RU2822664C2 - DOSES AND ADMINISTRATION OF ANTIBODIES AGAINST C5 FOR TREATMENT OF ATYPICAL HEMOLYTIC UREMIC SYNDROME (aHUS) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to methods of treating aHUS in an adult human patient aged 18 years or older using an anti-C5 antibody. Anti-C5 antibody is an antibody containing a heavy chain polypeptide with SEQ ID NO: 14, light chain polypeptide with SEQ ID NO: 11, or is ravulizumab. Anti-C5 antibody is administered in a specific dose amount based on the patient’s body weight and in accordance with a specific dose schedule.

EFFECT: inventions provide improved methods of treating patients with aHUS.

42 cl, 23 dwg, 22 tbl, 2 ex

Description

BACKGROUND OF THE INVENTION

The complement system works with the body's other immunological systems to protect against invading cellular and viral pathogens. There are at least 25 complement proteins, which are a complex collection of plasma proteins and membrane cofactors. Plasma proteins make up about 10% of globulins in vertebrate blood serum. Complement components perform their immune protective functions by interacting in a series of complex but precise enzymatic degradation and membrane binding processes. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions. A summary of the biological activities associated with complement activation is presented, for example, in The Merck Manual, ^16th Edition.

While a properly functioning complement system provides robust protection against infecting microbes, inappropriate regulation or activation of complement pathways has been implicated in the pathogenesis of various disorders, including atypical hemolytic uremic syndrome (aHUS). aHUS is an extremely rare disorder caused by chronic uncontrolled activation of complement. The resulting inflammation and cell damage lead to the devastating clinical manifestations of the disease.

Hemolytic uremic syndrome (HUS) is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. There are two types of HUS: diarrhea-associated (D+HUS; also called Shiga toxin-producing E. coli (STEC)-HUS or typical HUS) and nondiarrheal or atypical HUS (aHUS). D+HUS is the most common form, accounting for more than 90% of cases, and is caused by a preexisting infection with a Shiga-like toxin-producing bacterium, such as E coli O157:H7.

aHUS can be hereditary, acquired, or idiopathic. Inherited forms of aHUS may be associated with mutations in a number of human complement components, including, for example, complement factor H (CFH), membrane cofactor protein (MCP), complement factor I (CFI), C4b-binding protein (C4BP), complement factor B (CFB) and complement component 3 (C3). See, for example, Caprioli et al. (2006) Blood 108:1267–1279. Certain mutations in the gene encoding CD55, although not yet characterized as associated with aHUS, are associated with the severity of aHUS. See, for example, Esparza-Gordillo et al. (2005) Hum Mot Genet 14:703–712.

aHUS is rare and has a mortality rate of up to 25%. Many patients with the disease have permanent neurological or renal impairment, for example, in at least 50% of patients with aHUS, renal impairment progresses to end-stage chronic renal failure (ESRD). See, for example, Kavanagh et al. (2006) British Medical Bulletin 77 and 78:5-22. Until recently, treatment options for patients with aHUS were limited and often involved plasma infusion or plasma exchange. In some cases, patients with aHUS undergo unilateral or bilateral nephrectomy or kidney transplantation (see Artz et al. (2003) Transplantation 76:821-826). However, relapses of this disease in treated patients are common.

Patients with aHUS are at significant risk of morbidity and mortality. Accordingly, it is an object of this invention to provide improved methods for treating patients with aHUS.

BRIEF DESCRIPTION OF THE INVENTION

Provided herein are compositions and methods for treating aHUS in a human patient (e.g., an adult patient 18 years of age or older), comprising administering to said patient an anti-C5 antibody or an antigen-binding fragment thereof, wherein said anti-C5 antibody or antigen-binding fragment thereof administered (or intended to be administered) in accordance with a specific clinical regimen (ie, in a specific dosage amount and in accordance with a specific dosing schedule).

Any suitable anti-C5 antibody or antigen-binding fragment thereof may be used in the methods described herein. An exemplary anti-C5 antibody is ravulizumab (also known as ^ULTOMIRIS® , ALXN1210 and antibody BNJ441) containing heavy and light chains having the sequences set forth in SEQ ID NO: 14 and 11, respectively, or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises heavy and light chain complementarity determining regions (CDRs) or variable regions (VRs) of ravulizumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the ravulizumab heavy chain variable region (VH) having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of the ravulizumab light chain variable region (VL), having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 19, 18 and 3, respectively, and the light chain CDR1, CDR2 and CDR3 sequences, indicated SEQ ID NO: 4, 5 and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively. In another embodiment, the antibody comprises the heavy chain constant region set forth in SEQ ID NO: 13.

In another embodiment, the antibody comprises a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn), wherein the variant human Fc constant region CH3 contains the substitutions Met-429-Leu and Asn-435-Ser at residues corresponding to methionine 428 and asparagine 434 of the Fc constant region of native human IgG, each residue numbered according to the EU numbering.

In another embodiment, the antibody comprises the heavy chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NO: 19, 18 and 3, respectively, and the light chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NO: 4, 5 and 6, respectively , and a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn), wherein the variant human Fc constant region CH3 contains substitutions Met-429-Leu and Asn-435-Ser at residues corresponding to methionine 428 and asparagine 434 Fc constant region of native human IgG, each residue numbered according to EU numbering.

In another embodiment, the antibody binds to human C5 at pH 7.4 and 25°C with an affinity dissociation constant (K _D ) that ranges from 0.1 nM to 1 nM. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25°C with a K _D ≥ 10 nM. In yet another embodiment, the antibody index [(K _{D of} an antibody or antigen binding fragment thereof for human C5 at pH 6.0 and at 25°C)/(K _{D of} an antibody or antigen binding fragment thereof for human C5 at pH 7.4 and at 25 °C)] is more than 25.

Another exemplary anti-C5 antibody is antibody 7086, described in US Pat. No. 8,241,628 and US Pat. No. 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of antibody 7086 (see US Pat. No. 8,241,628 and US Pat. No. 8,883,158). In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 21, 22 and 23, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 24, 25 and 26, respectively. In another embodiment, the antibody or antigen binding fragment thereof comprises a VH region of antibody 7086 having the sequence set forth in SEQ ID NO: 27 and a VL region of antibody 7086 having the sequence set forth in SEQ ID NO: 28.

Another exemplary anti-C5 antibody is antibody 8110, also described in US Pat. No. 8,241,628 and US Pat. No. 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of antibody 8110. In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 29, 30 and 31 , respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 32, 33 and 34, respectively. In another embodiment, the antibody comprises the VH region of antibody 8110 having the sequence set forth in SEQ ID NO: 35 and the VL region of antibody 8110 having the sequence set forth in SEQ ID NO: 36.

Another exemplary anti-C5 antibody is the 305LO5 antibody described in US 2016/0176954 A1. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody. In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 37, 38 and 39, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively. In another embodiment, the antibody comprises a 305LO5 antibody VH region having the sequence set forth in SEQ ID NO: 43 and a 305LO5 antibody VL region having the sequence set forth in SEQ ID NO: 44.

Another exemplary anti-C5 antibody is the SKY59 antibody described in Fukuzawa T., et at, Rep. 2017 Apr 24;7(1): 1080). In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody. In another embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46.

Another exemplary anti-C5 antibody is antibody REGN3918 (also known as H4H12166PP) described in US 20170355757. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 47. 48. In another embodiment, the antibody comprises a heavy chain comprising SEQ ID NO: 49 and a light chain comprising SEQ ID NO: 50.

In another embodiment, the antibody competes for binding and/or binds to the same epitope on C5 as the above antibodies (e.g., eculizumab, ravulizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918). In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the aforementioned antibodies (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% variable region identity).

In one embodiment, the dose of the anti-C5 antibody or antigen-binding fragment thereof is based on the patient's body weight. For example, in one embodiment, 2400 mg or 3000 mg of an anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥40 to <60 kg. In another embodiment, 2700 mg or 3300 mg of an anti-C5 antibody or antigen binding fragment thereof is administered to a patient weighing ≥60 to <100 kg. In another embodiment, 3000 mg or 3600 mg of an anti-C5 antibody or antigen binding fragment thereof is administered to a patient weighing ≥ 100 kg. In certain embodiments, administration regimens are selected to provide the optimal desired response (eg, an effective response).

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered in one or more administration cycles. In one embodiment, the administration cycle is 26 weeks. In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered once on day 1 of the administration cycle, once on day 15 of the administration cycle, and every eight weeks thereafter. In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered every eight weeks after the dosing cycle during an extension period of up to and including two years (eg, at a dose of 3000 mg, 3300 mg, or 3600 mg).

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered in one or more administration cycles. In one embodiment, the administration cycle is 26 weeks. In another embodiment, the treatment consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 cycles. In another embodiment, treatment continues throughout the life of the human patient.

In another embodiment, a method is provided for treating a human patient with aHUS, the method comprising administering to the patient (e.g., during an administration cycle) an effective amount of an anti-C5 antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2, and CDR3 sequences, indicated by SEQ ID NOs: 19, 18 and 3, respectively, and the light chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NOs: 4, 5 and 6, respectively, wherein said anti-C5 antibody or antigen binding fragment thereof is administered:

(a) once on the 1st day at a dose of: 2400 mg to a patient weighing ≥ 40 to < 60 kg, 2700 mg to a patient weighing ≥ 60 to < 100 kg, or 3000 mg to a patient with body weight ≥ 100 kg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg - for a patient weighing ≥ 100 kg.

In another embodiment, a method is provided for treating a human patient with aHUS, the method comprising administering to the patient (e.g., during an administration cycle) an effective amount of an anti-C5 antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2, and CDR3 sequences, indicated by SEQ ID NOs: 19, 18 and 3, respectively, the light chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NOs: 4, 5 and 6, respectively, and a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn), wherein the variant CH3 human Fc constant region contains substitutions Met-429-Leu and Asn-435-Ser in residues corresponding to methionine 428 and asparagine 434 of the native human IgG Fc constant region, each residue is numbered according to EU numbering, while said anti-C5 antibody or antigen-binding fragment thereof is administered:

(a) once on the 1st day at a dose of: 2400 mg to a patient weighing ≥ 40 to < 60 kg, 2700 mg to a patient weighing ≥ 60 to < 100 kg, or 3000 mg to a patient with body weight ≥ 100 kg; And

(b) on the 15th day and thereafter - every eight weeks at a dose equal to:

3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg for a patient weighing ≥ 100 kg.

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥40 to <60 kg:

(a) once on the 1st day at a dose of: 2400 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose equal to:

3000 mg.

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥60 to <100 kg:

(a) once on the 1st day at a dose of: 2700 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose equal to:

3300 mg.

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥ 100 kg:

(a) once on the 1st day at a dose of: 3000 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose equal to:

3600 mg.

In some embodiments, the patient has not previously received treatment with a complement inhibitor (eg, the patient is one who has not previously received treatment with a complement inhibitor).

In other embodiments, the patient was previously treated with one anti-C5 antibody or antigen-binding fragment thereof and was then switched to another anti-C5 antibody or antigen-binding fragment thereof during the course of treatment. For example, in certain embodiments, various anti-C5 antibodies are administered during a course of treatment. In one embodiment, different anti-C5 antibodies are administered during separate treatment and extension periods. For example, in one embodiment, a patient is treated with eculizumab during a treatment period (eg, 26 weeks), followed by treatment with another anti-C5 antibody (eg, ravulizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918 ), for example during the renewal period. In another embodiment, eculizumab is administered to the patient at a dose of 600 mg on days 1, 8, 15, and 22 of the dosing cycle during the induction phase, followed by a maintenance dose of 900 mg of eculizumab on day 19 day of the administration cycle and every two weeks thereafter (eg, for a total of 26 weeks), followed by treatment with ravulizumab for an extension period of up to two years. In another embodiment, the patient is treated with ravulizumab (eg, for 26 weeks), followed by treatment with another anti-C5 antibody (eg eculizumab, 7086 antibody, 8110 antibody, 305LO5 antibody, SKY59 antibody or REGN3918 antibody) for, for example, an extension period.

Exemplary alternative anti-C5 antibodies include, but are not limited to: (i) ravulizumab, (ii) an antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2 and CDR3 domains containing SEQ ID NOs: 21, 22 and 23, respectively , and light chain CDR1, CDR2 and CDR3 domains comprising SEQ ID NO: 24, 25 and 26, respectively, (iii) an antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region chain containing SEQ ID NO: 28, (iv) an antibody or antigen binding fragment thereof containing the heavy chain CDR1, CDR2 and CDR3 domains containing SEQ ID NO: 29, 30 and 31, respectively, and the light chain CDR1, CDR2 and CDR3 domains containing SEQ ID NO: 32, 33 and 34, respectively, (v) an antibody or antigen binding fragment thereof, comprising a heavy chain variable region containing SEQ ID NO: 35 and a light chain variable region containing SEQ ID NO: 36, ( vi) an antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2 and CDR3 domains containing SEQ ID NOs: 37, 38 and 39, respectively, and light chain CDR1, CDR2 and CDR3 domains containing SEQ ID NOs: 40, 41 and 42, respectively, (vii) an antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 43 and a light chain variable region comprising SEQ ID NO: 44, (viii) an antibody or antigen binding fragment thereof comprising a heavy a chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46, respectively, (vii) an antibody or an antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region, comprising SEQ ID NO: 48, (x) an antibody or antigen binding fragment thereof, comprising a heavy chain containing SEQ ID NO: 49 and a light chain containing SEQ ID NO: 50.

In some embodiments, the patient has previously received treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months or at least 24 months with anti-C5 antibody or antigen-binding fragment thereof (eg, eculizumab) before switching to another anti-C5 antibody or antigen-binding fragment (eg, ravulizumab). In a specific embodiment, the patient has previously received treatment with eculizumab for at least 6 months.

In another embodiment, when a patient (eg, a patient with aHUS) is treated with a first anti-C5 antibody and is then switched to treatment with a second different anti-C5 antibody, especially when said second different anti-C5 antibody binds to an epitope on C5 different from the epitope , to which said first anti-C5 antibody binds, the administration schedules take into account the half-life of said first anti-C5 antibody. For example, to ensure that the first anti-C5 antibody is cleared (e.g., "washed out") from the patient before the second (other) anti-C5 antibody is administered (e.g., to avoid problems associated with aggregation, immune complex formation, etc. ), the half-life of said first anti-C5 antibody is taken into account. In one embodiment, the second (other) anti-C5 antibody is not administered until a period of time corresponding to 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 has passed. 6.5, 7 or 7.5 half-life of the first anti-C5 antibody after the final administration of said first anti-C5 antibody.

In another embodiment, the patient was previously treated with eculizumab and was then switched to treatment with a second (different) anti-C5 antibody (eg, ravulizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918). In one embodiment, when the first antibody administered is eculizumab, the second (other) anti-C5 antibody is not administered, for example, until at least 36, 45, 54, 63, 72, 81, 90, 99 , 108, 117 or 126 days after the final administration of eculizumab.

In another embodiment, the patient was previously treated with ravulizumab and then switched to treatment with another anti-C5 antibody (eg, eculizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918). In one embodiment, when the first antibody administered is ravulizumab, the second (other) anti-C5 antibody is not administered, for example, until at least 100, 125, 150, 175, 200, 225, 250, 275 , 300, 325, 375 or 400 days after the final administration of ravulizumab.

As an adjunct or alternative, techniques are used to eliminate or enhance the clearance of the first anti-C5 antibody before proceeding to treatment with a second (other) anti-C5 antibody. Exemplary techniques include, but are not limited to, plasmapheresis or blood transfusions. In another embodiment, an anti-first anti-C5 antibody (e.g., anti-eculizumab antibody, anti-ravulizumab antibody, anti-7086 antibody, anti-8110 antibody, anti-305LO5 antibody, anti-SKY59 antibody, or anti-REGN3918 antibody) is administered to clear or enhance the clearance of said first antibody anti-C5 before administration of the second (different) anti-C5 antibody.

In another embodiment, an anti-C5 antibody or antigen-binding fragment thereof (e.g., ravulizumab) is administered to a patient, wherein treatment (e.g., an administration cycle) begins after at least about two weeks, at least about three weeks, at least about four weeks , at least about six weeks, at least about seven weeks, or at least about eight weeks after the final dose of eculizumab is administered to said patient. In another embodiment, an anti-C5 antibody or antigen-binding fragment thereof (eg, ravulizumab) is administered to a patient, with treatment (eg, an administration cycle) beginning at least two weeks after the final dose of eculizumab is administered to said patient.

In some embodiments, patients treated in accordance with the methods described herein have been vaccinated against meningococcal diseases within 3 years before or at the time of initiation of treatment. In one embodiment, patients who received treatment less than 2 weeks after receiving the meningococcal vaccine also receive appropriate prophylactic antibiotics for 2 weeks after vaccination. In yet another embodiment, patients receiving treatment in accordance with the methods described herein are vaccinated against meningococcal serotypes A, C, Y, W135 and/or B.

In another aspect, the treatment regimens described herein are sufficient to maintain certain residual serum concentrations of the anti-C5 antibody or antigen-binding fragment thereof. For example, in one embodiment, treatment maintains a residual serum concentration of anti-C5 antibody or antigen-binding fragment thereof at 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 240, 25, 0, 255, 260, 265, 270, 280, 290, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 0, 395 or 400 mcg/ml or more during treatment. In one embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 100 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 150 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 200 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 250 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 300 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at a level of 100 μg/ml to 200 μg/ml. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at about 175 μg/ml.

In another embodiment, to obtain an effective response, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain at least 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg , 95 mcg, 100 mcg, 105 mcg, 110 mcg, 115 mcg, 120 mcg, 125 mcg, 130 mcg, 135 mcg, 140 mcg, 145 mcg, 150 mcg, 155 mcg, 160 mcg, 165 mcg, 170 mcg, 175 µg, 180 µg, 185 µg, 190 µg 195 µg, 200 µg, 205 µg, 210 µg, 215 µg, 220 µg, 225 µg, 230 µg, 235 µg, 240 µg, 245 µg, 250 µg, 255 µg or 260 mcg of antibody per milliliter of patient blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain between 50 μg and 250 μg of antibody per milliliter of the patient's blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain 100 μg to 200 μg of antibody per milliliter of the patient's blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain about 175 μg of antibody per milliliter of the patient's blood.

In another embodiment, to obtain an effective response, the anti-C5 antibody is administered to the patient in an amount and frequency that provides a minimum concentration of free C5. For example, in one embodiment, an anti-C5 antibody is administered to a patient in an amount and frequency to maintain a free C5 concentration of 0.2 μg/mL, 0.3 μg/mL, 0.4 μg/mL, 0.5 μg/mL. ml or lower. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain a free C5 concentration of 0.309 μg/mL to 0.5 μg/mL or lower. In another embodiment, the treatment described herein reduces the concentration of free C5 by more than 99% during the treatment period. In another embodiment, the treatment reduces the concentration of free C5 by more than 99.5% during the treatment period.

Antibodies against C5 or antigen binding fragments thereof can be administered to a patient by any suitable means. In one embodiment, the antibodies are formulated for intravenous administration.

The effectiveness of the treatments presented herein can be assessed using any suitable means. In one embodiment, for a patient with aHUS, treatment produces at least one therapeutic effect selected from the group consisting of reduction or cessation of severe hypertension, proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia, and renal dysfunction (eg , acute renal failure).

In other embodiments, the treatment results in inhibition of terminal complement.

In other embodiments, treatment causes a shift toward normal levels of a hemolysis-related hematologic biomarker selected from the group consisting of free hemoglobin, haptoglobin, reticulocyte count, PNH red cell clone, and D-dimer.

In another embodiment, the treatment causes an increase in hemoglobin stabilization compared to the patient's pre-treatment baseline level. In another embodiment, treatment results in an increase in hemoglobin levels of ≥20 g/L. In another embodiment, the treatment results in the prevention of a decrease in hemoglobin levels of ≥ 2 g/dL from baseline in the absence of transfusion from baseline to day 183.

In other embodiments, treatment results in normalization of platelet levels (≥ 150 x 10 ⁹ /L). In other embodiments, treatment results in normalization of platelet levels (≥ 150 x 10 ⁹ /L) for a period of at least 28 days (for example, at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in normalization of LDH levels (≤246 U/L). In other embodiments, treatment results in normalization of LDH levels (≤246 U/L) for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in an improvement in serum creatinine levels of ≥25% over baseline. In other embodiments, treatment results in an improvement in serum creatinine levels of ≥25% above baseline for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in a complete TMA response (i.e., normalization of platelet levels (≥150 x ¹⁰⁹ /L), normalization of LDH (≤246 U/L), and improvement in serum creatinine levels by ≥25% versus with the original level). In other embodiments, treatment results in a complete TMA response for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in a modified complete TMA response (ie, normalization of platelet levels (≥150 x ¹⁰⁹ /L), normalization of LDH levels (≤246 U/L), and the patient stops dialysis if he or she was on dialysis at baseline, or an improvement in serum creatinine ≥ 25% above baseline for a patient who was not on dialysis at baseline). In other embodiments, treatment results in a modified TMA complete response for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, the treatment reduces the need for blood transfusions. In another embodiment, treatment results in an increase in transfusion cessation by greater than 70%. In another embodiment, treatment results in discontinuation of transfusion during the period from baseline to day 183.

In other embodiments, the treatment results in resolution of breakthrough hemolysis during the treatment period. In another embodiment, treatment results in a decrease in breakthrough hemolysis compared to the baseline level of breakthrough hemolysis before treatment.

In other embodiments, treatment results in a reduction in major adverse vascular events (MAVE).

In other embodiments, treatment produces a change in quality of life from baseline, as assessed by the Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Score Version 4 and the 30-question European Organization for Research and Treatment of Malignant Tumors Quality of Life Questionnaire. In one embodiment, the treatment causes a change in quality of life, as measured by the FACIT fatigue score, of one or more (eg, 1, 2, or 3) points from baseline. In another embodiment, the treatment causes a change in quality of life from baseline, as assessed by the FACIT fatigue score, of 3 points after 150 days or more days (e.g., 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 days, 172 days, 173 days, 174 days, 175 days, 176 days, 177 days, 178 days, 179 days, 180 days, 181 days, 182 days 183 days, 184 days, 185 days, 186 days, 187 days , 188 days, 189 days, 190 days, 191 days, 192 days, 193 days, 194 days, 195 days, 196 days, 197 days, 198 days, 199 days, 200 days, 205 days, 210 days, 215 days, 220 days or 225 days) after the start of treatment.

Chronic kidney disease (CKD) stage is classified based on the US National Kidney Foundation stages of chronic kidney disease. The stages of CKD and the corresponding estimated glomerular filtration rate (eGFR) values are as follows: Stage 1: eGFR >= 90 (normal), Stage 2: eGFR = 60-89, Stage 3A: eGFR = 45-59, Stage 3B: eGFR = 30- 44, Stage 4: eGFR = 15–29 and stage 5: eGFR < 15 (including dialysis: End stage). Stage 1 is considered the best category. Stage 5 is considered the worst category. Improved eGFR (eg, >15) corresponds to improved CKD stage (eg, lower CKD stage). Accordingly, in other embodiments, the patient's chronic kidney disease (CKD) improves by one or more stages after initiation of treatment. For example, a patient's CKD improves by one, two, three, four or five stages). In another embodiment, the patient's CKD improves by one or more stages after 150 days or more days (e.g., 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 days, 172 days, 173 days, 174 days, 175 days, 176 days, 177 days, 178 days, 179 days, 180 days, 181 days, 182 days 183 days, 184 days, 185 days, 186 days, 187 days, 188 days, 189 days, 190 days, 191 days, 192 days, 193 days, 194 days, 195 days, 196 days, 197 days, 198 days, 199 days, 200 days, 205 days, 210 days, 215 days, 220 days or 225 days) after the start of treatment.

In other embodiments, treatment results in an increase in eGFR compared to baseline. In other embodiments, treatment results in a shift toward normal eGFR levels (eg, ≥90). In other embodiments, treatment results in an increase in eGFR compared to baseline and the patient's CKD improves by one or more stages. In other embodiments, treatment results in a shift toward normal eGFR levels (eg, ≥90) compared to baseline, and the patient's CKD improves by one or more stages.

In other embodiments, the treatment results in an EQ-5D-3L US Time Trade-Off (US TTO) value of >0.94.

In another aspect, there is provided an anti-C5 antibody or antigen binding fragment thereof comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the sequence set forth in SEQ ID NO: 12 and light chain variable region CDR1, CDR2 and CDR3 domains having the sequence specified in SEQ ID NO: 8, for administration to a patient having aHUS:

(a) once on the 1st day at a dose of: 2400 mg to a patient weighing ≥ 40 to < 60 kg, 2700 mg to a patient weighing ≥ 60 to < 100 kg, or 3000 mg to a patient with body weight ≥ 100 kg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg - for a patient weighing ≥ 100 kg.

In one embodiment, the antibody is determined to be safe, tolerable, and sufficiently non-immunogenic after multiple intravenous doses for use in patients with aHUS.

Also provided are kits that include a pharmaceutical composition containing an anti-C5 antibody or antigen-binding fragment thereof, such as ravulizumab, and a pharmaceutically acceptable carrier, in a therapeutically effective amount adapted for use in the methods described herein. In one embodiment, the kit contains:

(a) a dose of an anti-C5 antibody or an antigen-binding fragment thereof comprising the CDR1, CDR2 and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of a light chain variable region having the sequence, specified in SEQ ID NO: 8; And

(b) instructions for use of the anti-C5 antibody or antigen-binding fragment thereof in the methods described herein.

In one embodiment, 2400 mg or 3000 mg of an anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥40 to <60 kg. In another embodiment, 2700 mg or 3300 mg of an anti-C5 antibody or antigen binding fragment thereof is administered to a patient weighing ≥60 to <100 kg. In another embodiment, 3000 mg or 3600 mg of an anti-C5 antibody or antigen binding fragment thereof is administered to a patient weighing ≥ 100 kg.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows the study design for ALXN1210-aHUS-311.

Figure 2 summarizes the primary, secondary, and safety endpoints for ALXN1210-aHUS-311.

Figure 3 summarizes the inclusion and exclusion criteria for ALXN1210-aHUS-311.

Figure 4 shows the distribution of patients for ALXN1210-aHUS-311.

Figure 5 shows data for a derivation example with a confirmed complete TMA response.

Fig. 6 is a Venn diagram showing key efficacy data related to the primary complete response of TMA during the primary evaluation period.

Fig. 7 is a graph showing the time to complete TMA response.

Fig. 8 is a graph showing mean plasma concentrations (μg/mL) over time (linear scale). Weight-based dosing resulted in maximum, steady-state, and trough drug concentrations as predicted, without unexpected pharmacokinetic results.

Fig. 9 is a series of histograms displaying key efficacy data related to TMA primary complete response during the primary evaluation period through the end of data collection. 95% confidence intervals are represented by lines at the top of each column.

Figure 10 shows TMA complete response overall and by subgroup during the 26-week primary assessment period.

Figure 11 shows key efficacy outcomes for TMA complete response status over time (open circle), including normalization of platelet count (open triangle), normalization of hematology parameters (+), 25% increase in serum creatinine from baseline level (open square) and LDH normalization (X).

Figure 12 shows the mean change in eGFR (mL/min/1.73 m2) from baseline and 95% CI over time.

Figure 13 shows the shift in the stages of chronic kidney disease (CKD) on day 183 compared to baseline.

Figure 14 shows the observed and model-based mean change in platelet level (109/L) from baseline and 95% confidence interval over time.

Figure 15 shows the observed mean platelet level (109/L) and 95% confidence interval over time.

Figure 16 shows the observed and model-based mean changes in LDH levels (U/L) from baseline and 95% confidence interval over time.

Figure 17 shows the observed mean LDH level (U/L) and 95% confidence interval over time.

Figure 18 shows the observed and model-based mean changes in hemoglobin (Hb) (g/L) from baseline and 95% confidence interval over time.

Figure 19 shows the observed mean Hb level (g/L) and 95% confidence interval over time.

Figure 20 shows the mean change in FACIT fatigue score from baseline and 95% confidence interval over time. FACIT scores range from 0 to 52, with higher scores indicating less fatigue

Figure 21 shows the mean change in EQ-5D-3L from baseline and 95% confidence interval over time.

Figure 22 shows the average concentrations (μg/l) of the free component C5 of complement in the blood serum over time from the moment the initial level was determined to the 183rd day.

Figure 23 compares the study population and results between the ALXN1210-aHUS-311 study and the eculizumab adult study (C10-004).

DETAILED DESCRIPTION OF THE INVENTION

I. Antibodies against C5

Anti-C5 antibodies described herein bind to complement component C5 (eg, human C5) and inhibit the cleavage of C5 into C5a and C5b fragments. As described above, such antibodies also have, for example, improved pharmacokinetic properties compared to other anti-C5 antibodies (eg, eculizumab) used for therapeutic purposes.

The term "antibody" describes polypeptides containing at least one antigen binding site derived from an antibody (eg, VH/VL or Fv region, or CDR). Antibodies include known forms of antibodies. For example, the antibody may be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. The antibody may also be a Fab, Fab'2, ScFv, SMIP, affibody®, nanoantibody, or domain antibody. The antibody may also be any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD and IgE. The antibody may be a naturally occurring antibody or may be an antibody that has been modified by protein engineering techniques (eg, mutation, deletion, substitution, conjugation to a fragment other than the antibody). For example, an antibody may contain one or more variant amino acids (compared to a naturally occurring antibody) that alter a property (eg, a functional property) of the antibody. For example, numerous such changes are known in the art that affect, for example, the half-life, effector function, and/or immune responses to an antibody in a patient. The term antibody also includes artificial or engineered polypeptide constructs that contain at least one antibody-derived antigen binding site.

Antibodies against C5 (or VH/VL domains derived therefrom) suitable for use in this invention can be prepared using methods well known in the art. Alternatively, anti-C5 antibodies recognized in the art may be used. Antibodies that compete with any of these art-recognized antibodies for binding to C5 may also be used.

Eculizumab (also known as SOLIRIS®) is an anti-C5 antibody containing heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 4, 5 and 6, respectively. Eculizumab contains a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. Eculizumab variable regions are described in PCT/US1995/005688 and US Pat. No. 6355245, the contents of which are incorporated herein by reference. Eculizumab contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and a light chain having the amino acid sequence set forth in SEQ ID NO: 11. The complete heavy and light chains of eculizumab are described in PCT/US2007/006606, the contents of which are included into this document by reference.

An exemplary anti-C5 antibody is ravulizumab containing heavy and light chains having the sequences set forth in SEQ ID NO: 14 and 11, respectively, or antigen binding fragments and variants thereof. Ravulizumab (also known as ULTOMIRIS®, BNJ441 and ALXN1210) is described in PCT/US2015/019225 and US Patent No. 9079949, the contents of which are incorporated herein by reference. The terms ravulizumab, BNJ441 and ALXN1210 may be used interchangeably herein, but all refer to the same antibody. Ravulizumab selectively binds to human complement protein C5, inhibiting its cleavage to C5a and C5b during complement activation. This inhibition prevents the release of the proinflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9, while preserving the proximal or early complement activation components (e.g., C3 and C3b) required for opsonization of microorganisms and clearance of immune complexes.

In other embodiments, the antibody comprises heavy and light chain CDRs or variable regions of ravulizumab. For example, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ravulizumab having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of the VL region of ravulizumab having the sequence set forth in SEQ ID NO: 8 In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 19, 18 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID. NO: 4, 5 and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

Another exemplary anti-C5 antibody is BNJ421, which contains heavy and light chains having the sequences set forth in SEQ ID NOs: 20 and 11, respectively, or antigen binding fragments and variants thereof. BNJ421 (also known as ALXN1211) is described in PCT/US2015/019225 and US Patent No. 9079949, the contents of which are incorporated herein by reference.

In other embodiments, the antibody comprises heavy and light chain CDRs or BNJ421 variable regions. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the BNJ421 VH region having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of the BNJ421 VL region having the sequence set forth in SEQ ID NO: 8 In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 19, 18 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID. NO: 4, 5 and 6, respectively.

The exact boundaries of the CDR have been determined differently depending on different methods. In some embodiments, the positions of the CDRs or framework regions in the light or heavy chain variable domain may be as defined by Kabat et at. [(1991) "Sequences of Proteins of Immunological Interest." NIH Publication No. 91-3242, U.S. Department of Health and Human Services, Bethesda, MD]. In such cases, the CDRs may be referred to as “Kabat CDRs” (for example, “Kabat LCDR2” or “Kabat HCDR1”). In some embodiments, the CDR positions of the light and heavy chain variable regions may be as defined by Chothia et al. (1989) Nature 342:877–883. Accordingly, these regions may be referred to as “Chotiah CDRs” (eg, “Chotiah LCDR2” or “Chotiah HCDR3”). In some embodiments, the CDR positions of the light and heavy chain variable regions may be as determined by the combined Kabat-Chotia definition. In such embodiments, these regions may be referred to as "Kabat-Chotia Combined Definition CDRs." Thomas et al. [(1996) Mot Immunol 33(17/18): 1389-1401] illustrates the identification of CDR boundaries as defined by Kabat and Chotia.

In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively. In another embodiment, the antibody contains the heavy chain constant region set forth in SEQ ID NO: 13. In another embodiment, the antibody contains the heavy chain polypeptide set forth in SEQ ID NO: 14 and the light chain polypeptide set forth in SEQ ID NO: 11. In another embodiment, the antibody comprises a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn), wherein the variant human Fc constant region CH3 contains the substitutions Met-429-Leu and Asn-435-Ser at residues corresponding to methionine 428 and asparagine 434 of the Fc constant region of native human IgG, each residue numbered according to the EU numbering.

In another embodiment, the antibody comprises the heavy chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NO: 19, 18 and 3, respectively, and the light chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NO: 4, 5 and 6, respectively , and a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn), wherein the variant human Fc constant region CH3 contains substitutions Met-429-Leu and Asn-435-Ser at residues corresponding to methionine 428 and asparagine 434 Fc constant region of native human IgG, each residue numbered according to EU numbering.

In other embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR1 containing or consisting of the following amino acid sequence: GHIFSNYWIQ (SEQ ID NO: 19). In another embodiment, an anti-C5 antibody described herein comprises a heavy chain CDR2 containing or consisting of the following amino acid sequence: EILPGSGHTEYTENFKD (SEQ ID NO: 18).

In another embodiment, the antibody binds to human C5 at pH 7.4 and 25°C with an affinity dissociation constant (K _D ) that ranges from 0.1 nM to 1 nM. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25°C with a K _D ≥ 10 nM. In yet another embodiment, the antibody index [(K _{D of} an antibody or antigen binding fragment thereof for human C5 at pH 6.0 and at 25°C)/(K _{D of} an antibody or antigen binding fragment thereof for human C5 at pH 7.4 and at 25 °C)] is more than 25.

Another exemplary anti-C5 antibody is antibody 7086, described in US Pat. No. 8,241,628 and US Pat. No. 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of antibody 7086 (see US Pat. No. 8,241,628 and US Pat. No. 8,883,158). In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 21, 22 and 23, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 24, 25 and 26, respectively. In another embodiment, the antibody or antigen binding fragment thereof comprises a VH region of antibody 7086 having the sequence set forth in SEQ ID NO: 27 and a VL region of antibody 7086 having the sequence set forth in SEQ ID NO: 28.

Another exemplary anti-C5 antibody is antibody 8110, also described in US Pat. No. 8,241,628 and US Pat. No. 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of antibody 8110. In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 29, 30 and 31 , respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 32, 33 and 34, respectively. In another embodiment, the antibody comprises the VH region of antibody 8110 having the sequence set forth in SEQ ID NO: 35 and the VL region of antibody 8110 having the sequence set forth in SEQ ID NO: 36.

Another exemplary anti-C5 antibody is the 305LO5 antibody described in US 2016/0176954 A1. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody. In another embodiment, the antibody or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 37, 38 and 39, respectively, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively. In another embodiment, the antibody comprises a 305LO5 antibody VH region having the sequence set forth in SEQ ID NO: 43 and a 305LO5 antibody VL region having the sequence set forth in SEQ ID NO: 44.

Another exemplary anti-C5 antibody is the SKY59 antibody described in Fukuzawa T., et at, Rep. 2017 Apr 24;7(1): 1080). In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody. In another embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46.

Another exemplary anti-C5 antibody is antibody REGN3918 (also known as H4H12166PP) described in US 20170355757. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 47. 48. In another embodiment, the antibody comprises a heavy chain comprising SEQ ID NO: 49 and a light chain comprising SEQ ID NO: 50.

In another embodiment, the antibody competes for binding and/or binds to the same epitope on C5 as the above antibodies (e.g., eculizumab, ravulizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918). In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the aforementioned antibodies (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% variable region identity).

An anti-C5 antibody described herein may, in some embodiments, comprise a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn) with greater affinity than the affinity of the native human Fc constant region from which the variant constant region is derived. human Fc region. For example, the Fc constant region may contain one or more (eg, two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant Fc constant region was derived. Substitutions can increase the binding affinity of an IgG antibody containing a variant Fc constant region to FcRn at pH 6.0 while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art and are presented in the Experimental Examples. See, for example, PCT/US2015/019225 and US Patent No. 9079949, the disclosures of each of which are incorporated herein by reference in their entirety.

Substitutions that increase the binding affinity of the Fc constant region of an antibody to FcRn are known in the art and include, for example: (1) the M252Y/S254T/T256E triple substitution described in Dall' Acqua et al. (2006) J Biol Chem 281: 23514–23524; (2) M428L or T250Q/M428L substitutions described in Hinton et al. (2004) J Biol Chem 279:6213–6216 and Hinton et al. (2006) J Immunol 176:346–356; and (3) substitutions N434A or T307/E380A/N434A, described in Petkova et al. (2006) Int Immunol 18f 12): 1759-69. Additional pairs of substitutions - P257I/Q311I, P257I/N434H and D376V/N434H - are described, for example, in the work of Datta-Mannan et al. (2007) J Biol Chem 282(3): 1709-1717, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the variant constant region has a substitution at amino acid residue 255, numbered according to EU numbering, to valine. In some embodiments, the variant constant region has a substitution at amino acid residue 309, numbered according to EU numbering, to asparagine. In some embodiments, the variant constant region has a substitution at amino acid residue 312, numbered according to the EU numbering, to isoleucine. In some embodiments, the variant constant region has a substitution at amino acid residue 386 numbered according to EU numbering.

In some embodiments, the variant Fc constant region contains no more than 30 (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, nine, eight, seven, six, five, four, three or two) amino acid substitutions, insertions or deletions relative to the native constant region from which it was derived. In some embodiments, the variant Fc constant region contains one or more amino acid substitutions selected from the group consisting of: M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the human Fc variant constant region contains a methionine at position 428 and an asparagine at position 434, each residue numbered according to EU numbering. In some embodiments, the variant Fc constant region contains a double 428L/434S substitution, as described, for example, in US Pat. No. 8,088,376.

In some embodiments, the exact location of these mutations may be shifted from the position of the native human Fc constant region due to antibody engineering. For example, the 428L/434S double substitution when used in a chimeric IgG2/4 Fc may correspond to 429L and 435S, as in the M429L and N435S variants found in BNJ441 (ravulizumab) and described in US Pat. No. 9,079,949, the disclosure of which is incorporated herein by reference in full.

In some embodiments, the variant constant region contains a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 or 43 6 ( EU numbering) relative to the native human Fc constant region. In some embodiments, the substitution is selected from the group consisting of the following: methionine to glycine at position 237; alanine to proline at position 238; lysine on the series at position 239; isoleucine to lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine for threonine at position 250; phenylalanine, tryptophan or tyrosine to methionine at position 252; threonine on the series at position 254; glutamic acid to arginine at position 255; aspartic acid, glutamic acid or glutamine to threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine or valine to proline at position 257; histidine to glutamic acid at position 258; alanine to aspartic acid at position 265; phenylalanine to aspartic acid at position 270; alanine or glutamic acid to asparagine at position 286; histidine to threonine at position 289; alanine to asparagine at position 297; glycine on the series at position 298; alanine to valine at position 303; alanine to valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, valine, tryptophan or tyrosine to threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine or threonine to valine at position 308; alanine, aspartic acid, glutamic acid, proline or arginine to leucine or valine at position 309; alanine, histidine or isoleucine to glutamine at position 311; alanine or histidine to aspartic acid at position 312, lysine or arginine to leucine at position 314; alanine or histidine to asparagine at position 315; alanine to lysine at position 317; glycine to asparagine at position 325; valine to isoleucine at position 332; leucine to lysine at position 334; histidine to lysine at position 360; alanine to aspartic acid at position 376; alanine to glutamic acid at position 380; alanine to glutamic acid at position 382; alanine to asparagine or series at position 384; aspartic acid or histidine to glycine at position 385; proline to glutamine at position 386; glutamic acid to proline at position 387; alanine or series on asparagine at position 389; Alanine on the series at position 424; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan or tyrosine to methionine at position 428; lysine to histidine at position 433; alanine, phenylalanine, histidine, series, tryptophan or tyrosine to asparagine at position 434; and histidine to tyrosine or phenylalanine at position 436, each residue numbered according to EU numbering.

Suitable anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and/or a light chain polypeptide containing the amino acid sequence set forth in SEQ ID NO: 11. Alternatively, anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a heavy chain polypeptide containing the amino acid sequence set forth in SEQ ID NO: 20 and/or a light chain polypeptide containing amino acid sequence shown in SEQ ID NO: 11.

In one embodiment, the antibody binds to C5 at pH 7.4 and 25°C (and otherwise under physiological conditions) with an affinity dissociation constant (K _D ) that is at least 0.1 (e.g., at least at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55 , 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95 or 0.975 ) nM. In some embodiments, the K _D of the anti-C5 antibody or antigen-binding fragment thereof is no more than 1 (e.g., no more than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0. 3 or 0.2) nM.

In other embodiments, the [( _KD antibody for C5 at pH 6.0 at C)/( _KD antibody for C5 at pH 7.4 at 25°C)] is greater than 21 (e.g., greater than 22, 23 , 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, PO, 120, 130, 140 , 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 , 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500 or 8000).

Methods for determining whether an antibody binds to a protein antigen and/or determining the affinity of an antibody for a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantified using various techniques, such as, but not limited to: Western blotting, dot blotting, surface plasmon resonance (SPR) (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey, USA), or enzyme-linked immunosorbent assay (ELISA). See, for example, Benny K. S. Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); John et al. (1993) J Immunol Meth 160:191–198; Jonson et al. (1993) Ann Biol Clin 51:19–26; and Jonsson et al. (1991) Biotechniques 11:620-627. In addition, methods for measuring affinity (eg, dissociation and association constants) are presented in the experimental examples.

As used herein, the term "k _a " refers to the rate constant of association of an antibody with an antigen. The term "k _d " refers to the rate constant for the dissociation of an antibody from an antibody/antigen complex. And the term “K _D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction. The equilibrium dissociation constant is derived from the relationship between the kinetic rate constants: K _D = k _a /k _d . Such determinations are preferably carried out at 25°C or 37°C (see examples regarding the experimental part). For example, antibody binding kinetics to human C5 can be determined at pH 8.0, 7.4, 7.0, 6.5 and 6.0 by surface plasmon resonance (SPR) on a BIAcore 3000 instrument using the anti-Fc method capture to immobilize the antibody.

In one embodiment, an anti-C5 antibody or antigen-binding fragment thereof blocks the formation or activity of the active C5a and/or C5b fragments of the C5 protein (eg, human C5 protein). Due to this blocking effect, antibodies suppress, for example, the pro-inflammatory effects of C5a and the formation of the membrane attack complex (MAC) C5b-9 on the cell surface.

Methods for determining whether a particular antibody described herein inhibits C5 cleavage are known in the art. Inhibition of human complement component C5 may reduce the ability of complement to lyse cells in a subject's body fluids. This reduction in the ability of complement present in the body fluid(s) to lyse cells can be measured by methods well known in the art, such as, for example, a conventional hemolysis test, such as the hemolysis assay described in Kabat and Mayer (eds. ), "Experimental Immunochemistry, ^2nd Edition," 135-240, Springfield, IL, C. C. Thomas (1961), pages 135-139, or a conventional variant of this assay, such as the chicken erythrocyte hemolysis assay as described, for example, in eg , Hillman et al. (2004) N Engl J Med 350(6): 552. Methods for determining whether a candidate compound inhibits the cleavage of human C5 into the C5a and C5b forms are known in the art and are described in Evans et al. (1995) Mol Immunol 32(16): 1183–95. For example, the concentration and/or physiological activity of C5a and C5b in a body fluid can be measured by methods well known in the art. For C5b, hemolysis assays or soluble C5b-9 assays can be used, as discussed herein. Other assays known in the art may also be used. Using these or other suitable types of assays, candidate agents capable of inhibiting human complement component C5 can be screened.

Immunological techniques, such as, but not limited to, ELISA, can be used to measure the concentration of C5 protein and/or its degradation products to determine the ability of an anti-C5 antibody or antigen-binding fragment thereof to inhibit the conversion of C5 to biologically active products. In some embodiments, the formation of C5a is measured. In some embodiments, C5b-9 neo-epitope-specific antibodies are used to detect terminal complement formation.

Hemolysis assays can be used to determine the inhibitory activity of an anti-C5 antibody or its antigen-binding fragment upon complement activation. To determine the effect of an anti-C5 antibody or its antigen-binding fragment on hemolysis mediated by the classical complement pathway in a serum solution for in vitro testing, the target cells are, for example, hemolysin-coated sheep red blood cells or hemolysin-coated chicken red blood cells sensitized with an anti-red blood cell antibody. Chicken. The percentage of lysis is normalized by considering 100% lysis to be equal to the lysis occurring in the absence of the inhibitor. In some embodiments, the classical pathway of complement is activated by a human IgM antibody, for example, as used in the Classical Pathway Complement Kit from Wieslab® (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with an anti-C5 antibody or an antigen-binding fragment thereof in the presence of a human IgM antibody. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorogenic substrate and measuring the absorbance at the appropriate wavelength. As a control, the test serum is incubated in the absence of anti-C5 antibody or its antigen-binding fragment. In some embodiments, the test blood serum is C5-deficient blood serum reconstituted with a C5 polypeptide.

To determine the effect of an anti-C5 antibody or antigen-binding fragment thereof on hemolysis mediated by the alternative complement pathway, unsensitized rabbit or guinea pig erythrocytes can be used as target cells. In some embodiments, the serum solution tested is C5-deficient serum reconstituted with a C5 polypeptide. The percentage of lysis is normalized by considering 100% lysis to be equal to the lysis occurring in the absence of the inhibitor. In some embodiments, the alternative pathway of complement is activated by lipopolysaccharide molecules, for example, as used in the Alternative Pathway Complement Kit from Wieslab® (Wieslab® COMPL AP330, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with an anti-C5 antibody or an antigen-binding fragment thereof in the presence of lipopolysaccharide. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorogenic substrate and measuring the fluorescence at the appropriate wavelength. As a control, the test serum is incubated in the absence of anti-C5 antibody or its antigen-binding fragment.

In some embodiments, C5 activity or inhibition thereof is quantified using a CH50eq assay. The CH50eq assay is a method of measuring the overall activity of the classical complement pathway in serum. This lytic test is an assay that uses antibody-sensitized red blood cells as an activator of the classical complement pathway and various dilutions of the test serum to determine the amount required for 50% lysis (CH50). The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect assessment of terminal complement complex (TCC) formation, since the TCC itself is directly responsible for the hemolysis measured.

This analysis is well known and widely practiced by those skilled in the art. Briefly, to activate the classical complement pathway, undiluted blood serum samples (eg, reconstituted human serum samples) are added to microassay wells containing antibody-coated red blood cells to thereby generate TCC. The activated blood sera are then diluted into microassay wells that are coated with a capture reagent (eg, an antibody that binds to one or more TCC components). The TCC present in the activated samples binds to the monoclonal antibodies coating the surface of the microassay wells. The wells are washed and a detectably labeled detection reagent that recognizes bound TCC is added to each well. The detectable label may be, for example, a fluorescent label or an enzyme label. The results of this analysis are expressed in units of CH50 equivalents per milliliter (CH50, Ueq/mL).

Inhibition, for example, with respect to terminal complement activity includes at least 5 (e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, A 50, 55, or 60)% reduction in terminal complement activity, such as in a hemolysis assay or CH50eq assay, compared with the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and at an equimolar concentration. Significant inhibition, as that term is used herein, refers to inhibition of a given activity (e.g., terminal complement activity) by at least 40 (e.g., at least 45, 50, 55, 60, 65, 70, 75, 80 , 85, 90 or 95 or more)%. In some embodiments, the anti-C5 antibody described herein contains one or more amino acid substitutions relative to the eculizumab CDR (i.e., SEQ ID NOs: 1-6) but retains at least 30 (e.g., at least 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80 , 85, 90, or 95)% of the complement inhibitory activity of eculizumab in the hemolysis assay or CH50eq assay.

The anti-C5 antibody described herein has a half-life in human serum that is at least 20 (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55) days. In another embodiment, an anti-C5 antibody described herein has a half-life in human serum that is at least 40 days. In another embodiment, an anti-C5 antibody described herein has a half-life in human serum of about 43 days. In another embodiment, an anti-C5 antibody described herein has a half-life in human serum of 39-48 days. Methods for measuring serum half-life are known in the art. In some embodiments, an anti-C5 antibody or antigen-binding fragment thereof described herein has a serum half-life that is at least 20 (e.g., at least 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500)% greater than the serum half-life of eculizumab, for example, as measured in one of mouse model systems described in the examples relating to the experimental part (eg, C5-deficient/NOD/scid mouse or hFcRn transgenic mouse model system).

In one embodiment, the antibody competes for binding and/or binds to the same epitope on C5 as the antibodies described herein. The term “binds to the same epitope” when applied to two or more antibodies means that the antibodies bind to the same segment of amino acid residues as determined by this method. Techniques for determining whether antibodies bind to the “same epitope on C5” for the antibodies described herein include, for example, epitope mapping techniques such as crystal X-ray crystal analysis of antigen:antibody complexes, which provides atomic resolution of the epitope, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Other methods examine antibody binding to fragments of a peptide antigen or mutated variations of the antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered to be indicative of a component of the epitope. Additionally, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from phage display peptide combinatorial libraries. Antibodies having the same VH and VL sequences or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of another antibody to a target. Do two antibodies compete with each other for binding to the target, i.e. whether and to what extent one antibody inhibits the binding of another antibody to its target can be determined using known competitive binding experiments. In certain embodiments, the antibody competes with another antibody and inhibits its binding to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is the “blocking antibody” (ie, the unlabeled antibody that is first incubated with the target). Competing antibodies bind to the same epitope, overlapping epitope, or to adjacent epitopes (eg, as evidenced by steric hindrance).

The anti-C5 antibodies or antigen binding fragments thereof described herein used in the methods described herein can be prepared using a variety of techniques recognized in the art. Monoclonal antibodies can be produced using a variety of techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with the desired antigen are immortalized, usually by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for the production of antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells can be increased by various techniques, including injection into the peritoneal cavity of the vertebrate host. Alternatively, DNA sequences that encode a monoclonal antibody or a binding fragment thereof can be isolated by screening a DNA library from human B cells according to the general protocol set forth in Huse, et al., Science 246: 1275-1281 (1989) .

II. Compositions

Also provided herein are compositions comprising an anti-C5 antibody or an antigen-binding fragment thereof. In one embodiment, the composition comprises an anti-C5 antibody comprising CDR1, CDR2 and CDR3 domains in a heavy chain variable region having the sequence set forth in SEQ ID NO: 12. and CDR1, CDR2 and CDR3 domains in a light chain variable region having the sequence set forth in SEQ ID NO: 8. In another embodiment, the anti-C5 antibody comprises a heavy and light chain having the sequence set forth in SEQ ID NO: 14 and 11, respectively. In another embodiment, the anti-C5 antibody contains heavy and light chains having the sequences set forth in SEQ ID NO: 20 and 11, respectively.

The compositions may be formulated as a pharmaceutical solution, for example, for administration to a subject for the treatment or prevention of a complement disorder such as aHUS. The pharmaceutical compositions typically contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersing media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions may contain a pharmaceutically acceptable salt, for example, an acid addition salt or a base addition salt, sugars, carbohydrates, polyols and/or tonicity modifiers.

Compositions can be composed using standard methods. Pharmaceutical composition is a well-established art and is further described, for example, in Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems," 7th Edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and in Kibbe (2000) "Handbook of Pharmaceutical Excipients American Pharmaceutical Association," 3rd Edition (ISBN: 091733096X). In some embodiments, the composition may be formulated, for example, as a buffered solution of suitable concentration and suitable for storage at 2-8°C (eg, 4°C). In some embodiments, the composition may be formulated for storage at temperatures below 0°C (eg, -20°C or -80°C). In some embodiments, the composition may be formulated for storage for a period of up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1½ years or 2 years) at 2-8°C (for example, 4°C). Thus, in some embodiments, the compositions described herein are stable when stored for at least 1 year at 2-8°C (eg, 4°C).

Pharmaceutical compositions can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg, injections and infusions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends in part on the intended route of administration and therapeutic use. For example, compositions containing a composition intended for systemic or local delivery may be in the form of injections or infusions. Accordingly, the compositions may be formulated for administration by a parenteral route (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular injection). “Parenteral administration,” “administer parenterally,” and other grammatically equivalent phrases used herein refer to routes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, the following: intravenous , intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, tracarotid and intrasternal injections and infusion.

In one embodiment, the composition contains ravulizumab (also known as ULTOMIRIS®, antibody BNJ441 or ALXN1210) for injection. In one embodiment, the injection is a sterile, clear to translucent, slightly off-white, preservative-free solution for intravenous use. In another embodiment, each single dose vial contains 300 mg of ravulizumab injection at a concentration of 10 mg/ml with a pH of 7.0. In another embodiment, ravulizumab injection requires dilution to a final concentration of 5 mg/ml. In another embodiment, each ml further contains polysorbate 80 (0.2 mg) (vegetable source), sodium chloride (8.77 mg), dibasic sodium phosphate (1.78 mg), monobasic sodium phosphate (0.46 mg) and water for injections.

III. Treatment options

Provided herein are methods of treating aHUS in a human patient, comprising administering to said patient an anti-C5 antibody or an antigen-binding fragment thereof, wherein said anti-C5 antibody or antigen-binding fragment thereof is administered (or is intended to be administered) in accordance with a specific clinical regimen. (i.e., in a specific dose quantity and according to a specific dosing schedule).

In the context of this document, the terms “induction” and “induction phase” are used interchangeably and refer to the first phase of treatment in a clinical trial.

In the context of this document, the terms “maintenance” and “maintenance phase” are used interchangeably and refer to the second phase of treatment in a clinical trial. In certain embodiments, treatment is continued until clinical improvement is observed or until uncontrolled toxicity or disease progression occurs.

As used herein, the term “subject” or “patient” means a human patient (eg, a patient having a complement disorder). In one embodiment, the complement disorder is atypical hemolytic uremic syndrome (aHUS). Pathology and clinical manifestations in patients with aHUS are also caused by activation of terminal complement. More specifically, C5 activation and dysregulation of complement activation lead to endothelial damage, platelet consumption, and thrombotic microangiopathic (TMA) events characterized by thrombocytopenia, mechanical intravascular hemolysis, and renal injury. It is important to note that approximately 20% of patients also experience extrarenal manifestations of the disease, including manifestations in the central nervous system, heart, gastrointestinal tract, distal extremities, and severe systemic organ involvement (Loirat, et al., Orphanet. J Rare Dis. 2011; 6:60). Symptoms of aHUS are well known to those skilled in rare disease or kidney disease medicine and include, for example, severe hypertension, proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia, and impaired renal function (eg, acute renal failure).

aHUS can be hereditary, acquired, or idiopathic. aHUS may be considered hereditary when two or more (eg, three, four, five, or six, or more) members of the same family are affected by the disease at least six months apart and exposure to a common precipitating agent has been excluded, or when a subject has one or more mutations identified in a gene associated with aHUS (eg, one or more mutations in CFH, MCP/CD46, CFB, or CFI). For example, a subject may have CFH-associated aHUS, CFB-associated aHUS, CFI-associated aHUS, or MCP-associated aHUS. Up to 30% of familial aHUS is associated with mutations in CFH, 12% with mutations in MCP, 5-10% with mutations in CFI and less than 2% - with mutations in CFB. Hereditary aHUS can be multiplex (i.e., familial; when two or more family members have the disease) or simplex (i.e., occurring once in a family). aHUS can be considered acquired when an environmental agent (eg, drug, systemic disease, or viral agents or bacterial agents that do not produce Shiga-like exotoxins) or a trigger can be identified. aHUS can be considered idiopathic when there is no obvious trigger (genetic or environmental).

Laboratory tests may be performed to determine whether the human subject has thrombocytopenia, microangiopathic hemolytic anemia, or acute renal failure. Thrombocytopenia may be diagnosed by a health care professional as one or more of the following: (i) a platelet count that is less than 150,000/mm ³ (eg, less than 60,000/ ^{mm 3} ); (ii) decreased platelet survival time, reflecting increased platelet destruction in the bloodstream; and (iii) giant platelets observed on the peripheral blood smear, consistent with secondary activation of thrombocytopoiesis. Microangiopathic hemolytic anemia can be diagnosed by a health care professional as one or more of the following: (i) hemoglobin concentrations that are less than 10 mg/dL (eg, less than 6.5 mg/dL); (ii) elevated serum lactate dehydrogenase (LDH) concentrations (>460 U/L); (iii) hyperbilirubinemia, reticulocytosis, circulating free hemoglobin, and low or undetectable haptoglobin concentrations; and (iv) detection of fragmented red blood cells (schistocytes) with typical features of poikilocytes with styloid processes or helmeted red blood cells on a peripheral blood smear in combination with a negative Coombs test. See, for example, Kaplan et al. (1992) "Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura," Informa Health Care (ISBN 0824786637), and Zipfel (2005) "Complement and Kidney Disease," Springer (ISBN 3764371668). Blood concentrations of C3 and C4 can also be used as a measure of complement activation or dysregulation. In addition, the subject's condition may be further characterized by identifying the subject as a carrier of one or more mutations in a gene associated with aHUS, such as CFI, CFB, CFH, or MCP (see above). Suitable methods for detecting a mutation in a gene include, for example, DNA sequencing techniques and multiplex nucleic acid analysis techniques. See, for example, Breslin et al. (2006) Clin Am Soc Nephrol 1:88–99, and Goicoechea de Jorge et al. (2007) P'roc Natl Acad Sci USA 104:240–245.

As used herein, the term “effective treatment” refers to treatment that results in a beneficial effect, such as alleviation of at least one symptom of a disease or disorder. The positive effect may take the form of an improvement over baseline, i.e. improvements compared to the measurement or observation made before the start of therapy in accordance with the specified method. In the context of aHUS, for example, effective treatment may refer to the relief of one or more symptoms selected from the group consisting of severe hypertension, proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia, and/or renal dysfunction (eg , acute renal failure)).

The term "effective amount" refers to an amount of agent that provides a desired biological, therapeutic and/or prophylactic result. This result may be reduction, alleviation, temporary attenuation, reduction, delay and/or mitigation of one or more manifestations, symptoms or causes of a disease , or any other desired change in a biological system. In one example, an “effective amount” is that amount of an anti-C5 antibody or antigen-binding fragment thereof that is clinically proven to relieve at least one symptom of aHUS (eg, severe hypertension, proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia and renal dysfunction (eg, acute renal failure)). An effective amount can be administered through one or more administrations.

In one embodiment, the dose of the anti-C5 antibody or antigen-binding fragment thereof is based on the patient's body weight. For example, in one embodiment, 2400 mg or 3000 mg of an anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥40 to <60 kg. In another embodiment, 2700 mg or 3300 mg of an anti-C5 antibody or antigen binding fragment thereof is administered to a patient weighing ≥60 to <100 kg. In another embodiment, 3000 mg or 3600 mg of an anti-C5 antibody or antigen binding fragment thereof is administered to a patient weighing ≥ 100 kg. In certain embodiments, administration regimens are selected to provide the optimal desired response (eg, an effective response).

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered in one or more administration cycles. In one embodiment, the administration cycle is 26 weeks. In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered once on day 1 of the administration cycle, once on day 15 of the administration cycle, and every eight weeks thereafter. In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered every eight weeks after the dosing cycle during an extension period of up to and including two years (eg, at a dose of 3000 mg, 3300 mg, or 3600 mg).

In another embodiment, a method of treating a human patient with aHUS is provided, the method comprising administering to said patient an effective amount of an anti-C5 antibody or antigen binding fragment thereof comprising the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 19. 18 and 3, respectively, and the light chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NOs: 4, 5 and 6, respectively, wherein said anti-C5 antibody or antigen binding fragment thereof is administered:

(a) once on the 1st day at a dose of: 2400 mg to a patient weighing ≥ 40 to < 60 kg, 2700 mg to a patient weighing ≥ 60 to < 100 kg, or 3000 mg to a patient with body weight ≥ 100 kg; And

(b) on the 15th day and thereafter - every eight weeks at a dose equal to:

3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg for a patient weighing ≥ 100 kg.

In another embodiment, a method is provided for treating a human patient with aHUS, the method comprising administering to the patient (e.g., during an administration cycle) an effective amount of an anti-C5 antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2, and CDR3 sequences, indicated by SEQ ID NOs: 19, 18 and 3, respectively, the light chain CDR1, CDR2 and CDR3 sequences indicated by SEQ ID NOs: 4, 5 and 6, respectively, and a variant human Fc constant region that binds to the neonatal human Fc receptor (FcRn), wherein the variant constant region CH3 of human Fc contains substitutions Met-429-Leu and Asn-435-Ser in residues corresponding to methionine 428 and asparagine 434 of the native constant region Fc of human IgG, each residue is numbered according to EU numbering, while said anti-C5 antibody or antigen-binding fragment thereof is administered:

(a) once on the 1st day at a dose of: 2400 mg to a patient weighing ≥ 40 to < 60 kg, 2700 mg to a patient weighing ≥ 60 to < 100 kg, or 3000 mg to a patient with body weight ≥ 100 kg; And

(b) on the 15th day and thereafter - every eight weeks at a dose equal to:

3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg for a patient weighing ≥ 100 kg.

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥40 to <60 kg:

(a) once on the 1st day at a dose of: 2400 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg.

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥60 to <100 kg:

(a) once on the 1st day at a dose of: 2700 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3300 mg.

In another embodiment, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥ 100 kg:

(a) once on the 1st day at a dose of: 3000 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3600 mg.

In some embodiments, the patient has not previously received treatment with a complement inhibitor (eg, the patient is one who has not previously received treatment with a complement inhibitor).

In other embodiments, the patient was previously treated with one anti-C5 antibody or antigen-binding fragment thereof and was then switched to another anti-C5 antibody or antigen-binding fragment thereof during the course of treatment. For example, in certain embodiments, various anti-C5 antibodies are administered during a course of treatment. In one embodiment, different anti-C5 antibodies are administered during separate treatment and extension periods. For example, in one embodiment, a patient is treated with eculizumab during a treatment period (eg, 26 weeks), followed by treatment with another anti-C5 antibody (eg, ravulizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918 ), for example during the renewal period. In another embodiment, eculizumab is administered to the patient at a dose of 600 mg on days 1, 8, 15, and 22 of the dosing cycle during the induction phase, followed by a maintenance dose of 900 mg of eculizumab on day 19 day of the administration cycle and every two weeks thereafter (eg, for a total of 26 weeks), followed by treatment with ravulizumab for an extension period of up to two years. In another embodiment, the patient is treated with ravulizumab (eg, for 26 weeks), followed by treatment with another anti-C5 antibody (eg eculizumab, 7086 antibody, 8110 antibody, 305LO5 antibody, SKY59 antibody or REGN3918 antibody) for, for example, an extension period.

Exemplary alternative anti-C5 antibodies include, but are not limited to: (i) ravulizumab, (ii) an antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2 and CDR3 domains containing SEQ ID NOs: 21, 22 and 23, respectively , and light chain CDR1, CDR2 and CDR3 domains comprising SEQ ID NO: 24, 25 and 26, respectively, (iii) an antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region chain containing SEQ ID NO: 28, (iv) an antibody or antigen binding fragment thereof containing the heavy chain CDR1, CDR2 and CDR3 domains containing SEQ ID NO: 29, 30 and 31, respectively, and the light chain CDR1, CDR2 and CDR3 domains containing SEQ ID NO: 32, 33 and 34, respectively, (v) an antibody or antigen binding fragment thereof, comprising a heavy chain variable region containing SEQ ID NO: 35 and a light chain variable region containing SEQ ID NO: 36, ( vi) an antibody or antigen binding fragment thereof comprising heavy chain CDR1, CDR2 and CDR3 domains containing SEQ ID NOs: 37, 38 and 39, respectively, and light chain CDR1, CDR2 and CDR3 domains containing SEQ ID NOs: 40, 41 and 42, respectively, (vii) an antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 43 and a light chain variable region comprising SEQ ID NO: 44, (viii) an antibody or antigen binding fragment thereof comprising a heavy a chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46, respectively, (vii) an antibody or an antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region, comprising SEQ ID NO: 48, (x) an antibody or antigen binding fragment thereof, comprising a heavy chain containing SEQ ID NO: 49 and a light chain containing SEQ ID NO: 50.

In some embodiments, the patient has previously received treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months or at least 24 months with an anti-C5 antibody or antigen-binding fragment thereof (eg, eculizumab) before switching to another anti-C5 antibody or antigen-binding fragment (eg, ravulizumab). In a specific embodiment, the patient has previously received treatment with eculizumab for at least 6 months.

In another embodiment, when a patient (eg, a patient with aHUS) is treated with a first anti-C5 antibody and is then switched to treatment with a second different anti-C5 antibody, especially when said second different anti-C5 antibody binds to an epitope on C5 different from the epitope , to which said first anti-C5 antibody binds, the administration schedules take into account the half-life of said first anti-C5 antibody. For example, to ensure that the first anti-C5 antibody is cleared (e.g., "washed out") from the patient before the second (other) anti-C5 antibody is administered (e.g., to avoid problems associated with aggregation, immune complex formation, etc. ), the half-life of said first anti-C5 antibody is taken into account. In one embodiment, the second (other) anti-C5 antibody is not administered until a period of time corresponding to 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 has passed. 6.5, 7 or 7.5 half-life of the first anti-C5 antibody after the final administration of said first anti-C5 antibody.

In another embodiment, the patient was previously treated with eculizumab and was then switched to treatment with a second (different) anti-C5 antibody (eg, ravulizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918). In one embodiment, when the first antibody administered is eculizumab, the second (other) anti-C5 antibody is not administered, for example, until at least 36, 45, 54, 63, 72, 81, 90, 99 , 108, 117 or 126 days after the final administration of eculizumab.

In another embodiment, the patient was previously treated with ravulizumab and then switched to treatment with another anti-C5 antibody (eg, eculizumab, antibody 7086, antibody 8110, antibody 305LO5, antibody SKY59, or antibody REGN3918). In one embodiment, when the first antibody administered is ravulizumab, the second (other) anti-C5 antibody is not administered, for example, until at least 100, 125, 150, 175, 200, 225, 250, 275 , 300, 325, 375 or 400 days after the final administration of ravulizumab.

As an adjunct or alternative, techniques are used to remove or enhance the clearance of the first anti-C5 antibody before proceeding to treatment with a second (other) anti-C5 antibody. Exemplary techniques include, but are not limited to, plasmapheresis or blood transfusions. In another embodiment, an antibody against a first anti-C5 antibody is administered to clear or enhance the clearance of said first anti-C5 antibody (e.g., anti-eculizumab antibody, anti-ravulizumab antibody, anti-7086 antibody, anti-8110 antibody, anti-305LO5 antibody, anti-SKY59 antibody, or anti- REGN3918) before administration of the second (different) anti-C5 antibody.

In another embodiment, an anti-C5 antibody or antigen-binding fragment thereof (eg, ravulizumab) is administered to a patient, wherein the administration cycle begins after at least about two weeks, at least about three weeks, at least about four weeks, at least about six weeks, at least about seven weeks, or at least about eight weeks after the final dose of eculizumab is administered to said patient. In another embodiment, an anti-C5 antibody or antigen-binding fragment thereof (eg, ravulizumab) is administered to a patient, with treatment (eg, an administration cycle) beginning at least two weeks after the final dose of eculizumab is administered to said patient.

In some embodiments, patients treated in accordance with the methods described herein have been vaccinated against meningococcal diseases within 3 years before or at the time of initiation of treatment. In one embodiment, patients who received treatment less than 2 weeks after receiving the meningococcal vaccine also receive appropriate prophylactic antibiotics for 2 weeks after vaccination. In yet another embodiment, patients receiving treatment in accordance with the methods described herein are vaccinated against meningococcal serotypes A, C, Y, W135 and/or B.

As used herein, the term “residual serum level” refers to the lowest level at which a given agent (eg, anti-C5 antibody or antigen-binding fragment thereof) or drug is present in serum. In contrast, “peak serum level” refers to the highest serum level of a given agent. “Mean serum level” refers to the average serum level of a given agent over time.

In one embodiment, the described treatment regimens are sufficient to maintain certain residual serum concentrations of the anti-C5 antibody or antigen-binding fragment thereof. For example, in one embodiment, treatment maintains a residual serum concentration of anti-C5 antibody or antigen-binding fragment thereof at 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 240, 25, 0, 255, 260, 265, 270, 280, 290, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 0, 395 or 400 mcg/ml or more. In one embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 100 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 150 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 200 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 250 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at 300 μg/ml or greater. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at a level of 100 μg/ml to 200 μg/ml. In another embodiment, the treatment maintains a residual serum concentration of the anti-C5 antibody or antigen-binding fragment thereof at about 175 μg/ml.

In another embodiment, to obtain an effective response, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain at least 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg , 95 mcg, 100 mcg, 105 mcg, 110 mcg, 115 mcg, 120 mcg, 125 mcg, 130 mcg, 135 mcg, 140 mcg, 145 mcg, 150 mcg, 155 mcg, 160 mcg, 165 mcg, 170 mcg, 175 µg, 180 µg, 185 µg, 190 µg 195 µg, 200 µg, 205 µg, 210 µg, 215 µg, 220 µg, 225 µg, 230 µg, 235 µg, 240 µg, 245 µg, 250 µg, 255 µg or 260 micrograms of antibody per milliliter of patient blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain between 50 μg and 250 μg of antibody per milliliter of the patient's blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain 100 μg to 200 μg of antibody per milliliter of the patient's blood. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain about 175 μg of antibody per milliliter of the patient's blood.

In another embodiment, to obtain an effective response, the anti-C5 antibody is administered to the patient in an amount and frequency that provides a minimum concentration of free C5. For example, in one embodiment, an anti-C5 antibody is administered to a patient in an amount and frequency to maintain a free C5 concentration of 0.2 μg/mL, 0.3 μg/mL, 0.4 μg/mL, 0.5 μg/mL. ml or lower. In another embodiment, the anti-C5 antibody is administered to the patient in an amount and frequency to maintain a free C5 concentration of 0.309 μg/mL to 0.5 μg/mL or lower. In another embodiment, the treatment described herein reduces the concentration of free C5 by more than 99% during the treatment period. In another embodiment, the treatment reduces the concentration of free C5 by more than 99.5% during the treatment period.

IV. results

Provided herein are methods of treating aHUS in a patient, comprising administering to said patient an anti-C5 antibody or an antigen-binding fragment thereof.

Symptoms of aHUS include, but are not limited to, severe hypertension, proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia, and impaired renal function (eg, acute renal failure). Patients treated in accordance with the methods described herein preferably experience improvement in at least one of the manifestations of aHUS.

In other embodiments, the treatment results in inhibition of terminal complement.

In other embodiments, treatment causes a shift toward normal levels of a hemolysis-related hematologic biomarker selected from the group consisting of free hemoglobin, haptoglobin, reticulocyte count, PNH red cell clone, and D-dimer.

In another embodiment, the treatment causes an increase in hemoglobin stabilization compared to the patient's pre-treatment baseline level. In another embodiment, treatment results in an increase in hemoglobin levels of ≥20 g/L. In another embodiment, the treatment results in the prevention of a decrease in hemoglobin levels of ≥ 2 g/dL from baseline in the absence of transfusion from baseline to day 183.

In other embodiments, treatment results in normalization of platelet levels (≥ 150 x 10 ⁹ /L). In other embodiments, treatment results in normalization of platelet levels (≥ 150 x 10 ⁹ /L) for a period of at least 28 days (for example, at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in normalization of LDH levels (≤246 U/L). In other embodiments, treatment results in normalization of LDH levels (≤246 U/L) for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in an improvement in serum creatinine levels of ≥25% over baseline. In other embodiments, treatment results in an improvement in serum creatinine levels of ≥25% above baseline for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in a complete TMA response (i.e., platelet count normalization (≥150 x 10/L), LDH normalization (≤246 U/L), and serum creatinine level improvement ≥25% compared to initial level). In other embodiments, treatment results in a complete TMA response for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, treatment results in a modified complete TMA response (ie, normalization of platelet levels (≥150 x ¹⁰⁹ /L), normalization of LDH levels (≤246 U/L), and the patient stops dialysis if he or she was on dialysis at baseline, or an improvement in serum creatinine ≥ 25% above baseline for a patient who was not on dialysis at baseline). In other embodiments, treatment results in a modified TMA complete response for a period of at least 28 days (e.g., at least 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or two years).

In other embodiments, the treatment reduces the need for blood transfusions. In another embodiment, treatment results in an increase in transfusion cessation by greater than 70%. In another embodiment, treatment results in discontinuation of transfusion from baseline to day 183.

In other embodiments, the treatment results in resolution of breakthrough hemolysis during the treatment period. In another embodiment, the treatment results in a decrease in breakthrough hemolysis compared to the baseline level of breakthrough hemolysis before treatment.

In other embodiments, treatment results in a reduction in major adverse vascular events (MAVE).

In other embodiments, treatment produces a change in quality of life from baseline, as assessed by the Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Score Version 4 and the 30-question European Organization for Research and Treatment of Malignant Tumors Quality of Life Questionnaire. In one embodiment, the treatment causes a change in quality of life, as measured by the FACIT fatigue score, of one or more (eg, 1, 2, or 3) points from baseline. In another embodiment, the treatment causes a change in quality of life from baseline, as assessed by the FACIT fatigue score, of 3 points after 150 days or more days (e.g., 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 days, 172 days, 173 days, 174 days, 175 days, 176 days, 177 days, 178 days, 179 days, 180 days, 181 days, 182 days 183 days, 184 days, 185 days, 186 days, 187 days , 188 days, 189 days, 190 days, 191 days, 192 days, 193 days, 194 days, 195 days, 196 days, 197 days, 198 days, 199 days, 200 days, 205 days, 210 days, 215 days, 220 days or 225 days) after the start of treatment.

Chronic kidney disease (CKD) stage is classified based on the US National Kidney Foundation stages of chronic kidney disease. The stages of CKD and the corresponding estimated glomerular filtration rate (eGFR) values are as follows: Stage 1: eGFR >= 90 (normal), Stage 2: eGFR = 60-89, Stage 3A: eGFR = 45-59, Stage 3B: eGFR = 30- 44, Stage 4: eGFR = 15–29 and stage 5: eGFR < 15 (including dialysis: End stage). Stage 1 is considered the best category. Stage 5 is considered the worst category. Improved eGFR (eg, >15) corresponds to improved CKD stage (eg, lower CKD stage). Accordingly, in other embodiments, the patient's chronic kidney disease (CKD) improves by one or more stages after initiation of treatment. For example, a patient's CKD improves by one, two, three, four or five stages). In another embodiment, the patient's CKD improves by one or more stages after 150 days or more days (e.g., 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 days, 172 days, 173 days, 174 days, 175 days, 176 days, 177 days, 178 days, 179 days, 180 days, 181 days, 182 days 183 days, 184 days, 185 days, 186 days, 187 days, 188 days, 189 days, 190 days, 191 days, 192 days, 193 days, 194 days, 195 days, 196 days, 197 days, 198 days, 199 days, 200 days, 205 days, 210 days, 215 days, 220 days or 225 days) after the start of treatment.

In other embodiments, treatment results in an increase in eGFR compared to baseline. In other embodiments, treatment results in a shift toward normal eGFR levels (eg, ≥90). In other embodiments, treatment results in an increase in eGFR compared to baseline and the patient's CKD improves by one or more stages. In other embodiments, treatment results in a shift toward normal eGFR levels (eg, ≥90) compared to baseline, and the patient's CKD improves by one or more stages.

In other embodiments, the treatment results in an EQ-5D-3L US Time Trade-off Questionnaire (US TTO) value of >0.94.

V. Kits and unit dosage forms

Also provided herein are kits that include a pharmaceutical composition comprising an anti-C5 antibody or antigen-binding fragment thereof, such as ravulizumab, and a pharmaceutically acceptable carrier in a therapeutically effective amount adapted for use in the methods described herein. The kits may also optionally include instructions containing, for example, administration schedules allowing a practitioner (eg, physician, nurse, or patient) to administer the composition contained therein for administering the composition to a patient having aHUS. The kit may also include a syringe.

Optionally, the kits include multiple single-dose packages of pharmaceutical compositions, each of which contains an effective amount of an anti-C5 antibody or antigen-binding fragment thereof for single administration in accordance with the methods presented above. Instruments or devices necessary for administering the pharmaceutical composition(s) may also be included in these kits. For example, the kit may contain one or more pre-filled syringes containing an amount of anti-C5 antibody or antigen-binding fragment thereof.

In one embodiment, this invention provides a kit for treating aHUS in a human patient, comprising:

(a) a dose of an anti-C5 antibody or an antigen-binding fragment thereof comprising the CDR1, CDR2 and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2 and CDR3 domains of a light chain variable region having the sequence, specified in SEQ ID NO: 8; And

(b) instructions for use of an anti-C5 antibody or antigen-binding fragment thereof in accordance with any of the methods described herein.

In one embodiment, the kit contains a dose of an anti-C5 antibody or antigen-binding fragment thereof, wherein said anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥ 40 to < 60 kg:

(a) once on the 1st day at a dose of: 2400 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg.

In another embodiment, the kit contains a dose of an anti-C5 antibody or antigen-binding fragment thereof, wherein said anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥60 to <100 kg:

(a) once on the 1st day at a dose of: 2700 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3300 mg.

In another embodiment, the kit contains a dose of an anti-C5 antibody or antigen-binding fragment thereof, wherein said anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≥ 100 kg:

(a) once on the 1st day at a dose of: 3000 mg; And

(b) on the 15th day and thereafter - every eight weeks at a dose of: 3600 mg.

The following examples are merely illustrative and should not be construed as limiting the scope of this specification in any way, as many variations and equivalents will become apparent to those skilled in the art upon reading this specification.

The contents of all references, Genbank entries, patents and published patent applications cited in this application are expressly incorporated herein by reference.

EXAMPLES

EXAMPLE 1: Phase 3, single-arm, multicenter study of ravulizumab (ALXN1210) in complement inhibitor-naïve adults with atypical hemolytic uremic syndrome (aHUS)

A single-arm trial of ravulizumab (ALXN1210-aHUS-311) is being conducted in adult and adolescent patients with atypical hemolytic uremic syndrome (aHUS) who have not previously been treated with complement inhibitors. The design of this study is presented in Figure 1.

aHUS is a thrombotic microangiopathy (TMA) most often caused by mutations in genes encoding proteins involved in the alternative complement pathway (APP) or autoantibodies against APC regulatory proteins (Noris, et al., Clin. J. Am. Soc. Nephrol 2010;5:1844-59). Patients with aHUS are at risk for life-threatening disease manifestations resulting from endothelial damage, including thrombocytopenia, intravascular hemolysis, acute renal failure, and extrarenal tissue damage. It is important to note that approximately 20% of patients experience extrarenal manifestations of the disease, including manifestations in the central nervous system, heart, gastrointestinal tract, distal extremity, and severe systemic organ involvement (Loirat, et al, Orphanet J. Rare Dis. 2011; 6 :60 and Brodsky, Blood. 2015; 126:2459-65). Before the advent of eculizumab, mortality rates among patients with aHUS reached 15% during the acute progressive phase of the disease (Noris, et al., Clin. J. Am. Soc. Nephrol 2010;5:1844-59) and Sellier-Leclerc, J. Am. Soc. Nephrol 2007; 18:2392-2400). In at least 50% of patients, kidney failure progressed to end-stage renal disease (ESRD), often within a year of disease onset, and required dialysis or a kidney transplant to keep them alive. Chronic uncontrolled terminal complement activation, in particular activation of complement component 5 (C5) and dysregulation of complement activity, are central to the pathogenesis of aHUS and the devastating manifestations of this disease. As a consequence, targeted C5 blockade with selective inhibition of C5a and C5b-9 generation represents an important therapeutic mechanism for treatment.

1. Goals

The primary objective of this study is to evaluate the effectiveness of ravulizumab in adolescent and adult patients with aHUS not previously treated with complement inhibitors in inhibiting complement-mediated TMA, characterized by thrombocytopenia, hemolysis, and renal dysfunction.

The secondary objectives of this study are: (1) to characterize the safety and tolerability of ravulizumab in this patient population, (2) to evaluate the effectiveness of ravulizumab using additional indicators (eg, dialysis requirement status, time to TMA complete response, TMA complete response status over time , observed value and change from baseline in estimated glomerular filtration rate (eGFR), stage of chronic kidney disease (CKD) (based on assessment on individual target days and classified as improved, stable (no change) or worsened from baseline), observed value and change from baseline in hematological parameters (platelet, LDH, hemoglobin), increase in hemoglobin level ≥ 20 g/l from baseline (maintained for at least 2 consecutive measurements taken at least 4 weeks apart), changes from baseline in quality of life (QOL) (based on the European Quality of Life Questionnaire in 5 categories at 3 levels (EQ-5D-3L; all patients), Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Questionnaire version 4 (patients < 18 years of age) and Pediatric FACIT Fatigue Questionnaires (patients < 18 years of age)), (3) pharmacokinetics/pharmacodynamics (PK) characterization /PD) of ravulizumab by changes in serum ravulizumab concentrations over time and changes in free C5 concentrations over time, and (4) assessing the safety and efficacy of ravulizumab during long-term use. 2. Evaluation criteria

The primary, secondary, and safety endpoints for this study are summarized in Figure 2. The primary outcome measure is complete TMA response during the 26-week initial evaluation period, as demonstrated by normalization of hematologic parameters (platelet count and LDH) and an increase in serum creatinine ≥ 25% above baseline, as demonstrated by 2 successive measurements taken at intervals of at least 4 weeks.

The secondary efficacy measures in this study are as follows:

A. status of need for dialysis;

B. time to complete TMA response;

C. TMA complete response status over time;

D. observed value and change from baseline in eGFR;

E. CKD stage based on investigator assessment on specific target days and classified as improved, stable (no change), or worsened compared to baseline;

F. observed value and change from baseline in hematological parameters (platelet, LDH, hemoglobin);

G. an increase in hemoglobin levels of ≥ 20 g/L compared to baseline, persisting for at least 2 consecutive measurements taken at least 4 weeks apart;

H. change from baseline in QOL as measured by EQ-5D-3L (all patients), FACIT Version 4 Fatigue (patients ≥ 18 years of age), and Pediatric FACIT Fatigue (patients < 18 years of age) .

The pharmacokinetic (PK) and pharmacodynamic (PD) endpoints in this study are changes in serum ravulizumab concentrations over time and changes in free C5 concentrations over time.

The safety and tolerability of ravulizumab are assessed through physical examinations, vital signs, electrocardiograms (ECGs), laboratory tests, and the incidence of adverse events (AEs) and serious adverse events (SAEs). The relative number of patients who develop antidrug antibodies (ADAs) is also assessed.

Biomarkers of PD effect examined include, but are not limited to: change from baseline in levels of markers of complement dysregulation (eg, factor Ba), vascular inflammation (eg, soluble tumor necrosis factor receptor 1 [sTNFR1]), activation /endothelial damage (eg, soluble vascular adhesion molecule 1 [sVCAM1], thrombomodulin), coagulation (eg, D-dimer), and renal damage (eg, cystatin C). Additional assessments may include measurements of urinary ravulizumab excretion, chicken erythrocyte hemolysis, total C5, autoantibodies against complement proteins (eg, anti-factor H antibodies), and APC activity (eg, modified Ham test, complement deposition assay).

Experimental genetic studies can be performed to examine genetic variants in genes known to be associated with aHUS, as well as to identify new genetic variants associated with aHUS, complement dysregulation, metabolism, or ravulizumab efficacy. Patients may refuse to provide a sample for experimental genetic testing and still participate in the clinical trial.

3. Summary of study design

The ALXN1210-aHUS-311 study is a single-arm, single-arm, multicenter, phase 3 study to evaluate the safety and efficacy of ravulizumab administered by intravenous (IV) infusion in adolescent patients (ages 12 to <18 years) and adult patients (age 12 to <18 years). age ≥ 18 years) with aHUS. This study involves approximately 55 patients receiving ravulizumab. The design of this study is presented in Figure 1. All patients had never previously received complement inhibitor treatment and included at least 6 to 10 adolescents (aged 12 to <18 years at screening) and at least 10 to 25 patients with prior renal transplantation.

This study consists of a 7-day screening period, a 26-week initial assessment period, and an extension period of up to 2 years. Drug dosages are based on the patient's last recorded body weight at the study visit (Table 5). Patients receive a loading dose of ravulizumab IV (2400 mg for patients weighing ≥ 40 to < 60 kg, 2700 mg for patients weighing ≥ 60 to < 100 kg, 3000 mg for patients weighing ≥ 100 kg) on the 1st day, and then maintenance doses of ravulizumab intravenously (3000 mg for patients with body weight from ≥ 40 to < 60 kg, 3300 mg for patients with body weight from ≥ 60 to < 100 kg , 3600 mg - for patients with body weight ≥ 100 kg) on the 15th day and once every 8 weeks (1 day/8 weeks) thereafter for a total of 26 weeks of treatment. After the initial evaluation period, patients begin the extension period and receive ravulizumab until the drug is registered or approved (in accordance with country-specific regulations), or for a period of up to 2 years, whichever occurs first . Completion of a given clinical trial is defined as the last visit of the last patient.

This unblinded, single-arm, phase 3 study evaluates the safety and efficacy of ravulizumab treatment. Although no formal comparative analysis is planned for this study, data from patients treated with ravulizumab are evaluated in the context of data observed in a retrospective control group of patients treated with eculizumab. The retrospective control group consists of patients with aHUS who were treated with eculizumab in the prospective registration studies C08-002A/B, C10-003, and C10-004, for which the study design and conduct features of interest that may influence the effect size were similar this research. In addition, this control group is limited to patients aged ≥ 12 years who received plasma exchange/transfusion (PE/PT) 4 weeks or less before starting eculizumab treatment to further meet the inclusion criteria for this study.

The assessment schedule for the screening period and the initial assessment period is presented in Table 1. The assessment schedule for the extension period is presented in Table 2. Additional (unscheduled) visits beyond these visits are permitted at the discretion of the investigator. Procedures, analyzes and evaluations are at the discretion of the investigator. Any tests, procedures or assessments performed during unscheduled visits are recorded on electronic individual record cards (eCRFs). For tests during unscheduled visits, laboratory tests performed by a local laboratory or a central laboratory are used. However, if test results from a local laboratory must be used, duplicate samples are collected during unscheduled visits for testing at the central laboratory.

4. Study population

This study is enrolling a total of approximately 55 patients with documented aHUS who are being allocated to treatment with ravulizumab at approximately 200 study sites worldwide. This study enrolls at least 6 to 10 adolescent patients (aged 12 to <18 years at screening) and at least 10 to 25 patients with prior renal transplantation.

Subjects who do not meet the criteria for participation in this study (screening attrition) may be screened again. Patients can be screened again no more than 2 times. Prospective approval of protocol deviations regarding recruitment and enrollment criteria, also known as protocol deviations or exceptions, is not permitted.

A summary of the inclusion and exclusion criteria is presented in Figure 3 . Patients are eligible to participate in this study if they meet all of the following criteria and none of the exclusion criteria:

Male patients or female patients ≥ 12 years of age and weighing ≥ 40 kg at the time of consent.

• Signs of TMA, including thrombocytopenia, signs of hemolysis, and renal dysfunction, based on the following laboratory results from the screening visit: Platelet count < 150,000 per microliter (μL) and LDH ≥ 1.5 × upper limit of normal (ULN), and hemoglobin < age- and sex-matched lower limit of normal (LON), and a serum creatinine level ≥ ULN in adults (age ≥ 18 years) or ≥ ^97.5th percentile for age-matched screening in adolescents (age 12 to < 18 years) (patients requiring dialysis due to acute kidney injury may also participate).

• Among kidney transplant patients: history of aHUS prior to current kidney transplant or no history of aHUS, and persistent evidence of TMA after discontinuation of a calcineurin inhibitor ([CNI]; eg, cyclosporine, tacrolimus) or mammalian target of rapamycin inhibitor ([mTORi]; e.g. sirolimus, everolimus) for at least 4 days and at most 7 days.

• Among patients with onset of TMA in the postpartum period, persistent signs of TMA for ≥ 3 days after the day of delivery.

• To reduce the risk of meningococcal disease (Neisseria meningitidis), all patients should be vaccinated against meningococcal disease within 3 years before or at the time of starting study drug. Patients who receive meningococcal vaccination less than 2 weeks before starting ravulizumab treatment are treated with appropriate prophylactic antibiotics until 2 weeks after vaccination. Patients who have not been vaccinated before starting ravulizumab treatment receive prophylactic antibiotics before and for at least 2 weeks after meningococcal vaccination.

• Patients <18 years of age should be vaccinated against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae in accordance with national and local vaccination schedule recommendations.

• Patients of childbearing potential and patients with female partners of childbearing potential should follow protocol-specified contraceptive recommendations during treatment and for at least 8 months after the last dose of study drug.

• Willing and able to provide written informed consent and comply with the study visit schedule. For patients <18 years of age: the patient's legal guardian must be willing and able to provide written informed consent, and the patient must be willing and able to provide the minor's written informed consent.

Samples collected during screening can be tested either at a local laboratory or at a central laboratory. If local laboratory test results for LDH, platelet count, hemoglobin, and serum creatinine are used, duplicate samples are collected for testing at the central laboratory to ensure that baseline and post-baseline measurements for these tests are obtained from the central laboratory. laboratories. Although the results of tests performed by the local laboratory can be used to expedite the evaluation of eligibility for participation in this study, the final determination of these inclusion criteria should be based on the results obtained from the central laboratory.

Patients are excluded from participation in this study if they meet any of the following criteria:

A. Known deficiency (<5% activity) of "thrombospondin type 1 family member 13 disintegrin and metalloproteinase."

B. Shiga toxin-associated hemolytic-uremic syndrome (SH-HUS).

C. Hemolytic-uremic syndrome (HUS) associated with Streptococcus pneumoniae, as evidenced by a positive direct Coombs test and infection with Streptococcus pneumoniae (eg, culture, antigen test).

D. Known human immunodeficiency virus (HIV) infection.

E. Untreated systemic meningococcal meningitis.

F. Patients with a confirmed diagnosis of ongoing sepsis based on positive blood cultures within 7 days before screening and who have not received antibiotic treatment.

G. The presence or suspicion of an active and untreated systemic bacterial infection that, in the opinion of the investigator, makes accurate diagnosis of aHUS difficult or interferes with the ability to control aHUS disease.

H. Pregnancy or lactation.

I. Heart, lung, small intestine or liver transplant.

J. Among kidney transplant patients, any of the following:

a. acute renal dysfunction within 4 weeks of transplantation consistent with a diagnosis of acute antibody-mediated rejection (AMR) according to Banff 2013 criteria, or

b. acute renal dysfunction within 4 weeks of transplantation and elevated donor-specific antibodies (DSA) consistent with the clinical diagnosis of acute AOO.

c. History of polycystic kidney disease.

K. Among patients ≥ 18 years of age with systolic blood pressure

(SBP) ≥ 170 mm Hg. or patients aged 12 to <18 years with a clinical diagnosis of hypertension - any of the following:

A. Persistent signs of TMA (inclusion criterion No. 2) after less than 4 days of reduction in blood pressure (BP) to ≤ 140 mmHg.

b. Known left ventricular hypertrophy.

With. Known small and hyperechoic kidneys on ultrasound.

L. Detected HUS associated with drug exposure.

M. Receipt of CR/PT within 28 days or longer before screening for current TMA.

N. History of malignancy within 5 years of screening, excluding nonmelanoma skin cancer or cervical carcinoma in situ, which was treated without evidence of recurrence.

A. Bone marrow transplantation (BMT) / hematopoietic stem cell transplantation (HSCT) within the last 90 days before screening.

R. HUS associated with vitamin B12 deficiency.

Q. Known systemic sclerosis (scleroderma), systemic lupus erythematosus (SLE), or antiphospholipid antibody positivity, or antiphospholipid syndrome.

R. Chronic dialysis (defined as regular dialysis as renal replacement therapy for ESRD).

S. Patients chronically receiving intravenous immunoglobulin (IVIG) within 8 weeks before screening, unless unrelated to the disease being studied (eg, hypogammaglobulinemia); or chronic rituximab therapy within 12 weeks before screening.

T. Patients receiving other immunosuppressive therapies such as steroids, mTORi (eg, sirolimus, everolimus), CNI (eg, cyclosporine or tacrolimus) are excluded if: a) receiving established anti-rejection treatment after transplantation, or b ) the patient has confirmed complement factor antibodies requiring immunosuppressive therapy, or c) steroids are being used for a condition other than aHUS (eg, asthma).

U. Participation in another intervention study or receipt of any investigational therapy within 30 days before the start of the study drug on Day 1 of the study or within 5 half-lives of the study drug, whichever there are more values.

V. Previous use of eculizumab or other complement inhibitors.

W. Hypersensitivity to murine proteins or to one of the excipients.

X. Any medical or psychological condition that, in the opinion of the investigator, may increase the risk to the patient if the patient participates in this study or bias the results of this study.

Y. Known or suspected history of drug or alcohol abuse or dependence within 1 year prior to screening. Laboratory results for exclusion criterion number 1 may not be available until the first dose is administered. Late results under exclusion criterion number A may result in discontinuation of participation and replacement of that patient.

The patient has the right to discontinue participation in this study at any time. If the patient withdraws consent, the assessments indicated for the early termination (ED) visit are completed. Patients who discontinue participation in this study will not be replaced. A patient's study drug may be discontinued if the investigator or sponsor has reason to believe that it is in the patient's best interests to discontinue treatment.

Patients with a prior kidney transplant who develop AOO (C4d-positive kidney biopsy) and for whom rituximab is considered appropriate therapy should discontinue participation in this study and receive standard of care therapy. The root cause and any other reason(s) for termination are recorded in the eCRF.

If a patient discontinues participation in this study and has a current AE or a laboratory value that has not returned to normal that is significantly outside the reference range and is clinically significant, the investigator attempts to provide follow-up until a satisfactory clinical value for the laboratory value is achieved or adverse event.

The sponsor or competent authority may terminate this study for reasonable cause. Conditions that justify termination of this study include, but are not limited to: (1) discovery of an unexpected, serious, or unacceptable risk to patients enrolled in this study, (2) decision by the sponsor to suspend or terminate testing, evaluation, or development of an investigational drug, (3) the investigator's failure to comply with the approved protocol, applicable guidelines and/or regulations, and (4) the provision of false information by the investigator to the sponsor and/or regulatory authorities.

If at any time it is determined that a patient's screening data do not meet one or more of the following inclusion/exclusion criteria (inclusion criterion number 2 or exclusion criterion number 1), after receiving at least 1 dose of study drug (e.g., data patient analysis performed by local laboratories used to confirm inclusion criteria are subsequently determined by the central laboratory to no longer meet inclusion criteria), the patient's participation in this study is terminated and the patient may be replaced. Early termination procedures are performed for patients whose participation was terminated early, and all AEs are collected up to 60 days after the last dose of study drug was administered to the designated patient.

Study completion is defined as the date of the last patient visit during the extension period.

5. Investigational treatment

Ravulizumab, a humanized anti-C5 monoclonal antibody consisting of two heavy chains of 448 amino acids and two light chains of 214 amino acids, is an IgG2/4-kappa immunoglobulin consisting of human constant regions and mouse complementarity determining regions grafted onto a scaffold of variable regions of human light and heavy chains. Ravulizumab eculizumab has more than 99% primary amino acid sequence identity and very similar pharmacology.

The drug product ravulizumab is supplied for clinical studies as a sterile, preservative-free solution of 10 mg/mL in single-use vials, and is intended for infusion by dilution in commercially available saline (0.9% sodium chloride injection; country-specific pharmacopoeia ) for administration by intravenous (IV) infusion. Table 3 and the current investigator's brochure (BI) provide additional information.

Ravulizumab is packaged in USP/EU Pharmacopoeia (EP) type 1 borosilicate glass vials and sealed with a butyl rubber stopper with an aluminum cap and a flip-off combination cap. The study drug is supplied in kits. Ravulizumab is supplied to each study site after receipt of all necessary regulatory documents in accordance with applicable regulations.

Upon arrival of the investigational medicinal product kits at the study site, the pharmacist (or designated qualified person) immediately removes the investigational medicinal product kits from the shipping refrigerator and stores them in the original cartons under refrigerated conditions at a temperature of 2°C to 8°C (35°C). °F to 47°F) and away from light. Ravulizumab is not frozen. The study drug is stored in a secure, restricted access area and the temperature is monitored daily.

The drug is kept at room temperature before administration. The material is not heated by any means (such as a microwave oven or other heat source) other than ambient temperature.

Ravulizumab is not administered by IV bolus or bolus injection. Infusion solutions of the study drug are prepared under aseptic conditions. The patient's required dose of ravulizumab is further diluted in a commercially available saline solution (0.9% sodium chloride; country-specific pharmacopoeia) in the volume indicated in Table 4. The ravulizumab diluent solution is administered to the patient through an intravenous system using an infusion pump. For infusion it is necessary to use the filter built into the system.

Doses of the study drug are prepared and dispensed only by a pharmacist or qualified health care professional. The investigational medicinal product is dispensed only to registered patients with confirmed eligibility for participation in this study. Once the study drug is prepared for a patient, it is administered only to that patient. Study drug vials are for single use only, and any drug product remaining in the vial is not used for another patient. Any drug remaining in the infusion set or infusion bag is not used for another patient.

All clinical trial materials are stored in a secure location and distributed and dispensed by appropriately trained individuals. Detailed records are maintained of quantities of the study drug received, dispensed, and destroyed. Unless otherwise noted, empty vials and vials with residual materials are stored for review and reporting by the study monitor until they are destroyed or processed in accordance with local pharmaceutical standard operating procedures (SOPs) for medicinal products in clinical trials. To comply with regulatory drug accounting requirements, at the end of the study, all remaining ravulizumab inventory is reconciled and destroyed or returned to Alexion in accordance with applicable regulations.

Patients receive ravulizumab for 26 weeks. Ravulizumab is administered by slow IV infusion over about 2 hours. Ravulizumab is not administered by IV bolus or bolus injection.

The clinical regimen for ravulizumab use during the primary assessment period is based on the patient's last recorded body weight at the study visit (Table 5). Patients receive a loading dose of IV ravulizumab on day 1, followed by a maintenance dose of IV ravulizumab on day 15 and once/8 weeks (every eight weeks) thereafter.

After the initial assessment period, all patients enter an extension period of up to 2 years, during which all patients receive ravulizumab q.v./8 weeks. (every eight weeks). The actual time of administration of all doses is recorded in the patient's eCRF.

This study is unblinded. Patients who meet all criteria for participation in this study are assigned to study treatment with ravulizumab at the initial visit (day 1). An interactive voice or web response system (IxRS) is used to assign vials containing ravulizumab to each patient.

Infusion of other monoclonal antibodies has been associated with infusion reactions, which usually begin during infusions or shortly after completion of infusions.

Previously prescribed medications (including vitamins and herbal preparations), including those discussed in the exclusion criteria, and procedures (any therapeutic intervention such as surgery/biopsy or physical therapy) taken or undergone by the patient within 28 days (or 3 years - to document meningococcal vaccination) before the start of screening before the first dose of ravulizumab is administered, are recorded in the patient's eCRF.

For analytical purposes, any dialysis during the 14-day period immediately following the first dose of ravulizumab is not considered “new dialysis.”

All medications and procedures undertaken during the study are recorded in the original document/medical record and in the patient's eCRF. Recording this information includes all prescription drugs, herbal products, vitamins, minerals, over-the-counter drugs and current medications. Concomitant medications are recorded from the first infusion of study drug until 56 days after the patient's last dose of study drug. Any changes in concomitant medications are also recorded in the original document/medical record and in the patient's eCRF. Any concomitant medication that is considered necessary for the standard treatment of the patient during this study or for the treatment of any AE, along with the authorized medications described below, may be prescribed at the discretion of the investigator. However, it is the investigator's responsibility to ensure that all medications are fully documented in the original document/medical record and in the patient's eCRF.

Patients are prohibited from taking any of the following medications or procedures at any time after the first dose of investigational drug: eculizumab or other complement inhibitors, use of any other investigational drug or device in a clinical trial, IVIG (unless medically necessary not related to disease under investigation, e.g., hypogammaglobulinemia), rituximab, CR/PT after the first dose, and new dialysis within the first 48 hours after the first dose of ravulizumab, unless there is a compelling medical need assessed as: (1) hypervolemia unresponsive to diuretics, (2) refractory electrolyte imbalance, or (3) new-onset uremic encephalopathy. Exceptions must be approved by the sponsor prior to dialysis on a case-by-case basis.

The following concomitant medications and procedures are permitted under certain circumstances and with the following restrictions: use of other immunosuppressive therapies (such as steroids, mTORi [eg, sirolimus, everolimus], CNI [eg, cyclosporine or tacrolimus]) before screening or during this study not allowed if: a) is part of an established anti-rejection treatment regimen after transplantation, or b) the patient has confirmed antibodies against complement factors requiring immunosuppressive therapy, or c) steroids are used to treat a condition other than aHUS (eg, asthma) .

Any patients receiving other complement inhibitors (including eculizumab) or undergoing CR/PT after the first dose of study drug will discontinue participation in this study.

Due to its mechanism of action, the use of ravulizumab increases the patient's susceptibility to infection. To reduce the risk of infection, all patients are vaccinated against N. meningitidis, Hib, and Streptococcus pneumoniae.

Patients are vaccinated against N. meningitidis within 3 years before or at the time of the first dose of ravulizumab. Patients who receive study drug treatment less than 2 weeks after meningococcal vaccine administration are treated with appropriate prophylactic antibiotics until 2 weeks after vaccination. Vaccines against serotypes A, C, Y, W135 and B, where available, are recommended to prevent common pathogenic meningococcal serotypes. Patients are vaccinated or revaccinated according to current national vaccination guidelines or local practice for the use of complement inhibitor vaccination (eg, eculizumab).

It is recognized that some patients who have not been vaccinated against N. meningiditis within 3 years of receiving their first dose of ravulizumab may not be able to be vaccinated by the time they receive their first dose. Patients who have not been vaccinated before starting ravulizumab treatment receive prophylactic antibiotics before and for at least 2 weeks after meningococcal vaccination.

Vaccination may not be enough to prevent meningococcal disease. Official guidance and local practice on the appropriate use of antibacterial agents should be considered. All patients are monitored for early manifestations of meningococcal infection, immediately assessed if infection is suspected, and treated with appropriate antibiotics if necessary.

To increase awareness of risks and facilitate rapid identification of any potential signs or symptoms of infection that patients experience during this study, patients are provided with a safety card to carry with them at all times. Additional discussion and explanation of potential risks, signs and symptoms occur at specific time points as part of the patient safety record review and throughout the study as described in the assessment schedule (Table 1 and Table 2).

Patients are vaccinated against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae according to national and local vaccination schedule recommendations before or at the time of the first dose of ravulizumab. Vaccination status against N. meningitidis, Hib and S. pneumoniae is recorded in the patient's eCRF.

Patients are administered the study drug under controlled conditions under the supervision of the investigator or designee, thereby ensuring compliance with the study drug administration. The investigator or designee will ensure that all patients are adequately informed of the specific dosing regimen necessary to comply with the study protocol, ensure that the patient receives the appropriate dose at the designated time points during the study, and that appropriate safety monitoring is performed during infusions.

Before taking the study drug, patients who consider themselves to be postmenopausal must provide evidence of menopause based on the combination of amenorrhea for at least 1 year and elevated serum follicle-stimulating hormone (FSH) levels (>30 IU/L) (eg, in the absence of hormone replacement therapy, dietary phytoestrogens).

Patients of childbearing potential use a highly effective method of contraception (as defined below) starting at screening and continuing for at least 8 months after the last dose of study drug. Highly effective methods of contraception* include: hormonal contraception associated with ovulation inhibition, intrauterine device, intrauterine hormone-releasing system, bilateral tubal occlusion, partner vasectomy (provided the partner is the patient's only sexual partner), sexual abstinence (defined as abstinence from heterosexual sexual intercourse during the entire period of risk associated with treatment with the study drug; the reliability of sexual abstinence must be assessed in relation to the duration of the clinical study and the patient's preferred and usual lifestyle), the combination of a male condom with a cap, diaphragm or sponge with spermicide (double barrier contraceptive methods). ). Male patients who have a potentially fertile female spouse/partner, or a pregnant or breastfeeding spouse or partner, agree to use double barrier contraception (a male condom plus an appropriate barrier method for the female partner) during treatment and during for at least 8 months after the last dose of study drug. Double barrier contraception is required even when there is documented medical evaluation of a surgically successful vasectomy.

Male patients do not provide sperm during treatment and for at least 8 months after the last dose of study drug.

6. Performance evaluations

The primary outcome measure is complete TMA response during the 26-week primary assessment period. Criteria for a complete TMA response are: (1) normalization of platelet count, (2) normalization of LDH, and (3) increase in serum creatinine ≥ 25% from baseline.

Patients who meet all TMA complete response criteria, confirmed by 2 consecutive measurements obtained at least 4 weeks apart, are classified as meeting the primary efficacy endpoint.

The following secondary outcome measures are measured in this study:

A. status of need for dialysis;

B. time to complete TMA response;

C. TMA complete response status over time;

D. observed value and change from baseline in eGFR;

E. CKD stage based on investigator assessment on specific target days and classified as improved, stable (no change), or worsened compared to baseline;

F. observed value and change from baseline in hematological parameters (platelet, LDH, hemoglobin);

G. an increase in hemoglobin levels of ≥ 20 g/L compared to baseline, persisting for at least 2 consecutive measurements taken at least 4 weeks apart;

H. change from baseline in QOL as measured by EQ-5D-3L (all patients), FACIT Version 4 Fatigue (patients ≥ 18 years of age), and Pediatric FACIT Fatigue (patients < 18 years of age) .

7. Safety assessments

The investigator or designee meets with patients to discuss potential safety risks of ravulizumab and to provide the investigator with the opportunity to address any patient safety concerns related to the study.

Monitoring of the collection of AEs is carried out from the moment of obtaining informed consent until the completion of the study. Investigators monitor any AEs until they are resolved (resolved or stabilized). If a patient withdraws from this study, monitoring for AEs continues until the last visit of the last patient, if possible. The timing of clinical and laboratory assessments is determined in accordance with the assessment schedule (Tables 1 and 2). Any clinically significant abnormalities are monitored until they resolve or stabilize.

Conduct a review of demographic parameters, including age, gender, race, and ethnicity. A complete medical history is obtained and documented. Body weight and height are recorded. Height is measured only during screening.

The medical history of the patient with aHUS, including the onset of the first symptom of aHUS and the date of diagnosis, is documented at the screening visit.

The patient's medical history, including preexisting and concomitant medical conditions/disorders, is recorded at the screening visit. In addition to information on meningococcal vaccination, use of medications (prescription or over-the-counter, including vitamins and/or herbal supplements) in the 28 days (or 3 years to document meningococcal vaccination) prior to screening is also recorded.

The physical examination includes the following assessments: the patient's appearance; leather; head, ear, eye, nose and throat; neck; The lymph nodes; breast; heart; abdomen; limbs; central nervous system; and musculoskeletal system. The abbreviated physical examination consists of an examination of body systems based on the examiner's assessment and the patient's symptoms. Vital sign measurements are taken after the patient has rested for at least 5 minutes and include systolic and diastolic blood pressure (in millimeters of mercury [mmHg]), pulse oximetry, heart rate (beats per minute), frequency respirations (breaths per minute) and oral or tympanic temperature measurement (in degrees Celsius [°C] or degrees Fahrenheit [°F]).

Specimens for serum pregnancy tests, hematology studies, biochemical studies, coagulation tests, and urinalysis are collected within the time frames specified in the assessment schedule (Tables 1 and 2). Samples for laboratory testing are collected before each administration of the study drug.

Samples collected during screening can be tested either at a local laboratory or at a central laboratory. If local laboratory test results for LDH, platelet count, hemoglobin, and serum creatinine are used, duplicate samples are collected for testing at the central laboratory to ensure that baseline and post-baseline measurements for these tests are obtained from the central laboratory. laboratories. In case of duplicate samples from local and central laboratories, the results of the central laboratory are used for analysis.

It is expected that some laboratory values may be outside the normal range due to underlying disease. Investigators must use medical judgment in assessing the clinical significance of these indicators.

Clinical significance is defined as any variation in laboratory measurements that has medical significance and leads to a change in medical care. If clinically significant laboratory changes are noted compared to baseline, such changes are documented as AEs in the eCRF for AEs. The investigator assesses the relationship to study treatment for all clinically significant parameters outside the normal range. The investigator continues to monitor the patient through additional laboratory tests until: (1) parameters return to the normal range or baseline, or (2) in the opinion of the investigator, parameters that are outside the normal range are not related to the administration of the study drug. drug or other procedures determined by the protocol.

For women of childbearing potential, a serum or urine pregnancy test (ie, beta-human chorionic gonadotropin [β-hCG]) is performed according to the assessment schedule (Tables 1 and 2). Blood samples are analyzed for hematological parameters.

Blood samples are analyzed against serum biochemical parameters. Indirect bilirubin is calculated based on the values of total bilirubin and direct bilirubin; therefore, indirect bilirubin results are not available if direct bilirubin is below the quantitation limit. Serum FSH levels are measured during screening of postmenopausal patients to confirm their postmenopausal status.

Biochemical studies are carried out at the time points indicated in the assessment schedule (Tables 1 and 2). eGFR was calculated for all visits at which serum chemistry values were collected using the Modification of Diet for Kidney Disease formula for patients aged ≥ 18 years and the Schwartz Modification of Diet for Kidney Disease formula for patients aged < 18 years.

Blood samples are analyzed for blood clotting parameters.

Urine samples are analyzed. Microscopy of urine samples is performed if macroscopic results are not normal. Urine samples are also analyzed to measure protein and creatinine levels to calculate the ratio of total protein to creatinine in the urine.

Single-shot digital 12-lead ECGs are obtained for each patient according to the assessment schedule (Tables 1 and 2). Patients should lie on their back for about 5 to 10 minutes before taking the ECG and continue to lie on their back, but not asleep, during the ECG. The investigator or designee is responsible for analyzing the ECG to evaluate whether the ECG is within normal limits and to determine the clinical significance of the results. These assessments are indicated in the CRF.

Collect blood samples to test for the presence and titer of serum ALS to ravulizumab prior to study drug administration, as indicated in the assessment schedule (see Tables 1 and 2). If the results of this test are positive, this test may be repeated every 3 months until the results become negative or stabilize based on the measured titer and safety assessments. If necessary, further characterization of antibody production can be performed, including antibody binding and neutralization, PK/PD, safety and activity of ravulizumab.

An AE is any adverse medical event in a patient receiving a pharmaceutical drug that does not necessarily have a causal relationship with the treatment. Therefore, an AE can be any adverse or unexpected sign (eg, abnormal laboratory test), symptom, or illness temporarily associated with the use of a medicinal product, whether or not it is believed to be related to the medicinal product.

Situations in which an adverse medical event has not occurred (eg, hospitalization for elective surgery if planned before the start of this study, hospitalization for social reasons or for convenience), and expected daily fluctuations of pre-existing disease(s) or pathological Condition(s) that are present or detected at the start of this study and that do not worsen are not AEs.

Lack of effect from a drug is not an AE in clinical trials, since the purpose of a clinical trial is to establish the effect of the drug.

Medication error (including intentional misuse, abuse, and overdose of a medical product) or use other than as specified in the protocol is not considered an AE unless an adverse medical event occurs as a result of the medication error.

Pregnancies that occur during the period during which the mother or father is exposed to the study drug should be reported within 24 hours of informing the investigator/study site. Data on effects on the fetus and breastfeeding are collected for regulatory reporting and safety assessment.

Adverse events are recorded from the moment the consent is signed. An AE reported after informed consent but before administration of the study drug is considered to be a treatment-preceding AE.

The following are important identified risks in this study: meningococcal infections.

The severity of AEs is assessed using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 or higher. For each AE term, a rating scale (severity) is provided. Each CTCAE term is a Medical Dictionary for Regulatory Activities (MedDRA®) Low Level Term (LLT). Each LLT is coded according to MedDRA's preferred term. The score refers to the severity of the AE. CTCAE assigns a score from 1 to 5, with clear clinical descriptions of severity for each AE (Table 6).

Any change in the severity of an AE is documented based on the specific recommendations contained in the eCRF Completion Guide. Grade and seriousness are different: severity describes the intensity of an AE, while severity refers to an AE that meets specific criteria for a serious adverse event (SAE).

The investigator must provide an assessment of the certainty of causality (not related, unlikely related, possibly related, likely related, or definitely related) for all AEs (both serious and non-serious) based on the investigator's medical judgment and observed symptoms associated with the event ( table 7). This assessment is recorded on the eCRF and any additional forms as required.

A serious adverse event (SAE) is any adverse medical event that:

• Leads to death

• Is life-threatening (i.e. the patient was in danger of dying during the occurrence of this phenomenon)

• Requires hospitalization or extension of existing hospitalization.

• Leads to permanent or significant disability/disability

• Is a congenital anomaly/developmental defect

Important medical events that may not result in death, may not be immediately life-threatening or require hospitalization, may be considered serious adverse events when, based on appropriate medical judgment, they may place the patient at risk or may require interventions to prevent one of the consequences listed above.

Suspected serious unexpected adverse events (SUSARs) are serious events that are not listed in the BI and that the investigator determines to be related to the study drug or procedure. 21 CFR 312.32 and the European Union Clinical Trials Directive 2001/20/EC, and associated detailed guidance or national regulations in participating countries, require reporting of TEAEs.

All AEs (serious and non-serious) are collected from the moment of signing the informed consent form (IF) until 60 days after the last dose of the study drug for patients who terminated the study early, or until 56 days after the last dose of the study drug for patients who who completed the study. All AEs are recorded in the eCRF after the investigator or his/her staff becomes aware of their occurrence.

All SAEs are recorded regardless of the investigator's assessment of causality. There is no time limit for reporting SAEs believed to be causally related to the study drug. Investigators may report SAEs at any time, regardless of causality.

For all SAEs, the investigator must provide the following: relevant and requested additional information, causality of the SAE, treatment/intervention performed regarding the SAE, outcome of the SAE and supporting medical documentation, and laboratory/diagnostic information.

Pregnancy data are collected in this study for all female patients and female spouses/partners of male patients. Exposure during pregnancy (also called in utero exposure) may result from either maternal exposure or transfer of the drug through semen after paternal exposure. Pregnancy itself is not considered an AE unless there is a suspicion that the study drug could interfere with the effectiveness of contraceptive medications. However, pregnancy complications and abnormal pregnancy outcomes are AEs and may meet criteria for SAEs (eg, ectopic pregnancy, spontaneous abortion, intrauterine fetal death, neonatal death, or congenital malformation). Elective abortions without complications should not be reported as AEs.

8. Pharmacokinetics and pharmacodynamics assessments

Blood samples for determination of serum drug concentration and PD assessment are collected before and after administration of the study drug at the time points specified in the assessment schedule (see Tables 1 and 2). Record the actual date and time (in 24-hour format) of collection of each sample. The number of time points for PK sampling for any given patient does not exceed the currently planned number of time points.

Blood samples for PK and PD assessment are collected from the arm opposite the arm used for drug administration. PK/PD assessments are as follows: (1) changes in serum ravulizumab concentrations over time and (2) changes in free C5 concentrations.

9. Experimental estimates

For experimental biomarker studies, summary statistics of parameter value, change, and percentage change from baseline are provided.

The relationship between ravulizumab concentrations and experimental biomarkers or the correlation between clinical benefit and key experimental biomarkers can be assessed using a graphical representation. Experimental analyzes and analyzes of potential relationships between clinical outcomes, PK/PD, genetic profile, and biomarker levels can also be performed. Data on APC activity and autoantibody assay results, if assessed, are summarized.

Experimental genetic studies can be performed to examine genetic variants in genes known to be associated with aHUS, as well as to identify new genetic variants associated with aHUS, complement dysregulation, metabolism, or ravulizumab efficacy.

The investigator informs the patient or the patient's caregiver of genetic mutations of known clinical significance in aHUS and provides appropriate genetic counseling. Genetic variants of unknown clinical significance are not reported to patients or their investigator.

Additional signs or symptoms of aHUS are assessed using the Patient Resource Utilization Questionnaire and the Patient-Reported Symptoms of aHUS Questionnaire.

Components of extrarenal signs or symptoms of aHUS, including vital signs and clinical laboratory results, can be descriptively summarized for baseline time points, post-baseline time points, and relative changes from baseline. Patient lists can be provided.

Analyzes of signs, symptoms, and resource use may include standard approaches to categorical outcomes with or without repeated measures.

If the Day 1 score is not available, the screening score is used as the baseline score.

To assess TMA complete response during the 26-week primary assessment period (primary outcome measure), for patients who do not have an efficacy assessment that is part of the definition of TMA complete response at the time of study participation, the last documented value (LOCF methodology) is used. For patients who discontinue early participation in this study before week 26, their data up to the time of discontinuation from this study are used to assess TMA complete response.

Missing data for QoL determination systems are processed in accordance with the instructions for each determination system.

The interim analysis for this study is planned at the end of the 26-week primary assessment period after all patients have completed or exited the 26-week primary assessment period. In addition, a second analysis is performed at the end of the 2-year extension period to summarize long-term efficacy, safety, and PK outcomes.

EXAMPLE 2: Data from a single-arm, multicenter, phase 3 study of ravulizumab (ALXN1210) in complement inhibitor-naïve adults with atypical hemolytic uremic syndrome (aHUS)

The following is a summary of data from one study of ravulizumab (ALXN1210-aHUS-311) in complement inhibitor-naïve patients with atypical hemolytic uremic syndrome (aHUS), which was conducted essentially according to the protocol described in Example 1 above. The initial assessment period was 26 weeks, followed by an extension period of up to 2 years. The design of this study is presented in Fig. 1.

The purpose of this study was to evaluate the effectiveness of ravulizumab in complement inhibitor-naive adult patients with aHUS in inhibiting complement-mediated thrombotic microangiopathy (TMA), characterized by thrombocytopenia, hemolysis, and renal dysfunction. The primary outcome measure was complete TMA response within the 26-week initial evaluation period. The primary, secondary, and safety endpoints for this study are summarized in Figure 2. A summary of the inclusion and exclusion criteria is presented in Figure 3 .

Enrollment requirements were as follows: (1) at least 6 adolescents (deferred to ALXN1210-aHUS-312), (2) at least 10 patients with prior kidney transplant (8 enrolled), and (3) at least 30 patients meeting criteria for laboratory evaluation of TMA on day 1 based on central laboratory results (32 enrolled).

The scope of data collected includes data from the initial assessment period for all patients, as well as any available data from the extension period up to and including October 2019.

Fifty-eight (58) subjects were enrolled and received at least one dose (included in the safety analysis set), two subjects were enrolled and replaced (determined ineligible after the first dose and excluded as predefined in the protocol), fifty-six (56 )subjects were included in the full analysis sample, eleven (11) subjects discontinued treatment, and nine (9) subjects discontinued participation in this study. The distribution of patients is shown schematically in Figure 4 . Treatment compliance was 100%. Baseline demographics are listed in Table 8, baseline disease scores are listed in Table 9, and baseline laboratory scores are listed in Table 10.

Definitions of TMA response are listed in Table 11. Response was achieved when all criteria were met simultaneously and each criterion was met for at least 28 days. Platelet levels obtained from the day of blood platelet transfusion to day 3 after transfusion were excluded from all analyses. All serum creatinine levels obtained while the patient was on dialysis were excluded from all analyses. If the patient was on dialysis at baseline, the first acceptable creatinine value used as the baseline value was the first assessment ≥6 days after dialysis. If the patient was on dialysis for the entire 26-week initial assessment period, baseline creatinine levels were not calculated.

The key results of the performance analysis are shown in Tables 12-14. Figure 5 presents a derivation example showing the results of a confirmed TMA complete response at day 57, including platelet normalization at day 112, LDH normalization at day 35, and creatinine improvement of ≥ 25% at day 70.

TMA complete response criteria were achieved when all criteria were met simultaneously and each criterion was met for at least 28 days. TMA complete response components, as well as other secondary efficacy endpoints, indicate sustained response to treatment. As shown in Figure 9, 53.6% (30/56) of patients achieved a complete TMA response during the initial evaluation period. 33.9% (19/56) of patients had a partial response.

Normalization of hematological parameters includes simultaneous normalization of platelet counts and normalization of LDH levels. Compliance with each criterion lasted for at least 28 days. 73.2% (41/56) of subjects achieved normalization of hematological parameters during the initial evaluation period (see Table 12). 83.9% (47/56) of subjects achieved platelet count normalization during the initial assessment period (see Table 12, Figure 10, Figure 14 and Figure 15).

76.8% (43/56) of subjects achieved LDH normalization during the initial assessment period (see Table 12, Figure 10, Figure 16 and Figure 17).

As shown in Figure 6, there were thirty (30) subjects with a complete TMA response. Forty (40) of fifty-six (56) subjects (71.4%) achieved a hemoglobin (Hb) response (≥ 20 g/L increase). Of the thirty (30) complete TMA respondents, four (4) had no hemoglobin (Hb) response. Figure 10 shows TMA complete response overall and by subgroup during the 26-week primary assessment period.

In FIG. Figure 7 shows the time to complete TMA response. The median time to complete TMA response was 86 days. Patients who did not respond were screened on the day of the last visit or study termination.

Figure 11 shows TMA complete response status over time (open circle), including normalization of platelet count (open triangle), normalization of hematology parameters (+), 25% improvement in serum creatinine compared to baseline (open square) and normalization of LDH (X).

Seven (7) subjects had no response. Results for subjects who did not respond are shown in Table 13.

As shown in Table 14, 58.6% (17/29) of patients who were on dialysis at baseline were off dialysis at the time of last available follow-up. Of the 27 patients who were not on dialysis at baseline, 21 (78%) patients were still not on dialysis at last follow-up.

Regarding pharmacokinetics/pharmacodynamics, 99.53% of all free C5 concentration results obtained after the first dose during the primary evaluation period were ≤ 0.5 mg/ml, the established threshold for terminal complement inhibition (see Fig. 22) . Body weight-based dosing resulted in maximum, steady-state, and trough drug concentrations as predicted, without unexpected pharmacokinetic results (see FIG. 8).

Key findings from the efficacy analysis are shown in Table 15. The safety population included all patients who received ≥ 1 dose of study drug (N=58). Two of these patients were excluded from the per-protocol efficacy analysis due to ineligibility (STEC-HUS identification).

Four deaths occurred: 1 due to an adverse event prior to treatment (cerebral artery thrombosis) and 3 due to adverse events unrelated to treatment (2 septic shock, 1 intracranial hemorrhage). A summary of mortality information for patients who received at least one dose of ravulizumab is shown in Table 16.

As shown in Table 17, the most common adverse events were headache (N=21), diarrhea (N=18), vomiting (N=15), nausea (N=13), and hypertension (N=13). As shown in Table 18, the most common serious adverse event was pneumonia (N=3). Three subjects discontinued participation in the study due to an adverse event. There were no cases of meningococcal disease.

71.4% (40/56) of subjects achieved a hemoglobin (Hb) response during the primary assessment period (see Table 19 and Figure 6). Figure 18 shows the observed and model-based mean changes in Hb levels from baseline and 95% confidence intervals over time. Figure 19 shows the observed mean Hb level and 95% confidence interval over time.

Chronic kidney disease (CKD) stage is classified based on the US National Kidney Foundation stages of chronic kidney disease. The stages of CKD and the corresponding estimated glomerular filtration rate (eGFR) values are as follows: Stage 1: eGFR >= 90 (normal), Stage 2: eGFR = 60-89, Stage 3A: eGFR = 45-59, Stage 3B: eGFR = 30- 44, Stage 4: eGFR = 15–29 and stage 5: eGFR < 15 (including dialysis: End stage). Stage 1 is considered the best category. Stage 5 is considered the worst category. Improved eGFR (eg, >15) corresponds to improved CKD stage (eg, lower CKD stage).

Figure 12 shows the mean eGFR compared with baseline and the 95% confidence interval. Figure 13 shows the shift in CKD/eGFR categories from baseline to day 183. Data are presented as n (%). EGFR categories are shown in ml/min/1.73 m ² . Baseline values were calculated based on the last available eGFR value before treatment. The lower triangle (indicated by slashes) represents improvement from baseline to day 183, the upper triangle (indicated by dots) represents deterioration, and white cells represent no change.

In addition, as shown in Table 20 and Figure 13, thirty-two (32) of forty-seven (47) subjects had improved CKD stage by day 183 compared to baseline (six by 5 stages, seven - at stage 4, five - at stage 3, four - at stage 2 and ten - at stage 1). Thirteen (13) of forty-seven (47) subjects had no changes. The condition of two (2) of thirteen (13) worsened.

Table 21 and FIG. 20 show the change in fatigue scores over time. Data in FIG. 20 are presented as mean (95% CI error bars). There was a rapid improvement in fatigue scores, i.e. average improvement by 9 points by day 8. A clinically significant improvement in fatigue (≥ 3 points) was observed in 84.1% (37/44) of patients on day 183. The average increase was 20 points compared to the initial level by the 183rd day.

The EQ-5D-3L is scored using the ratio derived from the United States Time Trade-Off Questionnaire (US TTO) score as well as the Visual Analog Scale (VAS) response. US TTO > 0.94 indicates full health. The initial level represents the value for day 1. Figure 21 shows the observed mean EQ-5D-3L score and 95% confidence interval. Regarding immunogenicity, one patient had a treatment-related positive anti-drug antibody (ADA) test result with no neutralizing antibodies and no apparent effect on pharmacokinetics/pharmacodynamics (see Table 22).

Figure 22 shows serum concentrations of free complement C5 over time (semi-log scale). The dotted horizontal line represents a serum free C5 concentration of 0.5 μg/mL, with complete terminal complement inhibition defined as a serum free C5 concentration of ≤ 0.5 μg/mL. Subsequent samples of free C5 for day 1 were excluded because they were considered biologically implausible. Exceptions were confirmed by paired FC data because samples for PK and free C5 analysis were taken from the same blood draw. [N=2, samples from day 1 before dosing / N=1, sample from day 1 after completion of infusion]. As shown in FIG. 22, ravulizumab demonstrated immediate, complete, and sustained inhibition of terminal complement over an 8-week dosing interval.

Finally, the study population in this ravulizumab study (ALXN1210-aHUS-311) and the eculizumab adult study (C10-004) was similar at day 183. However, as shown in Figure 23, ravulizumab resulted in improved performance compared to eculizumab in the following secondary clinical parameters: (a) eGFR category/chronic kidney disease (CKD) stage and (b) increase in estimated glomerular filtration rate (eGFR). . Specifically, of patients treated with ravulizumab in this study, 68% achieved an improvement in the eGFR/CKD stage category of at least one stage, and the mean eGFR increase was 35 ± 35. In contrast, of patients treated with eculizumab in study C10-004, only 63% achieved an eGFR/CKD stage improvement of at least one stage, and the mean eGFR increase was 29 ± 24. “TMA Complete Response” in this study is equivalent to “modified TMA Complete Response” in C10-004.

Thus, ravulizumab provided immediate and complete C5 inhibition, which was maintained over the 8-week dosing interval. Complete TMA response was achieved in 54% of patients, which is similar to eculizumab (56% in study C10-004). There was a rapid improvement in platelet count. In addition, kidney function improved significantly. Specifically, 58.6% of patients on dialysis at baseline did not require dialysis at the end of this study. Moreover, no unexpected safety issues were identified. In summary, the results of this study support the use of ravulizumab at 8-week dosing intervals in adult patients with complement-mediated TMA.

--->

LIST OF SEQUENCES

<110> ALEXION PHARMACEUTICALS, INC.

<120> DOSES AND ADMINISTRATION OF ANTI-C5 ANTIBODIES FOR THE TREATMENT OF ATYPICAL HEMOLYTIC-UREMIC SYNDROME (AGUS)

<130>AXJ-257PC

<140> PCT/US2020/014998

<141> 2020-01-24

<160> 50

<170> Patent In version 3.5

<210> 1

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Gly Tyr Ile Phe Ser Asn Tyr Trp Ile Gln

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

1 5 10 15

Asp

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

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Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn

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Gly Ala Thr Asn Leu Ala Asp

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Gln Asn Val Leu Asn Thr Pro Leu Thr

15

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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

115 120

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

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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

100 105

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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 Asn Phe Gly Thr Gln Thr

65 70 75 80

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

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ser Leu Ser Leu Gly Lys

325

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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

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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 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

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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 His 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 His 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

115 120

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

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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 Asn Phe Gly Thr Gln Thr

65 70 75 80

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

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ser Leu Ser Leu Gly Lys

325

<210> 14

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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 His 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 His 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 Leu His Glu Ala

420 425 430

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

435 440 445

<210> 15

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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 Thr Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

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

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

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

290 295 300

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

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 16

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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 Thr 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 Tyr Ile Thr Arg

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg 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 Met Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Gln 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 Pro Gly Lys

435 440 445

<210> 17

<211> 11

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Gly Ala Ser Glu Asn Ile Tyr His Ala Leu Asn

1 5 10

<210> 18

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

1 5 10 15

Asp

<210> 19

<211> 10

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Gly His Ile Phe Ser Asn Tyr Trp Ile Gln

1 5 10

<210> 20

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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 His 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 His 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> 21

<211> 5

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Ser Tyr Ala Ile Ser

15

<210> 22

<211> 17

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Gly Ile Gly Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 23

<211> 7

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Asp Thr Pro Tyr Phe Asp Tyr

15

<210> 24

<211> 11

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Ser Gly Asp Ser Ile Pro Asn Tyr Tyr Val Tyr

1 5 10

<210> 25

<211> 7

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Asp Asp Ser Asn Arg Pro Ser

15

<210> 26

<211> 11

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<223> synthetic

<400> 26

Gln Ser Phe Asp Ser Ser Leu Asn Ala Glu Val

1 5 10

<210> 27

<211> 116

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 27

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Gly Gly Ile Gly Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

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

100 105 110

Thr Val Ser Ser

115

<210> 28

<211> 108

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

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

85 90 95

Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 29

<211> 4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 29

Asn Tyr Ile Ser

1

<210> 30

<211> 17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 30

Ile Ile Asp Pro Asp Asp Ser Tyr Thr Glu Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 31

<211> 8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 31

Tyr Glu Tyr Gly Gly Phe Asp Ile

15

<210> 32

<211> 11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 32

Ser Gly Asp Asn Ile Gly Asn Ser Tyr Val His

1 5 10

<210> 33

<211> 7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 33

Lys Asp Asn Asp Arg Pro Ser

15

<210> 34

<211> 9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 34

Gly Thr Tyr Asp Ile Glu Ser Tyr Val

15

<210> 35

<211> 116

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 35

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

1 5 10 15

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

20 25 30

Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly

35 40 45

Ile Ile Asp Pro Asp Asp Ser Tyr Thr Glu Tyr Ser Pro Ser Phe Gln

50 55 60

Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu

65 70 75 80

Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Tyr Glu Tyr Gly Gly Phe Asp Ile Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 36

<211> 106

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

Lys Asp Asn Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 37

<211> 6

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 37

Ser Ser Tyr Tyr Val Ala

15

<210> 38

<211> 17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 38

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

1 5 10 15

Gly

<210> 39

<211> 13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 39

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

1 5 10

<210> 40

<211> 11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 40

Gln Ala Ser Gln Asn Ile Gly Ser Ser Leu Ala

1 5 10

<210> 41

<211> 7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 41

Gly Ala Ser Lys Thr His Ser

15

<210> 42

<211> 12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 42

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

1 5 10

<210> 43

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ala Lys Gly Arg Phe 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 Gly Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

<211> 110

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 44

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

1 5 10 15

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

20 25 30

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

35 40 45

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

85 90 95

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

100 105 110

<210> 45

<211> 451

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 45

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 Ser Ala Ser Thr Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

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

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Pro

450

<210> 46

<211> 217

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 46

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 Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 47

<211> 120

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 47

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ser Ser Ser

20 25 30

Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Ser Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Glu Gly Asn Val Asp Thr Thr Met Ile Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 107

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 48

Ala 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 Arg Asn Asp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ala Gly

50 55 60

Arg 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 Leu Gln Asp Phe Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 447

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 49

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ser Ser Ser

20 25 30

Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Ser Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Glu Gly Asn Val Asp Thr Thr Met Ile Phe Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 50

<211> 214

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 50

Ala 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 Arg Asn Asp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ala Gly

50 55 60

Arg 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 Leu Gln Asp Phe Asn Tyr Pro Trp

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 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

<---

Claims (64)

1. A method of treating atypical hemolytic uremic syndrome (aHUS) in an adult human patient 18 years of age or older, comprising administering to said patient an effective amount of an anti-C5 antibody, wherein the anti-C5 antibody is an antibody comprising a heavy chain polypeptide containing an amino acid sequence set forth in SEQ ID NO: 14, a light chain polypeptide containing the amino acid sequence set forth in SEQ ID NO: 11, wherein said anti-C5 antibody is administered: (a) once on the 1st day at a dose of: 2400 mg - to a patient with a body weight from ≥ 40 to < 60 kg, 2700 mg - to a patient with a body weight from ≥ 60 to < 100 kg, or 3000 mg - to a patient with body weight ≥ 100 kg; And (b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg - for a patient weighing ≥ 100 kg. 2. A method of treating atypical hemolytic uremic syndrome (aHUS) in an adult human patient 18 years of age or older, comprising administering to said patient an effective amount of an anti-C5 antibody, wherein the anti-C5 antibody is ravulizumab, wherein said anti-C5 antibody is administered: (a) once on the 1st day at a dose of: 2400 mg - to a patient with a body weight from ≥ 40 to < 60 kg, 2700 mg - to a patient with a body weight from ≥ 60 to < 100 kg, or 3000 mg - to a patient with body weight ≥ 100 kg; And (b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg - for a patient weighing ≥ 100 kg. 3. The method according to claim 1 or 2, characterized in that said patient has previously received treatment with eculizumab. 4. The method according to any of the preceding claims, characterized in that treatment is started at least two weeks after the last dose of eculizumab has been administered to said patient. 5. The method according to any of the preceding claims, characterized in that said patient has received treatment with eculizumab for at least 6 months prior to the 1st day of treatment. 6. The method according to any of the preceding claims, characterized in that said patient has previously received treatment with eculizumab at a dose of 900 mg every 2 weeks. 7. The method according to any of the preceding claims, characterized in that said anti-C5 antibody binds to human C5 at pH 7.4 and 25°C with an affinity dissociation constant (K _D ) that ranges from 0.1 nM to 1 nM. 8. The method according to any of the preceding claims, characterized in that said anti-C5 antibody binds to human C5 at pH 6.0 and 25°C with a K _D ≥ 10 nM. 9. The method according to any of the preceding claims, characterized in that said anti-C5 antibody is administered to a patient weighing ≥ 40 to < 60 kg: (a) once on the 1st day at a dose of: 2400 mg; And (b) on the 15th day and thereafter - every eight weeks at a dose of 3000 mg. 10. Method according to any one of paragraphs. 1-8, characterized in that said anti-C5 antibody is administered to a patient with a body weight from ≥ 60 to < 100 kg: (a) once on the 1st day at a dose of: 2700 mg; And (b) on the 15th day and thereafter - every eight weeks at a dose of 3300 mg. 11. Method according to any one of paragraphs. 1-8, characterized in that said anti-C5 antibody is administered to a patient weighing ≥ 100 kg: (a) once on the 1st day at a dose of: 3000 mg; And (b) on the 15th day and thereafter - every eight weeks at a dose of 3600 mg. 12. A method according to any one of the preceding claims, wherein said treatment maintains a residual serum concentration of said anti-C5 antibody at 100 μg/ml or more during treatment. 13. A method according to any one of the preceding claims, wherein said treatment maintains a residual serum concentration of said anti-C5 antibody at 200 μg/ml or greater during treatment. 14. The method of any one of the preceding claims, wherein said treatment maintains the concentration of free C5 at a level of 0.309 to 0.5 μg/ml or lower. 15. A method according to any one of the preceding claims, characterized in that said treatment reduces the concentration of free C5 by more than 99% during the treatment period. 16. A method according to any one of the preceding claims, characterized in that said treatment reduces the concentration of free C5 by more than 99.5% during the treatment period. 17. A method according to any one of the preceding claims, wherein said anti-C5 antibody is administered at a dose of 3000 mg, 3300 mg or 3600 mg every eight weeks for a period of up to two years. 18. The method according to any of the preceding claims, characterized in that said anti-C5 antibody is formulated for intravenous administration. 19. The method according to any of the preceding claims, characterized in that said treatment is a total of 26 weeks of treatment. 20. A method according to any of the preceding claims, characterized in that said treatment results in inhibition of terminal complement. 21. A method as claimed in any one of the preceding claims, wherein said treatment results in a reduction in hemolysis compared to baseline based on an assessment of lactate dehydrogenase (LDH) levels. 22. A method according to any of the preceding claims, characterized in that said treatment results in normalization of LDH levels. 23. A method according to any of the preceding claims, characterized in that said treatment results in the elimination of breakthrough hemolysis during the treatment period. 24. The method of any one of the preceding claims, wherein said treatment causes a shift toward normal levels of a hemolysis-related hematologic biomarker selected from the group consisting of free hemoglobin, haptoglobin, reticulocyte count, PNH clone of red blood cells, and D-dimer. 25. The method according to any of the preceding claims, characterized in that said treatment produces at least one therapeutic effect selected from the group consisting of reduction or cessation of severe hypertension, proteinuria, uremia, lethargy, fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia and renal dysfunction compared with baseline. 26. The method of any of the preceding claims, wherein said treatment causes a shift toward normal levels of factor Ba, soluble tumor necrosis factor receptor 1 [sTNFR1], soluble vascular adhesion molecule 1 [sVCAM1], thrombomodulin, D-dimer and cystatin WITH. 27. A method according to any of the preceding claims, characterized in that said treatment causes an increase in hemoglobin stabilization compared to the initial level. 28. A method according to any of the preceding claims, characterized in that said treatment causes a reduction in the need for blood transfusions compared to the baseline. 29. A method according to any one of the preceding claims, characterized in that said treatment causes a reduction in the number of major adverse vascular events (MAVE - English: major adverse vascular events). 30. A method according to any of the preceding claims, characterized in that said treatment causes a change in quality of life compared to baseline, assessed using the Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Index version 4 and the European Organization for Research and Treatment questionnaire malignant tumors to assess quality of life from 30 questions. 31. A method according to any of the preceding claims, characterized in that said treatment leads to normalization of platelet levels. 32. A method as claimed in any one of the preceding claims, wherein said treatment results in an improvement in serum creatinine levels by ≥ 25% compared to baseline. 33. A method according to any of the preceding claims, characterized in that said treatment results in a complete response of the TMA. 34. A method according to any of the preceding claims, characterized in that said treatment results in a modified complete response of thrombotic microangiopathy (TMA). 35. The method of any one of the preceding claims, wherein the patient's chronic kidney disease (CKD) improves by one or more stages after initiation of treatment. 36. A method as claimed in any one of the preceding claims, wherein said treatment results in a shift toward normal eGFR levels. 37. The method of any one of the preceding claims, wherein said treatment results in an EQ-5D-3L US TTO value of >0.94. 38. Use of a kit for the treatment of atypical hemolytic uremic syndrome (aHUS) in an adult human patient aged 18 years or older, containing: (a) a dose of an anti-C5 antibody, wherein the anti-C5 antibody is ravulizumab; And (b) instructions for the use of ravulizumab according to the method according to claim 2, in this case, ravulizumab is administered 1) once on the 1st day at a dose of: 2400 mg - to a patient with a body weight from ≥ 40 to < 60 kg, 2700 mg - to a patient with a body weight from ≥ 60 to < 100 kg, or 3000 mg – for a patient weighing ≥ 100 kg; And 2) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg - for a patient with a body weight from ≥ 40 to < 60 kg, 3300 mg - for a patient with a body weight from ≥ 60 to < 100 kg, or 3600 mg – for a patient weighing ≥ 100 kg. 39. Use according to claim 38, characterized in that said anti-C5 antibody is administered to a patient with a body weight from ≥ 40 to < 60 kg: (a) once on the 1st day at a dose of 2400 mg; And (b) on the 15th day. 40. Use according to claim 38, characterized in that said anti-C5 antibody is administered to a patient with a body weight from ≥ 60 to < 100 kg: (a) once on the 1st day at a dose of 2700 mg; And (b) on the 15th day and thereafter - every eight weeks at a dose of 3300 mg. 41. Use according to claim 40, characterized in that said anti-C5 antibody is administered to a patient weighing ≥ 100 kg: (a) once on the 1st day at a dose of 3000 mg; And (b) on the 15th day and thereafter - every eight weeks at a dose of 3600 mg. 42. The use of an anti-C5 antibody for the treatment of atypical hemolytic uremic syndrome (aHUS) in an adult human patient aged 18 years or older, wherein the anti-C5 antibody is ravulizumab, wherein said anti-C5 antibody is administered: (a) once on the 1st day at a dose of: 2400 mg - to a patient with a body weight from ≥ 40 to < 60 kg, 2700 mg - to a patient with a body weight from ≥ 60 to < 100 kg, or 3000 mg - to a patient with body weight ≥ 100 kg; And (b) on the 15th day and thereafter - every eight weeks at a dose of: 3000 mg for a patient weighing ≥ 40 to < 60 kg, 3300 mg for a patient weighing ≥ 60 to < 100 kg, or 3600 mg - for a patient weighing ≥ 100 kg.