RU2789389C2 - Pharmaceutical composition intended for use for treatment or prevention of c5-related disease and method for treatment or prevention of c5-related disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to doses and schemes of administration of an antibody to C5. Pharmaceutical compositions intended for the use for treatment or prevention of C5-related disease and methods for treatment or prevention of C5-related disease are described. Doses and schemes of administration of an antibody to C5 or pharmaceutical compositions containing an antibody to C5 are also described.

EFFECT: obtainment of pharmaceutical compositions for treatment or prevention of C5-related disease.

15 cl, 11 ex, 4 tbl, 11 dwg

Description

The field of technology to which the invention belongs

The present invention relates to dosages and schedules for administering an anti-C5 antibody.

Prerequisites for the creation of the invention

The complement system plays a central role in immune complex clearance and immune responses to infectious agents, foreign antigens, virus-infected cells, and tumor cells. Approximately 25-30 complement proteins are known to exist as a complex group of plasma proteins and membrane cofactors. Complement components exercise their immune protective functions by participating in a series of complex enzymatic cleavages and membrane binding events. The resulting cascades of the complement system lead to products with opsonic, immunoregulatory and lytic functions.

It is now generally accepted that the complement system can be activated in three different ways: the classical pathway, the lectin pathway, and the alternative pathway. There are many components involved in these pathways, and although they differ in their initial stages, they then converge and include the same terminal components of the complement system (C5 to C9), which are responsible for the activation and destruction of target cells.

The classical pathway is usually activated by the formation of antigen-antibody complexes. Regardless, the first step in the activation of the lectin pathway is the binding of specific lectins such as mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL-11. In contrast, the alternative pathway undergoes spontaneous activation with a low level of turnover, which can easily rise on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue). These pathways converge at a point where complement component C3 is cleaved by an active protease to form C3a and C3b.

C3a is an anaphylatoxin. C3b binds to bacterial and other cells, as well as certain viruses and immune complexes, and marks them for removal from the bloodstream (plays the role of an opsonin). C3b also forms a complex with other components to form C5 convertase, which cleaves C5 to form C5a and C5b.

C5 is a protein with a molecular weight of 190 kDa, which is present in the serum of a healthy individual at a concentration of approximately 80 μg/ml (0.4 μm). C5 is glycosylated, with about 1.5-3% of its mass being carbohydrate. Mature C5 is a heterodimer in which the 115 kDa alpha chain is disulfide-linked to the 75 kDa beta chain. C5 is synthesized as a single chain precursor protein (precursor pro-C5) consisting of 1676 amino acids (see, for example, PTL1 and PTL2). The pro-C5 precursor is cleaved to release the beta chain as an amino-terminal fragment and the alpha chain as a carboxy-terminal fragment. Polypeptide fragments, representing the alpha chain and the beta chain, are connected to each other through a disulfide bond to form the mature C5 protein.

Mature C5 is cleaved to form a C5a fragment and a C5b fragment during the activation of complement pathways. As a result of the action of C5 convertase, C5a is cleaved from the C5 alpha chain in the form of an amino-terminal fragment containing the first 74 amino acids of the alpha chain. The remainder of the mature C5 is a C5b fragment which contains an alpha chain residue linked by a disulfide to the beta chain. Approximately 20% of the mass of C5a, which is 11 kDa, is accounted for by carbohydrate.

C5a is another anaphylatoxin. C5b combines with C6, C7, C8 and C9 to form a membrane attack complex (MAC, C5b-9, Terminal Complement Component Complex (TCC)) on the surface of the target cell. When sufficient MACs are incorporated into target cell membranes, the pores formed by the MAC mediate rapid osmotic lysis of the target cells.

As stated above, C3a and C5a are anaphylatoxins. They can trigger mast cell degranulation, which causes the release of histamine and other inflammatory mediators, which lead to smooth muscle contraction, increased vascular permeability, leukocyte activation, and other manifestations of inflammation, including cell proliferation leading to increased parenchymal cell counts. C5a also functions as a chemotactic peptide that serves to recruit granulocytes such as neutrophils, eosinophils, basophils, and monocytes to the site of complement activation.

C5a activity is regulated by the plasma enzyme carboxypeptidase N, which removes the carboxy-terminal arginine from C5a, resulting in the formation of the C5a-des-Arg derivative. C5a-des-Arg has only 1% of the anaphylactic activity and polymorphonuclear chemotactic activity inherent in unmodified C5a.

While a properly functioning complement system provides effective protection against infecting organisms, misregulation or activation of the complement system has been implicated in the pathogenesis of various disorders including, for example, rheumatoid arthritis (RA); lupus nephritis; damage caused by ischemia-reperfusion; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration (eg age-related macular degeneration (AMD)); syndrome: hemolysis, increased activity of liver enzymes and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; Pauci-immune (low-immune) vasculitis (vasculitis with hypocomplementemia); bullous epidermolysis; repeated loss of the fetus; multiple sclerosis (MS); traumatic brain injury and damage due to myocardial infarction, cardiopulmonary bypass and hemodialysis (see, for example, NPL1). Thus, inhibition of excessive or uncontrolled activation of the complement cascade may have a beneficial clinical effect on patients with these disorders.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder in which red blood cells are defective and therefore destroyed faster than healthy red blood cells. PNH is caused by clonal expansion of hematopoietic stem cells with somatic mutations in the PIG-A (class A phosphatidylinositolglycan) gene located on the X chromosome. Mutations in PIG-A result in early blockade of the synthesis of glycosylphosphatidylinositol (GPI), a molecule required to anchor many proteins to cell surfaces. As a result, in PNH, blood cells are deficient in GPI-anchored proteins, which include the complement-regulating proteins CD55 and CD59. Under normal circumstances, these complement-regulating proteins block the formation of MAC on cell surfaces, thereby preventing erythrocyte lysis. The absence of GPI-anchored proteins causes complement-mediated hemolysis in PNH.

PNH is characterized by hemolytic anemia (low red blood cell count), hemoglobinuria (the presence of hemoglobin in the urine, especially after sleep), and hemoglobinemia (the presence of hemoglobin in the bloodstream). PNH affected individuals are known to have paroxysms, which in this case are cases of dark colored urine. Hemolytic anemia is caused by intravascular destruction of red blood cells by components of the complement system. Other known symptoms include dysplasia, fatigue, erectile dysfunction, thrombosis, and recurring abdominal pain.

Eculizumab is a humanized anti-complement C5 monoclonal antibody and is the first-line therapeutic agent approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see e.g. NPL2). Eculizumab inhibits the cleavage of C5 by C5 convertase with the formation of C5a and C5b, which prevents the formation of the complex of the terminal components of the complement system C5b-9. Both C5a and C5b-9 induce terminal complex mediated events characteristic of PNH and aHUS (see also PTL3, PTL4, PTL5 and PTL6).

Several publications describe antibodies to C5. For example, PTL7 describes an anti-C5 antibody that binds to the C5 alpha chain but does not bind to C5a and blocks C5 activation, and PTL8 describes an anti-C5 monoclonal antibody that inhibits the formation of C5a. On the other hand, PTL9 describes an anti-C5 antibody that recognizes a proteolytic C5 convertase cleavage site on the C5 alpha chain and inhibits the conversion of C5 to C5a and C5b. PTL10 describes an anti-C5 antibody that has an affinity constant of at least 1×10 ⁷ M ^-1 .

Antibodies (IgG) bind to the neonatal Fc receptor (FcRn) and are characterized by long plasma retention times. IgG binding to FcRn typically occurs under acidic conditions (eg, pH 6.0) and rarely occurs under neutral conditions (eg, pH 7.4). Typically, IgGs are non-specifically incorporated into cells via endocytosis and returned to cell surfaces via binding to endosomal FcRn under acidic conditions in endosomes. The IgGs are then separated from the FcRn by dissociation under neutral conditions in the plasma. IgGs that fail to bind to FcRn are cleaved in lysosomes. If the ability of IgG to bind to FcRn under acidic conditions is eliminated by the introduction of mutations in its Fc region, then IgG does not return from endosomes to plasma, which leads to a marked decrease in plasma IgG retention. Published a method to increase its binding to FcRn under acidic conditions, designed to increase the retention of IgG in plasma. When IgG binding to FcRn under acidic conditions is increased by introducing an amino acid substitution into its Fc region, IgG is more efficiently returned from endosomes to plasma, and as a result, its plasma retention is increased. In addition, data have also been published showing that IgG with increased ability to bind to FcRn under neutral conditions is not separated by dissociation from FcRn under neutral conditions in plasma, even if it returns to the cell surface due to its binding to FcRn in acidic conditions. conditions in endosomes, and hence its plasma retention is not altered or rather deteriorated (see, for example, NPL3; NPL4; NPL5).

In recent years, antibodies have been described that bind to antigens in a pH dependent manner (see, for example, PTL11 and PTL12). Said antibodies bind strongly to antigens under neutral conditions in plasma and are separated by dissociation from antigens under acidic conditions which occur in endosomes. After separation by dissociation from antigens, antibodies regain the ability to bind to antigens when they are returned to plasma via FcRn. Thus, one antibody molecule can re-associate with several antigen molecules. In general, the plasma retention time of an antigen is much shorter than that of an antibody that has the above FcRn-mediated recycling mechanism. Thus, when an antigen is bound to an antibody, the antigen normally has a prolonged plasma retention, resulting in an increase in the plasma concentration of the antigen. On the other hand, it has been found that the antibodies described above, which bind to antigens in a pH-dependent manner, allow the elimination of antigens from plasma faster than typical antibodies, since they are separated by dissociation from antigens in endosomes during FcRn-mediated recycling. PTL13 also describes a computer simulation based assay demonstrating that an anti-C5 antibody having pH dependent binding can enhance antigen knockout.

Link List

Patent Literature

[PTL1 US patent No. 6355245,

[PTL2] US Pat. No. 7,432,356

[PTL3] W02005/074607,

[PTL4] W02007/106585,

[PTL5] W02008/069889,

[PTL6] W02010/054403,

[PTL7] WO 95/29697,

[PTL8] W02002/30985,

[PTL9] W02004/007553,

[PTL10] W02010/015608,

[PTL11] W02009/125825,

[PTL12] WO 2011/122011,

[PTL13] WO 2011/111007.

Non-Patent Literature

[NPL1] Holers et al., Immunol. Rev. 223, 2008, CC. 300-316,

[NPL2] Dmytrijuk et al., The Oncologist 13(9), 2008, pp. 993-1000,

[NPL3] Yeung et al., J Immunol. 182 (12), 2009, cc. 7663-7671,

[NPL4] Datta-Mannan et al., J Biol. Chem. 282 (3), 2007, cc. 1709-1717,

[NPL5] Dall'Acqua et al., J. Immunol. 169(9), 2002, pp. 5171-5180.

Brief summary of the invention

The invention provides pharmaceutical compositions for use in the treatment or prevention of a C5 related disease and methods of treating or preventing a C5 related disease. The invention also provides doses and regimens for administering anti-C5 antibody or pharmaceutical compositions containing anti-C5 antibody.

In the course of the invention, more effective and safe dosages and regimens for administering an anti-C5 antibody for use in the treatment and/or prevention of C5-associated diseases have been studied, which may also reduce patient burden. From this point of view, it has been found that, from this point of view, at least one administration of a lower dose before administration of higher doses at long intervals is preferable for the treatment or prevention of a C5-related disease by subcutaneous administration of an anti-C5 antibody.

When creating the invention also studied acceptable dosing conditions, including the dose of the antibody, the frequency and intervals of subcutaneous injections. In particular, the present invention provides: [1] A pharmaceutical composition for use in a method for treating or preventing a C5 related disease, which is in a subcutaneous injection form and contains an anti-C5 antibody, wherein the composition is administered subcutaneously using two phases, wherein in both phases there is an interval between every two subcutaneous injections, where each phase includes at least one interval and where in the first phase

I) at least one interval is shorter than at least one interval in the second phase, and

ii) the dose of antibody per administration is lower or the same as the dose of antibody per administration in the second phase;

[2] Pharmaceutical composition for use according to item [1], where at least one interval in the first phase is from 1 day to 2 months;

[3] Pharmaceutical composition for use according to item [1] or item [2], where at least one interval in the first phase is from 5 to 14 days;

[4] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[3], where at least one interval in the second phase is from 2 days to 6 months;

[5] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[4], where the specified at least one interval in the second phase is from 15 days to 3 months;

[6] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[5], where the dose of antibody for subcutaneous administration in the first phase is from 50 to 350 mg;

[7] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[6], wherein the subcutaneous dose of the antibody in the first phase is 150 to 200 mg and is lower than the subcutaneous dose in the second phase, preferably the dose of the antibody in the first phase is 170 mg;

[8] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[6], wherein the dose of antibody for subcutaneous administration in the first phase is from 300 to 350 mg and is the same as the dose of antibody for subcutaneous administration in the second phase;

[9] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[8], where the number of subcutaneous injections in the first phase is from 1 to 12;

[10] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[9], where the number of subcutaneous injections in the first phase is from 5 to 10, preferably the number is 8;

[11] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[10], where the dose of antibody per injection in the second phase is from 350 to 1000 mg or from 650 to 700 mg;

[12] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[7] and [9]-[11], where the dose of antibody to C5 per injection in the first phase is 3-5 times lower than the dose of antibody to C5 per injection in the second phase;

[13] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[12], where the pharmaceutical composition is prepared in a form for intravenous administration and the anti-C5 antibody contained therein is administered intravenously before the first subcutaneous administration in the first phase;

[14] A pharmaceutical composition for use according to [13], wherein the first subcutaneous administration in the first phase is carried out at an interval of 0 days to 1 month after the final administration of the intravenously administered pharmaceutical composition;

[15] Pharmaceutical composition intended for use according to item [13] or item [14], where the dose of antibodies for the specified intravenous administration is from 100 to 2000 mg;

[16] A pharmaceutical composition intended for use according to one of paragraphs. [1]-[15], wherein the C5 related disease is any disease selected from the group consisting of rheumatoid arthritis (RA); lupus nephritis; damage caused by ischemia-reperfusion; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration; syndrome: hemolysis, increased activity of liver enzymes and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; Pauci-immune vasculitis; bullous epidermolysis; repeated loss of the fetus; multiple sclerosis (MS); traumatic brain injury and damage due to myocardial infarction, cardiopulmonary bypass and hemodialysis;

[17] A method for treating or preventing a C5 related disease, wherein the method comprises subcutaneously administering to an individual a pharmaceutical composition that is formulated for subcutaneous injection and contains an anti-C5 antibody, wherein the composition is administered subcutaneously using two phases,

where in both phases there is an interval between every two subcutaneous injections, where each phase includes at least one interval and where in the first phase

I) at least one interval is shorter than at least one interval in the second phase, and

ii) the dose of antibody per administration is lower or the same as the dose of antibody per administration in the second phase;

[18] The use of an anti-C5 antibody for the preparation of a pharmaceutical composition for the treatment or prevention of a C5-associated disease, wherein the composition is prepared in a subcutaneous injection form and contains an anti-C5 antibody, and wherein the composition is administered subcutaneously using two phases,

where in both phases there is an interval between every two subcutaneous injections, where each phase includes at least one interval and where in the first phase

I) at least one interval is shorter than at least one interval in the second phase, and

ii) the dose of antibody per administration is lower or the same as the dose of antibody per administration in the second phase; And

[19] A product for treating or preventing a C5 related disease, which contains (I) a container; (II) a pharmaceutical composition in a container, wherein the pharmaceutical composition is formulated for subcutaneous injection and contains an anti-C5 antibody; and (III) a document providing instructions that the pharmaceutical composition is to be administered subcutaneously using two phases,

where in both phases there is an interval between every two subcutaneous injections, where each phase includes at least one interval and where in the first phase

I) at least one interval is shorter than at least one interval in the second phase, and

ii) the dose of antibody per administration is lower or the same as the dose of antibody per administration in the second phase.

Brief description of the drawings

The drawings show:

in FIG. 1 in panels A and B - time-dependent change in the concentration of 305LO15 in the plasma of cynomolgus monkeys with subcutaneous injection (A) or intravenous injection (B) of the 305LO15 antibody. Plasma 305LO15 concentration was assessed by ELISA. Each point corresponds to the mean of three males or three females (A) and the mean of six males or six females (B);

in FIG. 2 shows the evolution of plasma concentration of 305LO15 in cynomolgus monkeys as a function of time when the monkeys were treated with an initial intravenous injection and subsequent subcutaneous maintenance injections of the 305LO15 antibody. Plasma 305LO15 concentration was assessed by ELISA. Each point corresponds to the mean of five males or five females;

in FIG. 3A is a graph showing plasma concentrations of free C5 versus each plasma antibody concentration for each indicated dosage amount and route of administration. IV, intravenous injection; SC, subcutaneous injection;

in FIG. 3B is a graph showing measured complement activity levels versus each plasma antibody concentration for each indicated dosage amount and route of administration. IV, intravenous injection; SC, subcutaneous injection;

in FIG. 4A are simulated plasma concentration versus time profiles of 305LO15 for the dosing regimens planned for part 1 of the clinical study. IV, intravenous injection; SC, subcutaneous injection;

in FIG. 4B is a simulated plasma concentration versus time of 305LO15 for the dosing regimens planned for part 2 of the clinical study. SC, subcutaneous injection; QW, administration once every week;

in FIG. 4B are simulated plasma concentration versus time profiles of 305LO15 for the dosing regimens planned for Part 3 of the clinical study. SC, subcutaneous injection; QW, administration once every week; Q2W, administration once every two weeks; Q4W, given once every four weeks;

in fig. 5 is a profile of immunocomplexes formed by eculizumab (ECZ), human C5 (hC5) and/or 305LO15 analyzed in vitro by gel filtration at pH 7.4 (upper panel) and at pH 6.0 (lower panel);

in FIG. 6 are human measured and simulated plasma concentration versus time profiles of 305LO15 in healthy individuals who received 75 mg by IV infusion (over 60 minutes). The shaded gray area indicates the 95% prediction interval of the simulated 305LO15 concentration versus time profile. The solid line indicated by an arrow corresponds to the simulated time profile of the median 305LO15 concentration. The dotted line indicates the threshold dose of PD, which is 40 μg/g;

in FIG. 7 - Human measured and simulated plasma concentration versus time profiles of 305LO15 in healthy individuals who received 125 mg by IV infusion (over 60 minutes). The shaded gray area indicates the 95% prediction interval of the simulated 305LO15 concentration versus time profile. The solid line indicated by an arrow corresponds to the simulated time profile of the median 305LO15 concentration. The dotted line indicates the threshold dose of PD, which is 40 μg/g;

in fig. 8 are human measured and simulated plasma concentration versus time profiles of 305LO15 in healthy individuals who received 100 mg SC. The shaded gray area indicates the 95% prediction interval of the simulated 305LO15 concentration versus time profile. For simulations performed with a single 100 mg SC dose, a bioavailability of 90% is expected. The dotted line indicates the cut-off dose PD of 40 µg/g;

in FIG. 9 is the relationship between plasma concentrations of 305LO15 and hemolytic activity (LIA) after a single IV infusion or SC injection in healthy individuals. An ex vivo LIA dose-response curve was generated using human plasma samples treated with various concentrations of 305LO15;

in fig. 10A is a time profile of LDH (lactate dehydrogenase) serum levels in a PNH patient, Patient X, who received 375 mg IV dose of 305LO15 on day 1, 500 mg IV dose of 305LO15 on day 8, 1000 mg IV dose of 305LO15 on day 22 , 170 mg SC dose of 305LO15 on day 36 and 170 mg SC dose of 305LO15 on day 43 in Study Part 2;

in fig. 10B is a time profile of hemolytic activity (LIA) in a PNH patient, patient X, who received 375mg IV dose of 305LO15 on day 1, 500mg IV dose of 305LO15 on day 8, 1000mg IV dose of 305LO15 on day 22, SC -dose 170 mg 305LO15 on day 36 and SC dose 170 mg 305LO15 on day 43 in part 2 of the study;

in fig. 11 is the optimized SC dose regimen studied in part 3 of the BP39144 study.

Description of embodiments of the invention

The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are widely used using conventional methodologies such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001; Current Protocols in Molecular Biology, ed. F.M. Ausubel et al., 2003; Methods in Enzymology series, Academic Press, Inc.: PCR 2: A Practical Approach, ed. M.J. MacPherson, B.D. Hames and G.R. Taylor, 1995, Antibodies, A Laboratory Manual, ed. Harlow and Lane, 1988 and Animal Cell Culture, ed. R.I. Freshney, 1987; Oligonucleotide Synthesis, ed. M.J. Gait, 1984; Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook, ed. J.E. Cellis, 1998, from Academic Press; Animal Cell Culture, ed. R.I. Freshney, 1987; J.P. Mather and P.E. Roberts, Introduction to Cell and Tissue Culture, Plenum Press Publishing House, 1998; Cell and Tissue Culture: Laboratory Procedures, ed. A. Doyle, J.B. Griffiths and D.G. Newell, Publishing House J. Wiley and Sons, 1993-1998; Handbook of Experimental Immunology, ed. D.M. Weir and S.S. Blackwell; Gene Transfer Vectors for Mammalian Cells, ed. J.M. Miller and M.P. Calos, 1987; PCR: The Polymerase Chain Reaction, ed. Mullis et al., 1994; Current Protocols in Immunology, ed. J.E. Coligan et al., 1991; Short Protocols in Molecular Biology, Publishing House of Wiley and Sons, 1999; C.A. Janeway and P. Travers, Immunobiology, 1997; P. Finch, Antibodies, 1997; Antibodies: A Practical Approach, ed. D. Catty, IRL Press, 1988-1989; Monoclonal Antibodies: A Practical Approach, ed. P. Shepherd and S.dean, Publishing House Oxford University Press, 2000; E. Harlow and D. Lane, Using Antibodies: A Laboratory Manual, Publishing House of Cold Spring Harbor Laboratory Press, 1999; The Antibodies, ed. M. Zanetti and J. D. Capra, Publishing House Harwood Academic Publishers, 1995; and Cancer: Principles and Practice of Oncology, ed. V.T. DeVita et al., J.B. Lippincott Company, 1993.

I. Definitions

"Interval" (interval between separate administrations) means the interval between the administration of the n-th dose (n is an integer 1 or more) and the administration of the (n+1)-th dose.

II. Pharmaceutical composition for subcutaneous injection

What is provided is a pharmaceutical composition intended for use in a method for treating or preventing a C5-associated disease. The pharmaceutical composition is prepared in the form for subcutaneous injection and contains an antibody to C5, while the composition is administered subcutaneously using two phases,

where in both phases there is an interval between every two subcutaneous injections, where each phase includes at least one interval and where in the first phase

I) at least one interval is shorter than at least one interval in the second phase, and

ii) the dose of antibody per administration is lower or the same as the dose of antibody per administration in the second phase.

Subcutaneous injection can be performed using conventional devices and methods for introducing the pharmaceutical composition into the subcutaneous tissue by injection. You can also choose specific devices and methods for subcutaneous injection.

Examples of anti-C5 antibodies

The anti-C5 antibody is not limited to a specific variant, and can be selected accordingly from the antibodies known as anti-C5 antibodies. The term "antibody to C5" or "antibody that binds to C5" refers to an antibody that has the ability to bind to C5 with sufficient affinity so that the antibody can be used as a C5 targeting therapeutic agent.

In one aspect of the invention, anti-C5 antibodies inhibit C5 activation. In some embodiments, anti-C5 antibodies that prevent C5 cleavage resulting in C5a and C5b, thereby preventing C5a-associated anaphylactic activity and also preventing assembly of the C5b-9 membrane attack complex (MAC) associated with C5b. In some embodiments, anti-C5 antibodies block the conversion of C5 to C5a and C5b by C5 convertase. In some embodiments, anti-C5 antibodies block the access of the C5 convertase to the C5 cleavage site. In some embodiments, anti-C5 antibodies block hemolytic activity due to C5 activation. In other embodiments, anti-C5 antibodies inhibit C5 activation in a classical and/or alternative manner.

According to one aspect of the invention, the pharmaceutical compositions of the present invention can be used to treat or prevent a C5-associated disease based at least in part on the aforementioned anti-C5 antibody activities, such as inhibition of C5 activation.

In some embodiments of the invention, the activity of C5 can be assessed by its ability to carry out cell lysis in the total water of the individual. The ability of C5 to carry out cell lysis or its reduction can be assessed using methods well known in this field, for example, using a standard hemolytic assay, such as the hemolytic assay described in Experimental Immunochemistry, ed. Kabat and Mayer, 2nd ed., 135-240, CC Thomas, Springfield, IL, 1961, pp. 135 139, or a standard variant of this assay, such as the chicken erythrocyte hemolysis method described, for example, in Hillman et al., N. Engl. J. Med. 350(6), 2004, pp. 552-559. In some embodiments of the invention, the activity of C5 or its inhibition is quantified using CH50eq analysis. The CH50eq assay is a method for measuring the total activity of the classical complement pathway in serum. This test is a lytic activity assay that uses antibody-sensitized erythrocytes as an activator of the classical complement pathway and uses various dilutions of test serum to determine the amount needed to achieve 50% lysis (CH50). The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay is an indirect measure of terminal complement component complex (TCC) formation, since the TCC itself is directly responsible for the measured hemolysis. Inhibition of C5 activation can be detected and/or measured using the methods set forth and illustrated in the Experimental Examples. Using assays of these or other suitable types, candidate antibodies having the ability to inhibit C5 activation can be screened. In some embodiments, the inhibition of C5 activation comprises an assayed decrease in C5 activation of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, or more, compared to exposure to a negative control under similar conditions. In some embodiments, it refers to inhibition of C5 activation by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or more .

In preferred embodiments, the epitope of the anti-C5 antibody is different from that of eculizumab.

In some embodiments, an anti-C5 antibody of the present invention binds to an epitope in the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in the MG1-MG2 domain of the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in a fragment consisting of amino acids 19-180 of the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in the MG1 domain (amino acids 20-124 of SEQ ID NO: 1) of the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in a fragment consisting of amino acids 33-124 of the C5 beta chain (SEQ ID NO: 1).

In some embodiments, an anti-C5 antibody of the present invention binds to an epitope in the C5 beta chain that consists of the MG1 domain. In some embodiments, an anti-C5 antibody binds to an epitope in the beta chain (SEQ ID NO: 1) of C5 that contains at least one fragment selected from the group consisting of amino acids 47-57, 70-76, and 107- 110. In some embodiments, an anti-C5 antibody binds to an epitope in the beta chain fragment (SEQ ID NO: 1) of C5 that contains at least one amino acid selected from the group consisting of Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109 and His110. In some embodiments, an anti-C5 antibody binds to an epitope in the beta chain fragment (SEQ ID NO: 1) of C5 that contains at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. In some embodiments, the binding of an anti-C5 antibody to a C5 mutant is reduced compared to its binding to wild-type C5, where the C5 mutant has at least one amino acid substitution at a position selected from the group consisting of Glu48, Asp51, His72, and Lys109 . In another embodiment, the pH-dependent binding (described below) of an anti-C5 antibody to a C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, where the C5 mutant has at least one amino acid substitution at a position selected from the group, which consists of His70, His72 and His110. In a further embodiment of the invention, in the C5 mutant, the amino acid at a position selected from Glu48, Asp51 and Lys109 is changed to alanine and the amino acid at a position selected from His70, His72 and His110 is changed to tyrosine.

In another aspect of the invention, the anti-C5 antibodies of the present invention may have pH-dependent binding characteristics. In the context of the present description, the term "pH-dependent binding" means that the antibody has a "reduced ability to bind to C5 at acidic pH compared to its binding at neutral pH" (both terms can be used interchangeably for the purposes of this description). For example, antibodies with "pH dependent binding characteristics" include antibodies that bind to C5 with higher affinity at neutral pH than at acidic pH. In some embodiments, antibodies bind to C5 with an affinity of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times higher at neutral pH than at acidic pH. In some embodiments, antibodies bind to C5 with higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the antibodies bind to C5 with an affinity of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times higher at pH 7.4 than at pH 5.8.

For the purposes of this description, the "affinity" of an antibody to C5 is expressed in terms of the KD of the antibody. The KD of an antibody means the dissociation equilibrium constant of the antibody-antigen interaction. The higher the KD value, which characterizes the binding of an antibody to its antigen, the weaker its binding affinity for the specified specific antigen. Thus, in the context of the present description, the concept of "higher affinity at neutral pH than at acidic pH" (or the equivalent concept of "pH-dependent binding") means that the KD, which characterizes the binding of an antibody to C5 at acidic pH, is higher than the KD , which characterizes the binding of the antibody to C5 at neutral pH. For example, in the context of the present invention, an antibody is considered to bind to C5 with higher affinity at neutral pH than at acidic pH if the KD characterizing the binding of the antibody to C5 at acidic pH is at least 2 times higher than the KD characterizing binding of the antibody to C5 at neutral pH. Thus, anti-C5 antibodies of the present invention include antibodies whose binding to C5 at acidic pH is characterized by a KD value of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times the KD characterizing binding to C5 at neutral pH. In another embodiment, the KD value of the antibody at neutral pH may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 ,10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or more.

In other embodiments, an antibody is said to bind to C5 with a higher affinity at neutral pH than at acidic pH if the KD value characterizing the binding of the antibody to C5 at pH 5.8 is at least 2-fold higher than the KD , characterizing the binding of the antibody to C5 at pH 7.4. In some embodiments, antibody binding to C5 at pH 5.8 is characterized by a KD value that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the KD characterizing the binding of the antibody to C5 at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 M or more.

The binding characteristics of an antibody to a particular antigen can also be expressed as the kd of an antibody. The kd value of an antibody denotes the dissociation rate constant of an antibody relative to a particular antigen and is expressed as reciprocals of a second (ie, s ^-1 ). An increased kd value means weaker binding of the antibody to its antigen. Thus, the present invention includes antibodies whose binding to C5 has a higher kd value at acidic pH than at neutral pH. The antibodies of the present invention include antibodies whose binding to C5 at acidic pH is characterized by a kd value of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the kd characterizing the binding of the antibody to C5 at neutral pH. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more. Antibodies proposed in the present invention also include antibodies, the binding of which with C5 is characterized by a higher value of KD at a pH of 5.8 than at a pH of 7.4. Antibodies proposed in the present invention include antibodies, the binding of which is 5.8 from C5 at a pH of 5.8, which is at least 3, 10, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 45, 45, 45, 45, 45, 50, 50 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times higher than KD, characterizing the binding of antibodies with C5 at pH 7.4. In another embodiment of the invention, the value of KD antibodies at a pH of 7.4 can be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment of the invention, the value of KD antibodies at a pH of 5.8 can be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more.

In some cases, “a reduced ability to contact C5 in the pH compared to the ability to contact a neutral pH” is expressed in the form of a ratio of KD, which characterizes the binding of antibodies with C5 in pH, and the KD size characterizing the binding of antibodies with C5 with neutral pH ( or vice versa). For example, an antibody can be considered as a “reduced ability to contact C5 in the pH compared to the ability to contact a neutral pH” for the purposes of the present invention, if for antibodies the ratio of KD with acid/neutral pH is 2 or more. In some embodiments of the invention, the KD ratio at a pH of 5.8/pH 7.4 for antibodies is 2 or more. In some embodiments of the invention, the KD ratio with acid/neutral pH for antibodies can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 75, 75, 75, 75, 75, 75 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another version of the invention, the value of the KD antibodies with a neutral pH can be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 m or less. In another embodiment of the invention, the value of KD antibodies with sour pH can be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 m or more. In other cases, the antibody can be considered as a “reduced ability to contact C5 in the pH compared to the ability to contact a neutral pH”, if the antibodies is characterized by the ratio of KD at a pH of 5.8/pH 7.4, which is 2 or more. In some examples of the options for the invention, the KD ratio at the pH of 5.8/pH 7.4 for antibodies can be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment of the invention, the value of KD antibodies at a pH of 7.4 can be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 m or less. In another embodiment of the invention, the value of KD antibodies at a pH of 5.8 can be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 m or more.

In some cases, "reduced ability to bind to C5 at acidic pH compared to the ability to bind at neutral pH" is expressed as the ratio of the kd value characterizing the binding of the antibody to C5 at acidic pH, and the kd value characterizing the binding of the antibody to C5 at neutral pH ( or vice versa). For example, an antibody may be considered to have "reduced ability to bind to C5 at acidic pH compared to the ability to bind at neutral pH" for the purposes of the present invention if the antibody's acid/neutral pH kd ratio is 2 or more. In some embodiments, the kd ratio at pH 5.8/pH 7.4 for an anti-C5 antibody is 2 or more. In some exemplary embodiments of the invention, the acid/neutral pH kd ratio for an antibody may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In other exemplary embodiments of the invention, the ratio kd at pH 5.8/pH 7.4 for an antibody can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more. In some exemplary embodiments of the invention, the ratio kd at pH 5.8/pH 7.4 for an antibody may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 -6 1/s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 ,10 ^-2 , 10 ^-1 1/s or more.

In the context of the present description, the term "acidic pH" means a pH from 4.0 to 6.5. The term "acidic pH" includes one of the following pH values 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 , 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6 .2, 6.3, 6.4 and 6.5. In specific aspects of the invention, "acidic pH" corresponds to 5.8.

In the context of the present description, the term "neutral pH" means a pH from 6.7 to about 10.0. The term "neutral pH" includes one of the following pH values 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 , 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8 .9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In specific aspects of the invention, "neutral pH" corresponds to 7.4.

The KD and kd values described herein can be determined using a biosensor based surface plasmon resonance method to characterize antibody-antigen interactions. The KD values and kd values can be determined at 25°C or 37°C.

In another aspect of the invention, the anti-C5 antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VH sequence is at least 90%, 91%, 92 identical %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 2. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 2, including post-translational modifications of said sequence. In a specific embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 3, (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 4 , and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 5.

In the context of the present description, the term "HVR" means "hypervariable region", and the term "FR" means "framework region".

The term "framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically have the following location in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term "hypervariable region" or "HVR" in the context of the present description refers to each of the regions of the variable domain of an antibody, the sequences of which are hypervariable ("complementarity determining regions" or "CDRs") and/or form loops of a certain structure ("hypervariable loops") , and/or contain antigen-contacting residues ("antigen-contacts"). Typically, antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). As used herein, examples of HVRs include (a) hypervariable loops comprising amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196, 1987, pp. 901-917); (6) CDRs including amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH publication 91-3242); (c) antigen contact regions comprising amino acid residues 27 c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (HI), 47-58 (H2) and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262, 1996, pp. 732-745); and (d) combinations of residues specified in subparagraphs (a), (b) and/or (c), including amino acid residues HVR 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3). Residues in HVR and other residues in the variable domain (eg, residues in FR) are numbered according to Kabat et al., supra.

In another aspect of the invention, the anti-C5 antibody comprises a light chain variable domain sequence ((VL) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL sequence is at least 90%, 91% identical, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C5 antibody containing the specified sequence, retains the ability to bind to C5 In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 6. In some embodiments, the substitutions, insertions, or deletions affect regions outside HVR (i.e. FR). Optionally, the anti-C5 antibody contains the VL sequence shown in SEQ ID NO: 6, including post-translational modifications of said sequence. In a specific embodiment, the VL contains one, two, or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 7, (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 8 , and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 9.

In another embodiment, the antibody is a full-length antibody, such as an intact IgG1-, IgG2-, IgG3-, or IgG4-subclass antibody, or an antibody engineered to have regions of two or more IgGs selected from IgG1, IgG2 , IgG3 and IgG4, in the region(s) which/which are homologous among IgG subclasses, by recombination. The antibody may comprise a suitable Fc region containing the sequence of a human Fc region (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions).

Fc-region variants

In some embodiments, an anti-C5 antibody of the present invention comprises an Fc region variant in which one or more amino acid modification(s) are introduced into the antibody's native Fc region sequence. A variant Fc region may comprise a human Fc region sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions) comprising an amino acid modification (eg, substitution) at one or more amino acid positions.

Some embodiments of the invention provide a variant of an antibody that has some, but not all, effector functions, making it a promising candidate for applications where the in vivo half-life of the antibody is important, but some effector functions (such as complement-dependent cytotoxicity (CDC) and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks the ability to bind to Fc-gamma R (and therefore likely lacks ADCC activity), but retains the ability to bind to FcRn. Primary cells mediating ADCC, ie. NK cells express only Fc-gamma RIII while monocytes express Fc-gamma RI, Fc-gamma RII and Fc-gamma RIII. FcR expression data on hematopoietic cells are summarized in Table 3 on p. 464 by Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, pp. 457-492. Examples of in vitro assays for ADCC activity of a molecule of interest are, but not limited to, those described in US No. 5500362 (see, for example, Hellstrom et al., Proc. Natl. Acad. Sci. USA 83, 1986, cc 7059-7063 and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82, 1985, pp. 1499-1502); US No. 5821337 (see Bruggemann et al., J. Exp. Med. 166, 1987, cc. 1351-1361). Alternatively, non-radioactive assays can be used (see, for example, non-radioactive flow cytometry-based ACTI™ cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and non-radioactive CytoTox 96 cytotoxicity assay (Promega, Madison, Suitable effector cells for such assays are peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model, for example, as described in Clynes et al., Proc. Natl. Acad. Sci. USA 95, 1998, pp. 652-656 C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity (See e.g. WO 2006/029879 and WO 2005/100402 ELISA for assessing C1q and C3c binding.) b CDC analysis (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202, 1996, pp. 163-171; Cragg et al., Blood 101, 2003, pp. 1045-1052; and Cragg, Blood 103, 2004, pp. 2738-2743). Determination of FcRn binding and in vivo clearance/half-life can also be carried out using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18(12), 2006, cc. 1759- 1769).

Antibodies with reduced effector function include antibodies with the replacement of one or more of the following residues of the Fc region, such as 238, 265, 269, 270, 297, 327 and 329 (see, for example, US No. 6737056). These Fc mutants include Fc mutants with substitutions at two or more of the following amino acid positions: 265, 269, 270, 297, and 327, including the so-called "DANA" Fc region mutant carrying the substitution of residues 265 and 297 with alanine (US No. 7332581).

Some variants of antibodies with increased or decreased ability to bind to FcR have been described (see, for example, US No. 6737056; WO 2004/056312 and Shields et al., J. Biol. Chem. 276, 2001, cc. 6591-6604).

In specific embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering).

In some embodiments of the invention, changes are made in the Fc region that result in an altered (i.e., either increased or decreased) ability to bind C1q and/or complement dependent cytotoxicity (CDC), as described, for example, in US No. 6194551, WO 1999/51642 and Idusogie et al., J. Immunol. 164, 2000, pp. 4178-4184.

Antibodies with extended half-life and increased ability to bind to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the embryo (Guyer et al., J. Immunol. 117, 1976, pp. 587-593 and Kim et al. , J. Immunol. 24, 1994, pp. 2429-2434), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. These Fc region variants include variants with substitutions of one or more of the following Fc region residues: 378, 380, 382, 413, 424 or 434, for example, the replacement of residue 434 in the Fc region (US No. 7371826).

Additional examples regarding other variants of the Fc region are also described in Duncan, Nature 322, 1988, cc. 738-740; in US Nos. 5648260 and 5624821 and in WO 1994/29351.

In some embodiments, an anti-C5 antibody of the present invention comprises the VH shown above as one of the embodiments of the invention and a heavy chain constant region containing the amino acid sequence shown in one of SEQ ID NOs: 10, 11, 12 , 13, 14, and 15. In some embodiments, the anti-C5 antibody comprises the VL shown above as one of the embodiments of the invention and a light chain constant region containing the amino acid sequence shown in one of SEQ ID NOs: 16, 17 and 18.

In some embodiments, an anti-C5 antibody of the present invention is any of the antibodies described in WO 2016/098356, WO 2017/123636 and WO 2017/132259.

Subcutaneous administration

The composition for use according to the present invention is administered subcutaneously using two phases. In both phases there is an interval between every two subcutaneous injections. Each phase includes at least one interval. The last interval of the first phase is between the last subcutaneous introduction in the first phase and the first subcutaneous introduction in the second phase.

In some embodiments of the invention, at least one interval in the first phase is from 1 day to 2 months. A suitable interval in the first phase in an acceptable range can be determined depending on the conditions of the individual or patient. The specific interval between subcutaneous injections is 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days , 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days or 42 days. In preferred embodiments of the invention, the interval in the first phase is 4-35 days. In other preferred embodiments of the invention, the interval in the first phase is 5-14 days. In the most preferred embodiments of the invention, the interval in the first phase is 7 or 14 days. In the first phase, the pharmaceutical composition can be administered once every week (weekly) or once every 2 weeks (once every two weeks or at two-week intervals).

In some embodiments of the invention, at least one interval in the second phase is from 2 days to 6 months. Suitable duration of at least one interval in the second phase in an acceptable range can be determined depending on the conditions of the individual or patient. The exemplary duration of at least one interval in the second phase is 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days , 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days , 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, 100 days, 101 days, 102 days, 103 days, 104 days 105 days 106 days 107 days 108 days 109 days 110 days 111 days 112 days 113 days 114 days 115 days 116 days 117 days 118 days 119 days 120 days 121 day, 122 days, 123 days, 124 days, 125 days, 126 days, 127 days, 128 days, 129 days, 130 days, 131 days, 132 days, 133 days, 134 days, 135 days, 136 days, 137 days, 138 days, 139 days, 140 days, 141 days, 142 days, 143 days, 144 days, 145 days, 146 days, 147 days, 148 days, 149 days, 150 days, 151 days, 152 days, 153 days, 154 days , 155 days, 156 days, 157 days, 158 days, 159 days, 160 days, 161 days, 162 days, 163 days, 164 days, 165 days, 166 days, 167 days, 168 days, 169 days, 170 days, 171 day, 172 days, 173 days, 174 days, 175 days, 176 days, 177 days, 178 days, 179 days, 180 days, 181 days, 182 days, 183 days or 184 days. In preferred embodiments of the invention, the duration of at least one interval in the second phase is from 15 days to 3 months. In the second phase, the pharmaceutical composition can be administered once every month (monthly), once every two months (once every two months or at intervals of two months) or once every three months (every three months or at intervals of three months). A longer interval may reduce patient stress or suffering.

In some embodiments of the invention, the pharmaceutical composition is administered subcutaneously at a dose containing 17-6000 mg of antibody in the first and second phases. A suitable dose can be determined within an acceptable range depending on the conditions of the individual or patient. In the case where the pharmaceutical composition is administered by subcutaneous injection to the individual repeatedly, the doses are not required to be the same all the time, and they can be determined within an acceptable range. For example, the dose can be gradually reduced.

In some embodiments of the invention, the dose for subcutaneous administration in the first phase contains 50-350 mg of antibody. When the dose of antibody per subcutaneous administration in the first phase is lower than the dose of antibody per subcutaneous administration in the second phase, then the dose of antibody per subcutaneous administration in the first phase is preferably 150-200 mg. The specific preferred dose in this case is 170 mg. When the dose of antibody per subcutaneous administration in the first phase is the same as the dose of antibody per subcutaneous administration in the second phase, the dose per subcutaneous administration in the first phase preferably contains 300 to 350 mg of antibody. The specific preferred dose in this case is 340 mg. When the pharmaceutical composition is administered weekly (about 7 days apart) in the first phase, the dose is preferably 170 mg or 340 mg. When the pharmaceutical composition is administered in the first phase once every two weeks (about 14 days apart), the preferred dose is 340 mg.

In some embodiments of the invention, the dose of antibody per administration in the second phase is from 350 to 1000 mg. In preferred embodiments of the invention, the dose is from 650 to 700 mg. When the pharmaceutical composition is administered once every four weeks (about 28 days apart) or monthly, the preferred dose is 680 mg. Most preferred for subcutaneous administration is a dosing regimen of 680 mg of antibody once every four weeks or monthly, as this may reduce patient burden or suffering.

In some embodiments, the dose of anti-C5 antibody per administration in the first phase is 3-5 times lower than the dose of anti-C5 antibody per administration in the second phase. In preferred embodiments, the dose of antibody per subcutaneous injection in the first phase is 4 times lower than the dose of antibody per subcutaneous injection in the second phase.

In some embodiments, the number of subcutaneous injections in the first phase is from 1 to 12. In preferred embodiments, the number is from 5 to 10. In other preferred embodiments, the first phase is 8 subcutaneous injections.

In a specific embodiment of the invention, a pharmaceutically acceptable carrier suitable for a conventional subcutaneous injection composition may be included in the pharmaceutical composition. The type of carrier for subcutaneous injection may be selected from carriers generally known in the art.

In a specific embodiment of the invention, the pharmaceutical composition may be any one selected from the group that includes liquid, semi-solid and solid composition. The solid composition is obtained, as a rule, by lyophilization from a liquid formulation. The lyophilized composition is typically reconstituted with water or saline immediately prior to subcutaneous injection.

When the composition is administered subcutaneously, the concentration of anti-C5 antibody in the liquid formulation of the composition is within the generally accepted range known in the art. In a specific embodiment of the invention, the volume of subcutaneous injection is determined by the usual range known in this field.

In some embodiments, the C5-associated disease is a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In some embodiments, the C5-associated disease is at least one selected from the group consisting of rheumatoid arthritis (RA); lupus nephritis; damage caused by ischemia-reperfusion; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration; syndrome: hemolysis, increased activity of liver enzymes and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; Pauci-immune vasculitis; bullous epidermolysis; repeated loss of the fetus; multiple sclerosis (MS); traumatic brain injury and damage due to myocardial infarction, cardiopulmonary bypass and hemodialysis; resistant generalized myasthenia gravis (gMG); neuromyelitis of the optic nerve (NMO). In another preferred embodiment of the invention, the C5-associated disease is PNH.

In some objects of the invention, a pharmaceutical composition, which is prepared in the form for intravenous administration and contains antibodies to C5, is introduced before the first subcutaneous administration in the first phase. Preferably, the anti-C5 antibody in the intravenous formulation is the same as the anti-C5 antibody in the subcutaneous formulation. Intravenously administered pharmaceutical composition is the composition described below in the present description.

In some embodiments, the subcutaneous injection composition is administered subcutaneously on the same day or one or more days after the intravenous dose of the intravenous composition. One or more doses of the composition for intravenous injection may be administered to an individual or patient prior to administration of the first dose of the composition for subcutaneous injection. In a preferred embodiment of the invention, the first dose of the composition for subcutaneous injection is administered after the final dose of the composition for intravenous injection.

In some embodiments of the invention, the first subcutaneous administration in the first phase is carried out within a period of time ranging from 0 days to 1 month after the final administration of the intravenously administered pharmaceutical composition. Specifically, the time period between the first subcutaneous administration and the final intravenous administration is 0 days (i.e. within 24 hours), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days. In preferred embodiments of the invention, the period is from 0 days (ie, within 24 hours) to 14 days. In other preferred embodiments of the invention, the period is from 0 days (ie, within 24 hours) to 10 days. In the most preferred embodiments of the invention, the period is from 0 days (ie, within 24 hours) to 8 days.

III. Pharmaceutical composition for intravenous injection

In some aspects of the invention, in the first phase, the first subcutaneous administration of the pharmaceutical composition for use according to the present invention is carried out after the final administration of the composition for intravenous administration.

Thus, in one of the embodiments of the present invention, the composition, which is prepared in the form for intravenous administration and contains an antibody to C5, is administered intravenously before the first subcutaneous administration in the first phase. In a preferred embodiment, the anti-C5 antibody in the intravenous formulation is the same as the anti-C5 antibody in the subcutaneous formulation.

Preferably, no intravenous administrations are made after the first subcutaneous administration. Thus, according to a further embodiment of the invention, the last intravenous administration is carried out before the first subcutaneous administration.

The pharmaceutical composition formulated for intravenous injection according to the present invention is for use in the treatment or prevention of a C5 related disease, contains an anti-C5 antibody, and is administered intravenously. Intravenous injection can be performed using conventional devices and methods for introducing the pharmaceutical composition into a vein by injection. You can also choose specific devices and methods for intravenous injection.

Antibody to C5, used for intravenous injection, can be any antibody described above for pharmaceutical compositions for subcutaneous injection. A pharmaceutical composition for intravenous injection and a pharmaceutical composition for subcutaneous injection may contain the same antibody to C5 or can contain another antibody to C5. In preferred embodiments, the anti-C5 antibody used for intravenous injection is the same antibody as described above for subcutaneous injection.

In some embodiments of the invention, the pharmaceutical composition is administered intravenously at a dose containing 50-5000 mg of the antibody. A suitable dose can be determined within an acceptable range depending on the conditions of the individual or patient. In the case where the pharmaceutical composition is administered intravenously to an individual multiple times, the doses are not required to be the same all the time, and can be determined arbitrarily if the doses are within an acceptable range. For example, the dose can be gradually reduced. In preferred embodiments, the dosage range is from 55 to 4000 mg of the antibody. In other preferred embodiments of the invention, the dosage range is from 60 to 2500 mg of the antibody. In the most preferred embodiments of the invention, the dose range is from 100 to 2500 mg of antibody. Specific preferred doses are 75, 125, 150, 300, 375, 500 and 1000 mg. Other preferred doses are 375, 500 and 1000 mg, and among them, 1000 mg is the most preferred.

In some embodiments of the invention, the number of administrations of the pharmaceutical composition, which is administered by intravenous injection, is not specifically limited and may be one or more administrations. In preferred embodiments of the invention, the number of injections may be one, two or three injections. In other preferred embodiments of the invention, the number of injections is one or two injections. In the most preferred embodiment of the invention, the number of introductions is one introduction. Reducing the number of injections allows you to reduce the burden on the patient or his suffering.

In some embodiments, when the pharmaceutical composition is administered to an individual or patient intravenously multiple times, the composition is administered once an hour or every 14 days. A suitable interval between intravenous injections can be determined in an acceptable range depending on the conditions of the individual or patient.

The specific interval between intravenous injections is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours. , 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In preferred embodiments of the invention, the interval between injections is from 24 hours to 10 days. In other preferred embodiments of the invention, the interval between injections is from 48 hours to 7 days. In the most preferred embodiments of the invention, the interval between injections is from 3 to 5 days.

In a specific embodiment of the invention, a pharmaceutically acceptable carrier suitable for a conventional composition for intravenous injection may be included in the pharmaceutical composition. The type of carrier for intravenous injection may be selected from carriers generally known in the art.

In some embodiments, the formulation of the pharmaceutical composition may be any one selected from the group that includes liquid, semi-solid, and solid formulations. The solid composition is obtained, as a rule, by lyophilization from a liquid preparation. The lyophilized composition is typically reconstituted with water or saline immediately prior to intravenous injection. The formulation of the pharmaceutical composition for intravenous injection may be the same as or different from that of the pharmaceutical composition for subcutaneous injection. In preferred embodiments of the invention, the formulation of the pharmaceutical composition for intravenous injection is the same as the formulation of the pharmaceutical composition for subcutaneous injection, which helps to reduce the cost of production.

When the composition is administered intravenously, the concentration of anti-C5 antibody in the liquid formulation of the composition is within the generally accepted range known in the art. In a specific embodiment of the invention, the volume of intravenous injection is determined by the usual range known in this field.

In some objects of the invention, the dose of composition for intravenous injection is administered to the initial dose of another pharmaceutical composition, which is introduced subcutaneously. The pharmaceutical composition for subcutaneous injection is one of the compositions described above.

In some embodiments of the invention, the composition for intravenous injection is administered intravenously on the same day or one or more days before the first dose of the composition for subcutaneous injection, which is administered subcutaneously. One or more doses of composition for intravenous injection can be administered to an individual or patient before the first dose of the composition for subcutaneous injection. In a preferred embodiment of the invention, the final dose of the composition for intravenous injection is administered prior to the first dose of the composition for subcutaneous injection.

In some embodiments, the intravenous injection dose of the composition is administered on the same day or 1 day to 1 month prior to the first subcutaneous injection dose of the composition. The specific interval between administration of a dose of the composition for intravenous injection and the first dose of the composition for subcutaneous injection is 0 days (i.e. within 24 hours), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days. In preferred embodiments of the invention, the interval is from 0 days (ie, within 24 hours) to 14 days. In other preferred embodiments of the invention, the interval is from 0 days (ie, within 24 hours) to 10 days. In the most preferred embodiments of the invention, the interval is from 0 days (ie, within 24 hours) to 8 days.

IV. Switching from the use of another pharmacological product

According to another aspect of the invention, pharmaceutical compositions for subcutaneous or intravenous injection can be used to treat or prevent a C5 related disease in an individual who has been treated one or more times with at least one pharmacological product intended for use in the treatment or prevention of the disease. For example, the pharmaceutical compositions of the present invention can be used to treat a C5 related disease in a patient who has previously been treated with at least one pharmacological product intended to treat the disease, but who is expected to have an improved response to treatment with the pharmaceutical compositions. proposed in the present invention. In such cases, drug treatment can be switched from the pharmacological product to the pharmaceutical composition of the present invention. In preferred embodiments of the invention, the initial dose of the composition for intravenous injection proposed in the present invention is administered after the final dose of the pharmacological product used for the previous treatment.

In some embodiments, the pharmacological product contains an active substance that is different from the anti-C5 antibody used in the subcutaneous and intravenous injection composition described above. In some embodiments, the active ingredient of the pharmaceutical product is an siRNA targeting C5 mRNA, or an anti-C5 antibody other than the anti-C5 antibody contained in the subcutaneous and intravenous injection composition described above. In preferred embodiments, the pharmacological product contains an antibody that is different from the antibodies in the subcutaneous and intravenous injection composition described above. In the most preferred embodiment of the invention, the antibody included in the pharmacological product that was used for the previous treatment is eculizumab or its derivative.

In some embodiments of the invention, the initial dose of the composition for intravenous injection proposed in the present invention is administered on the same day or one or more days after administration of the final dose of the pharmacological product. The specific interval between administration of the final dose of the pharmacological product and the initial dose of the intravenous injection composition of the present invention is 0 days (meaning that the initial dose of the intravenous injection composition is administered on the same day as the final dose of the pharmacological product), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. In preferred embodiments of the invention, the composition for intravenous injection proposed in the present invention is administered 3 or more days after the final dose of the pharmacological product. In more preferred embodiments, the intravenous injection composition of the present invention is administered 7 or more days after the final dose of the pharmacological product. In other preferred embodiments of the invention, the composition for intravenous injection proposed in the present invention is administered 14 or more days after the final dose of the pharmacological product. In the most preferred embodiments of the invention, the composition for intravenous injection proposed in the present invention is administered 21 or more days after the final dose of the pharmacological product.

In some embodiments of the invention, the initial dose of the composition for intravenous injection proposed in the present invention is administered on the same day or one or more days after administration of the final dose of the pharmacological product. The specific interval between administration of the final dose of the pharmacological product and the initial dose of the intravenous injection composition of the present invention is 0 days (meaning that the initial dose of the intravenous injection composition is administered on the same day as the final dose of the pharmacological product), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. In preferred embodiments of the invention, the composition for intravenous injection proposed in the present invention is administered 3 or more days after the final dose of the pharmacological product. In more preferred embodiments, the intravenous injection composition of the present invention is administered 7 or more days after the final dose of the pharmacological product. In other preferred embodiments of the invention, the composition for intravenous injection proposed in the present invention is administered 14 or more days after the final dose of the pharmacological product. In the most preferred embodiments of the invention, the composition for intravenous injection proposed in the present invention is administered 21 or more days after the final dose of the pharmacological product. V. Method of treatment or prevention

Other objects of the invention are methods of treatment or prevention associated with C5 disease.

In one embodiment, the method comprises subcutaneously administering to an individual a pharmaceutical composition that is in a subcutaneous injection form and contains an anti-C5 antibody, wherein the composition is administered subcutaneously using two phases, wherein both phases have an interval between every two subcutaneous injections, where each phase includes at least one interval and where in the first phase

I) at least one interval is shorter than at least one interval in the second phase, and

ii) the dose of antibody per administration is lower or the same as the dose of antibody per administration in the second phase.

The subcutaneous pharmaceutical composition is the same as the composition described above.

In some embodiments of the invention, a pharmaceutical composition, which is prepared in the form for intravenous administration and contains antibodies to C5, is introduced to the first subcutaneous administration of the first phase. The intravenously administered pharmaceutical composition is the same as the composition described above.

The conditions and diseases targeted by the pharmaceutical composition for subcutaneous or intravenous injection in said method are the same as those mentioned above in sections "II. Pharmaceutical composition for subcutaneous injection" and "III. Pharmaceutical composition for intravenous injection.

VI. Vaccination

Infectious diseases, such as meningococcal infections, may occur in an individual administered with an anti-C5 antibody. To prevent the indicated infectious diseases of the individual, you can immunize using a vaccine in which the ability to treat or prevent the disease is known, before, after or when the composition is introduced for subcutaneous and intravenous injection.

VII. Product

Another object of the invention is an article or product containing/containing materials that can be used to treat or prevent associated with C5 diseases mentioned above. The article or product contains one or more containers and a label or package insert, which are placed on or enclosed in the container. Acceptable containers are, for example, jars, vials, syringes, SC injection syringes, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective in treating or preventing the condition and may have a sterile access port (for example, the container may be a vial provided with a stopper that can be pierced with a hypodermic needle). At least one active ingredient in the composition is an anti-C5 antibody as described above. The label, package insert or the like indicates that the composition may be used to treat the selected condition, for example any of the C5 related diseases described above.

In some embodiments of the invention, the composition in the container of the article or product is in the form of a subcutaneous injection. According to this embodiment of the invention, the article or product may additionally include a package insert leaflet with information on the method of administration indicated below. The pharmaceutical composition is administered subcutaneously using two phases, where both phases have an interval between every two subcutaneous injections. Each phase includes at least one interval. In the first phase, I) at least one interval is shorter than at least one interval in the second phase, and ii) the dose of antibody per administration is lower than or the same as the dose of antibody per administration in the second phase.

In some embodiments of the invention, a product or product may contain an additional container with a composition in it, which contains an additional therapeutic agent. According to this embodiment, the product can additionally contain a leaflet-liner in a package with information that the composition can be used to treat a specific state. Alternatively or additionally, the article of manufacture may further comprise another additional container with a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BVI), phosphate buffered saline, Ringer's solution and dextrose solution. In addition, it may include other products required from a commercial and consumer point of view, in particular other buffers, diluents, filters, needles and syringes.

Examples

Below are examples of methods and compositions proposed in the invention. As should be apparent, various other embodiments of the invention may be practiced based on the general description of the invention provided above.

Example 1

Creation of an antibody to C5

Antibody to C5 305LO15 described in WO 2016/098356 was obtained by a standard method. In general, the method was as follows. The genes encoding the 305LO15 heavy chain variable (VH) were fused with the genes encoding the SG115 modified variant (SEQ ID NO: 13) of the heavy chain constant domain (CH) of human IgG1. The genes encoding the light chain variable domain (VL) 305LO15 were combined with the genes encoding the human light chain constant domain (CL) (SKI, SEQ ID NO: 18). Antibodies were expressed in HEK293 cells co-transfected with a combination of heavy and light chain expression vectors and purified using protein A.

Example 2

Pharmaceuticals based on proteins, for example, drugs based on antibodies, are usually administered intravenously, since proteins are difficult to concentrate. Thus, eculizumab, which is currently the only antibody approved for treatment, is administered intravenously. If it becomes possible to use subcutaneous injection, then this should ease the burden on the patient, for example, in terms of outpatient treatment and the length of time spent in the clinic for intravenous injection, since self-injection becomes possible.

The 305LO15 antibody described in WO 2016/098356 was chosen to study the feasibility of subcutaneous administration of an anti-C5 antibody due to its high solubility. It was expected that the higher solubility would allow the administration of the protein-based pharmaceutical agent in a smaller volume, allowing the use of subcutaneous injection. Pharmaceutical formulations for intravenous injection and subcutaneous injection were prepared according to the general formulation described below:

170 mg/ml 305LO15,

30 mM histidine/aspartic acid,

100mM arginine hydrochloride,

0.05% Poloxamer 188,

pH 5.8.

To compare the pharmacokinetic characteristics of 305LO15 after subcutaneous injection or intravenous injection, cynomolgus monkeys were divided into two groups, a group treated by subcutaneous injection and a group treated by intravenous injection. 305LO15 was administered subcutaneously to the animals in the first group (three males and three females) once every week for a total of 22 times, each time administering the antibody at a dose of 40 mg/kg. The plasma concentration of 305LO15 in these animals was measured by ELISA at the time points indicated in FIG. 1a. 305LO15 was administered intravenously to the animals in the second group (six males and six females) once every week for a total of five times, each time administering the antibody at a dose of 40 mg/kg. Animal plasma concentration of 305LO15 was measured by ELISA at the time points shown in FIG. 1b.

As shown in FIG. 1A, the concentration of 305LO15 in the plasma of the monkeys Tsinomolgus increased during subcutaneous injections, this confirms that 305LO15 can be administered by subcutaneous injection for therapeutic purposes. The rate of initial increase in plasma concentration with subcutaneous injection (Fig. 1a) was lower than with intravenous injection (Fig. 1b).

Example 3

Then, a study of whether it was possible to compensate for the relatively low rate of increase in plasma concentration after the initial subcutaneous injection using intravenous injection 305LO15. Five male and five female cynomolgus monkeys received a single intravenous injection with an initial dose of 100 mg/kg 305LO15 antibody. One week after the initial intravenous administration, a subcutaneous injection of 305LO15 was started. Subcutaneous injection was performed a total of 26 times at a frequency of once every week, administering at each dosing antibody at a dose of 40 mg/kg.

As shown in FIG. 2, the rate of increase in plasma concentration of 305LO15 was greater when the initial dose of the antibody was administered by intravenous injection before maintenance doses by subcutaneous injection than when the initial dose was not administered by intravenous injection. The plasma concentration of 305LO15 reached a level comparable to that shown in FIG. 1a and maintained at this level. It can be assumed that this faster increase in plasma concentration of 305LO15 contributes to a more rapid achievement of the therapeutic effect of the drug.

Example 4

To assess the effective concentration of 305LO15 in plasma, the ratio between the concentration of free antigen (C5) in plasma or complement activity and the concentration of antibody (305LO15) in plasma was determined in cynomolgus monkeys. 305LO15 was administered intravenously (0.8, 4 or 20 mg/kg) or subcutaneously (4 mg/kg). Plasma antibody and free C5 concentrations were measured by ELISA. Complement activity was measured using a erythrocyte hemolytic assay (RBC). As shown in FIG. 3, the concentration of free C5 decreased to less than a few μg/ml when the plasma concentration of 305LO15 was maintained above 40 μg 305EO15/ml (Fig. 3a) and the plasma 305LO15 concentration was 40 μg/ml or higher, inhibited complement activity (hemolysis) to less than 20% of baseline (ie, in the absence of antibody) (FIG. 3b). Complement activity was found to be suppressed to less than 20% of baseline (ie, in the absence of antibody injection) when the plasma concentration of 305LO15 was maintained at or above 40 μg/mL.

Example 5

To determine acceptable doses and administration schedules of 305LO15 suitable for maintaining an effective concentration of 305LO15 in clinical trials, the dependence of the concentration of 305LO15 in human plasma was predicted by modeling. First, plasma concentrations of 305LO15 were measured as a function of time after antibody administration to cynomolgus monkeys at doses of 0.8, 4, 20 μg antibody/kg at each dose. PK parameters for cynomolgus monkeys were assessed by data analysis using a 2-compartment model. Human PK parameters were estimated from Cintomolgus monkey PK parameters using allometric scaling. Evaluated PK parameters for humans are presented in Table 1 (abbreviations used to designate parameters in Table 1: BW, body weight; CL, total drug clearance; F, bioavailability or relative amount of administered drug entering the bloodstream; Ka , absorption rate constant; mAb, monoclonal antibody; PK, pharmacokinetic parameter; Q, intercompartment clearance; SC, subcutaneous; Vc, volume of distribution in the central compartment; Vp, volume of distribution in the peripheral compartment).

The human PK profile was assessed for each cohort participating in the Phase 1/2 study (FIG. 4). Part 1 of the study was designed to include three groups of individuals. The first group was a group of individuals who were administered 305LO15 intravenously once at a dose of 75 mg/person. The second group was a group of individuals who were administered 305LO15 intravenously once at a dose of 150 mg/person. The third group was a group of individuals who were administered 305LO15 subcutaneously once at a dose of 170 mg/person. It was predicted that the plasma concentration of 305LO15 in individuals administered any one of the above doses would not be maintained above the threshold (40 μg/ml) for more than one week (FIG. 4a).

Part 2 of the study was designed to include a group of individuals who were administered 305LO15 intravenously three times (first at 300 mg/person, then 500 mg/person one week after the first dose, and finally 1000 mg/person two weeks after second injection) and two weeks after the final intravenous administration, 305LO15 was administered subcutaneously once a week at a dose of 170 mg/person. It was predicted that when using the dosing regimen planned for part 2 of the study, the concentration of 305LO15 would be maintained at a level above the threshold PD level (40 μg/ml) (Fig. 4b).

Part 3 of the study was designed to include three groups of individuals. First, 305LO15 was administered to individuals in all groups intravenously once at a dose of 500 mg/person. Starting from the day following the day of initial administration, 305LO15 was administered subcutaneously to individuals in the first group once every week at a dose of 170 mg/person, to individuals in the second group once every two weeks at a dose of 340 mg/person, and to individuals in the third group once every four weeks at a dose of 600 mg/person. The concentration of 305LO15 was predicted to be maintained above the PD threshold (40 μg/mL) in all three groups in Part 3 of the study (Fig. 4c).

Example 6

Eculizumab has been used to treat patients with PNH or aHUS. In the event that 305LO15 was predicted to be more beneficial than eculizumab in a patient with PNH or aHUS, the patient could be switched from eculizumab drug therapy to 305LO15 drug therapy. It should be taken into account that there is a possibility that 305LO15 forms an immune complex with C5 and eculizumab in the body, since the epitope for 305LO15 is different from the epitope for eculizumab, which means that 305LO15 binds to the C5 beta chain, while eculizumab binds with the C5 alpha chain. The formation of such an immune complex can cause abnormal activation and therefore it is desirable to avoid it as much as possible.

To investigate whether 305LO15 forms an immune complex with eculizumab (ECZ, heavy chain sequence shown in SEQ ID NO: 19 and light chain sequence shown in SEQ ID NO: 20) and human C5 (hC5, SEQ ID NO: 21), in vitro analysis was performed using gel filtration (SEC). hC5 was obtained according to the method described in WO 2016/098356. ECZ, hC5 and 305LO15 were dialyzed against D-PBS, pH 7.4, to exchange buffer. Then ECZ and hC5 were mixed and incubated at 37°C for 3-4 hours. After incubation, 305LO15 was added to the mixture containing ECZ and hC5. The final concentration of ECZ and hC5 was adjusted to 200 μg/ml, and the concentration of 305LO15 was adjusted to 0, 25, 125, 250, or 1250 μg/ml. After incubation of these mixtures at 37° C. for 3-4 hours, SEC analysis was performed at pH 7.4 and pH 6.0. The specific conditions for the SEC are listed below.

HPLC system: Waters Alliance e2695 HPLC system

column: TSKgel G4000SWXL

column temperature: 25°C

eluent: 50 mM Na-PB/300 mM NaCl, pH 7.4 or pH 6.0

flow rate: 0.5 ml/min

detection: UV absorption at 220 nm

injection: 10 µl

As a result, it was found that 305LO15 at concentrations from 125 to 1250 μg/ml had no effect on the SEC profile at pH 6.0, but had an effect on the profile at pH 7.4. More specifically, when 305LO15 was mixed with ECZ and hC5, peaks of large ECZ/hC5/305LO15 immune complexes that were not detected at pH 6.0 were detected at pH 7.4 (see peaks indicated by arrows in Fig. 5, top panel), in addition to the peaks corresponding to small complexes (see peaks labeled '2Ag+Ab' and 'Ag+Ab' in Fig. 5, top panel). At pH 6.0, only peaks corresponding to small complexes were detected (see peaks shown in FIG. 5, lower panel, corresponding to '2Ag+Ab' 'Ag+Ab' in FIG. 5, upper panel). This means that 305LO15 binds to the ECZ/hC5 complex at pH 7.4 but not at pH 6.0, which is consistent with the binding characteristics of 305LO15 (binding to hC5 at pH 7.4 but not at pH 6.0) . Based on these results, it was suggested that the simultaneous administration of 305LO15 and ECZ to an individual may result in the formation of large plasma immune complexes.

Example 7

Study BP39144 is an adaptive phase I/II study to evaluate the safety, efficacy, pharmacokinetic characteristics (PK) and pharmacodynamic characteristics (PD) of 305LO15 in healthy volunteers and patients with paroxysmal nocturnal hemoglobinuria (PNH).

Part 1 of Study BP39144 (Randomized, Investigator/Subject-Blinded, Adaptive, Placebo-Controlled, Parallel Group Study) assessed the safety and tolerability of single doses of 305LO15 in healthy volunteers. At each dose level in each cohort, a total of 5 healthy volunteers were randomly assigned to receive a single dose of 305LO15 or placebo intravenously (IV; cohorts 1 and 2) or subcutaneously (SC; cohort 3). The study had an adaptive design, according to which sufficient safety, tolerability, PK and PD were constantly assessed before starting the next administration. The target dose levels for cohorts 1, 2, and 3 were 75 mg IV, 150 mg IV, and 170 mg SC, respectively (Figure 4a). Based on a preliminary analysis of PK and PD data obtained for cohort 1, doses for cohorts 2 and 3 were reduced to 125 mg IV (cohort 2) and 100 mg SC (cohort 3).

When performing an IV infusion, blood samples were collected before dosing, after the end of the IV infusion (1 h), at 2, 6, 12, 24, 48, 72, 96, and 144 h after dosing, and on days 14, 21, 28 , 35, 42, 56, 84, and 91. For SC administration, blood samples were collected pre-dose, 12, 24, 48, 72, 96, and 144 hours post-dose, and on days 14, 21, 28, 35, 42, 56, 84 and 91.

Preliminary safety results from the BP39144 Part 1 study showed that 305LO15 was well tolerated at all dose levels studied (75 and 125 mg IV and 100 mg SC). Available clinical safety information did not raise significant safety concerns, and no data were obtained to suggest the presence of adverse events or abnormalities in laboratory tests. There were no noteworthy data or trends in vital signs or 12-lead electrocardiograms in all treatment groups.

Preliminary serum PK results and simulated profiles: 305bO15 concentration-time are shown in FIG. 6, fig. 7 and FIG. 8. These data indicate that the PK characteristics of 305LO15 in healthy individuals were consistent with the human PK characteristics previously predicted from cynomolgus PK data (Example 5). It appears that after IV dosing, exposure should be dose proportional in the range of 75 to 125 mg. The final half-life (t _1/2 ) for a typical 70 kg patient is estimated to be approximately 25 days. Preliminary PK results after SC administrations indicate that 305LO15 exposure peaked around day 7 and bioavailability was approximately 90%. After absorption, t _1/2 was comparable to t _1/2 in the case of IV infusion.

Preliminary estimates of the exposure-response relationship confirmed the previously obtained prediction, namely, that approximately 40 μg of 305LO15 per milliliter of blood is required to ensure complete complement inhibition (Fig. 9).

Example 8

Part 2 of the BP39144 study (an open-label, multi-dose, global, multicenter, intra-individual, dose-escalation study) assessed safety and tolerability over a total of 5 months, and the pharmacodynamic effects of multiple doses of 305LO15 on complement activity in the treatment of naive patients with PNH. This study included six patients with PNH. Study patients were not treated with any complement inhibitor or were previously treated but discontinued due to lack of efficacy due to a single heterozygous C5 missense mutation and had elevated serum LDH levels (>1.5x ULN) at screening (ULN: upper limit of normal range).

One patient (Patient X) of the six patients was a PNH patient and was a candidate for complement inhibitor treatment. Patient X was administered IV three single incremental doses; by a single infusion of 375 mg of 305LO15 on day 1, then by a single infusion of 500 mg of 305LO15 on day 8, and then by a single infusion of 1000 mg of 305LO15 on day 22. The first SC administration (170 mg) was started on day 36, then weekly ( QW) SC injections (170 mg) of 305LO15, which were to be continued throughout the treatment period of 5 months.

Preliminary results of pharmacodynamic evaluation for patient X are presented in Table 2 and in FIG. 10. Levels of LDH, which serves as a pharmacodynamic marker of hemolysis, decreased to levels within the normal range by day 15, continued to decrease further until day 36, and were maintained at a normalized level until day 43 (Table 2 and Fig. 10a). As shown in Table 2 and FIG. 10b, liposomal immunoassay (LIA) results indicate that complement activity was completely inhibited at the end of the infusion on study start day, day 1. The 375 mg dose of 305LO15 administered on day 1 maintained complete complement inhibition until the next dose was administered. 305LO15 dose of 500 mg on day 8. The 305LO15 dose of 500 mg given on day 8 maintained complete complement inhibition until the next dose of 305LO15 of 1000 mg on day 22. The 305LO15 dose of 1000 mg, which administered on day 22 maintained complete complement inhibition until the next 170 mg SC dose of 305LO15 was administered on day 36. The SC dose administered on day 36 maintained complete complement inhibition until day 43.

At 305LO15 was well tolerated with only mild abdominal pain unrelated to treatment.

Example 9

Part 3 of the BP39144 study (an open-label, multi-dose, global, multicenter study) assessed the safety and tolerability over a total of 5 months and the pharmacodynamic effects of multiple doses of 305LO15 on complement activity in PNH patients treated with eculizumab during the considered time period. This study included 18 patients with PNH. Patients participating in the study were treated continuously with eculizumab for at least 3 months prior to entry into the study and the patients were regularly infused with eculizumab.

One patient (Patient Y) of 18 patients was a PNH patient who was treated with eculizumab, with eculizumab administered last 14 days prior to the first IV infusion of 305LO15. Patient Y was given a loading dose by single infusion; administered a single infusion of 1000 mg 305LO15 on day 1. The first SC injection (680 mg) was administered on day 8, followed by SC injections (680 mg) of 305LO15 every 4 weeks (Q4W) which continued for the entire treatment period of 5 months.

Preliminary results of pharmacodynamic evaluation for patient Y are presented in Table 3. LDH levels, serving as a pharmacodynamic marker of hemolysis, were maintained at baseline from the first dose until day 43 (Table 3). As shown in Table 3, the results of the liposomal immunoassay (LIA) indicated that complement activity was completely inhibited at the end of the infusion on study start day, day 1. The 1000 mg dose of 305LO15 given on day 1 maintained complete complement inhibition up to until the SC dose of 680 mg 305LO15 on day 8. The SC dose on day 8 maintained complete complement inhibition until day 43.

Example 10

Determining Dosing in PNH Patients An open, multi-dose, global, multicenter study was conducted in PNH patients treated with eculizumab (see also Example 9). Approximately 18 adult men and women with PNH who were treated with eculizumab participated in this study. The aim of Part 3 of the study was to determine the optimal SC dosing regimen that provides a low drug burden for patients. For each dosing regimen studied, 305LO15 exposures should be achieved to inhibit terminal complement pathway activation throughout the entire dosing period. The required exposure was determined based on the available PK, PD and efficacy data from Part 2 of the study. For all patients in Part 3 of the study, 305LO15 dosing was started no later than 2 weeks after the patient received the last dose of eculizumab. Following IV loading, multiple maintenance SC doses were administered.

During Part 3, the studies modified some of the original SC dosing regimens to further improve safety. Three different SC dosing regimens (including weekly [QW], biweekly [Q2W] or monthly [Q1M] dosing) of 305LO15 were studied (see FIG. 11). Patients included in arm A received an SC dose of 170 mg QW 8 times in the first phase. Starting with the 9th SC dose (experimental day 64), representing the first administration in the second phase, patients included in the arm A began to undergo a maintenance regimen using a dose of 680 mg Q4W. An IV loading dose was administered to patients, for example, about 24 hours before the first SC dose. In part 3 of the study, patients were treated for a maximum of 5 months. In the event that a patient showed signs and symptoms of his/her underlying disease, such as breakthrough hemolysis, which may be the result of an acute event, such as an acute illness, trauma or surgery, etc., one or more additional IV doses of 305LO15. Prior to administration of this IV dose, an unscheduled PD biomarker sample should be performed to evaluate the underlying cause of breakthrough hemolysis.

Preliminary results of the pharmacodynamic study in patients Z1 and Z2 in arm A are presented in Table 4. LDH levels, serving as a pharmacodynamic marker of hemolysis, were maintained at baseline from the first dose to day 106 (Table 4). As shown in Table 4, the results of the liposomal immunoassay (LIA) indicated that complement activity was completely inhibited at the end of the infusion on study start day, day 1. The 1000 mg dose of 305LO15 administered on day 1 maintained complete complement inhibition up to to the first SC dose of 170 mg 305LO15 on day 8. Beginning with the 9th SC dose (experimental day 64), these patients received a maintenance regimen of 680 mg Q4W. Complete complement inhibition (LIA [u/ml] <10) in patient Z2 was maintained throughout the SC dosing period (day 64 to day 106). In patient Z1, complement inhibition was complete or sufficient throughout the SC dosing period (day 64 to day 106). The regimen used in study shoulder A was well tolerated with only mild adverse events (shoulder paresthesia in patient Z1, common cold in patient Z2) that were unrelated to treatment.

Example 11

Additional open study. OLE In PNH Patients The following is a description of OLE for patients in Part 3 of the study who have been shown to benefit from treatment with 305LO15. The number of patients included in the experience did not exceed the number of patients included in part 3 of the study described in example 10'. Patients who were transferred from the initial regimen used in Part 3 of the study initially maintained the same SC dosing regimen that they received in Part 3 of the study. Depending on the emerging safety issues, PK and PD data obtained in part 3 of the study, the SC dosing regimen for patients included in the OLE was adapted accordingly. The duration of treatment was a maximum of 2 years from entry into the OLE.

--->

SEQUENCE LIST

<110> CHUGAI SEYYAKU KABUSHIKI KAISHA

<120> PHARMACEUTICAL COMPOSITION INTENDED 5 FOR USE

FOR THE TREATMENT OR PREVENTION OF A C5-RELATED DISEASE,

AND METHOD OF TREATMENT OR PREVENTION OF C5 RELATED DISEASE

<130> C1-A1820P

<150> US 62/713,211

<151> 2018-08-01

<150> US 62/764,952

<151> 2018-08-17

<150> US 62/760,204

<151> 2018-11-13

<160> 21

<170>PatentIn version 3.5

<210> 1

<211> 673

<212> PRT

<213> Homo sapiens

<400> 1

Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr

1 5 10 15

Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg

20 25 30

Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu

35 40 45

Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe

50 55 60

Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln

65 70 75 80

Asn Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln

85 90 95

Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser

100 105 110

Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile

115 120 125

His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg

130 135 140

Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val

145 150 155 160

Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu Glu

165 170 175

Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser

180 185 190

Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp

195 200 205

Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu

210 215 220

Pro His Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr

225 230 235 240

Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr

245 250 255

Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg

260 265 270

Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln

275 280 285

Asn Thr Met Leu Ile Asn Gly Ile Ala Gln Val Thr Phe Asp Ser Glu

290 295 300

Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn

305 310 315 320

Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe

325 330 335

Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr

340 345 350

Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro

355 360 365

Tyr Pro Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln Leu Val Gly

370 375 380

Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu

385 390 395 400

Thr Ser Asp Leu Asp Pro Ser Lys Ser Val Thr Arg Val Asp Asp Gly

405 410 415

Val Ala Ser Phe Val Leu Asn Leu Pro Ser Gly Val Thr Val Leu Glu

420 425 430

Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala

435 440 445

Arg Glu Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr

450 455 460

Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu

465 470 475 480

His Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile

485 490 495

Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His Phe

500 505 510

Gly Thr Arg Glu Lys Phe Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile

515 520 525

Pro Val Thr Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr

530 535 540

Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp

545 550 555 560

Leu Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser

565 570 575

Pro Asp Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met

580 585 590

Ala Thr Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala

595 600 605

Val Tyr Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg Val Phe

610 615 620

Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu

625 630 635 640

Asn Asn Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr Asn

645 650 655

Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile

660 665 670

Leu

<210> 2

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> an artificially Synthesized Sequence

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser

20 25 30

Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Gly Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp

50 55 60

Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr

100 105 110

TRP Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 3

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> an artificially Synthesized Sequence

<400> 3

Ser Ser Tyr Tyr Met Ala

15

<210> 4

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> an artificially Synthesized Sequence

<400> 4

Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp Ala Lys

1 5 10 15

gly

<210> 5

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 5

Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr

1 5 10

<210> 6

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Glu Thr Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Thr Lys Val Gly Ser Ser

85 90 95

Tyr Gly Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 7

Arg Ala Ser Gln Gly Ile Ser Ser Ser Leu Ala

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 8

Gly Ala Ser Glu Thr Glu Ser

15

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 9

Gln Asn Thr Lys Val Gly Ser Ser Tyr Gly Asn Thr

1 5 10

<210> 10

<211> 325

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 10

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu

325

<210> 11

<211> 325

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 11

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Tyr Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 20 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Tyr His Val Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu

325

<210> 12

<211> 325

<122> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 12

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 20 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Pro

325

<210> 13

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 13

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 14

<211> 325

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

305 310 315 320

Leu Ser Leu Ser Pro

325

<210> 15

<211> 325

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 15

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

305 310 315 320

Leu Ser Leu Ser Pro

325

<210> 16

<211> 107

<122> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 16

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Cys

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<120> 17

<211> 107

<122> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 17

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 18

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> An artificially synthesized sequence

<400> 18

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 19

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> Eculizumab heavy chain

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 20

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Eculizumab light chain

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 21

<211> 1676

<212> PRT

<213> Homo sapiens

<400> 21

Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr

1 5 10 15

Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg

20 25 30

Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu

35 40 45

Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe

50 55 60

Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln

65 70 75 80

Asn Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln

85 90 95

Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser

100 105 110

Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile

115 120 125

His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg

130 135 140

Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val

145 150 155 160

Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu Glu

165 170 175

Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser

180 185 190

Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp

195 200 205

Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu

210 215 220

Pro His Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr

225 230 235 240

Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr

245 250 255

Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg

260 265 270

Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln

275 280 285

Asn Thr Met Leu Ile Asn Gly Ile Ala Gln Val Thr Phe Asp Ser Glu

290 295 300

Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn

305 310 315 320

Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe

325 330 335

Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr

340 345 350

Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro

355 360 365

Tyr Pro Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln Leu Val Gly

370 375 380

Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu

385 390 395 400

Thr Ser Asp Leu Asp Pro Ser Lys Ser Val Thr Arg Val Asp Asp Gly

405 410 415

Val Ala Ser Phe Val Leu Asn Leu Pro Ser Gly Val Thr Val Leu Glu

420 425 430

Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala

435 440 445

Arg Glu Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr

450 455 460

Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu

465 470 475 480

His Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile

485 490 495

Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His Phe

500 505 510

Gly Thr Arg Glu Lys Phe Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile

515 520 525

Pro Val Thr Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr

530 535 540

Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp

545 550 555 560

Leu Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser

565 570 575

Pro Asp Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met

580 585 590

Ala Thr Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala

595 600 605

Val Tyr Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg Val Phe

610 615 620

Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu

625 630 635 640

Asn Asn Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr Asn

645 650 655

Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile

660 665 670

Leu Arg Pro Arg Arg Thr Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala

675 680 685

Lys Tyr Lys His Ser Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Cys

690 695 700

Val Asn Asn Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu

705 710 715 720

Gly Pro Arg Cys Ile Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser

725 730 735

Gln Leu Arg Ala Asn Ile Ser His Lys Asp Met Gln Leu Gly Arg Leu

740 745 750

His Met Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr

755 760 765

Phe Pro Glu Ser Trp Leu Trp Glu Val His Leu Val Pro Arg Arg Lys

770 775 780

Gln Leu Gln Phe Ala Leu Pro Asp Ser Leu Thr Thr Trp Glu Ile Gln

785 790 795 800

Gly Val Gly Ile Ser Asn Thr Gly Ile Cys Val Ala Asp Thr Val Lys

805 810 815

Ala Lys Val Phe Lys Asp Val Phe Leu Glu Met Asn Ile Pro Tyr Ser

820 825 830

Val Val Arg Gly Glu Gln Ile Gln Leu Lys Gly Thr Val Tyr Asn Tyr

835 840 845

Arg Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu Gly

850 855 860

Ile Cys Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly Thr Lys Ser

865 870 875 880

Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser Ser Ser His Leu Val

885 890 895

Thr Phe Thr Val Leu Pro Leu Glu Ile Gly Leu His Asn Ile Asn Phe

900 905 910

Ser Leu Glu Thr Trp Phe Gly Lys Glu Ile Leu Val Lys Thr Leu Arg

915 920 925

Val Val Pro Glu Gly Val Lys Arg Glu Ser Tyr Ser Gly Val Thr Leu

930 935 940

Asp Pro Arg Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro

945 950 955 960

Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile

965 970 975

Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val Leu

980 985 990

Ser Gln Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser Ala

995 1000 1005

Glu Ala Glu Leu Met Ser Val Val Pro Val Phe Tyr Val Phe His

1010 1015 1020

Tyr Leu Glu Thr Gly Asn His Trp Asn Ile Phe His Ser Asp Pro

1025 1030 1035

Leu Ile Glu Lys Gln Lys Leu Lys Lys Lys Leu Lys Glu Gly Met

1040 1045 1050

Leu Ser Ile Met Ser Tyr Arg Asn Ala Asp Tyr Ser Tyr Ser Val

1055 1060 1065

Trp Lys Gly Gly Ser Ala Ser Thr Trp Leu Thr Ala Phe Ala Leu

1070 1075 1080

Arg Val Leu Gly Gln Val Asn Lys Tyr Val Glu Gln Asn Gln Asn

1085 1090 1095

Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr Gln Leu

1100 1105 1110

Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr Gln Pro Ile Lys

1115 1120 1125

Leu Gln Gly Thr Leu Pro Val Glu Ala Arg Glu Asn Ser Leu Tyr

1130 1135 1140

Leu Thr Ala Phe Thr Val Ile Gly Ile Arg Lys Ala Phe Asp Ile

1145 1150 1155

Cys Pro Leu Val Lys Ile Asp Thr Ala Leu Ile Lys Ala Asp Asn

1160 1165 1170

Phe Leu Leu Glu Asn Thr Leu Pro Ala Gln Ser Thr Phe Thr Leu

1175 1180 1185

Ala Ile Ser Ala Tyr Ala Leu Ser Leu Gly Asp Lys Thr His Pro

1190 1195 1200

Gln Phe Arg Ser Ile Val Ser Ala Leu Lys Arg Glu Ala Leu Val

1205 1210 1215

Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Asn Leu Gln

1220 1225 1230

His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val

1235 1240 1245

Glu Thr Thr Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp

1250 1255 1260

Ile Asn Tyr Val Asn Pro Val Ile Lys Trp Leu Ser Glu Glu Gln

1265 1270 1275

Arg Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp Thr Ile Asn Ala

1280 1285 1290

Ile Glu Gly Leu Thr Glu Tyr Ser Leu Leu Val Lys Gln Leu Arg

1295 1300 1305

Leu Ser Met Asp Ile Asp Val Ser Tyr Lys His Lys Gly Ala Leu

1310 1315 1320

His Asn Tyr Lys Met Thr Asp Lys Asn Phe Leu Gly Arg Pro Val

1325 1330 1335

Glu Val Leu Leu Asn Asp Asp Leu Ile Val Ser Thr Gly Phe Gly

1340 1345 1350

Ser Gly Leu Ala Thr Val His Val Thr Thr Val Val His Lys Thr

1355 1360 1365

Ser Thr Ser Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile Asp Thr

1370 1375 1380

Gln Asp Ile Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn Ser Asp

1385 1390 1395

Tyr Lys Arg Ile Val Ala Cys Ala Ser Tyr Lys Pro Ser Arg Glu

1400 1405 1410

Glu Ser Ser Ser Gly Ser Ser His Ala Val Met Asp Ile Ser Leu

1415 1420 1425

Pro Thr Gly Ile Ser Ala Asn Glu Glu Asp Leu Lys Ala Leu Val

1430 1435 1440

Glu Gly Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly

1445 1450 1455

His Val Ile Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp Phe Leu

1460 1465 1470

Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe Leu

1475 1480 1485

Ser Pro Ala Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys

1490 1495 1500

Gln Cys Thr Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile Gln Lys

1505 1510 1515

Val Cys Glu Gly Ala Ala Cys Lys Cys Val Glu Ala Asp Cys Gly

1520 1525 1530

Gln Met Gln Glu Glu Leu Asp Leu Thr Ile Ser Ala Glu Thr Arg

1535 1540 1545

Lys Gln Thr Ala Cys Lys Pro Glu Ile Ala Tyr Ala Tyr Lys Val

1550 1555 1560

Ser Ile Thr Ser Ile Thr Val Glu Asn Val Phe Val Lys Tyr Lys

1565 1570 1575

Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu

1580 1585 1590

Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val Thr Cys Thr Asn

1595 1600 1605

Ala Glu Leu Val Lys Gly Arg Gln Tyr Leu Ile Met Gly Lys Glu

1610 1615 1620

Ala Leu Gln Ile Lys Tyr Asn Phe Ser Phe Arg Tyr Ile Tyr Pro

1625 1630 1635

Leu Asp Ser Leu Thr Trp Ile Glu Tyr Trp Pro Arg Asp Thr Thr

1640 1645 1650

Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn Leu Asp Glu Phe Ala

1655 1660 1665

GLU ASP Ile PHE LEU ASN GLY CYS

1670 1675

<---

Claims (26)

1. A method of treating or preventing a C5 related disease, wherein the method comprises subcutaneously administering to an individual a pharmaceutical composition that is formulated for subcutaneous injection and contains an anti-C5 antibody, wherein the composition is administered subcutaneously using two phases, following intravenous administration of a single dose other pharmaceutical composition, where said other pharmaceutical composition is a pharmaceutical composition formulated for intravenous administration and containing an anti-C5 antibody, in which (i) the first subcutaneous administration of the first phase is carried out after intravenous administration, and the first subcutaneous administration of the first phase is carried out on any of days 0 (i.e. within 24 hours), 1 day or 2 days after administration of the pharmaceutical composition for intravenous administration and (ii) the dose of the antibody for intravenous administration is from 100 to 2000 mg, in which for subcutaneous administration in two phases in both phases there is an interval between every two subcutaneous injections and each phase includes at least one interval, in which in the first phase I) the interval is shorter than the interval in the second phase, and is 5-14 days, and ii) the dose of the antibody per administration is lower than the dose of the antibody per administration in the second phase, and is 50-350 mg, and where in the second phase (i) the dose of the antibody per administration is between 650 and 700 mg, and (ii) the interval is 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days or 35 days, and furthermore, the anti-C5 antibody comprises a heavy chain variable domain (VH) consisting of the amino acid sequence of SEQ ID NO: 2, a light chain variable domain (VL) consisting of the amino acid sequence of SEQ ID NO: 6, a heavy chain constant domain ( CH) consisting of the amino acid sequence of SEQ ID NO: 13, a light chain constant domain (CL) consisting of the amino acid sequence of SEQ ID NO: 18. 2. The method of claim 1, wherein one interval in the first phase is any of 5 days, 6 days, 7 days, or 8 days. 3. The method according to claim. 1 or 2, in which the subcutaneous administration in the first phase is carried out once a week (weekly). 4. Method according to any of the paragraphs. 1-3, wherein one interval in the second phase is any of 27 days, 28 days, or 29 days. 5. Method according to any of the paragraphs. 1-4, in which subcutaneous administration in the first phase is carried out once a week (weekly) with a dose of 340 mg of antibody. 6. Method according to any of the paragraphs. 1-5, in which the number of subcutaneous injections in the first phase is from 5 to 10. 7. Method according to any of the paragraphs. 1-6, in which subcutaneous administration in the second phase is carried out every four weeks or monthly with a dose of 680 mg of antibody. 8. Method according to any of the paragraphs. 1-7, in which the dose of antibody for intravenous administration is 1000 mg. 9. Method according to any of the paragraphs. 1-8, wherein the subject has been treated with at least one pharmacological product for the treatment or prevention of a C5 related disease, and one dose of the pharmaceutical composition for intravenous administration is administered after the administration of the last dose of the pharmacological product. 10. The method of claim 9, wherein one intravenous dose of the pharmaceutical composition is administered 3 or more days after the last dose of the pharmacological product. 11. The method of claim 9 or 10, wherein the pharmacological product comprises an siRNA targeting C5 mRNA or an anti-C5 antibody that is different from an anti-C5 antibody as defined in claim 1. 12. Method according to any of the paragraphs. 9-11, in which the pharmacological product contains eculizumab. 13. Method according to any of the paragraphs. 1-12, in which the anti-C5 antibody has pH-dependent binding characteristics. 14. Method according to any of the paragraphs. 1-13, wherein the C5 related disease is any disease selected from the group consisting of rheumatoid arthritis (RA); lupus nephritis; damage caused by ischemia-reperfusion; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration; syndrome (HELLP): hemolysis, increased activity of liver enzymes and thrombocytopenia; thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; Pauci-immune vasculitis; bullous epidermolysis; repeated loss of the fetus; multiple sclerosis (MS); traumatic brain injury and damage due to myocardial infarction, cardiopulmonary bypass and hemodialysis; generalized myasthenia gravis (gMG); neuromyelitis of the optic nerve (NMO). 15. The method of claim 14 wherein the C5 related disease is PNH, aHUS, gMG, or NMO.

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