylic acid functional group is part of a non-amino acid residue or a non-natural amino acid residue. In some cases, the bridging moiety may comprise a bond formed between residues including, but not limited to, (S)-2-amino-5-azidopentanoic acid (also referred to herein as "X02"), (S)-2-aminohept-6-enoic acid (also referred to herein as "X30"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X31"), and (S)-2-aminopent-4-enoic acid (also referred to herein as "X12"). The bridging moiety may be formed via a cyclization reaction using olefin metathesis. In some cases, such a bridging moiety may be formed between the X12 and X30 residues. In some embodiments, the bridging moiety comprises a disulfide bond formed between two thiol-containing residues. In some embodiments, the bridging moiety comprises one or more thioether bonds. Such thioether bonds may include those found in cyclosulfanyl compounds. These bonds are formed during a chemical cyclization reaction between a chloroacetic acid (also referred to herein as "X35") N-terminal modification group and a cysteine ​​residue. In some cases, the bridging portion comprises one or more triazole rings. Such triazole rings may include, but are not limited to, those formed by the cyclization reaction between X02 and X31. In some embodiments, the bridging portion comprises a non-protein or non-polypeptide-based portion, including but not limited to a cyclic ring (including but not limited to an aromatic ring structure (e.g., xylene)). Such bridging portions can be introduced by reaction with reagents containing a variety of reactive halogens, including but not limited to poly(bromomethyl)benzene, poly(bromomethyl)pyridine, poly(bromomethyl)alkylbenzene and / or (E)-1,4-dibromobut-2-ene. In some embodiments, the bridging portion of the present invention includes but is not limited to the following structures:

[0037]

[0038] wherein each X is independently N or CH such that no ring contains more than 2 N; each Z is independently absent or selected from a bond, NR, O, S, CH2, C(O)NR, NRC(O), S(O) v NR and NRS(O) v ; each m is independently selected from 0, 1, 2 and 3; each v is independently selected from 1 and 2; each R is independently selected from H and C1-C6; and each bridging portion is connected to the polypeptide by an independently selected bond or C1-C6 spacer.

[0039] In certain embodiments of the present invention, the polypeptide is made macrocyclic by forming a covalent bond between the atoms present in the linear polypeptide and the atoms of the bridging portion. This bridging portion serves the purpose of chemically tethering two reaction sites on the linear polypeptide, thereby supplying a cyclic polypeptide product. Embodiments of the present invention include polypeptides that are cyclized in the above manner and include a bridging portion containing an aromatic 6-membered ring. In these embodiments, the atoms of the linear polypeptide that form an explicit chemical bond with the bridging portion may be heteroatoms (including but not limited to nitrogen, oxygen and sulfur), or saturated or unsaturated carbon atoms. In each of these embodiments of the present invention, the atoms of the polypeptide side chain may be directly bonded to the carbon atoms in the aromatic ring of the bridging portion. In an alternative form, the atoms of the polypeptide side chain may be bonded to a saturated -CH2- group, which is directly bonded to the carbon atoms in the aromatic ring of the bridging portion. In some cases, the aromatic 6-membered ring in the bridging portion is benzene, such as in the following structure in which Z may be selected from NH, S, O and (CH)2:

[0040]

[0041] In alternative forms of the invention, the aromatic 6-membered ring comprising the bridging moiety is a heterocyclic ring and contains one or more nitrogen atoms. In these embodiments, the aromatic heterocyclic ring may be pyridine, which contains a single nitrogen atom in the aromatic ring [e.g., any of the structures below, wherein Z may be selected from NH, S, O, and (CH) 2]:

[0042]

[0043] The aromatic heterocycle may alternatively be a pyridazine, which contains two adjacent nitrogen atoms in a 1,2-orientation within the aromatic ring [such as any of the following structures, where Z may be selected from NH, S, O and (CH) 2 ]:

[0044]

[0045] In other embodiments, the aromatic heterocycle may be a pyrimidine, which contains two nitrogen atoms in a 1,3-orientation within the aromatic ring [such as any of the following structures, where Z may be selected from NH, S, O, and (CH) 2 ]:

[0046]

[0047] Alternatively, the aromatic heterocycle may be a pyrazine containing two nitrogen atoms in a 1,4-orientation within the aromatic ring [such as any of the following structures, wherein Z may be selected from NH, S, O and (CH) 2 ]:

[0048]

[0049] In alternative forms of the present invention, the polypeptide is macrocyclic due to the formation of covalent bonds between the atoms of the linear polypeptide and the atoms of the bridging portion consisting of a heterocyclic aromatic 5-membered ring. In these embodiments, the atoms of the linear polypeptide that form an explicit chemical bond with the bridging portion may be heteroatoms (including but not limited to nitrogen, oxygen and sulfur), or saturated or unsaturated carbon atoms. In each of these embodiments of the present invention, the atoms of the polypeptide side chain may be directly bonded to carbon atoms or nitrogen atoms in the aromatic ring of the bridging portion. In alternative forms, the atoms of the polypeptide side chain may be bonded to a saturated -CH2- group, which in turn is directly bonded to a carbon atom or nitrogen atom in the aromatic ring of the bridging portion. In some cases, the heterocyclic aromatic 5-membered ring in the bridging portion is 1,2,3-triazole. In these embodiments, the aromatic ring may be substituted at positions 1 and 4 with the chemical functional groups of the linear polypeptide being enclosed. Alternatively, the 1,2,3-triazole scaffold may be substituted at positions 1 and 4 with -CH2- groups directly bonded to the linear polypeptide atom being enclosed [e.g., any of the structures below, where Z can be selected from NH, S, O, and (CH)2]:

[0050]

[0051] In other embodiments of the invention, the heterocyclic aromatic 5-membered ring comprising the bridging moiety is a pyrazole. In these embodiments, the aromatic ring may be substituted at positions 1 and 3 or positions 1 and 4 with chemical functional groups of the linear polypeptide being enclosed. Alternatively, the pyrazole scaffold may be substituted at positions 1 and 3 or positions 1 and 4 with -CH2- groups directly bonded to atoms of the linear polypeptide being enclosed [e.g., any of the structures below, wherein Z may be selected from NH, S, O, and (CH)2]:

[0052]

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the cyclic polypeptides and methods presented herein, suitable methods and materials are described below.

[0054] Peptides as drugs

[0055] By virtue of their size and complexity, polypeptides are able to form numerous highly specific contacts with their biological targets and can display high levels of selectivity for the correct or desired target over closely related targets within the same family. Off-target effects (also known as side effects) often prevent highly effective drugs from passing regulatory approval due to safety concerns.

[0056] Numerous polypeptides (including but not limited to mimetic peptides) have been developed into effective drugs. These polypeptides include but are not limited to: insulin, glucagon-like peptide 1 (GLP-1), somatostatin, vasopressin, cyclosporine A, etc. The therapeutic polypeptide may be the same as a naturally occurring molecule (i.e., circulating in humans and considered "wild type" in the human population). In many other cases, due to a short circulation half-life often caused by metabolic instability in the body, the polypeptide is not suitable for therapeutic use or is suboptimal for therapeutic use. In these cases, modified or variant forms of the polypeptide (mimetic peptides) are used, resulting in improved pharmacokinetic and pharmacodynamic behavior. In other cases, polypeptides derived from natural sources have an equivalent mechanism of action and preferred drug profiles and can be used as a therapy. For example, exedin-4, a synthetic version of exedin-4, has similar biological properties to human glucagon-like peptide-1 (GLP-1), but has improved pharmacokinetics and has been approved by the FDA for the treatment of type 2 diabetes. As another example, salmon calcitonin, a calcitonin extracted from the posterior branchial glands of salmon, is similar to but more active than human calcitonin and can be used to treat postmenopausal osteoporosis, hypercalcemia, Paget's disease, bone metastases, and phantom limb pain.

[0057] Polypeptides are generally limited to non-oral administration routes. In almost all cases, polypeptides must be delivered by injection because even very short polypeptides (e.g., polypeptides with 4-10 amino acid residues) are unable or poorly able to pass through the cell membranes lining the intestinal tract. For effective oral availability, drugs generally need to pass through both the luminal membrane and the basolateral membrane of the intestinal epithelial cells in order to enter the systemic circulation. The poor membrane permeability and lack of oral bioavailability of polypeptides significantly limit their therapeutic use.

[0058] The efficacy of a peptide as a drug can be affected by its proteolytic stability. In vivo, peptides can be modified or degraded by enzymes, which can limit their effectiveness in interacting with their intended target.

[0059] The metabolic stability of polypeptides is important because it is related to the global flexibility, intramolecular fluctuations, various internal dynamic processes, and many biological functions of polypeptides. The metabolic stability of polypeptides can be critical in drug development, affecting parameters such as, but not limited to, the clearance rate, half-life, and bioavailability of the drug.

[0060] Maintaining a given level of a therapeutic polypeptide within the body or bloodstream may be difficult due to efflux. The rate at which a polypeptide effluxes from the body can vary and should be monitored when considering administration of a therapeutic polypeptide.

[0061] There remains a significant medical need for inhibitors of complement activation or complement activity, and for formulations of inhibitors that are highly potent and highly specific.

[0062] Discovery of Peptide Mimetics

[0063] Peptide mimics can be identified by various means. In some cases, naturally occurring peptides or sequences found in natural proteins are used as starting points. In these cases, the starting peptide sequence has been selected because it is known to interact with the desired target molecule in the body. A natural peptide can be selected because it is an agonist or antagonist for a receptor, inhibits an enzyme, or regulates a channel. A sequence found in a natural protein can be selected because it contains a domain involved in interacting with another protein or some other molecule in a human or animal. In many cases, structural data on interacting proteins can be obtained from public databases (e.g., RCSB Protein Data Bank; HMBerman, J. Westbrook, Z. Feng, G. Gilliland, TNBhat, H. Weissig, IN Shindyalov, PE Bourne (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242), and specific regions of proteins that interact with the desired target can be identified from crystallographic data on protein complexes. In other cases, polypeptides corresponding to various parts of a protein can be prepared and tested for binding to the target of interest. Once identified, chemical modifications are introduced to improve the stability and potency of the polypeptide, wherein the resulting peptidomimetic has improved pharmacokinetic or pharmacodynamic parameters.

[0064] In other cases, polypeptides are isolated by one of several methods for isolating polypeptide sequences from polypeptide libraries based on their affinity for specific target proteins, nucleic acids, carbohydrates, lipids, or whole cells. Such methods include phage display, mRNA display, ribosome display, DNA display, DNA-encoded assembly and two-hybrid screening, and their modifications (see, e.g., Takashashi, TT et al. (2003). Trends in Biochem. Sci. 28(3): 159-165; Kay, BK et al. (2001). Methods. 24: 240-246; He, M and Taussig, M (2002). Briefs in Functional Genomics and Proteomics. 1(2): 204-212; Rothe, A et al. (2006). The FASEB Journal. 20(10): 1599-1610, all of which are incorporated herein by reference in their entirety).

[0065] Polypeptides can adopt three-dimensional structures that are able to bind to other biomolecules with some degree of affinity and specificity. Some will bind with very high affinity and specificity. A library of random polypeptide sequences will consist of a population of molecules with a wide variety of three-dimensional structures. To isolate polypeptides with conformations that interact with a specific target protein, individual sequences from the library can be prepared and tested or screened for their affinity for the target. However, for very large libraries (>10 6Members), screening individual sequences for binding affinity is not feasible. To overcome this limitation, several techniques have been developed to select novel polypeptides from very large, complex mixtures based on their binding affinity to the target. Because high-affinity binding polypeptides are predicted to exist at very low frequencies within a population, these selection methods rely on maintaining a physical connection between the polypeptide and the genetic material encoding the polypeptide (usually a nucleic acid such as DNA or RNA) so that selection for the polypeptide automatically includes selection for the nucleic acid encoding it. The nucleic acid encoding the selected polypeptide can be amplified and sequenced to reveal the sequence of both the nucleic acid and the polypeptide. In one method, phage display (see Cwirla, SE et al. (1990). Proc. Natl. Acad. Sci. USA 87: 6378-6382; Dower, WJ and Cwirla, SE U.S. Pat. Nos. 5,427,908 and 5,580,717), each random polypeptide member of the library is displayed on the surface of a phage particle as part of a fusion protein between the polypeptide and one of the phage coat proteins. Phage particles provide a connection between a polypeptide and a coding DNA by co-locating the polypeptide and the coding DNA in the same physical entity, and the coding DNA can then be amplified by infecting bacteria with a selected phage. In another method, ribosome display (see Kawasaki, GH U.S. Patent Nos. 5,658,754 and 5,643,768), a mixture of messenger RNA (mRNA) molecules is translated in vitro in a manner that produces a stable complex of ribosomes, mRNA, and newly synthesized polypeptides extending from the ribosomes for each mRNA in the mixture. Stabilized complexes allow the complexes to be kept together while screening for polypeptides that are bound to the target target. The mRNA encoding the selected polypeptide can be amplified using polymerase chain reaction (PCR), and then characterized, for example, by sequencing.

[0066] In yet another method, mRNA display (see Szostak, JW and Roberts, RW, U.S. Patent No. 6,258,558, the contents of which are incorporated herein by reference in their entirety), each mRNA molecule in the library is modified by covalently adding a puromycin-like moiety at its 3' end. The puromycin-like moiety is an aminoacyl-tRNA acceptor stem analog that acts as a peptidyl acceptor and can be added to the growing polypeptide chain by the ribosomal peptidyl transferase activity of the translated mRNA. During in vitro translation, the mRNA and the encoded polypeptide become covalently linked via the puromycin-like moiety, thereby generating an RNA-peptide fusion. After selecting the fusion molecule by binding the polypeptide component of the fusion molecule to the target, the RNA component of the selected fusion molecule can be amplified using PCR and then characterized. Several other methods have been developed to create a physical link between a polypeptide and its encoding nucleic acid to facilitate selection and amplification (see Yanagawa, H., Nemoto, N., Miyamoto, E. and Husimi, Y., U.S. Pat. No. 6,361,943; Nemoto, H., Miyamoto-Sato, E., Husimi, H. and Yanagawa, H. (1997). FEBS Lett. 414: 405-408; Gold, L., Tuerk, C., Pribnow, D. and Smith, JD, U.S. Pat. Nos. 5,843,701 and 6,194,550; Williams, RB, U.S. Pat. No. 6,962,781; Baskerville, S. and Bartel, DP (2002). Proc. Natl. Acad. Sci. USA 99:9154-9159; Baskerville, DS and Bartel, DP, U.S. Pat. No. 6,716,973; Sergeeva, A. et al. (2006). Adv. Drug Deliv. Rev. 58:1622-1654; the contents of each of which are incorporated herein by reference in their entirety).

[0067] mRNA display is a particularly useful method for generating large libraries of polypeptides. Thus, provided herein are methods for selecting polypeptides (or mRNA encoding polypeptides) that interact with complement protein C5. The library typically contains at least 10 2 members, more preferably at least 10 6 members, and more preferably at least 10 9 In some embodiments, the library will include at least 10 12 members or at least 10 14members. In general, the members will be different from each other; however, it is expected that there will be a certain degree of redundancy in any library. The library may exist as a single mixture of all members, or may be divided into several groups held in separate containers or wells, each containing a subset of the library, or the library may be a collection of containers or wells on a plate, each container or well containing only one or a few members of the library.

[0068] Each mRNA in the library preferably comprises a translation initiation sequence, a start codon, and a variable polypeptide (e.g., protein or short peptide) coding region generated by, for example, random or semi-random assembly of nucleotides, and varies from mRNA to mRNA in the library (although there may be a certain degree of redundancy within the library). The translation initiation sequence, the start codon, and the variable polypeptide coding region may be flanked by known fixed sequences that may be used, for example, for PCR amplification of the mRNA after selection. Other fixed sequences that may be present include those corresponding to the following sequences: sequences encoding amino acids that may participate in chemical or enzymatic cross-linking reactions so that the resulting polypeptide may be modified or derivatized after translation, or sequences encoding fixed C-terminal extensions, such as polypeptide tags that may facilitate purification of peptide-mRNA fusions.

[0069] Once the mRNA library is generated by derivatization with puromycin, the library can be translated. The resulting polypeptides (eg, display polypeptides) will be linked to their corresponding mRNAs as described herein (eg, as mRNA-polypeptide complexes).

[0070] Numerous in vitro translation systems have been described in the literature. The most common systems utilize rabbit reticulocyte lysate, wheat germ extract, or Escherichia coli (E. coli) extracts, all of which are available in kit form from several commercial sources (e.g., Ambion, Austin, TX; Promega, Madison, WI; Novagen / EMD Chemicals, Gibbstown, NJ; Qiagen, Valencia, CA).

[0071] Unlike phage display or other systems that rely on intracellular translation, mRNA display can be suitable for directly generating a library of mimetic peptides by performing in vitro translation with non-natural or non-standard amino acids. 20 kinds of natural protein amino acids are identified and referred to herein by the following single-letter or three-letter names: aspartic acid (Asp: D), isoleucine (Ile: I), threonine (Thr: T), leucine (Leu: L), serine (Ser: S), tyrosine (Tyr: Y), glutamic acid (Glu: E), phenylalanine (Phe: F), proline (Pro: P), histidine (His: H), glycine (Gly: G), lysine (Lys: K), alanine (Ala: A), arginine (Arg: R), cysteine ​​(Cys: C), tryptophan (Trp: W), valine (Val: V), glutamine (Gln: Q), methionine (Met: M), asparagine (Asn: N). Naturally occurring amino acids exist in their left-handed (L) stereoisomer form. Unless otherwise indicated, amino acids referred to herein are the L-stereoisomers.

[0072] Unnatural amino acids have side chains or other features that are not present in the 20 naturally occurring amino acids listed above, and include, but are not limited to, N-methyl amino acids, N-alkyl amino acids, α, α-substituted amino acids, β-amino acids, α-hydroxy amino acids, D-amino acids, and other unnatural amino acids known in the art (see, e.g., Josephson et al., (2005) J. Am. Chem. Soc. 127: 11727-11735; Forster, AC et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357; Subtelny et al., (2008) J. Am. Chem. Soc. 130: 6131-6136; Hartman, MCT et al. (2007) PLoS ONE 2: e972; and Hartman et al., (2006) Proc. Natl. Acad. Sci. USA 101: 6353-6357). 103:4356-4361).

[0073] Essentially any amino acid that can be assembled into a polymer by native or mutant ribosomes can be used when attached to an appropriate tRNA (see Sando, S. et al., (2007) J. Am. Chem. Soc. 129:6180-6186; Dedkova, L. et al. (2003) J. Am. Chem. Soc. 125:6616-6617; Josephson, K., Hartman, MCT and Szostak, JW (2005) J. Am. Chem. Soc. 127:11727-11735; Forster, AC et al. (2003) Proc. Natl. Acad. Sci. USA 100:6353-6357; Subtelny, AO, Hartman, MCT and Szostak, JW (2008) J. Am. Chem. Soc. 130:6131-6136; and Hartman, MCT et al. (2007) PLoS ONE 2:e972).

[0074] When unnatural amino acids are required, it may be advantageous to use a purified translation system that lacks endogenous aminoacylated tRNA (Shimizu, Y. et al. (2001) Nat. Biotech. 19: 751-755; Josephson, K., Hartman, MCT and Szostak, JW (2005) J. Am. Chem. Soc. 127: 11727-11735; Forster, AC et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357). If unnatural amino acids are used in a lysate or extract-based in vitro translation system, it may be desirable to deplete the extract of endogenous tRNA, as described previously (see Jackson, RJ, Napthine, S. and Brierley, I. (2001) RNA 7: 765-773). Systems based on purified E. coli translation factors are commercially available (PUREXPRESS TM ; New England Biolabs, Ipswich, MA). These systems are particularly useful for generating peptidomimetics using translation of unnatural amino acids.

[0075] When natural amino acids are used in in vitro translation systems based on lysates or extracts, translation depends on the enzymatic loading of amino acids onto tRNAs by tRNA synthetases, all of which are components of the extracts. Alternatively, in vitro translation systems using purified translation factors and ribosomes or tRNA-depleted extracts require the provision of aminoacylated tRNAs. In these cases, purified or in vitro synthesized tRNAs can be charged with amino acids using chemical procedures (see Frankel, A., Millward, SW and Roberts, RW (2003) Chem. Biol. 10: 1043-1050) or enzymatic procedures (Josephson, K., Hartman, MCT and Szostak, JW (2005) J. Am. Chem. Soc. 127: 11727-11735; Murakami, H. et al. (2006) Nat. Methods 3: 357-359).

[0076] Numerous publications describe the recovery of mRNA displayed polypeptides from the translation complex, and these are suitable for use in the methods described herein (Liu, R. et al. (2000). Methods Enzymol. 318: 268-293; Baggio, R. et al. (2002). J. Mol. Recognit. 15: 126-134; U.S. Pat. No. 6,261,804). The recovery of mRNA displayed polypeptides can be facilitated by the use of various "tags" that are included in the polypeptide by translating a fixed sequence of the polypeptide coding sequence and are bound to a specific substrate or molecule. Numerous reagents for capturing such tags are commercially available, including reagents for capturing the following: His tag, FLAG tag, glutathione-S-transferase (GST) tag, strep tag, HSV tag, T7 tag, S tag, DsbA tag, DsbC tag, Nus tag, myc tag, hemagglutinin (HA) tag or Trx tag (Novagen, Gibbstown, NJ; Pierce, Rockford, IL). The polypeptide displayed by the mRNA can also be isolated by binding the poly A tail on the mRNA to a poly dT resin or a combination of the poly A tail and the His tag.

[0077] After the in vitro translation reaction has been carried out, and before the selection step, the mRNA portion of the functionalized RNA is usually reverse transcribed to produce RNA-DNA hybrid molecules. This serves to protect the RNA from degradation and also prevents the RNA from folding into a secondary structure that can bind to the selection target, which would result in the selection of inappropriate products (e.g., selecting RNA aptamers instead of polypeptide aptamers).

[0078] After in vitro translation and isolation of the polypeptide-mRNA fusion, the polypeptide portion can be modified by intramolecular or intermolecular cross-linking, chemical conjugation, enzymatic cleavage, truncation, or extension with additional amino acid monomers. One way to accomplish this is by incorporating non-natural amino acids with reactive side chains into the polypeptides that make up the library. After translation, the newly formed polypeptides can react with molecules that specifically react with the reactive side chains of the incorporated amino acids. For example, amino acids with terminal alkyne side chains can be incorporated into a polypeptide library and then reacted with azido sugars, thereby generating a library of display polypeptides with sugars attached to the alkynyl side chain position (Josephson, K., Hartman, MCT and Szostak, JW (2005) J. Am. Chem. Soc. 127: 11727-11735). A variety of reactive side chains can be used for such post-translational conjugation, including amines, carboxyls, azides, terminal alkynes, alkenes, and thiols.

[0079] A particularly useful modification is to generate a cyclic structure based on amino acid cross-linking. The cyclic region in the polypeptide contains a rigid domain that reduces conformational flexibility and rotational freedom, resulting in very high affinity binding to the target protein. Several methods for cyclizing polypeptides are available to those skilled in the art and are incorporated herein by reference. Generally, the chemical reactivity of a specific amino acid side chain and / or the carboxyl terminus or amino terminus of a polypeptide is fully utilized to cross-link two sites of a polypeptide to generate a cyclic molecule. In one method, the thiol group of a cysteine ​​residue is cross-linked with another cysteine ​​residue to form a disulfide bond. In some embodiments, the thiol group of a cysteine ​​residue reacts with the bromomethyl group of a poly(bromomethyl)benzene molecule to form a stable connection (see Timmerman, P. et al., (2005) Chem Bio Chem 6: 821-824, the contents of which are incorporated herein by reference in their entirety). The poly(bromomethyl)benzene molecules of the present invention may include, but are not limited to, 1,2-bis(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene, and 1,4-bis(bromomethyl)benzene. For example, bis-, tri- and tetra(bromomethyl)benzene molecules can be used to generate bridging moieties to generate bridging moieties having one, two or three rings, respectively. The bromomethyl groups of the poly(bromomethyl)benzene molecules can be arranged on adjacent ring carbons (ortho or ortho), with one ring carbon separating two groups (meta or meta), or on opposite ring carbons (para or para) on the benzene ring. In some embodiments, meta-bis(bromomethyl)benzene (also referred to herein as meta-dibromoxylene) is used in the formation of cyclic polypeptides. In some embodiments, ortho-bis(bromomethyl)benzene (also referred to herein as ortho-dibromoxylene) or para-bis(bromomethyl)benzene (also referred to herein as para-dibromoxylene) is used in the formation of cyclic polypeptides. In some embodiments, the thiol group of the cysteine ​​residue reacts with other reagents comprising one or more bromine functional groups to form a stable connection. Such agents may include, but are not limited to, poly(bromomethyl)pyridines (including but not limited to 2,6-bis(bromomethyl)pyridine), poly(bromomethyl)alkylbenzenes (including but not limited to 1,2-bis(bromomethyl)-4-alkylbenzene), and / or (E)-1,4-dibromobut-2-ene.

[0080] In another exemplary method, the side chain amino group and the terminal amino group are cross-linked with disuccinimidyl glutarate (see Millward, SW et al., J. Am. Chem. Soc. 127: 14142-14143, 2005). In other methods, cyclization is accomplished by forming a thioether bond between two sites on the polypeptide (see Timmerman, P. et al., (2005) Chem Bio Chem 6: 821-824; which is incorporated herein by reference in its entirety). Enzymatic methods rely on the reaction between (1) cysteine ​​and (2) dehydroalanine or dehydro α-aminobutyric acid groups, catalyzed by lantibiotic synthetase, to produce a thioether bond (see Levengood, MR and Van der Donk, WA, Bioorg. and Med. Chem. Lett. 18: 3025-3028, 2008). Dehydrogenation functional groups can also be generated chemically by oxidation of selenoamino acid side chains incorporated during translation (see Seebeck, FP and Szostak, JWJ Am. Chem. Soc. 2006).

[0081] Libraries of mRNA-polypeptide fusions (also referred to herein as mRNA display libraries) generated using the methods described above, and which may or may not have undergone post-translational modifications (such as polypeptide cyclization, as described above), can be subjected to a batch selection step to isolate those complexes that display the desired polypeptide.

[0082] Typically, C5 is bound to a solid matrix, such as agarose or synthetic polymer microbeads. Numerous methods can be used to fix C5 to a solid support. In a particularly useful method, C5 is bound to biotin and streptavidin microbeads are used to immobilize the protein. Microbeads containing immobilized C5 are mixed with an mRNA display library and incubated under conditions (e.g., temperature, ionic strength, divalent cations, and competing binding molecules) that allow specific members of the library to bind to the target. Alternatively, the biotinylated enzyme is not present in the solution, and after binding to the appropriate polypeptide, the mRNA-polypeptide fusion bound to C5 is captured by appropriately modified microbeads.

[0083] The binding conditions can be varied to change the stringency of the selection. For example, a low concentration of a competitive binding agent can be added to ensure that the selected polypeptide has a relatively higher affinity. Alternatively, the incubation period can be selected to be very brief so that only polypeptides with a high k are isolated. onIn some embodiments, the incubation conditions are used to determine the polypeptide of the selected polypeptide. In this way, the incubation conditions play an important role in determining the properties of the selected polypeptide. Negative selection can also be used. In this case, the selection of polypeptides with affinity for the matrix (e.g., Sehoraose) bound to the target is performed by applying the display library to the matrix microbeads lacking the target protein. This step can remove mRNA and its encoded polypeptide that are not specific to the target protein. Numerous references are available that describe how to perform selection experiments (see, e.g., U.S. Pat. No. 6,258,558, Smith, GP and Petrenko, VA, (1997) Chem. Rev. 97:391-410; Keefe, AD and Szostak, JW (2001) Nature 15:715-718; Baggio, R. et al. (2002) J. Mol. Recog. 15:126-134 and Sergeeva, A. et al. (2006) Adv. Drug Deliv. Rev. 58:1622-1654; the contents of each of which are incorporated herein by reference in their entirety).

[0084] The frequency of binding molecule presence in the library of random sequence is expected to be extremely low. Therefore, in the initial selection step, the very few polypeptides (and its associated mRNA) that reach the selection standard should be recovered. Usually, the selection is repeated with the mRNA selected from the first round of selection. This is accomplished by using PCR to amplify the mRNA selected in the first round or the corresponding cDNA, followed by in vitro transcription to produce a new library of mRNA. Use PCR primers corresponding to the 5' end and 3' end of the mRNA in the library. Usually, 5' primers will extend beyond the end of the mRNA in the 5' direction so that a bacterial promoter such as a T7 promoter is added to the 5' end of each amplified molecule. Once amplified, double-stranded DNA can be used in an in vitro transcription reaction to produce mRNA for subsequent rounds of selection.

[0085] The selection process typically involves several rounds or cycles, wherein the population of selected molecules is increasingly enriched in a particular set of sequences at the end of each round. The selection conditions may be the same for each round, or the conditions may be varied, for example, to increase the stringency of selection in later rounds. The progression of selection may be accomplished by using, for example, 35The amount of radiolabeled polypeptide bound to the target is monitored by isotope-labeled amino acids such as S-methionine. The amount of radiolabeled polypeptide bound to the target in each round is measured, and the progressive increase in the recovered radiolabel indicates the progressive enrichment of RNA molecules encoding polypeptides with affinity for target binding. After any round, the PCR product can be cloned and sequenced. In general, after a round in which a considerable amount of radiolabeled polypeptide is recovered in the target binding population (e.g., >2% of the background of microbeads lacking fixed C5), cloning and sequencing are performed. Sequences found in a variety of isolates are candidates for encoding polypeptides that specifically bind to the target. Alternatively, high-throughput sequencing of tens of thousands of clones can be performed after the first or subsequent rounds. Sequences that increase in frequency between, for example, the third and fourth rounds are candidates for encoding polypeptides that specifically bind to the target. Polypeptides encoded by any sequence can be translated or synthesized and tested for their binding affinity to the original target protein used in the selection.

[0086] The libraries and methods of the invention can be used to optimize the function or properties of polypeptides. In one approach, once a population is enriched for polypeptides having a certain level of binding affinity, mutagenic PCR (Keefe, AD and Szostak, JW (2001). Nature 15: 715-718) is used to introduce sequence changes into the library. Alternatively, a single RNA sequence encoding a polypeptide having defined binding properties can be replicated but with a defined level of mutation, or mutagenic PCR can be performed to generate a population of mutant molecules. Upon in vitro translation, the resulting mixture of mRNA molecules generated from this population is expected to encode a set of polypeptides having improved, similar or reduced affinities compared to the starting sequence, and selection of mRNA from this population can be expected to identify polypeptides having improved affinity if appropriate stringency conditions are used during the selection.

[0087] In the second method, optimization is carried out in a directed manner. The sequence encoding the binding or functional properties determined is subjected to site-directed mutagenesis, whereby a series of sequences are produced, wherein each sequence has a codon replaced with, for example, an alanine codon. The number of sequences in the group is equal to the number of amino acid residues to be mutated. After in vitro translation, the binding or functional properties of the polypeptide product of each "alanine scanning" mutant are tested. Sites where alanine replacement affects the binding or functional properties of the polypeptide are considered to be key residues. Similarly, N-methyl scanning can be performed so that each residue is replaced with an N-methyl derivative, and the position of the N-methyl substitution can be tolerated in the polypeptide main chain.

[0088] Alternatively, the sequence can be concentrated, subjected to one or more rounds of high stringency selection, and the group of sequences representing high-affinity binding polypeptides is separated. The key residues are identified as those that cannot be replaced by alanine residues without loss of activity after DNA sequencing of the recovered DNA. Once the key residues are identified, groups of mRNA molecules encoding various natural (or non-natural) amino acids at each key position are generated. The resulting group is subjected to one or more rounds of high stringency selection (with a suitable mixture of tRNA loaded with natural or non-natural amino acids), and the sequence representing high-affinity binding polypeptides is separated after in vitro translation. In this way, the optimal polypeptide can be identified. Because the optimal sequence may not necessarily be identified by combining the optimal residues at individual sites, it is useful to test mutations in combination at multiple sites.

[0089] Both alanine scanning and N-methyl scanning can also be performed using chemical synthesis methods such as solid phase polypeptide synthesis (see, e.g., Coin, I et al. (2007); Nature Protocols 2(12):3247-56, the contents of which are incorporated herein by reference in their entirety).

[0090] Once groups, populations or subgroups of polypeptides are identified, the polypeptides can be evaluated for therapeutic or diagnostic applications, including improved pharmacokinetic and / or pharmacodynamic properties.

[0091] In one embodiment, the polypeptide is evaluated for one or more of: target binding affinity, activity in a biochemical or cell-based assay, protease resistance, in vitro or in vivo permeability, properties related to suitability for use as a pharmaceutical agent such as plasma protein binding, metabolism (in microsomes, hepatocytes or plasma), P-glycoprotein (Pgp) inhibition and cytochrome P450 inhibition. The polypeptides of the invention may also be tested for oral bioavailability, toxicity, human ether-a-go-go related gene product (hERG) inhibition, circulation half-life, other pharmacokinetic and pharmacodynamic parameters, and efficacy in animal models of disease.

[0092] Polypeptides of the present invention

[0093] According to the present invention, once a single polypeptide or a group of candidate polypeptide molecules are identified, they can be subjected to one or more rounds of structure-activity relationship (SAR) optimization using standard chemical techniques and polypeptide synthesis techniques. Such optimization may include considerations such as avoiding charged polar side chains (Asp, Glu, Arg, Lys) that can inhibit cell penetration, avoiding side chains that cause metabolic liabilities (Tyr, Met, Trp, Cys), improving solubility, avoiding unnecessary molecular weight, avoiding rotatable bonds, and altering lipophilicity.

[0094] In one embodiment, it is an object of the present invention to provide cyclic peptidomimetics that are designed to be metabolically stable, cell permeable and / or orally available.

[0095] Amino acid variants

[0096] As used herein, the term "amino acid" includes residues of natural amino acids and non-natural amino acids. The term also includes amino acids with conventional amino protecting groups (e.g., acetyl or benzyloxycarbonyl), and natural and non-natural amino acids protected at the carboxyl terminus (e.g., as (C1-C6) alkyl, phenyl or benzyl esters or amides; or as α-methylbenzylamide). Other suitable amino and carboxyl protecting groups are known to those skilled in the art (see, e.g., Greene, TW; Wutz, PGM, Protecting Groups In Organic Synthesis; Second Edition, 1991, New York, John Wiley & sons, and documents cited therein). The polypeptides and / or polypeptide compositions of the present invention may also include modified amino acids.

[0097] Unnatural amino acids that can be used to optimize the polypeptides and / or polypeptide compositions of the invention include, but are not limited to, 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-2,3-hydro-1H-indene-1-carboxylic acid, homolysine, homoarginine, homoserine, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 5-aminovaleric acid, 5-aminohexanoic acid, 6-aminohexanoic acid, 2-aminovaleric acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, desmosine, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N -methylpentylglycine, naphthylalanine, ornithine, pentylglycine, thioproline, norvaline, tert-butylglycine, phenylglycine, azatryptophan, 5-azatryptophan, 7-azatryptophan, 4-fluorophenylalanine, penicillamine, sarcosine, homocysteine, 1-aminocyclopropanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 4-aminotetrahydro-2H-pyran-4-carboxylic acid, (S)-2-amino-3-(1H-tetrazol-5-yl)propionic acid, cyclopentylglycine, cyclohexylglycine, cyclopropylglycine, η-ω-methylarginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluorotryptophan, 5-chlorotryptophan, citrulline, 4-chlorohomophenylalanine,

[0098] homophenylalanine, 4-aminomethylphenylalanine, 3-aminomethylphenylalanine, octylglycine, norleucine, tranexamic acid, 2-aminopentanoic acid, 2-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, 2-aminodecanoic acid, 2-aminoundecanoic acid, 2-aminododecanoic acid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, pipecolic acid, 2-carboxyazetidine, hexafluoroleucine, 3-fluorovaline, 2-amino-4,4-difluoro-3-methylbutyric acid, 3-fluoroisoleucine, 4-fluoroisoleucine, 5-fluoroisoleucine, 4-methylphenylglycine, 4-ethylphenylglycine, 4-isopropylphenylglycine, ( (S)-2-amino-5-azidopentanoic acid (also referred to herein as "X02"), (S)-2-aminohept-6-enoic acid (also referred to herein as "X30"), (S)-2-aminopent-4-ynoic acid (also referred to herein as "X31"), (S)-2-aminopent-4-enoic acid (also referred to herein as "X12"), (S)-2-amino-5-(3-methylguanidino)pentanoic acid, (S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid, (S)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid, (S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butanoic acid, (S)-leucinol, (S) -valinol, (S)-tert-leucinol, (R)-3-methylbutan-2-amine, (S)-2-methyl-1-phenylpropan-1-amine, and (S)-N,2-dimethyl-1-(pyridin-2-yl)propan-1-amine, (S)-2-amino-3-(oxazol-2-yl)propanoic acid, (S)-2-amino-3-(oxazol-5-yl)propanoic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propanoic acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)propanoic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and (S)-2-amino-3-(1H -indazol-3-yl)propanoic acid, (S)-2-amino-3-(oxazol-2-yl)butanoic acid, (S)-2-amino-3-(oxazol-5-yl)butanoic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)butanoic acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)butanoic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)butanoic acid, and (S)-2-amino-3-(1H-indazol-3-yl)butanoic acid, 2-(2'MeOphenyl)-2-aminoacetic acid, tetrahydro 3-isoquinolinecarboxylic acid, and their stereoisomers (including but not limited to D and L isomers).

[0099] Other non-natural amino acids that can be used for optimization of the polypeptide or polypeptide composition of the present invention include, but are not limited to, fluorinated amino acids in which one or more carbon-bound hydrogen atoms are replaced by fluorine. The number of fluorine atoms included can range from 1 to up to and including all hydrogen atoms. Examples of such amino acids include, but are not limited to, 3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline, 4,4-difluoroproline, 3,4-difluoroproline (3,4-difluroproline), 3,3,4,4-tetrafluoroproline, 4-fluorotryptophan, 5-fluorotryptophan (5-flurotryptophan), 6-fluorotryptophan, 7-fluorotryptophan, and stereoisomers thereof.

[0100] Other non-natural amino acids that can be used for the optimization of polypeptides or polypeptide compositions of the present invention include, but are not limited to, those that are disubstituted at the α-carbon. These include amino acids in which the two substituents on the α-carbon are identical, such as α-aminoisobutyric acid, and 2-amino-2-ethylbutyric acid, and those in which the substituents are different, such as α-methylphenylglycine and α-methylproline. In addition, the substituents on the α-carbon can be combined to form a ring, such as 1-aminocyclopentanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 3-aminotetrahydrofuran-3-carboxylic acid, 3-aminotetrahydropyran-3-carboxylic acid, 4-aminotetrahydropyran-4-carboxylic acid, 3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylic acid, 4-aminopiperidinone-4-carboxylic acid (4-aminopiperidinnne-4-carboylix acid), and their stereoisomers.

[0101] Other non-natural amino acids that can be used for optimization of polypeptides or polypeptide compositions of the present invention include, but are not limited to, analogs of tryptophan in which the indole ring system is replaced by another 9-membered or 10-membered bicyclic ring system comprising 0, 1, 2, 3 or 4 heteroatoms independently selected from N, O or S. Each ring system may be saturated, partially unsaturated or fully unsaturated. The ring system may be substituted with 0, 1, 2, 3 or 4 substituents at any substitutable atom. Each substituent is independently selected from H, F, Cl, Br, CN, COOR, CONRR', oxygen, OR, NRR'. Each R and R' is independently selected from H, C1-C20 alkyl, C1-C20 alkyl-O-C1-20 alkyl.

[0102] In some embodiments, analogs of tryptophan (also referred to herein as "tryptophan analogs") that can be used to optimize the polypeptides or polypeptide compositions of the present invention include, but are not limited to: 5-fluorotryptophan [(5-F)W], 5-methyl-O-tryptophan [(5-MeO)W], 1-methyltryptophan [(1-Me-W) or (1-Me)W], D-tryptophan (D-Trp), azatryptophan (including but not limited to 4-azatryptophan, 7-azatryptophan and 5-azatryptophan), 5-chlorotryptophan, 4-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, and stereoisomers thereof. Unless otherwise indicated, as used herein, the term "azatryptophan" and its abbreviation "azaTrp" refer to 7-azatryptophan.

[0103] Modified amino acid residues that can be used for optimization of polypeptides and / or polypeptide compositions of the present invention include, but are not limited to, those that are chemically blocked reversibly or irreversibly, or chemically modified on its N-terminal amino group or its side chain group, or chemically modified in the amide backbone, for example, N-methylated, D (non-natural amino acid) and L (natural amino acid) stereoisomers or residues, wherein the side chain functional group is chemically modified to another functional group. For example, modified amino acids include, but are not limited to: methionine sulfoxide; methionine sulfone; aspartic acid-(β-methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a modified amino acid of glycine; or alanine carboxamide, and a modified amino acid of alanine. Non-natural amino acids can be purchased from Sigma-Aldrich (St.Louis, MO), Bachem (Torrance, CA) or other suppliers. Non-natural amino acids may further include any of those listed in Table 2 of U.S. Patent Publication US2011 / 0172126, the contents of which are incorporated herein by reference in their entirety.

[0104] In some embodiments, the amino acid sequence of a polypeptide and / or polypeptide composition of the present invention may comprise only naturally occurring amino acids. Although the terms peptide, polypeptide and / or their fragments are known in the art to imply relative sizes, unless otherwise noted, these terms used herein should not be considered to be restrictive relative to the sizes of various polypeptide-based molecules mentioned herein and encompassed within the present invention. In some embodiments of the present invention, a polypeptide may comprise both naturally occurring and non-naturally occurring and / or modified amino acids, or may consist solely of non-naturally occurring amino acids.

[0105] Peptide variants

[0106] According to the present invention, any amino acid-based molecule (natural or non-natural) may be referred to as a "polypeptide", and this term includes "peptide", "peptide mimetic" and "protein". Polypeptide is also a category of protein, and is conventionally considered to range in size from about 4 to about 50 amino acids. Dipeptides, those with two amino acid residues, are a category of polypeptides such as tripeptides (polypeptides comprising three amino acids). Polypeptides greater than about 50 amino acids are generally referred to as "proteins". The polypeptide sequence may be linear or cyclic. For example, cyclic polypeptides may be prepared or may be produced by forming a disulfide bond between two cysteine ​​residues in the sequence. Polypeptides may be cyclized via the carboxyl terminus, the amino terminus or via any other convenient binding point, such as via any side chain of cysteine ​​or amino acid residues or other connections including but not limited to maleimide connection, amide connection, ester connection, ether connection, thioether connection, hydrazone connection or acetamide connection. In some embodiments, a cyclic polypeptide is formed when a molecule acts as a bridging moiety to connect two or more regions of a polypeptide.

[0107] The term "amino acid sequence variant" refers to a polypeptide having some differences in the amino acid sequence of the polypeptide compared to a starting, reference or native sequence. Amino acid sequence variants may have substitutions, deletions and / or insertions at certain positions within the amino acid sequence. Typically, variants will have at least about 70% homology to the native or starting sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to the native or starting sequence.

[0108] When applied to amino acid sequences, "homology" is defined as the percentage of residues in a candidate amino acid sequence that are identical to the residues in the amino acid sequence of a second sequence, after the sequences have been aligned and gaps have been introduced, where necessary, to achieve the maximum percent homology. Methods and computer programs for alignment are well known in the art. It will be appreciated that homology is determined by calculation of percent identity, but may differ in value due to gaps and penalties introduced in the calculation.

[0109] When applied to amino acid sequences, "homologous" means that the corresponding sequence in another species has substantial identity to a second sequence in the second species.

[0110] "Analogs" are meant to include amino acid sequence variants that differ by one or more amino acid changes, such as substitutions, additions or deletions of amino acid residues, while retaining one or more of the properties of the parent or starting polypeptide.

[0111] The present invention encompasses several types of compositions including polypeptides including variants and derivatives. These include substitutions, insertions, deletions and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant" and refers to a molecule that has been modified or altered in any way relative to a reference molecule or starting molecule.

[0112] Thus, included within the scope of the present invention are polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications. For example, sequence tags or amino acids such as one or more lysines may be added to the polypeptide sequences of the present invention (e.g., at the N-terminus or C-terminus). Sequence tags may be used for polypeptide purification or localization. Lysine may be used to increase polypeptide solubility or allow site-specific modifications such as, but not limited to, biotinylation or PEGylation. In some cases, the polypeptide may be desulfobiotinylated. As used herein, a desulfobiotinylated polypeptide may include a desulfobiotin (Dtb) moiety that is bound to the ε-amino group of a lysine residue. In some cases, such lysine residues may be C-terminal residues. Alternatively, amino acid residues located in the carboxyl and amino terminal regions of the amino acid sequence of the polypeptide may be optionally deleted to provide a truncated sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) may be deleted alternatively depending on the purpose of the sequence, for example, the sequence is expressed as a portion of a larger sequence that is soluble or connected to a solid support.

[0113] When referring to polypeptides, "substitution variants" are those in which at least one amino acid residue in the native or starting sequence is removed and a different amino acid is inserted at the same position in the place of the removed amino acid. The substitution may be single, in which only one amino acid in the molecule has been replaced, or multiple, in which two or more amino acids in the same molecule have been replaced.

[0114] As used herein, the term "conservative amino acid substitution" refers to replacing an amino acid normally present in a sequence with a different amino acid of similar size, charge or polarity. Examples of conservative substitutions include replacing another non-polar residue with a non-polar (hydrophobic) residue such as isoleucine, valine and leucine. Similarly, examples of conservative substitutions include replacing another with a polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, replacing another with a basic residue such as lysine, arginine or histidine, or replacing another with an acidic residue such as aspartic acid or glutamic acid is another example of conservative substitution. Examples of non-conservative substitutions include replacing a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine with a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine, and / or replacing a non-polar residue with a polar residue.

[0115] An "isostere" is one of two or more molecules that exhibit some similarity in biological properties by having the same number of total electrons or valence electrons in the same arrangement and that are composed of different atoms, not necessarily the same number of atoms. There are two types of isosteres, classical and nonclassical. Classical isosteres have the same number of atoms and / or the same number of valence electrons, while nonclassical isosteres are molecules that produce similar biological effects in vivo but do not have the same number of atoms and / or valence electrons.

[0116] According to the present invention, "peptide bond isosteres" are defined as isosteres with similar properties to peptide bonds. Peptide bond isosteres may be linear types comprising at least one peptide bond replacement, or may be cyclic and comprise amine and carboxylic acid functions. Such replacements may utilize any portion of the physicochemical, structural or functional properties of the improved molecule. Replacement of peptide bonds may increase the metabolic stability of the polypeptide and reduce or increase flexibility. The peptide bond isosteres described herein may be mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona- or deca-peptide bond isosteres, meaning that at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptide bonds may be replaced. For amide (peptide) bonds, non-limiting examples of linear dipeptide bond isosteres include thioamide, sulfonamide, sulfonate, phosphonamide), phosphonate, thiophosphothioate, phosphinate, alkane, 1 or 2 hydroxyethylene, dihydroxyethylene, C-C single bond (alkane), C-C double bond (alkene), C-C triple bond (alkyne), C-O bond (methyleneoxy), O-N or N-O bond (methylenemino), triazole, hydrazide, urea, ketone, urethane bond, (di)haloalkene, methylenethio, methyleneamino, trifluoroethylamino, hydrazide, amideoxy, and others known to those skilled in the art.

[0117] Peptide bond isosteres can also be cyclic molecules decorated with amine and carboxylic acid functionalities. Non-limiting examples of cyclic peptide bond isosteres with varying ring sizes include carbacycles, nitrogen heterocycles, and oxygen heterocycles. Nitrogen heterocycles can be based on alkaloid cores that form bicyclic structure isosteres. Examples of nitrogen heterocyclic isosteres include isosteres based on triazole rings formed by copper-catalyzed azide-alkyne cycloaddition. The cyclic peptide bond isosteres described herein can be di-, tri-, tetra-, penta-, hexa-, hepta-, octa-nine-ten-peptide cyclic isosteres.

[0118] When referring to polypeptides, "insertion variants" are those with one or more amino acids inserted just adjacent to the amino acid at a particular position in the native or starting sequence. "Just adjacent" to an amino acid means attached to the α-carboxyl or α-amino functional group of the amino acid.

[0119] When referring to polypeptides, "deletion variants" are those in which one or more amino acids are removed from the native or starting amino acid sequence. Typically, deletion variants will have one or more amino acids deleted in a specific region of the molecule.

[0120] "Truncation variants" when referring to polypeptides are those in which one or more amino acids in the native or starting amino acid sequence are removed from either terminus of the polypeptide.

[0121] According to the present invention, the polypeptide may be modified by the addition of one or more conjugate groups. In some embodiments, the polypeptide may be administered in combination with one or more additional molecules.

[0122] As used herein, "conjugate" refers to any molecule or part attached to another molecule. In the present invention, the conjugate may be based on or not based on a polypeptide (amino acid). The conjugate may include a lipid, a small molecule, RNA, DNA, a polypeptide, a polymer or a combination thereof. Functionally, the conjugate may serve as a target molecule or may serve as a payload to be delivered to a cell, an organ or a tissue. The conjugate is generally a covalent modification introduced by reacting a target amino acid residue or a polypeptide end with an organic derivatizing agent, which can react with a selected side chain or terminal residue. Such modifications are within the scope of those of ordinary skill in the art and are not carried out without excessive experimentation.

[0123] The conjugation process may involve PEGylation, lipidation, albumination, biotinylation, desulfobiotinylation, addition of other polypeptide tails, or grafting to an antibody Fc domain, a CDR region of an intact antibody, or an antibody domain generated by any number of means. The conjugate may include an anchor comprising a cholesterol oleate moiety, a cholesterol laurate moiety, an alpha-tocopherol moiety, a phytol moiety, an oleate moiety, or an unsaturated cholesterol ester moiety or a lipophilic compound selected from acetanilide, anilide, aminoquinoline, benzhydryl compound, benzodiazepine, benzofuran, cannabinoid, cyclic polypeptide, dibenzazepine, digitalis glycoside, ergot alkaloid, flavonoid, imidazole, quinoline, macrolide, naphthalene, opiate (such as but not limited to morphinan or other psychotropic drugs), oxazine, oxazole, phenylalkylamine, piperidine, polycyclic aromatic hydrocarbons, pyrrolidine, pyrrolidone, stilbene, sulfonylurea, sulfone, triazole, tropane, and vinca alkaloid.

[0124] As used herein, when referring to a polypeptide, the term "covalent derivative" includes modification of a natural or starting polypeptide with an organic proteinaceous or non-proteinaceous derivatizing agent, and / or post-translational modification. Covalent modifications are routinely introduced by reacting the target amino acid residue of the polypeptide with an organic derivatizing agent that can react with a selected side chain or terminal residue, or by a constraint mechanism of post-translational modification that works in a selected recombinant host cell. The resulting covalent derivative can be used in a program for identifying residues that are important for biological activity, immunoassays, or the preparation of anti-protein antibodies for immunoaffinity purification of recombinant proteins. Such modifications are within the scope of those of ordinary skill in the art and are performed without excessive experimentation.

[0125] Certain post-translational modifications are the result of the action of the recombinant host cell on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mild acidic conditions. Any form of these residues may be present in the polypeptides produced according to the present invention.

[0126] Other post-translational modifications include: hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of tyrosinyl, seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (Creighton, TE, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86).

[0127] Covalent modification specifically includes non-protein polymer bonding to the polypeptide of the present invention. Non-protein polymers may include hydrophilic synthetic polymers, i.e., polymers not otherwise found in nature. However, polymers present in nature and produced by recombinant or in vitro methods are available, and polymers separated from nature are also available. Hydrophilic polyvinyl polymers fall within the scope of the present invention, such as polyvinyl alcohol and polyvinyl pyrrolidone. Polypeptide can be connected to various non-protein polymers such as polyethylene glycol, polypropylene glycol or polyoxyalkylene by the method described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337.

[0128] When referring to polypeptides, "features" are defined as components of the molecule based on distinct amino acid sequences. Features of the polypeptides of the invention include surface appearance, local conformational shape, fold, loop, half-loop, domain, half-domain, site, terminus, or any combination thereof.

[0129] As used herein, when referring to polypeptides, the term "fold" refers to the resulting conformation of an amino acid sequence when energy is minimized. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary folds include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed by the aggregation or separation of physicochemically distinct regions. Regions formed in this manner include hydrophobic and hydrophilic pockets, among others.

[0130] As used herein, the term "turn" when relating to protein conformation means a bend that changes the direction of the polypeptide backbone and may involve one, two, three or more amino acid residues.

[0131] As used herein, when referring to a polypeptide, the term "loop" refers to a structural feature of a polypeptide that can be used to reverse the direction of the polypeptide backbone. In cases where a loop is found in a polypeptide and only changes the direction of the backbone, the loop may contain four or more amino acid residues. Oliva et al. have identified at least five types of protein loops (Oliva, B. et al., J Mol Biol. 1997 Mar 7; 266(4): 814-30; the contents of which are incorporated herein by reference in their entirety). Loops may be open or closed. When two amino acids are connected by a bridging moiety, a closed loop or "annular" loop may be formed. Annular loops contain amino acids that are present between the bridging amino acids along the polypeptide. Annular loops may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0132] As used herein, when referring to a polypeptide, the term "half-ring" refers to a portion of an identified ring having at least half the number of amino acid residues compared to the ring from which it is derived. It is understood that a ring may not always contain an even number of amino acid residues. Therefore, in those cases where a ring contains or is identified to contain an odd number of amino acids, the half-ring of the odd number of rings will contain the integer portion of the ring or the next integer portion (number of amino acids in the ring / 2+ / -0.5 amino acids). For example, a ring identified as a 7 amino acid ring can produce a half-ring of 3 amino acids or 4 amino acids (7 / 2=3.5+ / -0.5 is 3 or 4).

[0133] As used herein, the term "region" when referring to a protein refers to a zone or general area. In some embodiments, when referring to a protein, a region may include a linear sequence of amino acids along a protein or may include a specific secondary or tertiary structure and / or one or more features or protein domains.

[0134] As used herein, when referring to a protein, the term "domain" refers to a motif of a polypeptide that has one or more identifiable structural (such as secondary or tertiary structure) or functional characteristics or properties (e.g., binding ability, serving as a site for protein-protein interaction).

[0135] As used herein, when referring to a protein, the term "half-domain" refers to a portion of an identified domain having at least half the number of amino acid residues compared to the domain from which it is derived. It is understood that a domain may not always contain an even number of amino acid residues. Thus, in those cases where a domain contains or is identified to contain an odd number of amino acids, a half-domain of the odd-numbered domain will contain an integer portion of the domain or the next integer portion (number of amino acids in the domain / 2 + / - 0.5 amino acids). For example, a domain identified as a 7 amino acid domain may produce a half-domain of 3 amino acids or 4 amino acids (7 / 2 = 3.5 + / - 0.5 is 3 or 4). It is also understood that subdomains may be identified within a domain or half-domain that have properties that are less than all of the structural or functional properties identified in the domain or half-domain from which it is derived. It is also understood that the amino acids comprising any of the domain types herein need not be contiguous along the polypeptide backbone (i.e., non-adjacent amino acids may structurally fold to produce a domain, half-domain, or subdomain).

[0136] As used herein, when referring to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when related to amino acid-based embodiments. A site refers to a position within a polypeptide that can be modified, manipulated, altered, derivatized or varied within a polypeptide-based molecule of the invention.

[0137] As used herein, when referring to a polypeptide, the term "terminus" or "end" refers to the extreme end of the polypeptide. Such extreme ends are not limited to the first or last site of the polypeptide, but may include other amino acids in the terminal region. The polypeptide-based molecules of the present invention can be characterized as having both an N-terminus and a C-terminus. The polypeptides and / or polypeptide compositions of the present invention are composed of multiple polypeptide chains (polymers, oligomers) in some cases, and the polypeptide chains are brought together by disulfide bonds or by non-covalent forces. These categories of proteins will have multiple N-termini and C-termini. Alternatively, the polypeptide termini may be modified so that, depending on the possible circumstances, the polypeptide begins or ends with a non-polypeptide-based portion such as an organic binder.

[0138] In one embodiment, the polypeptide of the present invention may include a terminal region. As used herein, a "terminal region" is an amino acid terminal region that may include cysteine. The terminal region may be an N-terminal region and / or a C-terminal region. In some embodiments, a bridging portion may be used to connect the terminal region to a parent polypeptide. As used herein, a "parent polypeptide" refers to a polypeptide portion that does not include a terminal region. The terminal region may be separated from the parent polypeptide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues.The added residue may be selected from, but is not limited to, any natural or unnatural amino acid, any N-methylated form of a natural or unnatural amino acid, any D-stereoisomer of a natural or unnatural amino acid, norvaline, tert-butylglycine, phenylglycine, azatryptophan, 7-azatryptophan, 4-fluorophenylalanine, penicillamine, sarcosine, homocysteine, 1-aminocyclopropanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 4-aminotetrahydro-2H-pyran-4-carboxylic acid, aminoisobutyric acid, (S)-2-amino-3-(1H-tetrazol-5-yl)propionic acid, cyclopentylglycine , cyclohexylglycine, cyclopropylglycine, η-ω-methylarginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluorotryptophan, 5-chlorotryptophan, citrulline, 4-chlorohomophenylalanine, homophenylalanine, 4-aminomethylphenylalanine, 3-aminomethylphenylalanine, octylglycine, norleucine, tranexamic acid, 2-aminopentanoic acid, 2-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, 2-aminodecanoic acid, 2-aminoundecanoic acid, 2-aminododecanoic acid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, pipecolic acid, 2-carboxyazetidine, hexafluoroleucine, 3-Fluorovaline, 2-amino-4,4-difluoro-3-methylbutyric acid, 3-fluoroisoleucine, 4-fluoroisoleucine, 5-fluoroisoleucine, 4-methylphenylglycine, 4-ethylphenylglycine, 4-isopropylphenylglycine, (S)-2-amino-5-(3-methylguanidino)pentanoic acid, (S)-2-amino-3-(4-(aminomethyl)phenyl)propionic acid, (S)-2-amino-3-(3-(aminomethyl)phenyl)propionic acid, (S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butyric acid, (S)-leucinol, (S)-valinol, (S)-tert-leucinol, (R)-3 -methylbutan-2-amine, (S)-2-methyl-1-phenylpropan-1-amine, and (S)-N,2-dimethyl-1-(pyridin-2-yl)propan-1-amine, (S)-2-amino-3-(oxazol-2-yl)propanoic acid, (S)-2-amino-3-(oxazol-5-yl)propanoic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propanoic acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)propanoic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and (S)-2-amino-3-(1H-indazol-3-yl)propanoic acid.

[0139] Other non-natural amino acids that can be used for optimization of the polypeptides of the present invention include, but are not limited to, fluorinated amino acids in which one or more carbon-bound hydrogen atoms are replaced by fluorine. The number of fluorine atoms included can range from 1 to up to and including all hydrogen atoms. Examples of such amino acids include, but are not limited to: 3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline, 4,4-difluoroproline, 3,4-difluoroproline, 3,3,4,4-tetrafluoroproline, 4-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, and stereoisomers thereof.

[0140] Other non-natural amino acids that can be used for optimization of polypeptides of the present invention include, but are not limited to, those that are disubstituted at the α-carbon. These include amino acids in which the two substituents on the α-carbon are identical, such as α-aminoisobutyric acid, and 2-amino-2-ethylbutyric acid, as well as those in which the substituents are different, such as α-methylphenylglycine and α-methylproline. In addition, the substituents on the α-carbon can be combined to form a ring, such as 1-aminocyclopentanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 3-aminotetrahydrofuran-3-carboxylic acid, 3-aminotetrahydropyran-3-carboxylic acid, 4-aminotetrahydropyran-4-carboxylic acid, 3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylic acid, 4-aminopiperidone-4-carboxylic acid, and their stereoisomers.

[0141] Other non-natural amino acids that can be used for optimization of the polypeptides of the present invention include, but are not limited to, analogs of tryptophan in which the indole ring system is replaced by another 9-membered or 10-membered bicyclic ring system containing 0, 1, 2, 3 or 4 heteroatoms independently selected from N, O or S. Each ring system may be saturated, partially unsaturated or fully unsaturated. The ring system may be substituted by 0, 1, 2, 3 or 4 substituents at any substitutable atom. Each substituent is independently selected from H, F, Cl, Br, CN, oxygen, COOR, CONRR', OR, NRR'. Each R and R' is independently selected from H, C1-C20 alkyl, C1-C20 alkyl-O-C1-20 alkyl.

[0142] In some embodiments, analogs of optimized tryptophan (also referred to herein as "tryptophan analogs") that can be used in the polypeptides of the present invention include, but are not limited to, 5-fluorotryptophan [(5-F)W], 5-methyl-O-tryptophan [(5-MeO)W], 1-methyltryptophan [(1-Me-W) or (1-Me)W], D-tryptophan (D-Trp), 7-azatryptophan (including but not limited to 4-azatryptophan, 7-azatryptophan and 5-azatryptophan), 5-chlorotryptophan, 4-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, and stereoisomers thereof. Unless otherwise indicated, as used herein, the term "azatryptophan" and its abbreviation "azaTrp" refer to 7-azatryptophan.

[0143] In one embodiment, the polypeptides of the invention may include terminal modifications with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues at the N-terminus or C-terminus and / or cysteine ​​in the terminal region. The added residues may be selected from, but are not limited to, any natural or unnatural amino acid, any N-methylated form of any natural or unnatural amino acid, any D-stereoisomer of a natural or unnatural amino acid, norvaline, tert-butylglycine, phenylglycine, azatryptophan, 7-azatryptophan, 4-fluorophenylalanine, penicillamine, sarcosine, homocysteine, 1-aminocyclopropanecarboxylic acid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 4-aminotetrahydro-2H-pyran-4-carboxylic acid, aminoisobutyric acid, acid), (S)-2-amino-3-(1H-tetrazol-5-yl)propionic acid, cyclopentylglycine, cyclohexylglycine, cyclopropylglycine, η-ω-methylarginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluorotryptophan, 5-chlorotryptophan, citrulline, 4-chlorohomophenylalanine, homophenylalanine, 4-aminomethylphenylalanine, 3-aminomethylphenylalanine, octylglycine, norleucine, tranexamic acid, 2-aminovaleric acid, 2-aminohexanoic acid, 2-amino 2-aminoheptanic acid, 2-aminooctanoic acid, 2-aminononanoic acid, 2-aminodecanoic acid, 2-aminoundecanoic acid, 2-aminododecanoic acid, aminopentanoic acid, and 2-(2-aminoethoxy)acetic acid, pipecolic acid, 2-carboxyazetidine, hexafluoroleucine, 3-fluorovaline, 2-amino-4,4-difluoro-3-methylbutyric acid, 3-fluoroisoleucine, 4-fluoroisoleucine, 5-fluoroisoleucine, 4-methylphenylglycine, 4-ethylphenylglycine, 4-isopropylphenylglycine, (S)-2-amino -5-(3-methylguanidino)pentanoic acid, (S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid, (S)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid, (S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butanoic acid, (S)-leucinol, (S)-valinol, (S)-tert-leucinol, (R)-3-methylbutan-2-amine, (S)-2-methyl-1-phenylpropan-1-amine, and (S)-N,2-dimethyl-1-(pyrrolidone)-1-amine. (S)-2-amino-3-(oxazol-2-yl)propanoic acid, (S)-2-amino-3-(oxazol-5-yl)propanoic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propanoic acid, (S)-2-amino-3-(1,2,4-oxadiazol-3-yl)propanoic acid, (S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and (S)-2-amino-3-(1H-indazol-3-yl)propanoic acid.

[0144] The polypeptides of the present invention can be bound to polypeptides that increase or decrease plasma protein binding, including but not limited to those described in: Dennis, MS et al., Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J Biol Chem. 2002 Sep 20;277(38):35035-43; Nguyen, A. et al., The pharmacokinetics of an albumin-binding Fab (AB Fab) can be modulated as a function of affinity for albumin. Protein Eng Des Sel. 2006 Jul;19(7):291-7 and Langerheim, JF et al., Improving the pharmacokinetics / pharmacodynamics of prolactin, GH, and their antagonists by fusion to a synthetic albumin-binding polypeptide. J Endocrinol. 2009 Dec;203(3):375-87. In some embodiments, such polypeptides bind to serum albumin (referred to herein as "albumin binding polypeptides"). In some embodiments, albumin binding polypeptides are cyclized by forming disulfide bonds between cysteine ​​residues present in their polypeptide sequences. In some embodiments, albumin binding polypeptides are bound by the N-terminus or the C-terminus. In some embodiments, binding to an albumin binding polypeptide regulates the amount of time that a polypeptide of the present invention remains intact in a subject. In a preferred embodiment, binding to an albumin binding polypeptide increases the amount of time that a polypeptide of the present invention remains in the blood of a subject. The polypeptides of the present invention may be bound to polypeptides having cell penetrating properties (referred to herein as "cell penetrating polypeptides"), including but not limited to those disclosed in Milletti, F., Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today. 2012 August; 17(15-16): 850-60. Other cell penetrating polypeptides are known to those skilled in the art. The polypeptides of the present invention may be conjugated to any of the polypeptide conjugates taught in, for example, US Patent Publication No. US20110172126 or US20030040472, the contents of which are incorporated herein by reference in their entirety.The polypeptides of the invention may be conjugated to lipophilic molecules that increase plasma protein binding, such as liphophilic substituents as taught in U.S. Pat. No. 6,268,343 or U.S. Publication No. US2013 / 0053311, the contents of each of which are incorporated herein by reference in their entirety.

[0145] Once any of the features have been identified or defined as a desired component of a polypeptide, any of several manipulations and / or modifications of these features can be performed by moving, exchanging, inverting, deleting, randomizing, or repeating. In addition, it is to be understood that manipulation of features can produce the same effects as modifications to the molecules of the invention. For example, manipulations involving deletion of domains will produce changes in the length of the molecule, just as modification of a nucleic acid to encode less than a full-length molecule would produce.

[0146] Modification and manipulation can be accomplished by methods known in the art such as, but not limited to, site-directed mutagenesis. The activity of the resulting modified molecule can then be tested using in vitro or in vivo assays, such as those described herein or any other suitable screening assays known in the art.

[0147] According to the present invention, a polypeptide may comprise a consensus sequence discovered through several rounds of experiments.As used herein, a "consensus" sequence is a single sequence that represents a collective population of sequences, allowing for variability at one or more sites.

[0148] The term "identity" as known in the art refers to the relationship between two or more polypeptide sequences as determined by comparing sequences. In the art, identity also means the degree of sequence relatedness between polypeptides, as determined by the number of matches between two or more amino acid residue strings. Using gap comparisons (if any) processed by a specific mathematical model or computer program (i.e., "algorithm"), identity measures the percentage of identical matches between two or more smaller sequences. The identity of related polypeptides can be easily calculated by known methods. Such methods include, but are not limited to, those previously described by others (Lesk, AM, ed., Computational Molecular Biology, Oxford University Press, New York, 1988; Smith, DW, ed., Biocomputing: Informatics and Genome Projects, Academic Press, New York, 1993; Griffin, AM et al., eds., Computer Analysis of Sequence Data, Part 1, Humana Press, New Jersey, 1994; von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, 1987; Gribskov, M. et al., eds., Sequence Analysis Primer, M. Stockton Press, New York, 1991; and Carillo et al., Applied Math, SIAM J, 1988, 48, 1073).

[0149] In some embodiments, a polypeptide variant may have the same or similar activity as a reference polypeptide. Alternatively, a variant may have an activity that is altered (e.g., increased or decreased) relative to a reference polypeptide. In general, variants of a specific polypeptide of the present invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity compared to the sequence identity of a specific reference polypeptide, using sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite of programs (Altschul, SF et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 1997, 25: 3389-3402). Other tools are described herein, particularly in the definition of "identity".

[0150] Default parameters in the BLAST algorithm include, for example, an expectation threshold of 10, a word size of 28, match / mismatch scores of 1, -2, and a gap penalty of linear. Any filtering may be applied, as well as selection of species-specific replicates, e.g., Homo sapiens.

[0151] Abbreviations used in peptides

[0152] As used herein, the abbreviations have the following meanings: "Ac" and "NH2" refer to the acetyl and amidated termini, respectively; "Nvl" represents norvaline; "Phg" represents phenylglycine; "Tbg" represents tert-butylglycine; "Chg" represents cyclohexylglycine; "(N-Me)X" represents the N-methylated form of an amino acid, with the variable "X" being substituted for the letter or three-letter amino acid code to indicate the amino acid, written as N-methyl-X [e.g., (N-Me)A or (N-Me)Ala represent the N-methylated form of alanine or N-methyl-alanine]; "azaTrp" represents azatryptophan; "(4-F)Phe" represents 4-fluorophenylalanine; "Tyr( "OMe)" refers to O-methyltyrosine, "Aib" refers to aminoisobutyric acid; "(homo)F ((homo)F)" or "(homo)Phe ((homo)Phe)" refers to homophenylalanine; "(2-OMe)Phg" refers to 2-O-methylphenylglycine; "(5-F)W" refers to 5-fluorotryptophan; "DX" refers to the D-stereoisomer of a given amino acid "X" [e.g., (D-Chg) refers to D-cyclohexylglycine]; "(5-MeO)W" refers to 5-methyl-O-tryptophan; "homoC (homoC)" refers to homocysteine; "(1-Me-W)" or "(1-Me)W" refers to 1-methyltryptophan; " "Nle" refers to norleucine; "Tiq" refers to tetrahydroisoquinoline residue; "Asp(T)" refers to (S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid; "(3-Cl-Phe)" refers to 3-chlorophenylalanine; "[(N-Me-4-F)Phe]" or "(N-Me-4-F)Phe" refers to N-methyl-4-fluorophenylalanine; "(m-Cl-homo)Phe((m-Cl-high)Phe)" refers to meta-chlorohomophenylalanine; "(des-amino)C((deamino)C)" refers to 3-thiopropanoic acid; "(alpha-methyl)D((α-methyl)D)" refers to α-methyl L-aspartic acid; "2Nal" refers to 2-naphthylalanine; "(3-aminomethyl)Phe" refers to 3-aminomethyl L-phenylalanine (phenyalanine); "Cle" refers to cycloleucine; "Ac-Pyran" refers to 4-amino-tetrahydro-pyran-4-carboxylic acid; "(Lys-C16)" refers to N-ε-palmitoyl lysine; "(Lys-C12)" refers to N-ε-lauryl lysine; "(Lys-C10)" refers to N-ε-decanoyl lysine; "(Lys-C8)" refers to N-ε-octanoyl lysine;"[xXylyl(y,z)]([xXylyl(y,z)]) refers to a xylyl bridging moiety between two thiol-containing amino acids, where x can be m, p, or o to indicate the use of meta-, para-, or ortho-dibromoxylene (respectively) to create the bridging moiety, and the numerical identifiers y and z locate the amino acid positions within the polypeptide with the amino acid participating in the cyclization; "[cyclo(y,z)]([cyclo(y,z)])" refers to the formation of a bond between two amino acid residues, where the numerical identifiers y and z locate the positions of the residues participating in the bond; "[cyclo-olefinyl(y,z)]([cycloolefinyl(y,z)])" refers to the formation of a bond between two amino acid residues by olefin metathesis, where the numerical identifiers y and z locate the positions of the residues participating in the bond; "[ "cyclo-thioalkyl(y,z)]([cyclothioalkyl(y,z)])" refers to the formation of a thioether bond between two amino acid residues, wherein the numerical identifiers y and z locate the positions of the residues participating in the bond; "cyclo-triazolyl(y,z)]([cyclotriazolyl(y,z)])" refers to the formation of a triazole ring between two amino acid residues, wherein the numerical identifiers y and z locate the positions of the residues participating in the bond; "B20" refers to N-ε-(PEG2-γ-glutamic acid-N-α-octadecandioic acid)lysine [also known as (1S,28S)-1-amino-7,16,25,30-tetraoxo-9,12,18,21-tetraoxa-6,15,24,29-tetraazahexadecane-1,28,46-tricarboxylic acid].

[0153] B20

[0154]

[0155] "B28" refers to N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine.

[0156] B28

[0157]

[0158] "K14" refers to N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine. All other symbols refer to the standard single letter amino acid code.

[0159] Antibody

[0160] In some embodiments, the compounds and / or compositions of the present invention may include antibodies or fragments thereof. As used herein, the term "antibody" refers to the broadest sense, and various embodiments are explicitly covered, including but not limited to: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two complete antibodies), and antibody fragments such as double antibodies, as long as the antibodies exhibit the desired biological activity. The antibodies of the present invention may also include human antibodies or humanized antibodies. Antibodies are primarily amino acid-based molecules, but may also include one or more modifications (including but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.).

[0161] As used herein, the term "antibody fragment" refers to any portion of an intact antibody. In some embodiments, an antibody fragment comprises an antigen-binding region from an intact antibody. Examples of antibody fragments may include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. A residual "Fc" fragment is also produced, the name of which reflects its ability to crystallize easily. Pepsin treatment produces a F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link antigens. The compounds and / or compositions of the present invention may comprise one or more of these fragments. For purposes herein, an "antibody" may comprise a heavy chain variable region and a light chain variable region and an Fc region.

[0162] As used herein, the term "natural antibody" refers to a glycoprotein of about 150,000 daltons, usually heterotetrameric, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, but the number of disulfide bonds varies in the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (V H ), followed by several constant regions. Each light chain has a variable region (V L ) and a constant region at the other end; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the light chain variable region is aligned with the variable region of the heavy chain.

[0163] As used herein, the term "variable region" refers to specific antibody regions that vary extensively in sequence and are used in the binding and specificity of each particular antibody for its particular antigen.

[0164] As used herein, the term "Fv" refers to an antibody fragment that contains complete antigen recognition and antigen binding sites. These regions consist of a dimer of one heavy chain and one light chain variable region in tight, non-covalent association.

[0165] As used herein, the term "light chain" refers to the component of antibodies from any vertebrate species that is assigned to one of two clearly distinct types, called kappa and lambda, based on the constant region amino acid sequence.

[0166] Antibodies can be assigned to different classes, depending on the amino acid sequence of the constant region of their heavy chains. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2.

[0167] As used herein, the term "single-chain Fv" or "scFv" refers to a V H and V L A fusion protein of antibody regions in which these regions are linked together into a single polypeptide chain. In some embodiments, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.

[0168] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites. Diabodies contain a light chain variable region V linked to a light chain variable region V in the same polypeptide chain. L The heavy chain variable region V H By using a linker that is too short to allow pairing between the two regions on the same chain, the regions are forced to pair with the complementary regions of another chain and create two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al. (Hollinger, P. et al., "Diabodies": Small bivalent and bispecific antibody fragments. PNAS. 1993. 90: 6444-8), the contents of each of which are incorporated herein by reference in their entirety.

[0169] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous cells (or clones), i.e., comprising the same and / or individual antibodies of the population that bind to the same epitope, except that possible variants may occur during the production of the monoclonal antibody, such variants are generally present in smaller amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0170] The modifier "monoclonal" refers to the antibody characteristic of being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include: "chimeric" antibodies (immunoglobulins), in which portions of the heavy and / or light chains are identical 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 one or more chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies.

[0171] As used herein, the term "humanized antibody" refers to a chimeric antibody that comprises a minimal portion from one or more non-human (e.g., murine) antibody sources, while the remainder is derived from one or more human immunoglobulin sources. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient antibody are replaced by residues from a hypervariable region of an antibody (donor antibody) of a non-human species such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and / or capacity.

[0172] As used herein, the term "hypervariable region" refers to the region within the antigen binding domain of an antibody that contains the amino acid residues responsible for antigen binding. The amino acids present in the hypervariable region determine the structure of the complementarity determining region (CDR). As used herein, the term "CDR" refers to the region of an antibody that contains a structure that is complementary to its target antigen or epitope.

[0173] In some embodiments, the compounds and / or compositions of the present invention may be antibody mimics or include antibody mimics. As used herein, the term "antibody mimics" refers to any molecule that simulates the function or effect of an antibody and specifically and highly avidly binds to its molecular target. In some embodiments, an antibody mimic may be a monobody, which is designed to insert a fibronectin type III domain (Fn3) as a protein scaffold (US 6,673,901 and US 6,348,584, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, antibody mimics may include those known in the art, including but not limited to: affibody molecules, affilin, affitin, anticalin, avimer (affinity polymer), Centyrin, DARPINS TM , Fynomers, Adnectins, and Kunitz domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.

[0174] As used herein, the term "antibody variant" refers to a biological molecule that is structurally and / or functionally similar to an antibody, which contains some differences in amino acid sequence, composition or structure compared to a native antibody.

[0175] Whether monoclonal or polyclonal, the preparation of antibodies is known in the art. The techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999.

[0176] In some embodiments, the polypeptide sequences provided herein can be used in the production of one or more antibodies. In some cases, such polypeptide sequences can be inserted into antibody variable regions. Such variable regions can be introduced into antibodies, antibody mimetics or antibody variants.

[0177] Small molecules

[0178] In some embodiments, the compounds of the invention may be small molecules. Such compounds may comprise a size of about 100 to about 2000 Daltons (e.g., about 100 to about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 Daltons). Small molecules may be non-peptide, or share some or many of the properties of polypeptides and cyclic polypeptides, including amide bonds, cyclic structures, and amino acid-like substituents.

[0179] Aptamer

[0180] In some embodiments, the compounds of the present invention may include aptamers (Keefe, AD, Pai, S. and Ellington, A. (2010). Nat. Rev. Drug Discovery 9: 537-550). As used herein, the term "aptamer" refers to an oligonucleic molecule or polypeptide molecule that is capable of binding to a specific target molecule. Some aptamers can adopt a three-dimensional conformation that is capable of binding to such target molecules with high affinity and specificity.

[0181] Isotopic variant

[0182] The polypeptides of the present invention may contain one or more atoms that are isotopes. As used herein, the term "isotope" refers to a chemical element that has one or more extra neutrons. In one embodiment, the polypeptides of the present invention may be deuterated. As used herein, the term "deuterated" refers to a substance that has one or more hydrogen atoms replaced by a deuterium isotope. A deuterium isotope is an isotope of hydrogen. The nucleus of a hydrogen atom contains one proton, while the nucleus of a deuterium atom contains both a proton and a neutron. The compounds and pharmaceutical compositions of the present invention may be deuterated in order to change physical properties such as stability, or to allow them to be used in diagnostic and experimental applications.

[0183] Formulation and delivery

[0184] The term "pharmaceutical composition" refers to a composition comprising at least one active ingredient, such as, for example, a polypeptide, in a form and amount such that the active ingredient is therapeutically effective.

[0185] The polypeptide formulations of the present invention include controlled duodenal release formulations, delayed release formulations, osmotic controlled release delivery systems, microemulsions, microspheres, liposomes, nanoparticles, patches, pumps, drug reservoirs, etc. Specifically included in the present invention are solid oral dosage forms such as powders, soft gels, capsules, pills, and tablets.

[0186] The pharmaceutical compositions of the present invention may be administered by any route that produces a therapeutically effective effect. These include, but are not limited to, enteral, enteral, epidural, oral, epidural, intracerebral (into the brain), intratracheal (into the airways for delivery to the lungs), intraventricular (into the ventricles), epidermal (applied to the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion ( The drug may be administered intravenously, intrathecally, intraperitoneally, (infusion or injection into the peritoneum), intravesical, intravitreal, (into the posterior chamber of the eye), intracavernous, (into the base of the penis), intravaginal, intrauterine, extra-amniotic, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), insufflation (inhalation), buccal, sublingual, sublabial, enema, eye drops (onto the conjunctiva), or as ear drops.

[0187] In some embodiments, the polypeptide of the present invention is formulated into a sterile aqueous solution. In some embodiments, the polypeptide of the present invention is formulated into a lipid or non-lipid formulation. In another embodiment, the polypeptide of the present invention is formulated into a cationic or non-cationic lipid formulation. In either embodiment, the sterile aqueous solution may contain other active or inactive components. Inactive components, also referred to herein as "excipients", may include but are not limited to physiologically compatible salts, sugars, fillers, surfactants or buffers.

[0188] The polypeptide and / or polypeptide composition of the present invention may comprise one or more carrier agents or be combined with one or more carrier agents to prepare or deliver. As used herein, the term "carrier" refers to a substance that assists the polypeptide and / or polypeptide composition of the present invention in delivering or improving the efficacy of the polypeptide and / or polypeptide composition of the present invention. The carrier agent may be a naturally occurring substance, such as: a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, budding pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or a lipid. The carrier molecule may also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include poly-L-lysine (PLL), poly-L-aspartic acid, and poly-L-glutamic acid, as well as polymers comprising the D-stereoisomers of these amino acids. Other carriers include poly(L-lactide-co-glycolide) copolymers, polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(2-ethylacryllic acid) and N-isopropylacrylamide polymers. Other useful carrier molecules can be identified by conventional methods.

[0189] In some embodiments, the compounds of the present invention can be combined with one or more pharmaceutically acceptable excipients to form a pharmaceutical composition. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of good medical judgment without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio. As used herein, the term "pharmaceutically acceptable excipient" refers to any ingredient (e.g., a carrier capable of suspending or dissolving the active compound) other than the inventive compounds described herein and has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example: antiadhesives, antioxidants, adhesives, coatings, compression aids, disintegrants, dyes (colors), softeners, emulsifiers, fillers (diluents), film formers or coatings, fragrances, flavors, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and bound water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, (divalent) calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl pyridone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, (corn) starch, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. In some embodiments, the pharmaceutical compositions comprise one or more active polypeptide ingredients, together with ethanol, corn oil-mono-di-triglycerides, hydrogenated castor oil, DL-tocopherol, propylene glycol, gelatin, glycerin, colorants, flavors, and sweeteners.

[0190] In other embodiments, the pharmaceutical composition comprises one or more active polypeptide ingredients, together with a delivery agent such as 4-(2-hydroxy-4-methoxybenzamido)butyric acid (or any of the delivery agents described in U.S. Pat. No. 7,744,910 B2, the contents of which are incorporated herein by reference in their entirety), a pharmaceutically acceptable buffer, a disintegrant, a detergent, hydroxypropyl methylcellulose, a colorant, a flavor, and a sweetener.

[0191] In other embodiments, the pharmaceutical composition comprises one or more active polypeptide ingredients together with ethanol, soy phosphatidylcholine, and dioleylglycerol injected into an excess of saline solution as described in U.S. Patent Application 2008 / 0146490A1, the contents of which are incorporated herein by reference in their entirety.

[0192] Delivery of one or more polypeptides to a subject in need can be achieved in several different ways. In vivo delivery can be performed directly by administering a composition comprising one or more polypeptides to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the polypeptides.

[0193] Local delivery avoids intestinal permeability and systemic exposure. For example, the polypeptides and / or polypeptide compositions of the present invention can be used as drops in the eye or by direct injection in the posterior segment of the eye. They can be applied to the target enzyme in the intestine. They can be applied topically by skin application (e.g., creams, ointments, transdermal patches).

[0194] The polypeptide and / or polypeptide composition of the present invention may contain or be formulated with one or more fusogenic agents. As used herein, the term "fusogenic agent" refers to an agent that responds to changes, such as changes in pH in the environment. When encountering the pH of the endosomal body, the fusogenic agent may cause physical changes, such as changes in the permeability properties that interfere with or increase the permeability of the endosomal membrane. Preferably, the fusogenic agent changes charge at a pH below the physiological range, such as becoming protonated. For example, the fusogenic agent may become protonated at pH 4.5-6.5. The fusogenic agent may be used to release the polypeptide into the cytoplasm of the cell, such as via endocytosis, after the cell absorbs the composition, thereby increasing the cell concentration of the polypeptide in the cell.

[0195] In some embodiments, the fusogenic agent may have a moiety, such as an amino group, which will undergo, for example, a charge change, such as protonation, when exposed to a specified pH range. The charge change of the fusogenic agent can trigger a change, such as an osmotic change, in a vesicle, such as an intracellular vesicle, such as an endosome. For example, the fusogenic agent, when exposed to the pH environment of the endosomal body, will cause a solubility or osmotic change sufficient to increase the porosity of the endosomal membrane (preferably, sufficient to rupture the endosomal membrane).

[0196] The fusogenic agent may be a polymer, preferably a polyamino chain, such as polyethyleneimine (PEI). PEI may be linear, branched, synthetic or natural. PEI may be, for example, an alkyl-substituted PEI or a lipid-substituted PEI.

[0197] In other embodiments, the fusogenic agent may be polyhistidine, polyimidazole, polypyridine, polypropylene imine, metformin, or a polyacetal substance such as a cationic polyacetal. In some embodiments, the fusogenic agent may have an alpha helical structure. The fusogenic agent may be a membrane disruptor such as melittin. Other suitable fusogenic agents may be tested and identified by a technician.

[0198] The polypeptide and / or polypeptide composition of the present invention may contain or be formulated with one or more coagulants. The coagulants of the compositions described herein may interact with the polypeptide (e.g., attract, retain or bind to the polypeptide) and act to (a) coagulate, e.g., reduce the size or charge of the polypeptide and / or (b) protect the polypeptide, e.g., protect the polypeptide from degradation. The coagulant may include, e.g., a portion of a charged portion that may interact with the polypeptide through ionic interactions. The coagulant will preferably be a charged polymer, e.g., a polycationic chain. The coagulant may be polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidines, protamines, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0199] In some embodiments, the polypeptide of the invention may be a bicyclic polypeptide. As used herein, the term "bicyclic polypeptide" refers to a polypeptide having two rings. As a non-limiting example, a bicyclic polypeptide inhibitor of C5 can be generated in a combinatorial library. Each ring of a bicyclic polypeptide can have 2, 3, 4, 5, 6 or more amino acids.

[0200] In some embodiments, polypeptides and / or polypeptide compositions of the present invention can be provided as prodrugs. As used herein, the term "prodrug" refers to a drug provided in an inactive form that becomes active at a certain point after administration. In some embodiments in which the polypeptide is administered in the form of a prodrug, amino acids critical to the polypeptide's inhibitory activity cannot be used to interact with the target due to reversible chemical bonds such as ester bonds. When administered, such prodrugs may be subjected to the rupture of reversible chemical bonds, such as enzymatic or acid hydrolysis in the stomach, blood, and / or a given target tissue cell.

[0201] C5 inhibitor

[0202] Some polypeptides and / or polypeptide compositions of the present invention inhibit complement activation at the level of complement component C5, referred to herein as "C5 inhibitors". Some C5 inhibitors work by preventing C5 from being cleaved into cleavage products C5a and C5b, and such inhibitors are referred to herein as "C5 cleavage inhibitors". In some embodiments, the methods of the present invention may include inhibiting C5 cleavage in a system. As used herein, "system" refers to a group of related parts that work together. Such systems include those that contain C5, referred to herein as "C5 systems". C5 systems may include, but are not limited to, solutions, matrices, cells, tissues, organs, and body fluids (including but not limited to blood). In some cases, the C5 system may be a cell system. As used herein, the term "cell system" refers to a system comprising one or more cells or one or more cell components or cell products. In some cases, the C5 system may include an in vivo system, an in vitro system, and an ex vivo system. The in vivo C5 system may include a subject or be contained in a subject. As used herein, the term "subject" refers to any organism to which a compound according to the present invention may be administered, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans).

[0203] In some cases, a C5 inhibitor of the invention may include any of the polypeptides listed in Table 1.

[0204] Table 1. Compounds of the present invention

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] In the C5 system, C5 and other system components may be in solution or may be fixed in, for example, a measurement well. The C5 system may further comprise other components of complement, including in some cases all components necessary to form a membrane attack complex (MAC). In some cases, the polypeptides and / or polypeptide compositions of the present invention may be used to inhibit C5 cleavage in human subjects. Such polypeptides and / or polypeptide compositions may be used to treat various complement-related disorders and / or diseases and associated inflammatory conditions. Certain C5 inhibitors are known in the art and are taught in U.S. Patent Nos. 7,348,401 and 6,355,245; the above patents are incorporated herein by reference in their entirety.

[0220] C5 cleavage produces the proteolytic products C5a and C5b. The C5 cleavage site that cleaves to produce these products is referred to herein as the C5a-C5b cleavage site. C5b contributes to the formation of the membrane attack complex (MAC), while C5a stimulates the immune system and inflammatory responses. In some embodiments, the polypeptides and / or polypeptide compositions of the present invention prevent C5 cleavage and can therefore be used in the treatment of inflammation via inhibition of inflammatory events, including but not limited to chemotaxis and activation of inflammatory cells (e.g., macrophages, mast cells, neutrophils, and platelets), proliferation of endothelial cells, and edema.

[0221] Many complement system components, including but not limited to C3, C4, and C5, are functionally inert in their native state until targeted for cleavage into multiple active components. Cleavage of C3 or C4 results in a conformational change that exposes an internal thioester domain. Within the domain, the internal thioester bond between the side chains of cysteine ​​and glutamine residues is a chemically labile bond that confers the ability of C3 and C4 to bind to cell surfaces and / or biomolecules. Cleavage of C3 and C4 also provides the components of the C5 convertase, C3bC4bC2a or (C3b)2Bb. (Law, SK et al. (1997). Protein Science. 6: 263-274; van den Elsen, JMH, (2002). J. Mol. Biol. 322: 1103-1115; the contents of each of the foregoing are incorporated herein by reference in their entirety).

[0222] The multiple domain structures of C5 are similar to those of C3 and C4. C5 convertase cleaves C5 into components C5a and C5b. C5 cleavage results in a conformational change that exposes the C5b-like thioester domain, which plays a role in C5 binding to C6, followed by interaction with C7 and C8 to form a cytolytic MAC. The domain structure of C5 contains regulatory features that are critical for complement processing and downstream activities. (Fredslund, F. et al. (2008). Nature. 9: 753-760; Hadders, MA et al. (2012). Cell Reports. 1: 200-207).

[0223] In some embodiments, compounds of the invention can bind to C5 and prevent the cleavage of C5 into C5a and C5b cleavage products.

[0224] Recently, a new paradigm for complement activation was proposed based on the discovery that thrombin generates a previously unidentified C5 product that supports the terminal complement activation pathway (Krisinger et al. (2014). Blood. 120(8): 1717-1725).

[0225] Thrombin plays a role in a second circulation-based process, the coagulation cascade, by which the organism is able to limit bleeding, restore vascular integrity, and promote healing in response to injury. Following vascular damage, tissue factor is exposed to the circulation, initiating a cascade of proteolytic reactions leading to the generation of the central coagulation enzyme thrombin, which converts fibrinogen into a fibrin clot.

[0226] Historically, the complement activation pathway has been viewed as separate from the coagulation cascade; however, the mutual influence of these two systems deserves reconsideration. Coagulation and complement are synergistically activated in response to common pathophysiological stimuli in an overlapping spatiotemporal manner to maintain homeostasis, and disease occurs when there is unchecked activation of innate immunity and coagulation responses, as demonstrated by, for example, atherosclerosis, stroke, coronary heart disease, diabetes, ischemia-reperfusion injury, trauma, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, and atypical hemolytic uremic syndrome. In fact, the introduction of complement inhibitors has been found to simultaneously treat inflammatory and thrombotic disorders associated with some of these conditions.

[0227] As noted above, the complement system is activated via three major pathways, all of which converge on proteolytic activation of the central complement component C3. Subsequently, the formation of the C5 convertase results in the cleavage of C5 at arginine 751 (R751) to release the chemotactic and anaphylatoxin C5a fragment and generate C5b. C5b is the initiator for the assembly of the C5b-dependent lytic membrane attack complex (MAC; also known as C5b-9), which is responsible for the destruction of damaged cells and pathogens.

[0228] Several molecular connections between complement and coagulation have been identified. The most notable of these is the description of a novel complement activation pathway, in which thrombin was found to be able to directly promote complement activation by presumably cleaving C5 at R751, thereby releasing C5a in the absence of C3 (Huber-Lang et al., 2006. Nature Med. 12(6): 682-687). However, these studies did not compare thrombin to the true C5 convertase and only performed limited biochemical analysis; therefore, the physiological relevance of this pathway could not be assessed.

[0229] By using a purified and plasma-based system, the effects of thrombin and C5 convertase on C5 were evaluated by measuring the release of the anaphylatoxin C5a and the generation of the MAC component C5b. It was found that while thrombin cleaved C5 poorly at R751, generating minimal C5a and C5b, it efficiently cleaved C5 at the newly identified, highly conserved site R947, generating a previously undescribed intermediate C5 T and C5b T Tissue factor-induced coagulation of plasma results in proteolysis of C5 at a thrombin-sensitive site corresponding to this new R947 site rather than R751. Combined treatment of C5 with thrombin and C5 convertase generates C5a and C5b T , which forms C5b, which has significantly greater lytic activity than C5b-9. T -9 membrane attack complex. Thus, a new paradigm for complement activation has been proposed, in which thrombin is an invariant and critical partner of the C5 convertase in initiating the formation of the more active MAC via the formation of a previously unidentified C5 product, which is generated via cooperative proteolysis of the two enzymes. In the context of coagulation activation occurring in many diseases, these findings provide new insights into the regulation of innate immunity. (Krisinger et al., (2014). Blood. 120(8): 1717-1725).

[0230] In some embodiments, the polypeptides and / or polypeptide compositions of the present invention can inhibit thrombin-induced complement activation. Therefore, such polypeptides and / or polypeptide compositions can be used to treat hemolysis caused by thrombin-induced complement activation.

[0231] In view of the discovery of a molecular connection between complement and the coagulation pathway, it is believed that complement can be activated by other components of the coagulation and / or inflammatory cascade. For example, other serine proteases with slightly different substrate specificities may function in a similar manner. Huber-Lang et al. (2006) showed that thrombin not only cleaves C5, but also cleaves in vitro produced C3a when incubated with native C3 (Huber-Lang et al., 2006. Nature Med. 12 (6): 682-687; the contents of which are incorporated herein by reference in their entirety). Similarly, other components of the coagulation pathway, such as FXa, FXIa and plasmin, have been found to cleave both C5 and C3.

[0232] Specifically, in a mechanism similar to that observed for activation via thrombin, plasmin, FXa, FIXa, and FXIa have been observed to be able to cleave C5 to generate C5a and C5b (Amara et al., (2010). J. Immunol. 185: 5628-5636; Amara et al., (2008) "Interaction Between the Coagulation and Complement System" in Current Topics in Complement II, J.D. Lambris (ed.), pp. 71-79). The anaphylatoxins produced were found to be biologically active as shown by dose-dependent chemotactic responses of neutrophils and HMC-1 cells, respectively. Plasmin-induced cleavage activity can be dose-dependently blocked by the serine protease inhibitors aprotinin and leupeptin. These findings suggest that various serine proteases belonging to the coagulation system are able to activate the complement cascade independently of established pathways. In addition, functional C5a and C3a were produced (as detected by immunoblotting and ELISA), both known to be critically involved in inflammatory responses.

[0233] In some embodiments, polypeptides and / or polypeptide compositions of the invention can inhibit C5 activation by plasmin, FXa, FIXa, FXIa, and other proteases of the coagulation pathway.

[0234] Human leukocyte elastase (HLE), an enzyme secreted by neutrophils and macrophages during inflammation, has long been known to also release chemotactic C5a-like fragments from C5. However, this C5a-like fragment is not identical to C5a, as HLE does not cleave the peptide bond at the cleavage site that normally cleaves C5 into C5a and C5b after exposure to complement convertases. In contrast, cleavage of complement C5 by HLE has also been found to generate functionally active C5b-like molecules that are able to participate in MAC formation (Vogt, (1999). Immunobiology. 201: 470-477).

[0235] In some embodiments, polypeptides and / or polypeptide compositions of the invention can inhibit C5 activation by HLE and other proteases of the inflammatory cascade.

[0236] In some embodiments, polypeptides and / or polypeptide compositions of the present invention can be used to treat diseases, disorders and / or conditions, wherein C5 cleavage leads to the progression of the disease, disorder and / or condition. Such diseases, disorders and / or conditions may include, but are not limited to, immune and autoimmune, neurological, cardiovascular, pulmonary and ocular diseases, disorders and / or conditions. Immune and autoimmune diseases and / or conditions may include, but are not limited to: acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, acute antibody-mediated rejection after organ transplantation, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune familial dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune Myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, bacterial sepsis and septic shock, Balo's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, Trauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie virus myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), type I diabetes, discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental Allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrotic alveolitis, Giant cell arteritis (temporal arteritis), Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with polyangiitis (GPA) see Wegener's, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia (including atypical hemolytic uremic syndrome and atypical hemolytic uremic syndrome with antiplasma therapy),Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, large vessel vasculopathy, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), systemic lupus erythematosus (SLE), Lyme disease, Meniere's disease ) disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple endocrine neoplasia syndrome, multiple sclerosis, multifocal motor neuropathy, myositis, myasthenia gravis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, osteoarthritis, relapsing rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parrot's syndrome, Parson-Turner syndrome, pars planitis (peripheral uveitis ), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes type I, II, and III, polyendocrine gland disease, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, simple red cell aplasia, Raynaud's phenomenon, reactive arthritis, sympathetic reflex dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis Neoplasia, Schmidt syndrome, Scleritis, Scleroderma, Shiga toxin-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS), Sjögren's syndrome, Small vessel vasculopathy, Sperm and testicular autoimmunity, Stiff-man syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis / giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Tubular autoimmune disorders, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vesicobullous dermatosis, Vasculitis,Vitiligo and Wegener's granulomatosis (also known as granulomatosis with polyangiitis (GPA)). Neurological diseases, disorders and / or conditions may include, but are not limited to, Alzheimer's disease, Parkinson's disease, Lewy body dementia, and multiple sclerosis. Cardiovascular diseases, disorders and / or conditions may include, but are not limited to, atherosclerosis, myocardial infarction, stroke, vasculitis, trauma and conditions caused by cardiovascular interventions (including, but not limited to, heart bypass surgery, arterial grafts, and angioplasty). Pulmonary diseases, disorders and / or conditions may include, but are not limited to, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), and adult respiratory distress syndrome. Ocular related applications include, but are not limited to, age-related macular degeneration, allergic conjunctivitis and giant papillary conjunctivitis, Behcet's disease, choroidal inflammation, complications associated with intraocular surgery, corneal transplant rejection, corneal ulcers, cytomegalovirus retinitis, dry eye syndrome, endophthalmitis, Fuch's disease, glaucoma, immune complex vasculitis, inflammatory conjunctivitis, ischemic retinal disease, keratitis, macular edema, parasitic infection / migration of the eye, retinitis pigmentosa, scleritis, Stargardt's disease, subretinal fibrosis, uveitis, vitreo-retinal inflammation, and Vogt-Koyanagi-Harada disease.

[0237] The polypeptides and / or polypeptide compositions of the present invention may be particularly useful in the treatment of patients with PNH who exhibit resistance to, for example, PNH due to mutations in the C5 gene that prevent antibodies from binding to C5. Poor response to monoclonal antibody therapy for treatment of leukemia (Nishimura, JI. (2012). 54th ASH Annual Meeting, Abstract 3197).

[0238] The polypeptides and / or polypeptide compositions of the present invention can be used to treat infectious diseases, disorders and / or conditions, for example in a subject suffering from an infection. In some preferred embodiments, the subject suffers from an infection and is at risk of developing sepsis or a septic syndrome. The polypeptides and / or polypeptide compositions of the present invention are particularly useful in the treatment of sepsis.

[0239] The polypeptides and / or polypeptide compositions of the present invention may also be administered to improve the results of clinical procedures in which complement inhibition is required. Such procedures may include, but are not limited to, grafting, transplantation, implantation, catheterization, intubation, etc. In some embodiments, the polypeptides and / or polypeptide compositions of the present invention are used to coat the equipment, materials, and / or biomaterials used in such procedures. In some embodiments, the inner surface of the tube may be coated with the polypeptide and / or polypeptide composition to prevent complement activation in the body fluid passing through the tube in vivo or in vitro (e.g., extracorporeal shunts, such as dialysis and cardiac bypass).

[0240] How to use

[0241] Treatment Indications

[0242] The present invention specifically relates to the use of polypeptides (e.g., peptidomimetics and cyclic polypeptides) and compositions containing at least one polypeptide for treating disorders, conditions or diseases. In some cases, the compounds and compositions of the present invention can be used to treat subjects with paroxysmal nocturnal hemoglobinuria (PNH). Subjects with PNH cannot synthesize functional versions of complement regulatory proteins CD55 and CD59 on hematopoietic stem cells. This leads to complement-mediated hemolysis and various downstream complications. Other complement-related disorders and diseases include, but are not limited to, autoimmune diseases and disorders, neurological diseases and disorders, blood diseases and disorders, and infectious diseases and disorders. Experimental evidence shows that many complement-related disorders are alleviated via inhibition of complement activity.

[0243] Acquired mutations in the class A phosphatidylinositol glycan anchor biosynthesis (PIG-A) gene derived from pluripotent hematopoietic stem cells lead to a rare disease called paroxysmalnocturnal hemoglobinuria (PNH) (Pu, JJ et al., Paroxysmalnocturnal hemoglobinuria from bench to bedside. Clin Transl Sci. 2011 Jun; 4(3): 219-24). PNH is characterized by bone marrow disorders, hemolytic anemia and thrombosis. The PIG-A gene product is necessary for the production of the glycolipid anchor-glycosylphosphatidylinositol (GPI) used to tether proteins to the plasma membrane. Two complement regulatory proteins CD55 and CD59 become non-functional in the absence of GPI. This leads to complement-mediated destruction of these cells. The polypeptides and / or polypeptide compositions of the present invention are particularly useful in the treatment of PNH. As used herein, the term "treatment" and the like refers to the alleviation or reduction of a pathological process. In the context of the present invention, the terms "treat", "treat" and the like, so far as they relate to any of the other conditions detailed herein below, mean: alleviating or alleviating at least one symptom associated with such condition; or slowing or reversing the progression or expected progression of such condition, such as slowing the progression of a malignancy or cancer; or increasing the clearance rate of infectious organisms to alleviate / reduce symptoms caused by infection, for example hepatitis caused by infection with a hepatitis virus or reducing red blood cell destruction due to paroxysmal nocturnal hemoglobinuria (as measured by reduced transfusion requirements or increased hematocrit or hemoglobin levels).

[0244] In the context of a disease marker or symptom, "reduced" or "reduced" means a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably as low as levels accepted as being within the normal range for individuals not suffering from such a disorder.

[0245] In the context of a disease marker or symptom, "increase" or "elevation" means a statistically significant increase in such levels. The increase can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably up to a level accepted as being within the normal range for individuals not suffering from such a disorder.

[0246] As used herein, the words "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a pathological process or a significant symptom of one or more pathological processes. The specific amount that is therapeutically effective can be readily determined by an ordinary practitioner and can vary depending on factors known in the art such as the type of pathological process, the patient's medical history and age, the stage of the pathological process, and the administration of other agents that inhibit the pathological process.

[0247] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a polypeptide and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to the amount of a polypeptide that effectively produces an intended pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 10% change (increase or decrease) in a measurable parameter associated with a disease or condition, then the therapeutically effective amount of the drug used to treat the disease or condition is the amount necessary to achieve at least a 10% change in the parameter. For example, a therapeutically effective amount of a polypeptide may be an amount that changes the binding of a target to its natural binding partner by at least 10%.

[0248] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term explicitly excludes cell culture media. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, will generally be magnesium stearate, stearic acid, or talc. When necessary, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The medicaments included in the pharmaceutical preparations are further described herein below.

[0249] The efficacy of disease treatment or improvement can be evaluated, for example, by measuring disease progression, disease alleviation, symptom severity, reduction of pain, quality of life, dosage required to maintain therapeutic effect, disease marker levels, or any other measurable parameter appropriate for a given disease to be treated or targeted for prevention. It is entirely within the capabilities of those skilled in the art to monitor the efficacy of treatment or prevention by measuring any one of such parameters or any combination of parameters. With respect to the administration of a polypeptide or its pharmaceutical composition, "effective resistance" to a disease or condition refers to the administration in a clinically appropriate manner to produce beneficial effects such as the following on at least a portion of the patients: symptom improvement, cure, reduction of disease load, reduction of tumor mass or cell number, extension of life, improvement of quality of life, reduction of transfusion requirements, or other effects generally considered positive by doctors familiar with the treatment of a specific type of disease or condition.

[0250] The therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when a symptom that would otherwise be expected fails to worsen or develop. As an example, at least 10%, and preferably at least 20%, 30%, 40%, 50% or more favorable changes in the measurable parameters of the disease can indicate effective treatment. The efficacy of a given polypeptide drug or pharmaceutical preparation can also be judged using experimental animal models known in the art for a given disease. When using an experimental animal model, therapeutic efficacy is demonstrated when a statistically significant modulation in a marker or symptom is observed.

[0251] The polypeptide and other therapeutic agent can be administered in combination in the same composition, eg, parenterally, or the other therapeutic agent can be administered as part of a separate composition or by another method described herein.

[0252] Inflammatory Indications

[0253] In some embodiments, the compounds and compositions of the present invention can be used to treat subjects with diseases, disorders and / or conditions associated with inflammation. Inflammation can be upregulated during the proteolytic cascade of the complement system. Although inflammation can have beneficial effects, excessive inflammation can lead to various pathologies (Markiewski et al. 2007. Am J Pathol. 17: 715-27). Therefore, the compounds and compositions of the present invention can be used to reduce or eliminate inflammation associated with complement activation.

[0254] Sterile inflammation

[0255] In some embodiments, the compounds and compositions of the present invention can be used to treat, prevent or delay the development of sterile inflammation. Sterile inflammation is inflammation that occurs in response to stimuli other than infection. Sterile inflammation can be a common response to genomic stress, hypoxia stress, nutritional stress or endoplasmic reticulum stress such as caused by physical, chemical or metabolic harmful stimuli. Sterile inflammation can contribute to the onset of many diseases such as but not limited to the following: ischemic induced injury, rheumatoid arthritis, acute lung injury, drug-induced liver injury, inflammatory bowel disease and / or other diseases, disorders or conditions. Mechanisms of sterile inflammation and methods and compositions for treating, preventing and / or delaying symptoms of sterile inflammation may include any of those taught by Rubartelli et al., Frontiers in Immunology, 2013, 4:398-99, Rock et al., Annu Rev Immunol. 2010, 28:321-342, or U.S. Pat. No. 8,101,586, the contents of each of which are incorporated herein by reference in their entirety.

[0256] Systemic Inflammatory Response (SIRS) and Sepsis

[0257] In some embodiments, the compounds and compositions of the present invention can be used to treat and / or prevent systemic inflammatory response syndrome (SIRS). SIRS is an inflammation that affects the whole body. In the case of SIRS caused by infection, the SIRS is called sepsis. SIRS can also be caused by non-infectious events such as trauma, injury, burns, ischemia, hemorrhage and / or other conditions. During sepsis and SIRS, complement activation leads to excessive production of complement activation products, which can cause multiple organ failure (MOF) in the subject. The compounds and compositions of the present invention can be used to control and / or balance complement activation to prevent and treat SIRS, sepsis and / or MOF. The method of applying complement inhibitors to treat SIRS and sepsis may include those taught by Rittirsch et al. in Clin Dev Immunol, 2012, 962927, in U.S. Publication No. US2013 / 0053302, or in U.S. Patent No. 8,329,169, and the contents of each of the above references are incorporated herein by reference in their entirety.

[0258] Acute respiratory distress syndrome (ARDS)

[0259] In some embodiments, the compounds and compositions of the present invention can be used to treat and / or prevent the development of acute respiratory distress syndrome (ARDS). ARDS is a generalized inflammation of the lungs and can be caused by trauma, infection (e.g., sepsis), severe pneumonia, and / or inhalation of harmful substances. ARDS is usually a severe, life-threatening complication. Studies have shown that neutrophils can contribute to the development of ARDS by affecting the accumulation of polymorphonuclear cells in damaged alveoli and interstitial lung tissue. Therefore, the compounds and compositions of the present invention can be used to reduce and / or prevent the production of tissue factor in alveolar neutrophils. The compounds and compositions of the present invention can be further used to treat, prevent, and / or delay ARDS according to any of the methods taught in International Publication No. WO2009 / 014633 in some cases, and the above-mentioned published content is incorporated herein by reference in its entirety.

[0260] Periodontitis

[0261] In some embodiments, the compounds and compositions of the present invention can be used to treat or prevent the development of periodontitis and / or related conditions. Periodontitis is a widespread, chronic inflammation that causes destruction of periodontal tissues, which are tissues that support and surround teeth. Conditions also involve loss of alveolar bone (bone that supports teeth). Periodontitis can be caused by lack of oral hygiene that causes bacteria, also known as dental plaque, to accumulate at the gum line. Certain health conditions such as diabetes or malnutrition and / or habits such as smoking can increase the risk of periodontitis. Periodontitis can increase the risk of stroke, myocardial infarction, atherosclerosis, diabetes, osteoporosis, premature births, and other health problems. Studies have shown the correlation between periodontitis and local complement activity. Periodontal bacteria can inhibit or activate certain components of the complement cascade. Therefore, the compounds and compositions of the present invention can be used to prevent and / or treat periodontitis and related diseases and conditions. Complement activation inhibitors and treatment methods may include any of those taught by Hajishengallis in Biochem Pharmacol. 2010, 15;80(12):1 and Lambris or US Publication No. US2013 / 0344082, the contents of each of which are incorporated herein by reference in their entirety.

[0262] Trauma and Injury

[0263] The compounds and compositions of the present invention can be used to treat and / or promote the healing of different types of wounds and / or injuries. As used herein, the term "injury" generally refers to physical trauma, but may include local infection or disease processes. Injury can be characterized by injury, damage or destruction caused by external events affecting body parts and / or organs. Trauma is associated with cuts, blows, burns and / or other impacts to the skin that break or damage the skin. Trauma and injury are often acute, but may lead to chronic complications and / or inflammation if they are not properly healed.

[0264] Trauma and burn wounds

[0265] In some embodiments, the compounds and compositions of the present invention can be used to treat wounds and / or promote wound healing. Healthy skin provides a waterproof protective barrier against pathogens and other environmental influences. The skin also controls body temperature and fluid evaporation. These functions are disturbed when the skin is injured, making skin healing a challenge. Trauma triggers a set of physiological processes related to the immune system that repairs and regenerates tissues. Complement activation is one of these processes. Complement activation studies have identified several complement components involved in wound healing, as taught by van de Goot et al. in J Burn Care Res 2009, 30: 274-280 and Cazander et al. Clin Dev Immunol, 2012, 2012: 534291, and the contents of each of the above references are incorporated herein by reference in their entirety. In some cases, complement activation may be excessive, causing cell death and enhanced inflammation (leading to weakened wound healing and chronic wounds). In some cases, the compounds and compositions of the present invention can be used to reduce or eliminate such complement activation to promote wound healing. Treatment with the compounds and compositions of the present invention may be performed according to any of the methods for treating wounds disclosed in International Publication No. WO2012 / 174055, the contents of which are incorporated herein by reference in their entirety.

[0266] Head trauma

[0267] In some embodiments, the compounds and compositions of the present invention can be used to treat head trauma and / or promote healing of head trauma. Head trauma includes damage to the scalp, skull or brain. Examples of head trauma include, but are not limited to, concussions, contusions, skull fractures, traumatic brain injury and / or other injuries. Head trauma can be mild or severe. In some cases, head trauma can lead to long-term physical and / or psychological complications or death. Studies have shown that head trauma can induce inappropriate intracranial complement cascade activation, which can lead to local inflammatory responses that contribute to secondary brain damage by developing cerebral edema and / or neuronal death (Stahel et al. in Brain Research Reviews, 1998, 27: 243-56, the contents of which are incorporated herein by reference in their entirety). The compounds and compositions of the present invention can be used to treat head trauma and / or reduce or prevent related secondary complications. Methods of controlling complement cascade activation in head trauma using the compounds and compositions of the invention may include any of those taught by Holers et al. in US Pat. No. 8,911,733, the contents of which are incorporated herein by reference in their entirety.

[0268] Crush Injury

[0269] In some embodiments, the compounds and compositions of the present invention can be used to treat crush injuries and / or promote the healing of crush injuries. Crush injuries are injuries caused by forces or pressures applied to the body that cause bleeding, bruising, fractures, nerve damage, trauma, and / or other damage to the body. The compounds and compositions of the present invention can be used to reduce complement activation after crush injuries, thereby promoting healing after crush injuries (e.g., by promoting nerve regeneration, promoting fracture healing, preventing or treating inflammation and / or other related complications). The compounds and compositions of the present invention can be used to promote healing according to any of the methods taught in U.S. Patent No. 8,703,136, International Publication No. WO2012 / 162215, WO2012 / 174055, or U.S. Publication No. US2006 / 0270590, and the contents of each of the above patents or publications are incorporated herein by reference in their entirety.

[0270] Autoimmune diseases

[0271] The compounds and compositions of the present invention can be used to treat subjects with autoimmune diseases and / or conditions. The immune system can be divided into an innate system and an adaptive system, which refer to nonspecific immediate defense mechanisms and more complex antigen-specific systems, respectively. The complement system is part of the innate immune system, identifying and eliminating pathogens. In addition, complement proteins can regulate adaptive immunity, connecting innate and adaptive responses. Autoimmune diseases and conditions are immune abnormalities that cause the system to target self-tissues and substances. Autoimmune diseases may involve certain tissues or organs of the body. The compounds and compositions of the present invention can be used to regulate complement in the treatment and / or prevention of autoimmune diseases. In some cases, such compounds and compositions can be used according to the methods presented in Ballanti et al. Immunol Res (2013) 56: 477-491, which are incorporated herein by reference in their entirety.

[0272] Antiphospholipid syndrome (APS) and catastrophic antiphospholipid syndrome (CAPS)

[0273] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat antiphospholipid syndrome (APS) by controlling complement activation. APS is an autoimmune condition caused by antiphospholipid antibodies that cause blood to clot. APS can lead to recurrent venous or arterial thrombosis in organs, as well as complications in the placental circulation, causing pregnancy-related complications such as miscarriage, stillbirth, preeclampsia, premature birth and / or other complications. Catastrophic antiphospholipid syndrome (CAPS) is an extreme and acute version of a similar condition that causes simultaneous blockage of veins in several organs. Studies have shown that complement activation can contribute to APS-related complications, including pregnancy-related complications, thrombotic (coagulation) complications, and vascular complications. The compounds and compositions of the present invention can be used to treat APS-related conditions by reducing or eliminating complement activation. In some cases, the compounds and compositions of the present invention can be used to treat APS and / or APS-related complications according to the methods taught by Salmon et al. Ann Rheum Dis 2002;61(Suppl II):ii46-ii50 and Mackworth-Young in Clin Exp Immunol 2004, 136:393-401, the contents of which are incorporated herein by reference in their entirety.

[0274] Cold agglutinin disease

[0275] In some embodiments, the compounds and compositions of the present invention can be used to treat cold agglutinin disease (CAD), also known as cold agglutinin-mediated hemolysis. CAD is an autoimmune disease caused by the interaction of high concentrations of IgM antibodies with red blood cells at low range body temperatures [Engelhardt et al. Blood, 2002, 100 (5): 1922-23]. CAD can lead to symptoms such as anemia, fatigue, dyspnea, hemoglobinuria and / or acrocyanosis. CAD is associated with robust complement activation, and studies have shown that CAD can be treated with complement inhibitor therapy. Therefore, the present invention provides methods for treating CAD using the compounds and compositions of the present invention. In some cases, the compounds and compositions of the present invention can be used to treat CAD according to the methods taught by Roth et al. in Blood, 2009, 113: 3885-86 or in International Publication No. WO2012 / 139081, the contents of each of which are incorporated herein by reference in their entirety.

[0276] Vascular Indications

[0277] In some embodiments, the compounds and compositions of the invention can be used to treat vascular indications affecting blood vessels (e.g., arteries, veins, and capillaries). Such indications can affect blood circulation, blood pressure, blood flow, organ function, and / or other body functions.

[0278] Thrombotic microangiopathy (TMA)

[0279] In some embodiments, the compounds and compositions of the present invention can be used to treat and / or prevent thrombotic microangiopathy (TMA) and related diseases. Microangiopathy affects the body's small blood vessels (capillaries), making the capillary walls thick, weak and prone to bleeding, and slowing blood circulation. TMA tends to lead to the development of vascular thrombosis, endothelial cell damage, thrombocytopenia and hemolysis. Organs such as the brain, kidneys, muscles, gastrointestinal system, skin and lungs may be affected. TMA may be caused by medical procedures and / or conditions, including but not limited to hematopoietic stem cell transplantation (HSCT), kidney disease, diabetes and / or other conditions. TMA may be caused by underlying complement system dysfunction, as described by Meri et al. in European Journal of Internal Medicine, 2013, 24: 496-502, the contents of the above references are incorporated herein by reference in their entirety. In general, TMA may be caused by increased levels of certain complement components that lead to thrombosis. In some cases, this may be caused by mutations in complement proteins or related enzymes. The resulting complement dysfunction can result in complement targeting to endothelial cells and platelets, leading to increased thrombosis. In some embodiments, TMA can be prevented and / or treated with the compounds and compositions of the present invention. In some cases, methods for treating TMA using the compounds and compositions of the present invention can be performed according to those described in U.S. Publication Nos. US2012 / 0225056 or US2013 / 0246083, the contents of each of which are incorporated herein by reference in their entirety.

[0280] Disseminated intravascular coagulation (DIC)

[0281] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat disseminated intravascular coagulation (DIC) by controlling complement activation. DIC is a pathological condition in which the coagulation cascade in the blood is widely activated and causes blood clot formation, especially in capillaries. DIC can cause tissue blood flow obstruction and can eventually damage organs. In addition, DIC affects the normal process of coagulation and can cause severe bleeding. The compounds and compositions of the present invention can be used to treat, prevent or reduce the severity of DIC by regulating complement activity. In some cases, the compounds and compositions of the present invention can be used according to any of the DIC treatment methods taught in U.S. Patent No. 8,652,477, the contents of which are incorporated herein by reference in their entirety.

[0282] Vasculitis

[0283] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat vasculitis. In general, vasculitis is a condition associated with inflammation of blood vessels, including veins and arteries, characterized by white blood cells attacking tissues and causing blood vessel swelling. Vasculitis can be associated with infections such as Rocky Mountain spotted fever or autoimmunity. An example of an autoimmune-related vasculitis is anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. ANCA vasculitis is caused by abnormal antibodies that attack the body's own cells and tissues. ANCA attacks the cytoplasm of certain white blood cells and neutrophils, causing these cells to attack the walls of blood vessels in certain organs and tissues of the body. ANCA vasculitis can affect the skin, lungs, eyes, and / or kidneys. Studies have shown that ANCA disease activates the alternative complement pathway and produces certain complement components that cause an inflammatory amplification loop that leads to vascular damage (Jennette et al. 2013, Semin Nephrol. 33(6): 557-64, the contents of which are incorporated herein by reference in their entirety). In some cases, the compounds and compositions of the invention may be used to prevent and / or treat ANCA vasculitis by inhibiting complement activation.

[0284] Neurological Indications

[0285] The compounds and compositions of the present invention can be used to prevent, treat and / or alleviate the symptoms of neurological indications including, but not limited to, neurodegenerative diseases and related conditions. Neurodegeneration is generally associated with loss of structure or function of neurons, including neuronal death. These conditions can be treated by inhibiting the effects of complement on neuronal cells using the compounds and compositions of the present invention. Neurodegeneration-related conditions include, but are not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson's disease, and Alzheimer's disease.

[0286] Amyotrophic lateral sclerosis (ALS)

[0287] In some embodiments, the compounds and compositions of the present invention can be used to prevent, treat and / or alleviate the symptoms of ALS. ALS is a fatal motor neuron disease characterized by degeneration of spinal cord, brainstem and motor cortical neurons. ALS causes loss of muscle strength, ultimately leading to respiratory failure. Complement dysfunction can contribute to ALS, and therefore can be prevented, treated and / or reduced symptoms by using the therapy of the compounds and compositions of the present invention targeting complement activity. In some cases, the compounds and compositions of the present invention can be used to promote neural regeneration. In some cases, the compounds and compositions of the present invention can be used as any one of the complement inhibitors according to the methods taught in U.S. Publication Nos. US2014 / 0234275 or US2010 / 0143344, and the contents of each of the above publications are incorporated herein by reference in their entirety.

[0288] Alzheimer's disease

[0289] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat Alzheimer's disease by controlling complement activity. Alzheimer's disease is a chronic neurodegenerative disease with symptoms including disorientation, memory loss, mood swings, behavioral problems, and eventual loss of physical function. Alzheimer's disease is believed to be caused by extracellular brain deposition of amyloid, which is associated with inflammation-related proteins such as complement proteins (Sjoberg et al. 2009. Trends in Immunology. 30 (2): 83-90, the contents of which are incorporated herein by reference in their entirety). In some cases, the compounds and compositions of the present invention can be used as complement inhibitors according to any of the methods taught in U.S. Publication No. US2014 / 0234275, the contents of which are incorporated herein by reference in their entirety.

[0290] Kidney-related indications

[0291] The compounds and compositions of the present invention can be used to treat certain diseases, disorders and / or conditions associated with the kidney by inhibiting complement activity in some cases. The kidney is an organ responsible for removing metabolic waste products from the bloodstream. The kidney regulates blood pressure, urinary system and homeostasis functions, and is therefore necessary for various body functions. Due to unique structural features and exposure to blood, the kidney can be more severely affected by inflammation (compared to other organs). The kidney also produces autologous complement proteins that can be activated during infection, renal disease and renal transplantation. In some cases, the compounds and compositions of the present invention can be used as complement inhibitors in the treatment of certain renal diseases, conditions and / or disorders according to the methods taught by Quigg, J Immunol 2003; 171: 3319-24, and the contents of the above references are incorporated herein by reference in their entirety.

[0292] Lupus nephritis

[0293] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat lupus nephritis by inhibiting complement activity. Lupus nephritis is a renal inflammation caused by an autoimmune disease called systemic lupus erythematosus (SLE). Symptoms of lupus nephritis include high blood pressure; foamy urine; swelling of the legs, feet, hands, or face; joint pain; muscle pain; fever; and rash. Lupus nephritis can be treated by inhibitors that control complement activity including the compounds and compositions of the present invention. Methods and compositions for preventing and / or treating lupus nephritis by complement inhibition may include any of those taught in U.S. Publication No. US2013 / 0345257 or U.S. Patent No. 8,377,437, and the contents of each of the above publications or patents are incorporated herein by reference in their entirety.

[0294] Membranous Glomerulonephritis (MGN)

[0295] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat membranous glomerulonephritis (MGN) by inhibiting the activation of certain complement components. MGN is a kidney disease that can cause inflammation and structural changes. MGN is caused by antibodies binding to soluble antigens in the renal capillaries (glomeruli). MGN can affect renal function such as filtering fluid and can lead to renal failure. The compounds and compositions of the present invention can be used according to the methods for preventing and / or treating MGN by complement inhibition as taught in U.S. Publication No. US2010 / 0015139 or International Publication No. WO2000 / 021559, and the contents of each of the above publications are incorporated herein by reference in their entirety.

[0296] Complications of hemodialysis

[0297] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat complications associated with hemodialysis by inhibiting complement activation. Hemodialysis is a medical procedure used to maintain renal function in subjects with renal failure. In hemodialysis, the removal of waste products such as creatinine, urea, and free water from the blood is performed externally. A common complication of hemodialysis treatment is chronic inflammation caused by contact between the blood and the dialysis membrane. Another common complication is thrombosis, which refers to the formation of blood clots that block blood circulation. Studies have shown that these complications are associated with complement activation. Hemodialysis can be combined with complement inhibitor therapy to provide a way to control inflammatory responses and pathology and / or prevent or treat thrombosis in subjects undergoing hemodialysis due to renal failure. Methods of using the compounds and compositions of the present invention to treat hemodialysis complications can be carried out according to any of the methods taught by DeAngelis et al. in Immunobiology, 2012, 217(11): 1097-1105 or by Kourtzelis et al. Blood, 2010, 116(4): 631-639, the contents of each of which are incorporated herein by reference in their entirety.

[0298] Eye diseases

[0299] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat certain eye-related diseases, disorders and / or conditions. In healthy eyes, the complement system is activated at low levels and is continuously regulated by membrane-bound and soluble intraocular proteins that protect against pathogens. Therefore, complement activation plays an important role in several complications associated with the eye, and controlling complement activation can be used to treat such diseases. The compounds and compositions of the present invention can be used as complement inhibitors in the treatment of eye diseases according to any of the methods taught by Jha et al. in Mol Immunol. 2007; 44 (16): 3901-3908 or in U.S. Pat. No. 8,753,625, the contents of each of which are incorporated herein by reference in their entirety.

[0300] Age-related macular degeneration (AMD)

[0301] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat age-related macular degeneration (AMD) by inhibiting complement activation in the eye. AMD is a chronic eye disease that causes blurred central vision, blind spots in central vision, and / or eventual loss of central vision. Central vision affects the ability to read, drive a vehicle, and / or recognize faces. AMD is generally divided into two types, non-exudative (dry) and exudative (wet). Dry AMD refers to the deterioration of the spots, which are tissues in the center of the retina. Wet AMD refers to the failure of blood vessels under the retina, resulting in blood and fluid leakage. Several human and animal studies have identified complement proteins associated with AMD, and new treatment strategies include controlling complement activation pathways, as discussed by Jha et al. in Mol Immunol. 2007; 44 (16): 3901-8. Methods of the invention involving the use of the compounds and compositions of the invention to prevent and / or treat AMD may include any of those taught in US Publication Nos. US2011 / 0269807 or US2008 / 0269318, the contents of each of which are incorporated herein by reference in their entirety.

[0302] Corneal diseases

[0303] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat corneal diseases by inhibiting ocular complement activation. The complement system plays an important role in protecting the cornea from pathogenic particles and / or inflammatory antigens. The cornea is the outermost anterior portion of the eye that covers and protects the iris, pupil, and anterior chamber, and is therefore exposed to external factors. Corneal diseases include, but are not limited to: keratoconus, keratitis, ocular herpes, and / or other diseases. Corneal complications can cause pain, blurred vision, tearing, redness, photosensitivity, and / or corneal scarring. The complement system is critical for corneal protection, but complement activation can cause damage to corneal tissue after the infection is cleared due to the large expression of certain complement compounds. The methods of the present invention for regulating complement activity in the treatment of corneal diseases may include any of those taught by Jha et al. in Mol Immunol. 2007; 44 (16): 3901-8, the contents of which are incorporated herein by reference in their entirety.

[0304] Autoimmune uveitis

[0305] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat uveitis, which is an inflammation of the uveal layer of the eye. The uvea is the pigmented area of ​​the eye that contains the choroid, iris, and ciliary body of the eye. Uveitis causes redness, blurred vision, pain, iris adhesions, and can ultimately cause blindness. Studies have shown that complement activation products are present in the eyes of patients with autoimmune uveitis and that complement plays an important role in the development of the disease. In some cases, the compounds and compositions of the present invention can be used to treat and / or prevent uveitis according to any of the methods identified in Jha et al., Mol Immunol. 2007. 44(16): 3901-8, the contents of which are incorporated herein by reference in their entirety.

[0306] Diabetic retinopathy

[0307] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat diabetic retinopathy, a disease caused by changes in the retinal blood vessels in diabetic patients. Retinopathy can cause blood vessel swelling and fluid leakage and / or abnormal blood vessel growth. Diabetic retinopathy affects vision and can ultimately lead to blindness. Studies have shown that complement activation plays an important role in the development of diabetic retinopathy. In some cases, the compounds and compositions of the present invention can be used according to the methods for treating diabetic retinopathy described in Jha et al. Mol Immunol. 2007; 44(16): 3901-8, the above reference is incorporated herein by reference in its entirety.

[0308] Preeclampsia and HELLP syndrome

[0309] In some embodiments, the compounds and compositions of the present invention can be used to prevent and / or treat pre-eclampsia and / or HELLP (an abbreviation for the syndrome features of 1) hemolysis, 2) elevated liver enzymes, and 3) low platelet count) syndrome by complement inhibitor therapy. Pre-eclampsia is a pregnancy disorder with symptoms including elevated blood pressure, swelling, shortness of breath, renal dysfunction, impaired liver function, and / or low platelet count. Pre-eclampsia is usually diagnosed by high urine protein levels and high blood pressure. HELLP syndrome is a combination of hemolysis, elevated liver enzymes, and low platelet conditions. Hemolysis is a disease involving the rupture of red blood cells, resulting in the release of hemoglobin from the red blood cells. Elevated liver enzymes can indicate pregnancy-induced liver conditions. Low platelet levels lead to reduced clotting ability, creating a risk of excessive bleeding. HELLP is associated with pre-eclampsia and liver disorders. HELLP syndrome usually occurs during the later stages of pregnancy or after delivery. It is usually diagnosed by a blood test that indicates the presence of the three conditions it involves. HELLP is usually treated by induced delivery.

[0310] Studies have shown that complement activation occurs during HELLP syndrome and pre-eclampsia, and that certain complement components are present at elevated levels during HELLP and pre-eclampsia. Complement inhibitors can be used as therapeutic agents to prevent and / or treat these conditions. The compounds and compositions of the present invention can be used according to the methods for preventing and / or treating HELLP and pre-eclampsia as taught by Heager et al. in Obstetrics & Gynecology, 1992, 79(1): 19-26 or International Publication No. WO201 / 078622, the contents of each of which are incorporated herein by reference in their entirety.

[0311] Dosage and Administration

[0312] For use as a treatment for human subjects, the polypeptide can be formulated as a pharmaceutical composition. Depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or treatment), the polypeptide is formulated in a manner consistent with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, J. Swarbrick and JC Boylan, eds., 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0313] The compositions of the present invention are preferably provided in a therapeutically effective amount, which can be, for example, about 0.1 mg to about 100 mg, about 0.5 mg to about 200 mg, about 1 mg to about 300 mg, about 5 mg to about 500 mg, about 10 mg to about 750 mg, about 50 mg to about 1000 mg, or at least 1000 mg per day. In one embodiment, the pharmaceutical composition comprises a capsule, for example, in unit dosage form.

[0314] Unit dosage form

[0315] The polypeptide of the present invention can be present in an amount of 0.1% by weight to 95% by weight of the total weight of the composition. The composition can be provided in a dosage form suitable for oral administration. Therefore, the pharmaceutical composition can be in the form of, for example, a hard capsule (e.g., a hard gelatin capsule or a hard hydroxypropyl methylcellulose capsule), a soft gelatin capsule, a tablet, a capsule, an enteric-coated tablet, a chewable tablet, an enteric-coated hard gelatin capsule, an enteric-coated soft gelatin capsule, a capsule, a lozenge, a film, a strip, a capsule, a dragee, a solution, an emulsion, a suspension, a syrup, or a spray.

[0316] The subject may be administered a therapeutic amount of the polypeptide, such as 0.01 mg / kg, 1.0 mg / kg or 15 mg / kg. For administration to a human subject, the dosage of the polypeptide of the invention is typically 0.01 mg / kg to 15 mg / kg, more preferably 3 mg / kg to 5 mg / kg. However, the dosage level may be highly dependent on the nature of the condition, the efficacy of the drug, the patient's condition, the doctor's judgment, and the frequency and mode of administration.

[0317] In other embodiments, polypeptide is administered at a frequency such as every 4 hr, every 6 hr, every 12 hr, every 18 hr, every 24 hr, every 36 hr, every 72 hr, every 84 hr, every 96 hr, every 5 days, every 7 days, every 10 days, every 14 days, every 3 weeks, or longer. Compositions can be administered once daily, or polypeptide can be administered or delivered via controlled release formulations as two, three or more sub-doses at appropriate intervals throughout a day. In that case, the polypeptide contained in each sub-dosage must be correspondingly smaller in order to achieve a total daily dose. Dosage units can also be compounded to deliver within a few days, for example, using conventional sustained release formulations that provide polypeptide sustained release within a few days.

[0318] Sustained release preparations are well known in the art, and are particularly useful for delivering medicaments to specific sites, such as can be used together with peptide compositions of the present invention. The effect of a single dose can be long-acting, so that subsequent doses are used at intervals of no more than 3 days, 4 days or 5 days or at intervals of no more than 1 week, 2 weeks, 3 weeks or 4 weeks.

[0319] Polypeptide can be used by intravenous infusion in a period of time, such as in 5 minutes, 10 minutes, 15 minutes, 20 minutes or 25 minutes period of time.Use can be repeated for example on a conventional basis, such as once every two weeks (that is, every two weeks) for one month, two months, three months, four months or longer.After the initial treatment scheme, treatment can be used on a less frequent basis.For example, after once every two weeks for three months, use can be repeated once a month, for six months or one year or longer.The use of polypeptide or composition can, for example, reduce, decrease, increase or change at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more in combination or any physiological harmful process in the patient's cell, tissue, blood, urine or other compartment.

[0320] Before using a full dose of polypeptide and / or polypeptide composition, the patient can use a smaller dose, such as 5% of the full dose, and monitor the patient's adverse effects such as allergic reactions or infusion reactions or the lipid levels or blood pressure that rise. In another example, the patient's unwanted immunostimulatory effects, such as elevated cytokine (e.g., TNF-α, Il-1, Il-6 or Il-10) levels can be monitored.

[0321] Genetic predisposition plays a role in the development of some diseases or conditions. Therefore, patients in need of polypeptides and / or polypeptide compositions can be identified by obtaining a family history or, for example, screening for one or more genetic markers or variants. A health care provider such as a doctor, nurse, or family member can obtain a family history before prescribing or administering the therapeutic composition of the present invention.

[0322] Reagent test kit

[0323] Any of the compositions described herein may be included in a kit. In a non-limiting example, a polypeptide may be included in a kit to treat a disease. The kit may include a sterile bottle, a dry polypeptide powder, a sterile solution for dissolving the dry powder, and a syringe for an infusion device for administering the polypeptide.

[0324] When the polypeptide is provided as a dry powder, it is contemplated that between 10 micrograms and 1000 milligrams of the polypeptide, or at least or at most those amounts, will be provided in the kits of the invention.

[0325] The container means will generally include at least one vial, test tube, flask, bottle, syringe and / or other container means, in which the polypeptide formulation is placed, preferably suitably dispensed. The kit may also include a second container means to hold a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0326] The kit may include instructions for utilizing the kit components and for using any other reagents not included in the kit. The instructions may include variations that may be implemented.

[0327] While various embodiments of the present invention have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the invention as defined by the following claims.

[0328] Equivalents and scope

[0329] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth as in the appended claims.

[0330] In the claims, articles such as "a", "an", and "the" may mean one or more than one, unless indicated to the contrary or otherwise apparent from the context. Claims or descriptions that include "or" between one or more members of a group are considered to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise apparent from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0331] Also note that the term "comprising" is intended to be open ended and allows but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting of" and "or including" are also encompassed and disclosed thereby.

[0332] In the case of a given range, the endpoints are included. In addition, it will be understood that unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, the values ​​expressed as ranges may take any specific value or local range within the range specified in different embodiments of the present invention, up to the tenth unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0333] In addition, it will be understood that any specific embodiment of the present invention that is a prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not explicitly stated herein. Regardless of whether it is related to the existence of prior art, any specific embodiment of the composition of the present invention (e.g., any nucleic acid or protein encoded thereby; any production method; any method of use, etc.) may be excluded from any one or more claims for any reason.

[0334] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated into this application by reference, even if not specifically stated in the reference. In the event of a conflict between a cited source and the statements of this application, the statements in this application shall prevail.

[0335] Section and table headings are not intended to be limiting. Example

[0336] Example 1. Preparation of biotinylated C5

[0337] C5 to biotin with an effective final molar ratio of 1:4 was used for large-scale biotinylation. A 10mM solution of EZ-Link Sulfo-NHS-LC biotin (Thermo Scientific, Billerica, MA) was prepared according to the manufacturer's instructions. 2.1 μl of 10mM biotin solution was added to 1mg of 1mg / ml C5 (Complement Tech, Tyler TX) and incubated on ice for 2 hours. After adding 100 μl of 1M Tris HCl pH 7.5, the reaction was quenched at 4°C for 30 minutes. The reaction was dialyzed overnight against cold PBST (phosphate buffered saline (PBS) + 0.1% Tween 80). Biotinylated C5 was aliquoted and stored at -80°C. Biotinylated C5 was characterized by SDS-PAGE under reducing and non-reducing conditions and activity was characterized by a red blood cell hemolysis test. The recovery of biotinylated C5 was also checked on streptavidin microbeads (Invitrogen, Grand Island, NY). Capture was performed using the conditions recommended by the manufacturer. To capture 4 μg of biotinylated C5 from a 100 nM solution, 40 μl of bead slurry was used and incubated for 1 hour at 4° C. The concentration of captured biotinylated C5 was calculated by running known amounts of C5 on NuPage 4-12% Bis-Tris gels (Invitrogen, Grand Island, NY).

[0338] Example 2. Human hemolysis assay for biotinylated C5 QC

[0339] Hemolytic assays were performed using C5-depleted serum and biotinylated C5 and non-biotinylated C5 to compare the lytic activity of C5 before and after biotinylation. 8 Antibody-sensitized sheep erythrocytes (Complement Technology, Tyler TX) in a 100 μl solution of 10 cells / ml were centrifuged at 2,090× gravity for 3 min and resuspended in GVB++ buffer (Complement Technology, Tyler TX). C5-depleted human serum (Complement Technology, Tyler TX) was rapidly thawed at 37°C and placed on ice until diluted in GVB++. Non-biotinylated C5 protein (Complement Technology, Tyler TX) and biotinylated C5 protein (in house biotinylated) were rapidly thawed at 37°C and placed on a wet ice slurry until diluted in GVB++. 100 μl cells (final concentration 2.5×10 7 Cells / ml) were combined with C5-depleted human serum and 50 μl of biotinylated C5 or non-biotinylated C5 (with a final concentration of 10 μg / ml, 3 μg / ml or 1 μg / ml) in a tissue culture treated clear 96-well microtiter plate (USA Scientific, Ocala, FL). The plate was incubated at 37°C for 1 hour. After incubation, the plate was then centrifuged at 2,090× gravity for 2 minutes, after which 100 μl of the supernatant was transferred to a new microtiter plate. The absorbance was read at 412 nm, and the percentage of lytic activity of non-biotinylated C5 and biotinylated C5 was compared.

[0340] Example 3. Selection of polypeptides that bind to C5

[0341] C5 inhibitors were identified via several rounds of mRNA display and selection. mRNA display was generally performed as described (Roberts, RW and Szostak, JW (1997). Proc. Natl. Acad. Sci. USA 94, 12297-12302; WO2009067191; incorporated herein by reference in its entirety) with modifications as described herein. RNA populations were generated by in vitro transcription from DNA synthesized with fixed N-terminal methionine and cysteine ​​codons, followed by three positions of a sixteen-codon phosphoramidite mixture, followed by eight positions of a second codon mixture also containing cysteine ​​codons. The resulting mRNA library had a fixed start methionine, followed by a cysteine ​​residue, followed by three positions lacking cysteine, followed by eight positions where cysteine ​​appeared at a frequency of 12.5%. For selection, the first round of enrichment included a first step where the RNA population containing a 3' end UV cross-linked, puromycin-containing oligonucleotide was in vitro translated with purified translation components listed in Table 2. Translation was performed under two independent conditions to generate two unique libraries based on amino acid changes. The first condition utilized only the 20 natural amino acids, while the second condition utilized natural amino acids (0.1 mM histidine, threonine, proline, lysine, asparagine, tyrosine, glutamate, and cysteine), unnatural amino acids (2 mM tert-butylglycine (Tbg), 0.8 mM 7-azatryptophan (abbreviated as "azaTrp" in this example), and 1 mM norvaline (Nvl), azaleucine, and phenylglycine (Phg)), and N-methyl amino acids (a 450 μM mixture of N-methylated serine [(N-Me)S], alanine [(N-Me)A], glycine [(N-Me)G], and 4-fluoro-N-methylphenylalanine [(N-Me-4-F)Phe]). 35 S-labeled cysteine ​​residues were included in both conditions to allow monitoring of polypeptide enrichment at each round.

[0342] Table 2. In vitro translation components

[0343]

[0344]

[0345] tRNA is enzymatically loaded with its corresponding amino acid using tRNA synthetase.Four N-methyl tRNAs are preloaded, while all other tRNAs are enzymatically loaded during in vitro translation reactions.tRNA synthetase is added on a volume basis, without considering its concentration.Add 0.1 μl of each tRNA synthetase (except methionine tRNA synthetase, which adds 0.4 μl per 25 μl translation reaction) to load in situ during translation for 25 μl translation reactions.Add cross-linked mRNA at 0.75 μM final concentration.Keep the translation reaction at 37 °C for 1 hour.After translation, the fusion of the translated polypeptide and its corresponding mRNA is carried out by adding high salt to the translation mixture and incubating at 37 °C for 1.5 hours.The library selected for natural polypeptides is prepared from eight separate libraries with fixed cysteine ​​codons in position 5-11. Random positions in these libraries with all 20 amino acids possible were prepared combinatorially using repeating codon units of NNS (N is A, G, C or T; S is G or C) (Devlin, JJ et al., (1990). Science 249, 404-406). Translation of these libraries into native polypeptides was performed using a rabbit reticulocyte in vitro translation kit rather than the above-mentioned recombinant system.

[0346] The recovery of mRNA display polypeptides was performed using both oligo dT and Ni-NTA affinity to isolate fusion molecules containing both poly A mRNA and His-tagged polypeptides. The polypeptides bound to oligo dT beads were then cyclized using dibromoxylene as described by others (J. Am. Chem. Soc. 127: 1 1727 (2005)).

[0347] Then direct selection of polypeptides was performed by target affinity. The mRNA display polypeptides were bound to biotinylated C5 at 4°C for 1 hour in a PBST solution of 100nM biotinylated C5. The RNA corresponding to the affinity selected polypeptides was reverse transcribed and PCR amplified to produce a double-stranded DNA population. The DNA population was transcribed in vitro to produce mRNA, and the produced mRNA was cross-linked at its 3' end with a puromycin-containing oligonucleotide as before. The mRNA-puromycin fusion was translated in vitro to produce a second round of libraries, which are now enriched for polypeptides that bind complement component C5. The selection cycle was repeated for six rounds. After the sixth round, the DNA population representing the selected polypeptides was cloned and sequenced, and the amino acid sequence of the candidate C5 inhibitor was determined based on the DNA sequence. The identified polypeptide sequences are listed in Table 3.

[0348] As used in all of the following tables and in the sequence listing, the abbreviations have the following meanings: "Ac" and "NH2" refer to acetyl and amidated termini, respectively; "Nvl" represents norvaline; "Phg" represents phenylglycine; "Sar" represents sarcosine; "Tbg" represents tert-butylglycine; "Trt" represents trityl or triphenylmethyl; "Chg" represents cyclohexylglycine; "(N-Me)X" represents the N-methylated form of an amino acid, indicated by the letter or three-letter abbreviation for the amino acid in place of the variable "X", written as (N-Me)X. as)N-methyl-X [e.g., (N-Me)A and (N-Me)Ala both represent the N-methylated form of alanine or N-methylalanine]; where "azaTrp" is indicated, 7-azatryptophan is incorporated; "(4-F)Phe" represents 4-fluorophenylalanine; "Tyr(OMe)" represents O-methyltyrosine; "Aib" represents aminoisobutyric acid; "(homo)F" or "(homo)Phe" represents homophenylalanine; "(2-OMe)Phg" refers to 2-O-methylphenylglycine; "Pro pargylGly (propargylGly)" refers to propargylglycine; "(5-F)W" or "(5-F)Trp" refers to 5-fluorotryptophan; "DX" refers to the D-stereoisomer of a given amino acid "X", where the amino acid may be abbreviated using a single-letter or three-letter code [e.g., (D-Chg) represents D-cyclohexylglycine, and (DW) represents D-tryptophan]; "(5-MeO)W" or "(5-MeO)Trp" refers to 5-methyl-O-tryptophan; "homoC" refers to homocysteine; " "(1-Me-W)" or "(1-Me)W" or "(1-Me-Trp)" or "(1-Me)Trp" refers to 1-methyltryptophan; "Nle" refers to norleucine; where "Tiq" is indicated, 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is incorporated; "Asp(T)" refers to (S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid; "(3-Cl-Phe)" refers to 3-chlorophenylalanine; "[(N-Me-4-F)Phe]" or "(N-Me-4-F)Ph "e" refers to N-methyl-4-fluorophenylalanine; "Boc" is a tert-butyloxyhydroxy protecting group; "[xXylyl(y,z)]" refers to a xylyl bridging moiety between two cysteines, where x can be m, p, or o to indicate the use of meta-, para-, or ortho-dibromoxylene (respectively) to generate the bridging moiety, and the numerical identifiers y and z locate the amino acid positions within the polypeptide having the cysteines participating in the cyclization; "[ring(y,z)]" refers to the formation of a bond between two residues, where the numerical identifiers y and z locate the positions of the residues participating in the bond;"[mXylyl-bicyclo]([m-xylyl-bicyclo])" refers to a polypeptide containing two cyclic rings and the bridging moiety is generated by reaction with m-dibromoxylene. All other symbols refer to the standard single letter amino acid code. In addition, polypeptides containing PEG2000 or BODIPY-TMR-X sequence tags are indicated. ;

[0349] Table 3. Peptide sequences

[0350]

[0351] Example 4. Peptide Synthesis

[0352] The peptides were synthesized using a standard solid phase Fmoc / tBu method. The synthesis was usually performed on a Liberty automatic microwave peptide synthesizer (CEM, Matthews NC) using a standard procedure using Rink amide resin, but other automatic synthesizers without microwave capabilities may also be used. Unless otherwise indicated, all amino acids were obtained from commercial sources. The coupling reagent used was 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3,-tetramethylammonium hexafluorophosphate (HCTU), and the base was diisopropylethylamine (DIEA). The peptide was cleaved from the resin for 3 hours with 95% TFA, 2.5% TIS, and 2.5% water, and separated by precipitation with ether. The crude peptide was purified in 30 min using a C18 column on a reverse phase preparative HPLC with a 20%-50% acetonitrile / water 0.1% TFA gradient. The fractions containing the pure peptide were collected and lyophilized, and all peptides were analyzed by LC-MS.

[0353] Example 5. Formation of disulfide cyclized polypeptides

[0354] To generate disulfide cyclized polypeptides, the linear polypeptide was dissolved in a mixture of water and DMSO, and the resulting solution was vigorously stirred for 12 hours under an air atmosphere.

[0355] Example 6. Cyclization of dibromoxylene polypeptide

[0356] A 100 mL flask was loaded with acetonitrile (12 mL) and water (24 mL) and degassed with argon for about 5 min. Linear peptide (0.01 mmol) and 200 mM ammonium bicarbonate (6 mL) were added, followed by 0.012 mmol or 1,3-bis(bromomethyl)benzene, 1,2-bis(bromomethyl)benzene, 1,4-bis(bromomethyl)benzene, 2,6-bis(bromomethyl)pyridine or (E)-1,4-dibromobut-2-ene. The reaction mixture was stirred at room temperature under argon for about 2 hours and monitored by LC-MS. After the reaction was complete, the reaction solution was frozen and lyophilized. HPLC purification of the crude lyophilized product followed by lyophilization of the portion containing the pure peptide produced the final cyclized product as a white powder.

[0357] Example 7. Lactam polypeptide cyclization

[0358] The polypeptide cyclization using the lactam moiety was performed in the solid phase. The polypeptides were first synthesized by standard Fmoc chemistry on a solid supported Wang resin. Fmoc-ASP (allyl) -OH and Fmoc-LYS (allyloxycarbonyl) -OH were incorporated into the polypeptide at the indicated positions as two precursor monomers for lactam bridge formation. After complete extension, the resin was washed with dry dichloromethane (3×) and purged with dry nitrogen for 10 min. To remove the allyl and allyloxycarbonyl protecting groups, the resin was treated with a 5-fold molar excess of phenylsilane and purged with nitrogen for 10 min. A catalytic amount of tetra-Pd(0) was dissolved in dry dichloromethane and added to the resin suspension. After one hour, the resin was washed sequentially with dichloromethane (3×), dimethylformamide (3×), sodium diethyldithiocarbamate trihydrate (3×), dimethylformamide (3×), and dichloromethane (3×). Lactam cyclization was achieved in dimethylformamide (DMF) by treating the resin containing the deprotected polypeptide with PyAOP ((3-hydroxy-3H-1,2,3-triazolo[4,5-b]pyridine-O)tri-1-pyrrolidinyl-phosphorus hexafluorophosphate) and diisopropylethylamine and allowing the reaction to proceed overnight. The resin was rinsed with DMF and treated with fresh PyAOP and diisopropylethylamine at 45°C for an additional 60 minutes. The resin was rinsed and washed five times with dimethylformamide. The polypeptide was cleaved and purified as described in Example 4.

[0359] Example 8. Triazole polypeptide cyclization

[0360] Cyclization of polypeptides containing azide and alkyne moieties was performed on a solid phase. The resin containing the polypeptide was treated with dichloromethane (0.05 mmol) and allowed to swell for 10 min. The solvent was then exchanged to DMF (3-5 mL) and a Cu-TBTA ligand solution (125 μL of a 20 mM solution) was added after 10 min. The suspension was purged with argon and ascorbic acid (5 μ moles) was then added. The solution was shaken for 2 h and excess reagents were removed, and the resin was washed with a DMF solution of EDTA to remove excess copper. The polypeptide was cleaved and purified as described in Example 4.

[0361] Example 9. Polypeptides of the present invention

[0362] The polypeptides of the invention were synthesized. These include the compounds listed in Table 4.

[0363] Table 4. Compounds of the present invention

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379] Polypeptides R3183 (SEQ ID NO: 184) and R3193 (SEQ ID NO: 194) were synthesized according to the amino acid sequence of R3176 (SEQ ID NO: 177), except that Nvl-NH2 was replaced by Lys. The side chain amine group of the Lys residue was modified with different lipophilic moieties to generate lipidated polypeptides.

[0380] Example 10. Optimization and testing of C5 inhibitors

[0381] The polypeptides selected according to Example 3 and listed in Table 3 were tested for their ability to inhibit complement-mediated cell lysis. In addition, a variety of optimized polypeptides were synthesized according to the methods of Examples 4-8 and tested (see Table 4). The optimized polypeptide sequences include those obtained by various truncations, deletions, additions and / or substitutions of compounds R3002 (SEQ ID NO: 3), R3008 (SEQ ID NO: 9) and R3021 (SEQ ID NO: 11) or by forming a hybrid polypeptide comprising a combination of regions selected from any of the above three.

[0382] Human Hemolysis Test (RBC Lysis Assay Using Whole Human Serum)

[0383] The inhibitory activity of the peptides listed in Table 3 and their optimized derivatives (see Table 4) was evaluated using a red blood cell hemolysis assay. Antibody-sensitized sheep red blood cells (Complement Technology, Tyler, TX) were lysed with whole human serum (Complement Technology, Tyler, TX) and the peptides at 2.5 × 10 7 10 cells / well were plated to determine the inhibitory effect of the peptides on red blood cell lysis. The cells were centrifuged at 2,090× gravity for 3 minutes and resuspended in fresh GVB++ buffer (Complement Technology, Tyler TX). Human serum was rapidly thawed at 37°C and then stored on ice until diluted in GVB++. Ten 6-fold serial dilutions of the peptide (10 mM stock, DMSO) were made in DMSO and then added to the buffer. 50 μl of each peptide dilution was combined with serum and 100 μl of cells in a single well of a 96-well tissue culture treated clear microtiter plate (USA Scientific, Ocala, FL) and resuspended by pipetting. The samples were incubated at 37°C for one hour. After incubation, the plate was centrifuged at 2,090× gravity for 2 minutes. 100 μl of the supernatant was transferred to a new plate and the absorbance was read at 412 nm. The data were fit using the log-logit formula to generate dose response curves and IC 50 As used herein, the term “IC 50 ” refers to the half maximal inhibitory concentration, which is a value used to indicate the amount of inhibitor required to reduce a given reaction or process by half. The tested compounds are listed in Table 5.

[0384] Table 5. Compounds analyzed

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391] Example 11. Alternative Human Hemolytic Assay Using C5-Depleted Serum

[0392] Human C5-depleted serum and purified human C5 were used instead of intact human serum to test the functional activity of the polypeptides listed in Table 6 in the red blood cell hemolysis assay. To evaluate activity, antibody-sensitized sheep red blood cells (Complement Technology, Tyler, TX) were plated with 1.5% human C5-depleted serum (Complement Technology, Tyler, TX) and 0.5 nM purified human C5 (Complement Technology, Tyler, TX) to 2.5 × 10 7 Cells / well. Antibody-sensitized sheep erythrocytes were centrifuged at 2,090× gravity for 3 minutes and then resuspended in fresh GVB++ (Complement Technology, Tyler, TX). Human C5-depleted serum and purified human C5 were rapidly thawed at 37°C and then stored on ice or wet ice. The peptide stock solution (10 mM, DMSO) was serially diluted in DMSO to obtain 10 6-fold dilutions and GVB++ was then added to them. 50 μl of each peptide dilution was combined with 25 μl C5-depleted serum, 25 μl purified human C5 and 100 μl cells in a single well of a 96-well tissue culture treated clear microtiter plate (USA Scientific, Ocala, FL) and resuspended by pipetting. The samples were incubated at 37°C for one hour. At the completion of the incubation, the plate was centrifuged at 2,090× gravity for 2 minutes. 100 μl of supernatant was transferred to a new plate and the absorbance was read at 412 nm. The data were fitted using the log-logit equation to generate dose-response curves and IC 50 .

[0393] Table 6. Compounds analyzed

[0394]

[0395]

[0396] Example 12. Enzyme immunoassay for evaluating C5 inhibition

[0397] C5 inhibitory activity was evaluated by enzyme immunoassay (EIA). Inhibition of C5a and membrane attack complex (MAC) production was measured using the MicroVue EIA kit (Quidel Corporation, San Diego, CA).

[0398] C5a EIA

[0399] Supernatants from human RBC hemolysis assays of R3002 (SEQ ID NO: 3) and R3008 (SEQ ID NO: 9) were diluted 1:50 and assayed by C5a EIA ( Figure 1). Both peptides inhibited C5a formation. R3002 (SEQ ID NO: 3) had an IC of 5.4 nM 50 , R3008 (SEQ ID NO: 9) had an IC of 54.5 nM 50 .

[0400] Membrane attack complex (MAC) EIA

[0401] MAC EIA was performed on diluted supernatant (1:5) of R3008 (SEQ ID NO: 9) from human RBC hemolysis assay ( Figure 2 ). This peptide was shown to inhibit the formation of MAC with an IC of 33 nM 50 .

[0402] Example 13. Characterization of Peptide Mimetic Binding by Fluorescence Polarization

[0403] Fluorescence polarization (FP) allows the measurement of binding events in homogeneous solutions (Banks, P. et al., Impact of ared-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors. J Biomol Screen. 2000 Oct; 5(5): 329-34 and Parker, GJ et al., Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase / phosphatase assays. J Biomol Screen. 2000 Apr; 5(2): 77-88). The key concept of FP is that the degree to which a fluorophore polarizes light is inversely related to its molecular rotation (Lea, WA et al., Fluorescence polarization assays in smallmolecule screening. Expert Opin Drug Discov. 2011 Jan; 6(1): 17-32), and that a fluorophore bound to a much larger target protein rotates more slowly than an unbound fluorophore, resulting in a quantifiable increase in polarization. FP has been used in high-throughput activities as a tool to measure ligand-target binding (Parker, GJ et al., Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase / phosphatase assays. J Biomol Screen. 2000 April; 5(2): 77-88), for equilibrium dissociation constants (K D) assay (Prystay, L. et al., Determination of equilibrium dissociation constants in fluorescence polarization. J Biomol Screen. 2001 Jun;6(3):141-50) was increasingly used and led to the discovery of a competitive binding assay (Tian, ​​W. et al., Development of novel fluorescence polarization-based assay for studying the β-catenin / Tcf4 interaction. J Biomol Screen. 2012 Apr;17(4):530-4).

[0404] Materials and methods

[0405] FP was used to screen for competitive peptide inhibitors of C5 protein. Probe R3076 (SEQ ID NO: 40) was generated by incubating the parent peptide R3072 (SEQ ID NO: 20) with BODIPY-TMR-X, SE (Life Technologies, Grand Island, NY) in DMF (Sigma, Saint Louis, MO) for 4 hours. BODIPY-TMR dye was attached to the C-terminal lysine of the protein, and the subsequently labeled probe was purified by HPLC.

[0406] The equilibrium dissociation constant (K) of R3076 (SEQ ID NO: 40) binding to human C5 protein (Complement Technology, Tyler, TX) was determined by incubating 25 nM R3076 (SEQ ID NO: 40) solutions with increasing concentrations of C5 protein. D Polarization was measured over time until binding reached equilibrium. K was determined using Graphpad Prism (using "Saturation binding curve, single site specific binding with Hill slope" as curve fit). D ) to determine K D After 10 minutes, equilibrium is reached, where K D The values ​​of the ramp slope and the maximum binding remained stable within 60 min. The K D The final K value was determined to be 8.07 nM DValue (standard deviation 0.53). Based on this information, 25nM and 50nM concentrations of R3076 (SEQ ID NO: 40) and C5 protein were selected for competition assays, respectively. These concentrations represent the approximate protein levels necessary for 95% probe binding to C5 protein. R3023 (SEQ ID NO: 104) is a scrambled polypeptide variant of R3002 (SEQ ID NO: 3) and was included in all assays as a negative control.

[0407] Human C5 protein was diluted to 200 nM in assay buffer consisting of TBS (EMD Millipore, Billerica, MA) + 0.005% Triton-X (Sigma, Saint Louis, MO). 10 μl of assay buffer was added to all wells of a black non-binding 384-well assay plate (Greiner, Monroe, NC), and 10 μl of diluted C5 protein stock was added to the experimental wells and designated control wells.

[0408] Probe R3076 (SEQ ID NO: 40) was diluted 1 to 10 in DMSO (Life Technologies, Grand Island, NY), and 30 μl of this stock was diluted in 3 ml of assay buffer to produce a 100 nM stock. 10 μl of this working stock was then added to each well in the assay plate. The assay plate was incubated in the dark at room temperature for 20 minutes to allow binding to reach equilibrium.

[0409] The test articles listed in Table 7 were then diluted in DMSO, then assay buffer containing 10 2-fold dilutions, and then rapidly added to the assay plate in triplicate. The assay plate was then incubated at 25°C for 60 minutes in a Paradigm (Molecular Devices, Sunnyvale, CA) plate reader.

[0410] After 60 minutes of incubation, the plates were read using the Paradigm FP protocol (Molecular Devices, Sunnyvale, CA) and the raw polarization values ​​were imported into Graphpad Prism. K was determined in Graphpad i (Using unit point K i Curve fitting model, probe concentration = 25 nM, K D = 8.07 nM, with baseline limited to the mean of 0% binding controls) and IC 50 (Logarithm of inhibitor ratio response, 4 parameter curve fit).

[0411] result

[0412] All test products were able to compete with the labeled probe for binding to human C5 protein ( Figure 3 , Table 7). R3003 (SEQ ID NO: 4) was the most potent polypeptide tested, with a K of 9.54 nM. i No binding of R3023 (SEQ ID NO: 104) was detected at the highest concentration tested.

[0413] Table 7. Competitive fluorescence polarization data

[0414]

[0415] The data shown in Table 7 were obtained from curve fitting analysis performed by Graphpad Prism software as described above. Three replicates were averaged to generate the data points presented in each experiment. Among the polypeptides tested, R3003 (SEQ ID NO: 4) was initially identified by mRNA display selection. The affinity of R3003 (SEQ ID NO: 4) for C5 was determined by displaying a low K i IC 50 The inhibitors R3011 (SEQ ID NO: 31) and R3050 (SEQ ID NO: 23) also showed relatively strong affinity for C5. The control polypeptide R3023 (SEQ ID NO: 104) did not show affinity for C5, while the inhibitors R3014 (SEQ ID NO: 55) and R3043 (SEQ ID NO: 50) showed weak affinity.

[0416] Example 14. Analysis of compound stability in plasma

[0417] The stability of the compounds in human plasma was determined under the following conditions. Human plasma was obtained from Bioreclamation (Westbury, NY) and collected in sodium heparin. The plasma was adjusted to pH 7.4. A 10 mM DMSO stock solution was prepared for the test compound. An aliquot of the DMSO solution was added to 1 mL of plasma pre-warmed to 37°C to a final concentration of 10 μM of the test compound. The vials were kept on the benchtop for the duration of the experiment. (Eppendorf, Hauppauge, NY). Aliquots (100 μL) were taken out at each time point and added to a 96-well plate pre-filled with 300 μL of acetonitrile solution containing a mixture of internal standards (metoprolol, propranolol and warfarin, 500 ng / mL each). The samples were stored at 4°C until the end of the experiment. After sampling at the last time point, the plate was mixed and then centrifuged at 3,000 rpm for 10 minutes. Aliquots of the supernatant were removed and analyzed by LC-HRAMS. The liquid chromatography settings are listed in Table 8, and the mass spectrometry settings are listed in Table 9.

[0418] Table 8. Liquid chromatography settings

[0419]

[0420]

[0421] Table 9. Mass spectrometry settings

[0422]

[0423] The concentration of the test compound R3050 (SEQ ID NO: 23) was determined by comparison with a previously determined calibration curve (Table 10).

[0424] Table 10. Stability distribution

[0425]

[0426] Under these conditions, R3050 (SEQ ID NO: 23) was shown to be highly stable.

[0427] Example 15. Polypeptide variants comprising tryptophan analogs

[0428] In some embodiments, the polypeptides of the invention comprise 7-azatryptophan. To determine the importance of this residue in C5 inhibition, amino acid substitution analyses were performed in which 7-azatryptophan was replaced with natural tryptophan as well as various other tryptophan analogs including 5-fluorotryptophan [(5-F)W], 1-methyltryptophan [(1-Me)W], D-tryptophan, and 5-methyl-O-tryptophan [(5-MeO)W]. Similar polypeptides with non-tryptophan substitutions were also analyzed.

[0429] Polypeptide variants of R3002 (SEQ ID NO: 3) and R3008 (SEQ ID NO: 9) were synthesized and tested for their ability to inhibit red blood cell lysis as described in Example 10 (see Tables 11 and 12). Of the variants tested, all variants with 7-azatryptophan residue substitutions exhibited increased average IC 50The reduced ability to inhibit red blood cell lysis is indicated by the half-maximal inhibitory concentration (a measure of the half-maximal inhibitory concentration, which is used to indicate the amount of inhibitor required to reduce a given reaction or process by half).

[0430] Table 11. 7-azatryptophan variant polypeptides of R3002 (SEQ ID NO: 3) analyzed by human hemolysis assay

[0431]

[0432]

[0433] Table 12. 7-azatryptophan variant polypeptides of R3008 (SEQ ID NO: 9) analyzed by human hemolysis assay

[0434]

[0435]

[0436] Example 16. Analysis of the effects of polypeptide truncation and amino acid deletion by human hemolysis assay

[0437] C-terminally truncated polypeptide variants of R3021 (SEQ ID NO: 11) were synthesized and tested for their ability to inhibit C5-dependent erythrocyte lysis by the human hemolysis assay as described in Example 10. The average IC 50 The values ​​(a measure of half-maximal inhibitory concentration, which indicates the amount of inhibitor required to reduce a given reaction or process by half) are listed in Table 13. Truncated polypeptides exhibited reduced ability to inhibit red blood cell lysis (e.g., IC 50 ), with those variants lacking tryptophan having the largest IC 50 value.

[0438] In addition, polypeptide variants of R3021 (SEQ ID NO: 11) with internal amino acid deletions were synthesized and tested for their ability to inhibit C5-dependent red blood cell lysis according to the method described in Example 10 (see Table 14). In addition, the N-terminal methionine in these variants was replaced by an acetyl group. The average IC of each tested polypeptide was 50 The values ​​are listed in Table 14. Interestingly, replacement of the N-terminal methionine [R3048 (SEQ ID NO: 19)] with an acetyl group alone increased the ability of the polypeptide to inhibit red blood cell lysis. Removal of internal residue D [R3124 (SEQ ID NO: 130)] or internal residue DVY [R3125 (SEQ ID NO: 131), corresponding to residues 8, 9 and 10 of R3021 (SEQ ID NO: 11)] resulted in a decrease in the ability of the polypeptide to inhibit red blood cell lysis.

[0439] Table 13. C-terminally truncated polypeptide variants of R3021 (SEQ ID NO: 11) analyzed by human hemolysis assay

[0440]

[0441]

[0442] Table 14. Polypeptide variants of R3021 (SEQ ID NO: 11) with internal amino acid deletions analyzed by human hemolytic assay

[0443]

[0444] Example 17. Introduction of albumin binding polypeptide

[0445] The polypeptide is conjugated to one or more polypeptides that modulate plasma protein binding. These polypeptides, referred to herein as "albumin binding polypeptides," are listed in Table 15.

[0446] Table 15. Albumin binding peptides

[0447]

[0448]

[0449] The albumin binding polypeptide is cyclized via disulfide bond formation at a cysteine ​​residue. In some embodiments, the albumin binding polypeptide is bound via its N-terminus or C-terminus and thus has a slightly different structure (eg, no acetyl group).

[0450] Example 18. Introduction of cell-penetrating polypeptides

[0451] The polypeptides are conjugated to polypeptides having cell penetrating properties. These polypeptides are listed in Table 16 and described in Milletti, F., Cell-penetrating polypeptides: classes, origin, and current landscape. Drug Discov Today. 2012 Aug; 17(15-16): 850-60.

[0452] Table 16. Cell penetrating peptides

[0453] Cell Penetrating Peptides SEQ ID NO RKKRRRESRKKRRRES 205 RKKRRQRRR 206 RQIKIWFQNRRMKWKK 207 AAVLLPVLLAAP 208 VPTLK 209 PLILLRLLRGQF 210

[0454] Example 19. Analysis of a polypeptide mixture containing amino acid stereoisomers

[0455] Polypeptides R3136 (SEQ ID NO: 137) and R3137 (SEQ ID NO: 138) were synthesized based on the amino acid sequences of R3085 (SEQ ID NO: 90) and R3082 (SEQ ID NO: 116), respectively, except that D-Phg was used to replace Phg in each polypeptide (see Table 17). The ability of compositions comprising R3136 (SEQ ID NO: 137) and R3085 (SEQ ID NO: 90) or R3137 (SEQ ID NO: 138) and R3082 (SEQ ID NO: 116) to inhibit red blood cell lysis was analyzed according to the human hemolysis assay described in Example 10. Compositions comprising R3136 (SEQ ID NO: 137) and R3085 (SEQ ID NO: 90) produced an average IC of >50,000. 50 (nM), while the composition comprising R3137 (SEQ ID NO: 138) and R3082 (SEQ ID NO: 116) produced an average IC of >100,000 50 (nM).

[0456] Table 17. Compounds used in amino acid stereoisomer peptide mixtures

[0457]

[0458] Example 20. Pharmacokinetic studies in non-human primates

[0459] Pharmacokinetic studies were conducted in non-human primates using the compounds listed in Table 18. In the table, "Cmpd" refers to compound, and "Avg" refers to average.

[0460] Table 18. Compounds tested in in vivo studies

[0461]

[0462] The plasma concentration of the polypeptide R3152 (SEQ ID NO: 153) was determined in cynomolgus monkeys after a single intravenous (IV) administration. Three male animals received 3 mg / kg of the polypeptide and the plasma concentration of the polypeptide was determined using LC / MS-MS after acetonitrile precipitation and extraction on Sirocco protein precipitation plates (Waters Corporation, Milford, MA.). The time course of the combined plasma concentration of R3152 (SEQ ID NO: 153) (see Figure 4) Pharmacokinetic (PK) parameters were calculated from immediately after administration until 48h after administration. Plasma drug levels dropped rapidly during the initial distribution phase (<1 hour) and then reached a plateau, and could be detected continuously until 48 hours. R3152 (SEQ ID NO: 153) has an average terminal half-life of 10.9±0.8 hours. The average clearance was 0.129±0.0122 L / hr / kg, which is approximately 5% of the liver blood flow (2.6 L / hr / kg) of a typical monkey. The average distribution volume was 1.49±0.152 L / kg, which is approximately twice the total body water (0.7 L / kg) of a typical monkey. The average AUC∞ was 23319±2120 hr*ng / mL.

[0463] R3152 (SEQ ID NO: 153) binds to the primate C5 protein with high affinity and blocks the complement pathway by preventing the production of C5a and C5b products and the generation of multimeric membrane attack complexes (MACs). Inhibition of complement-mediated MAC formation in plasma samples from the above PK studies was examined using an established ex vivo assay (see the human hemolysis assay described in Example 10) where the plasma was diluted 1:100 and incubated with activated sheep red blood cells (Complement Technology, Tyler, TX). At each time point, hemolytic activity was measured as an indicator of active serum complement (see Figure 4 In plasma containing >200 ng / mL R3152 (SEQ ID NO: 153), there was a significant inhibition of complement-mediated hemolysis, indicating blockade of MAC formation. Exogenous R3152 (SEQ ID NO: 153) added to normal cynomolgus monkey plasma had an IC 50 =2-20 ng / mL. Hemolytic activity returned to normal levels 48 hours after administration as the plasma level of R3152 (SEQ ID NO: 153) dropped to less than 100 ng / mL.

[0464] Example 21. Pharmacokinetic studies in rats

[0465] R3152 (SEQ ID NO: 153) was delivered to male rats at 2 mg / kg and 30 mg / kg, either intravenously (IV) or subcutaneously (SC). Following IV administration, R3152 (SEQ ID NO: 153) was monitored by using LC / MS-MS after acetonitrile precipitation as described above and extraction on Sirocco protein precipitation plates (Waters Corporation, Milford, MA). The combined plasma concentration time course of R3152 (SEQ ID NO: 153) and its equivalent C-terminal deamidated metabolite R3201 (SEQ ID NO: 211) was obtained (see Figure 5) to calculate pharmacokinetic (PK) parameters.

[0466] R3152 (SEQ ID NO: 153) / R3201 (SEQ ID NO: 211) exhibited a fast distribution phase followed by a phase with t 1 / 2 = Slow clearance of 5.3 hours. Similar clearance rates were observed after SC dosing at 30 mg / kg, with approximately 65% ​​dosing bioavailability based on AUC. The 4-hour T seen with SC dosing max and prolonged drug exposure allows for an extended coverage of therapeutic concentrations in plasma. Since R3152 (SEQ ID NO: 153) and R3201 (SEQ ID NO: 211) do not bind to rat C5, little inhibitory activity was observed in the ex vivo hemolytic assay.

[0467] The pharmacokinetic properties of lipidated and non-lipidated compounds R3183 (SEQ ID NO: 184) and R3176 (SEQ ID NO: 177) were evaluated in male Sprague-Dawley rats following intravenous or subcutaneous administration, respectively. Figure 6 The results are shown. Figure 6 The left panel shows male Sprague-Dawley rats (n=3) injected intravenously with a single 2 mg / kg dose. Blood samples were collected at the indicated time points, processed into plasma, and analyzed by LC-MS for the indicated compounds. Black circles: R3176 (SEQ ID NO: 177) (non-lipidated compound); open circles: R3183 (SEQ ID NO: 184) (C16 lipidated compound). Figure 6 The right panel shows male Sprague-Dawley rats (n=3) injected subcutaneously with a single 15 mg / kg dose. Blood samples were collected at the indicated time points, processed into plasma, and analyzed by LC-MS for the indicated compounds. Black circles: R3176 (SEQ ID NO: 177) (non-lipidated compound); open circles: R3183 (SEQ ID NO: 184) (C16 lipidated compound). Lipidation resulted in increased exposure, as assessed by measuring the area under the curve (AUC) 2.1-fold by the intravenous route and 2.7-fold by the subcutaneous route.

[0468] Example 22. Inhibition of hemolysis in the thrombin-inducible complement pathway

[0469] Thrombin can cleave C5 into C5 T To induce complement activity, C5 T It is subsequently cleaved into C5a and C5b T .C5b TLike C5b, it will bind to C6 and the remaining terminal components of the complement pathway, C7, C8, and C9, which will lead to the formation of the membrane attack complex (MAC) that causes erythrocyte lysis (Krisinger et al., (2014). Blood. 120(8): 1717-1725). Therefore, R3183 and C5b were tested via the thrombin-inducible complement pathway. Similar anti-C5 monoclonal antibodies have the ability to inhibit hemolysis.

[0470] To evaluate inhibitory activity, C5 (Complement Technology, Tyler, TX) was added to reach a concentration of 400 nM and in the presence of R3183 or Samples were incubated at 37°C for 30 minutes with C6 (Complement Technology, Tyler, TX) at a final concentration of 600 nM and thrombin (Enzyme Research Laborites, South Bend, IN) at a concentration of 50 nM in the presence of similar anti-C5 monoclonal antibodies or in the absence of inhibitors. The reaction was stopped with hirudin (CellSciences, Canton, MA) added to 150 nM in GVB+EDTA buffer (Complement Technology, Tyler, TX) and incubated at room temperature for 5 minutes. These diluted samples were mixed with antibody-sensitized sheep erythrocytes (Complement Technology, Tyler, TX) in a 96-well microtiter plate (USA Scientific, Ocala, FL) and incubated at 37°C for 5 minutes. C7 (Complement Technology, Tyler, TX) was then added to the wells to reach a concentration of 15 nM, and the plates were returned to 37°C for 15 minutes. A complex of C8 (10 nM; Complement Technology, Tyler, TX) and C9 (25 nM; Complement Technology, Tyler, TX) was then added to the assay mixture, and the samples were incubated at 37°C for 30 minutes. After incubation, the plate was centrifuged at 1000×g, and 100 μl of the supernatant was transferred to a new microtiter plate and the absorbance was read at 412 nm. The resulting data are shown in Figure 7 R3183 was found to inhibit hemolysis via the thrombin-inducible complement pathway at concentrations above 6 ng / mL, whereas anti-C5 monoclonal antibodies did not.

Claims

1. A composition comprising compound R3191 or a variant thereof, wherein compound R3191 consists of SEQ ID NO: 192 or a variant thereof, wherein the variant consists of a sequence having at least 70% identity with SEQ ID NO:

192.

2. A composition as claimed in claim 1, wherein compound R3191 or a variant thereof is modified by adding one or more conjugate groups.

3. The composition of claim 2, wherein compound R3191 or a variant thereof is bound to a water soluble polymer.

4. The composition of claim 3, wherein the water-soluble polymer is a hydrophilic polymer selected from the group consisting of polyalkylene oxide homopolymers, polypropylene glycol, polyethylene glycol and polyoxyethylated polyols.

5. The composition of claim 3, wherein the water-soluble polymer is a lipid moiety.

6. The composition of claim 5, wherein the lipid portion is selected from the group consisting of fatty acids, phospholipids and sterols.

7. The composition of claim 5 or 6, wherein the lipid portion: a) directly binds to compound R3191 or a variant thereof; or b) linked to compound R3191 or a variant thereof via polyethylene glycol.

8. The composition of claim 2, wherein compound R3191 or a variant thereof is bound to a cholesterol oleate moiety, a cholesterol laurate moiety, an α-tocopherol moiety, a phytol moiety, an oleate moiety, an unsaturated cholesterol ester moiety, or a lipophilic compound.

9. The composition of claim 8, wherein the lipophilic compound is selected from the group consisting of acetanilide, anilide, aminoquinoline, benzhydryl compound, benzodiazepine, benzofuran, cannabinoid, cyclic polypeptide, dibenzazepine, digitalis glycoside, ergot alkaloid, flavonoid, imidazole, quinoline, macrolide, naphthalene, opiate, oxazine, oxazole, phenylalkylamine, piperidine, polycyclic aromatic hydrocarbon, pyrrolidine, pyrrolidone, stilbene, sulfonylurea, sulfone, triazole, tropane and vinca alkaloid.

10. The composition of any one of claims 1 to 9, wherein the variant of compound R3191 consists of a sequence having at least 93% identity to SEQ ID NO:

192.

11. The composition of any one of claims 2 to 9, wherein the composition comprises compound R3191.

12. A combination comprising: a) compound R3191 or a sequence having at least 70% identity to SEQ ID NO: 192, wherein compound R3191 consists of SEQ ID NO: 192; and b) Conjugate group.

13. Use of the composition according to any one of claims 1 to 11 or the conjugate according to claim 12 in the preparation of a medicament.

14. Use of the composition according to any one of claims 1 to 11 or the conjugate according to claim 12 in the preparation of a medicament for reducing complement-mediated cell lysis by preventing the cleavage of C5 into C5a and C5b fragments.

15. Use of the composition according to any one of claims 1 to 11 or the conjugate according to claim 12 in the preparation of a medicament for treating a complement-related disorder.

16. The use of claim 15, wherein the complement-related disorder is paroxysmal nocturnal hemoglobinuria, myasthenia gravis, vasculitis, pre-eclampsia or HELLP syndrome.

17. The use according to any one of claims 13 to 16, wherein administration to the subject is selected from the group consisting of buccal, intranasal, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal and intravitreal.

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