Dosing regimens, and related compositions and methods

Abstract

To provide a complement inhibitor and a method of using the same.SOLUTION: In some aspects, the present invention provides cell-reactive compstatin analogs and compositions comprising cell-reactive compstatin analogs. In some aspects, the invention further provides methods of using cell-reactive compstatin analogs, e.g., treating a complement-mediated disorder, e.g., to inhibit complement-mediated damage to a cell, tissue, or organ. In some aspects, the invention provides long-acting compstatin analogs and compositions comprising long-acting compstatin analogs. In some aspects, the invention further provides methods of using long-acting compstatin analogs, e.g., to treat a complement-mediated disorder, e.g., to inhibit complement-mediated damage to a cell, tissue, or organ.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 483,295, filed April 7, 2017, and U.S. Provisional Patent Application No. 62 / 485,343, filed April 13, 2017, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Complement is a system of over 30 plasma- and cell-bound proteins that plays a significant role in both innate and adaptive immunity. The proteins of the complement system act in a series of enzyme cascades through diverse protein interactions and cleavage events. Complement activation occurs via three major pathways: the antibody-dependent classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway. Inappropriate or excessive complement activation is an underlying cause or contributing factor to many serious diseases and conditions, and considerable efforts have been made in the past few decades to explore various complement inhibitors as therapeutic agents. Summary of the Invention

[0003] Summary of the Invention In certain embodiments, the present invention provides and / or relates to certain long-acting compstatin analogs. For example, the present invention provides and / or relates to such long-acting compstatin analogs, compositions comprising them, and methods for making, identifying, characterizing, and / or using them. In certain embodiments, the present invention provides and / or relates to physiologically acceptable compositions comprising long-acting compstatin analogs. In certain embodiments, the present invention provides and / or relates to pharmaceutical-grade compositions comprising long-acting compstatin analogs. In particular, in certain embodiments, the present disclosure describes particularly useful long-acting compstatin analogs, and further provides specific doses, administration formats, dosing regimens, unit dose compositions, and other technologies for administering long-acting compstatin analogs to human subjects, e.g., certain human subjects having and / or susceptible to one or more certain diseases, disorders, or conditions.

[0004] In certain embodiments, the present invention provides methods of treating a complement-mediated disorder in a subject in need of treatment, which may include administering to the subject a long-acting compstatin analog using a particular dose, administration format (e.g., a unit dose composition and / or a particular formulation), and / or administration regimen (determined as desired for the treatment of a particular disease, disorder, or condition, and in certain embodiments, e.g., route of administration, timing of administration, etc.).

[0005] In some embodiments, the complement-mediated disorder treated according to the present invention is paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), or other disorders associated with complement-mediated hemolysis. In some embodiments, the disorder is ischemia / reperfusion (I / R) injury (e.g., myocardial infarction, thromboembolic stroke, or surgery-related). In some embodiments, the disorder is trauma. In some embodiments, the disorder is transplant rejection. In some embodiments, the disorder is a chronic respiratory disorder, such as asthma or COPD.

[0006] All references, books, patent applications, patents, other publications, websites, and databases mentioned herein are incorporated herein by reference. In the event of a conflict between the statements herein and any of the incorporated materials, the statements herein (including any amendments thereto) shall control. Unless otherwise specified, art-recognized meanings of terms and abbreviations are used herein. The practice of certain embodiments described herein may employ conventional techniques of molecular biology, cell culture, recombinant nucleic acid (e.g., DNA) technology, immunology, and / or nucleic acid and polypeptide synthesis, detection, manipulation, and quantification, which are within the ordinary skill of the art. See, for example, Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, NY, e.g., the current edition as of January 2010 or any successor edition; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001 or 4 th See ed., 2012. [Brief explanation of the drawings]

[0007] [Figure 1]1 is a plot showing the percent inhibition of complement activation for compstatin analog CA28 (SEQ ID NO: 28) and three long-acting compstatin analogs (CA28-1, CA28-2, CA28-3) as a function of peptide concentration (μM). Inhibition of complement activation was tested in vitro using a classical complement inhibition assay. The plot shows values obtained by averaging the results of duplicate determinations. CA28 (circles; red), CA28-1 (crosses; blue); CA28-2 (triangles, green), CA28-3 (squares, purple).

[0008] [Figure 2] Figure 1 shows a plot of the percent inhibition of complement activation for CA28 and the long-acting compstatin analogs CA28-2 and CA28-3 as a function of compound concentration (µM). CA28 (squares, light gray), CA28-2 (diamonds, black), and CA28-3 (circles, dark gray). CA28-3 is a compound containing multiple peptide moieties. While the activity per peptide moiety is lower than that of individual CA28 molecules, the total activity of CA28-3 exceeds that of CA28 on a molar basis.

[0009] [Figure 3] Figure 1 shows a plot of plasma concentration versus time for CA28 and the long-acting compstatin analogs CA28-2 and CA28-3 after a single intravenous injection in cynomolgus monkeys. CA28 was administered at 200 mg / kg. CA28-2 and CA28-3 were each administered at 50 mg / kg. For dose calculations for these experiments, the administered CA28-2 and CA28-3 materials were estimated to consist of 80 wt% active compound on a dry weight basis. However, during sample analysis, the standard curve was estimated to be 100 wt% compound on a dry weight basis, and was estimated at 30%. Therefore, the Cmax values are an overestimate of the actual Cmax. CA28 (squares, light gray), CA28-2 (triangles, black), and CA28-3 (circles, dark gray).

[0010] [Figure 4]1 is a plot showing the percent inhibition of complement activation for CA28 and the long-acting compstatin analog CA28-4 as a function of compound concentration (μM). Inhibition of complement activation was tested using an in vitro classical complement inhibition assay. The plot shows values obtained by averaging the results of four determinations of CA28-4. CA28 (squares, light gray), CA28-4 (crosses, black).

[0011] [Figure 5] Figure 1 shows a plot of plasma concentration versus time for CA28 and the long-acting compstatin analogs CA28-2, CA28-3, and CA28-4 after a single intravenous injection in cynomolgus monkeys. CA28 was administered at 200 mg / kg. CA28-2, CA28-3, and CA28-4 were each administered at 50 mg / kg. For dose calculations for these experiments, the administered CA28-2 and CA28-3 materials were estimated to consist of 80 wt% active compound on a dry weight basis. However, during sample analysis, the standard curve was estimated to represent 100 wt% compound on a dry weight basis. Therefore, the Cmax values are estimated to be approximately 30% higher than the Cmax achieved when these compounds were administered at the indicated doses on a dry weight basis. CA28 (square, light gray), CA28-2 (triangle, black), CA28-3 (circle, dark gray), CA28-4 (inverted triangle, black).

[0012] [Figure 6] 1 is a representative chromatogram showing ultraviolet (UV) detection of a PEG-based long-acting compstatin analog using reverse-phase HPLC. The peak with a retention time (RT) of 33.68 minutes represents the PEGylated compstatin analog and has a relative area of 96%.

[0013] [Figure 7]1 is a plot showing the percent complement activation inhibitory activity of CA28 and long-acting compstatin analogs, CA28-2CS, CA28-2GS, CA28-2HS, and CA28-2TS, as a function of compound concentration (μM): CA28-2CS (diamonds, red); CA28-2GS (crosses, blue); CA28-2HS (triangles, green); CA28-2TS (squares, black).

[0014] [Figure 8] 1 is a plot showing the percent complement activation inhibitory activity of CA28 and the bifunctional long-acting compstatin analog CA28-2GS-BF as a function of compound concentration (μM). CA28 (open circles, blue); CA28-2G-SBF (filled circles, red).

[0015] [Figure 9] Figure 1 shows a plot of plasma concentration versus time for CA28 and the long-acting compstatin analog CA28-2GS-BF in cynomolgus monkeys after a single intravenous injection (CA28 (squares, red) and CA28-2GS-BF (circles, purple)) or once-daily subcutaneous injection for 7 days (CA28-2GS-BF only, asterisk, blue). CA28-2GS-BF was administered at 25 mg / ml. The dose volume was 2 ml / kg intravenously and 0.28 ml / kg / day subcutaneously. CA28 data was from a separate experiment in which the compound was also in 5% dextrose and formulated at 20 mg / ml in a 10 ml / kg dose volume. The vehicle in each example was an aqueous solution of 5% dextrose.

[0016] [Figure 10(A)] Plots showing the percent complement activation inhibitory activity of CA28 and the bifunctional long-acting compstatin analog, CA28-2TS-BF, as a function of compound concentration (μM). (A) Classical pathway inhibition by CA28 (circles, red) and CA28-2TS-BF (crosses, blue).

[0017] [Figure 10(B)](B) Plot showing the percent complement activation inhibitory activity of CA28 and the bifunctional long-acting compstatin analog, CA28-2TS-BF, as a function of compound concentration (μM). (C) Alternative pathway inhibition. CA28 (circles, red) and CA28-2TS-BF (crosses, blue).

[0018] [Figure 10(C)] The structure of CA28-2TS-BF (assuming the PEG moiety is 40 kD) is shown.

[0019] [Figure 11] 1 is a plot showing the plasma concentration versus time of CA28 and the long-acting compstatin analog CA28-2TS-BF in cynomolgus monkeys after a single intravenous injection of CA28 at 200 mg / kg (squares, red), a single intravenous injection of CA28-2TS-BF at 7 mg / kg (asterisk, purple), a single subcutaneous injection of CA28-2TS-BF at 7 mg / kg (circles, blue), or 7 consecutive daily subcutaneous injections of CA28-2TS-BF at 7 mg / kg (inverted triangles, green). The vehicle in each example is 5% dextrose in water.

[0020] [Figure 12(A)] Figure 1 shows flow cytometry analysis of C3 deposition on red blood cells from patients with PNH exposed to activated complement in a modified Ham test.Figure 2 shows the results of a dilution experiment demonstrating the effect of CA28 on C3 deposition.

[0021] [Figure 12(B)] 1 shows flow cytometry analysis of C3 deposition on red blood cells from patients with PNH exposed to activated complement in a modified Ham test. Results of a dilution experiment showing the effect of CA28-2GS-BF on C3 deposition are shown. Compound concentrations used are indicated on and above each panel.

[0022] [Figure 13]Flow cytometric analysis of C3 deposition on red blood cells from patients with PNH exposed to activated complement in a modified Ham test in the absence of complement inhibitors (left panel), in the presence of the anti-C5 monoclonal antibody eculizumab (center panel), and in the presence of CA28-2GS-BF (right panel).

[0023] [Figure 14] 1 shows a plot of ex vivo serum-induced hemolysis observed in a multiple ascending dose clinical trial of a long-acting compstatin analog containing 40 kD PEG in healthy subjects.

[0024] [Figure 15] A schematic diagram of the target-mediated drug disposition (TMDD) model is shown.

[0025] [Figure 16] Figure 1 shows observed and predicted trough LACA-40 serum concentrations over the baseline time period after dosing, separated by dosing regimen, for both single-dose (SD) and 28-day multiple-dose (MD, once daily) studies in healthy subjects. Black circles represent the mean PK concentration at each baseline time point, overlaid with the TMDD model prediction, represented by a solid line. Each dosing regimen is represented by a different color. LACA-40 was administered subcutaneously.

[0026] [Figure 17] Figure 1 shows predicted trough PK serum concentrations over time following multiple once-daily (Q1D), subcutaneous (SC) administration of LACA-40 at three dose levels: 180 mg, 270 mg, and 360 mg Q1D. Model predictions were based on the TMDD model.

[0027] [Figure 18] Figure 1 shows predicted trough PK serum concentrations over time following multiple once-daily (Q1D) and three times-weekly (Mon / Wed / Fri) SC administrations of various doses of LACA-40. Each dosing regimen is represented by a different colored line, and model predictions were based on the TMDD model.

[0028] [Figure 19] Figure 1 shows predicted trough PK serum concentrations after multiple once-daily (Q1D) and twice-weekly (Mon / Thurs) SC administrations of various doses of LACA-40. Each dosing regimen is represented by a different colored line, and model predictions were based on the TMDD model.

[0029] [Figure 20] Figure 1 shows predicted trough PK serum concentrations over time following multiple once-daily (Q1D) and once-weekly (Q1W) SC administrations of various doses of LACA-40. Each dosing regimen is represented by a different colored line, and model predictions were based on the TMDD model.

[0030] [Figure 21] Figure 1 shows predicted trough PK serum concentrations over time following multiple once-daily (Q1D) and once-weekly (Q1W) SC administrations of various doses of LACA-40. Each dosing regimen is represented by a different colored line, and model predictions were based on the TMDD model.

[0031] [Figure 22] Figure 1 shows predicted serum concentrations over time after multiple once-daily (qd), twice-weekly (biw), or once-weekly (qw) SC administrations of various doses of LACA-40 over a 28-day administration period, corresponding to the administration regimens for Cohorts 1, 2, and 3 in the clinical trial described in Example 30. Each administration regimen is represented by a different colored line, and model predictions were based on the TMDD model. For the twice-weekly regimen, the plot reflects administration at alternating 3-day and 4-day intervals (i.e., administration on days 1, 4, 8, 11, 15, 18, 22, and 25).

[0032] [Figure 23] 1 shows projections extended to 35 days for LACA-40 concentrations shown for daily and twice-weekly administration over a 28-day period.

[0033] [Figure 24]1 shows the individual and mean summary observed LACA-40 serum concentrations for Cohorts 1 and 2 in the clinical trial described in Example 30.

[0034] [Figure 25] 1 shows the individual and mean summary observed LACA-40 serum concentrations for Cohort 1 in the clinical trial described in Example 30, as well as the predicted serum concentrations for this dosing regimen based on the TMDD model.

[0035] [Figure 26] 1 shows the individual and mean summary observed LACA-40 serum concentrations for Cohort 2 in the clinical trial described in Example 30, as well as the predicted serum concentrations for this dosing regimen based on the TMDD model.

[0036] [Figure 27] 1 shows the mean summary observed LACA-40 serum concentrations and predicted serum concentrations for these dosing regimens based on the TMDD model for Cohorts 1 and 2 in the clinical trial described in Example 30. The plot further shows that the observed PK data are consistent with predictions from the TMDD model.

[0037] [Figure 28] 1 shows the mean summary observed LACA-40 serum concentrations for Cohorts 1 and 2 in the clinical trial described in Example 30, as well as serum concentrations for Cohort 3 based on the TMDD model. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description of Certain Embodiments of the Invention I. Definition The terms "approximately" or "about" in connection with a numerical value generally include numbers within ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.5% of that numerical value, unless otherwise specified or otherwise clear from the context (except when such numerical value impermissibly exceeds 100% of the possible numerical values).

[0039] "Complement components" or "complement proteins" (CRPs) are proteins involved in the activation of the complement system or mediate one or more complement-mediated activities. Components of the classical complement pathway include, for example, C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex (also known as the membrane attack complex (MAC)) and active fragments or enzymatic cleavage products of any of the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components of the alternative pathway include, for example, factor B, factor D, and properdin. Components of the lectin pathway include, for example, MBL2, MASP-1, and MASP-2. Complement components also include cell-bound receptors for soluble complement components, which mediate one or more of the biological activities of such soluble complement components following their binding. Such receptors include, for example, C5a receptor (C5aR), C3a receptor (C3aR), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3, also known as CD45), etc. It will be recognized that the term "complement component" is not intended to include molecules and molecular structures that act as "triggers" for complement activation, such as antigen-antibody complexes, foreign structures found on microbial or artificial surfaces, etc.

[0040] A "complement-mediated disorder" is any disorder in which complement activation is known or suspected to be a contributing factor and / or at least partially causative in at least some subjects having the disorder, e.g., a disorder in which complement activation leads to tissue damage. Non-limiting examples of complement-mediated disorders include, but are not limited to, (i) various disorders characterized by hemolysis or hemolytic anemia, such as atypical hemolytic uremic syndrome, warm antibody autoimmune hemolytic anemia, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, and transfusion reactions; (ii) graft rejection (e.g., hyperacute or acute graft rejection) or graft dysfunction; (iii) disorders involving ischemia / reperfusion injury, such as trauma, surgery (e.g., aneurysm repair), myocardial infarction, and ischemic stroke; (iv) disorders of the respiratory system, such as asthma and chronic obstructive pulmonary disease (COPD); (v) arthritis, such as rheumatoid arthritis; and (vi) ophthalmic disorders, such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, and uveitis. "Disorder" is used herein interchangeably with "disease," "condition," and similar terms to refer to any impaired health or abnormal functional state of an organism, e.g., any condition for which a medical and / or surgical response is indicated or for which the subject seeks appropriate medical and / or surgical treatment. It should also be understood that listing particular disorders within particular categories is for convenience and is not intended to limit the invention. It is understood that certain disorders are appropriately exemplified in multiple categories.

[0041] "Complement regulatory proteins" (CRPs) are proteins involved in regulating complement activity. Complement regulatory proteins can downregulate complement activity, for example, by inhibiting complement activation or by inactivating or accelerating the degradation of one or more activated complement proteins. Examples of complement regulatory proteins include C1 inhibitor, C4-binding protein, clusterin, vitronectin, CFH, factor I, and the cell-binding proteins CD46, CD55, CD59, CR1, CR2, and CR3.

[0042] As used herein, "isolated" means 1) separated from at least some components that normally accompany it in nature; 2) produced or purified by human intervention; and / or 3) not found in nature, e.g., present in an artificial environment. Generally, unless otherwise specified or clearly demonstrated, any object, product, drug, composition, etc., can be considered "isolated," as the case may be.

[0043] "Linked" as used herein in connection with two or more moieties means that the moieties are physically bound or linked to one another to form a molecular structure that is sufficiently stable so that the moieties remain linked under the conditions under which the bond is formed and, preferably, under the conditions under which the new molecular structure is utilized, e.g., physiological conditions. In certain preferred embodiments of the present invention, the bond is covalent. In other embodiments, the bond is non-covalent. Moieties can be linked directly or indirectly. When two moieties are directly linked, they are covalently bound to one another or are in sufficient proximity that intermolecular forces between the two moieties maintain their association. When two moieties are indirectly linked, they are covalently or non-covalently bound to a third moiety that maintains the association of the two moieties. In general, when two moieties are said to be linked by a "linking moiety" or "linking site," the bond between the two linked moieties is indirect, and typically each of the linked moieties is covalently bound to the linking moiety. Two moieties may be linked using a "linker." The linker may be any suitable moiety that reacts with the substance to be attached under conditions consistent with the stability of the substance (some of which may be appropriately protected), for a reasonable time, and in an amount sufficient to obtain a reasonable yield. Typically, the linker contains at least two functional groups, one of which reacts with the first substance and the other of which reacts with the second substance. It is recognized that after the linker reacts with the substance to be attached, the term "linker" may refer to the portion of the resulting structure derived from the linker, or at least the portion not contained in the reacted functional group. The attachment moiety may include a portion that does not participate in bonding with the substance to be attached, the primary purpose of which may be to spatially separate these substances from each other. Such a portion may be referred to as a "spacer."

[0044] As used herein, "physiological conditions" refers to a set of conditions, such as temperature, salt concentration, and pH, that at least partially mimic those typically found in a living subject, e.g., a mammalian subject. In some embodiments, physiological conditions refer to an aqueous medium, e.g., a medium containing at least 90%, 95%, 96%, 97%, 97%, 99%, or about 100% water on a volume / volume basis. In some embodiments, other fluids, if present, do not substantially affect protein secondary or tertiary structure. In some embodiments, physiological conditions at least partially mimic those found in bodily fluids, such as blood or extracellular fluid, e.g., interstitial fluid, of a mammalian subject. For example, a variety of physiological conditions useful for in vitro assays are known in the art. Generally, a medium under physiological conditions contains a physiological concentration of salt, e.g., sodium chloride. In some embodiments, physiological concentrations of salt refer to concentrations in the range of about 250 mOsm / L to about 350 mOsm / L, e.g., about 275 mOsm / L to about 325 mOsm / L, e.g., about 300 mOsm / L. In some embodiments, physiological conditions are approximately isotonic with a body fluid, e.g., blood or extracellular fluid, e.g., interstitial fluid. In some embodiments, physiological conditions include a pH in the range of about 6.5 to about 7.8, e.g., about 7.0 to about 7.5. In some embodiments, the physiological medium includes a buffer substance that helps maintain the pH of the medium within a physiological range. In some embodiments, physiological conditions include conditions under which typical mammalian proteins, e.g., proteins typically found in body fluids such as blood or extracellular fluid, substantially maintain the secondary and, if applicable, tertiary structure that they possess in the body fluids in which they are normally found. In some embodiments, the components of a physiological medium are typically substantially non-toxic to mammalian cells at the concentrations present in the physiological medium. A variety of physiological media (sometimes referred to as "buffers") are listed in various standard references, such as those cited above (e.g., Sambrook, et al., Protocols series). In some embodiments, physiological temperatures range from about 25°C to about 38°C, e.g., from about 30°C to about 37°C, e.g., from 35°C to 37°C.

[0045] As used herein, a "polypeptide" refers to a polymer of amino acids, which may optionally contain one or more amino acid analogs. A protein is a molecule composed of one or more polypeptides. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length, e.g., 8 to 40 amino acids in length. The terms "protein," "polypeptide," and "peptide" may be used interchangeably. As used herein, a polypeptide may include amino acids naturally found in proteins, amino acids not naturally found in proteins, and / or non-amino acid amino acid analogs. As used herein, an amino acid "analog" may be another amino acid structurally similar to an amino acid, or a compound other than an amino acid structurally similar to an amino acid. Many art-recognized analogs of the 20 amino acids commonly found in proteins (the "standard amino acids") are known. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of chemicals such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modification. Some non-limiting suitable analogs and modification methods are described in WO2004026328 and further discussed herein. Polypeptides may be, for example, acetylated at the N-terminus and amidated, for example, at the C-terminus.

[0046] As used herein, the term "purified" refers to a material that has been separated from at least some or most of the components that naturally accompany it, or that, if synthetically derived, accompany it prior to purification. Generally, such purification involves human intervention. A purified product may be partially purified, substantially purified, or pure. Such a product may be, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. In certain embodiments, a nucleic acid, polypeptide, or small molecule is purified to constitute at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the total nucleic acid, polypeptide, or small molecule material, respectively, present in the preparation. In some embodiments, organic substances, such as nucleic acids, polypeptides, or small molecules, are purified so that they constitute at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the total organic substances present in the preparation. Purity can be based, for example, on dry weight, peak size of chromatographic traces (GC, HPLC, etc.), molecular abundance, electrophoresis method, band intensity on a gel, spectral data (e.g., NMR), elemental analysis, high-throughput sequencing, mass spectrometry, or any art-accepted quantitative method. In some embodiments, water, buffer substances, ions, and / or small molecules (e.g., synthetic precursors such as nucleotides or amino acids) may optionally be present in the purified preparation. Purified drugs can be produced by separating them from other substances (e.g., other cellular substances) or by preparing them in a manner that achieves the desired degree of purity. In certain embodiments, "partially purified" with respect to a molecule produced by a cell means that the molecule produced by the cell is no longer present within the cell, e.g., the cell has been lysed and, optionally, at least some of the cellular material (e.g., cell wall, cell membrane, organelles) has been removed and / or the molecule has been separated or isolated from at least some of the same type of molecules (proteins, RNA, DNA, etc.) that were present in the lysate.

[0047] "Recombinant host cell," "host cell," and other such terms refer to a prokaryotic or eukaryotic cell or cell line that contains foreign nucleic acid (typically DNA), such as an expression vector containing a nucleic acid encoding a polypeptide of interest. Such terms are understood to include the progeny of the original cell into which the vector or other nucleic acid has been introduced. Suitable host cells include any cell routinely used in the art to express polynucleotides (e.g., for production of a polypeptide encoded by such polynucleotide), including prokaryotes such as Escherichia, other bacteria such as Lactobacillus, Bacillus (e.g., Bacillus subtilis), Salmonella, Pseudomonas, Streptomyces, Staphylococcus, etc., and eukaryotes, including fungi such as yeast (e.g., Pichia (e.g., Pichia pastoris), Kluyveromyces, e.g., Kluyveromyces lactis, Hansenula, e.g., Hansenula polymorpha). Other examples of fungal cells include cells of filamentous fungi, such as Aspergillus, Neurospora, Fusarium, or Trichoderma, e.g., strains of Aspergillus oryzae, Aspergillus nidulans, or Aspergillus niger; insect cells (e.g., Sf9); plant and animal cells, e.g., CHO, R1.1, BW, LM, African green monkey kidney cells (e.g., COS-1, COS-7, BSC-1, BSC-40, and BMT-10), and mammalian cells such as cultured human cells. Also included are genetically engineered cells in genetically engineered (e.g., transgenic) plants or animals, in which recombinant polypeptides are produced in at least some of such cells. The polypeptides may be secreted in milk or obtained from plant material. The exogenous nucleic acid may be stably maintained as an episome, such as a plasmid, or integrated, at least in part, into the host cell's genome, optionally after replication or reverse transcription. Terms such as "host cell" are also used to refer to cells or cell lines that can be used as recipients of exogenous nucleic acids prior to production of the nucleic acid. A "recombinant polynucleotide" is a polynucleotide that generally contains nucleic acid sequences that are not known to be directly linked to each other in nature.For example, the nucleic acid sequences may be present in different genes or different species, or one or more of the sequences may be variants of a naturally occurring sequence, or may be artificial sequences in which at least a portion is not homologous to a naturally occurring sequence. A "recombinant polypeptide" is generally a polypeptide that is produced, at least in part, by transcription and translation of a foreign nucleic acid by a recombinant host cell or a cell-free in vitro expression system and / or that contains amino acid sequences that are not known to be directly linked to each other in nature. In the latter case, the recombinant polypeptide may be referred to as a "chimeric polypeptide." The amino acid sequences of a chimeric polypeptide may, for example, be present, over a significant portion of its length, in different genes or different species, or one or more of the sequences may be variants of a naturally occurring sequence, or may be artificial sequences in which at least a portion is not identical to, or in some embodiments not homologous to, a naturally occurring sequence. It is understood that a chimeric polypeptide may comprise more than one polypeptide. For example, the first and second polypeptides A and B of a chimeric polypeptide may be directly linked (AB or BA) or separated by a third polypeptide moiety C (ACB or BCA). In some embodiments, the C portion represents a polypeptide linker, which may be, for example, multiple glycine and / or serine residues or various other amino acids. In some embodiments, two or more polypeptides may be linked by a non-polypeptide linker. As used herein, "recombinant" includes, in some embodiments, polypeptides produced by linking (e.g., chemical or enzymatic) short recombinant polypeptides that may be produced in a recombinant host cell. In some embodiments, a recombinant polypeptide may include a signal sequence that directs secretion of the polypeptide or a sequence that targets the polypeptide to a specific compartment or organelle. Suitable sequences are known in the art. An appropriate sequence may be selected for the desired host cell type (e.g., bacterial, fungal, mammalian, plant, etc.). In some embodiments, the signal sequence may be located at or near (e.g., within up to 10-50 amino acids) the N-terminus or C-terminus. In some embodiments, the polypeptide includes a tag. A tag may be useful for facilitating detection and / or purification of a protein containing it.Examples of tags include polyhistidine tags (e.g., 6X-His tags), glutathione-S-transferase, maltose-binding protein, NUS tags, SNUT tags, Strep tags, and epitope tags, such as V5, HA, Myc, or FLAG. In certain embodiments, a protease cleavage site is located in the region between the tag and the polypeptide, allowing the polypeptide to be separated from the tag by exposure to a protease. In certain embodiments, the polynucleotide encoding the recombinant polypeptide is at least partially codon-optimized for expression in a desired host cell (e.g., bacterial, fungal, mammalian, plant, etc.). The tag can be located at or near the N-terminus or C-terminus of the polypeptide (e.g., within up to 10-50 amino acids), in various arrangements. The recombinant polypeptide can be isolated, purified, etc., using any of a variety of methods. See, e.g., Sambrook, Protocols series, or other standard references. Methods of use may include, for example, dialysis (e.g., using membranes with defined pore sizes), chromatography, precipitation, gel purification, or affinity-based methods, which may, in certain embodiments, utilize specific binding agents such as tags or antibodies.

[0048] As used herein, "reactive functional group" refers to an olefin, acetylene, alcohol, phenol, ether, oxide, halide, aldehyde, ketone, carboxylic acid, ester, amide, cyanate, isocyanate, thiocyanate, isothiocyanate, amine, hydrazine, hydrazone, hydrazide, diazo, diazonium, nitro, nitrile, mercaptan, sulfide, disulfide, sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal, ketal, anhydride, sulfur, Examples of functional groups include, but are not limited to, esters, sulfenic acids, isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, allenes, orthoesters, sulfites, enamines, yneamines, ureas, pseudoureas, semicarboazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds, N-hydroxysuccinimide esters, maleimides, sulfhydryls, and the like. Methods for preparing each of these functional groups are well known in the art, and their application or modification for a particular purpose is within the ability of one of ordinary skill in the art (see, e.g., Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989 and Hermanson, G., Bioconjugate Techniques, 2000). nd ed., Academic Press, San Diego, 2008).

[0049] "Specific binding" generally refers to the physical association between a target polypeptide (or, more specifically, a target molecule) and a binding molecule, such as an antibody or a ligand. This binding typically depends on the presence of a particular structural feature of the target, such as an antigenic determinant, epitope, binding pocket, or cleft, recognized by the binding molecule. For example, if an antibody is specific for epitope A, the presence of a polypeptide containing epitope A or the presence of free, unlabeled A in a reaction containing both free, labeled A and a binding molecule that binds to it will reduce the amount of labeled A that binds to the binding molecule. It should be understood that specificity need not be absolute and generally refers to the circumstances under which binding occurs. For example, it is well known in the art that many antibodies cross-react with epitopes other than those present on the target molecule. Such cross-reactivity may be acceptable depending on the application for which the antibody is used. One skilled in the art can select an antibody or ligand with a sufficient degree of specificity to properly perform a given application (e.g., for target molecule detection, therapeutic purposes, etc.). It is also understood that specificity can be evaluated in terms of additional factors, such as the affinity of the binding molecule for the target relative to the affinity of the binding molecule for another target, e.g., a competitor. If a binding molecule exhibits high affinity for the target molecule desired to be detected and low affinity for non-target molecules, the antibody is likely to be an acceptable reagent. Once the specificity of a binding molecule in one or more situations has been established, it can be used in other, preferably similar, situations without re-evaluating its specificity. In some embodiments, the affinity (measured as the equilibrium dissociation constant, Kd) of two molecules exhibiting specific binding is less than 10 under the conditions tested, e.g., physiological conditions. -3 M or less, e.g., 10 -4 M or less, e.g., 10 -5 M or less, e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less or 10 -9 It is below M.

[0050] A "subject" treated according to the present invention is typically a human, non-human primate, or lower animal (e.g., a mouse or rat) that expresses or contains at least one primate (e.g., human) complement component C3, and optionally one or more additional primate complement components. In certain embodiments, the subject is male. In certain embodiments, the subject is female. In certain embodiments, the subject is an adult, e.g., a human at least 18 years of age, e.g., 18-100 years of age. In certain embodiments, the human subject is at least 12 years of age. In certain embodiments, the subject is an adult, e.g., a human at least 18 years of age, e.g., 18-100 years of age. In certain embodiments, the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age. In certain embodiments, the subject is a child, e.g., a human aged 0-4 years or 5-11 years.

[0051] As used herein, "treatment" in reference to treating a subject refers to providing treatment, i.e., providing some type of medical or surgical procedure to a subject. Treatment can be provided to eliminate, reduce, inhibit, prevent, or reduce the likelihood of a disease, or to eliminate, reduce, inhibit, or prevent, prevent, or reduce the likelihood of one or more symptoms or manifestations of a disease. "Prevention" refers to preventing a disease or symptom or manifestation of a disease from occurring in at least some individuals for at least a certain period of time. Treatment can involve administering a compound or composition to a subject after the onset of one or more symptoms or manifestations indicative of a disease, for example, to eliminate, reduce, reduce the severity, and / or inhibit or prevent the progression of a disease and / or to eliminate, reduce, reduce the severity, and / or prevent one or more symptoms or manifestations of a disease. The compound or composition can be administered to a subject who has a disease or who is at increased risk of developing a disease compared to members of the general population. The compound or composition can be administered to a subject who has developed a disease and is at an increased risk of developing one or more specific symptoms or signs of the disease or of the disease progression, compared to other individuals diagnosed with the disease or compared to the subject's typical or average risk of such symptoms or signs or progression.For example, the subject may be exposed to a "trigger" that increases the subject's risk of experiencing progression (e.g., a temporarily increased risk).The compound or composition can be administered prophylactically, i.e., before the onset of any symptoms or signs of the disease.Typically, in this case, the subject is at risk of developing the disease, for example, compared to members of the general population, optionally matched for age, sex, and / or other demographic variables.

[0052] A "vector" can be any of a variety of nucleic acid molecules, viruses, or portions thereof, that can mediate the insertion, e.g., introduction, transport, etc., of a nucleic acid of interest between genetic environments or into cells. The nucleic acid of interest can be joined, e.g., inserted, into a vector using, e.g., restriction and ligation. Vectors include, for example, nucleic acids that can be packaged in DNA or RNA plasmids, cosmids, naturally occurring or modified viral genomes or portions thereof, viral capsids, minichromosomes, artificial chromosomes, etc. Plasmid vectors typically contain an origin of replication (e.g., for replication in prokaryotic cells). Plasmids contain part or all of a viral genome (e.g., viral promoters, enhancers, processing or packaging signals, and / or sequences sufficient to generate a nucleic acid that can be integrated into a host cell genome and / or generate an infectious virus). A virus or portion thereof that can be used to introduce a nucleic acid into a cell can be called a viral vector. Viral vectors include, for example, adenoviruses, adeno-associated viruses, retroviruses (e.g., lentiviruses, vaccinia viruses and other poxviruses, herpes viruses (e.g., herpes simplex viruses), and others. Baculoviruses, for example, are for use in insect cells. A wide variety of plant viral vectors are known, including, for example, those based on or comprising cauliflower mosaic virus, tobacco mosaic virus, or one or more genetic elements thereof (e.g., the cauliflower mosaic virus 35S promoter). A viral vector may or may not contain sufficient viral genetic information for the production of infectious virus when introduced into a host cell; i.e., the viral vector can be replication competent or replication defective. In certain embodiments, for example, when sufficient information for the production of infectious virus is lacking, e.g., if viral production is desired, this information can be supplied by the host cell or by other vectors introduced into the cell. In certain embodiments, for example, if viral production is not desired, such information is not supplied. The introduced nucleic acid can be incorporated into a naturally occurring or modified viral genome or part thereof, or can be present within the viral capsid as a separate nucleic acid molecule.A vector may contain one or more nucleic acids encoding a marker suitable for identifying and / or selecting cells that have taken up the vector. Markers include, for example, various proteins that increase or decrease resistance or sensitivity to antibiotics or other drugs (e.g., proteins that confer resistance to antibiotics such as puromycin, hygromycin, or blasticidin), enzymes whose activities are detectable by assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and proteins or RNAs that detectably affect the phenotype of the expressing cell (e.g., fluorescent proteins). Vectors often have one or more suitable sites for restriction enzymes, which can be used to facilitate insertion of a nucleic acid, e.g., a nucleic acid to be expressed, into the vector. An expression vector is one into which a desired nucleic acid has been inserted or can be inserted such that it is operably linked to control elements (also called "control sequences," "expression control elements," or "expression control sequences") and can be expressed as an RNA transcript (e.g., mRNA, which can be translated into protein or non-coding RNA). An expression vector contains sufficient control sequences, e.g., expression control sequences, to direct transcription of an operably linked nucleic acid under at least certain conditions; other elements necessary or supporting expression may be supplied, for example, by the host cell or an in vitro expression system. Such control sequences typically include a promoter and may include enhancer sequences or upstream activator sequences. In certain embodiments, a vector may include sequences encoding 5' and / or 3' untranslated regions, which may include cleavage and / or polyadenylation signals. Generally, control elements may be included in the vector prior to insertion of the nucleic acid desired to be expressed, included in the insert nucleic acid, or inserted into the vector after insertion of the nucleic acid desired to be expressed. As used herein, a nucleic acid and a control element are "operably linked" when they are covalently linked such that expression or transcription of the nucleic acid is under the influence or control of the control element. For example, a promoter region is operably linked to a nucleic acid if the promoter region is capable of affecting transcription of the nucleic acid.Those skilled in the art will understand that the exact nature of the regulatory sequences useful for gene expression will vary from species to species or cell type to cell type, but will generally include sequences involved in transcription initiation, RNA processing, or translation initiation, as appropriate. The selection and design of appropriate vectors and regulatory elements is within the ability and discretion of those skilled in the art. For example, one of skill in the art will select an appropriate promoter (or other expression control sequence) for expression in the desired species (e.g., prokaryotic (bacterial) or eukaryotic (e.g., fungi, plants, mammalian species) or cell type. The vector can include a suitable viral promoter from, for example, cytomegalovirus (CMV), retrovirus, simian virus (e.g., SV40), papillomavirus, herpesvirus, or other viruses that infect mammalian cells, or a promoter capable of directing expression in mammalian cells, such as mammalian promoters derived from genes such as EF1 alpha, ubiquitin (e.g., ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK), etc., or a composite promoter such as the CAG promoter (a combination of the CMV early enhancer element and the chicken beta-actin promoter). In certain embodiments, a human promoter can be used. In certain embodiments, a promoter that normally directs transcription by eukaryotic RNA polymerase I ("pol I") can be used. In certain embodiments, a promoter that normally directs transcription by eukaryotic RNA polymerase II (a "pol II promoter"), or a functional variant thereof, can be used. In certain embodiments, a promoter that normally directs transcription by eukaryotic RNA polymerase III (a "pol III promoter"), for example, a promoter for transcription of a ribosomal RNA (other than 5S rRNA) or a functional variant thereof, can be used. One of skill in the art will select an appropriate promoter to direct transcription of a sequence of interest. Examples of expression vectors that can be used in mammalian cells include, for example, the pcDNA vector series, pSV2 vector series, pCMV vector series, pRSV vector series, pEF1 vector series, Gateway® vectors, and the like.In some embodiments, a controllable (e.g., inducible or repressible) expression regulatory element, such as a controllable promoter, is used to regulate expression, e.g., activate, increase, block, or decrease. In some embodiments, a vector comprises a polynucleotide sequence encoding a polypeptide, wherein the polynucleotide sequence is positioned in frame with a nucleic acid inserted into the vector to create an N-terminal or C-terminal fusion. In some embodiments, the polypeptide encoded by the polynucleotide sequence may comprise a signal sequence (directing secretion of the protein) or a sequence that directs the expressed protein to a specific organelle or intracellular location, such as the nucleus or mitochondria. In some embodiments, the polypeptide comprises a tag. Tags can be useful for facilitating detection and / or purification of proteins containing them. Examples of tags include polyhistidine tags (e.g., 6X-His tags), glutathione-S-transferase, maltose-binding protein, NUS tags, SNUT tags, Strep tags, and epitope tags, such as V5, HA, Myc, or FLAG. In some embodiments, the protease cleavage site is located in a region between the protein encoded by the inserted nucleic acid and the polypeptide, allowing the polypeptide to be separated from the tag by exposure to a protease. The vector can be introduced into a host cell using methods known in the art. Those skilled in the art will select an appropriate method based on, for example, the vector, cell type, etc. Suitable methods include, for example, calcium phosphate-mediated transfection, lipid-based or non-lipid-based transfection using any of a variety of commercially available reagents, such as FuGENE, Lipofectamine, TurboFect, etc.; electroporation; biolistic bombardment; etc. Such methods are described in detail in Sambrook, Protocols series, and other standard references.

[0053] As used herein, the term "aliphatic" refers to a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched-chain, or cyclic (including fused, bridged, and spiro-fused polycyclics), and may be fully saturated or contain one or more units of unsaturation, but is not aromatic. Unless otherwise specified, aliphatic groups contain 1-30 carbon atoms. In some embodiments, aliphatic groups contain 1-10 carbon atoms. In other embodiments, aliphatic groups contain 1-8 carbon atoms. In yet other embodiments, aliphatic groups contain 1-6 carbon atoms, and in yet other embodiments, aliphatic groups contain 1-4 carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain alkyl, alkenyl, and alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0054] As used herein, "alkyl" refers to a saturated straight-chain, branched-chain, or cyclic hydrocarbon having from about 1 to about 22 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 12 or from about 1 to about 7 carbon atoms being preferred in certain embodiments of the invention. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0055] As used herein, "halo" refers to F, Cl, Br, or I.

[0056] As used herein, "alkanoyl" refers to an optionally substituted, straight-chain or branched aliphatic acyclic residue having about 1 to 10 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms), e.g., about 1 to 7 carbon atoms, connected by a single bond to a terminal C=O group, as will be recognized (sometimes referred to as an "acyl group"). Alkanoyl groups include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl, 2-methylbutyryl, 2,2-dimethoxypropionyl, hexanoyl, heptanoyl, octanoyl, and the like; for purposes of the present invention, a formyl group is considered an alkanoyl group. "Lower alkanoyl" refers to an optionally substituted, straight-chain or branched aliphatic acyclic residue having about 1 to about 5 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms). Such groups include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl.

[0057] As used herein, "aryl" refers to an optionally substituted monocyclic or bicyclic aromatic ring system having about 5 to about 14 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), preferably about 6 to about 10 carbon atoms. Non-limiting examples include, but are not limited to, phenyl and naphthyl.

[0058] As used herein, "aralkyl" refers to an alkyl radical having an aryl substituent and having from about 6 to about 22 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 6 to about 12 carbon atoms being preferred in some embodiments. Aralkyl groups can be optionally substituted. Non-limiting examples include, for example, benzyl, naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.

[0059] The terms "alkoxy" and "alkoxyl," as used herein, refer to an optionally substituted alkyl-O- group, where alkyl is defined as above. Examples of alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, and heptoxy.

[0060] As used herein, "carboxy" refers to the group --C(.dbd.O)OH. As used herein, "alkoxycarbonyl" refers to a -C(=O)O-alkyl group, where alkyl is defined above.

[0061] As used herein, "aroyl" refers to the group -C(=O)-aryl, where aryl is as defined above. Examples of aroyl groups include benzoyl and naphthoyl.

[0062] The term "cyclic ring system" refers to non-aromatic, partially unsaturated or fully saturated 3- to 10-membered ring systems, including monocyclic rings sized from 3 to 8 atoms, as well as bicyclic and tricyclic ring systems that may contain an aromatic 5- or 6-membered aryl or heterocyclic group fused to a non-aromatic ring. Such heterocyclic rings include heterocyclic rings having 1 to 3 heteroatoms independently selected from the group consisting of oxygen, sulfur, and nitrogen. In certain embodiments, the term heterocyclic refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group in which at least one ring atom is a heteroatom selected from the group consisting of O, S, and N, including, but not limited to, a bicyclic or tricyclic group containing a fused 6-membered ring having 1 to 3 heteroatoms independently selected from the group consisting of oxygen, sulfur, and nitrogen. In certain embodiments, "ring system" refers to a cycloalkyl group, and as used herein, a cycloalkyl group refers to a group having 3 to 10, e.g., 4 to 7, carbon atoms. Cycloalkyl includes, but is not limited to, optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. In certain embodiments, "ring system" refers to an optionally substituted cycloalkenyl or cycloalkynyl moiety.

[0063] Typically, a substituted chemical moiety contains one or more substituents replacing hydrogen. Examples of substituents include, for example, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl (-OH), alkoxyl, cyano (-CN), carboxyl (-COOH), -C(=O)O-alkyl, aminocarbonyl (-C(=O)NH), -N-substituted aminocarbonyl (-C(=O)NHR"), CF, CFCF, and the like. For the above substituents, each moiety R" can independently be, for example, H, alkyl, cycloalkyl, aryl, or aralkyl.

[0064] As used herein, "L-amino acid" refers to either a naturally occurring levorotatory alpha amino acid that occurs naturally in proteins or an alkyl ester of such an alpha amino acid. The term "D-amino acid" refers to a dextrorotatory alpha amino acid. Unless otherwise specified, all amino acids referred to herein are L-amino acids.

[0065] As used herein, an "aromatic amino acid" refers to an amino acid that contains at least one aromatic ring, e.g., an aromatic amino acid contains an aryl group.

[0066] As used herein, an "aromatic amino acid analog" is an amino acid analog that contains at least one aromatic ring, e.g., it contains an aryl group.

[0067] II. Complement system To facilitate understanding of the present invention, and without intending to limit the present invention in any way, this section provides a brief overview of complement and its activation pathways. For a more detailed description, see, for example, Kuby Immunology, 6 th ed., 2006; Paul, WE, Fundamental Immunology, Lippincott Williams & Wilkins; 6 th ed., 2008; and Walport MJ., Complement. First of two parts. N Engl J Med., 344(14): 1058-66, 2001.

[0068] Complement is part of the innate immune system and plays a key role in protecting the body from infectious agents. The complement system includes more than 30 serum and cellular proteins involved in three major pathways, known as the classical, alternative, and lectin pathways. The classical pathway is typically initiated by the binding of C1 to complexes of antigen and IgM or IgG antibodies (although certain other activators can also initiate this pathway). Activated C1 cleaves C4 and C2 into C4a and C4b and C2a and C2b. C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3a and C3b. Binding of C3b to C3 convertase generates C5 convertase, which cleaves C5 into C5a and C5b. C3a, C4a, and C5a are anaphylotoxins that mediate multiple reactions in acute inflammatory responses. C3a and C5a also act as chemotactic factors that attract immune system cells, such as neutrophils.

[0069] The alternative pathway is initiated and amplified by, for example, microbial surfaces and various complex polysaccharides. In this pathway, low-level spontaneous hydrolysis of C3 to C3(H2O) leads to binding of factor B, which is cleaved by factor D to generate a fluid-phase C3 convertase, which activates complement by cleaving C3 into C3a and C3b. C3b binds to targets such as cell surfaces and forms a complex with factor B, which is subsequently cleaved by factor D to generate the C3 convertase. The surface-bound C3 convertase then cleaves and activates another C3 molecule, which is rapidly deposited near the activation site, forming yet another C3 convertase, which then generates yet another C3b. This process results in a cycle of C3 cleavage and C3 convertase formation that greatly amplifies the reaction. C3 cleavage and binding of another C3b molecule to the C3 convertase generates the C5 convertase. The C3 and C5 convertases of this pathway are regulated by the host cell molecules CR1, DAF, MCP, CD59, and fH. The mechanisms of action of these proteins include disruption of activation promotion (i.e., their ability to dissociate the convertases), their ability to act as cofactors in the degradation of C3b or C4b by factor I, or both. Normally, the presence of complement regulatory proteins on the host cell surface prevents significant complement activation from occurring at the cell surface.

[0070] The C5 convertases produced by both pathways cleave C5 to produce C5a and C5b. C5b then combines with C6, C7, and C8 to form C5b-8, which catalyzes the polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The MAC penetrates the target cell membrane, causing cell lysis. Small amounts of MAC on the cell membrane can have a variety of consequences other than cell death.

[0071] The lectin complement pathway is initiated by carbohydrate binding by mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs). The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein present in the intermediate region between the endoplasmic reticulum and the Golgi apparatus. The MBL-2 gene encodes a soluble mannose-binding protein present in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, resulting in the C3 convertases mentioned above.

[0072] Complement activity is regulated by a variety of mammalian proteins called complement control proteins (CCPs) or regulators of complement activation (RCA) proteins (U.S. Patent 6,897,290). These proteins differ in their ligand specificity and mechanisms of complement inhibition. They accelerate the normal decay of convertases and / or function as cofactors for factor I, enzymatically cleaving C3b and / or C4b into smaller fragments. CCPs are characterized by the presence of multiple (usually 4-56) homologous motifs, approximately 50-70 amino acids in length, containing a conserved motif with four disulfide-bonded cysteines (two disulfide bonds), proline, tryptophan, and numerous hydrophobic residues, known as short consensus repeats (SCRs), complement control protein (CCP) modules, or SUSHI domains. The CCP family includes complement receptor 1 (CR1; C3b:C4b receptor), complement receptor 2 (CR2), membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF), complement factor H (fH), and C4b-binding protein (C4bp). CD59 is a membrane-bound complement regulatory protein that is structurally unrelated to CCPs. Complement regulatory proteins normally limit complement activation that would otherwise occur on cells and tissues of a mammalian, e.g., human, host. Thus, "self" cells are normally protected from the harmful effects that would follow complement activation on these cells if it proceeded. Deficiency or deficiency of complement regulatory proteins is involved in the pathogenesis of a variety of complement-mediated disorders, such as those described herein.

[0073] III. Compstatin Analogs Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. U.S. Patent 6,319,897 describes a peptide having the sequence Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr (SEQ ID NO: 1), in which the disulfide bond between the two cysteines is represented by brackets. The name "compstatin" is not used in U.S. Patent 6,319,897, but it is understood to have been subsequently adopted in the scientific and patent literature (see, e.g., Morikis, et al., Protein Sci., 7(3): 619-27, 1998) to refer to a peptide having the same sequence as SEQ ID NO: 2 disclosed in U.S. Patent 6,319,897, but with a C-terminal amidation (SEQ ID NO: 8), as shown in Table 1. The term "compstatin" is used consistently in this specification (i.e., when referring to SEQ ID NO: 8). Compstatin analogs with higher complement inhibitory activity than compstatin have been developed. See, for example, WO2004 / 026328 (PCT / US2003 / 029653), Morikis, D., et al., Biochem Soc Trans. 32(Pt 1): 28-32, 2004, Mallik, B., et al., J. Med. Chem., 274-286, 2005; Katragadda, M., et al., J. Med. Chem., 49: 4616-4622, 2006; See WO2007062249 (PCT / US2006 / 045539); WO2007044668 (PCT / US2006 / 039397), WO / 2009 / 046198 (PCT / US2008 / 078593); WO / 2010 / 127336 (PCT / US2010 / 033345) and the discussion below.

[0074] A compstatin analog can be, for example, acetylated or amidated at the N-terminus and / or C-terminus. For example, a compstatin analog can be acetylated at the N-terminus and amidated at the C-terminus. As used in the art, "compstatin" as used herein and the activity of compstatin analogs described herein relative to the activity of compstatin refer to C-terminally amidated compstatin (Mallik, 2005, supra).

[0075] Concatamers or multimers of compstatin or its complement-inhibiting analogs also find use in the present invention.

[0076] As used herein, the term "compstatin analog" includes compstatin and all complement-inhibiting analogs thereof. The term "compstatin analog" includes compstatin and other compounds designed or identified based on compstatin, whose complement inhibitory activity is at least 50% as strong as that of compstatin, as measured, for example, using any art-recognized complement activation assay or an assay substantially similar or equivalent thereto. Certain suitable assays are described in U.S. Patent 6,319,897, WO2004 / 026328, Morikis, supra, Mallik, supra, Katragadda 2006, supra, WO2007062249 (PCT / US2006 / 045539); WO2007044668 (PCT / US2006 / 039397), WO / 2009 / 046198 (PCT / US2008 / 078593); and / or WO / 2010 / 127336 (PCT / US2010 / 033345). The assay may, for example, measure alternative or classical pathway-mediated erythrocyte lysis or may be an ELISA assay. In one embodiment, the assay described in WO / 2010 / 135717 (PCT / US2010 / 035871) is used.

[0077] The activity of compstatin analogs is related to their IC 50(the concentration of a compound that inhibits complement activation by 50%), and is, as recognized in the art, known as the IC 50 A lower IC indicates greater activity. The activity of preferred compstatin analogs for use in the present invention is at least as great as that of compstatin. It should be noted that certain modifications known to reduce or negate complement inhibitory activity may be expressly excluded from any embodiment of the present invention. The IC of compstatin 50 has been measured as 12 μM using an alternative pathway-mediated erythrocyte lysis assay (WO2004 / 026328). The exact IC measured for certain compstatin analogs is 50 It is understood that values will vary depending on experimental conditions (e.g., serum concentration used in the assay). For example, IC values for multiple different compounds under substantially identical conditions. 50 Comparative values obtained from experiments determining the IC of a compstatin analog are useful. 50 is the IC of compstatin 50 In some embodiments of the invention, the activity of the compstatin analog is 2 to 99 times that of compstatin (i.e., the IC 50 is the IC of compstatin 50 (2-99 times less than the activity of compstatin). For example, the activity may be as much as 10-50 times greater than the activity of compstatin, or as much as 50-99 times greater than the activity of compstatin. In certain embodiments of the invention, the activity of the compstatin analog is 99-264 times greater than the activity of compstatin. For example, the activity may be 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, or 264-fold greater than the activity of compstatin. In certain embodiments, the activity is 250-300-fold, 300-350-fold, 350-400-fold, or 400-500-fold greater than the activity of compstatin. The present invention further contemplates compstatin analogs that have 500 to 1000 times or more the activity of compstatin. In certain embodiments, the IC of a compstatin analog is 50In some embodiments, the IC of the compstatin analog is about 0.2 to about 0.5 μM. 50 In some embodiments, the IC of the compstatin analog is about 0.1 to about 0.2 μM. 50 In some embodiments, the IC of a compstatin analog is about 0.05 to about 0.1 μM. 50 is about 0.001 to about 0.05 μM.

[0078] K of compstatin binding to C3 d can be measured using isothermal titration calorimetry (Katragadda, et al., J. Biol. Chem., 279(53), 54987-54995, 2004). As is recognized in the art, there is a correlation between the binding affinity of various compstatin analogs to C3 and their activity, with the K d A lower K indicates a higher binding affinity. A linear correlation between binding affinity and activity has been observed for certain analogs tested (Katragadda, 2004, supra; Katragadda 2006, supra). In certain embodiments of the invention, compstatin analogs have a K of 0.1-1.0 μM, 0.05-0.1 μM, 0.025-0.05 μM, 0.015-0.025 μM, 0.01-0.015 μM, or 0.001-0.01 μM. d It binds to C3.

[0079] Compounds "designed or identified based on compstatin" include, but are not limited to, compounds comprising an amino acid chain whose sequence is obtained by (i) modifying the sequence of compstatin (e.g., replacing one or more amino acids in the sequence of compstatin with a different amino acid or amino acid analog, inserting one or more amino acids or amino acid analogs into the sequence of compstatin, or deleting one or more amino acids from the sequence of compstatin); (ii) randomizing one or more amino acids in compstatin, optionally by selection from a phage display peptide library further modified according to method (i); or (iii) identifying by screening compounds that compete with compstatin or any analog obtained by method (i) or (ii) for binding to C3 or a fragment thereof. Many useful compstatin analogs contain a hydrophobic cluster, a β-turn, and a disulfide bridge.

[0080] In certain embodiments of the invention, the sequence of a compstatin analog comprises or consists essentially of a sequence obtained by making one, two, three, or four substitutions in the sequence of compstatin, i.e., replacing one, two, three, or four amino acids in the sequence of compstatin with different standard amino acids or non-standard amino acids. In certain embodiments of the invention, the amino acid at position 4 is altered. In certain embodiments of the invention, the amino acid at position 9 is altered. In certain embodiments of the invention, the amino acids at positions 4 and 9 are altered. In certain embodiments of the invention, only the amino acids at positions 4 and 9 are altered. In certain embodiments of the invention, the amino acid at positions 4 or 9 is altered, or in certain embodiments, both the amino acids at positions 4 and 9 are altered, and up to two amino acids at positions selected from positions 1, 7, 10, 11, and 13 are altered. In certain embodiments of the invention, the amino acids at positions 4, 7, and 9 are altered. In certain embodiments of the invention, the amino acids at positions 2, 12, or both are altered, but the alterations preserve the ability of the compound to cyclize. Such changes at positions 2 and / or 12 may be in addition to changes at positions 1, 4, 7, 9, 10, 11, and / or 13. Optionally, any compstatin analog sequence resulting from the replacement of one or more amino acids in the compstatin sequence includes up to one, two, or three additional amino acids at the C-terminus. In one embodiment, the additional amino acid is Gly. Optionally, any compstatin analog sequence resulting from the replacement of one or more amino acids in the compstatin sequence further includes up to five or up to ten additional amino acids at the C-terminus. Unless otherwise specified or apparent from the context, it should be understood that a compstatin analog may have any one or more features or characteristics of the various embodiments described herein, and that a feature or characteristic of any embodiment may further characterize any other embodiment described herein.In certain embodiments of the invention, the sequence of the compstatin analog comprises or consists essentially of a sequence identical to that of compstatin except for positions corresponding to positions 4 and 9 of the compstatin sequence.

[0081] Compstatin and certain compstatin analogs that are somewhat more active than compstatin contain only standard amino acids ("standard amino acids" are glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartic acid, asparagine, glutamic acid, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine, and histidine). Certain compstatin analogs with improved activity incorporate one or more non-standard amino acids. Useful non-standard amino acids include mono- and polyhalogenated (e.g., fluorinated) amino acids, D-amino acids, homoamino acids, N-alkylamino acids, dehydroamino acids, aromatic amino acids (except phenylalanine, tyrosine, and tryptophan), ortho-, meta-, or para-aminobenzoic acids, phospho-amino acids, methoxylated amino acids, and α,α-disubstituted amino acids. In certain embodiments of the invention, compstatin analogs are designed by replacing one or more L-amino acids of the compstatin analogs described elsewhere herein with the corresponding D-amino acids. Such compounds and methods of use thereof are an aspect of the invention.Examples of useful non-standard amino acids are 2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI), 2-indanylglycine carboxylic acid (2Igl), dihydrotryptophan (Dht), 4-benzoyl-L-phenylalanine (Bpa), 2-α-aminobutyric acid (2-Abu), 3-α-aminobutyric acid (3-Abu), 4-α-aminobutyric acid (4-Abu), cyclohexylalanine (Cha), homocyclohexylalanine (hCha), 4-fluoro-L-tryptophan (4fW), 5-fluoro-L-tryptophan (5fW), 6-fluoro-L-tryptophan (6fW), These include 4-hydroxy-L-tryptophan (4OH-W), 5-hydroxy-L-tryptophan (5OH-W), 6-hydroxy-L-tryptophan (6OH-W), 1-methyl-L-tryptophan (1MeW), 4-methyl-L-tryptophan (4MeW), 5-methyl-L-tryptophan (5MeW), 7-aza-L-tryptophan (7aW), α-methyl-L-tryptophan (αMeW), β-methyl-L-tryptophan (βMeW), N-methyl-L-tryptophan (NMeW), ornithine (orn), citrulline, norleucine, and γ-glutamic acid.

[0082] In certain embodiments of the invention, a compstatin analog contains one or more Trp analogs (e.g., at positions 4 and / or 7 of the compstatin sequence). Examples of Trp analogs are described above. See also Beene, et al., Biochemistry 41: 10262-10269, 2002 (depicting, among other things, mono- and poly-halogenated Trp analogs); Babitzke & Yanofsky, J. Biol. Chem. 270: 12452-12456, 1995 (depicting, among other things, methylated Trp, halogenated Trp, and other Trp analogs, as well as indole analogs); and U.S. Patents 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750, and 4,299,838. Other Trp analogs include variants substituted (e.g., with methyl groups) at the α or β carbon of the indole ring and optionally at one or more positions. Amino acids containing two or more aromatic rings are included as Trp analogs, including substituted, unsubstituted, or substituted variants in other embodiments. In certain embodiments of the present invention, for example, at position 4, the Trp analog is 5-methoxy, 5-methyl, 1-methyl, or 1-formyl-tryptophan. In certain embodiments of the present invention, Trp analogs (e.g., analogs at position 4) containing 1-alkyl substituents, e.g., lower alkyl (e.g., C1-C5) substituents, are used. In certain embodiments, N(α)methyltryptophan or 5-methyltryptophan is used. In certain embodiments, analogs containing 1-alkanoyl substituents, e.g., lower alkanoyl (e.g., C1-C5), are used. Examples include 1-acetyl-L-tryptophan and L-β-tryptophan.

[0083] In some embodiments, the Trp analog has increased hydrophobicity compared to Trp. For example, the indole ring can be substituted with one or more alkyl (e.g., methyl) groups. In some embodiments, the Trp analog participates in hydrophobic interactions with C3. Such a Trp analog can be located, for example, at position 4 in the sequence of compstatin. In some embodiments, the Trp analog contains a substituted or unsubstituted bicyclic aromatic ring moiety or two or more substituted or unsubstituted monocyclic aromatic ring moieties.

[0084] In some embodiments, the Trp analog has an increased tendency to form hydrogen bonds with C3 compared to Trp, but does not have increased hydrophobicity compared to Trp. The Trp analog may have increased polarity compared to Trp and / or an increased ability to participate in electrostatic interactions with hydrogen bond donors on C3. Particular exemplary Trp analogs with increased propensity to form hydrogen bonds include those containing electronegative substituents on the indole ring. Such Trp analogs may be located, for example, at position 7 in the sequence of compstatin.

[0085] In some embodiments of the invention, a compstatin analog contains one or more Ala analogs (e.g., at position 9 relative to the compstatin sequence), Ala analogs that are identical to Ala except that they contain one or more CH2 groups in the side chain. In some embodiments, the Ala analogs are unbranched, single-methyl amino acids such as 2-Abu. In some embodiments of the invention, a compstatin analog contains one or more Trp analogs (e.g., at positions 4 and / or 7 in the compstatin sequence) and an Ala analog (e.g., at position 9 in the compstatin sequence).

[0086] In some embodiments of the present invention, the compstatin analog is (X'aa) n -Gln-Asp-Xaa-Gly-(X”aa) m(SEQ ID NO: 2), wherein each X'aa and each X"aa is independently selected from an amino acid or amino acid analog, Xaa is Trp or an analog of Trp, and n > 1, m > 1, and n + m is 5 to 21. The peptide has the core sequence Gln-Asp-Xaa-Gly, wherein Xaa is Trp or an analog of Trp, e.g., an analog of Trp that has an increased tendency to form hydrogen bonds with H-bond donors relative to Trp, but in some embodiments does not have increased hydrophobicity relative to Trp. For example, the analog can be The indole ring of Trp may be substituted with an electronegative group, e.g., a halogen such as fluorine. In one embodiment, Xaa is 5-fluorotryptophan. Unless otherwise indicated, those skilled in the art will recognize that any non-natural peptide that contains this core sequence and inhibits complement activation and / or binds to C3 may be designed based on the compstatin sequence. In another embodiment, Xaa is an amino acid or amino acid analog other than a Trp analog that allows the Gln-Asp-Xaa-Gly peptide to form a β-turn.

[0087] In some embodiments of the present invention, the peptide has the core sequence X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa and Xaa are selected from Trp and analogs of Trp. In some embodiments of the present invention, the peptide has the core sequence X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa and Xaa are selected from Trp, analogs of Trp, and other amino acids or amino acid analogs containing at least one aromatic ring. In some embodiments of the present invention, the core sequence forms a β-turn in the context of the peptide. The β-turn can be flexible, allowing the peptide to assume two or more conformations, as assessed, for example, using nuclear magnetic resonance (NMR). In some embodiments, X'aa is a substituted or unsubstituted bicyclic aromatic ring moiety or an analog of Trp containing two or more substituted or unsubstituted monocyclic aromatic ring moieties. In some embodiments of the present invention, X'aa is selected from the group consisting of 2-naphthylalanine, 1-naphthylalanine, 2-indanylglycinecarboxylic acid, dihydrotryptophan, and benzoylphenylalanine. In some embodiments of the present invention, X'aa is an analog of Trp having increased hydrophobicity compared to Trp. For example, X'aa can be 1-methyltryptophan. In some embodiments of the present invention, Xaa is an analog of Trp having increased propensity to form hydrogen bonds compared to Trp, but in some embodiments, not increased hydrophobicity compared to Trp. In some embodiments of the present invention, the analog of Trp having increased propensity to form hydrogen bonds compared to Trp contains a modification, for example, at the 5-position of the indole ring of Trp, such as replacing the H atom at the 5-position with a halogen atom. For example, Xaa can be 5-fluorotryptophan.

[0088] In certain embodiments of the invention, the peptide has the core sequence X'aa-Gln-Asp-Xaa-Gly-X"aa (SEQ ID NO: 4), where X'aa and Xaa are each independently selected from Trp and an analog of Trp, and X"aa is selected from His, Ala, an analog of Ala, an analog of Phe, and an analog of Trp. In certain embodiments of the invention, X'aa is 1-methyltryptophan or another Trp analog having an alkyl substituent on the indole ring (e.g., at position 1, 4, 5, or 6), which has increased hydrophobicity relative to Trp. In certain embodiments, X'aa is an analog of Trp containing a substituted or unsubstituted bicyclic aromatic ring moiety or two or more substituted or unsubstituted monocyclic aromatic ring moieties. In certain embodiments of the invention, X'aa is selected from the group consisting of 2-naphthylalanine, 1-naphthylalanine, 2-indanylglycine carboxylic acid, dihydrotryptophan, and benzoylphenylalanine. In some embodiments of the present invention, Xaa is an analog of Trp that has an increased tendency to form a hydrogen bond with C3 compared to Trp, but in some embodiments, does not have increased hydrophobicity compared to Trp. In some embodiments of the present invention, the analog of Trp that has an increased tendency to form a hydrogen bond compared to Trp includes a modification, for example, at position 5 of the indole ring of Trp, such as replacing the H atom at position 5 with a halogen atom. For example, Xaa can be 5-fluorotryptophan. In certain embodiments, X"aa is an analog of Ala, such as Ala or Abu, or another unbranched monomethyl amino acid. In certain embodiments of the invention, the peptide has the core sequence X'aa-Gln-Asp-Xaa-Gly-X"aa (SEQ ID NO: 4), where X'aa and Xaa are each independently selected from Trp, an analog of Trp, and an amino acid or amino acid analog containing at least one aromatic side chain, and X"aa is selected from His, Ala, an analog of Ala, Phe, and Trp. In certain embodiments, X"aa is selected from an analog of Trp, an aromatic amino acid, and an aromatic amino acid analog.

[0089] In a preferred embodiment of the invention, the peptide is cyclic.n and the other is (X”aa) m The peptide may be cyclized by a bond between any two amino acids located within the amino acid sequence. In certain embodiments, the cyclic portion of the peptide is 9-15 amino acids in length, e.g., 10-12 amino acids in length. In certain embodiments, the cyclic portion of the peptide is 11 amino acids in length, with a bond (e.g., a disulfide bond) between the amino acids at positions 2 and 12. For example, the peptide may be 13 amino acids in length, with a bond between the amino acids at positions 2 and 12 resulting in a cyclic portion 11 amino acids in length.

[0090] In certain embodiments, the peptide comprises or consists of the sequence X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X"aa1-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO: 5). In certain embodiments, X'aa4 and Xaa are selected from Trp and analogs of Trp, and X'aa1, X'aa2, X'aa3, X"aa1, X"aa2, X"aa3, X"aa4 and X"aa5 are independently selected from amino acids and amino acid analogs. In certain embodiments, X'aa4 and Xaa are selected from aromatic amino acids and aromatic amino acid analogs. Any one or more of X'aa1, X'aa2, X'aa3, X"aa1, X"aa2, X"aa3, X"aa4, and X"aa5 can match the amino acid at the corresponding position in compstatin. In one embodiment, X"aa1 is Ala or a monomethyl unbranched amino acid. The peptide can be cyclized by a covalent bond between (i) X'aa1, X'aa2, or X'aa3 and (ii) X"aa2, X"aa3, X"aa4, or X"aa5. In one embodiment, the peptide is cyclized by a covalent bond between X'aa2 and X"aa4. In one embodiment, each covalently linked amino acid is Cys, and the covalent bond is a disulfide (SS) bond. In another embodiment, the covalent bond is a CC, CO, CS, or CN bond. In some embodiments, one covalently linked residue is an amino acid or amino acid analog having a side chain containing a primary or secondary amine, the other covalently linked residue is an amino acid or amino acid analog having a side chain containing a carboxylic acid group, and the covalent bond is an amide bond. Amino acids or amino acid analogs having a side chain containing a primary or secondary amine include lysine and amino acids having the general structure NH2(CH2) nDiaminocarboxylic acids of CH(NH2)COOH include, for example, 2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid (daba), and ornithine (orn), where n = 1(dapa), 2(daba), and 3(orn), respectively. Examples of amino acids with a side chain containing a carboxylic acid group include dicarboxylic amino acids such as glutamic acid and aspartic acid. Analogs such as β-hydroxy-L-glutamic acid can also be used. In some embodiments, the peptide is cyclized by a thioether bond, as described in, for example, PCT / US2011 / 052442 (WO2012 / 040259). For example, in some embodiments, the disulfide bond of any peptide is replaced with a thioether bond. In some embodiments, cystathionine is formed. In some embodiments, the cystathionine is δ-cystathionine or γ-cystathionine. In certain embodiments, the modification comprises replacing the Cys-Cys disulfide bond between the cysteines at X'aa2 and X"aa4 of SEQ ID NO:5 (or the corresponding position in other sequences) with the addition of a CH2 to form a homocysteine at X'aa2 or X"aa4, and introducing a thioether bond to form cystathionine. In one embodiment, the cystathionine is γ-cystathionine. In another embodiment, the cystathionine is δ-cystathionine. Another modification provided herein comprises replacing the disulfide bond with a thioether bond without adding a CH2 to form lantithionine. In certain embodiments, compstatin analogs having a thioether bond in place of a disulfide bond exhibit increased stability, at least under some conditions, compared to compstatin analogs having a disulfide bond.

[0091] In some embodiments, the compstatin analog has the sequence Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2 * -Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO: 6) [During the ceremony, Xaa1 is Ile, Val, Leu, B 1-Ile, B 1 -Val, B 1 -Leu or Gly-Ile or B 1 -Gly-Ile dipeptide, B 1 represents the first blocking portion; Xaa2 and Xaa2 * is independently selected from Trp and an analog of Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp or an analog of Trp; Xaa4 is a dipeptide selected from L-Thr, D-Thr, Ile, Val, Gly, Thr-Ala, and Thr-Asn, or a tripeptide containing Thr-Ala-Asn, wherein the carboxy-terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn is optionally linked to a second blocking moiety B 2 has been replaced by; The two Cys residues are linked by a disulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe, or a dipeptide or tripeptide Xaa5-Ala-Asn selected from Xaa5-Ala and Xaa5-Asn, where Xaa5 is selected from Leu, Nle, His, or Phe, and the carboxy-terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn is optionally linked to a second blocking moiety B 2 The two Cys residues are linked by a disulfide bond.

[0092] In other embodiments, Xaa1 is absent or is any amino acid or amino acid analog, and Xaa2, Xaa2 * , Xaa3 and Xaa4 are as defined above. If Xaa1 is absent, the N-terminal Cys residue has a blocking moiety B attached thereto. 1 It has.

[0093] In other embodiments, Xaa4 is any amino acid or amino acid analog, and Xaa1, Xaa2, Xaa2 * and Xaa3 is as defined above. In other embodiments, Xaa4 is a dipeptide selected from the group consisting of Thr-Ala and Thr-Asn, wherein the carboxy-terminal —OH or Ala or Asn is optionally a second blocking moiety B 2 has been replaced by.

[0094] In any embodiment of the compstatin analog of SEQ ID NO: 6, Xaa2 can be Trp.

[0095] In any embodiment of the compstatin analog of SEQ ID NO: 6, Xaa2 can be an analog of Trp containing a substituted or unsubstituted bicyclic aromatic ring moiety or two or more substituted or unsubstituted monocyclic aromatic ring moieties. For example, the analog of Trp can be selected from 2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI), 2-indanylglycine carboxylic acid (IgI), dihydrotryptophan (Dht), and 4-benzoyl-L-phenylalanine.

[0096] In any embodiment of the compstatin analog of SEQ ID NO: 6, Xaa2 can be an analog of Trp having increased hydrophobicity relative to Trp. For example, the analog of Trp can be selected from 1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan, and 6-methyltryptophan. In one embodiment, the analog of Trp is 1-methyltryptophan. In one embodiment, Xaa2 is 1-methyltryptophan and Xaa2 * is Trp, Xaa3 is Ala, and the other amino acids are identical to those of compstatin.

[0097] In any embodiment of the compstatin analog of SEQ ID NO: 6, Xaa2 *can be an analog of Trp, e.g., an analog of Trp that has an increased tendency to form a hydrogen bond with C3 relative to Trp, but in some embodiments does not have increased hydrophobicity relative to Trp. In some embodiments, the analog of Trp contains an electronegative substituent on the indole ring. For example, the analog of Trp can be selected from 5-fluorotryptophan and 6-fluorotryptophan.

[0098] In one embodiment of the invention, Xaa2 is Trp, and Xaa2 * is an analog of Trp that has an increased propensity to form a hydrogen bond with C3 relative to Trp, but in some embodiments does not have increased hydrophobicity relative to Trp. In some embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2 is an analog of Trp that has increased hydrophobicity relative to Trp, such as an analog of Trp selected from 1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan, and 6-methyltryptophan, and Xaa2 * is an analog of Trp that has an increased propensity to form a hydrogen bond with C3 relative to Trp, but in some embodiments does not have increased hydrophobicity relative to Trp. For example, in one embodiment, Xaa2 is methyltryptophan and Xaa2 * is 5-fluorotryptophan.

[0099] In any of the above embodiments, Xaa3 is Ala. In any of the above embodiments, Xaa3 is a monomethyl unbranched amino acid, such as Abu.

[0100] The present invention further provides a compstatin analog of SEQ ID NO: 6 above, comprising Xaa2 and Xaa2 * are independently selected from Trp, an analog of Trp, and other amino acids or amino acid analogs containing at least one aromatic ring, and Xaa3 is His, Ala, or an analog of Ala, Phe, Trp, an analog of Trp, or another aromatic amino acid or aromatic amino acid analog.

[0101] In certain embodiments of the invention, the blocking moiety present at the N-terminus or C-terminus of any compstatin analog described herein is any moiety that stabilizes the peptide against degradation that may occur in the blood or interstitial fluid of a mammal (e.g., a human or non-human primate). For example, blocking moiety B 1 can be any moiety that alters the structure of the N-terminus of the peptide so as to inhibit cleavage of the peptide bond between the N-terminal amino acid of the peptide and the amino acid adjacent to it. 2 can be any moiety that alters the structure of the C-terminus of the peptide such that cleavage of the peptide bond between the C-terminal amino acid of the peptide and the amino acid adjacent to it is inhibited. Any suitable blocking moiety known in the art can be used. In one embodiment of the present invention, blocking moiety B 1 comprises an acyl group (i.e., the moiety remaining after removal of the -OH group from a carboxylic acid). The acyl group typically contains 1 to 12 carbons, e.g., 1 to 6 carbons. For example, in one embodiment of the invention, the blocking moiety B 1 is selected from the group consisting of formyl, acetyl, proprionyl, butyryl, isobutyryl, valeryl, isovaleryl, and the like. 1 is an acetyl group, ie, Xaa1 is Ac-Ile, Ac-Val, Ac-Leu or Ac-Gly-Ile.

[0102] In one embodiment of the invention, blocking moiety B 2 is a primary or secondary amine (-NH2 or -NHR where R is an organic moiety such as an alkyl group) 1 )

[0103] In one embodiment of the invention, blocking moiety B 1 is any moiety that neutralizes or reduces the negative charge that may be present at the N-terminus at physiological pH. In one embodiment of the invention, the blocking moiety B 2 is any moiety that neutralizes or reduces the negative charge that may be present at the C-terminus at physiological pH.

[0104] In some embodiments of the invention, the compstatin analog is acetylated or amidated at the N-terminus and / or C-terminus, respectively. The compstatin analog may be acetylated at the N-terminus, amidated at the C-terminus, or both acetylated at the N-terminus and amidated at the C-terminus. In some embodiments of the invention, the compstatin analog contains an alkyl or aryl group at the N-terminus rather than an acetyl group.

[0105] In some embodiments, the compstatin analog has the sequence Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2 * -Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO: 7) [During the ceremony, Xaa1 is a dipeptide containing Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu, or Gly-Ile or Ac-Gly-Ile; Xaa2 and Xaa2 * is independently selected from Trp and an analog of Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp or an analog of Trp; Xaa4 is a dipeptide selected from L-Thr, D-Thr, Ile, Val, Gly, Thr-Ala, and Thr-Asn, or a tripeptide containing Thr-Ala-Asn, wherein the carboxy-terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn is optionally replaced with —NH2; The two Cys residues are linked by a disulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe, or a dipeptide or tripeptide Xaa5-Ala-Asn selected from Xaa5-Ala and Xaa5-Asn, where Xaa5 is selected from Leu, Nle, His, or Phe, and the carboxy-terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn is optionally linked to a second blocking moiety B 2 has been replaced by; The two Cys residues are linked by a disulfide bond.

[0106] In one embodiment, Xaa1, Xaa2, Xaa2 * , Xaa3, and Xaa4 are as described above for various embodiments of SEQ ID NO: 6. For example, in one embodiment, Xaa2 * is Trp. In some embodiments, Xaa2 is an analog of Trp that has increased hydrophobicity relative to Trp, such as 1-methyltryptophan. In some embodiments, Xaa3 is Ala. In some embodiments, Xaa3 is a monomethyl unbranched amino acid.

[0107] In one embodiment of the invention, Xaa1 is Ile and Xaa4 is L-Thr.

[0108] In one embodiment of the invention, Xaa1 is Ile and Xaa2 * is Trp and Xaa4 is L-Thr.

[0109] The present invention further provides a compstatin analog of SEQ ID NO: 7 above, comprising Xaa2 and Xaa2 * are independently selected from Trp, an analog of Trp, other amino acids or aromatic amino acid analogs, and Xaa3 is His, Ala or an analog of Ala, Phe, Trp, an analog of Trp, or another aromatic amino acid or aromatic amino acid analog.

[0110] In some embodiments of any of the compstatin analogs described herein, an analog of Phe is used rather than Phe.

[0111] Table 1 provides a non-limiting list of compstatin analogs useful in the present invention. Each analog is abbreviated in the left-hand column, indicating the specific modification at the designated position (positions 1-13) compared to the parent peptide, compstatin. As used herein, "compstatin" and the activities of the compstatin analogs described herein relative to the activity of compstatin refer to C-terminally amidated compstatin peptides. Unless otherwise specified, the peptides in Table 1 are C-terminally amidated. Bold type is used to indicate specific modifications. Activity relative to compstatin is based on published data and assays described therein (WO2004 / 026328, WO2007044668, Mallik, 2005; Katragadda, 2006). When multiple publications reporting a single activity are referenced, the most recently published value was used, but it is recognized that values may be adjusted if there are discrepancies between assays. It is also understood that, in certain embodiments of the present invention, when the peptides listed in Table 1 are used in the therapeutic compositions and methods of the present invention, they are cyclized via a disulfide bond between two Cys residues. Alternative means of cyclizing peptides are also within the scope of the present invention. As noted above, in various embodiments of the present invention, one or more amino acids of a compstatin analog (e.g., any of the compstatin analogs disclosed herein) can be an N-alkylamino acid (e.g., an N-methylamino acid). For example, but not limited to, at least one amino acid within the cyclic portion of the peptide, at least one amino acid at the N-terminus of the cyclic portion, and / or at least one amino acid at the C-terminus of the cyclic portion can be an N-alkylamino acid, e.g., an N-methylamino acid. In certain embodiments of the present invention, for example, a compstatin analog includes an N-methylglycine, e.g., at a position corresponding to position 8 of compstatin and / or a position corresponding to position 13 of compstatin. In certain embodiments, one or more compstatin analogs of Table 1 contain one or more N-methylglycines, eg, at a position corresponding to position 8 of compstatin and / or at a position corresponding to position 13 of compstatin.In certain embodiments, one or more compstatin analogs in Table 1 contain at least one N-methylisoleucine, e.g., at a position corresponding to position 13 of compstatin. For example, an N-methyl Ile can replace a Thr at or near the C-terminus of a peptide whose sequence is listed in Table 1 or any other compstatin analog sequence. As will be appreciated, in certain embodiments, the N-methylated amino acid comprises an N-methyl Gly at position 8 and an N-methyl Ile at position 13. In certain embodiments, the N-methylated amino acid comprises an N-methyl Gly in a core sequence such as SEQ ID NO:3 or SEQ ID NO:4. In certain embodiments, the N-methylated amino acid comprises an N-methyl Gly in a core sequence such as SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0112] [Table 1] NA=Not available

[0113] In certain embodiments of the compositions and methods of the invention, the compstatin analog has a sequence selected from sequences 9-36. In certain embodiments of the compositions and methods of the invention, the compstatin analog has a sequence selected from SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, and 36. In certain embodiments of the compositions and / or methods of the invention, the compstatin analog has a sequence selected from SEQ ID NOs: 30 and 31. In one embodiment of the compositions and methods of the invention, the compstatin analog has the sequence of SEQ ID NO: 28. In one embodiment of the compositions and methods of the invention, the compstatin analog has the sequence of SEQ ID NO: 32. In one embodiment of the compositions and methods of the invention, the compstatin analog has the sequence of SEQ ID NO: 34. In one embodiment of the compositions and methods of the invention, the compstatin analog has the sequence of SEQ ID NO: 36.

[0114] In some embodiments, blocking moiety B 1 In some embodiments, the blocking moiety B 2In some embodiments, the blocking moiety B 1 and / or B. 2 includes non-standard amino acids such as D-amino acids, N-alkyl amino acids (e.g., N-methyl amino acids). In some embodiments, the blocking moiety B 1 and / or B. 2 includes non-standard amino acids that are analogs of standard amino acids. In some embodiments, the amino acid analogs include lower alkyl, lower alkoxy, or halogen substituents relative to the standard amino acid that they are analogs of. In some embodiments, the substituents are in the side chain. In some embodiments, the substituents are in the alpha carbon atom. In some embodiments, the blocking moiety B includes an amino acid, e.g., a non-standard amino acid. 1 is further divided into part B 1a For example, blocking portion B 1 is B 1a In some embodiments, B 1a may neutralize or reduce the positive charge that may otherwise be present at the N-terminus at physiological pH. 1a may comprise or consist of, for example, an acyl group containing, for example, 1 to 12 carbons, for example, 1 to 6 carbons. 1a is selected from the group consisting of formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, and the like. In some embodiments, a blocking moiety B comprising an amino acid, e.g., a non-standard amino acid, is 2 is further divided into part B 2a For example, blocking moiety B 2 is XaaN-B 2a where N represents the appropriate number of amino acids (according to the numbering used in the peptide backbone). 2a may neutralize or reduce the negative charge that may otherwise be present at the C-terminus at physiological pH. 2a comprises or consists of a primary or secondary amine (e.g., NH). Moiety B 1a -Xaa0 and / or XaaN-B 2aIt is understood that the blocking activity of B is provided by either or both components of the moiety in various embodiments. In some embodiments, the blocking moiety or a portion thereof, e.g., an amino acid residue, contributes to the affinity of the compound for C3 or C3b and / or improves the activity of the compound. In some embodiments, the contribution of an amino acid residue to affinity or activity may be at least as important as its contribution to blocking activity. For example, in some embodiments, B 1a -Xaa0 and / or XaaN-B 2a Xaa0 and / or XaaN in may function primarily to increase the affinity or activity of the compound, but B 1a and / or B. 2a may prevent digestion and / or charge neutralization of the peptide. In certain embodiments, the compstatin analog has the amino acid sequence of any of SEQ ID NOS: 5-36, wherein SEQ ID NOS: 5-36 are further extended at the N-terminus and / or C-terminus. In certain embodiments, the sequence is 1a -Xaa0-sequence-XaaN-B 2a where the sequence represents any one of SEQ ID NOs: 5 to 36, and B 1a and B 2a may or may not be independently present. For example, in some embodiments, the compstatin analog is 1a -Xaa0-X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X”aa1-X”aa2-X”aa3-X”aa4-X”aa5-XaaN-B 2a (SEQ ID NO: 37A), wherein X'aa1-X'aa2-X'aa3-X'aa4, Xaa, X"aa1, X"aa2, X"aa3, X"aa4 and X"aa5 are as set forth in SEQ ID NO: 5 above.

[0115] In some embodiments, the compstatin analog is B 1a -Xaa0-Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2 * -Gly-Xaa3-His-Arg-Cys-Xaa4-XaaN-B 2a(SEQ ID NO: 38A), wherein Xaa1, Xaa2, Xaa2 * , Xaa3 and Xaa4 are as set forth in SEQ ID NO: 6 above, or Xaa1, Xaa2, Xaa2 * , Xaa3 and Xaa4 are as shown in SEQ ID NO:6 or SEQ ID NO:7.

[0116] In some embodiments, the compstatin analog is B 1a -Xaa0-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-XaaN-B 2a (SEQ ID NO: 39A), wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12 and Xaa13 are identical to the amino acids at positions 1 to 13 of any of SEQ ID NOs: 9 to 36.

[0117] In some embodiments, Xaa0 and / or XaaN in any compstatin analog sequence comprise an amino acid comprising an aromatic ring bearing one or more alkyl substituents. In some embodiments, the alkyl substituent is a lower alkyl substituent. For example, in some embodiments, the alkyl substituent is a methyl or ethyl group. In some embodiments, the substituent is located at any position that does not disrupt the aromaticity of the compound. In some embodiments, the substituent is located at any position that does not disrupt the aromaticity of the ring to which the substituent is attached. In some embodiments, the substituent is located at position 1, 2, 3, 4, or 5. In some embodiments, Xaa0 comprises an O-methyl analog of tyrosine, 2-hydroxyphenylalanine, or 3-hydroxyphenylalanine. For purposes of the present invention, a lowercase "m" followed by a three-letter amino acid abbreviation may be used to specifically indicate that the amino acid is an N-methyl amino acid. For example, the abbreviation "mGly" when used herein means N-methylglycine (also known as sarcosine or Sar). In some embodiments, Xaa0 is or comprises mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), Cha, mPhe, mVal, mIle, mAla, DTyr, DPhe, DArg, DTrp, DThr, DTyr(Me), mPhe, mVal, mIle, DAla, or DCha. For example, in some embodiments, a compstatin analog has the sequence B 1 -Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B 2 (SEQ ID NO: 40A) or B 1 -Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B 2 (SEQ ID NO: 41A). The two Cys residues are linked by a disulfide bond in the active compound. In some embodiments, the peptide is acetylated at the N-terminus and / or amidated at the C-terminus. In some embodiments, the peptide is selected from the group consisting of B, BH ... 1 is B 1a-Contains Xaa0 and / or B 2 is XaaN-B 2a For example, in one embodiment, B 1 comprises or consists of Gly, mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), mPhe, mVal, mIle, mAla, DTyr, DPhe, DTrp, DCha, DAla, and B 2 contains an NH, e.g., the carboxy terminal OH of mIle is replaced with an NH. In some embodiments, B 1 comprises or consists of mGly, Tyr, DTyr or Tyr(Me), B 2 contains an NH, e.g., the carboxy-terminal OH of mIle is replaced with an NH. In one embodiment, Ile at position Xaa1 is replaced with Gly. The complement inhibitory ability and / or C3b binding parameters of selected compstatin analogs are described in WO / 2010 / 127336 (PCT / US2010 / 033345) and / or Qu, et al., Immunobiology (2012), doi: 10.1016 / j.imbio.2012.06.003.

[0118] In certain embodiments, a blocking moiety or a portion thereof, e.g., a single amino acid residue, may contribute to increasing the affinity of the compound for C3 or C3b and / or improve the activity of the compound. In certain embodiments, the contribution of the amino acid or amino acid analog to affinity or activity may be more significant than the blocking activity.

[0119] In certain embodiments of the compositions and methods of the present invention, the compstatin analog has a sequence as set forth in Table 1, except that the Ac group is replaced by a blocking moiety B, as described above. 1 In some embodiments, the -NH group is replaced by another blocking moiety B, as described herein. 2 has been replaced by.

[0120] In one embodiment, the compstatin analog binds to a region of the beta chain of human C3 that is substantially the same as the region to which compstatin binds. In one embodiment, the compstatin analog is a compound that binds to a fragment of the C-terminal portion of the beta chain of human C3, with a molecular weight of approximately 40 kDa, to which compstatin binds (Soulika, AM, et al., Mol. Immunol., 35: 160, 1998; Soulika, AM, et al., Mol. Immunol. 43(12): 2023-9, 2006). In some embodiments, the compstatin analog is a compound that binds to the binding site of compstatin determined in a compstatin-C3 structure, for example, a crystal structure or a 3D structure by NMR. In some embodiments, the compstatin analog is a compound that can replace compstatin in the compstatin-C3 structure and form substantially the same intermolecular contact with C3 as compstatin. In certain embodiments, the compstatin analog is a compound that binds to the binding site of a peptide having a sequence set forth in Table 1, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, 37, 37A, 38A, 39A, 40A, or 41A, or other compstatin analog sequences described herein, within the peptide-C3 structure, e.g., the crystal structure. In certain embodiments, the compstatin analog is a compound that binds to the binding site of a peptide having SEQ ID NO: 30 or 31, within the peptide-C3 structure, e.g., the crystal structure. In certain embodiments, a compstatin analog is a compound that can replace a peptide of SEQ ID NO:9-36, e.g., a peptide of SEQ ID NO:14, 21, 28, 29, 32, 33, 34, or 36, 37, 37A, 38A, 39A, 40A, or 41A, or other compstatin analog sequences described herein, in a peptide-C3 structure and form substantially the same intermolecular contacts with C3 as the peptide. In certain embodiments, a compstatin analog is a compound that can replace a peptide of SEQ ID NO:30 or 31 in a peptide-C3 structure and form substantially the same intermolecular contacts with C3 as the peptide.

[0121] Those skilled in the art can easily determine whether a compstatin analog binds to a fragment of the C-terminal portion of the β-chain of C3 using routine experimental methods. For example, those skilled in the art can synthesize a photocrosslinkable compstatin analog by incorporating a photocrosslinking amino acid, such as p-benzoyl-L-phenylalanine (Bpa), at the C-terminus of the sequence of the compound (Soulika, AM, et al., supra). Optionally, additional amino acids, such as an epitope tag, such as a FLAG tag or an HA tag, can be incorporated to facilitate detection of the compound, for example, by Western blotting. The compstatin analog is incubated with the fragment to initiate crosslinking. Colocalization of the compstatin analog and the C3 fragment indicates binding. Surface plasmon resonance may be used to determine whether a compstatin analog binds to the compstatin binding site of C3 or a fragment thereof. One skilled in the art can use molecular modeling software programs to predict whether a compound will form substantially the same intermolecular contacts with C3 as a peptide having the sequence of compstatin or any of the peptides listed in Table 1, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34 or 36, or in certain embodiments, SEQ ID NO: 30 or 31, 37, 37A, 38A, 39A, 40A or 41A, or other compstatin analog sequences described herein.

[0122] Compstatin analogs can be prepared by various synthetic methods of peptide synthesis known in the art through condensation of amino acid residues, for example, by expression in vitro or in living cells from an appropriate nucleic acid sequence encoding it, using methods known in the art, following conventional peptide synthesis techniques. For example, peptides can be synthesized using standard solid-phase methods as described in Malik, supra, Katragadda, supra, WO2004026328 and / or WO2007062249. Potentially reactive moieties, such as amino and carboxyl groups, reactive functional groups, and the like, can be protected and then deprotected using various protecting groups and methodologies known in the art. For example, see "Protective Groups in Organic Synthesis," 3 rd See, e.g., Greene, TW and Wuts, PG, Eds., John Wiley & Sons, New York: 1999). Peptides can be purified using standard methods, such as reverse-phase HPLC. If desired, separation of diastereomeric peptides can be performed using known methods, such as reverse-phase HPLC. If desired, the preparation can be lyophilized and then dissolved in a suitable solvent, e.g., water. The pH of the resulting solution can be adjusted, e.g., to physiological pH, using a base such as NaOH. If desired, the peptide preparation can be characterized by mass spectrometry to confirm mass and / or disulfide bond formation. See, e.g., Mallik, 2005, and Katragadda, 2006.

[0123] In some embodiments, the compstatin analog may be or may contain a cell-reactive compstatin analog. A cell-reactive compstatin analog is a compound comprising a compstatin analog moiety and a cell-reactive functional group that can react with a functional group exposed on a cell surface, for example, under physiological conditions, to form a covalent bond. Thus, the cell-reactive compstatin analog becomes covalently bound to a cell. Without wishing to be bound by any particular theory, the cell-tethered compstatin analog protects cells from complement-mediated damage, for example, by binding to C3 (which may be in the form of C3(H2O)) on the cell surface and / or around the cell, inhibiting C3 cleavage and activation, and / or by binding to C3b, preventing its deposition in the cell or its participation in the complement activation cascade. In some embodiments of the present invention, isolated cells are contacted with the cell-reactive compstatin analog ex vivo (outside the body). In some embodiments of the present invention, the cells are present in an isolated tissue or organ, for example, a tissue or organ to be transplanted into a subject. In some embodiments of the present invention, cells are contacted with a cell-reactive compstatin analog in vivo by administering the cell-reactive compstatin analog to a subject. The cell-reactive compstatin analog becomes covalently bound to the cells in vivo. In some embodiments, the present invention protects cells, tissues, and / or organs from the harmful effects of complement activation for at least two weeks without the need for treatment during that time.

[0124] In certain embodiments, the present invention provides and / or utilizes compstatin analogs that include a targeting moiety that noncovalently binds to a target molecule present on the surface of a cell or tissue or to extracellular material not bound to a cell or tissue. Such compstatin analogs are referred to herein as "targeted compstatin analogs." The target molecule is often a protein or carbohydrate that is bound to a cell membrane and exposed on the cell surface. The targeting moiety targets the compstatin analog to a cell, tissue, or location susceptible to complement activation. In certain embodiments of the present invention, isolated cells are contacted with the targeted compstatin analog ex vivo (outside the body). In certain embodiments of the present invention, the cells are present in an isolated tissue or organ, e.g., a tissue or organ to be transplanted into a subject. In certain embodiments of the present invention, the targeted compstatin analog is administered to a subject and becomes noncovalently bound to the cell, tissue, or extracellular material in vivo. In certain embodiments, the methods of the present invention protect cells, tissues, and / or organs from the harmful effects of complement activation for at least two weeks without the need for treatment during that time. In some embodiments, the targeted compstatin analog contains both a targeting moiety and a cell-reactive moiety. The targeting moiety targets the compstatin analog to a specific cell type, for example, by non-covalently binding to a molecule on such cells. The cell-reactive moiety then covalently binds to cells or extracellular substances. In other embodiments, the targeted compstatin analog does not contain a cell-reactive moiety.

[0125] In some embodiments, the compstatin analog may be or include a long-acting compstatin analog, where the long-acting compstatin analog includes a moiety such as polyethylene glycol (PEG) that extends the compound's lifespan in the body (e.g., by reducing clearance from the blood). In some embodiments, the long-acting compstatin analog does not include a targeting moiety or a cell-reactive moiety. In some embodiments, the long-acting compstatin analog includes a targeting moiety and / or a cell-reactive moiety.

[0126] A compstatin analog, optionally conjugated to a cell-reactive or targeting moiety, can be modified by the addition of, for example, polyethylene glycol (PEG) or similar molecules to stabilize the compound, reduce its immunogenicity, extend its longevity in the body, increase or decrease its solubility, and / or increase its resistance to degradation. Methods of PEGylation are well known in the art (Veronese, FM & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, FF, Adv. Drug Deliv. Rev. 54, 457-458, 2002); Hinds, KD & Kim, SW Adv. Drug Deliv. Rev. 54, 505-530 (2002; Roberts, MJ, Bentley, MD & Harris, JM Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, YS et al. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymers, such as PEG and modified PEG, including derivatized PEG, to which polypeptides can be appropriately conjugated are described in the Nektar Advanced Pegylation 2005-2006 Product Catalog, Nektar Therapeutics, San Carlos, CA, which also provides details of suitable conjugation methods. In other embodiments, the compstatin analog is fused to an immunoglobulin Fc domain or a portion thereof. In other embodiments, the compstatin analog is conjugated to an albumin moiety or an albumin-binding peptide. Thus, in some embodiments, the compstatin analog is modified with one or more polypeptide or non-polypeptide moieties, e.g., the compstatin analog is pegylated or conjugated to another moiety. In some embodiments, the moiety is not an immunoglobulin Fc domain or a portion thereof.Compstatin analogs can be obtained as multimers, which can include a single molecular species or multiple different molecular species (eg, multiple different analogs), or as part of a supramolecular complex.

[0127] In certain embodiments, the compstatin analog is a polyvalent compound comprising multiple compstatin analog moieties covalently or non-covalently attached to a polymer backbone or scaffold. The compstatin analog moieties can be the same or different. In certain embodiments of the invention, the polyvalent compound comprises multiple instances or copies of a single compstatin analog moiety. In other embodiments of the invention, the polyvalent compound comprises two or more different compstatin analog moieties, e.g., one or more instances of each of three, four, five, or more different compstatin analog moieties. In certain embodiments of the invention, the number of compstatin analog moieties ("n") is 2 to 6. In other embodiments of the invention, n is 7 to 20. In other embodiments of the invention, n is 20 to 100. In other embodiments, n is 100 and 1,000. In other embodiments of the invention, n is 1,000 to 10,000. In other embodiments, n is 10,000 to 50,000. In other embodiments, n is 50,000 to 100,000. In other embodiments, n is 100,000 to 1,000,000.

[0128] The compstatin analog moiety may be attached directly to the polymer scaffold or may be attached via a linking moiety that connects the compstatin analog moiety to the polymer scaffold. The linking moiety may be attached to a single compstatin analog moiety and the polymer scaffold. Alternatively, the linking moiety may have multiple compstatin analog moieties attached thereto such that the linking moiety attaches multiple compstatin analog moieties to the polymer scaffold.

[0129] In some embodiments, the compstatin analog contains an amino acid having a side chain containing a primary or secondary amine, such as a Lys residue. For example, a Lys residue or a sequence containing a Lys residue is added to the N-terminus and / or C-terminus of the compstatin analog. In some embodiments, the Lys residue is separated from the cyclic portion of the compstatin analog by a rigid or flexible spacer. The spacer can include, for example, a substituted or unsubstituted saturated or unsaturated alkyl chain, an oligo(ethylene glycol) chain, and / or other moieties, such as those described in Section VI with respect to linkers. The chain length can be, for example, 2 to 20 carbon atoms. In other embodiments, the spacer is a peptide. A peptide spacer can be, for example, 1 to 20 amino acids in length, e.g., 4 to 20 amino acids in length. Suitable spacers can include or consist of, for example, multiple Gly residues, Ser residues, or both. Optionally, the amino acid having a side chain containing a primary or secondary amine and / or at least one amino acid in the spacer is a D-amino acid. Any of a variety of polymer backbones or scaffolds can be used. For example, the polymer backbone or scaffold can be a polyamide, polysaccharide, polyanhydride, polyacrylamide, polymethacrylic acid, polypeptide, polyethylene oxide, or dendrimer. Suitable methods and polymer backbones are described, for example, in WO98 / 46270 (PCT / US98 / 07171) or WO98 / 47002 (PCT / US98 / 06963). In one embodiment, the polymer backbone or scaffold contains multiple reactive functional groups, such as carboxylic acid groups, anhydride groups, or succinimide groups. The polymer backbone or scaffold is reacted with a compstatin analog. In one embodiment, the compstatin analog contains any of a number of different reactive functional groups, such as carboxylic acid groups, anhydride groups, or succinimide groups, which are reacted with appropriate groups on the polymer backbone. Alternatively, monomer units that can be linked together to form the polymer backbone or scaffold can first be reacted with a compstatin analog, and the resulting monomers are polymerized.In another embodiment, short chains are polymerized and functionalized, and then a mixture of short chains of different compositions is assembled into a long polymer.

[0130] IV. Compstatin Mimetics The structure of compstatin is known in the art, as are the NMR structures of numerous compstatin analogs that have greater activity than compstatin (Malik, supra). Structural information can be used to design compstatin mimetics.

[0131] In one embodiment, a compstatin mimetic is any compound that competes with compstatin or any compstatin analog (e.g., a compstatin analog with a sequence listed in Table 1) for binding to C3 or a fragment thereof (such as the 40 kD fragment of the β-chain to which compstatin binds). In some embodiments, a compstatin mimetic has activity that is equal to or greater than that of compstatin. In some embodiments, a compstatin mimetic is more stable, orally available, or more bioavailable than compstatin. A compstatin mimetic can be a peptide, nucleic acid, or small molecule. In some embodiments, a compstatin mimetic is a compound that binds to the binding site of compstatin as determined in a compstatin-C3 structure, e.g., a crystal structure or a 3-D structure obtained by NMR experiments. In some embodiments, a compstatin mimetic is a compound that can replace compstatin in the compstatin-C3 structure and form substantially the same intermolecular contacts with C3 as compstatin. In certain embodiments, a compstatin mimetic is a compound that binds to the binding site of a peptide having a sequence set forth in Table 1 within a peptide-C3 structure, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, or in certain embodiments, SEQ ID NO: 30 or 31, or other compstatin analog sequence. In certain embodiments, a compstatin mimetic is a compound that can replace a peptide having a sequence set forth in Table 1 within a peptide-C3 structure, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, or in certain embodiments, SEQ ID NO: 30 or 31, or other compstatin analog sequence, and can form substantially the same intermolecular contacts with C3 as the peptide. In certain embodiments, a compstatin mimetic has a non-peptide backbone but with side chains arranged in a designed sequence based on the sequence of compstatin.

[0132] Those skilled in the art will understand that once a specific desired conformation of a short peptide has been identified, methods for designing peptides or peptidomimetics that fit that conformation are well known.See, for example, GR Marshall (1993), Tetrahedron, 49: 3547-3558; Hruby and Nikiforovich (1991), in Molecular Conformation and Biological Interactions, P. Balaram & S. Ramasehan, eds., Indian Acad. of Sci., Bangalore, PP. 429-455), Eguchi M, Kahn M., Mini Rev Med Chem., 2(5): 447-62, 2002.With particular reference to the present invention, for example, as reported in the art, inter alia, for compstatin and its analogs, the design of peptide analogs can be further refined by taking into account the contribution of various side chains of amino acid residues to functional group effects or conformational considerations.

[0133] Those skilled in the art will appreciate that peptidomimetics can serve equally well as peptides in providing the specific backbone conformation and side chain functional groups necessary to bind C3 and inhibit complement activation. Thus, it is considered within the scope of the present invention to produce and utilize compounds that bind C3 and inhibit complement by using any naturally occurring amino acid, amino acid derivative, amino acid analog, or non-amino acid molecule that can combine to form the appropriate backbone conformation. Non-peptide analogs, or analogs containing both peptidic and non-peptidic components, may be referred to herein as "peptidomimetics" or "isostere mimetics" to refer to peptide replacements or derivatives that have comparable backbone conformational properties and / or most other functionalities to the exemplary peptides in inhibiting complement activation. More generally, a compstatin mimetic is any compound whose pharmacophore, despite a different backbone, can be positioned similarly to that of compstatin.

[0134] The use of peptidomimetics for the development of high-affinity peptide analogs is well known in the art. Analogs containing non-amino acid moieties can be analyzed to identify conformational motifs, assuming approximately the same rotational constraints as amino acid residues in a peptide, using Ramachandran plots, among other known techniques (Hruby & Nikiforovich 1991).

[0135] Those skilled in the art can easily establish suitable screening assays to identify additional compstatin mimetics and select those with the desired inhibitory activity. For example, compstatin or its analogs can be labeled (e.g., with a radioactive or fluorescent label) and contacted with C3 in the presence of various concentrations of a test compound. The ability of the test compound to reduce the binding of a compstatin analog to C3 is evaluated. Test compounds that significantly reduce the binding of a compstatin analog to C3 are considered to be candidate compstatin mimetics. For example, test compounds that reduce the steady-state concentration of the compstatin analog-C3 complex or reduce the formation rate of the compstatin analog-C3 complex by at least 25% or at least 50% are considered to be candidate compstatin mimetics. Those skilled in the art will recognize that numerous variations of this screening assay can be used. Compounds to be screened include natural products, aptamer libraries, phage display libraries, compound libraries synthesized using combinatorial chemistry, and the like. The present invention encompasses synthesizing a combinatorial library of compounds based on the core sequence and screening the library to identify compstatin mimetics. Any of these methods can also be used to identify new compstatin analogs with greater inhibitory activity than previously tested compstatin analogs. It is understood that compstatin mimetics can be used in the cell-reactive compounds of the present invention, and that the present invention provides such cell-reactive compstatin mimetics.

[0136] V. Cell-Reactive or Long-Acting Compstatin Analogs As noted above, the present invention provides and / or utilizes various cell-reactive compstatin analogs. In some aspects, the cell-reactive compstatin analogs include compounds of formula ALM, where A is a moiety containing a cell-reactive functional group J, L is an optional linker, and M comprises a compstatin analog moiety. The compstatin analog moiety can comprise any compstatin analog in various embodiments, such as any of the compstatin analogs described above. Formula ALM includes embodiments in which AL is present at the N-terminus of the compstatin analog moiety, AL is present at the C-terminus of the compstatin analog moiety, AL is attached to the side chain of an amino acid in the compstatin analog moiety, and the same or different ALs are present at both ends of M. It is understood that when a particular compstatin analog is present as a compstatin analog moiety in a compound of formula ALM, a functional group of the compstatin analog reacts with a functional group of L to form a covalent bond with A or L. For example, a compstatin analog in which the compstatin analog moiety includes an amino acid having a side chain containing a primary amine (NH2) group (the compstatin analog has the formula R 1 Cell-reactive compstatin analogs containing a compound represented by formula R (which can be represented by -(NH)) have the formula R, where a new covalent bond (e.g., N-C) is formed with L and a hydrogen is lost. 1The term "compstatin analog moiety" may have the molar activity -NH-LA. Thus, the term "compstatin analog moiety" encompasses molecular structures in which at least one atom of a compstatin analog is involved in a covalent bond to a second moiety (e.g., which may be a side chain modification). The same considerations apply to compstatin analog moieties present in the polyvalent compounds described above. In certain embodiments, a blocking moiety at the N-terminus or C-terminus of a compstatin analog, e.g., a compstatin analog described in Section IV above, is replaced with A-L in the structure of the cell-reactive compstatin analog. In certain embodiments, A or L comprises a blocking moiety. In certain embodiments, the cell-reactive compstatin analog has at least about 10%, 20%, or 30%, e.g., 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, or more, of the molar activity of a corresponding compstatin analog having the same amino acid sequence (and one or more blocking moieties, if applicable) but not including the cell-reactive moiety. In certain embodiments in which the cell-reactive compstatin analog comprises multiple compstatin analog moieties, the molar activity of the cell-reactive compstatin analog is at least about 10%, 20%, or 30%, e.g., 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% or more of the sum of the activities of the compstatin analog moieties.

[0137] The cell-reactive moiety A can comprise any of a variety of different cell-reactive functional groups J in various embodiments. Generally, the cell-reactive functional group can be selected, at least in part, based on factors such as: (a) the specific functional group targeted; (b) the ability of the reactive functional group to react with the target functional group under physiologically acceptable ex vivo conditions (e.g., physiologically acceptable pH and osmolality) and / or in vivo conditions (e.g., in blood); (c) the specificity of the reaction that occurs between the reactive functional group and the target functional group under physiologically acceptable ex vivo and / or in vivo conditions; (d) the stability (e.g., under in vivo conditions) of the covalent bond that may result from the reaction of the reactive functional group with its target functional group; and (e) the ease of synthesis of a cell-reactive compstatin analog that includes the reactive functional group. In certain embodiments, a reactive functional group is selected that reacts with the target chemical group without dissociating a leaving group. In certain embodiments, a reactive functional group is selected that results in dissociation of a leaving group upon reaction with the target. Compounds containing such groups can be useful, for example, for monitoring the progress and / or extent of a reaction. In some embodiments, the leaving group is physiologically acceptable to cells, tissues or organs in the amount produced (e.g., based on the concentration and / or absolute amount produced) and / or medically acceptable to a subject in the amount produced (e.g., based on the concentration in relevant body fluids such as blood and / or based on the absolute amount produced).In some embodiments, at least a portion of the leaving group produced ex vivo is removed, for example, by washing cells or washing or perfusing tissues or organs, for example, with saline solution.

[0138] In many embodiments, the cell-reactive functional groups used in the present invention react with the side chains of amino acid residues and / or the N-terminal amino group or C-terminal carboxyl group of proteins. In some embodiments, the cell-reactive functional groups react with sulfhydryl (-SH) groups present in the side chains of cysteine residues. In some embodiments, maleimide groups are used. Maleimide groups react with sulfhydryl groups of cysteine residues of proteins at physiological pH to form stable thioether bonds. In some embodiments, haloacetyl groups such as iodoacetyl or bromoacetyl groups are used. Haloacetyl groups react with sulfhydryl groups at physiological pH. The reaction of iodoacetyl groups proceeds by nucleophilic substitution of iodine with the sulfhydryl group's sulfur atom, resulting in a stable thioether bond. In other embodiments, iodoacetamide groups are used. In some embodiments, the cell-reactive functional groups react with amino (-NH) groups present at the N-terminus of proteins and amino groups present in the side chains of lysine residues (ε-amino groups). In some embodiments, an active ester, such as a succinimidyl ester (i.e., an NHS ester), is used. For example, N-hydroxysuccinimide (NHS) or its water-soluble analog (sulfo-NHS) is used in the synthesis, and the resulting cell-reactive compstatin analog contains an NHS ester. In some embodiments, the cell-reactive functional group reacts with the carboxyl (-COOH) groups present at the C-terminus of proteins and in the side chains of various amino acid residues. In some embodiments, the cell-reactive compstatin analog reacts with the hydroxyl (-OH) groups present in the side chains of various amino acids and in the carbohydrate moieties of glycosylated proteins.

[0139] In general, the linking moiety L can contain any one or more aliphatic and / or aromatic moieties consistent with the formation of a stable compound connecting the moieties to which it is attached. As used herein, the term "stable" preferably refers to a compound that is stable enough to be manufacturable, e.g., that maintains its integrity for a period of time that makes it useful for one or more of the purposes described herein. In certain embodiments, L comprises a saturated or unsaturated, substituted or unsubstituted, branched or unbranched aliphatic chain having a length of 1 to 30, 1 to 20, 1 to 10, 1 to 6, or 5 or less carbon atoms, where length refers to the number of C atoms in the main chain (longest chain). In certain embodiments, the aliphatic chain comprises one or more heteroatoms (O, N, S), which may be independently selected. In certain embodiments, at least 50% of the atoms in the main chain of L are carbon atoms. In certain embodiments, L comprises a saturated alkyl moiety (CH2). n wherein n is 1 to 30.

[0140] In some embodiments, L contains one or more heteroatoms and has a length of 1 to 1000, 1 to 800, 1 to 600, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 1 to 30, or 1 to 10 total carbon atoms in the chain. In some embodiments, L is an oligo(ethylene glycol) moiety (-(O-CH-CH-) n ), wherein n is 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 10 to 200, 200 to 300, 100 to 200, 40 to 500, 30 to 500, 20 to 500, 10 to 500, 1 to 40, 1 to 30, 1 to 20, or 1 to 10.

[0141] In certain embodiments, L comprises an unsaturated moiety such as -CH=CH- or -CH-CH=CH-; a moiety containing a non-aromatic ring system (e.g., a cyclohexyl moiety); an aromatic moiety (e.g., an aromatic ring system such as a phenyl moiety); an ether moiety (-COC-); an amide moiety (-C(=O)-N-); an ester moiety (-CO-O-); a carbonyl moiety (-C(=O)-); an imine moiety (-C=N-); a thioether moiety (-CSC-); an amino acid residue; and / or any moiety that can be formed by the reaction of two compatible reactive functional groups. In certain embodiments, one or more portions of the binding moiety or cell-reactive moiety are substituted by independently replacing one or more hydrogen (or other) atoms on the moiety with one or more moieties, including, but not limited to, aliphatic; aromatic, aryl; alkyl, aralkyl, alkanoyl, aroyl, alkoxy; thio; F; Cl; Br; I; —NO 2 ; —CN; —CF 3 ; —CH 2 CF 3 ; —CHCl 2 ; —CH 2 OH; —CH 2 CH 2 OH; —CH 2 NH 2 ; —CH 2 SO 2 CH 3 ; — or —GRG 1 , where G is —O—, —S—, —NRG 2 —, —C(═O)—, —S(═O)—, —SO 2 —, —C(═O)O—, —C(═O)NRG 2 — , -OC(=O)-, -NRG2C(=O)-, -OC(=O)O-, -OC(=O)NRG2-, -NRG2C(=O)O-, -NRG2C(=O)NRG2-, -C(=S)-, -C(=S)S-, -SC(=S)-, -SC(=S)S-, -C(=NRG2)-, -C(=NRG2)O-, -C(=NRG2)NRG3-, -OC(=NRG2)-, -NRG2C(=NRG3)-, -NRG2SO2-, -NRG2SO2NRG3- or -SON2NRG2-, wherein RG1, RG2 and RG3 each independently include, but are not limited to, hydrogen, halogen, or an optionally substituted aliphatic, aromatic, or aryl moiety. It is understood that when a ring system is present as a substituent, it may optionally be attached via a linear moiety.Combinations of substituents and variables contemplated by the present invention are preferably those that result in stable compounds useful in any one or more of the methods described herein, e.g., useful for treating one or more of the disorders described herein and / or contacting cells, tissues, or organs, and / or useful as intermediates in the preparation of one or more such compounds.

[0142] In various embodiments, L can comprise any one or more of the moieties described in the preceding paragraph. In certain embodiments, L comprises two or more distinct moieties that are linked together to form a structure typically having a length of 1 to about 60 atoms, 1 to about 50 atoms, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 6 atoms, where length refers to the number of atoms in the main chain (longest chain). In certain embodiments, L comprises two or more distinct moieties that are linked together to form a structure typically having a main chain (longest chain) of 1 to about 40 carbon atoms, e.g., 1 to 30, e.g., 1 to 20, 1 to 10, or 1 to 6 carbon atoms. The structure of such cell-reactive compstatin analogs generally has the formula A-(L Pj )jM, where j is typically 1 to 10, and L Pj are each independently selected from the moieties described in the preceding paragraph. In many embodiments, L comprises one or more carbon-containing chains, such as -(CH2)n- and / or -(O-CH2-CH2-)n, which are covalently bonded to each other and / or to the cell-reactive functional group or compstatin analog, e.g., by a moiety resulting from the reaction of two compatible reactive functional groups (e.g., an amide moiety, an ester moiety, or an ether moiety). In some embodiments, L comprises an oligo(ethylene glycol) moiety and / or a saturated alkyl chain. In some embodiments, L is -(CH2) m -C(=O)-NH-(CH2CH2O) n (CH2) p C(=O)- or -(CH2) m -C(=O)-NH-(CH2) p (OCH2CH2) nC(═O)—. In certain embodiments, m, n, and p are selected so that the number of carbon atoms in the chain is 1 to 500, e.g., 2 to 400, 2 to 300, 2 to 200, 2 to 100, 2 to 50, 4 to 40, 6 to 30, or 8 to 20. In certain embodiments, m is 2 to 10, n is 1 to 500, and / or p is 2 to 10. In certain embodiments, m is 2 to 10, n is 1 to 400, and / or p is 2 to 10. In certain embodiments, m is 2 to 10, n is 1 to 300, and / or p is 2 to 10. In certain embodiments, m is 2 to 10, n is 1 to 200, and / or p is 2 to 10. In certain embodiments, m is 2 to 10, n is 1 to 100, and / or p is 2 to 10. In certain embodiments, m is 2-10, n is 1-50, and / or p is 2-10. In certain embodiments, m is 2-10, n is 1-25, and / or p is 2-10. In certain embodiments, m is 2-10, n is 1-8, and / or p is 2-10. Optionally, at least one -CH2- is replaced with CH-R, where R can be any substituent. Optionally, at least one -CH2- is replaced with a heteroatom, a ring system, an amide moiety, an ester moiety, or an ether moiety. In certain embodiments, L does not include alkyl groups with a longest chain of more than 3 carbon atoms. In certain embodiments, L does not include alkyl groups with a longest chain of 4, 5, 6, 7, 8, 9, 10, or 11 carbon atoms.

[0143] In one embodiment of the present invention, A comprises a cell-reactive functional group J and a linker L P1 a linker L comprising 1 and a reactive functional group that reacts with a compstatin analog to produce AM. In some embodiments, the compound comprises two reactive functional groups and a linker, L P2 a bifunctional linker L comprising 2 The reactive functional group of L reacts with the appropriate reactive functional groups of A and M to produce the cell-reactive compstatin analog ALM. In some embodiments, the compstatin analog is linked to the linker L.P3 a linker L comprising 3 For example, as described below, a linker containing a reactive functional group may be present at the N-terminus or C-terminus, or a moiety containing a reactive functional group may be attached to the N-terminus or C-terminus via a linker. Thus, L may be a linker containing multiple linking moieties L, provided, for example, by A, the linker used to connect A and M, and / or the compstatin analog. P If present in the structure ALM, L 1 , L 2 , L 3 It is understood that certain reactive functional groups present in, for example, L, undergo reaction, and only a portion of the reactive functional groups are present in the final structure ALM, and the compound includes the moieties formed by the reaction of the functional groups. Generally, when a compound includes more than one linking moiety, the linking moieties can be the same or different and can be independently selected in various embodiments. P can be linked together to form a larger linker moiety L, at least some of which can have one or more compstatin analogs and / or cell-reactive functional groups attached thereto. In molecules containing multiple compstatin analogs, the compstatin analogs can be the same or different, and if different, can be independently selected. The same applies to linkers and reactive functional groups. The present invention encompasses the use of multivalent compstatin analogs containing one or more cell-reactive functional groups, as well as the use of concatemers of compstatin analogs containing one or more cell-reactive functional groups. In certain embodiments, at least one bond is a stable non-covalent bond, such as a biotin / (strept)avidin bond or other non-covalent bond of approximately the same strength.

[0144] In some embodiments, the cell-reactive compstatin analog comprises a compstatin analog of any of SEQ ID NOS: 3-36, 37A, 38A, 39A, 40A, or 41A extended by one or more amino acids at the N-terminus, C-terminus, or both, where at least one of the amino acids has a side chain containing a reactive functional group, such as a primary or secondary amine, a sulfhydryl group, a carboxyl group (which may be present as a carboxylic acid group), a guanidino group, a phenol group, an indole ring, a thioether, or an imidazole ring. In some embodiments, the amino acids are L-amino acids. In some embodiments, any one or more amino acids are D-amino acids. If multiple amino acids are added, the amino acids can be selected independently. In some embodiments, a reactive functional group (e.g., a primary or secondary amine) is used as a target for the addition of a moiety containing a cell-reactive functional group. Amino acids with a side chain containing a primary or secondary amine include lysine (Lys) and amino acids with the general structure NH2(CH2). nDiaminocarboxylic acids of CH(NH2)COOH include, for example, 2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid (daba), and ornithine (orn) (where n = 1(dapa), 2(daba), and 3(orn), respectively). In some embodiments, at least one amino acid is cysteine, aspartic acid, glutamic acid, arginine, tyrosine, tryptophan, methionine, or histidine. Cysteine has a side chain containing a sulfhydryl group. Aspartic acid and glutamic acid have side chains containing a carboxyl group (which becomes a carboxylic acid group when ionized). Arginine has a side chain containing a guanidino group. Tyrosine has a side chain containing a phenol group (which becomes a phenolate group when ionized). Tryptophan has a side chain containing an indole ring, e.g., with tryptophan. Methionine has a side chain containing a thioether group, e.g., with methionine. Histidine has a side chain containing an imidazole ring. A wide variety of non-standard amino acids, including naturally occurring and non-naturally occurring amino acids, are available with side chains containing one or more such reactive functional groups. See, for example, Hughes, B. (ed.), Amino Acids, Peptides and Proteins in Organic Chemistry, Volumes 1-4, Wiley-VCH (2009-2011); Blaskovich, M., Handbook on Syntheses of Amino Acids: General Routes to Amino Acids, Oxford University Press, 2010. The present invention encompasses embodiments in which one or more non-standard amino acids are used as targets for the addition of moieties containing cell-reactive functional groups. If necessary, any one or more amino acids may be protected during the synthesis of the compound. For example, one or more amino acids may be protected during reactions involving the targeted amino acid side chain. In certain embodiments where a sulfhydryl-containing amino acid is used as a target for the attachment of a moiety containing a cell-reactive functional group, the sulfhydryl is protected during cyclization of the compound by forming an intramolecular disulfide bond with another amino acid, such as cysteine.

[0145] This paragraph uses amino acids having side chains containing amine groups as examples. The present invention encompasses similar embodiments using amino acids having side chains containing different reactive functional groups. In some embodiments, an amino acid having a side chain containing a primary or secondary amine is directly attached to the N- or C-terminus of any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A via a peptide bond. In some embodiments, an amino acid having a side chain containing a primary or secondary amine is attached to the N- or C-terminus of any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A via a linkage that may include any one or more of the linkage moieties described above. In some embodiments, at least two amino acids are attached to one or both termini. The two or more attached amino acids may be attached to each other via a peptide bond, or at least some of the attached amino acids may be attached to each other via a linkage that may include any one or more of the linkage moieties described herein. Thus, in certain embodiments, a cell-reactive compstatin analog comprises a compstatin analog moiety M of the formula B1-R1-M1-R2-B2, where M1 represents any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A; either R1 or R2 is optional; at least one of R1 and R2 comprises an amino acid having a side chain containing a primary or secondary amine; and B1 and B2 are optional blocking moieties. R1 and / or R2 may be bonded to M1 by a peptide bond or a non-peptide bond. R1 and / or R2 may be bonded to a linker L. P3 For example, R1 may include a group of formula M2-L P3 and / or R2 has the formula L P3 -M2, wherein L P3 is a linker, and M2 comprises at least one amino acid having a side chain containing a primary or secondary amine. For example, M2 can be Lys or an amino acid chain containing Lys. In some embodiments, L P3may contain or consist of one or more amino acids. For example, L P3 is 1 to about 20 amino acids in length, for example, 4 to 20 amino acids in length. P3 comprises or consists of multiple Gly, Ser and / or Ala residues. P3 does not contain amino acids containing reactive SH groups, such as Cys. P3 contains an oligo(ethylene glycol) moiety and / or a saturated alkyl chain. In some embodiments, L P3 is linked to the N-terminal amino acid of M1 via an amide bond. P3 is attached to the C-terminal amino acid of M1 via an amide bond. The compound may be further extended at one or both ends by the addition of additional linkages and / or amino acids. The amino acids may be the same or different, and if different, may be independently selected. In some embodiments, two or more amino acids having side chains containing reactive functional groups are used, and the reactive functional groups may be the same or different. Two or more reactive functional groups can be used as targets for the attachment of two or more moieties. In some embodiments, two or more cell-reactive moieties are added. In some embodiments, a cell-reactive moiety and a targeting moiety are added. In some embodiments, a linker and / or cell-reactive moiety is attached to the amino acid side chain after the amino acid is incorporated into the peptide chain. In some embodiments, a linker and / or cell-reactive moiety is already attached to the amino acid side chain before the amino acid is used in the synthesis of a cell-reactive compstatin analog. For example, a Lys derivative with a linker attached to its side chain can be used. The linker may contain a cell-reactive functional group or may be later modified to contain a cell-reactive functional group.

[0146] Specific cell-reactive compstatin analogs are described in further detail below. In the following discussion, exemplary compstatin analog moieties include those having the amino acid sequence Ile-Cys *-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr (SEQ ID NO: 37) (corresponding to the compstatin analog of SEQ ID NO: 28, where in the sequence of SEQ ID NO: 37, the asterisk represents a cysteine bonded by a disulfide bond in the active compound, and (1Me)Trp represents 1-methyl-tryptophan); maleimide (abbreviated as Mal) is used as an example of a cell-reactive functional group; and (CH2) is used as an example of a binding moiety. n and (O-CH2-CH2) n lysine is used as an example of an amino acid containing a reactive functional group (in some compounds), and acetylation and amidation (in italics, Ac and NH2, respectively) at the N-terminus and C-terminus, respectively, are used as examples of blocking moieties that are optionally present in some compounds. It is understood that the compounds can be prepared using a variety of synthetic methods and a variety of precursors. The following description of various synthetic methods and precursors is not intended to limit the invention. In general, any feature of any compound described below can be freely combined with the features of other compounds described below or herein or elsewhere in this specification, and the invention encompasses such embodiments.

[0147] In some embodiments, the cell-reactive moiety is provided by a cell-reactive compound containing a maleimide group (as the cell-reactive functional group) and an alkanoic acid (RCOOH) (where R is an alkyl group), such as 6-maleimidocaproic acid (Mal-(CH)-COOH) shown below. [ka] can be used.

[0148] In some embodiments, the cell-reactive moiety is provided by a derivative of an alkanoic acid in which the carboxylic acid moiety has been activated, e.g., the OH moiety has been converted to a more favorable leaving group. For example, the carboxyl group of Compound I can be reacted with EDC, followed by NHS (optionally provided as water-soluble sulfo-NHS) to produce an N-hydroxysuccinimide ester derivative of 6-maleimidocaproic acid, i.e., 6-maleimidohexanoic acid N-hydroxysuccinimide (NHS) ester (see below). [ka] can be obtained.

[0149] The N-terminus and / or C-terminus of the compound of SEQ ID NO: 37 can be modified to provide a cell-reactive compstatin analog. For example, compound II can be used to provide the following cell-reactive compstatin analog by reaction with the N-terminal amino group of Ile: Maleimido-(CH2)5-C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 38) In SEQ ID NO: 38, it is understood that the -C(=O) moiety is linked to the adjacent C-terminal amino acid (Ile) via a C-N bond (where N is part of the amino acid and is not shown).

[0150] In another embodiment, a maleimide group is attached to the C-terminal Thr to provide the following cell-reactive compstatin analog: Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-(C=O)-(CH)-maleimide (SEQ ID NO: 39)

[0151] In some embodiments, cell-reactive compstatin analogs can be synthesized using bifunctional linkers (e.g., heterobifunctional linkers). (CH2-CH2-O) n Part and (CH2) m An example of a heterobifunctional linker comprising a moiety where m=2 is shown below: [ka]

[0152] Compound III contains a maleimide group as a cell-reactive functional group and an NHS ester moiety that readily reacts with amino groups (eg, N-terminal amino groups or amino groups on amino acid side chains).

[0153] Using the embodiment of Compound III where n=2, the compstatin analog of SEQ ID NO: 37 can be used to obtain the following cell-reactive compstatin analogs: Maleimido-(CH2)2-C(=O)-NH-CH2CH2OCH2CH2OCH2CH2C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 40)

[0154] In the compound of SEQ ID NO: 40, it is understood that the -C(=O) moiety is attached to the N-terminal amino acid (Ile residue) via a C-N bond (where N is part of the amino acid and is not shown). In some embodiments, the linker has the formula of compound III, where n is 1 or greater. (CH2-CH2-O) n Examples of values for the portion n are given here.

[0155] In certain embodiments, the alkyl chain connecting the maleimide moiety to the remainder of the molecule contains more or fewer methylene units, the oligo(ethylene glycol) moiety contains more or fewer ethylene glycol units, and / or there are more or fewer methylene units adjacent to one or both ends of the oligo(ethylene glycol) moiety compared to the compound of SEQ ID NO: 39 or SEQ ID NO: 40. To illustrate such variations, examples of cell-reactive compstatin analogs are shown below (SEQ ID NOs: 41-46).

[0156] Maleimido-(CH2)2-C(=O)-NH-CH2CH2OCH2CH2C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 41) Maleimido-(CH2)3-C(=O)-NH-CH2CH2OCH2CH2OCH2C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 42) Maleimido-(CH2)5-C(=O)-NH-CH2CH2OCH2CH2OCH2C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 43) Maleimido-(CH2)4-C(=O)-NH-CH2CH2OCH2CH2OCH2CH2C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 44) Maleimido-(CH2)2-C(=O)-NH-CH2CH2OCH2CH2OCH2CH2C(=O)-Ile-Cys *-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 45) Maleimido-(CH2)5-C(=O)-NH-CH2CH2OCH2CH2OCH2C(=O)-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2 (SEQ ID NO: 46)

[0157] In certain embodiments, SEQ ID NO: 37 is extended to include a Lys residue at the N- or C-terminus of the peptide, for example as shown below for a C-terminal linkage. Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-Lys-NH2 (SEQ ID NO: 47)

[0158] In certain embodiments, a Lys residue is attached to the N- or C-terminus of SEQ ID NO: 37 via a peptide linker, for example as shown below for a C-terminal attachment. Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-(Gly)5-Lys-NH2 (SEQ ID NO: 48)

[0159] In some embodiments, a linker containing a primary or secondary amine is attached to the N-terminus or C-terminus of the compstatin analog. In some embodiments, the linker contains an alkyl chain and / or an oligo(ethylene glycol) moiety. For example, NH2(CH2CH2O) nCHC(=O)OH (e.g., 8-amino-3,6-dioxaoctanoic acid (AEEAc) or 11-amino-3,6,9-trioxaundecanoic acid) or its NHS ester (e.g., the NHS ester of 8-amino-3,6-dioxaoctanoic acid or 11-amino-3,6,9-trioxaundecanoic acid) can be used. In some embodiments, the resulting compound is as follows (where the portion provided by the linker is shown in bold): [ka] [ka]

[0160] In some embodiments, the Lys residue is linked to the N-terminus or C-terminus of SEQ ID NO: 37 via a linker containing a non-peptide moiety. For example, the linker can include an alkyl chain, an oligo(ethylene glycol) chain, and / or a ring system. In some embodiments, 8-AEEAc or its NHS ester is used to obtain the following compound (where the portion represented by 8-AEEAc is shown in bold) (when Lys is linked at the C-terminus): [ka]

[0161] In SEQ ID NOs: 49 and 50, it is understood that the -C(=O) moiety is attached to the adjacent He residue via a C-N bond (where N is part of an amino acid and is not shown). Similarly, in SEQ ID NO: 51, the -C(=O) moiety is attached to the adjacent Lys residue via a C-N bond (where N is part of an amino acid and is not shown). It is also understood that in SEQ ID NO: 51, the NH moiety is attached to the adjacent N-terminal amino acid (Thr) via a C-N bond (where C is the carbonyl carbon of the amino acid and is not shown).

[0162] The primary amine groups of the compounds of SEQ ID NOs: 47-51 can be readily modified to provide cell-reactive compstatin analogs. For example, the compounds of SEQ ID NOs: 47-51 (or other compounds containing a primary or secondary amine and a compstatin analog moiety) can be reacted with 6-maleimidocaproic acid N-succinimidyl ester to provide the following cell-reactive compstatin analogs: Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-Lys-(C(=O)-(CH2)5-Mal)-NH2 (SEQ ID NO: 52) Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-(Gly)5-Lys--(C(=O)-(CH2)5-Mal)-NH2 (SEQ ID NO: 53) [ka] [ka] [ka]

[0163] In other embodiments, the cell-reactive compstatin analog is Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-Lys-C(=O)-CH2(OCH2CH2)2NH(C(=O)-(CH2)5-Mal)-NH2 (SEQ ID NO: 57).

[0164] The present invention provides variants of SEQ ID NOs: 38 to 57, which variants are -Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys *-Thr- is replaced by an amino acid sequence containing any other compstatin analog, for example, any of the amino acid sequences of SEQ ID NOs: 3-27, or 29-36, 37, 37A, 38A, 39A, 40A, or 41A, except that the blocking moieties present at the N- and / or C-termini of the compstatin analog are absent, replaced by a linker (which may contain a blocking moiety), or linked to different N- or C-terminal amino acids present in the corresponding variant.

[0165] Other bifunctional crosslinkers containing a maleimide as the cell-reactive moiety and an NHS ester as the amine-reactive moiety that are useful in various embodiments of the present invention include, for example, succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC); and N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). Addition of a sulfonic acid to the NHS ring provides water-soluble analogs, such as sulfo-succinimidyl (4-iodoacetyl)-aminobenzoate (sulfo-SIAB), sulfo-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC), sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), and sulfo-N-γ-maleimidobutyryl-oxysuccinimide ester (sulfo-GMBS), eliminating the need for organic solvents. In some embodiments of the invention, long-chain versions of any of the above are used, including a spacer arm between the NHS ester moiety and the rest of the molecule. The spacer can include, for example, an alkyl chain. An example is succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate].

[0166] In some embodiments, bifunctional linker is used, which comprises NHS ester (as amine reactive moiety) and iodoacetyl group (reacts with sulfhydryl group).Such linker includes, for example, N-succinimidyl (4-iodoacetyl)-aminobenzoate (SIAB);Succinimidyl 6-[(iodoacetyl)-amino] hexanoate (SIAX);Succinimidyl 6-[6-(((iodoacetyl)amino)-hexanoyl)amino] hexanoate (SIAXX);Succinimidyl 4-((iodoacetyl)amino)methyl)-cyclohexane-1-carboxylate (SIAC);Succinimidyl 6-((((4-(iodoacetyl)amino)methyl-cyclohexane-1-carbonyl)amino) hexanoate (SIACX).

[0167] In certain embodiments, bifunctional linkers containing an NHS ester (as the amine-reactive moiety) and a pyridyl disulfide group (as the cell-reactive moiety that reacts with sulfhydryl groups) are used. Examples include N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP); succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), and versions that contain a sulfonic acid on the NHS ring and / or a spacer containing an alkyl chain between the NHS ester moiety and the remainder of the molecule (e.g., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate) (LC-SPDP). Variations of such linkers containing additional or different moieties can be used. For example, longer or shorter alkyl chains can be used in the spacer, or an oligo(ethylene glycol) moiety can be used in place of the alkyl chain.

[0168] In general, cell-reactive compstatin analogs can be synthesized using a variety of methods. Cell-reactive compounds containing a cell-reactive functional group and a linker are often commercially available as preformed building blocks. For example, 6-maleimidocaproic acid and 6-maleimidocaproic acid N-hydroxysuccinimide ester can be purchased from various suppliers. Alternatively, such compounds can be synthesized using methods known in the art. See, for example, Keller O, Rudinger J. Helv Chim Acta. 58(2): 531-41, 1975 and Hashida S, et al., J Appl Biochem., 6(1-2): 56-63, 1984. For further discussion of methods and reagents useful for conjugate synthesis, see Hermanson, G. supra and references therein. The present invention generally encompasses any method for producing a compound containing a compstatin analog moiety and a cell-reactive functional group, and the resulting compounds.

[0169] In some embodiments, amino acids having linkers attached to their side chains are used in the synthesis of linear peptides. Linear peptides can be synthesized using standard methods for peptide synthesis known in the art, such as standard solid-phase peptide synthesis. The linear peptide is then cyclized (e.g., by oxidizing a Cys residue to form an intramolecular disulfide). This cyclic compound can then be reacted with a linker containing a cell-reactive functional group. In other embodiments, a moiety containing a cell-reactive functional group is reacted with the linear compound before cyclization. Generally, reactive functional groups can be protected as appropriate to prevent undesired reactions between themselves during the synthesis of cell-reactive compstatin analogs. Cell-reactive functional groups, any amino acid side chains, and / or one or both ends of the peptide can be protected during the reaction and subsequently deprotected. For example, SH groups on Cys residues and / or SH-reactive moieties, such as maleimides, can be protected until after cyclization to prevent reactions between them. Reaction conditions are selected based, at least in part, on the needs of the particular reactive functional group to obtain a reasonable yield in a reasonable time. Temperature, pH, and reagent concentrations can be adjusted to obtain the desired extent or rate of reaction. See, e.g., Hermanson, supra. The desired product can be purified to remove, for example, unreacted compounds containing cell-reactive functional groups, unreacted compstatin analogs, linkers, products of the reaction other than the desired cell-reactive compstatin analog, and other substances present in the reaction mixture. Compositions and methods for producing cell-reactive compstatin analogs and synthetic intermediates are aspects of the present invention.

[0170] In certain embodiments of the present invention, the above-described linkers are used to prepare compstatin analogs containing moieties, such as polyethylene glycol (PEG) chains or other polymers, that stabilize the compound, extend its bioavailability, increase its solubility, reduce its immunogenicity, and / or increase its resistance to degradation. Without limiting the present invention in any way, such moieties may be referred to herein as "clearance-reducing moieties" (CRMs), and compstatin analogs containing such moieties may be referred to as "long-acting compstatin analogs" (LACAs). In certain embodiments, the long-acting compstatin analogs have a mean plasma half-life of at least 1 day, e.g., 1-3 days, 3-7 days, 7-14 days, or 14-28 days, when administered intravenously to a human or non-human primate at a dose of 10 mg / kg or at a dose of about 1-3 mg / kg, 3-5 mg / kg, 5-10 mg / kg, e.g., 7 mg / kg. In certain embodiments, the long-acting compstatin analog has a mean plasma half-life of at least 1 day, e.g., 1-3 days, 3-7 days, 7-14 days, or 14-28 days, when administered subcutaneously to a human or non-human primate, e.g., at a dose of about 1-3 mg / kg, 3-5 mg / kg, 5-10 mg / kg, e.g., 7 mg / kg. In certain embodiments, the long-acting compstatin analog has a mean plasma half-life (e.g., terminal half-life) of about 4 to 10 days, 5 to 9 days, 5 to 8 days, 6 to 9 days, 7 to 9 days, or 8 to 9 days, e.g., about 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days, when administered intravenously to a human or non-human primate, e.g., at a dose of about 1 to 3 mg / kg, 3 to 5 mg / kg, or 5 to 10 mg / kg, e.g., 7 mg / kg.In certain embodiments, a long-acting compstatin analog, when administered subcutaneously to a human or non-human primate at a dose of, e.g., about 1-3 mg / kg, 3-5 mg / kg, 5-10 mg / kg, e.g., 7 mg / kg, has a mean plasma half-life (e.g., terminal half-life) of about 4-10 days, 5-9 days, 5-8 days, 6-9 days, 7-9 days, or 8-9 days, e.g., about 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. In certain embodiments, a long-acting compstatin analog is characterized by being extensively absorbed from the administration site over a period of time following subcutaneous injection, achieving blood levels comparable to those achieved by the same amount of compound administered intravenously, e.g., on or about day 1-2 after administration. In certain embodiments, blood levels at or about 2, 3, 4, 5, 6, 7, 8, or more days after subcutaneous administration are about 5%, 10%, 15%, 20%, or 25% of the blood levels achieved with the same amount of compound administered intravenously. See, for example, Figure 11, which illustrates the pharmacokinetics of intravenous and subcutaneous administration of exemplary compounds described herein after about 1-2 days of administration. In certain embodiments, the mean plasma half-life of a long-acting compstatin analog following intravenous administration of 10 mg / kg to a human or non-human primate is increased by at least 2-fold, e.g., 2-5-fold, 5-10-fold, 10-50-fold, or 50-100-fold, or 100-150-fold, or 150-200-fold, compared to a corresponding compstatin analog having the same amino acid sequence (and, if applicable, one or more blocking moieties) but without a CRM. It is understood that in various embodiments, such increased half-life may also be observed following other routes of administration, such as subcutaneous administration, and / or using other doses, e.g., other doses described herein, e.g., 20 mg / kg.

[0171] As noted above, in certain embodiments, a compstatin analog of any of SEQ ID NOS: 3-36, 37, 37A, 38A, 39A, 40A, or 41A is extended at the N-terminus, C-terminus, or both by one or more amino acids, at least one of which has a side chain containing a reactive functional group, such as a primary or secondary amine, a sulfhydryl group, a carboxyl group (which may be present as a carboxylic acid group), a guanidino group, a phenol group, an indole ring, a thioether, or an imidazole ring, that facilitates attachment of the CRM to the reactive functional group that connects the compstatin analog. It is understood that a corresponding compstatin analog that does not contain a CRM may lack one or more such amino acids present in the corresponding long-acting compstatin analog. Thus, a corresponding compstatin analog comprising any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A, and lacking a CRM, is understood to "have the same amino acid sequence" as SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A, respectively. For example, a corresponding compstatin analog comprising SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, or 3, and lacking a CRM, is understood to "have the same amino acid sequence" as SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, or 36, respectively.

[0172] In some embodiments, the plasma half-life refers to the terminal half-life after a single intravenous administration. In some embodiments, the plasma half-life refers to the terminal half-life after reaching steady state after multiple intravenous administrations. In some embodiments, the long-acting compstatin analog has a plasma C value of 0.01% or less after a single intravenous administration or multiple intravenous administrations to primates. max Plasma C of the corresponding compstatin analog without CRM max In some embodiments, the long-acting compstatin analog has a plasma C of at least 5 times, e.g., 5 to 50 times, after a single intravenous dose or multiple intravenous doses in primates. max Plasma C of the corresponding compstatin analog without CRM maxIn one embodiment, the primate is a human.

[0173] In certain embodiments, the primate is a non-human primate, eg, a monkey such as a cynomolgus monkey or a rhesus monkey.

[0174] In certain embodiments, a dose of 10 mg / kg or 20 mg / kg to a human or non-human primate results in at least a 2-fold, e.g., 2-5-fold, 5-10-fold, 10-50-fold, 50-100-fold, 100-150-fold, or 150-200-fold, reduction in renal clearance compared to the corresponding compstatin analog. It is understood that in various embodiments, such reductions in renal clearance may also be observed following other routes of administration, such as subcutaneous administration, and / or using other doses, e.g., other doses described herein, e.g., 20 mg / kg.

[0175] The concentration of compstatin analogs can be measured in blood and / or urine samples using, for example, UV, HPLC, mass spectrometry (MS), or antibodies against CRM, or a combination of such methods, such as LC / MS or LC / MS / MS. Pharmacokinetic parameters such as half-life and clearance can be determined using methods known to those skilled in the art. For example, pharmacokinetic analysis can be performed using WinNonlin software v 5.2 (Pharsight Corporation, St. Louis, MO).

[0176] In some embodiments, the CRM is stable under physiological conditions for at least 24 hours or more. In some embodiments, the CRM is stable in mammalian, e.g., primate, e.g., human or non-human primate (e.g., monkey) blood, plasma, or serum for at least 24 hours. In various embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the CRM molecules remain intact after incubation under physiological conditions for 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, or more. In various embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the CRM molecules remain intact in blood, plasma, or serum after incubation at 37°C for 48, 72, 96, 120, 144, 168, or more hours. In various embodiments, incubation can be performed using CRM at a concentration of 1 μL / ml to about 100 mg / ml. Samples can be analyzed at various time points. Size or integrity can be assessed, for example, using chromatography (e.g., HPLC), mass spectrometry, Western blot, or any other suitable method. Such stability characteristics can be conferred by the moiety attached to the CRM. In various embodiments, a long-acting compstatin analog containing a CRM can have any of the stability characteristics described above. In some embodiments, integrity with respect to a long-acting compstatin analog means that the compstatin analog moiety remains bound to the CRM and the CRM size remains about the same as at the start of incubation or administration.

[0177] In certain embodiments, a long-acting compstatin analog has a molar activity that is at least about 10%, 20%, 30%, e.g., 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% or more of the activity of a corresponding compstatin analog having the same amino acid sequence (and one or more blocking moieties, if applicable) but without a CRM. In certain embodiments in which a long-acting compstatin analog comprises multiple compstatin analog moieties, the molar activity of the long-acting compstatin analog is at least about 10%, 20%, or 30%, e.g., 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% or more of the sum of the activities of the compstatin analog moieties.

[0178] In some embodiments, the polyethylene glycol (PEG) is a (CH2CH2O) having a molecular weight of at least 500 daltons. n Includes parts. In some embodiments, the linker has an average molecular weight of about 500; 1,000; 1,500; 2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; to 100,000 daltons (CHCHO). n Includes parts.

[0179] In some embodiments, the average molecular weight of PEG is at least 20,000 daltons, up to about 100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons. "Average molecular weight" refers to the number average molecular weight. In some embodiments, (CH2CHO) n The polydispersity D of the moiety is 1.0005 to 1.50, for example, 1.005 to 1.10, 1.15, 1.20, 1.25, 1.30, 1.40 or 1.50, or any value between 1.0005 and 1.50.

[0180] In some embodiments, (CH2CH2O) n The moiety is monodisperse, (CH2CH2O)n The polydispersity of the moiety is 1.0. Such monodisperse (CH2CH2O) n Moieties are known in the art and commercially available from Quanta BioDesign (Powell, Ohio), and non-limiting examples include monodisperse moieties where n is 2, 4, 6, 8, 12, 16, 20, or 24.

[0181] In some embodiments, the compound is a compound having a plurality of (CH2CH2O) n The (CH2CH2O) moiety n The total molecular weight of the moieties is about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; to 100,000 daltons. In some embodiments, the compound or (CH2CHO) n The average combined molecular weight of the moieties is at least 20,000 daltons, and up to about 100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons. In some embodiments, the compounds are (CH2CHO) 2-amino-3-methyl-1-methyl-2 ... n In some embodiments, the compound comprises a plurality of (CH2CH2O) moieties of defined length. n The moiety is (CH2CH2O) n In some embodiments, the compound (CH2CH2O) n The average total molecular weight of the moieties is at least 20,000 daltons, up to about 100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons. In some embodiments, n is from about 30 to about 3000.

[0182] In some embodiments, a compstatin analog moiety is attached to each end of a linear PEG. For example, as described above, a bifunctional PEG can be used, with a reactive functional group at each end of the chain. In some embodiments, the reactive functional groups are the same, while in some embodiments, different reactive functional groups are present at each end.

[0183] In some embodiments, a plurality of (CH2CH2O) n The moieties are provided in a branched structure. The branches may be attached to a linear polymer backbone (e.g., a comb structure) or may originate from one or more core groups (e.g., a star structure). In some embodiments, the branched molecule is (CH2CH2O) n In some embodiments, the branched molecule has 3 to 10 chains. n In some embodiments, the branched molecule has 4 to 8 chains. n The star molecule may have 10, 9, 8, 7, 6, 5, 4, or 3 chains. In some embodiments, the star molecule originates from a core group (CH2CH2O) n The long-acting compstatin analog has 10 to 100, 10 to 50, 10 to 30, or 10 to 20 chains. Thus, in some embodiments, the long-acting compstatin analog has a (CH2CH2O) n The long-acting compstatin analog may comprise, for example, 3 to 10, e.g., 4 to 8, compstatin analog moieties attached to the chain. In some embodiments, the long-acting compstatin analog is a (CH2CH2O) analog, each of which is attached to the chain via a functional group at the end of the chain. n The chain may contain 10 to 100 compstatin analog moieties attached thereto. In some embodiments, the branches (sometimes called "arms") of a branched or star-shaped PEG contain approximately the same number of (CH2CHO) moieties. In some embodiments, at least some of the branches may be of different lengths. In some embodiments, one or more (CH2CHO) nIt is understood that a chain does not have a compstatin analog moiety attached thereto. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the chains have a compstatin analog moiety attached thereto.

[0184] In the classes and compounds described herein, the polyethylene glycol moiety is depicted with the oxygen atom to the right of the repeating unit or to the left of the repeating unit. Even if only one orientation is depicted, the present invention relates to the polyethylene glycol moiety in both orientations of a given compound or class of compounds (i.e., (CHCHO) n and (OCH2CH2) n ), and when a compound or group of compounds contains multiple polyethylene glycol moieties, all combinations of orientations are encompassed by the present disclosure.

[0185] Some exemplary formulas of monofunctional PEGs containing reactive functional groups are illustrated below. For illustrative purposes, formulas are shown in which the reactive functional group includes an NHS ester, although other reactive functional groups, such as those described above, can also be used. In one embodiment, (CH2CHO) n is depicted as terminating at the left end in a methoxy group (OCH), it is understood that the chains depicted below or elsewhere herein may terminate in a different OR moiety (e.g., an aliphatic group, an alkyl group, a lower alkyl group, or any other suitable PEG terminal group) or an OH group. Also, in various embodiments, moieties other than those depicted may be (CHCHO). n It is also understood that the moiety and the NHS group can be attached.

[0186] In some embodiments, the monofunctional PEG has the formula: [ka] is a PEG of the formula: "Reactive functional group" and n are defined above and as described in the classes and subclasses herein; R 1is hydrogen, aliphatic, or any suitable terminal group; T is a covalent bond or one or more carbon units of T are optionally independently -O-, -S-, -N(R x )-, -C(O)-, -C(O)O-, -OC(O)-, -N(R x )C(O)-, -C(O)N(R x )-, -S(O)-, -S(O)2-, -N(R x )SO2- or SO2N(R x )- replaced by C 1-12 is a linear or branched hydrocarbon chain; R x are each independently hydrogen or C 1-6 It is aliphatic.

[0187] An example of a monofunctional PEG of formula A is [ka] Includes.

[0188] In Formula I, the moiety containing the reactive functional group has the general structure -CO-(CH) m In some embodiments, the monofunctional PEG has the structure of Formula I, where m is 1 to 10, e.g., 1 to 5. For example, in some embodiments, m is 3, as shown below. [ka]

[0189] [ka] In Formula II, the moiety containing the reactive functional group has the general structure -(CH) m In some embodiments, the monofunctional PEG has the structure of Formula II, where m is 1 to 10 (e.g., m is 5, as shown in Formula III below), or m is 0 (as shown in Formula IIIa below). [ka] [ka]

[0190] In some embodiments, a bifunctional linear PEG contains moieties containing reactive functional groups at both ends. The reactive functional groups may be the same (homobifunctional) or different (heterobifunctional). In some embodiments, the bifunctional PEG may have a symmetric structure, in which the reactive functional groups and -(CHCHO) n The oxygen atoms at each end of the chain are connected using the same moiety. In some embodiments, the two reactive functional groups and the PEG portion of the molecule are connected using different moieties. The structure of an exemplary bifunctional PEG is illustrated below. For illustrative purposes, a formula is shown in which the reactive functional group comprises an NHS ester, although other reactive functional groups can be used.

[0191] In one embodiment, the bifunctional linear PEG has the formula B [ka] wherein T and the "reactive functionality" are each independently as defined above and described in the classes and subclasses herein, and n is as defined above and described in the classes and subclasses herein.

[0192] An example of a bifunctional PEG of formula B is [ka] Includes.

[0193] In Formula IV, the moiety containing the reactive functional group has the general structure -(CH) mIn some embodiments, the bifunctional PEG has the structure of Formula IV, where m is 1 to 10, e.g., 1 to 5. In some embodiments, m is 0, e.g., in some embodiments, the moiety containing the reactive functional group has the general structure -COO-NHS. For example, in some embodiments, the bifunctional PEG has the structure of Formula IVa, shown below: [ka]

[0194] [ka]

[0195] In Formula V, the moiety containing the reactive functional group has the general structure -CO-(CH) m In some embodiments, the bifunctional PEG has the structure of Formula V, where m is 1 to 10, e.g., 1 to 5. In some embodiments, m is 2, for example, as shown below. [ka]

[0196] In some embodiments, the present invention provides a compstatin analog conjugate linked to a polymer. In some embodiments, the present invention provides compstatin analog conjugates of the PEG-containing compounds and compounds described herein. In some embodiments, such conjugates are prepared by reacting a functional group (e.g., an amine group, a hydroxyl group, or a thiol group) on a compstatin analog with a PEG-containing compound having a "reactive functional group" as described herein. By way of example, Formulas III and IV may each have the following structure: [ka] or [ka] A compstatin analog conjugate having the formula: [ka] represents the point of attachment of an amine group on a compstatin analog. In some embodiments, the amine group is a lysine side chain group.

[0197] It is understood that any of the PEG-containing compounds and compounds described herein can be used to form corresponding conjugates, depending on the selection of the reactive functional group and / or the compstatin functional group. For example, Formulas IVa and Va can form compstatin analog conjugates having the following structures, respectively: [ka] [ka]

[0198] In certain embodiments, the PEG component of such conjugates has an average molecular weight of about 20 kD to 100 kD, about 20 kD to 90 kD, about 20 kD to 80 kD, about 20 kD to 70 kD, about 20 kD to 60 kD, about 20 kD to 50 kD, about 30 kD to 80 kD, about 30 kD to 70 kD, about 30 kD to 60 kD, about 30 kD to 50 kD, about 30 kD to 45 kD, about 35 kD to 50 kD, about 35 kD to 45 kD, about 36 kD to 44 kD, about 37 kD to 43 kD, about 38 kD to 42 kD, or about 39 kD to 41 kD. In certain embodiments, the PEG component of such conjugates has an average molecular weight of about 40 kD.

[0199] The terms "bifunctional" or "bifunctionalized" may be used to refer to a compound comprising two compstatin analog moieties linked to a CRM. Such compounds may be designated by the letters "BF." In certain embodiments, a bifunctional compound is symmetrical. In certain embodiments, the bond between the CRM and each compstatin analog moiety of a bifunctional compound is the same. In certain embodiments, each bond between the CRM and the compstatin analog of a bifunctional compound comprises a carbamate. In certain embodiments, each bond between the CRM and the compstatin analog of a bifunctional compound comprises a carbamate and does not comprise an ester. In certain embodiments, each compstatin analog of a bifunctional compound is directly linked to a CRM via a carbamate. In certain embodiments, each compstatin analog of a bifunctional compound is directly linked to a CRM via a carbamate, and the bifunctional compound has the following structure: [ka]

[0200] In certain embodiments and embodiments of the formulae described herein, [ka] is the structure [ka] where the symbol "~" represents the point of attachment of the chemical moiety to the remainder of the molecule or chemical formula.

[0201] In some embodiments, the branched comb or star PEG has a reactive functional group-containing moiety, each of which is a plurality of -(CH2CH2O) n The reactive functional groups may be the same or there may be at least two different groups. In some embodiments, the branched, comb, or star PEG has the formula: [ka] [ka] [ka] [ka] [ka] [ka] wherein R 2 are each independently a "reactive functional group" or R 1 where T, n, and "reactive functionality" are each independently as defined above and as described in the classes and subclasses herein. The structure of an example branched PEG (having 8 arms or branches) containing an NHS moiety as a reactive functionality is illustrated below.

[0202] [ka] [ka]

[0203] The structure of an example branched PEG (having four arms or branches) containing an NHS moiety as a reactive functional group is illustrated below. [ka] [ka]

[0204] The number of branches emanating from the backbone can vary. For example, the number of branches in Formulas VI and VII above, which is 4, can be changed in various embodiments to any other integer from 0 to 10. In some embodiments, one or more branches do not contain a reactive functional group, and the branch terminates in a —CHCHOH group or a —CHCHOR group, as described above.

[0205] In certain embodiments, the branched PEG has the structure of Formula VII, VIII, or IX (or variants thereof with different numbers of branches), where x is [ka] is.

[0206] In certain embodiments, the branched PEG has the structure of Formula VII, VIII, or IX (or variants thereof with different numbers of branches), provided that x is [ka] is.

[0207] Of course, the methylene (CH2) group in the x portion may be replaced by a longer alkyl chain (CH2). m (where m is 2, 3, 4, 5, 6, 8, 10, 20, or 30 or less), or may include one or more other moieties described herein.

[0208] In one embodiment, an example of a branched PEG having an NHS reactive group or a maleimide reactive group is illustrated as follows: [ka] [ka] In certain embodiments, variants of formula X or XI are used in which three of the four branches or each branch contains a reactive functional group.

[0209] Yet another example of PEG is represented as follows: [ka] [ka]

[0210] As described above and as described herein, in various embodiments, the peptide component of a long-acting compstatin analog and (CH2CH2O) n It is understood that any of a variety of moieties, such as linear alkyl, ester, amide, aromatic ring (e.g., substituted or unsubstituted phenyl), substituted or unsubstituted cycloalkyl structure, or combinations thereof, can be incorporated between the -R moiety. In certain embodiments, such moieties increase the compound's susceptibility to hydrolysis, which can cause the peptide portion of the compound to dissociate from the CRM. In certain embodiments, such dissociation can increase in vivo tissue permeability and / or activity of the compound. In certain embodiments, the hydrolysis is general (e.g., acid-base) hydrolysis. In certain embodiments, the hydrolysis is enzyme-catalyzed, for example, esterase-catalyzed. Of course, both types of hydrolysis can occur. Examples of PEGs containing one or more such moieties and an NHS ester as a reactive functional group include: [ka] [ka] [ka]

[0211] In certain embodiments, the branched (multi-arm) or star-shaped PEG comprises a pentaerythritol nucleus, a hexaglycerol nucleus, or a tripentaerythritol nucleus, it being understood that in certain embodiments, the branches may not all originate from a single point.

[0212] Monofunctional, bifunctional, branched, and other PEGs containing one or more reactive functional groups can, in certain embodiments, be obtained from, for example, NOF America Corp. White Plains, NY or BOC Sciences 45-16 Ramsey Road Shirley, NY 11967, USA, among others, or prepared using methods known in the art.

[0213] In some embodiments, the bond between the CRM and the compstatin analog comprises a carbamate. In some embodiments, the compstatin analog is directly bonded to the CRM via a carbamate. In some embodiments, the bond between the CRM and the compstatin analog does not comprise an ester. In some embodiments, the bond between the CRM and the compstatin analog comprises a carbamate and does not comprise an ester. In some embodiments, the bond between the CRM and the compstatin analog comprises a carbamate and does not comprise a bond that is more susceptible to hydrolysis than a carbamate in an aqueous medium. In some embodiments, the CRM comprises or consists of a PEG moiety.

[0214] In some embodiments, the bond between the CRM and the compstatin analog comprises an amide. In some embodiments, the compstatin analog is directly bonded to the CRM via an amide. In some embodiments, the bond between the CRM and the compstatin analog comprises an amide and does not comprise an ester. In some embodiments, the bond between the CRM and the compstatin analog comprises an amide and does not comprise a bond that is more susceptible to hydrolysis than an amide in an aqueous medium. In some embodiments, the CRM comprises or consists of a PEG moiety.

[0215] In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to a CRM via a carbamate-containing bond. In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to a CRM via a bond that does not contain an ester. In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to a CRM via a carbamate-free bond. In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to a CRM via a bond that does not contain a bond that is more susceptible to hydrolysis than a carbamate in aqueous media. In some embodiments, each compstatin analog of a multifunctionalized compound (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compound) is linked directly to a CRM via a carbamate.

[0216] In some embodiments, the CRM comprises or consists of a PEG moiety. In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to the CRM through an amide-containing bond. In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to the CRM through an amide-containing bond that does not contain an ester. In some embodiments, one or more of the compstatin analogs that are multifunctionalized compounds (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compounds) are linked to the CRM through an amide-containing bond that does not contain a bond that is more susceptible to hydrolysis than an amide in aqueous media. In some embodiments, each compstatin analog of a multifunctionalized compound (e.g., bifunctionalized, trifunctionalized, or more extensively functionalized compound) is directly linked to the CRM through an amide. In some embodiments, the CRM comprises or consists of a PEG moiety.

[0217] In some embodiments, the present invention provides a compstatin analog conjugated to a polymer other than PEG. In some embodiments, the polymer is polyoxazoline (POZ). Examples of mono- and poly-functionalized polyoxazoline derivatives for direct conjugation or conjugation via a linker are shown below. ZT-[N(COR x )CH2CH2] n -TR 1 ; R 1 -{[N(CO-TZ)CH2CH2] m -[N(COR x )CH2CH2] n} a -TR 1 ; R 1 -{[N(CO-TZ 1 )CH2CH2] p -[N(COR x )CH2CH2] n -[N(CO-TZ 2 )CH2CH2] m} a -TR 1 ; R 1 -{[N(CO-TZ 1 )CH2CH2] p -[N(COR x )CH2CH2] n -[N(CO-TZ 2 )CH2CH2] m} a -TZ; R 1 -[N(COR x )CH2CH2] n -TB(-R 1 )(-TZ)-T-[N(COR x )CH2CH2] m -R 1 [During the ceremony Z, Z 1 and Z 2 each of which is a reactive functional group as defined above and described in groups or subgroups herein; T, R x and R 1 each of which is defined above and described in groups or subgroups herein; Each of m, n, and p is an integer between 0 and 1000, provided that the sum of m, n, and p in each formula is not 0; a is "ran" indicating a random copolymer or "block" indicating a block copolymer; B is a branching moiety that is attached to other parts of the polymer with or without a linker.

[0218] Other examples of functionalized polyoxazoline derivatives for attachment include, but are not limited to, those described in PCT Patent Application Publication Nos. WO / 2010 / 006282, WO / 2009 / 089542, WO / 2009 / 043027, and WO / 2008 / 106186, the contents of each of which are incorporated herein by reference.

[0219] An example of a compstatin analog conjugate with a polyoxazoline polymer is shown below. [ka] [ka] [ka] [ka] wherein each variable is as defined above and described in groups or subgroups herein.

[0220] In some embodiments, the present invention provides a polymer-linked compstatin analog, wherein the compstatin analog is linked to the polymer via one or more linkers. In some embodiments, the polymer is a PEG-containing compound and group described above and in the groups or subgroups. In some embodiments, the present invention provides a compstatin analog conjugate of a PEG-containing compound and group described herein, wherein the compstatin analog is linked to the PEG-containing moiety via one or more linkers. Mono- and polyfunctional PEGs containing one or more reactive functional groups for linkage are defined above and described herein in groups or subgroups, including, but not limited to, those of formula A, I, Ia, II, III, IIIa, B, IV, IVa, V, Va, C, D, E, F, G, H, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, or XVI.

[0221] Suitable linkers for linking a compstatin analog to a polymer moiety, such as PEG or polyoxazoline, are broadly described above and in groups and subgroups herein. In some embodiments, the linker has multiple functional groups, where one functional group is attached to the compstatin analog and the other is attached to the polymer moiety. In some embodiments, the linker is a bifunctional compound. In some embodiments, the linker is NH2(CH2CH2O) n The linker has the structure CHC(=O)OH (where n is 1 to 1000). In some embodiments, the linker is 8-amino-3,6-dioxaoctanoic acid (AEEAc). In some embodiments, the linker is activated for conjugation with a functional group of a polymer moiety or a compstatin analog. For example, in some embodiments, the carboxyl group of AEEAc is activated with the amine group of the side chain of a lysine group prior to conjugation.

[0222] In some embodiments, a suitable functional group (e.g., an amine group, a hydroxyl group, a thiol group, or a carboxylic acid group) on the compstatin analog is used for conjugation to the polymer moiety directly or via a linker. In some embodiments, the compstatin analog is conjugated to the PEG moiety via an amine group via a linker. In some embodiments, the amine group is the alpha amino group of an amino acid residue. In some embodiments, the amine group is the amine group of a lysine side chain. In some embodiments, the compstatin analog is conjugated to the PEG moiety via NH(CHCHO). n The compstatin analog is attached to the PEG moiety through the amino group of a lysine side chain (ε-amino group) via a linker having the structure CHC(=O)OH (where n is 1 to 1000). In some embodiments, the compstatin analog is attached to the PEG moiety through the amino group of a lysine side chain via an AEEAc linker. In some embodiments, the compstatin analog is attached to the PEG moiety through the amino group of a lysine side chain via an AEEAc linker. n The CH2C(=O)OH linker, after attachment, attaches -NH(CH2CH2O) to the compstatin lysine side chain. n Inserts a -CH2C(=O)- moiety. In certain embodiments, the AEEAc linker, after attachment, inserts a -NH(CH2CH2O)2CH2C(=O)- moiety into a compstatin lysine side chain.

[0223] In some embodiments, the compstatin analog is attached to the polymer moiety via a linker, wherein the linker comprises an AEEAc moiety and an amino acid residue. In some embodiments, the compstatin analog is attached to the polymer moiety via a linker, wherein the linker comprises an AEEAc moiety and a lysine residue. In some embodiments, the polymer is PEG. In some embodiments, the C-terminus of the compstatin analog is attached to the amino group of AEEAc, and the C-terminus of AEEAc is attached to the lysine residue. In some embodiments, the C-terminus of the compstatin analog is attached to the amino group of AEEAc, and the C-terminus of AEEAc is attached to the alpha-amino group of the lysine residue. In some embodiments, the C-terminus of the compstatin analog is attached to the amino group of AEEAc, and the C-terminus of AEEAc is attached to the alpha-amino group of the lysine residue, and a polymer moiety such as a PEG moiety is attached via the epsilon-amino group of the lysine residue. In some embodiments, the C-terminus of the lysine residue is modified. In some embodiments, the C-terminus of the lysine residue is modified by amidation. In some embodiments, the N-terminus of the compstatin analog is modified. In certain embodiments, the N-terminus of the compstatin analog is acetylated.

[0224] Examples of conjugates comprising an AEEAc linker and a polymer are described below, where: [ka] indicates the point of attachment of the amine group on the compstatin analog; [ka] represents a compstatin analog linked through its C-terminus, and each of the other variables is independently as defined above and described in groups and subgroups herein. In certain embodiments, the amine group is a lysine side chain amino group. [ka] [ka]

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[0225] In certain embodiments, a compstatin analog can be represented as M-AEEAc-Lys-B2, where B2 is a blocking moiety, e.g., NH2, and M is any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A, provided that the C-terminal amino acid of any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A is attached to AEEAc-Lys-B2 via a peptide bond. The NHS moiety of a mono- or polyfunctional (e.g., bifunctional) PEG reacts with the free amine of a lysine side chain to produce a mono-functionalized (single compstatin analog moiety) or poly-functionalized (multiple compstatin analog moieties) long-acting compstatin analog. In various embodiments, any amino acid containing a side chain containing a reactive functional group can be used in place of (or in addition to) Lys. Mono- or polyfunctional PEGs containing suitable reactive functional groups can be reacted with such side chains in a manner analogous to the reaction of NHS-ester activated PEG with Lys.

[0226] It is understood that in any of the above formulas and structures, embodiments in which the compstatin analog component comprises any of the compstatin analogs described herein, e.g., any of the compstatin analogs set forth in SEQ ID NOS: 3-36, 37, 37A, 38A, 39A, 40A, and 41A, are expressly disclosed. By way of example, and without limitation, a compstatin analog may comprise the amino acid sequence of SEQ ID NO: 28. An example of a long-acting compstatin analog in which the compstatin analog component comprises the amino acid sequence of SEQ ID NO: 28 is shown in Figure 10(C). It is understood that the PEG moiety, in various embodiments, can have a variety of different molecular weights or average molecular weights, as described herein. For example, as described herein, individual PEG chains in a formulation may differ in molecular weight and / or different formulations may have different average molecular weights and / or polydispersities. In some embodiments, the PEG moiety in the compound of Figure 10(C) has an average molecular weight of about 20 to 100 kD, about 20 to 90 kD, about 20 to 80 kD, about 20 to 70 kD, about 20 to 60 kD, about 20 to 50 kD, about 30 to 80 kD, about 30 to 70 kD, about 30 to 60 kD, about 30 to 50 kD, about 30 to 45 kD, about 35 to 50 kD, about 35 to 45 kD, about 36 to 44 kD, about 37 to 43 kD, about 38 to 42 kD, or about 39 to 41 kD. In some embodiments, the PEG moiety in the compound of Figure 10(C) has an average molecular weight of about 30 to about 50 kD, e.g., about 35 to about 45 kD, or about 37.5 to about 42.5 kD. In some embodiments, the compound has an average molecular weight of about 40 kD, e.g., 37.5-42.5 kD, 38 kD, 39 kD, 40 kD, 41 kD, or 42 kD, and is sometimes referred to herein as CA28-2TS-BF. In some embodiments, the CRM, e.g., a compound comprising a PEG moiety, has an average molecular weight of about 40 kD, e.g., 37.5-42.5 kD, 38 kD, 39 kD, 40 kD, 41 kD, or 42 kD, and when administered intravenously or subcutaneously to a non-human primate or human, e.g., at a dose of about 1-3 mg / kg, 3-5 mg / kg, or 5-10 mg / kg, has a terminal half-life of at least about 5 days, e.g., about 5-10 days, e.g., about 5, 6, 7, 8, or 9 days.

[0227] In some embodiments, the present invention relates to the use of click chemistry in connection with compstatin analogs. "Click chemistry" is well known in the art and is useful in certain embodiments of the present invention. In some embodiments, click chemistry embodies a versatile cycloaddition reaction between azides and alkynes, enabling many useful applications. Methods for performing click chemistry are known in the art and are described in Kolb, HC; Sharpless, KB, Drug Disc. Today, 2003, 1128-1137; Moses, JE; Moorhouse, AD; Chem. Soc. Rev., 2007, 1249-1262, each of which is incorporated herein by reference in its entirety. Click chemistry is a popular method for bioconjugation due to its high reactivity and selectivity, even in biological media. See Kolb, HC; Finn, MG; Sharpless, KB Angew. Chem. Int. Ed. 2001, 40, 2004-2021; and Wang, Q.; Chan, TR; Hilgraf, R.; Fokin, VV; Sharpless, KB; Finn, MGJ Am. Chem. Soc. 2003, 125, 3192-3193. Furthermore, currently available recombinant technologies and synthetic methods allow for the introduction of azide- and alkyne-containing nonclassical amino acids into peptides, proteins, cells, viruses, bacteria, and other protein- or protein-displaying biological materials. Link, AJ; Vink, MKS; Tirrell, DAJ Am. Chem. Soc. 2004, 126, 10598-10602; Deiters, A.; Cropp, TA; Mukherji, M.; Chin, JW; Anderson, C.; Schultz, PGJ Am. Chem. Soc. 2003, 125, See 11782-11783.

[0228] As used herein, the term "click chemistry group" may be used to refer to a reactive functional group that can participate in a click chemistry reaction with an appropriate second reactive functional group, where the second reactive functional group is also a click chemistry group. The first and second click chemistry groups or substances (e.g., molecules) containing such groups may be referred to as complementary. The first and second substances, e.g., molecules, containing complementary click chemistry groups may be referred to as click chemistry partners. A substance or molecule containing a click chemistry group may be referred to as "click functionalized." The bond formed by the reaction of complementary click chemistry partners may be referred to as a "click chemistry bond."

[0229] In some embodiments, the present invention provides click-functionalized compstatin analogs, e.g., for conjugation to a complementary moiety on a partner molecule or biomolecule. In some embodiments, the complementary partner molecule or biomolecule is a polymer, peptide, protein, or molecule that functions as a clearance-reducing moiety. In some embodiments, the "click-functionalized" moiety is an alkyne or alkyne derivative that can undergo a [3 + 2] cycloaddition reaction with complementary azide-containing molecules and biomolecules. In other embodiments, the "click-functionalized" functionality is an azide or azide derivative that can undergo a [3 + 2] cycloaddition reaction (i.e., click chemistry) with complementary alkyne-containing molecules and biomolecules.

[0230] In some embodiments, the click-functionalized compstatin analog has an azide group on any of the side groups of the compstatin analog, hi some embodiments, the click-functionalized compstatin analog has an azide group on a lysine side group.

[0231] In some embodiments, the click-functionalized compstatin analog has an alkyne group on one of the side groups of the compstatin analog. In some embodiments, the click-functionalized compstatin analog has an alkyne group on a lysine side group.

[0232] In some embodiments, the present invention provides a compstatin conjugate comprising a compstatin analog, a molecule that functions as a clearance-reducing moiety, and a triazole linker. In some embodiments, the triazole linker is the result of click-linking chemistry between the compstatin conjugate and the molecule that functions as a clearance-reducing moiety. In some embodiments, the CRM can be any CRM disclosed herein. For example, the CRM can be PEG, a polypeptide, or POZ.

[0233] In certain embodiments, the present invention provides a compstatin conjugate comprising a compstatin analog, a PEG moiety, and a triazole linker. In certain embodiments, the triazole linker is the result of click-linking chemistry between the compstatin conjugate and the PEG moiety.

[0234] In some embodiments, the present invention provides a compstatin conjugate comprising a compstatin analog, a polyoxazoline moiety, and a triazole linker. In some embodiments, the triazole linker is the result of click-linking chemistry between the compstatin conjugate and the polyoxazoline moiety.

[0235] In some embodiments, click chemistry between a compstatin analog and another moiety is catalyzed by a transition metal. Copper-containing molecules that catalyze the "click" reaction include, but are not limited to, copper wire, copper bromide (CuBr), copper chloride (CuCl), copper sulfate (CuSO), copper sulfate pentahydrate (CuSO·5H0), copper acetate (Cu(AcO), copper iodide (CuI), [Cu(MeCN)](OTf), [Cu(MeCN)](PF), colloidal copper sources, and immobilized copper sources. In some embodiments, other metals are used, such as ruthenium. Reducing agents and organic and inorganic metal-binding ligands can be used with the metal catalyst, including, but not limited to, sodium ascorbate, tris(triazolyl)amine ligands, tris(carboxyethyl)phosphine (TCEP), bathophenanthroline sulfonic acid ligands, and benzimidazole-based ligands.

[0236] In some embodiments, compstatin analogs are coupled to other moieties using metal-free click chemistry (also known as copper-free click chemistry) to yield metal-free compositions or conjugates. In contrast to standard click chemistry, also known as copper-assisted click chemistry (CuACC), metal-free click chemistry occurs between strained, cyclic alkynes or alkyne precursors, such as oxanorbornadiene, and azide groups. As the name suggests, no metal catalyst is required for the reaction to occur. Examples of such chemistries include reactions involving cyclooctyne derivatives (Codelli, et. al. J. Am. Chem. Soc., 2008, 130, 11486-11493; Jewett, et. al. J. Am. Chem. Soc., 2010, 132, 3688-3690; Ning, et. al. Angew. Chem. Int. Ed., 2008, 47, 2253-2255), difluoro-oxanorbornene derivatives (van Berkel, et. al. ChemBioChem, 2007, 8, 1504-1508), or nitrile oxide derivatives (Lutz, et. al. Macromolecules, 2009, 42, 5411-5413). In some embodiments, the metal-free click chemistry reaction is a metal-free [3+2] cycloaddition reaction, a Diels-Alder reaction, or a thiol-alkene radical addition reaction. Examples of click chemistry reactions and click chemistry groups are described, for example, in Joerg Lahann, Click Chemistry for Biotechnology and Materials Science, 2009, John Wiley & Sons Ltd, ISBN 978-0-470-69970-6; Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900-4908. In some embodiments, the click chemistry group comprises a diarylcyclooctyne.

[0237] Certain examples of metal-free click chemistry are shown in the scheme below. [ka]

[0238] Certain metal-free click moieties are known in the literature. An example is 4-dibenzocyclooctynol (DIBO). [ka] (Ning et. al; Angew Chem Int Ed, 2008, 47, 2253); difluorocyclooctynes (DIFO or DFO) [ka] (from Codelli, et. al.; J. Am. Chem. Soc. 2008, 130, 11486-11493); biarylazacyclooctynone (BARAC) [ka] (Jewett et. al.; J. Am. Chem. Soc. 2010, 132, 3688); or bicyclononyne (BCN) [ka] (Dommerholt, et. al.; Angew Chem Int Ed, 2010, 49, 9422-9425) or dibenzylcyclooctyne (DBCO) [ka] Includes.

[0239] The reaction scheme involving the reaction of DBCO with azide is shown below. [ka]

[0240] In the above scheme, in various embodiments, A may comprise or consist of a compstatin analog moiety and B may comprise or consist of a CRM, e.g., a polymer or polypeptide such as PEG or POZ, or B may comprise or consist of a compstatin analog moiety and A may comprise or consist of a CRM, e.g., a polymer or polypeptide such as PEG or POZ.

[0241] In certain embodiments, the "metal-free click-functionalized" moiety is an acetylene or acetylene derivative that can undergo a [3+2] cycloaddition reaction with complementary azide-containing molecules and biomolecules without the use of a metal catalyst.

[0242] In some embodiments, the R and R' groups of the metal-free click chemistry reagent can be a compstatin analog or any molecule described herein to which a compstatin analog can be attached. In some embodiments, such compstatin analogs have a click-functionalized moiety on a lysine side chain. In some embodiments, such compstatin analogs are attached to a click-functionalized moiety via a linker. In some embodiments, such compstatin analogs are attached to a click-functionalized moiety via AEEAc.

[0243] In some embodiments, the click chemistry reagent comprises DBCO. Examples of the reagent and its use are provided below. [ka] DBCO-acid. In certain embodiments, DBCO-acid may be used for reaction with an amine-containing moiety.

[0244] [ka] DBCO-NHS ester (above) or DBCO-sulfo-NHS ester (below), The DBCO functionality can be used for incorporation into amine-containing molecules such as compstatin analogs or polypeptides containing lysine residues. [ka]

[0245] [ka] DBCO-PEG4-NHS ester. In some embodiments, such reagents are useful for incorporating DBCO moieties by reaction with available amine functionality. In some embodiments, the presence of a PEG chain as a hydrophilic spacer can be useful, for example, to increase solubility or provide flexibility.

[0246] [ka] DBCO-amine. In certain embodiments, the click chemistry reagent comprises a carbonyl / carboxyl reactive dibenzylcyclooctyne that can react with acids, active esters, and / or aldehydes.

[0247] In some embodiments, the click chemistry reaction involves cyclooctyne: [ka]

[0248] In certain embodiments, click chemistry reactions include the reaction of nitrones with cyclooctynes (see, e.g., Ning, Xinghai; Temming, Rinske P.; Dommerholt, Jan; Guo, Jun; Ania, Daniel B.; Debets, Marjoke F.; Wolfert, Margreet A.; Boons, Geert-Jan et al. (2010). "Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition". Angewandte Chemie International Edition 49 (17): 3065), oxime / hydrazone formation from aldehydes and ketones, and tetrazine ligation (see, e.g., Blackman, Melissa L.; Royzen, Maksim; Fox, Joseph M. (2008). "The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity". Journal of the American Chemical Society 130 (41): 13518-9), tetrazole ligation, isonitrile-based click reactions (see, e.g., Stackmann, Henning; Neves, Andre A.; Stairs, Shaun; Brindle, Kevin M.; Leeper, Finian J. (2011). “Exploring isonitrile-based click chemistry for ligation with biomolecules”. Organic & Biomolecular Chemistry 9 (21): 7303), and quadricyclane ligation (see, e.g., Sletten, Ellen M.; Bertozzi, Carolyn R. (2011). “A Bioorthogonal Quadricyclane Ligation”.(See Journal of the American Chemical Society 133(44):17570-3). In one embodiment, the click chemistry reaction is Staudinger ligation (phosphine-azide).

[0249] Any compstatin analog can be modified to incorporate a click chemistry group in various embodiments. For example, a compstatin analog containing any of the sequences set forth in SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A can be so modified. In certain embodiments, any such sequence further contains a lysine residue or an AEEAc-Lys moiety, for example, at the C-terminus. In certain embodiments, the click chemistry group is inserted after peptide synthesis. For example, to insert an azide moiety as a click chemistry group, the Lys side chain can be reacted with azidoacetic acid. In certain embodiments, the click chemistry group is inserted after cyclization, and in certain embodiments, after addition of a blocking moiety at the N-terminus and / or C-terminus. In certain embodiments, the click chemistry group is inserted during peptide synthesis. For example, an amino acid containing a side chain containing a click chemistry group can be used in the synthesis of a compstatin analog. A variety of such amino acids are commercially available from many sources, e.g., AAPPTec (Louisville, KY), Jena Bioscience GmbH (Jena, Germany). In certain embodiments, methods for preparing click chemistry-functionalized compstatin analogs are provided herein.

[0250] In some embodiments, a composition is provided comprising a compstatin analog and a click chemistry reagent. The click chemistry reagent can be any molecule capable of reacting with an amino acid side chain or terminus of a compound comprising a compstatin analog to introduce a click chemistry group, e.g., any click chemistry group known in the art. In some embodiments, the composition can be incubated under appropriate conditions to functionalize the compstatin analog with the click chemistry functionality, which may include providing a suitable catalyst, light (e.g., UV), etc. In some embodiments, the present invention provides a compstatin analog comprising any click chemistry group, including, but not limited to, those described herein. In some embodiments, a method for producing a long-acting compstatin analog is provided. In some embodiments, the method comprises mixing a compstatin analog comprising a first click chemistry group with a CRM comprising a complementary click chemistry group under conditions suitable for the click chemistry reaction to occur. The resulting conjugate may further comprise a step of purifying the conjugate. In some embodiments, purification comprises removing at least some unreacted components, e.g., with a suitable scavenger.

[0251] In some embodiments, click chemistry is used to conjugate two or more CRMs, at least two of which are conjugated with compstatin analog moieties. The compstatin analog moieties may be the same or different in various embodiments. The compstatin analog moieties may or may not be conjugated to a CRM via click chemistry. For example, in some embodiments, a first heterobifunctional PEG containing a first click chemistry group at its first end and an NHS ester at its second end is coupled to the compstatin analog moiety via an NHS ester. In a separate reaction, a second heterobifunctional PEG containing a second click chemistry group at its first end and an NHS ester at its second end is coupled to the compstatin analog moiety via an NHS ester. The resulting two compounds are then reacted via click chemistry to form a large molecule containing two compstatin analog moieties. Although PEG is described as an example of a CRM, it should be understood that this method can be used with any CRM. For example, in some embodiments, a CRM containing a polypeptide, such as HSA or a portion thereof, or albumin or an albumin-binding peptide, or an antibody or a portion thereof, can be used. In some embodiments, POZ may be used in this manner.

[0252] Compstatin analogs containing click chemistry groups can have a variety of uses. In some embodiments, a compstatin analog containing a first click chemistry group is reacted with any substance containing a complementary click chemistry group. Substances containing complementary click chemistry groups can include, for example, labels (e.g., fluorophores, fluorescent proteins, radioisotopes, etc.), affinity reactants, antibodies, targeting moieties, metals, particles, etc. In some embodiments, the click chemistry group is used to attach the compstatin analog moiety to a surface, which contains or has been functionalized to contain the complementary click chemistry group. In some embodiments, the surface is a sensor, e.g., a surface or sensor for capturing / detecting C3. In some embodiments, the surface forms part of a medical device, tubing, membrane, reservoir, implant, or other material that may come into contact with blood (e.g., outside the body) or that may be temporarily or indefinitely implanted within a subject's body (e.g., a prosthetic organ or drug delivery device). In some embodiments, the surface is functionalized with a compstatin analog to reduce complement activation thereon. In some embodiments, the device or tubing is used in blood circulation, e.g., for dialysis, during surgery, etc. In some embodiments, the device is a hemodialyzer or extracorporeal circulation support unit. Such compstatin analog-functionalized devices and methods for their manufacture are provided herein.

[0253] In some embodiments of the invention, a compstatin analog comprises both a cell-reactive functional group and a CRM. In some embodiments, the invention provides a compstatin analog (CH2CHO) in which the cell-reactive functional group or moiety has a molecular weight of at least 500 daltons, e.g., at least 1,500 daltons, up to about 100,000 daltons (e.g., an average molecular weight of about 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; or 100,000 daltons). n Molecular variants of any of the above cell-reactive compstatin analogs are provided in which a moiety (e.g., any of the PEGs described herein) or other polymer (e.g., POZ, polypeptide) is substituted. In some embodiments, the compound or (CH2CH2O)n The moiety (or other polymer, e.g., POZ or polypeptide) has an average molecular weight of at least 20,000 daltons up to about 100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons. Thus, the teachings herein regarding cell-reactive compstatin analog moieties, e.g., compstatin analog moieties used and linkages by which compstatin analog moieties are attached to cell-reactive moieties, can be applied to long-acting compstatin analogs, which may have any of the structures depicted by ALM, as described above, where A is a clearance-reducing moiety (e.g., any of the clearance-reducing moieties described herein), and further wherein L (or L P1 , L P2 , or L P3There can be one, two, or more (e.g., 3, 4, 5, 6, 7, 8) compstatin analog moieties M attached to A by linkages shown as (A, B, C, C, D, E, E, F ... Additionally, when a compstatin analog moiety comprising any of SEQ ID NOS: 3-36, 37, 37A, 38A, 39A, 40A, or 41A, or variants thereof, is extended at the N-terminus, C-terminus, or both termini by one or more amino acids, wherein at least one of the amino acids has a side chain containing a reactive functional group, such one or more amino acid extensions can extend from the cyclic portion of the compstatin analog moiety by, for example, a substituted or unsubstituted, saturated or unsaturated alkyl chain, an oligo(ethylene glycol) chain, and / or ... an unsaturated alkyl chain, wherein L (or L) P1 , L P2 , or L P3 It is further understood that the nuclei may be separated by flexible or rigid spacer moieties, including any of the other moieties shown as .

[0254] Examples of long-acting compstatin analogs in which n is sufficient to provide an average molecular weight of about 500, 1,000, 1,500, 2,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or up to 100,000 daltons are described below. In some embodiments, n is sufficient to provide an average molecular weight of about 20,000 daltons, up to about 100,000, 120,000, 140,000, 160,000, 180,000, or 200,000 daltons.

[0255] (CH2CH2O) n C(=O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH) (SEQ ID NO: 58) Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C(=O)-Lys-C(=O)-(CH2CH2O) n -NH2 (SEQ ID NO: 59) Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-Lys-C(=O)-(CH2CH2O) n -NH2 (SEQ ID NO: 60). Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-(Gly)5-Lys-C(=O)-(CH2CH2O) n -NH2 (SEQ ID NO: 61) Ac-(CH2CH2O) n C(=O)Lys-(Gly)5-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH2) (SEQ ID NO: 62) Ac-(CH2CH2O) n C(=O)Lys-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH 2) (SEQ ID NO: 63)

[0256] In SEQ ID NO: 58, (CH2CH2O) n is bound to the N-terminal amino acid via an amide bond. In SEQ ID NOs: 59 to 63, (CH2CH2O) n The (CH2CHO) moiety is linked to the Lys side chain via an amide bond. Thus, as described above, the C-terminal NH2 of SEQ ID NOs: 59, 60, and 61 is understood to represent amidation of the C-terminus of the peptide, and the N-terminal Ac of SEQ ID NOs: 62 and 63 is understood to represent acetylation of the N-terminus of the peptide. n The free end of the moiety is usually the underlined O, which represents the O atom of the terminal (CH2CH2O) group ( O It is understood by those skilled in the art that (OR (underlined O)) often ends in a hydroxyl ( O H) group or methoxy (- O Although the moiety is an Ac-Ile-Cys group, other groups (e.g., other alkoxy groups) can be used. Thus, for example, SEQ ID NO: 59 is an Ac-Ile-Cys group. * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C(=O)-Lys-(C(=O)-(CH2CH2O) n-R)-NH2 (SEQ ID NO: 64), where R is, e.g., H or CH3 for linear PEGs. In the case of bifunctional, branched, or star PEGs, R represents the remainder of the molecule. Furthermore, it is understood that the moiety containing the reactive functionality can vary as described herein (e.g., according to any of the formulas described herein). For example, a long-acting compstatin analog comprising the same peptide sequence as SEQ ID NO: 64, where the moiety containing the reactive functionality comprises an ester and / or alkyl chain, can be represented as follows: Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C(=O)-Lys-(C(=O)-(CH2) m -(CH2CH2O) n -R)-NH2 (SEQ ID NO: 65); Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C(=O)-Lys-(C(=O)-(CH2) m- C(=O)-(CH2CH2O) n -R)-NH2 (SEQ ID NO: 66) Ac-Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr-NH-CH2CH2OCH2CH2OCH2-C(=O)-Lys-(C(=O)-(CH2) m- C(=O)-(CH2)j (CH2CH2O) n -R)-NH2 (SEQ ID NO: 67)

[0257] In SEQ ID NOs:65-67, m can range from 1 up to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in various embodiments. In SEQ ID NO:67, j can range from 1 up to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in various embodiments. Also, as described herein, in various embodiments, Lys-(C(=O)-) and (CHCHO) n It is also understood that other moieties can be incorporated between -R, such as an amide, an aromatic ring (e.g., substituted or unsubstituted phenyl), or a substituted or unsubstituted cycloalkyl structure.

[0258] The present invention provides variants of SEQ ID NOs: 58 to 67, which variants are -Ile-Cys * -Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * -Thr- is replaced by an amino acid sequence containing any other compstatin analog, for example, any of the amino acid sequences of SEQ ID NOs: 3-27, or 29-36, 37, 37A, 38A, 39A, 40A, or 41A, except that the blocking moieties present at the N- and / or C-termini of the compstatin analog are absent, replaced by a linker (which may contain a blocking moiety), or linked to different N- or C-terminal amino acids present in the corresponding variant.

[0259] Any compstatin analog, e.g., any compound comprising any of SEQ ID NOs: 3-37, 37A, 38A, 39A, 40A, or 41A, can, in various embodiments, be directly or indirectly linked via or near its N-terminus or C-terminus (e.g., via the side chain of an amino acid at or near its N- or C-terminal amino acid) to any moiety containing a reactive functional group, e.g., a compound of any of Formulas I-XVI or Formulas A-H.

[0260] In some embodiments, the CRM comprises a polypeptide found in human serum or a fragment thereof, or a variant substantially similar to the polypeptide or fragment thereof. In some embodiments, the polypeptide, fragment, or variant has a molecular weight of 5-150 kD, e.g., at least 5 kD, 10 kD, 20 kD, 30 kD, 40 kD, 50 kD, 60 kD, 70 kD, 80 kD, 90 kD, 100 kD, or more, e.g., 100-120 kD or 120-150 kD. In some embodiments, preparing a long-acting compstatin analog comprises reacting a compstatin analog containing a reactive functional group with one or more amino acid side chains of a polypeptide, wherein the side chains contain compatible functional groups. In some embodiments, preparing a long-acting compstatin analog comprises reacting a compstatin analog containing a reactive functional group with the N-terminal amine and / or C-terminal carboxyl group of a polypeptide. In some embodiments, the preparation of a long-acting compstatin analog involves reacting a compstatin analog containing an amine-reactive functional group with an amino acid having a side chain containing a primary amine (e.g., lysine) and / or the N-terminal amine of a polypeptide. In some embodiments, the preparation of a long-acting compstatin analog involves reacting a compstatin analog containing a carboxyl-reactive functional group with the C-terminal carboxyl group of a polypeptide. In some embodiments, a compstatin analog moiety is attached to each end of the polypeptide, and optionally attached to the side chain of one or more internal amino acids. In some embodiments, the preparation of a long-acting compstatin analog involves reacting a compstatin analog containing a sulfhydryl-reactive functional group with one or more sulfhydryl groups of a polypeptide.

[0261] In some embodiments, at least one reactive functional group is introduced into a polypeptide. For example, in some embodiments, at least one side chain of the polypeptide is modified to convert a first reactive functional group into a different reactive functional group before reacting with a compstatin analog. In some embodiments, a thiol is introduced. Several methods can be used to introduce a thiol into a biomolecule, including reduction of endogenous disulfides and conversion of amine, aldehyde, or carboxylic acid groups to thiol groups. Disulfide bridges of cystine in proteins can be reduced to cysteine residues using dithiothreitol (DTT), tris-(2-carboxyethyl)phosphine (TCEP), or tris-(2-cyanoethyl)phosphine. Amines can be indirectly thiolated by reacting with succinimidyl 3-(2-pyridyldithio)propionate (SPDP) followed by reduction of the 3-(2-pyridyldithio)propionyl conjugate with DTT or TCEP. Amines can be indirectly thiolated by reaction with succinimidyl acetylthioacetate followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine at approximately neutral pH. Amines can be directly thiolated by reaction with 2-iminothiolane, introducing a free thiol while preserving the overall charge of the molecule. Tryptophan residues in thiol-free proteins can be oxidized to mercaptotryptophan residues, which can then be modified with iodoacetamide or maleimide. Polypeptides containing one or more thiols and Ac-Ile-Cys can be oxidized to mercaptotryptophan residues. * -Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys * Long-acting compstatin analogs may be prepared by reacting with a compstatin analog containing a maleimide group, such as -Thr-AEEAc-Lys-(C(=O)-(CH2)5-Mal)-NH2 (SEQ ID NO: 68).

[0262] In some embodiments, the polypeptide is recombinantly produced. In some embodiments, the polypeptide is at least partially recombinantly produced (e.g., in bacteria or in the cells of a eukaryotic host, such as a fungus, insect, plant, or vertebrate) and / or at least partially produced using chemical synthesis. In some embodiments, the polypeptide is purified. For example, in some embodiments, the polypeptide is purified from a host cell lysate or from the culture medium where it is secreted by the host cell. In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide is unglycosylated. In some embodiments, the polypeptide is human serum albumin (HSA). In some embodiments, a substantially similar variant of a polypeptide is similar enough to the polypeptide that it is not recognized as foreign by the normal immune system of a subject, e.g., a human subject. In some embodiments, changes in the sequence of the substantially similar variant compared to the polypeptide from which the variant is derived are selected so as to avoid the occurrence of an MHC class I epitope. Various methods known in the art can be used to predict whether a sequence contains an MHC class I epitope.

[0263] In some embodiments, one or more amino acids in a polypeptide, linker, or composition may be selected to be hydrophobic or hydrophilic, or to confer high hydrophilicity, or in some embodiments, high hydrophobicity, to a compound comprising the amino acid. As known in the art, the terms "hydrophilic" and "hydrophobic" are used to refer to the degree of affinity a substance has for water. In some embodiments, a hydrophilic substance has a strong affinity for water and tends to dissolve in, mix with, or become wet with water, while a hydrophobic substance substantially lacks affinity for water, tends to repel or not absorb water, and tends not to dissolve in, mix with, or become wet with water. Amino acids are classified based on hydrophobicity, as known in the art. Examples of "hydrophilic amino acids" are arginine, lysine, threonine, alanine, asparagine, glutamine, aspartic acid, glutamic acid, serine, and glycine. Examples of "hydrophobic amino acids" are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine. In certain embodiments, analogs of standard amino acids are used, which have increased or decreased hydrophilicity or hydrophobicity relative to the amino acid from which the analog is derived.

[0264] The present invention further provides multimers, e.g., concatamers, comprising two or more (e.g., 2-10) CRM-containing compstatin analogs, wherein the resulting molecules (or their CRM components) have an average molecular weight of 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; to 100,000 daltons. In certain embodiments, any of the above-described linking moieties can be used to link CRM-containing compstatin analogs.

[0265] In some embodiments, the total molecular weight of a long-acting compstatin analog containing a compstatin analog portion does not exceed 50 kD. For example, in the case of an LACA containing 40 kD PEG, in some embodiments, the molecular weight contributed by the remaining portion of the compound containing the compstatin analog portion does not exceed 10 kD, e.g., 1.5 kD to 5.0 kD or 5.0 kD to 10 kD. In some embodiments, the total molecular weight of an LACA containing a compstatin analog portion is 45 kD to 50 kD. In some embodiments, the total molecular weight of an LACA containing a compstatin analog portion is 40 kD to 45 kD, 15 kD to 40 kD, e.g., 15 kD to 25 kD, 25 kD to 35 kD, or 35 kD to 40 kD. Thus, when the present invention refers to a compstatin analog comprising a polymer or CRM having a particular molecular weight or within a particular range, in certain embodiments, the total molecular weight of the compstatin analog can be, for example, 1.5 kD to 5 kD greater than the molecular weight of the polymer or CRM, or in certain embodiments, 5 kD to 10 kD greater than the molecular weight of the polymer. The molecular weight of a compound, e.g., a compound comprising a polymer, can refer to the average molecular weight of the molecules of such compound in a composition.

[0266] A wide variety of methods and assays useful for detecting polymers, e.g., PEG, POZ, and / or polypeptides and / or measuring the physical and / or structural properties of polymers, e.g., PEG, POZ, and / or polypeptides, are known in the art and may be used, in some cases, to detect compstatin analogs, e.g., cell-reactive, long-acting, targeted compstatin analogs or compstatin analog moieties. For example, methods and assays useful for measuring properties such as aggregation, solubility, size, structure, melting characteristics, purity, presence of degradation products or contaminants, water content, hydrodynamic radius, etc., are available. Such methods include, for example, analytical centrifugation, various types of chromatography, such as liquid chromatography (e.g., HPLC-ion exchange, HPLC-molecular sieve, HPLC-reverse phase), light scattering, capillary electrophoresis, circular dichroism, isothermal calorimetry, differential scanning calorimetry, fluorescence, infrared (IR), nuclear magnetic resonance (NMR), Raman spectroscopy, refractometry, UV / visible spectroscopy, mass spectrometry, immunological methods, etc. It is understood that methods may be combined. In some embodiments, a cell-reactive, long-acting, or targeted compstatin analog (or a composition comprising a cell-reactive, long-acting, or targeted compstatin analog) has one or more of the properties described herein, as assessed using any of the above methods. In some embodiments, methods useful for detecting and / or quantifying long-acting compstatin analogs are described herein.

[0267] VI. Targeted Compstatin Analogs The present invention provides and / or utilizes targeted compstatin analogs comprising a targeting moiety and a compstatin analog moiety, wherein the targeting moiety non-covalently binds to a target molecule. In some aspects, the present invention provides targeted compstatin analogs similar to the cell-reactive compstatin analogs described in Section VI, wherein the compound comprises a targeting moiety in addition to or instead of the cell-reactive moiety. The targeting moiety can comprise, for example, an antibody, polypeptide, peptide, nucleic acid (e.g., an aptamer), carbohydrate, small molecule, or supramolecular complex that specifically binds to the target molecule. In some embodiments, the affinity of the targeting moiety (as measured by the equilibrium dissociation constant Kd) for the target molecule (as measured by the equilibrium dissociation constant Kd) is greater than or equal to 10 under test conditions, e.g., physiological conditions. -3 M or less, e.g. 10 -4 M or less, e.g. 10 -5 M or less, e.g. 10 -6 M or less, 10 -7 M or less, 10 -8 M or less or 10 -9 It is below M.

[0268] In embodiments of the invention in which the targeting moiety is an antibody, the antibody can be any immunoglobulin or derivative thereof that retains binding ability, or any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources or can be partially or wholly synthetically produced (e.g., using recombinant DNA technology, chemical synthesis, etc.). The antibody can be from any species, e.g., human, rodent, rabbit, goat, chicken, etc. The antibody can be a member of any immunoglobulin class, including all of the human classes: IgG, IgM, IgA, IgD, and IgE. In various embodiments of the invention, the antibody can be an antibody fragment such as Fab', F(ab')2, scFv (single-chain variable region), or other fragment that retains the antigen-binding site, or a recombinantly produced scFv fragment, including a recombinantly produced fragment. See, e.g., Allen, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references cited therein. Monovalent, bivalent, or polyvalent antibodies can be used. Antibodies can be chimeric, e.g., by fusing rodent-derived variable domains with human-derived constant domains, thereby retaining the specificity of rodent antibodies. In some embodiments, human antibodies or portions thereof are produced using display technologies such as phage display, e.g., in rodents with human immunoglobulin genes integrated into their genomes. In some embodiments, humanized antibodies are produced by grafting one or more complementarity-determining regions from a non-human species (e.g., mouse) into human antibody sequences. Antibodies can be partially or fully humanized. For example, see Almagro JC, Fransson J., Humanization of antibodies. Front Biosci. 13: 1619-33 (2008), for a review of various methods for obtaining humanized antibodies that can be used to obtain targeting moieties useful in the present invention. Antibodies can be polyclonal or monoclonal, although monoclonal antibodies are generally preferred for purposes of the present invention.In certain embodiments of the invention, F(ab')2 or F(ab')2 fragments are used, and in other embodiments, antibodies comprising an Fc domain are used. Methods for producing antibodies that specifically bind to almost any molecule of interest are known in the art. For example, monoclonal or polyclonal antibodies can be purified from natural sources, e.g., from the blood or ascites fluid of an animal that produces the antibody (e.g., after immunization with the molecule or an antigenic fragment thereof), or can be produced recombinantly in cell culture. Methods for producing antibody fragments, e.g., by digestion, disulfide reduction, or synthesis, are known in the art.

[0269] In various embodiments of the present invention, a targeting moiety can be any molecule that specifically binds to a target molecule by a mechanism other than an antigen-antibody interaction. Such a targeting moiety is referred to as a "ligand." For example, in various embodiments of the present invention, a ligand can be a polypeptide, peptide, nucleic acid (e.g., DNA or RNA), carbohydrate, lipid or phospholipid, or small molecule. In some embodiments, a small molecule is an organic compound, whether natural or artificially produced, that is relatively low in molecular weight, is not a protein, polypeptide, nucleic acid, or lipid, typically has a molecular weight less than about 1500 g / mol, and typically has multiple carbon-carbon bonds. In general, an aptamer is an oligonucleotide (e.g., RNA or DNA, optionally containing modified nucleosides (e.g., bases or sugars other than the five standard bases (A, G, C, T, U) or sugars (ribose and deoxyribose) most commonly found in RNA and DNA) or modified internucleoside linkages (e.g., non-phosphodiester linkages) that stabilize the molecule, for example, by increasing its resistance to degradation by nucleases) that binds to a specific protein. In some embodiments, the oligonucleotide is up to about 100 nucleosides in length, e.g., 12 to 100 nucleosides in length. Aptamers can be obtained using an in vitro evolution method called SELEX, and methods for obtaining aptamers specific to proteins of interest are known in the art. See, e.g., Brody EN, Gold L. J Biotechnol. 2000 March; 74(1): 5-13. In some embodiments, peptide nucleic acids or locked nucleic acids are used.

[0270] In some embodiments of the invention, the targeting moiety comprises a peptide. In some embodiments, display technologies such as phage display, ribosome display, yeast display, etc. are used to identify peptides that bind to a target molecule of interest.

[0271] Small molecules can be used as ligands. Methods for identifying such ligands are known in the art. For example, in vitro or computational screening of small molecule libraries, including combinatorial libraries, to identify low molecular weight organic compounds that bind to protein pockets can identify small molecule ligands for many proteins of interest (Huang, Z., Pharm. & Ther. 86: 201-215, 2000).

[0272] In some embodiments of the present invention, the targeting moiety is not a protein or molecule that is typically used as a carrier and conjugated with an antigen for the purpose of eliciting antibodies. Examples include carrier proteins or molecules such as bovine serum albumin, keyhole limpet hemocyanin, bovine gamma globulin, and diphtheria toxin. In some embodiments of the present invention, the targeting moiety is not the Fc portion of an immunoglobulin molecule. In some embodiments, the targeting moiety is part of a complex that includes one or more additional moieties to which it is covalently or non-covalently bound.

[0273] In various embodiments of the present invention, a target molecule can be any molecule produced by a cell (including any form expressed on the cell surface or modified forms thereof that are at least partially due to extracellular modification). In certain embodiments, a target molecule is an extracellular substance present in or on a tissue. In certain embodiments, a target molecule is characteristic of a particular disease or physiological condition or is characteristic of one or more cell or tissue types. Target molecules are often molecules (e.g., transmembrane or membrane-bound proteins) that are at least partially present on the cell surface such that at least a portion of the molecule is accessible to binding by an extracellular binding agent such as an antibody. Target molecules can, but need not, be cell-type specific. For example, cell-type-specific target molecules are often proteins, peptides, mRNAs, lipids, or carbohydrates that are present at higher levels on or in one or more specific types of cells than on or in many other types of cells. In some cases, cell-type-specific target molecules are present at detectable levels only in or on cells of a specific type of interest. However, it is understood that a useful cell-type-specific target molecule need not be completely specific for the cell type of interest to be considered cell-type specific. In some embodiments, a cell type-specific target molecule of a particular cell type is expressed within that cell type at a level at least three times higher than a reference cell population, e.g., a mixture of approximately equal amounts of cells from multiple (e.g., 5-10 or more) different tissues or organs. In some embodiments, the cell type-specific target molecule is present at a level at least 4-5 times, 5-10 times, or 10 times or more higher than its average expression level in the reference population. In some embodiments, detecting or measuring a cell type-specific target molecule allows one skilled in the art to distinguish one or more cell types of interest from many, most, or all other cell types.In general, the presence, absence, and / or quantity of most target molecules can be determined using one or more standard techniques, such as Northern blotting, in situ hybridization, RT-PCR, sequencing, immunological methods (such as immunoblotting, immunodetection (e.g., by immunohistochemistry) or fluorescent detection (e.g., using FACS) after staining with fluorescently labeled antibodies), oligonucleotide microarrays, cDNA microarrays, membrane arrays, protein microarray analysis, mass spectrometry, etc.

[0274] In some embodiments, the target molecule is a channel, transporter, receptor, or other molecule at least a portion of which is exposed on the cell surface, hi some embodiments, the target molecule is an anion transporter or water channel (e.g., an aquaporin protein).

[0275] In certain embodiments, the target molecule is a protein, at least a portion of which is exposed on the surface of red blood cells, such as glycophorin (eg, glycophorin A, B, C, or D) or band 3.

[0276] In some embodiments, the target molecule is a protein at least a portion of which is exposed on the surface of an endothelial cell. In some embodiments, the target molecule is present on the surface of normal, healthy vasculature. In some embodiments, the target molecule is present on the surface of activated endothelial cells. In some embodiments, the target molecule is present on the surface of activated endothelial cells, but not on the surface of non-activated endothelial cells. In some embodiments, the target molecule is a molecule whose expression or exposure is induced by a stimulus such as injury or inflammation. In some embodiments, the target molecule can be recognized as "non-self" by a recipient of a graft containing cells expressing the target molecule. In some embodiments, the target molecule is a carbohydrate xenoantigen against which antibodies are commonly found in humans. In some embodiments, the carbohydrate comprises a blood group antigen. In some embodiments, the carbohydrate comprises a xenoantigen. For example, the alpha-gal epitope (Gal alpha 1-3Gal beta 1-(3)4GlcNAc-R) (see, e.g., Macher BA and Galili U. The Galalpha1, 3Galbeta1, 4GlcNAc-R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance. Biochim Biophys Acta. 1780(2): 75-88 (2008)).

[0277] In some embodiments of the invention, a compstatin analog comprises both a targeting moiety and a CRM. In some embodiments, the targeted compstatin analog comprises multiple targeting moieties, which can be the same or different. The different targeting moieties can bind to the same target molecule or different target molecules. The present invention provides targeted compstatin analogs that are multivalent with respect to the targeting moiety, the compstatin analog, or both.

[0278] In general, the present invention encompasses any method for preparing a compound comprising a compstatin analog moiety and a targeting moiety, and the resulting compounds. In some embodiments, targeted compstatin analogs can be prepared using methods generally similar to those described in Section VI, but using a targeting moiety instead of or in addition to the cell-reactive moiety. In some embodiments, targeted compstatin analogs comprising a peptide as the targeting moiety are synthesized as a polypeptide chain comprising the compstatin analog moiety and the peptide targeting moiety. Optionally, the polypeptide chain includes one or more spacer peptides between the compstatin analog moiety and the targeting moiety.

[0279] In certain embodiments, a targeted compstatin analog has a molar activity of at least about 10%, 20%, or 30%, e.g., 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, or more, of the activity of a corresponding compstatin analog having the same amino acid sequence (and one or more blocking moieties, if applicable) but lacking a targeting moiety. In certain embodiments, where a targeted compstatin analog comprises multiple compstatin analog moieties, the molar activity of the targeted compstatin analog is at least about 10%, 20%, or 30%, e.g., 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, or more, of the sum of the activities of the compstatin analog moieties. Compositions and methods of making and synthetic intermediates for targeted compstatin analogs are aspects of the present invention.

[0280] VII. Use Cell-reactive, long-acting, or targeted compstatin analogs have a wide variety of uses. While not limiting the invention in any way, specific uses of cell-reactive, long-acting, or targeted compstatin analogs and related embodiments of the invention are described herein. In one embodiment, a cell-reactive, long-acting, or targeted compstatin analog is administered to a subject suffering from or at risk of complement-mediated damage to an organ, tissue, or cell. In one embodiment, the cell-reactive compstatin analog contacts and covalently binds to an organ, tissue, or cell ex vivo. The organ, tissue, or cell is then introduced into the subject and protected from damage that may be caused by the recipient's complement system.

[0281] For the purposes described herein, compstatin analogs that do not covalently bind to cells can be used. For example, compstatin analogs modified with moieties that extend the compound's longevity in the body and / or compstatin analogs that include a moiety that targets the compstatin analog to a cell type or site susceptible to complement activation can be used, and the present invention encompasses such uses. In some embodiments, long-acting compstatin analogs are used. In some embodiments, compstatin analogs that include a targeting moiety are used. In some embodiments, compstatin analogs that include both a moiety that extends the compound's longevity in the body and a targeting moiety are used. When cell-reactive compstatin analogs are referred to below, the present invention provides similar compositions and methods related to targeted compstatin analogs, as well as embodiments that (at least in embodiments involving administering a compstatin analog to a subject) use a compstatin analog that does not include a targeting moiety or a cell-reactive moiety, optionally a long-acting compstatin analog, instead of or in addition to the cell-reactive compstatin analog.

[0282] Intended uses include: (1) protecting red blood cells (RBCs) from complement-mediated damage in patients with disorders such as paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome, or other disorders characterized by complement-mediated RBC lysis; (2) protecting transplanted organs, tissues, and cells from complement-mediated damage; (3) reducing ischemia / reperfusion (I / R) injury (e.g., in patients with trauma, vascular occlusion, myocardial infarction, or other conditions where I / R injury may occur); and (4) protecting various body structures (e.g., retina) or membranes (e.g., synovium) that may be exposed to complement components from complement-mediated damage in any of a variety of different complement-mediated disorders. The beneficial effects of inhibiting complement activation on the surface of cells or other body structures are not limited to those that result directly from protecting the cells or structures themselves from complement-mediated damage (e.g., preventing cell lysis). For example, inhibition of complement activation using cell-reactive compstatin analogs may have beneficial effects on distant organ systems or the entire body by reducing the production of anaphylotoxins and the resulting influx / activation of neutrophils and other pro-inflammatory events, and / or by reducing the release of potentially damaging cellular contents.

[0283] A. Blood cell protection In some embodiments of the present invention, a cell-reactive compstatin analog, a cell-targeted compstatin analog, and / or a non-targeted compstatin analog (e.g., a long-acting non-targeted compstatin analog) is used to protect blood cells from complement-mediated damage. Blood cells can be any cellular component of blood, such as red blood cells (RBCs), white blood cells (WBCs), and / or platelets. In some embodiments, the cell-targeted compstatin analog is targeted to a target molecule, such as glycophorin or band 3, exposed on the cell surface of RBCs. Many disorders result from complement-mediated damage to blood cells. Such disorders can arise, for example, from deficiencies or abnormalities in one or more intracellular or soluble CRPs in a patient, such as those due to (a) mutations in genes encoding such proteins; (b) mutations in one or more genes required for the production or proper function of CRPs, and / or (c) the presence of autoantibodies against one or more CRPs. Complement-mediated RBC lysis can result from the presence of autoantibodies to RBC antigens, which can arise from a variety of causes (often idiopathic). Patients with such mutations in the gene encoding CRP and / or antibodies to CRP or their own RBCs are at increased risk for disorders involving complement-mediated RBC injury. Patients who have experienced one or more episodes of symptoms characteristic of the disorder are at increased risk of recurrence.

[0284] Paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disorder involving acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure, and thrombophilia (the tendency to form blood clots). The disorder affects an estimated 16 per million people worldwide and can affect both men and women of any age, but is more common in young adults (Bessler, M. & Hiken, J., Hematology Am Soc Hematol Educ Program, 104-110 (2008); Hillmen, P. Hematology Am Soc Hematol Educ Program, 116-123 (2008)). PNH is a chronic, debilitating disease punctuated by acute hemolysis, resulting in significant morbidity and a shortened life expectancy. In addition to anemia, many patients experience abdominal pain, dysphagia, erectile dysfunction, and pulmonary hypertension, and are at increased risk for renal failure and thromboembolism.

[0285] Although PNH was first described as an isolated disease in the 1800s, the cause of hemolysis in PNH was not firmly established until the 1950s, when an alternative pathway of complement activation was discovered (Parker CJ. Paroxysmal nocturnal hemoglobinuria: an historical overview. Hematology Am Soc Hematol Educ Program. 93-103 (2008)). CD55 and CD59 are normally associated with the cell membrane via glycosylphosphatidylinositol (GPI) anchors, glycolipid structures that anchor specific proteins to the cell membrane. PNH results from nonmalignant clonal proliferation of hematopoietic stem cells that have developed somatic mutations in the PIGA gene, which encodes a protein involved in GPI anchor synthesis (Takeda J, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 73: 703-711 (1993)). The progeny of these stem cells lack GPI-anchored proteins, including CD55 and CD59. This defect renders the cells susceptible to complement-mediated RBC lysis. Diagnosis is often achieved by flow cytometry analysis using antibodies against GPI-anchored proteins. This assay detects the loss of GPI-anchored proteins on the cell surface and can reveal the extent of the loss and the proportion of affected cells (Brodsky RA. Advances in the diagnosis and therapy of paroxysmal nocturnal hemoglobinuria. Blood Rev. 22(2): 65-74 (2008)). PNHIII RBCs lack all GPI-anchored proteins, making them highly sensitive to complement, whereas PNHII RBCs lack some GPI-anchored proteins, making them relatively insensitive to complement.FLAER is a fluorescently labeled inactive variant of proaerolysin (a bacterial toxin that binds to GPI anchors) and is increasingly used in conjunction with flow cytometry to diagnose PNH. Absence of FLAER binding to granulocytes indicates PNH. In one embodiment, a cell-reactive compstatin analog protects PNH RBCs from C3b deposition.

[0286] In one embodiment, a cell-reactive, long-acting, or targeted compstatin analog is administered to a subject suffering from atypical hemolytic syndrome (aHUS). aHUS is a chronic disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is caused by abnormal complement activation, often due to mutations in genes encoding complement regulatory proteins (Warwicker, P., et al., Kidney Int 53, 836-844 (1998); Kavanagh, D. & Goodship, T. Pediatr Nephrol 25, 2431-2442 (2010)). Mutations in the complement factor H (CFH) gene are the most common genetic abnormality in aHUS patients, and 60-70% of these patients die or develop end-stage renal failure within one year of disease onset (Kavanagh & Goodship, supra). Other mutations described include those in factor I, factor B, C3, factor H-related proteins 1-5, and thrombomodulin. Other causes of aHUS include autoantibodies to complement regulatory proteins, such as CFH. In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is administered to a subject identified as having a mutation in factor I, factor B, C3, factor H-related proteins 1-5, or thrombomodulin, or an antibody to a complement regulatory protein, e.g., CFH, or a convertase (e.g., C3Nef), or a complement factor.

[0287] Complement-mediated hemolysis is also seen in a diverse group of other conditions, including autoimmune hemolytic anemias associated with antibodies that bind to RBCs and cause complement-mediated hemolysis. For example, such hemolysis can occur in primary chronic cold agglutinin disease, warm antibody autoimmune hemolytic anemia (wAIHA), and certain reactions to drugs and other foreign substances (Berentsen, S., et al., Hematology 12, 361-370 (2007); Rosse, WF, Hillmen, P. & Schreiber, AD Hematology Am Soc Hematol Educ Program, 48-62 (2004)). In one embodiment, a cell-reactive compstatin analog is administered to a subject suffering from or at risk of suffering from chronic cold agglutinin disease. In one embodiment of the present invention, a cell-reactive compstatin analog is administered to a subject suffering from or at risk of wAIHA. In other embodiments, cell-reactive compstatin analogs are used to treat subjects suffering from or at risk of suffering from HELLP syndrome, a disease defined by hemolysis, elevated liver enzymes, and decreased platelet counts, which in at least some patients is caused by mutations in complement regulatory proteins (Fakhouri, F., et al., 112: 4542-4545 (2008)).

[0288] In other embodiments, cell-reactive compstatin analogs are used to protect RBCs or other cellular components of blood transfused into a subject. Some examples of such uses are further described below. As described above, targeted compstatin analogs and / or long-acting compstatin analogs can be used in the above methods of inhibiting complement-mediated hemolysis and / or RBC injury. In some embodiments, long-acting compstatin analogs containing a (CH2CHO) moiety are used to treat PNH or aHUS.

[0289] B. Transplantation Transplantation is an increasingly important therapeutic method, providing a means of replacing organs and tissues damaged by trauma, disease, or other conditions. Kidneys, liver, lungs, pancreas, and heart are among the organs that have been successfully transplanted. Tissues frequently transplanted include bone, cartilage, tendons, cornea, skin, heart valves, and blood vessels. Pancreatic islet or islet cell transplantation is a promising method for treating diabetes, e.g., type 1 diabetes. For purposes of the present invention, an organ, tissue, or cell (or group of cells) that is to be transplanted, being transplanted, or has already been transplanted may be referred to as a "graft." For purposes of this specification, blood transfusions are considered a "graft."

[0290] During transplantation, the graft is exposed to a variety of injurious events and stimuli that can contribute to graft dysfunction and potentially graft failure. For example, ischemia-reperfusion (I / R) injury is a common and important cause of morbidity and mortality in many transplants, particularly solid organ transplants, and can be a major determinant of the likelihood of graft survival. Transplant rejection is one of the major risks associated with transplantation between genetically distinct individuals and can lead to graft failure and the need to remove the graft from the recipient.

[0291] In certain embodiments of the present invention, cell-reactive compstatin analogs, cell-targeted compstatin analogs, and / or long-acting compstatin analogs are used to protect grafts from complement-mediated injury. Cell-reactive compstatin analogs react with and covalently bind to cells of the graft, inhibiting complement activation. Cell-targeted compstatin analogs bind to target molecules of the graft (e.g., those expressed by endothelial or other cells of the graft) and inhibit complement activation. Target molecules can be, for example, molecules whose expression is induced or stimulated by stimuli such as injury or inflammation, molecules that can be recognized as "non-self" by the recipient, carbohydrate xenoantigens for which antibodies are commonly found in humans, such as blood group antigens or xenoantigens, e.g., molecules containing the alpha-gal epitope. In certain embodiments, a decrease in complement activation can be indicated by a decrease in the mean level of C4d deposition in blood vessels of grafts contacted with a compstatin analog, e.g., a cell-reactive compstatin analog, compared to the mean level of C4d deposition in grafts not contacted with the compstatin analog (e.g., in subjects matched for the graft and other treatments received).

[0292] In various embodiments of the present invention, a cell-reactive, long-acting, or targeted compstatin analog can be contacted with a graft before, during, and / or after transplantation. For example, a liquid containing a cell-reactive, long-acting, or targeted compstatin analog can be contacted with a graft removed from a donor before transplantation. For example, the graft can be immersed in the solution and / or perfused with the solution. In other embodiments, a cell-reactive, long-acting, or targeted compstatin analog is administered to the donor before the graft is removed. In some embodiments, a cell-reactive, long-acting, or targeted compstatin analog is administered to the recipient during and / or after the introduction of the graft. In some embodiments, a cell-reactive compstatin, long-acting, or targeted analog is delivered locally to the transplanted graft. In some embodiments, a cell-reactive compstatin analog is administered systemically, for example, by intravenous administration.

[0293] The present invention provides a composition comprising an isolated graft (a) and a cell-reactive, long-acting, or targeted compstatin analog (b). In some embodiments, the composition further comprises a solution suitable for contacting (e.g., suitable for rinsing, washing, immersing, perfusing, maintaining, or storing) a graft (e.g., an organ), such as an isolated graft removed from a donor and awaiting transplantation into a recipient. In some embodiments, the present invention provides a composition comprising (a) a solution suitable for contacting a graft (e.g., an organ) and (b) a cell-reactive, long-acting, or targeted compstatin analog. The solution can be any solution that is physiologically acceptable to the graft (e.g., of appropriate osmotic composition and non-cytotoxic), medically acceptable with respect to the subsequent introduction of the graft into the recipient (e.g., preferably sterile or at least reasonably free of microbial or other contaminants), and compatible with cell-reactive compstatin analogs (i.e., does not impair the reactivity of the compstatin analog) or compatible with long-acting or targeted compstatin analogs. In certain embodiments, the solution is any solution known in the art for any such purpose. In some embodiments, the solution is Marshall's solution or hypertonic citrate solution (Soltran®, Baxter Healthcare), University of Wisconsin (UW) solution (ViaSpan™, Bristol Myers Squibb), histidine-tryptophan-ketoglutarate (HTK) solution (Custodial®, Kohler Medical Limited), EuroCollins (Fresenius), and Celsior® (Sangstat Medical), Polysol, IGL-1, or AQIX® RS-1. Of course, other solutions within the scope of physiologically acceptable compositions can also be used, e.g., solutions containing the same or nearly the same components at the same or different concentrations. In some embodiments, the solution does not contain components to which a cell-reactive compstatin analog would be expected to produce a significant response, and any solution can be modified or designed to be free of such components.In some embodiments, the cell-reactive compstatin analog is present in a solution compatible with the graft, for example, at a concentration of 0.01-100 mg / ml, or may be added to this solution to reach such a concentration.

[0294] In some embodiments, the present invention provides a kit comprising (a) a cell-reactive, long-acting, or targeted compstatin analog and (b) a graft-compatible solution or solid (e.g., powder) component thereof. The cell-reactive, long-acting, or targeted compstatin analog may be provided in solid form (e.g., powder) or at least partially dissolved in a solution. In some embodiments, the cell-reactive, long-acting, or targeted compstatin analog and / or the graft-compatible solution are provided in predetermined amounts such that, when combined, they form a solution of a concentration suitable for contacting the graft with the cell-reactive, long-acting, or targeted compstatin analog. In many embodiments, the cell-reactive, long-acting, or targeted compstatin analog and the graft-compatible solution or solid (e.g., powder) component thereof are contained in separate containers within the kit. In some embodiments, the cell-reactive compstatin analog and the graft-compatible solution components are both contained in separate containers or mixed together and provided in solid (e.g., powder) form. In certain embodiments, the kit includes instructions for use, e.g., instructions for adding the cell-reactive, long-acting, or targeted compstatin analog to a solution compatible with the transplant and / or instructions for contacting the transplant with the cell-reactive compstatin analog. Optionally, the kit includes labeling approved by a government agency responsible for regulating products used in transplantation, cell therapy, and / or blood transfusion.

[0295] The present invention further provides a method for covalently linking a compstatin analog to an isolated graft, comprising contacting the isolated graft with a cell-reactive compstatin analog. The present invention also provides an isolated graft having a covalently linked compstatin analog. The isolated graft typically has multiple compstatin analog molecules bound thereto. In some embodiments, the graft is or comprises a solid organ such as a kidney, liver, lung, pancreas, or heart. In some embodiments, the graft is or comprises bone, cartilage, fascia, tendon, ligament, cornea, sclera, pericardium, skin, heart valve, blood vessel, amniotic membrane, or dura mater. In some embodiments, the graft comprises multiple organs, such as a heart-lung or pancreatic-kidney graft. In some embodiments, the graft comprises less than an entire organ or tissue. For example, the graft can comprise a portion of an organ or tissue, such as a liver lobe, a portion of a blood vessel, a skin flap, or a heart valve. In some embodiments, the graft comprises a preparation comprising isolated cells or tissue fragments, such as pancreatic islets, that have been isolated from the original tissue but retain at least some of the tissue structure. In some embodiments, the preparation comprises isolated cells that are not bound to each other via connective tissue, e.g., hematopoietic stem or progenitor cells derived from peripheral blood and / or umbilical cord blood or whole blood, or any cell-containing blood product, such as red blood cells (RBCs) or platelets. In some embodiments, the graft is obtained from a deceased donor (e.g., a "donate after brain death" (DBD) donor or a "donate after cardiac death" donor). In some embodiments, depending on the specific type of graft, the graft is obtained from a living donor. Types of grafts that can often be obtained from living donors without imposing undue risk on the donor and without violating good medical practice include, for example, kidneys, liver segments, and blood cells.

[0296] In some embodiments, the transplant is a xenotransplant (i.e., the donor and recipient are of different species). In some embodiments, the transplant is an autologous transplant (i.e., transplantation from one part of the body to another within the same individual). In some embodiments, the transplant is a syngeneic transplant (i.e., the donor and recipient are genetically matched). In most embodiments, the transplant is an allogeneic transplant (i.e., the donor and recipient are of the same species but genetically distinct). In an allogeneic transplant, the donor and recipient may or may not be genetically related (e.g., family members). Typically, the donor and recipient are blood type-compatible (at least ABO compatible, and optionally compatible for Rh, Kell, and / or other blood cell antigens). The recipient's blood may have been screened for alloantibodies to the graft and / or recipient and donor, as the presence of alloantibodies can result in hyperacute rejection (i.e., rejection that begins almost immediately, e.g., within minutes, of the graft coming into contact with the recipient's blood). A subject's serum can be screened for anti-HLA antibodies using a complement-dependent cytotoxicity (CDC) test. The serum is incubated with a panel of lymphocytes of known HLA phenotype. If the serum contains antibodies against HLA molecules on the target cells, cell death occurs by complement-mediated lysis. Using a selected panel of target cells, it is possible to determine the specificity of the detected antibodies. Other techniques useful for determining the presence or absence of anti-HLA antibodies, and optionally their HLA specificity, include ELISA assays, flow cytometry assays, and microbead array technology (e.g., Luminex technology). The methodologies for performing these assays are well known, and a variety of kits for performing them are commercially available.

[0297] In certain embodiments, cell-reactive, long-acting, or targeted compstatin analogs inhibit complement-mediated rejection. For example, in certain embodiments, cell-reactive, long-acting, or targeted compstatin analogs inhibit hyperacute rejection. Hyperacute rejection is caused, at least in part, by antibody-mediated activation of the recipient's complement system via the classical pathway and the resulting deposition of MAC on the graft. Hyperacute rejection is usually due to the presence of pre-existing antibodies in the recipient that react with the graft. While it is desirable to attempt to avoid hyperacute rejection by adequate matching before transplantation, this is not always feasible, for example, due to time and / or resource constraints. Furthermore, some recipients (e.g., patients who have received multiple blood transfusions, patients with previous transplants, women with multiple pregnancies) have a large number of preformed antibodies, which may include antibodies against antigens not normally tested for, making it difficult or nearly impossible to reliably obtain a timely and compatible graft. These patients are at increased risk of hyperacute rejection.

[0298] In some embodiments, a cell-reactive, long-acting, or targeted compstatin analog inhibits acute rejection or graft failure. As used herein, "acute rejection" refers to rejection occurring within at least 24 hours after transplantation, usually at least several days to a week, and up to six months after transplantation. Acute antibody-mediated rejection (AMR) is often accompanied by a rapid increase in donor-specific alloantibodies (DSA) in the first few weeks after transplantation. Without wishing to be bound by any theory, it is possible that pre-existing plasma cells and / or newly converted memory B cells may play a role in the increased production of DSA. Such antibodies can cause complement-mediated damage to the graft, which can be inhibited by contacting the graft with a cell-reactive compstatin analog. Without wishing to be bound by any theory, inhibiting complement activation in the graft may reduce leukocyte (e.g., neutrophil) infiltration, another cause of acute graft failure.

[0299] In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs inhibit complement-mediated I / R injury to grafts. As discussed further below, I / R injury can occur upon reperfusion of tissues whose blood supply is temporarily interrupted, such as in transplanted organs. Reduction of I / R injury can reduce the likelihood or severity of acute graft dysfunction and reduce the likelihood of acute graft failure.

[0300] In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs suppress chronic rejection and / or chronic graft failure. As used herein, "chronic rejection or chronic graft failure" refers to rejection or failure that occurs at least six months after transplantation, e.g., six months to one, two, three, four, five, or more years after transplantation, often after the graft has functioned normally for several months to several years. This is due to a chronic inflammatory immune response against the graft. For purposes of this specification, chronic rejection may include chronic allograft vasculopathy, a term used to refer to fibrosis of the internal blood vessels of the transplanted tissue. Because the incidence of acute rejection has decreased with immunosuppressive therapy, chronic rejection has become more prominent as a cause of graft dysfunction and failure. There is evidence that B cell alloantibody production is an important factor in the development of chronic rejection and chronic graft failure (Kwun J. and Knechtle SJ, Transplantation, 88(8): 955-61 (2009)). Initial injury to the graft can contribute to chronic processes such as fibrosis that can ultimately lead to chronic rejection. Thus, inhibiting this initial injury with a cell-reactive compstatin analog can delay the onset of chronic graft rejection and / or reduce the likelihood or severity of chronic graft rejection.

[0301] In certain embodiments, a long-acting compstatin analog is administered to a transplant recipient to inhibit transplant rejection and / or transplant failure.

[0302] C. Ischemia / Reperfusion Injury Ischemia-reperfusion (I / R) injury is an important cause of tissue injury after trauma and in other conditions involving temporary interruption of blood flow such as myocardial infarction, stroke, severe infection, vascular disease, aneurysm repair, cardiopulmonary bypass surgery, and transplantation.

[0303] In traumatic situations, systemic hypoxemia, hypotension, and localized interruption of blood supply due to contusion, compartment syndrome, and vascular injury result in ischemia, which damages metabolically active tissues. Restoration of blood supply induces a strong systemic inflammatory response that is often more detrimental than the ischemia itself. Upon reperfusion of the ischemic area, locally produced and released factors enter the circulation and reach distant sites, potentially causing significant injury to organs such as the lungs and intestine that were not affected by the original ischemic injury, resulting in single- and multiorgan dysfunction. Complement activation occurs immediately after reperfusion and is a key mediator of postischemic injury, both directly and through its chemotactic and stimulatory effects on neutrophils. All three major complement pathways are activated and, acting cooperatively or independently, contribute to I / R-related adverse events affecting multiple organ systems. In one embodiment of the present invention, a cell-reactive, long-acting, or targeted compstatin analog is administered to a subject who has recently (e.g., within the past 2, 4, 8, 12, 24, or 48 hours) suffered a traumatic injury, such as systemic hypoxemia, hypotension, and / or localized blood supply disruption, placing the subject at risk for I / R injury. In one embodiment, the cell-reactive compstatin analog is administered intravascularly, optionally into a blood vessel supplying the injured body part, or directly to the body part. In one embodiment, the subject is a patient with spinal cord injury, traumatic brain injury, burn, and / or hemorrhagic shock.

[0304] In some embodiments, a cell-reactive, long-acting or targeted compstatin analog is administered to a subject before, during or after a surgical procedure, such as a surgical procedure that is expected to temporarily block blood flow to tissue, organ or part of the body.Examples of such procedures include cardiopulmonary bypass, angioplasty, heart valve repair / replacement, aneurysm repair or other vascular surgery.Cell-reactive compstatin analogs can be administered before, after and / or during the time overlapping with the surgical procedure.

[0305] In some embodiments, a cell-reactive, long-acting, or targeted compstatin analog is administered to a subject suffering from MI, thromboembolic stroke, deep vein thrombosis, or pulmonary embolism. The cell-reactive compstatin analog may be administered in combination with a thrombolytic agent, such as tissue plasminogen activator (tPA) (e.g., alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase)), anistreplase (Eminase), streptokinase (Kabikinase, Streptase), or urokinase (Abbokinase). The cell-reactive, long-acting, or targeted compstatin analog may be administered before, after, and / or during the overlapping period with the thrombolytic agent.

[0306] In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is administered to a subject to treat I / R injury.

[0307] D. Other Complement-Mediated Disorders In some embodiments, cell-reactive, long-acting or targeted compstatin analogs are introduced into the eye to treat eye disorders such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma or uveitis. For example, cell-reactive, long-acting or targeted compstatin analogs are introduced into the vitreous cavity (e.g., by intravitreal injection) to treat subjects suffering from AMD or at risk of having AMD. In some embodiments, AMD is neovascular (wet) AMD. In some embodiments, AMD is dry AMD. As those skilled in the art will recognize, dry AMD includes geographic atrophy (GA), intermediate AMD and early AMD. In some embodiments, subjects with GA are treated to delay or stop disease progression. For example, in some embodiments, treating subjects with GA reduces the rate of retinal cell death. The reduction in the rate of retinal cell death can be demonstrated by the reduction in the growth rate of GA lesions in patients treated with LACA compared with controls (e.g., sham-injected patients). In some embodiments, the subject has intermediate AMD. In some embodiments, the subject has early AMD. In some embodiments, the subject with intermediate or early AMD is treated to delay or stop the progression of the disease. For example, in some embodiments, the treatment of the subject with intermediate AMD can delay or prevent the progression to advanced forms of AMD (neovascular AMD or GA). In some embodiments, the treatment of the subject with early AMD can delay or prevent the progression to intermediate AMD. In some embodiments, the eye has both GA and neovascular AMD. In some embodiments, the eye has GA but not wet AMD. In some embodiments, the cell-reactive, long-acting or targeted compstatin analog is injected intravitreally to treat glaucoma, uveitis (e.g., posterior uveitis) or diabetic retinopathy. In some embodiments, the cell-reactive, long-acting or targeted compstatin analog is introduced into the anterior chamber to treat, for example, anterior uveitis.

[0308] In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is used to treat a subject suffering from or at risk for an autoimmune disease, e.g., an autoimmune disease mediated at least in part by antibodies against one or more autoantigens.

[0309] Cell-reactive, long-acting, or targeted compstatin analogs can be introduced, for example, into the synovial cavity of a subject suffering from arthritis (e.g., rheumatoid arthritis). Of course, they can also be administered systemically.

[0310] In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is used to treat a subject suffering from or at risk for intracerebral hemorrhage.

[0311] In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is used to treat a subject suffering from or at risk for myasthenia gravis.

[0312] In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is used to treat a subject suffering from or at risk for neuromyelitis optica (NMO).

[0313] In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is used to treat a subject suffering from or at risk for membranoproliferative glomerulonephritis (MPGN), e.g., type I MPGN, type II MPGN, or type III MPGH.

[0314] In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects suffering from or at risk of suffering from neurodegenerative diseases. In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects suffering from or at risk of developing neuropathic pain. In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects suffering from or at risk of developing rhinosinusitis or nasal polyps. In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects suffering from or at risk of cancer. In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects suffering from or at risk of sepsis. In certain embodiments, a cell-reactive, long-acting, or targeted compstatin analog is used to treat a subject suffering from or at risk for adult respiratory distress syndrome.

[0315] In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects who have anaphylactic reactions or infusion reactions, or who are at risk of anaphylaxis or infusion reactions.For example, in some embodiments, subjects may be pretreated before, during, or after administration of a drug or solvent that can cause anaphylaxis or infusion reactions.In some embodiments, cell-reactive, long-acting, or targeted compstatin analogs are used to treat subjects who are at risk of, or who are experiencing, anaphylaxis due to food (e.g., peanuts, shellfish, or other food allergens) or insect stings (e.g., honeybees, wasps).

[0316] In various embodiments of the present invention, the cell-reactive, long-acting, or targeted compstatin analogs may be administered locally or systemically.

[0317] In some embodiments, cell-reactive, long-acting or targeted compstatin analogs are used to treat respiratory diseases, such as asthma or chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis.In various embodiments, for example, cell-reactive, long-acting or targeted compstatin analogs can be administered to the airways by inhalation, for example, as dry powder or by nebulization, or by injection, for example, intravenously, intramuscularly or subcutaneously.In some embodiments, cell-reactive, long-acting or targeted compstatin analogs are used to treat severe asthma, for example, asthma that is not adequately controlled by bronchodilators and / or inhaled corticosteroids.

[0318] In one embodiment, a method for treating a complement-mediated disorder, e.g., a chronic complement-mediated disorder, is provided, comprising administering a long-acting complement inhibitor to a subject in need of such treatment. The long-acting compstatin analog, in various embodiments, can be any long-acting compstatin analog described herein. In one embodiment, a method for treating a Th17-associated disorder is provided, comprising administering a long-acting complement inhibitor to a subject in need of such treatment.

[0319] In some embodiments, a "chronic disorder" is one that persists for at least three months and / or is recognized in the art as a chronic disorder. In many embodiments, a chronic disorder persists for at least six months, e.g., at least one year or more, e.g., permanently. One of skill in the art will recognize that at least some manifestations of various chronic disorders may be intermittent and / or may wax and wane in severity over time. Chronic disorders may be progressive, e.g., becoming more severe or widespread over time. Many chronic complement-mediated disorders are described herein. A chronic complement-mediated disorder may be, for example, any chronic disorder that implicates complement activation (e.g., excessive or inappropriate complement activation) as a causative and / or at least partially causative factor. For convenience, disorders may be classified by reference to the organ or system that is most frequently affected in subjects afflicted with the disorder. It is recognized that many disorders may affect multiple organs or systems, and such classification is in no way limiting. Furthermore, many manifestations (e.g., symptoms) may occur in subjects with any of many different disorders. Non-limiting information regarding disorders of interest herein can be found, for example, in standard reference texts of internal medicine, such as Cecil Textbook of Medicine (e.g., 23rd edition), Harrison's Principles of Internal Medicine (e.g., 17th edition), and / or standard reference texts that focus on particular areas of medicine, particular body systems or organs, and / or particular disorders.

[0320] In one embodiment, the chronic complement-mediated disorder is a Th2-associated disorder. As used herein, a Th2-associated disorder is a disorder characterized by an excessive number and / or excessive or inappropriate activity of CD4+ helper T cells of the Th2 subtype ("Th2 cells") in the body or a portion of the body, e.g., at least one tissue, organ, or structure. For example, Th2 cells may predominate over CD4+ helper T cells of the Th1 subtype ("Th1 cells") in, e.g., at least one tissue, organ, or structure affected by the disorder. As is known in the art, Th2 cells typically secrete characteristic cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), while Th1 cells typically secrete interferon-γ (IFN-γ) and tumor necrosis factor β (TNFβ). In certain embodiments, a Th2-associated disorder is characterized, for example, by excessive production and / or amount of IL-4, IL-5 and / or IL-13, e.g., in relation to IFN-γ and / or TNFβ, in at least one tissue, organ or structure.

[0321] In some embodiments, the chronic complement-mediated disorder is a Th17-associated disorder. In some embodiments, as described in further detail in PCT / US2012 / 043845, filed June 22, 2012, entitled "Method of Treating Chronic Disorders with Complement Inhibitors," complement activation and Th17 cells participate in a cycle involving dendritic cells and antibodies, contributing to the maintenance of a pathological immune microenvironment underlying a range of disorders. Without wishing to be bound by any theory, once established, the pathological immune microenvironment is self-sustaining and contributes to cell and tissue injury. In some embodiments, long-acting compstatin analogs are useful for treating Th17-associated disorders.

[0322] As used herein, a Th17-associated disorder is a disorder characterized by an excessive number and / or excessive or inappropriate activity of CD4+ helper T cells of the Th17 subtype ("Th17 cells") in the body or a portion of the body, e.g., at least one tissue, organ, or structure. For example, there can be a predominance of Th17 cells over Th1 and / or Th2 cells in, e.g., at least one tissue, organ, or structure affected by the disorder. In certain embodiments, the predominance of Th17 cells is a relative predominance, e.g., the ratio of Th17 cells to Th1 cells and / or the ratio of Th17 cells to Th2 cells is elevated relative to normal values. In certain embodiments, the ratio of Th17 cells to regulatory T cells (CD4 + CD25 +Regulatory T cells, also known as "Treg cells," are elevated relative to normal levels. Th17 cell formation and / or Th17 cell activation are promoted by various cytokines, e.g., interleukin 6 (IL-6), interleukin 21 (IL-21), interleukin 23 (IL-23), and / or interleukin 1β (IL-1β). Th17 cell formation involves the differentiation of precursor T cells, e.g., naive CD4+ T cells, toward a Th17 phenotype and their differentiation into functional Th17 cells. In certain embodiments, Th17 cell formation encompasses certain aspects of Th17 cell development, proliferation (expansion), survival, and / or maturation. In certain embodiments, a Th17-associated disorder is characterized by excessive production and / or amounts of IL-6, IL-21, IL-23, and / or IL-1β. Th17 cells typically secrete characteristic cytokines such as interleukin-17A (IL-17A), interleukin-17F (IL-17F), interleukin-21 (IL-21), and interleukin-22 (IL-22). In certain embodiments, a Th17-associated disorder is characterized by excessive production and / or amounts of Th17 effector cytokines, e.g., IL-17A, IL-17F, IL-21, and / or IL-22. In certain embodiments, the excessive production or amount of cytokines is detectable in the blood. In certain embodiments, the excessive production or amount of cytokines is detectable locally, e.g., in at least one tissue, organ, or structure. In certain embodiments, a Th17-associated disorder is associated with a decreased number of Tregs and / or a decreased amount of Treg-associated cytokines. In one embodiment, the Th17 disorder is any chronic inflammatory disease, which term encompasses a range of conditions characterized by self-perpetuating immune insults to various tissues, which appear to be dissociated from the initial insult (which may be unknown) that caused the condition. In one embodiment, the Th17-associated disorder is any autoimmune disease. Many, if not most, "chronic inflammatory diseases" are in fact autoimmune diseases.Examples of Th17-related disorders include inflammatory skin diseases such as psoriasis and atopic dermatitis; systemic sclerosis and sclerosis; inflammatory bowel disease (IBD) (for example, Crohn's disease and ulcerative colitis); Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; rheumatoid arthritis (RA), Sjögren's syndrome, multiple sclerosis, vasculitis; central nervous system (CNS) inflammatory disorders, chronic hepatitis; chronic pancreatitis, glomerulonephritis; sarcoidosis; thyroiditis, pathological immune response to tissue / organ transplantation (for example, graft rejection); COPD, asthma, bronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), periodontal disease and gingivitis.In some embodiments, Th17 disease is a long-standing autoimmune disease such as type I diabetes or psoriasis. In certain embodiments, the Th17-associated disorder is age-related macular degeneration.

[0323] In some embodiments, the chronic complement-mediated disorder is an IgE-related disorder. As used herein, an "IgE-related disorder" is a disorder characterized by excessive and / or inappropriate production and / or amounts of IgE, excessive or inappropriate activity of IgE-producing cells (e.g., IgE-producing B cells or plasma cells), and / or excessive and / or inappropriate activity of IgE-responsive cells such as eosinophils or mast cells. In some embodiments, an IgE-related disorder is characterized by elevated levels of total IgE and / or, in some embodiments, allergen-specific IgE in the plasma and / or locally in a subject.

[0324] In some embodiments, chronic complement-mediated disorders are characterized by the presence of autoantibodies and / or immune complexes in the body, which can activate complement, for example, via the classical pathway. Autoantibodies can, for example, bind to autoantigens on cells or tissues in the body. In some embodiments, autoantibodies can bind to antigens in blood vessels, skin, nerves, muscles, connective tissue, heart, kidney, thyroid, etc. In some embodiments, the subject has neuromyelitis optica and produces autoantibodies (e.g., IgG autoantibodies) against aquaporin 4. In certain embodiments, the subject has pemphigoid and produces autoantibodies (e.g., IgG or IgE autoantibodies) against structural elements of hemidesmosomes (e.g., transmembrane collagen XVII (BP180 or BPAG2) and / or plakin family protein BP230 (BPAG1). In certain embodiments, the subject has a polyneuropathy, e.g., a chronic polyneuropathy such as multifocal motor neuropathy or an acute polyneuropathy, and produces anti-ganglioside antibodies that bind to gangliosides present in nerve cell membranes. In certain embodiments, the chronic complement-mediated disorder is not characterized by autoantibodies and / or immune complexes.

[0325] In some embodiments, the chronic complement-mediated disorder is a respiratory disorder. In some embodiments, the chronic respiratory disorder is asthma or chronic obstructive pulmonary disease (COPD). In some embodiments, the chronic respiratory disorder is pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis (also known as allergic alveolitis), eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener's granulomatosis, or bronchiolitis obliterans. In some embodiments, the present invention provides a method for treating a chronic respiratory disorder in a subject in need of treatment, such as asthma, COPD, pulmonary fibrosis, radiation-induced lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis (also known as allergic alveolitis), eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener's granulomatosis, or bronchiolitis obliterans, comprising administering a long-acting complement inhibitor to the subject in need of treatment.

[0326] In certain embodiments, the chronic complement-mediated disorder is allergic rhinitis, rhinosinusitis, or nasal polyposis. In certain embodiments, the present invention provides a method for treating allergic rhinitis, rhinosinusitis, or nasal polyposis in a subject in need of treatment, comprising administering a long-acting complement inhibitor to the subject in need of treatment.

[0327] In some embodiments, the chronic complement-mediated disorder is a disorder affecting the musculoskeletal system. Examples of such disorders include inflammatory joint conditions (e.g., arthritis such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthropathies, Reiter's syndrome, and gout). In some embodiments, the musculoskeletal disorder results in symptoms such as pain, stiffness, and / or limited movement in the affected body part. Inflammatory myopathies include dermatomyositis, polymyositis, and various other disorders of chronic muscle inflammation of unknown etiology that cause muscle weakness. In some embodiments, the chronic complement-mediated disorder is myasthenia gravis. In some embodiments, the present invention provides a method for treating any of the aforementioned musculoskeletal disorders, comprising administering a long-acting complement inhibitor to a subject in need of such treatment.

[0328] In some embodiments, the chronic complement-mediated disorder is a disorder affecting the integumentary system. Examples of such disorders include, for example, atopic dermatitis, psoriasis, pemphigoid, pemphigus, systemic lupus erythematosus, dermatomyositis, scleroderma, sclerodermatomyositis, Sjogren's syndrome, and chronic urticaria. In some embodiments, the present invention provides a method for treating any of the above-mentioned disorders affecting the integumentary system, comprising administering a long-acting complement inhibitor to a subject in need of such treatment.

[0329] In certain embodiments, the chronic complement-mediated disorder affects the nervous system, e.g., the central nervous system (CNS) and / or the peripheral nervous system (PNS). Examples of such disorders include, for example, multiple sclerosis, other chronic demyelinating diseases (e.g., neuromyelitis optica), acute or chronic neuropathies associated with autoantibodies against neuronal components, such as anti-ganglioside antibodies (e.g., Guillain-Barré syndrome, multifocal motor neuropathy), amyotrophic lateral sclerosis, chronic pain, stroke, allergic neuritis, Huntington's disease, Alzheimer's disease, and Parkinson's disease. In certain embodiments, the present invention provides a method for treating any of the disorders affecting the nervous system, comprising administering to a subject in need of such treatment a complement inhibitor, e.g., a permanent, ring-acting, targeted, or cell-reactive compstatin analog.

[0330] In some embodiments, the chronic complement-mediated disorder occurs in the circulatory system. For example, in some embodiments, the disorder is vasculitis or other vascular inflammation, e.g., a disorder associated with inflammation of blood vessels and / or lymphatic vessels. In some embodiments, the vasculitis is polyarteritis nodosa, Wegener's granulomatosis, giant cell arteritis, Churg-Strauss syndrome, microscopic polyangiitis, Henoch-Schönlein purpura, Takayasu's arteritis, Kawasaki disease, or Behçet's disease. In some embodiments, the subject, e.g., a subject in need of treatment for vasculitis, is antineutrophil cytoplasmic antibody (ANCA)-positive.

[0331] In some embodiments, the chronic complement-mediated disorder occurs in the digestive system. For example, the disorder can be inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In some embodiments, the present invention provides a method for treating a chronic complement-mediated disorder occurring in the digestive system, comprising administering a long-acting complement inhibitor to a subject in need of treatment for the disorder.

[0332] In certain embodiments, the chronic complement-mediated disorder is thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease, postpartum thyroiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis, glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis), or panniculitis.

[0333] In some embodiments, the chronic complement-mediated disorder affects the kidney. For example, the disorder can be glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis). In some embodiments, such disorders are characterized by the deposition of complement fragments in the kidney, which may be present along with antibodies. For example, in some embodiments, the disorder affecting the kidney is C3 glomerulopathy, including dense deposition disease (DDD) and C3 glomerulonephritis (C3G). Patients suffering from C3 glomerulopathy exhibit staining for C3 fragments (e.g., iC3b, C3c, C3dg) in glomeruli, which can be detected using antibodies. Such patients may exhibit overactivation of the alternative complement pathway, for example, by autoantibodies that stabilize C3 convertase, such as C3Nef or anti-factor ...

Claims

1. A physiologically acceptable or pharmaceutical grade composition comprising a cell-reactive compstatin analog.

2. 10. The composition of claim 1, which is physiologically acceptable.

3. 10. The composition of claim 1, which is a pharmaceutical grade composition.

4. 10. The composition of claim 1, which is pharmaceutically acceptable for administration to a human.

5. 10. The composition of claim 1, wherein the cell-reactive compstatin analog comprises a cell-reactive functional group capable of covalently binding to a mammalian cell.

6. 10. The composition of claim 1, wherein the cell-reactive compstatin analog comprises a cell-reactive moiety that comprises a cell-reactive functional group.

7. 10. The composition of claim 1, wherein the compstatin analog comprises a cell-reactive functional group that reacts with a sulfhydryl (SH) group to form a covalent bond.

8. 10. The composition of claim 1, wherein the compstatin analog comprises a cell-reactive functional group that reacts with an amine group to form a covalent bond.

9. 10. The composition of claim 1, wherein the cell-reactive compstatin analog comprises a maleimide group.

10. 10. The composition of claim 1, wherein the cell-reactive compstatin analog comprises a cell-reactive moiety, and the cell-reactive moiety comprises a cell-reactive functional group.

11. 2. The composition of claim 1, wherein the cell-reactive compstatin analog comprises a cell-reactive functional group, a compstatin analog portion, and a linking portion that separates the cell-reactive functional group from the compstatin analog portion of the cell-reactive compstatin analog.

12. 10. The composition of claim 1, wherein the cell-reactive compstatin analog comprises an amino acid whose side chain comprises a group of the formula (NH)-R, where R represents a moiety that contains a cell-reactive functional group.

13. 2. The composition of claim 1, wherein the cell-reactive compstatin analog is a compound comprising a cyclic peptide having the core sequence X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa and Xaa are selected from Trp and analogs of Trp, and the compound comprises a cell-reactive moiety.

14. 2. The composition of claim 1, wherein the compstatin analog is a compound comprising a cyclic peptide having the core sequence X'aa-Gln-Asp-Xaa-Gly-X"aa (SEQ ID NO: 4), where X'aa and Xaa are each independently selected from Trp and an analog of Trp, and X"aa is selected from His, Ala, a monomethyl unbranched amino acid, Phe, Trp, and an analog of Trp, and wherein the compound comprises a cell-reactive moiety.

15. 2. The composition of claim 1, wherein the peptide has the sequence X'aa1-X'aa2-X'aa3-X'aa-Gln-Asp-Xaa-Gly-X"aa-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO: 5), where X'aa1, X'aa2, X'aa3, X"aa2, X"aa3-X"aa4, and X"aa5 correspond to the amino acids at the corresponding positions in compstatin, and the compound comprises a cell-reactive moiety.

16. 2. The composition of claim 1, wherein the compstatin analog is a compound comprising a cyclic peptide having the sequence X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X"aa1-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO: 5), wherein X'aa4 and Xaa are selected from Trp and analogs of Trp, and X'aa1, X'aa2, X'aa3, X"aa1, X"aa2, X"aa3, X"aa4 and X"aa5 are independently selected from amino acids and amino acid analogs, the peptide is cyclized via the bond between X'aa2 and X"aa4, and the compound comprises a cell-reactive moiety.

17. 17. The composition of claim 16, wherein X'aa2 and X"aa4 are Cys, and X"aa1 is optionally Ala or a monomethyl unbranched amino acid, and the compound comprises a cell-reactive moiety.

18. The composition of claim 16, wherein X'aa2 and X"aa4 are Cys, and any one or more of X'aa1, X'aa2, X'aa3, X"aa2, X"aa3, X"aa4 and X"aa5 correspond to the amino acids at the corresponding positions in compstatin, and X"aa1 is Ala or a monomethyl unbranched amino acid, and the compound comprises a cell-reactive moiety.

19. The compstatin analog has the sequence Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2 ＊ -Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO: 6), wherein Xaa1 is Ile, Val, Leu, B １ -Ile, B １ -Val, B １ -Leu or Gly-Ile or B １ -Gly-Ile dipeptide, B １ represents the first blocking portion, Xaa2 and Xaa2 ＊ is independently selected from Trp and an analog of Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp or an analog of Trp; Xaa4 is a dipeptide selected from L-Thr, D-Thr, Ile, Val, Gly, Thr-Ala, and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, and the carboxy terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn is optionally a second blocking moiety B ２ has been replaced by a compound comprising a cyclic peptide in which two Cys residues are linked by a disulfide bond, the compound comprises a cell-reactive moiety; The composition of claim 1.

20. Xaa1 is a dipeptide containing Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu, Gly-Ile, or Ac-Gly-Ile, Xaa2 and Xaa2 ＊ are each independently selected from Trp and an analog of Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp or an analog of Trp; Xaa4 is a dipeptide selected from L-Thr, D-Thr, Ile, Val, Gly, Thr-Ala, and Thr-Asn, or a tripeptide containing Thr-Ala-Asn, and the carboxy terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn is optionally replaced with —NH ２ has been replaced by 20. The composition of claim 19.

21. 20. The composition of claim 19, wherein Xaa2 is an analog of Trp that has increased hydrophobicity relative to Trp.

22. 20. The composition of claim 19, wherein Xaa2 is an analog of Trp containing a substituted or unsubstituted bicyclic aromatic ring moiety or two or more substituted or unsubstituted monocyclic aromatic ring moieties.

23. Xaa2 ＊ 20. The composition of claim 19, wherein is an analog of Trp having electronegative substituents on the indole ring and not having increased hydrophobicity relative to Trp.

24. Xaa2 ＊ 20. The composition of claim 19, wherein is an analog of Trp containing a lower alkoxy or lower alkyl substituent at the 1- or 5-position of tryptophan or a halogen substituent at the 5- or 6-position of tryptophan.

25. Xaa2 ＊ is an analog of Trp containing a lower alkoxy or lower alkyl substituent at the 1- or 5-position of tryptophan or a halogen substituent at the 5- or 6-position of tryptophan, ＊ 20. The composition of claim 19, wherein is Trp.

26. 20. The composition of claim 19, wherein the compstatin analog comprises a cyclic peptide having a sequence selected from the group consisting of SEQ ID NOs: 9-36 and a cell-reactive moiety.

27. 20. The composition of claim 19, wherein the compstatin analog comprises a cyclic peptide having a sequence selected from the group consisting of SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, or 36 and a cell-reactive moiety.

28. 20. The composition of claim 19, wherein the compstatin analog comprises a cyclic peptide having the sequence of SEQ ID NO: 28, 32, or 34.

29. 2. The composition of claim 1, wherein the compstatin analog is a compound comprising a cyclic peptide having the sequence X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X"aa1-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO: 5), wherein X'aa4 and Xaa are selected from Trp and analogs of Trp, X'aa1, X'aa2, X'aa3, X"aa1, X"aa2, X"aa3, X"aa4 and X"aa5 are independently selected from amino acids and amino acid analogs, X'aa2 and X"aa4 are not Cys, the peptide is cyclized via the bond between X'aa2 and X"aa4, and the compound comprises a cell-reactive moiety.

30. The composition of claim 29, wherein any one or more of X'aa1, X'aa3, X"aa2, X"aa3 and X"aa5 correspond to the amino acids at the corresponding positions of the peptides of any one of claims 18 to 28, and X"aa1 is Ala or a monomethyl unbranched amino acid.

31. 30. The composition of claim 29, wherein one of X'aa2 and X"aa4 is an amino acid or amino acid analog having a side chain containing a primary or secondary amine, and the other of X'aa2 and X"aa4 is an amino acid or amino acid analog having a side chain containing a carboxylic acid group, and the bond is an amide bond.

32. 30. The composition of claim 29, wherein X'aa1, X'aa3, X"aa1, X"aa2, X"aa3 and X"aa5 correspond to the amino acids at the corresponding positions of the cyclic peptide of any one of claims 18 to 28, and optionally one of X'aa2 and X"aa4 is an amino acid or amino acid analogue having a side chain containing a primary or secondary amine, and the other of X'aa2 and X"aa4 is an amino acid or amino acid analogue having a side chain containing a carboxylic acid group, and the bond is an amide bond.

33. The composition of any of claims 10 to 32, wherein the cyclic peptide is acetylated at the N-terminus, amidated at the C-terminus, or acetylated at the N-terminus and amidated at the C-terminus.

34. An isolated cell or organ having a covalently attached compstatin analog.

35. 35. The isolated cell or organ of claim 34, which is a human cell or organ.

36. 35. The isolated cell or organ of claim 34, wherein the isolated cell is a blood cell or the isolated organ is a heart, kidney, liver, lung or pancreas.

37. A method for reducing the susceptibility of a cell or organ to complement-dependent injury, comprising contacting the cell with a cell-reactive compstatin analog, wherein the cell-reactive compstatin analog covalently binds to the cell or organ.

38. 38. The method of claim 37, wherein the cell or organ is a human cell or organ.

39. 38. The method of claim 37, wherein the cell is a blood cell or the organ is a heart, kidney, liver, lung, or pancreas.

40. 38. The method of claim 37, wherein the cell or organ is an isolated cell or organ to be transplanted into a subject, and the method comprises contacting the cell or organ with the cell-reactive compstatin analog prior to transplantation.

41. 38. The method of claim 37, wherein the cell or the organ is transplanted into the subject, and the method comprises contacting the organ with the cell-reactive compstatin analog after transplantation.

42. 38. The method of claim 37, comprising perfusing the organ with a fluid containing the cell-reactive compstatin analog.

43. 38. The method of claim 37, comprising contacting the cell or the organ with the cell-reactive compstatin analog at the time of transplantation of the organ into the subject.

44. 38. The method of claim 37, comprising administering the cell-reactive compstatin analog to the subject after transplanting the cell or the organ into the subject.

45. 38. The method of claim 37, comprising administering the cell-reactive compstatin analog to the subject after transplanting the organ into the subject, wherein the cell-reactive compstatin analog is administered locally to the transplanted organ.

46. 38. The method of claim 37, wherein the cells or organ have been or are to be transplanted into a subject at high risk of developing a hyperacute or acute complement-mediated transfusion reaction or complement-mediated organ rejection.

47. 1. A method of treating a subject in need of treatment for a complement-mediated disorder, comprising administering to the subject a cell-reactive compstatin analog.

48. 48. The method of claim 47, wherein the cell-reactive compstatin analog is administered locally to a site at risk for or experiencing complement-mediated injury.

49. 48. The method of claim 47, wherein the injury results in complement-mediated injury to red blood cells and the cell-reactive compstatin analog is administered intravascularly.

50. 48. The method of claim 47, wherein the subject has a defect in complement regulation.

51. 48. The method of claim 47, wherein the subject is in need of treatment for transplant rejection.

52. 48. The method of claim 47, wherein the subject is in need of treatment for ischemia / reperfusion injury.

53. 48. The method of claim 47, wherein the subject is in need of treatment for hemolytic anemia.

54. A long-acting compstatin analog comprising one or more compstatin analog moieties and a polymer having a molecular weight of 10 kilodaltons (kD) to 45 kD as a clearance-reducing moiety (CRM).

55. 55. The long-acting compstatin analog of claim 54, comprising as the CRM a polymer having a molecular weight of 20 kilodaltons (kD) to 45 kD.

56. 56. The long-acting compstatin analog of claim 54 or 55, comprising a polymer with a molecular weight of 30 kilodaltons (kD) as the clearance-reducing moiety.

57. 56. The long-acting compstatin analog of claim 54 or 55, comprising a polymer with a molecular weight of 40 kilodaltons (kD) as the clearance-reducing moiety.

58. 58. The long-acting compstatin analog of any of claims 54-57, wherein the polymer comprises polyethylene glycol (PEG).

59. 59. The long-acting compstatin analog of any of claims 54-58, wherein the polymer comprises a linear PEG.

60. 59. The long-acting compstatin analog of any of claims 54-58, wherein the polymer comprises a branched PEG.

61. 60. The long-acting compstatin analog of claim 59, wherein the polymer comprises a linear PEG and comprises a compstatin analog moiety attached to each end of the linear PEG.

62. 61. The long-acting compstatin analog of claim 60, wherein the polymer comprises a branched PEG having 3 to 10 branches.

63. 63. The long-acting compstatin analog of claim 62, wherein the polymer comprises a branched PEG having 3 to 10 branches, at least about 50% of the branches having a compstatin analog moiety attached thereto.

64. 64. The long-acting compstatin analog of claim 63, wherein the polymer comprises a branched PEG having 3 to 10 branches, at least about 75% of the branches having a compstatin analog moiety attached thereto.

65. 58. The long-acting compstatin analog of any of claims 54-57, wherein the polymer comprises human serum albumin.

66. 66. The long-acting compstatin analog of any of claims 54-65, comprising 2 to 10 compstatin analog moieties.

67. 67. The long-acting compstatin analog of any of claims 54-66, comprising 2 to 100 compstatin analog moieties.

68. 68. The long-acting compstatin analog of any of claims 54 to 67, having a plasma half-life of at least 2 days when intravenously injected into a primate.

69. 69. The long-acting compstatin analog of any of claims 54 to 68, having a plasma half-life of at least 3 days when intravenously injected into a primate.

70. 70. The long-acting compstatin analog of any of claims 54 to 69, having a plasma half-life of at least 4 days when intravenously injected into a primate.

71. 71. The long-acting compstatin analog of any of claims 54-70, having a molar activity that is at least about 20% of the activity of a corresponding compstatin analog having the same amino acid sequence but that does not include the clearance-reducing moiety.

72. 72. The long-acting compstatin analog of any of claims 54-71, having a molar activity that is at least about 30% of the activity of the corresponding compstatin analog that does not include the clearance-reducing moiety.

73. 73. The long-acting compstatin analog of any of claims 54-72, comprising multiple compstatin analog moieties and having a molar activity approximately equal to the sum of the activities of the compstatin analog moieties.

74. 74. The long-acting compstatin analog of any of claims 54-73, having a terminal half-life that is at least 5 times that of a corresponding compstatin analog without an equivalent amount of said CRM.

75. C ｍａｘ is at least 10 times that of an equivalent amount of a corresponding compstatin analog without the clearance-reducing moiety.

76. 76. The long-acting compstatin analog of any of claims 54-75, having a plasma half-life of at least 2 days when injected subcutaneously in a primate.

77. 77. The long-acting compstatin analog of claim 76, having a plasma half-life of at least 3 days upon subcutaneous injection in a primate.

78. 77. The long-acting compstatin analog of claim 76, having a plasma half-life of at least 4 days upon subcutaneous injection in a primate.

79. 79. The long-acting compstatin analog of any of claims 76-78, having a molar activity that is at least about 20% of the activity of a corresponding compstatin analog that does not include a clearance-reducing moiety.

80. 80. The long-acting compstatin analog of any of claims 76-79, having a molar activity that is at least about 30% of the activity of a compstatin analog comprising the same amino acid sequence but without the clearance-reducing moiety.

81. 80. The long-acting compstatin analog of any of claims 76-79, comprising a plurality of compstatin analog moieties and having a molar activity that is at least about 10% of the sum of the activities of the compstatin analog moieties.

82. C ｍａｘ is at least 10-fold greater than an equivalent amount of a corresponding compstatin analog without the clearance-reducing moiety.

83. at least about 30% of the activity and at least 10-fold higher C than a corresponding compstatin analog that does not contain an equivalent amount of the clearance-reducing moiety. ｍａｘ and having a plasma half-life of at least 3 days.

84. 84. The long-acting compstatin analog of any of claims 54-83, wherein the compstatin analog portion comprises a cyclic peptide having the core sequence X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa and Xaa are selected from Trp and analogs of Trp.

85. 84. The long-acting compstatin analog of any of claims 54-83, wherein the compstatin analog moiety comprises a cyclic peptide having the core sequence X'aa-Gln-Asp-Xaa-Gly-X"aa (SEQ ID NO: 4), where X'aa and Xaa are each independently selected from Trp and analogs of Trp, and X"aa is selected from His, Ala, mono-methyl unbranched amino acids, Phe, Trp, and analogs of Trp.

86. 84. The long-acting compstatin analog of any of claims 54-83, wherein the compstatin analog portion comprises a cyclic peptide having the sequence X'aa1-X'aa2-X'aa3-X'aa-Gln-Asp-Xaa-Gly-X"aa-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO: 5), wherein X'aa1, X'aa2, X'aa3, X"aa2, X"aa3-X"aa4, and X"aa5 correspond to the amino acids at the corresponding positions in compstatin.

87. 84. The long-acting compstatin analog of any of claims 54-83, wherein the compstatin analog portion comprises a cyclic peptide having the sequence X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X"aa1-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO:5), wherein X'aa4 and Xaa are selected from Trp and analogs of Trp, and X'aa1, X'aa2, X'aa3, X"aa1, X"aa2, X"aa3, X"aa4 and X"aa5 are independently selected from amino acids and amino acid analogs, and the peptide is cyclized via the bond between X'aa2 and X"aa4.

88. 84. The long-acting compstatin analog of any of claims 54-83, wherein X'aa2 and X"aa4 are Cys, and X"aa1 is optionally Ala or a monomethyl unbranched amino acid.

89. 84. The long-acting compstatin analog of any of claims 54 to 83, wherein X'aa2 and X"aa4 are Cys, and any one or more of X'aa1, X'aa2, X'aa3, X"aa2, X"aa3, X"aa4, and X"aa5 correspond to the amino acids at the corresponding positions in compstatin, and X"aa1 is Ala or a monomethyl unbranched amino acid.

90. The compstatin analog moiety is Sequence Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2 ＊ -Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO: 6) wherein Xaa1 is Ile, Val, Leu, B １ -Ile, B １ -Val, B １ -Leu or Gly-Ile or B １ -Gly-Ile dipeptide, B １ represents the first blocking portion, Xaa2 and Xaa2 ＊ is independently selected from Trp and an analog of Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp or an analog of Trp; Xaa4 is a dipeptide selected from L-Thr, D-Thr, Ile, Val, Gly, Thr-Ala, and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, and the carboxy terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn is optionally a second blocking moiety B ２ has been replaced by A cyclic peptide in which two Cys residues are linked by a disulfide bond.

84. The long-acting compstatin analog of any of claims 54-83, comprising:

91. Xaa1 is a dipeptide containing Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu, Gly-Ile, or Ac-Gly-Ile, Xaa2 and Xaa2 ＊ is independently selected from Trp and an analog of Trp; Xaa3 is His, Ala or an analog of Ala, Phe, Trp or an analog of Trp; Xaa4 is a dipeptide selected from L-Thr, D-Thr, Ile, Val, Gly, Thr-Ala, and Thr-Asn, or a tripeptide containing Thr-Ala-Asn, and the carboxy terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn is optionally replaced with —NH ２ 91. The long-acting compstatin analog of claim 90, wherein:

92. 91. The long-acting compstatin analog of claim 90, wherein Xaa2 is an analog of Trp that has increased hydrophobicity relative to Trp.

93. 91. The long-acting compstatin analog of claim 90, wherein Xaa2 is an analog of Trp containing a substituted or unsubstituted bicyclic aromatic ring moiety or two or more substituted or unsubstituted monocyclic aromatic ring moieties.

94. Xaa2 ＊ 91. The long-acting compstatin analog of claim 90, wherein is an analog of Trp having electronegative substituents on the indole ring and not having increased hydrophobicity relative to Trp.

95. Xaa2 ＊ 91. The long-acting compstatin analog of claim 90, wherein is an analog of Trp containing a lower alkoxy or lower alkyl substituent at the 1- or 5-position of tryptophan or a halogen substituent at the 5- or 6-position of tryptophan.

96. Xaa2 ＊ is an analog of Trp containing a lower alkoxy or lower alkyl substituent at the 1- or 5-position of tryptophan, or a halogen substituent at the 5- or 6-position of tryptophan, and Xaa2 ＊ 91. The long-acting compstatin analog of claim 90, wherein is Trp.

97. 91. The long-acting compstatin analog of claim 90, comprising a compstatin analog portion comprising a cyclic peptide having a sequence selected from the group consisting of SEQ ID NOs: 9-36, and optionally further comprising an amino acid having a side chain comprising a primary or secondary or sulfhydryl reactive group.

98. 91. The long-acting compstatin analog of claim 90, wherein the compstatin analog moiety comprises a cyclic peptide having a sequence selected from the group consisting of SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, or 36.

99. 91. The long-acting compstatin analog of claim 90, wherein the compstatin analog portion comprises a cyclic peptide having the sequence of SEQ ID NO: 28, 32, or 34.

100. 84. The long-acting compstatin analog of any of claims 54-83, comprising a compstatin analog moiety comprising a cyclic peptide having the sequence X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X"aa1-X"aa2-X"aa3-X"aa4-X"aa5 (SEQ ID NO: 5), wherein X'aa4 and Xaa are selected from Trp and analogs of Trp, and X'aa1, X'aa2, X'aa3, X"aa1, X"aa2, X"aa3, X"aa4 and X"aa5 are independently selected from amino acids and amino acid analogs, X'aa2 and X"aa4 are not Cys, and the peptide is cyclized via the bond between X'aa2 and X"aa4.

101. 101. The long-acting compstatin analog of claim 100, wherein any one or more of X'aa1, X'aa3, X"aa2, X"aa3, and X"aa5 corresponds to the amino acid at the corresponding position in the peptide of any one of claims 90 to 99, and X"aa1 is Ala or a monomethyl unbranched amino acid.

102. The long-acting compstatin analog of claim 100, wherein one of X'aa2 and X"aa4 is an amino acid or amino acid analog having a side chain containing a primary or secondary amine, and the other of X'aa2 and X"aa4 is an amino acid or amino acid analog having a side chain containing a carboxylic acid group, and the bond is an amide bond.

103. 101. The long-acting compstatin analog of claim 100, wherein X'aa1, X'aa3, X"aa1, X"aa2, X"aa3, and X"aa5 correspond to the amino acids at the corresponding positions of the cyclic peptide of any of claims 90 to 99, and optionally one of X'aa2 and X"aa4 is an amino acid or amino acid analog having a side chain containing a primary or secondary amine, and the other of X'aa2 and X"aa4 is an amino acid or amino acid analog having a side chain containing a carboxylic acid group, and the bond is an amide bond.

104. 104. The long-acting compstatin analog of any of claims 84-103, wherein the cyclic peptide is acetylated at the N-terminus, amidated at the C-terminus, or both acetylated at the N-terminus and amidated at the C-terminus.

105. A long-acting compstatin analog comprising: (i) a compound comprising a cyclic peptide having a sequence selected from the group consisting of SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, or 36; and (ii) a polymer having a molecular weight of 10 kD to 45 kD.

106. 106. The long-acting compstatin analog of any of claims 84-105, comprising a compound of any of formulas I-XVI or A-H in which at least one NHS ester has reacted with a side chain or terminal amino group of the compstatin analog moiety, provided that the compound of any of formulas I-XVI or A-H has a molecular weight of 10 kD to 45 kD.

107. 1. A method for preparing a long-acting compstatin analog, comprising reacting a compound of any of Formulas I-XVI or A-H with a compstatin analog moiety, wherein the compound of any of Formulas I-XVI or A-H has a molecular weight of 10 kD to 45 kD.

108. 1. A method for producing a long-acting compstatin analog, comprising reacting a compound of any of Formulas I-XVI or Formulas A-H with a compstatin analog moiety comprising an amino acid sequence of any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40a, or 41A, wherein the compound of any of Formulas I-XVI or Formulas A-H has a molecular weight of 10 kD to 45 kD.

109. 109. A long-acting compstatin analog prepared according to or structurally identical to the method of any of claims 107-108.

110. 165. The long-acting compstatin analog of any of claims 84-106, 109, or 145-164, further comprising a targeting moiety.

111. 165. A composition comprising the long-acting compstatin analog of any of claims 54-105, 109, or 145-164 and a pharmaceutically acceptable carrier.

112. 165. A pharmaceutical composition comprising a long-acting compstatin analog of any of claims 54-106, 109, or 145-164.

113. 165. A pharmaceutical composition comprising a long-acting compstatin analog of any of claims 54-106, 109, or 145-164 and a pharmaceutically acceptable carrier.

114. 165. A method of reducing the susceptibility of a cell or organ to complement-dependent injury, comprising contacting said cell with a long-acting compstatin analog or composition of any of claims 54-106, 109-113, or 144-165.

115. 115. The method of claim 114, wherein the cell or organ is a human cell or organ.

116. 115. The method of claim 114, wherein the cell is a blood cell or the organ is a heart, kidney, liver, lung, or pancreas.

117. 115. The method of claim 114, comprising administering to a subject the long-acting compstatin analog or the composition.

118. 174. A method of treating a subject in need of treatment for a complement-mediated disorder, comprising administering to the subject a long-acting compstatin analog or composition of any of claims 54-106, 109-113, 144-165, or 167-174.

119. 119. The method of claim 118, wherein the long-acting compstatin analog is administered locally to a site at risk for or experiencing complement-mediated injury.

120. 119. The method of claim 118, wherein the injury causes complement-mediated injury to red blood cells.

121. 119. The method of claim 118, wherein the injury results in complement-mediated injury to red blood cells and the long-acting compstatin analog is administered intravenously or subcutaneously.

122. 119. The method of claim 118, wherein the subject has a defect in complement regulation.

123. 119. The method of claim 118, wherein the subject is in need of treatment for transplant rejection.

124. 119. The method of claim 118, wherein the subject is in need of treatment for ischemia / reperfusion injury.

125. 119. The method of claim 118, wherein the subject is in need of treatment for hemolytic anemia.

126. 119. The method of claim 118, wherein the subject is in need of treatment for an autoimmune disease.

127. 119. The method of claim 118, wherein the subject is in need of treatment for neuropathic pain.

128. 119. The method of claim 118, wherein the subject is in need of treatment for MPGN.

129. 119. The method of claim 118, wherein the subject is in need of treatment for neuromyelitis optica.

130. 119. The method of claim 118, wherein the subject is in need of treatment for spinal cord injury.

131. 119. The method of claim 118, wherein the subject is in need of treatment for asthma, COPD, or idiopathic pulmonary fibrosis.

132. 132. The method of any of claims 118-131, wherein the long-acting compstatin analog is administered subcutaneously.

133. 132. The method of any of claims 118-131, wherein the long-acting compstatin analog is administered subcutaneously one or more times daily.

134. 132. The method of any of claims 118-131, wherein the long-acting compstatin analog is administered transdermally.

135. 132. The method of any of claims 118-131, wherein the long-acting compstatin analog is administered subcutaneously using a pen device.

136. 132. The method of any of claims 118-131, wherein the long-acting compstatin analog is administered intramuscularly.

137. 132. The method of any of claims 118-131, wherein the long-acting compstatin analog comprises a polymer having a molecular weight of about 40 kD and is administered subcutaneously once or twice daily, wherein the total daily dose is between 90 mg and 360 mg, optionally between 180 mg / day and 270 mg / day.

138. 109. The method of claim 107 or 108, comprising a method comprising: combining a compound of any of Formulas I-XVI or Formulas A-H and a compstatin analog moiety with a click functionality and undergoing a click chemistry reaction, respectively.

139. Compstatin analogues containing click chemistry groups.

140. 140. The compstatin analog of claim 139, wherein the compstatin analog comprises a compound comprising any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A.

141. 141. The compstatin analog of claim 139 or 140, wherein the click chemistry group comprises an azide, alkyne, octyne, or dibenzoarylcyclooctyne.

142. 141. The compstatin analog of claim 140, wherein the click chemistry group is DBCO, DIBO, DIFO, BARAC, or BCN.

143. 141. The compstatin analog of claim 140, wherein the click chemistry group is suitable for copper-free click chemistry reactions.

144. 144. A composition comprising the compstatin analog of any of claims 139-143 and a CRM, wherein the CRM comprises a polymer having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, for example 40 kD.

145. 144. A conjugate formed by reacting a compstatin analog of any of claims 139-143 with a CMR comprising a complementary click chemistry group, wherein the CRM comprises a polymer having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, e.g., 40 kD.

146. A long-acting compstatin analog comprising a compstatin analog moiety and a CRM linked via a click chemistry bond, wherein the CRM comprises a polymer having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, for example 40 kD.

147. A long-acting compstatin analog comprising a compstatin analog moiety and a CRM, wherein the CRM comprises POZ having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, for example 40 kD.

148. A long-acting compstatin analog comprising a CRM comprising at least two compstatin analog moieties and a polymer having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, e.g., 40 kD, and further optionally each compstatin analog moiety comprising any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A or 41A.

149. 149. The long-acting compstatin analog of claim 148, having at least 90% or at least 100% of the activity on a molar basis of a compstatin analog having the same sequence as the compstatin analog portion.

150. 149. The long-acting compstatin analog of claim 148, wherein the CRM comprises PEG.

151. 149. The long-acting compstatin analog of claim 148, wherein the CRM comprises POZ.

152. 149. The long-acting compstatin analog of claim 148, wherein the CRM comprises a polypeptide.

153. 153. The long-acting compstatin analog of any of claims 148-152, comprising two compstatin analog moieties.

154. 153. The long-acting compstatin analog of any of claims 148-152, comprising three compstatin analog moieties.

155. 153. The long-acting compstatin analog of any of claims 148-152, comprising 2 to 8 compstatin analog moieties.

156. 149. The long-acting compstatin analog of claim 148, comprising two compstatin analog moieties and a linear PEG, wherein a compstatin analog moiety is optionally attached to each end of the linear PEG by a carbamate or ester bond.

157. 149. The long-acting compstatin analog of claim 148, comprising three compstatin analog moieties and PEG.

158. 149. The long-acting compstatin analog of claim 148, comprising 2 to 8 compstatin analog moieties and PEG.

159. 149. The long-acting compstatin analog of claim 148, comprising two compstatin analog moieties and POZ.

160. 149. The long-acting compstatin analog of claim 148, comprising three compstatin analog moieties and POZ.

161. 149. The long-acting compstatin analog of claim 148, comprising 2 to 8 compstatin analog moieties and POZ.

162. 149. The long-acting compstatin analog of claim 148, comprising two compstatin analog moieties and a polypeptide.

163. 149. The long-acting compstatin analog of claim 148, comprising three compstatin analog moieties and a polypeptide.

164. 149. The long-acting compstatin analog of claim 148, comprising 2 to 8 compstatin analog moieties and a polypeptide.

165. 165. A composition comprising a compstatin analog of any of claims 139-164, optionally a pharmaceutical composition.

166. 119. The method of claim 118, wherein a long-acting compstatin analog is administered for the treatment of a Th17-associated disease.

167. 175. The long-acting compstatin analog of any of claims 54-106, 109-113, 144-165, or 167-174, having a molecular weight of at least about 30 kD, a terminal half-life of at least about 3 days when administered to a primate, and exhibiting at least 80% of the activity, on a molar basis, of a compstatin analog that contains the same compstatin analog sequence as the compstatin analog portion but is not bound to a CRM.

168. 168. The long-acting compstatin analog of claim 167, having a molecular weight of at least about 40 kD.

169. 169. The long-acting compstatin analog of claim 167 or 168, having a terminal half-life of at least about 4 days.

170. 169. The long-acting compstatin analog of claim 167 or 168, having a terminal half-life of at least about 5 days.

171. 169. The long-acting compstatin analog of claim 167 or 168, which exhibits at least 90% of the activity, on a molar basis, of a compstatin analog that contains the same compstatin analog sequence as the compstatin analog portion but is not bound to a CRM.

172. 169. The long-acting compstatin analog of claim 167 or 168, which has at least the same activity, on a molar basis, as a compstatin analog that contains the same compstatin analog sequence as the compstatin analog portion but is not bound to a CRM.

173. 169. The long-acting compstatin analog of claim 167 or 168, wherein the compstatin analog portion comprises any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A.

174. 174. The long-acting compstatin analog of any of claims 167-173, wherein the CRM comprises PEG, POZ, or a polypeptide.

175. 174. A method of treating a complement-mediated eye disorder, comprising administering to a subject in need thereof a long-acting compstatin analog of any of claims 54-106, 109-113, 144-165, or 167-174.

176. 176. The method of claim 175, wherein the eye disorder is AMD.

177. 176. The method of claim 175, wherein the eye disorder is geographic atrophy.

178. 176. The method of claim 175, wherein the eye disorder is intermediate AMD.

179. 179. The method of any of claims 175-178, wherein the long-acting compstatin analog is administered by intravitreal injection.

180. 180. The method of claim 179, wherein LACA is administered monthly or bimonthly.

181. 181. The method of claim 179 or 180, wherein the administered dose is between 10 mg and 20 mg.

182. 181. The method of claim 179 or 180, wherein the administered dose is 15 mg.

183. A unit dose of LACA comprising a polymer having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, for example, 40 kD, wherein the amount of the unit dose is 45 mg to 360 mg, optionally 180 mg to 270 mg, for example, 180 mg or 270 mg.

184. A unit dose for intravitreal administration of LACA, wherein the LACA comprises a polymer having a molecular weight of 10 kD to 45 kD, optionally 35 kD to 45 kD, for example, 40 kD, wherein the amount of the unit dose is 10 mg to 20 mg, optionally 15 mg.

185. 185. The unit dose of claim 183 or 184, wherein the polymer has a molecular weight of about 30 kD.

186. 185. The unit dose of claim 183 or 184, wherein the polymer has a molecular weight of about 40 kD.

187. 187. The unit dose of any of claims 183-186, further comprising a pharmaceutically acceptable carrier.

188. 188. A syringe or container containing a unit dose according to any one of claims 183 to 187.

189. 189. The unit dose, syringe or container of claim 187 or 188, wherein the LACA is present in a concentration of 125 mg / ml to 200 mg / ml in a pharmaceutically acceptable carrier.

190. 190. The unit dose, syringe or container of claim 189, wherein the LACA is present in a pharmaceutically acceptable carrier at a concentration of 140 mg / ml to 180 mg / ml, for example 150 mg / ml.

191. 189. A unit dose, syringe or container according to claim 187 or 188, wherein the LACA is present in a pharmaceutically acceptable carrier at a concentration of 80 mg / ml to 125 mg / ml, for example 100 mg / ml.

192. 192. The unit dose, syringe or container of any of claims 183-191, wherein the polymer is PEG.

193. 193. The unit dose, syringe, or container of any of claims 183-192, wherein the LACA comprises two compstatin analog moieties, wherein one compstatin analog moiety is attached to each end of the polymer by an unsaturated alkyl moiety, a moiety comprising a non-aromatic cyclic ring system, an aromatic moiety, an ether moiety, an amide moiety, an ester moiety, a carbonyl moiety, an imine moiety, a thioether moiety, and / or a moiety comprising an amino acid residue.

194. 194. The unit dose, syringe, or container of any of claims 183-193, wherein the LACA comprises two compstatin analog moieties, wherein one compstatin analog moiety is attached to each end of the polymer by an ester or carbamate bond.

195. 195. The unit dose, syringe or container of any of claims 183-194, wherein the compstatin analog portion comprises any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A.

196. 196. The unit dose, syringe, or container of any of claims 183-195, wherein the compstatin analog moiety comprises a peptide comprising a cyclic moiety, the peptide extended at the N-terminus, C-terminus, or both termini by one or more amino acids, wherein at least one of the amino acids has a side chain comprising a reactive cyclic group, and wherein the one or more amino acid extensions are optionally separated from the cyclic moiety of the compstatin analog moiety by a spacer.

197. 197. The unit dose, syringe or container of claim 196, wherein the spacer comprises a substituted or unsubstituted, saturated or unsaturated alkyl chain, or an oligo(ethylene glycol) chain.

198. 198. The unit dose, syringe or container of claim 197, wherein the spacer comprises an AEEAc moiety.

199. 199. The unit dose, syringe or container of any of claims 183-198, wherein the LACA is CA28-2TS-BF or CA28-2GS-BF.

200. A unit dose of LACA comprising a polymer having a molecular weight of 35 kD to 45 kD, the unit dose being in an amount of 545 mg to 5040 mg.

201. 201. The unit dose of claim 200, wherein the polymer has a molecular weight of 37.5 kD to 42.5 kD.

202. 202. The unit dose of claim 200 or 201, wherein the polymer has a molecular weight of 40 kD.

203. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 545 mg and 1690 mg, optionally wherein the unit dose is for subcutaneous administration three times per week.

204. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 630 mg and 930 mg, optionally wherein the unit dose is for subcutaneous administration three times per week.

205. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 795 mg and 885 mg, optionally wherein the unit dose is for subcutaneous administration three times per week.

206. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 585 mg and 2510 mg, optionally wherein the unit dose is for subcutaneous administration twice a week.

207. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 900 mg and 1395 mg, optionally wherein the unit dose is for subcutaneous administration twice a week.

208. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 990 mg and 1215 mg, optionally wherein the unit dose is for subcutaneous administration twice a week.

209. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 1215 mg and 1395 mg, optionally wherein the unit dose is for subcutaneous administration twice a week.

210. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 1080 mg and 5040 mg, optionally wherein the unit dose is for once-weekly subcutaneous administration.

211. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 2160 mg and 2520 mg, optionally wherein the unit dose is for once-weekly subcutaneous administration.

212. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 2520 mg and 2880 mg, optionally wherein the unit dose is for once-weekly subcutaneous administration.

213. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 2880 mg and 3240 mg, optionally wherein the unit dose is for once-weekly subcutaneous administration.

214. 203. The unit dose of any one of claims 200 to 202, wherein the amount is between 3240 mg and 3600 mg, optionally wherein the unit dose is for once-weekly subcutaneous administration.

215. 215. The unit dose of any one of claims 200 to 214, further comprising a pharmaceutically acceptable carrier.

216. 217. A syringe or container comprising a unit dose according to any one of claims 200 to 216.

217. 217. The unit dose, syringe or container of any one of claims 200 to 216, wherein the LACA is present in a concentration of 25 mg / ml to 150 mg / ml in the pharmaceutically acceptable carrier.

218. 218. The unit dose, syringe or container of claim 217, wherein the LACA is present in a concentration of 25 mg / ml to 50 mg / ml in the pharmaceutically acceptable carrier.

219. 218. The unit dose, syringe or container of claim 217, wherein the LACA is present in a concentration of 50 mg / ml to 75 mg / ml in the pharmaceutically acceptable carrier.

220. 218. The unit dose, syringe or container of claim 217, wherein the LACA is present in a concentration of 75 mg / ml to 100 mg / ml in the pharmaceutically acceptable carrier.

221. 218. The unit dose, syringe or container of claim 217, wherein the LACA is present in a concentration of 100 mg / ml to 125 mg / ml in the pharmaceutically acceptable carrier.

222. 222. The unit dose, syringe or container of any one of claims 200-221, wherein the polymer is PEG.

223. 223. The unit dose, syringe, or container of any one of claims 200-222, wherein the polymer is a linear polymer and the LACA comprises two compstatin analog moieties, one compstatin analog attached to each end of the polymer by an unsaturated alkyl moiety, a moiety comprising a non-aromatic ring system, an aromatic moiety, an ether moiety, an amide moiety, an ester moiety, a carbonyl moiety, an imine moiety, a thioether moiety, and / or a moiety comprising an amino acid residue.

224. 224. The unit dose, syringe or container of claim 223, wherein one compstatin analog moiety is attached to each end of the polymer by an ester or carbamate bond.

225. 225. The unit dose, syringe or container of claim 223 or 224, wherein the LACA comprises two compstatin analog moieties, one compstatin analog moiety attached to each end of the polymer by a carbamate bond.

226. 226. The unit dose, syringe or container of any one of claims 200-225, wherein the compstatin analog moiety comprises any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A or 41A.

227. 227. The unit dose, syringe or container of any one of claims 200-226, wherein the compstatin analog moiety comprises a peptide extended by one or more amino acids at the N-terminus, C-terminus or both termini comprising a cyclic moiety, said one or more stretches of amino acids optionally being separated from the cyclic moiety of the compstatin analog moiety by a spacer.

228. 228. The unit dose, syringe or container of claim 227, wherein the cyclic peptide is extended by an amino acid sequence comprising at least one amino acid having a side chain comprising a primary or secondary amine.

229. 229. The unit dose, syringe or container of claim 227 or 228, wherein at least one amino acid having a side chain containing a primary or secondary amine is a lysine attached at the C-terminus of the cyclic peptide.

230. 230. The unit dose, syringe, or container of any one of claims 227-229, wherein the spacer comprises a substituted or unsubstituted, saturated or unsaturated alkyl chain, or an oligo(ethylene glycol) chain.

231. The oligo(ethylene glycol) portion is (-(O-CH2-CH2-) ｎ and n is 1 to 10.

232. The spacer is a covalently linked -(CH2) ｍ - and -(O-CH2-CH2-) ｎ 232. The method of any one of claims 227 to 231, comprising:

233. 233. The unit dose, syringe or container of claim 232, wherein the spacer comprises 8-amino-3,6-dioxaoctanoic acid (AEEAc) or 11-amino-3,6,9-trioxaundecanoic acid.

234. 234. The unit dose, syringe or container of claim 233, wherein the spacer comprises an AEEAc moiety.

235. 235. The unit dose, syringe or container of any one of claims 200-234, wherein the LACA is CA28-2TS-BF or CA28-2GS-BF.

236. 236. A method of inhibiting complement activation in a subject, comprising subcutaneously administering to the subject a unit dose according to any one of claims 200-235.

237. 237. The method of claim 236, wherein the unit dose is administered using a syringe pump.

238. 237. The method of claim 236, wherein the unit dose is administered using an internal delivery device.

239. 239. The method of any one of claims 236 to 238, wherein the unit dose is administered in a volume of from 10 ml to 50 ml.

240. 240. The method of any one of claims 236 to 239, wherein the unit dose is administered in a volume of from 20 ml to 40 ml.

241. 241. The method of any one of claims 236 to 240, wherein the unit dose is administered three times per week.

242. 241. The method of any one of claims 236-240, wherein the unit dose is administered twice weekly.

243. 241. The method of any one of claims 236 to 240, wherein the unit dose is administered once a week.

244. 244. The method of any one of claims 236-243, wherein the subject is suffering from or at risk of a complement-mediated disorder.

245. 236. A method of treating a subject in need of treatment for a complement-mediated disorder, comprising subcutaneously administering to the subject a unit dose according to any one of claims 200-235.

246. 246. The method of claim 245, wherein the unit dose is administered using a syringe pump.

247. 246. The method of claim 245, wherein the unit dose is administered using an internal delivery device.

248. 248. The method of any one of claims 245 to 247, wherein the unit dose is administered in a volume of from 10 ml to 50 ml.

249. 248. The method of any one of claims 245 to 247, wherein the unit dose is administered in a volume of between 20 ml and 40 ml.

250. 250. The method of any one of claims 245 to 249, wherein the unit dose is administered three times per week.

251. 250. The method of any one of claims 245 to 249, wherein the unit dose is administered twice weekly.

252. 250. The method of any one of claims 245 to 249, wherein the unit dose is administered once a week.

253. 253. The method of any one of claims 244 to 252, wherein the complement-mediated disorder is hemolytic anemia.

254. 253. The method of any one of claims 244 to 252, wherein the complement-mediated disorder is PNH.

255. 253. The method of any one of claims 244-252, wherein the complement-mediated disorder is autoimmune hemolytic anemia, and optionally the hemolytic anemia is cold agglutinin disease or warm autoimmune hemolytic anemia.

256. 253. The method of any one of claims 244-252, wherein the complement-mediated disorder is myasthenia gravis.

257. 253. The method of any one of claims 244 to 252, wherein the complement-mediated disorder is NMO.

258. 253. The method of any one of claims 244 to 252, wherein the complement-mediated disorder is a polyneuropathy, or wherein the complement-mediated disorder is a neuropathy, or wherein the complement-mediated disorder is a vasculitis.

259. A method for inhibiting complement activation in a subject, comprising subcutaneously administering to the subject LACA comprising a polymer having a molecular weight of 35 kD to 45 kD on a dosing schedule of three times a week, two times a week, or once a week.

260. 260. The method of claim 259, wherein the polymer has a molecular weight of 37.5 kD to 42.5 kD.

261. 261. The method of claim 259 or 260, wherein the polymer has a molecular weight of 40 kD.

262. The method of any one of claims 259 to 261, wherein LACA is administered three times a week in an amount of 545 mg to 1690 mg per dose.

263. The method of any one of claims 259 to 261, wherein LACA is administered three times a week in an amount of 630 mg to 930 mg per dose.

264. The method of any one of claims 259 to 261, wherein LACA is administered three times a week in an amount of 795 mg to 885 mg per dose.

265. The method of any one of claims 259 to 261, wherein LACA is administered twice a week in an amount of 585 mg to 2510 mg per dose.

266. 262. The method of any one of claims 259 to 261, wherein LACA is administered twice a week in an amount of 900 mg to 1395 mg per dose.

267. 262. The method of any one of claims 259 to 261, wherein LACA is administered twice a week in an amount of 990 mg to 1215 mg per dose.

268. The method of any one of claims 259 to 261, wherein LACA is administered twice a week in an amount of 1215 mg to 1395 mg per dose.

269. The method of any one of claims 259 to 261, wherein LACA is administered once a week in an amount of 1080 mg to 5040 mg per dose.

270. The method of any one of claims 259 to 261, wherein LACA is administered once a week in an amount of 2160 mg to 2520 mg per dose.

271. The method of any one of claims 259 to 261, wherein LACA is administered once a week in an amount of 2520 mg to 2880 mg per dose.

272. The method of any one of claims 259 to 261, wherein LACA is administered once a week in an amount of 2880 mg to 3240 mg per dose.

273. The method of any one of claims 259 to 261, wherein LACA is administered once a week in an amount of 3240 mg to 3600 mg per dose.

274. 262. The method of any one of claims 259 to 261, wherein LACA is administered as a composition comprising LACA and a pharmaceutically acceptable carrier.

275. 275. The method of any one of claims 259 to 274, wherein LACA is administered at a concentration of 25 mg / ml to 150 mg / ml.

276. 275. The method of any one of claims 259 to 274, wherein LACA is administered at a concentration of 25 mg / ml to 50 mg / ml.

277. 275. The method of any one of claims 259 to 274, wherein LACA is administered at a concentration of 50 mg / ml to 75 mg / ml.

278. 275. The method of any one of claims 259 to 274, wherein LACA is administered at a concentration of 75 mg / ml to 100 mg / ml.

279. 275. The method of any one of claims 259 to 274, wherein LACA is administered at a concentration of 100 mg / ml to 125 mg / ml.

280. 280. The method of any one of claims 259 to 279, wherein the polymer is PEG.

281. 281. The method of any one of claims 259-280, wherein the polymer is a linear polymer and the LACA comprises two compstatin analog moieties, one compstatin analog attached to each end of the polymer by an unsaturated alkyl moiety, a moiety comprising a non-aromatic ring system, an aromatic moiety, an ether moiety, an amide moiety, an ester moiety, a carbonyl moiety, an imine moiety, a thioether moiety, and / or a moiety comprising an amino acid residue.

282. 282. The method of any one of claims 259-281, wherein the LACA comprises two compstatin analog moieties, one compstatin analog moiety attached to each end of the polymer by an ester or carbamate bond.

283. 283. The method of any one of claims 259-282, wherein the LACA comprises two compstatin analog moieties, one compstatin analog moiety attached to each end of the polymer by a carbamate bond.

284. 284. The method of any one of claims 259-283, wherein the compstatin analog moiety comprises any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A.

285. 285. The method of any one of claims 259-284, wherein the compstatin analog moiety comprises a peptide extended by one or more amino acids at the N-terminus, C-terminus or both termini comprising a cyclic moiety, wherein the one or more stretches of amino acids may optionally be separated from the cyclic moiety of the compstatin analog moiety by a spacer.

286. 286. The unit dose, syringe or container of claim 285, extended by an amino acid sequence comprising at least one amino acid having a side chain comprising a primary or secondary amine.

287. 287. The method of claim 285 or 286, wherein at least one amino acid having a side chain containing a primary or secondary amine is a lysine attached at the C-terminus of the cyclic peptide.

288. 288. The method of any one of claims 285 to 287, wherein the spacer comprises a substituted or unsubstituted, saturated or unsaturated alkyl chain, or an oligo(ethylene glycol) chain.

289. The oligo(ethylene glycol) portion is (-(O-CH2-CH2-) ｎ and n is 1 to 10.

290. The spacer is a covalently linked -(CH2) ｍ - and -(O-CH2-CH2-) ｎ 290. The method of any one of claims 285 to 289, comprising:

291. 291. The method of claim 290, wherein the spacer comprises 8-amino-3,6-dioxaoctanoic acid (AEEAc) or 11-amino-3,6,9-trioxaundecanoic acid.

292. 292. The method of claim 291, wherein the spacer comprises an AEEAc moiety.

293. The method of any one of claims 259 to 292, wherein the LACA is CA28-2TS-BF or CA28-2GS-BF.

294. 294. The method of any one of claims 259 to 293, wherein the subject is suffering from a complement-mediated disorder.

295. A method of treating a subject in need of treatment for a complement-mediated disorder, comprising subcutaneously administering to the subject LACA comprising a polymer having a molecular weight of 35 kD to 45 kD on a dosing schedule of three times a week, two times a week, or once a week.

296. 296. The method of claim 295, wherein the polymer has a molecular weight of 37.5 kD to 42.5 kD.

297. 297. The method of claim 295 or 296, wherein the polymer has a molecular weight of 40 kD.

298. The method of any one of claims 295 to 297, wherein LACA is administered three times a week in an amount of 545 mg to 1690 mg per dose.

299. The method of any one of claims 295 to 297, wherein LACA is administered three times a week in an amount of 630 mg to 930 mg per dose.

300. The method of any one of claims 295 to 297, wherein LACA is administered three times a week in an amount of 795 mg to 885 mg per dose.

301. The method of any one of claims 295 to 297, wherein LACA is administered twice a week in an amount of 585 mg to 2510 mg per dose.

302. The method of any one of claims 295 to 297, wherein LACA is administered twice a week in an amount of 900 mg to 1395 mg per dose.

303. The method of any one of claims 295 to 297, wherein LACA is administered twice a week in an amount of 990 mg to 1215 mg per dose.

304. The method of any one of claims 295 to 297, wherein LACA is administered twice a week in an amount of 1215 mg to 1395 mg per dose.

305. The method of any one of claims 295 to 297, wherein LACA is administered once a week in an amount of 1080 mg to 5040 mg per dose.

306. The method of any one of claims 295 to 297, wherein LACA is administered once a week in an amount of 2160 mg to 2520 mg per dose.

307. The method of any one of claims 295 to 297, wherein LACA is administered once a week in an amount of 2520 mg to 2880 mg per dose.

308. The method of any one of claims 295 to 297, wherein LACA is administered once a week in an amount of 2880 mg to 3240 mg per dose.

309. The method of any one of claims 295 to 297, wherein LACA is administered once a week in an amount of 3240 mg to 3600 mg per dose.

310. 298. The method of any one of claims 295 to 297, wherein LACA is administered as a composition comprising LACA and a pharmaceutically acceptable carrier.

311. 311. The method of any one of claims 295 to 310, wherein LACA is administered at a concentration of 25 mg / ml to 150 mg / ml.

312. 311. The method of any one of claims 295 to 310, wherein LACA is administered at a concentration of 25 mg / ml to 50 mg / ml.

313. The method of any one of claims 295 to 310, wherein LACA is administered at a concentration of 50 mg / ml to 75 mg / ml.

314. 311. The method of any one of claims 295 to 310, wherein LACA is administered at a concentration of 75 mg / ml to 100 mg / ml.

315. 311. The method of any one of claims 295 to 310, wherein LACA is administered at a concentration of 100 mg / ml to 125 mg / ml.

316. The method of any one of claims 295 to 315, wherein the polymer is PEG.

317. 317. The method of any one of claims 295-316, wherein the polymer is a linear polymer and the LACA comprises two compstatin analog moieties, one compstatin analog attached to each end of the polymer by an unsaturated alkyl moiety, a moiety comprising a non-aromatic ring system, an aromatic moiety, an ether moiety, an amide moiety, an ester moiety, a carbonyl moiety, an imine moiety, a thioether moiety, and / or a moiety comprising an amino acid residue.

318. The method of any one of claims 295-317, wherein the LACA comprises two compstatin analog moieties, one compstatin analog moiety attached to each end of the polymer by an ester or carbamate bond.

319. The method of any one of claims 295-318, wherein the LACA comprises two compstatin analog moieties, one compstatin analog moiety attached to each end of the polymer by a carbamate bond.

320. 320. The method of any one of claims 295-319, wherein the compstatin analog moiety comprises any of SEQ ID NOs: 3-36, 37, 37A, 38A, 39A, 40A, or 41A.

321. The method of any one of claims 295-320, wherein the compstatin analog moiety comprises a peptide extended by one or more amino acids at the N-terminus, C-terminus or both termini comprising a cyclic moiety, wherein the one or more stretches of amino acids may optionally be separated from the cyclic moiety of the compstatin analog moiety by a spacer.

322. 322. The unit dose, syringe or container of claim 321, extended by an amino acid sequence comprising at least one amino acid having a side chain comprising a primary or secondary amine.

323. 323. The method of claim 321 or 322, wherein at least one amino acid having a side chain containing a primary or secondary amine is a lysine attached at the C-terminus of the cyclic peptide.

324. 334. The method of any one of claims 321-333, wherein the spacer comprises a substituted or unsubstituted, saturated or unsaturated alkyl chain, or an oligo(ethylene glycol) chain.

325. The oligo(ethylene glycol) portion is (-(O-CH2-CH2-) ｎ and n is 1 to 10.

326. The spacer is a covalently linked -(CH2) ｍ - and -(O-CH2-CH2-) ｎ The method of any one of claims 321 to 325, comprising:

327. 327. The method of claim 326, wherein the spacer comprises 8-amino-3,6-dioxaoctanoic acid (AEEAc) or 11-amino-3,6,9-trioxaundecanoic acid.

328. 328. The method of claim 327, wherein the spacer comprises an AEEAc moiety.

329. The method of any one of claims 295 to 328, wherein the LACA is CA28-2TS-BF or CA28-2GS-BF.

330. 330. The method of any one of claims 294 to 329, wherein the complement-mediated disorder is hemolytic anemia.

331. 330. The method of any one of claims 294 to 329, wherein the complement-mediated disorder is PNH.

332. 330. The method of any one of claims 294-329, wherein the complement-mediated disorder is autoimmune hemolytic anemia, and optionally the hemolytic anemia is cold agglutinin disease or warm autoimmune hemolytic anemia.

333. 330. The method of any one of claims 294-329, wherein the complement-mediated disorder is myasthenia gravis.

334. The method of any one of claims 294 to 329, wherein the complement-mediated disorder is NMO.

335. 330. The method of any one of claims 294 to 329, wherein the complement-mediated disorder is a polyneuropathy, or wherein the complement-mediated disorder is a neuropathy, or wherein the complement-mediated disorder is a vasculitis.

336. 336. The unit dose, syringe, container or method of any one of claims 183-335, wherein the compstatin analog comprises SEQ ID NO:28.

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