CN109312326A - Anti- C5 antibody and application method - Google Patents

Anti- C5 antibody and application method Download PDF

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Publication number
CN109312326A
CN109312326A CN201780036916.4A CN201780036916A CN109312326A CN 109312326 A CN109312326 A CN 109312326A CN 201780036916 A CN201780036916 A CN 201780036916A CN 109312326 A CN109312326 A CN 109312326A
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antibody
seq
amino acid
hvr
acid sequence
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CN109312326B (en
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类家庆直
三瓶全次郎
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Chugai Pharmaceutical Co Ltd
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Chugai Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The present invention provides anti-C5 antibody and its application method.In some embodiments, the anti-C5 antibody of separation of the invention combines the affinity of the epitope in the β chain of C5 higher than at acidic under neutral ph.The present invention also provides the nucleic acid of the coding of separation anti-C5 antibody of the invention.The present invention also provides host cell, the host cell includes nucleic acid of the invention.The present invention also provides the methods for preparing antibody, and the method includes cultivating host cell of the invention to prepare the antibody.The present invention also provides the methods for preparing anti-C5 antibody, and the method includes the polypeptide immune animals of the MG1-MG2 structural domain for the β chain comprising C5.Anti- C5 antibody of the invention may be used as drug.

Description

Anti- C5 antibody and application method
Technical field
The present invention relates to anti-C5 antibody and its application methods.
Background technique
Complement system is in the removing of immune complex and to infective agent, exogenous antigen, and the cell and tumour of virus infection are thin It plays a crucial role in the immune response of born of the same parents.In the presence of about 25-30 kind complement protein, it is found to be plasma protein and film co-factor Compound set.Complement component realizes that it is immune anti-and interacting in the digestion of a series of complex and film binding events Imperial function.Generated complement cascade causes to generate the product with opsonin, immunological regulation and bacteriolyze function.
Currently, being widely accepted, complement system can be activated by three kinds of different approach: classical pathway, be coagulated Collect plain approach and alternative pathway.These approach share many components, although and its initial step it is different, it is concentrated and is total to There is the late complement component (C5 to C9) of identical responsible activation and destruction target cell.
Classical pathway is usually activated and forming antigen-antibody complex.Independently, the of lectin pathway activation One step is specific agglutinin such as mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin and C- The combination of type agglutinin CL-11.On the contrary, the low-level revolution activation of the spontaneous carry out of alternative pathway, this can be easily in external source Or it is amplified on other abnormal surfaces (bacterium, yeast, the cell of virus infection, or the tissue of damage).These approach converge at A bit, i.e. complement component C3 is cut to generate C3a and C3b by active protease.
C3a is a kind of anaphylatoxin.C3b combination bacterium and other cells and certain viruses and immune complex, and It is marked as removing (being known as opsonic effect) in self-loopa.C3b forms compound with other components also to be formed C5 is cut into C5a and C5b by C5 convertase, the enzyme.
C5 is the albumen of the 190kDa of the about 80 μ g/ml (0.4 μM) found in normal serum.About 1.5-3% in C5 The quality for being attributed to carbohydrate be glycosylated.Mature C5 be disulfide bond connection 115kDa α chain and 75kDa β chain it is different Source dimer.C5 be synthesized into 1676 amino acid single chain precursor protein (pro-C5 precursor) (see, for example, PTL1 and PTL2).Pro-C5 precursor generates the β chain as n terminal fragment and the α chain as carboxyl-terminal fragment by cutting.α chain and β Chain polypeptide fragment is connected to each other via disulfide bond and constitutes mature C5 albumen.
Mature C5 is cut into C5a and C5b segment during the activation of complement pathway.C5a is by C5 convertase from the α of C5 Chain cutting, is the n terminal fragment as preceding 74 amino acid comprising α chain.The remainder of mature C5 is segment C5b, It contains the remaining α chain and β chain connected through disulfide bond.About 20% in the 11kDa molecular weight of C5a is attributed to carbohydrate.
C5a is another anaphylatoxin.C5b and C6, C7, C8 and C9 are combined and are formed membrane attack complex (MAC, C5b- 9, in the final complement complex (TCC) of target cell surface).When in sufficient amount of MAC insertion target cell membrane, formed MAC aperture is to mediate the rapid osmotic lysis of target cell.
As mentioned above, C3a and C5a is anaphylatoxin.It can trigger mast cell threshing, this discharges histamine and its His inflammatory mediator leads to smooth muscle contraction, and vascular permeability increases, leukocyte activation and other inflammatory phenomenons, including leads to cell Excessive cell Proliferation.C5a acts also as Chemotactic Peptide, is used to attract granulocyte such as neutrophil cell, eosinocyte, basophilic Property granulocyte and monocyte are to complement activation site.
The activity of C5a is adjusted by plasma enzymes carboxypeptidase N, and the enzyme removes carboxyl terminal arginine from C5a, to form C5a- Des-Arg derivative.C5a-des-Arg shows 1% anaphylactic activity and polymorphonuclear chemotactic activity of only unmodified C5a.
The strong defence for microbial infection of normally functioning complement system offer, and the unsuitable adjusting of complement Or activation involves the morbidity of various diseases, the illness includes, for example, rheumatoid arthritis (rheumatoid Arthritis, RA)；Lupus nephritis (lupus nephritis)；Ischemical reperfusion injury (ischemia-reperfusion injury)；Paroxysmal nocturnal hemoglobinuria (paroxysmal nocturnal hemoglobinuria, PNH)；Atypia Hemolytic uremic syndrome (atypical hemolytic uremic syndrome, aHUS)；Dense sediment disease (dense deposit disease, DDD)；Macular degeneration is (for example, age-related macular degeneration (age-related Macular degeneration, AMD))；Haemolysis (hemolysis), liver enzyme increase (elevated liver enzymes), With low platelet (HELLP) syndrome；Thrombotic thrombocytopenic purpura (thrombotic thrombocytopenic Purpura, TTP)；Spontaneous abortion (spontaneous fetal loss)；Skeptophylaxis vasculitis (Pauci-immune vasculitis)；Epidermolysis bullosa (epidermolysis bullosa)；Recidivity miscarriage (recurrent fetal loss)；Multiple sclerosis (multiple sclerosis, MS)；Traumatic brain injury (traumatic brain injury)； With by myocardial infarction (myocardial infarction), cardiopulmonary bypass (cardiopulmonary bypass) and blood It dialyses and damages (see for example, NPL 1) caused by (hemodialysis).Therefore, inhibit excessive or uncontrolled complement activation Cascade can provide clinical benefit for the patient with the illness.
Paroxysmal nocturnal hemoglobinuria (PNH) is a kind of uncommon hematologic disease, wherein red blood cell it is impaired and because This is quickly destroyed than normocyte.PNH is due to having PIG-A (the glypican A being located on X chromosome Type) gene body mutation candidate stem cell clonal expansion caused by.The mutation of PIG-A leads to glycosyl-phosphatidyl inositol (GPI) The early stage of synthesis blocks, which is that many albumen are anchored to required for cell surface.Therefore, PNH haemocyte lacks GPI- The albumen of anchoring, the albumen include complement-regulatory protein CD55 and CD59.Under normal operation, these complement-regulatory proteins The formation of MAC on cell surface is blocked, red blood cell lysis is thus prevented.In PNH, there is no the albumen of GPI- anchoring to cause to mend Body-mediation haemolysis.
PNH is characterized as being hemolytic anemia (reduction of red blood cell quantity), and (there are hemoglobins in urine, sleep for hemoglobinuria It is especially apparent after dormancy) and haemoglobinaemia (there are hemoglobins in blood flow).The known individual disturbed by PNH has prominent So breaking-out is defined here as the generation of dark urine.Hemolytic anemia is the blood due to the red blood cell by complement component Caused by being destroyed in pipe.Other known symptom includes dysphasia, fatigue, erectile dysfunction, thrombosis and recurrent Abdominal pain.
It according to the Humanized monoclonal antibodies that library pearl monoclonal antibody (Eculizumab) is for complement protein C5, and is the first It is approved for the therapy for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and Atypical Hemolytic Uremic Syndrome (aHUS) (see for example, NPL 2).Inhibit C5 convertase that C5 is cut into C5a and C5b according to library pearl monoclonal antibody, this prevents final complement Compound C5b-9.C5a and C5b-9 result in the advanced stage complement-mediated for being characterized as PNH and aHUS event (also see, PTL 3, PTL 4, PTL 5 and PTL 6).
Anti- C5 antibody has been described in several reports.For example, PTL 7 describes the α chain in conjunction with C5 but does not combine C5a And the anti-C5 antibody of the activation of C5 is blocked, and PTL 8 describes the anti-C5 monoclonal antibody for inhibiting C5a to be formed.Another party Face, PTL 9 describe the proteolysis sites of the C5 convertase on the α chain of identification C5 and C5 are inhibited to convert to C5a and C5b Anti- C5 antibody.It is at least 1x10 that PTL 10, which describes affinity constant,7M-1Anti- C5 antibody.
Antibody (IgG) combines neonatal Fc receptor (FcRn), and has long blood plasma retention time.Typically in acidity The combination of IgG and FcRn are observed under condition (for example, pH 6.0), and are almost seen at neutrallty condition (for example, pH 7.4) Examine less than.Typically, IgG is non-specifically entered in cell via encytosis, and by combining in acid condition Inner body FcRn in inner body and return to cell surface.Then, IgG and FcRn is dissociated in neutral conditions in blood plasma.Do not tie The IgG for closing FcRn is decomposed in lysosome.When eliminating its FcRn under acid condition by introducing mutation in the area Fc to IgG When binding ability, IgG is not recycled in blood plasma from inner body, this significant damage for causing the blood plasma of IgG to retain.In order to improve The blood plasma of IgG retains, and enhances the method that its FcRn in acid condition is combined and has been reported.When by drawing to the area Fc of IgG Enter amino acid replacement come when improving its FcRn combination in acid condition, IgG is more effectively recycled to blood plasma from inner body, and And thus show that the blood plasma of raising retains.Meanwhile it is also reported that, the IgG that there is the FcRn of enhancing to combine in neutral conditions exists Do not dissociated in neutral conditions with FcRn in blood plasma, even its via its in inner body in acid condition in conjunction with FcRn And when returning to cell surface, and its blood plasma remains unchanged later, or is deteriorated (see for example, NPL 3；NPL4； NPL5)。
Recently, the antibody in a manner of pH dependence in conjunction with antigen has been reported (see for example, PTL11 and PTL 12).These Antibody consumingly combines antigen under the conditions of Plasma Neutral and dissociates under inner body acid condition with antigen.It is dissociated with antigen Afterwards, the antibody becomes able to recombine antigen when being recycled to blood plasma via FcRn.Therefore, single antibody molecule can be with Repeatedly combine multiple antigen molecules.In general, the blood plasma of antigen retains resisting than the recycling mechanism with above-mentioned FcRn mediation The blood plasma of body retains much shorter.Therefore, when antigen is in conjunction with antibody, antigen usually shows that extended blood plasma retains, to lead Cause the increase of antigen plasma concentration.On the other hand, it has been reported that, it is above-mentioned in a manner of pH dependence compared with typical antibody Antigen is eliminated from blood plasma more quickly in conjunction with the antibody of antigen, because including it is during the process recycling that FcRn is mediated It is dissociated in vivo with antigen.PTL 13 also describes computer modeling analysis, described analysis shows that having the pH dependence for C5 In conjunction with antibody can extend antigen and strike and subtract (knockdown).
Reference listing
Patent document
PTL 1: U.S. Patent number 6,355,245
PTL 2: U.S. Patent number 7,432,356
PTL 3:WO 2005/074607
PTL 4:WO 2007/106585
PTL 5:WO 2008/069889
PTL 6:WO 2010/054403
PTL 7:WO 95/29697
PTL8:WO 02/30985
PTL 9:WO 2004/007553
PTL 10:WO 2010/015608
PTL 11:WO 2009/125825
PTL12:WO 2011/122011
PTL 13:WO 2011/111007
Non-patent literature
NPL 1:Holers etc., Immunol.Rev.223:300-316 (2008)
(2008) NPL 2:Dmytrijuk etc., The Oncologist 13 (9): 993-1000
NPL 3:Yeung etc., J Immunol.182 (12): 7663-7671 (2009)
NPL 4:Datta-Mannan etc., J Biol.Chem.282 (3): 1709-1717 (2007)
NPL 5:Dall ' Acqua etc., J.Immunol.169 (9): 5171-5180 (2002)
Summary of the invention
Technical problem
It is an object of the present invention to provide anti-C5 antibody and its application methods.
Solution to problem
The present invention provides anti-C5 antibody and its application method.
In some embodiments, the epitope in the β chain of the anti-C5 antibody combination C5 of the invention of separation.In some realities It applies in scheme, the epitope in the MGl-MG2 structural domain of the β chain of the anti-C5 antibody combination C5 of the invention of separation.In some implementations In scheme, what the anti-C5 antibody of the invention of separation was formed in conjunction with the amino acid 33-124 of the β chain (SEQ ID NO:40) by C5 Epitope in segment.In some embodiments, the anti-C5 antibody combination C5 of the invention of separation β chain (SEQ ID NO: 40) epitope in, the epitope include the segment that at least one is selected from the group being made up of: amino acid 47-57,70-76, and 107-110.In some embodiments, the piece of the β chain (SEQ ID NO:40) of the anti-C5 antibody combination C5 of the invention of separation Epitope in section, the epitope include the amino acid residue that at least one is selected from the group being made up of: SEQ ID NO:40's Glu48, Asp51, His70, His72, Lys109 and His110.In a further embodiment, the antibody is under neutral ph Affinity in conjunction with C5 is higher than at acidic.In a further embodiment, the antibody combines the parent of C5 at pH7.4 It is higher in pH5.8 with power ratio.In another embodiment, antibody described in the anti-C5 antibody of the invention Yu table 2 of separation In conjunction with identical epitope.In a further embodiment, the antibody combines identical as antibody described in table 2 at pH7.4 Epitope affinity ratio it is higher in pH5.8.In another embodiment, anti-C5 antibody of the invention and institute in table 7 or 8 The antibody stated combines identical epitope.In a further embodiment, the antibody at pH7.4 combine with described in table 7 or 8 The identical epitope of antibody affinity ratio it is higher in pH5.8.
In certain embodiments, anti-C5 antibody of the invention and the antibody competition comprising VH selected from the following and VL couples In conjunction with C5:(a) VL of the VH and SEQ ID NO:11 of SEQ ID NO:1；(b) VH the and SEQ ID NO:15 of SEQ ID NO:5 VL；(c) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；(d) VH the and SEQ ID NO:16 of SEQ ID NO:6 VL；(e) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；(f) VL of the VH and SEQ ID NO:13 of SEQ ID NO:3； (g) VL of the VH and SEQ ID NO:19 of SEQ ID NO:9；(h) VL of the VH and SEQ ID NO:17 of SEQ ID NO:7；(i) The VL of the VH and SEQ ID NO:18 of SEQ ID NO:8；(j) VL of the VH and SEQ ID NO:20 of SEQ ID NO:10.? In other embodiments, anti-C5 antibody combines the affinity of C5 higher than at acidic under neutral ph.In other reality It applies in scheme, affinity ratio of the anti-C5 antibody at pH7.4 in conjunction with C5 is higher in pH5.8.
In some embodiments, the anti-C5 antibody of the invention of separation has the feature selected from the group being made up of: (a) the amino acid D51 and K109 of antibody contact C5 (SEQ ID NO:39)；(b) antibody to C5 (SEQ ID NO: 39) affinity is greater than the affinity for the C5 mutant that the antibody forms the E48A displacement by SEQ ID NO:39；Or (c) The antibody combines the C5 albumen being made of the amino acid sequence of SEQ ID NO:39 at pH7.4, but does not tie at pH7.4 Close the C5 albumen being made of the amino acid sequence of the SEQ ID NO:39 replaced with H72Y.In a further embodiment, institute State antibody combines the affinity of C5 higher than at acidic under neutral ph.In a further embodiment, the antibody exists Affinity ratio under pH7.4 in conjunction with C5 is higher in pH5.8.
In some embodiments, the anti-C5 antibody of the invention of separation inhibits the activation of C5.In some embodiments In, the anti-C5 antibody of the invention of separation inhibits the activation of C5 variant R885H.In some embodiments, the isolated present invention Anti- C5 antibody be monoclonal antibody.In some embodiments, the anti-C5 antibody of the invention of separation is people, humanization, Or chimeric antibody.In some embodiments, the anti-C5 antibody of the invention of separation is the antibody fragment in conjunction with C5.Some In embodiment, the anti-C5 antibody of the invention of separation is overall length IgG1 or IgG4 antibody.
In some embodiments, the anti-C5 antibody of the invention of separation includes (a) HVR-H3, and the HVR-H3 includes Amino acid sequence DX1GYX2X3PTHAMX4X5, wherein X1It is G or A, X2It is V, Q or D, X3It is T or Y, X4It is Y or H, X5It is L or Y (SEQ ID NO:128), (b) HVR-L3, the HVR-L3 include amino acid sequence QX1TX2VGSSYGNX3, wherein X1It is S, C, N or T, X2It is F or K, X3It is A, T or H (SEQ ID NO:131), and (c) HVR-H2, the HVR-H2 include amino acid sequence X1IX2TGSGAX3YX4AX5WX6KG, wherein X1It is C, A or G, X2It is Y or F, X3It is T, D or E, X4It is Y, K or Q, X5It is S, D or E, X6It is A or V (SEQ ID NO:127).
In some embodiments, the anti-C5 antibody of the invention of separation includes (a) HVR-H1, and the HVR-H1 includes SSYYX1X2, wherein X1It is M or V, X2It is C or A (SEQ ID NO:126), (b) HVR-H2, the HVR-H2 include amino acid sequence Arrange X1IX2TGSGAX3YX4AX5WX6KG, wherein X1It is C, A or G, X2It is Y or F, X3It is T, D or E, X4It is Y, K or Q, X5S, D or E, X6It is A or V (SEQ ID NO:127), and (c) HVR-H3, the HVR-H3 include amino acid sequence DX1GYX2X3PTHAMX4X5, wherein X1It is G or A, X2It is V, Q or D, X3It is T or Y, X4It is Y or H, X5Be L or Y (SEQ ID NO: 128).In a further embodiment, the antibody includes (a) HVR-L1, and the HVR-L1 includes amino acid sequence X1ASQX2IX3SX4LA, wherein X1It is Q or R, X2It is N, Q or G, X3It is G or S, X4It is D, K or S (SEQ ID NO:129)；(b) HVR-L2, the HVR-L2 include amino acid sequence GASX1X2X3S, wherein X1It is K, E or T, X2It is L or T, X3It is A, H, E or Q (SEQ ID NO:130)；(c) HVR-L3, the HVR-L3 include amino acid sequence QX1TX2VGSSYGNX3, wherein X1It is S, C, N or T, X2It is F or K, X3It is A, T or H (SEQ ID NO:131).
In some embodiments, the anti-C5 antibody of the invention of separation includes (a) HVR-L1, and the HVR-L1 includes Amino acid sequence X1ASQX2IX3SX4LA, wherein X1It is Q or R, X2It is N, Q or G, X3It is G or S, X4Be D, K or S (SEQ ID NO: 129)；(b) HVR-L2, the HVR-L2 include amino acid sequence GASX1X2X3S, wherein X1It is K, E or T, X2It is L or T, X3It is A, H, E or Q (SEQ ID NO:130)；(c) HVR-L3, the HVR-L3 include amino acid sequence QX1TX2VGSSYGNX3, Middle X1It is S, C, N or T, X2It is F or K, X3It is A, T or H (SEQ ID NO:131).
In some embodiments, the anti-C5 antibody of the invention of separation includes heavy-chain variable domains frame FR1, institute State the amino acid sequence that FR1 includes any of SEQ ID NO:132-134；FR2, the FR2 include SEQ ID NO:135- Any of 136 amino acid sequence；FR3, the FR3 include the amino acid sequence of any of SEQ ID NO:137-139； And FR4, the FR4 include the amino acid sequence of any of SEQ ID NO:140-141.In some embodiments, it separates Anti- C5 antibody of the invention include light variable domains frame FRi, the FR1 include SEQ ID NO:142-143 in appoint One amino acid sequence；FR2, the FR2 include the amino acid sequence of any of SEQ ID NO:144-145；FR3, institute State the amino acid sequence that FR3 includes any of SEQ ID NO:146-147；And FR4, the FR4 include SEQ ID NO:148 Amino acid sequence.
In some embodiments, the anti-C5 antibody of the invention of separation includes (a) VH sequence, the VH sequence and ammonia It is any in base acid sequence SEQ ID NO:10,106-110 that there is at least 95% sequence identity；(b) VL sequence, the VL sequence There is at least 95% sequence identity with any in amino acid sequence SEQ ID NO:20,111-113；Or (c) the VH sequence in (a) VL sequence in column and (b).In a further embodiment, the antibody includes SEQ ID NO:10, any of 106-110 VH sequence.In a further embodiment, the antibody includes the VL sequence of SEQ ID NO:20, any of 111-113.
The present invention provides antibody, and the antibody includes the VH sequence and SEQ of SEQ ID NO:10, any of 106-110 The VL sequence of ID NO:20, any of 111-113.
The present invention also provides isolated nucleic acid, encode anti-C5 antibody of the invention.The present invention also provides host cell, It includes nucleic acid of the invention.The present invention also provides the methods for preparing antibody, and the method includes the host for cultivating of the invention is thin Born of the same parents are to prepare the antibody.
The present invention also provides the methods for preparing anti-C5 antibody.In some embodiments, the method includes being directed to polypeptide Immune animal, the polypeptide include the MG1-MG2 structural domain (SEQ ID NO:43) of the β chain of C5.In some embodiments, institute State method include for polypeptide immune animal, the polypeptide include the β chain (SEQ ID NO:40) corresponding to C5 position 33 to The region of amino acid at 124.In some embodiments, the method includes being directed to polypeptide immune animal, the polypeptide packet The segment of amino acid 47-57,70-76 and 107-110 containing at least one β chain (SEQ ID NO:40) for being selected from C5.Some In embodiment, the method includes being directed to polypeptide immune animal, the polypeptide includes the piece of the β chain (SEQ ID NO:40) of C5 Section, the segment include the amino acid that at least one is selected from Glu48, Asp51, His70, His72, Lys109 and His110.
The present invention also provides pharmaceutical preparation, the pharmaceutical preparation includes anti-C5 antibody and pharmaceutical carrier of the invention.
Anti- C5 antibody of the invention may be used as drug.Anti- C5 antibody of the invention can be used for treating complement-mediated Disease is related to the illness of excessive or uncontrolled C5 activation.Anti- C5 antibody of the invention can be used for enhancing C5 from blood plasma Removing.
Anti- C5 antibody of the invention can be used for preparing drug.In some embodiments, the drug is mended for treating The disease or be related to the illness of excessive or uncontrolled C5 activation that body mediates.In some embodiments, the drug is used for Enhance removing of the C5 from blood plasma.
The disease of complement-mediated is suffered from the present invention also provides treatment or is related to the illness of excessive or uncontrolled C5 activation Individual method.In some embodiments, the method includes to apply a effective amount of anti-C5 of the invention to the individual Antibody.The present invention also provides the methods for enhancing the removing of C5 from blood plasma in individual.In some embodiments, the method packet Containing applying a effective amount of anti-C5 antibody of the invention to the individual to enhance removing of the C5 from blood plasma.
Brief description
[Fig. 1]
Fig. 1 shows the epitope frame of anti-C5 antibody simultaneously (binning), as described in embodiment 2.2.It is classified into identical table Antibody in position frame (bin) is added frame with thick line.
[Fig. 2A]
Fig. 2A shows the anti-C5 antibody for assessing pH- dependence pH7.4's (solid line) and pH5.8 (dotted line) BIACORE (registered trademark) influence chart, as described in embodiment 3.2.CFA0305, CFA0307, CFA0366, CFA0501, CFA0538 and CFA0599 is the antibody being grouped into epitope C, as described in embodiment 2.2.
[Fig. 2 B]
Fig. 2 B shows the anti-C5 antibody for assessing pH- dependence pH7.4's (solid line) and pH5.8 (dotted line) BIACORE (registered trademark) influence chart, as described in embodiment 3.2.CFA0666, CFA0672 and CFA0675 are to be grouped into Antibody in epitope C, CFA0330 and CFA0341 are the antibody being grouped into epitope B, as described in embodiment 2.2. 305LO5 is the humanized antibody of CFA0305, as described in embodiment 2.3.
[Fig. 3]
Fig. 3 is shown for segment (the amino acid 1 9- of SEQ ID NO:40 derived from the C5 beta chain with GST- tag fusion 180,161-340,321-500 and 481-660) western blot analysis, as described in embodiment 4.1.CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are to be grouped into table Antibody in the C of position.Anti- GST antibody is positive control.Fusion has the position arrow mark of the C5 segment (46-49kDa) of GST.
[Fig. 4]
Fig. 4 show anti-C5 antibody to BIACORE (registered trademark) influence chart of the MG1-MG2 structural domain of C5 beta chain, strictly according to the facts It applies described in example 4.3.Upper figure shows CFA0305 (solid line), CFA0307 (dotted line), CFA0366 (pecked line), and according to library pearl list The result of anti-(dotted line).Intermediate figure shows CFA0501 (solid line), CFA0599 (dotted line), CFA0538 (pecked line), He Yiku The result of pearl monoclonal antibody (dotted line).Following figure shows CFA0666 (solid line), CFA0672 (dotted line), CFA0675 (pecked line), and According to the result of library pearl monoclonal antibody (dotted line).CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are the antibody being grouped into epitope C.It is the anti-C5 antibody of control according to library pearl monoclonal antibody.
[Fig. 5 A]
Fig. 5 A is shown for the peptide fragment (ammonia of SEQ ID NO:40 derived from the MG1-MG2 structural domain with GST tag fusion Base acid 33-124,45-124,52-124,33-111,33-108 and 45-111) western blot analysis, in embodiment 4.4 It is described.Anti- GST antibody is used as reaction antibody.Fusion has the position arrow mark of the C5 segment (35-37kDa) of GST.
[Fig. 5 B]
Fig. 5 B show for peptide fragment derived from MG1-MG2 structural domain with GST- tag fusion (SEQ ID NO:40's Amino acid 33-124,45-124,52-124,33-111,33-108 and 45-111) western blot analysis, such as embodiment 4.4 Described in.CFA0305 is used as reaction antibody.
[Fig. 5 C]
Fig. 5 C outlines the association reaction of segment derived from anti-C5 antibody and C5 beta chain, as described in Example 4.4.With Be grouped into epitope C anti-C5 antibody (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675) combine segment with gray display, segment not in connection is with white displays.
[Fig. 6]
Fig. 6 shows the western blot analysis for C5 point mutation body, E48, the D51 of beta chain in the mutant, and K109 is replaced as alanine (respectively E48A, D51A and K109A), as described in embodiment 4.5.In left figure, according to library pearl Monoclonal antibody (anti-C5 antibody, α-link mixture) is used as reaction antibody, and the position arrow of α-chain (about 113kDa) of C5 Label.In right figure, CFA0305 (being grouped into epitope C, beta chain bonding agent) is used as reaction antibody, and the β-of C5 The position arrow mark of chain (about 74kDa).
[Fig. 7]
Fig. 7 shows BIACORE (registered trademark) influence chart, the figure show according to library pearl monoclonal antibody-F760G4 (above) or The interaction of 305LO5 (following figure) and C5 mutant, as described in embodiment 4.6.Influence chart passes through respectively that C5-wt is (solid Curve), C5-E48A (short imaginary curve), C5-D51A (long imaginary curve) and C5-K109A (thin solid-line curve) be injected at be fixed with according to It is obtained on the sensor surface of library pearl monoclonal antibody-F760G4 or 305LO5.It is the anti-C5 antibody of control according to library pearl monoclonal antibody.305LO5 is CFA0305 humanized antibody (is grouped into epitope C), as described in embodiment 2.3.
[Fig. 8]
Fig. 8 shows the BIACORE of the interaction of display 305LO5 and C5 His mutant for assessing pH- dependence (registered trademark) influence chart, as described in embodiment 4.7.Influence chart is by the way that by C5-wt (thick solid curve), C5-H70Y is (long respectively Imaginary curve), C5-H72Y (short imaginary curve), C5-H110Y (point curve) and C5-H70Y+H110Y (thin solid-line curve) are injected at admittedly Surely it is obtained on the sensor surface for having 305LO5.Antibody/antigen compound is allowed to dissociate in pH7.4, it is further in pH5.8 later Dissociation (arrow is signified) is to assess the interaction of pH- dependence.
[Fig. 9 A]
Fig. 9 A shows the inhibition of the liposome dissolving of the complement activation by anti-C5 antibody, as described in embodiment 5.1. Show CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, the CFA0666 being grouped into epitope C, CFA0672 and CFA0675's as a result, as described in embodiment 2.2.
[Fig. 9 B]
Fig. 9 B shows the inhibition of the liposome dissolving of the complement activation by anti-C5 antibody, as described in embodiment 5.1. Show the antibody CFA0330 that is grouped into epitope B and CFA0341's as a result, as described in embodiment 2.2.
[Figure 10 A]
Figure 10 A shows the inhibition of the C5a generation by anti-C5 antibody, as described in embodiment 5.2.It describes in figure 9 a Liposome dissolving measurement during quantify C5a concentration in the supernatant that obtains.
[Figure 10 B]
Figure 10 B shows the inhibition of the C5a generation by anti-C5 antibody, as described in embodiment 5.2.It describes in figures 9 b and 9 Liposome dissolving measurement during quantify C5a concentration in the supernatant that obtains.
[Figure 11]
Figure 11 shows the inhibition of the haemolysis of the complement activation by anti-C5 antibody, as described in embodiment 5.3.Complement warp It is activated by classical pathway.
[Figure 12]
Figure 12 shows the inhibition of the haemolysis of the complement activation by anti-C5 antibody, as described in embodiment 5.4.Complement warp It is activated by alternative pathway.
[Figure 13]
Figure 13 is shown in the individual people C5 or people C5 of application and Anti-Human C5 antibody in the mouse medium sized vein that assessment C5 is removed The time-histories of blood plasma people's C5 concentration afterwards, as described in embodiment 6.2.CFA0305,CFA0307,CFA0366,CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are the antibody being grouped into epitope C, and CFA0330 and CFA0341 is the antibody being grouped into epitope B, as described in embodiment 2.2.
[Figure 14]
Figure 14 is shown in the individual people C5 of application and Anti-Human C5 antibody in the mouse medium sized vein of assessment antibody pharmacokinetics The time-histories of blood plasma Anti-Human's C5 antibody concentration afterwards, as described in embodiment 6.3.CFA0305,CFA0307,CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are the antibody being grouped into epitope C, and CFA0330 and CFA0341 is the antibody being grouped into epitope B, as described in embodiment 2.2.
[Figure 15]
Figure 15 shows the inhibition of the liposome dissolving of the complement activation by anti-C5 antibody, as described in embodiment 9.1. Show antibody 305LO15-SG422,305LO16-SG422,305LO18-SG422,305LO19-SG422,305LO20- The result of SG422 and 305LO20-SG115.
[Figure 16]
Figure 16 shows the inhibition of the liposome dissolving of the complement activation by anti-C5 antibody, as described in embodiment 9.1. Show the result of antibody 305LO15-SG115 and 305LO23-SG429.
[Figure 17]
Figure 17 shows the inhibition of the liposome dissolving of the complement activation by anti-C5 antibody, as described in embodiment 9.1. Show antibody 305LO22-SG115, the result of 305LO22-SG422,305LO23-SG115 and 305LO23-SG422.
[Figure 18]
Figure 18 shows the inhibition of the C5a generation by anti-C5 antibody, as described in embodiment 9.2.Described in Figure 15 Quantify C5a concentration in the supernatant that liposome dissolving obtains during measuring.
[Figure 19]
Figure 19 shows the inhibition of the C5a generation by anti-C5 antibody, as described in embodiment 9.2.Described in Figure 16 Quantify C5a concentration in the supernatant that liposome dissolving obtains during measuring.
[Figure 20]
Figure 20 shows the inhibition of the complement activity in the monkey blood plasma by anti-C5 antibody, as described in embodiment 9.3.It is anti- C5 antibody is applied in machin (cynomolgus monkey), and the complement in monkey blood plasma is measured in haemolysis measurement Activity.
[Figure 21]
Figure 21 show by the wild type C5 (WT) and C5 variant of anti-C5 antibody (according to library pearl monoclonal antibody) (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D) biological activity inhibition, as described in embodiment 9.4.
[Figure 22]
Figure 22 show by the wild type C5 (WT) and C5 variant of anti-C5 antibody (305 variant) (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D) biological activity inhibition, as described in embodiment 9.4.
[Figure 23]
Figure 23 shows the inhibition of the liposome dissolving of the complement activation by anti-C5 antibody (BNJ441 and 305 variants), such as Described in embodiment 9.5.
[Figure 24]
The blood plasma machin C5 that Figure 24 is shown in the machin medium sized vein that assessment C5 is removed after application Anti-Human C5 antibody is dense The time-histories of degree, as described in embodiment 10.2.
[Figure 25]
Figure 25 is shown in the blood plasma in the machin medium sized vein of assessment antibody pharmacokinetics after application Anti-Human C5 antibody The time-histories of Anti-Human's C5 antibody concentration, as described in embodiment 10.3.
[Figure 26]
Figure 26 A and 26B show the crystal structure of the 305Fab in conjunction with people C5 (hC5)-MG1 structural domain, such as embodiment Described in 11.6.Figure 26 A shows asymmetric unit.MG1 with surface representations are shown and 305Fab to be shown as ribbon (dark-grey Color: heavy chain, light grey: light chain).Figure 26 B shows stacked molecule 1 and 2 (Dark grey: molecule 1, light gray: molecule 2).
[Figure 27 A]
Figure 27 A shows the epitope of the contact zone 305Fab on MG1 structural domain, as described in embodiment 11.6.Figure 27 A is shown Epitope mapping in MG1 amino acid sequence (Dark grey: the degree of approach is less than 3.0 angstroms, and light grey: the degree of approach is less than 4.5 angstroms).
[Figure 27 B]
Figure 27 B shows the epitope of 305 contact zones Fab on MG1 structural domain, as described in embodiment 11.6.Figure 27 B is aobvious Show the epitope mapping in crystal structure (dark-grey chromosphere: the degree of approach is less than 3.0 angstroms, light grey rod: the degree of approach is less than 4.5 angstroms).
[Figure 28 A]
Figure 28 A shows the feature of the interaction of E48, D51 and K109 (rod representation) and 305Fab (surface representations) View, as described in embodiment 11.7.
[Figure 28 B]
Figure 28 B shows E48 and its environment (Dark grey dotted line: the hydrogen bond with Fab, light grey dotted line: the hydrogen bond that water mediates) Between interaction, as described in embodiment 11.7.
[Figure 28 C]
Figure 28 C shows the interaction (Dark grey dotted line: the hydrogen bond with Fab) between D51 and its environment, such as embodiment Described in 11.7.
[Figure 28 D]
Figure 28 D shows interaction (the Dark grey dotted line: the hydrogen bond with Fab, light gray color dot between K109 and its environment Line: the salt bridge with H-CDR3_D95), as described in embodiment 11.7.
[Figure 29 A]
Figure 29 A shows the feature of the interaction of H70, H72 and H110 (rod representation) and 305 Fab (surface representations) View orients identical as Figure 28 A as described in embodiment 11.8.
[Figure 29 B]
Figure 29 B shows the interaction between H70 and its environment, as described in embodiment 11.8.The histidine residues with Rod and the instruction of net representation.Hydrogen bond is indicated with dotted line.
[Figure 29 C]
Figure 29 C shows the interaction between H72 and its environment, as described in embodiment 11.8.The histidine residues with Rod and the instruction of net representation.Hydrogen bond is indicated with dotted line.
[Figure 29 D]
Figure 29 D shows the interaction between H110 and its environment, as described in embodiment 11.8.The histidine residues with Rod and the instruction of net representation.The distance between H110 and H-CDR3_H100c are shown with dotted line.
Specific embodiment
Described herein or reference technology and methods are that those skilled in the art are usually managed very well using conventional methodologies It solves and conventional use of, e.g., for example, widely used method Sambrook etc., Molecular described in following The 3rd edition (2001) Cold Spring Harbor Laboratory Press of Cloning:A Laboratory Manual, Cold Spring Harbor, N.Y.；Current Protocols in Molecular Biology (F.M.Ausubel etc., Eds., (2003))；Serial Methods in Enzymology (Academic Press, Inc.): PCR 2:A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I.Freshney, ed. (1987))；Oligonucleotide Synthesis (M.J.Gait, ed., 1984)；Methods in Molecular Biology, Humana Press；Cell Biology:A Laboratory Notebook (J.E.Cellis, ed., 1998) Academic Press；Animal Cell Culture (R.I.Freshney), ed., 1987)；Introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press；Cell and Tissue Culture:Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell, eds., 1993-8)J.Wiley&Sons；Handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell, eds.)；Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos, eds., 1987)；PCR:The Polymerase Chain Reaction, (Mullis etc., eds., 1994)； Current Protocols in Immunology (J.E.Coligan etc., eds., 1991)；Short Protocols in Molecular Biology (Wiley&Sons, 1999)；Immunobiology (C.A.Janeway and P.Travers, 1997)；Antibodies (P.Finch, 1997)；Antibodies:A Practical Approach (D.Catty., ed., IRL Press, 1988-1989)；Monoclonal Antibodies:A Practical Approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000)；Using Antibodies:A Laboratory Manual (E.Harlow and D.Lane Cold Spring Harbor Laboratory Press, 1999)；The Antibodies (M.Zanetti and J.D.Capra, eds., Harwood Academic Publishers, 1995)；And Cancer:Principles and Practice of Oncology (V.T.DeVita etc., eds., J.B.Lippincott Company, 1993).
I. it defines
Unless otherwise defined, technical and scientific term used herein has and the common skill of the technical field of the invention Art personnel's is generally understood identical meaning.Singleton etc., Dictionary of Microbiology and Molecular Biology 2nd ed., J.Wiley&Sons (New York, N.Y.1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley&Sons (New York, N.Y.1992) provides the general guide to numerous terms use herein to those skilled in the art.This All documents (including patent application and publication) quoted in text are completely combined by reference.
In order to explain the application, defined below to be applicable in and when appropriate, the term that odd number uses also will include plural number And vice versa.It is to be understood that technology used herein is merely to describe special embodiment, and be not intended to It is restrictive.Conflict if any definition given below has with any document being incorporated herein, with Under subject to the definition that provides.
It is comprising sharing frame from human immunoglobulin(HIg) frame or people with " the receptor people frame " of purpose in this article The frame of the amino acid sequence of light variable domains (VL) frame or heavy-chain variable domains (VH) frame of frame, such as following institute It limits.Receptor people's frame that " deriving from " human immunoglobulin(HIg) frame or people share frame may include its identical amino acid sequence It arranges or it can change containing amino acid sequence.In some embodiments, amino acid variation number be 10 hereinafter, 9 with Under, 8 hereinafter, 7 hereinafter, 6 hereinafter, 5 hereinafter, 4 hereinafter, 3 hereinafter, or 2 or less.In some embodiments, VL receptor people frame Sequence it is identical as VL human immunoglobulin(HIg) Frame sequence or human consensus framework sequence.
" affinity " refer to molecule (for example, antibody) single binding site and its binding partners (for example, antigen) it Between noncovalent interaction strength summation.Unless otherwise noted, as used in this article, " binding affinity " refers to intrinsic knot Close affinity, 1: 1 interaction between member's (for example, antibody and antigen) of reflection combination pair.Molecule X is to its gametophyte The affinity of Y can usually be represented by dissociation constant (Kd).Affinity can be measured by conventional method as known in the art, The method includes those of described herein.The specific illustrative and exemplary implementation of measurement binding affinity is described below Scheme.
" affinity maturation " antibody refers to, and there are one or more changes (to compare in one or more hypervariable regions (HVR) In the parental antibody for not having such change) antibody, such change causes antibody to improve the affinity of antigen.
Term " anti-C5 antibody " and " antibody for combining C5 " refer to that such antibody, the antibody can be with enough parents With power combination C5 so that the antibody can be used as the diagnosticum and/or therapeutic agent for targeting C5.In one embodiment, Anti- C5 antibody is less than about 10% of the antibody in conjunction with C5 with the combination degree of incoherent, non-C5 albumen, such as example passes through Radioimmunoassay (RIA) measurement.In certain embodiments, in conjunction with the dissociation constant (Kd) of the antibody of C5 are as follows: 1 μM with Under, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less or 0.001nM or less be (for example, 10-8M with Under, for example, 10-8M to 10-13M, for example, 10-9M to 10-13M).In certain embodiments, the epitope of anti-C5 antibody combination C5, The epitope is conservative between the C5 from different plant species.
Term " antibody " is used and with broadest including various antibody structures, including but not limited to monoclonal herein Antibody, polyclonal antibody, multi-specificity antibody (for example, bispecific antibody) and antibody fragment, as long as anti-needed for its display Original combines activity.
" antibody fragment " refers to the molecule different from complete antibody, is combined it includes complete antibody combination complete antibody The part of antigen.The example of antibody fragment includes but is not limited to Fv, Fab, Fab ', Fab '-SH, F (ab ')2；Double antibody；Linearly Antibody；Single-chain antibody molecules (for example, scFv)；With the multi-specificity antibody formed by antibody fragment.
Refer to that such antibody, the antibody block in competition assay with reference antibody " antibody for combining same epitope " The combination of reference antibody and its antigen, and/or on the contrary, reference antibody blocks the antibody and its antigen in competition assay In conjunction with.Exemplary competition assay provides herein.
Term " chimeric " antibody refers to such antibody, and wherein a part of heavy chain and/or light chain derives from particular source Or species, and the remainder of heavy chain and/or light chain derives from different sources or species.
" classification " of antibody refers to the type of constant domain possessed by its heavy chain or constant region.Mainly there are five classes anti- Body: IgA, IgD, IgE, IgG and IgM, and some in these can be further divided into subclass (allograft), example Such as, IgG1, IgG2, IgG3, IgG4, IgA1And IgA2.The heavy chain constant domain of immunoglobulin corresponding to different types It is hereinafter referred to as α, δ, ε, γ and μ.
As used in this article, term " cytotoxic agent " refers to inhibition or prevents cell function and/or cause cell dead The substance died or damaged.Cytotoxic agent includes but is not limited to radioactive isotope (for example, At211, I131, I125, y90, Re186, Re188, Sm153, Bi212, p32, pb212With the radioactive isotope of Lu)；Chemotherapeutics or drug are (for example, methotrexate (MTX) (methotrexate), adriamycin (adriamycin), vinca alkaloids (vinca alkaloid) (vincristine (vincristine), vincaleukoblastinum (vinblastine), etoposide (etoposide)), Doxorubicin (doxorubicin), Melphalan (melphalan), mitomycin C (mitomycin C), Chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other are fitted into agent)；Growth inhibitor；Enzyme and its segment such as hydrolase nucleic acid；Antibiotic；Toxin is for example small The enzyme activity toxin of molecule toxin or bacterium, fungi, plant or animal origin, including its segment and/or variant；And it is following Disclosed various antitumor agents or anticancer agent.
" effector function ", which refers to, is attributable to those of antibody Fc district biological activity, becomes with antibody allotype Change.The example of antibody mediated effect subfunction includes: that C1q is combined and complement-dependent cytotoxicity (CDC)；Fc receptor combines；Antibody Dependent cell mediated cytotoxicity (ADCC)；Phagocytosis；The downward of cell surface receptor (for example, B-cell receptor)；With And B cell activation.
" effective quantity " of reagent (for example, pharmaceutical preparation) refers to dosage necessary to the treatment or prevention result needed for realizing With the effective quantity of time.
Term " epitope " includes any determinant that can be selectively bound by the antibody.Epitope is that antigen is targeted the antigen The region that antibody combines, and the specific amino acids including directly being contacted with antibody.Epitopic determinants may include molecule such as ammonia Base acid, carbohydrate side chain, the chemical active surface gathering of phosphoryl or sulfonyl, and can have specific three-dimensional structural feature, And/or specific charge characteristic.Typically for special target antigen have specificity antibody will preferentially identify albumen and/or The epitope on target antigen in the composite mix of macromolecular.
Herein, term " area Fc " be used to limit at least part of heavy chain immunoglobulin containing constant region C- end regions.The term includes the area native sequences Fc and the area variant Fc.In one embodiment, the area human IgG heavy chain Fc from Cys226 or Pro230 extends to the c-terminus of heavy chain.However, there may be may also for the end the C- lysine (Lys447) in the area Fc It is not present.Unless otherwise indicated herein, the numbering amino acid residues in the area Fc or constant region be according to EU numbering system, Referred to as EU index, such as Kabat, Sequences of Proteins of Immunological Interest, 5th It is limited in Ed.Public Health Service, National Institutes of Health, Bethesda, MD, 1991 Fixed.
" frame " or " FR " refers to the variable domains residue different from hypervariable region (HVR) residue.The FR of variable domains It is usually made of four FR structural domains: FR1, FR2, FR3 and FR4.Therefore, in VH (or VL) HVR and FR sequence usually with Lower sequence occurs: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.
Term " full length antibody ", " complete antibody " and " whole antibody " are convertibly used to indicate such resist herein Body, the antibody is with the structure substantially similar with native antibody structure or with the weight for containing the area Fc as defined herein Chain.
Term " host cell ", " host cell line " and " host cell cultures " are convertibly used to indicate and are introduced into The cell of exogenous nucleic acid, the offspring including such cell.Host cell includes " transformant " and " cell of conversion " comprising is turned The primary cell of change and offspring derived therefrom (not considering passage number).The nucleic acid content of offspring can be endless with parental cell It is exactly the same, and mutation can be contained.It herein include having to screen or select identical with the cell for original transformation Function or biological activity Mutant progeny.
" human antibody " is such antibody, the amino acid sequence having correspond to the antibody generated by people or people's cell or From the amino acid sequence of the antibody using human antibody library or the inhuman source of other people antibody coding sequences.Human antibody is somebody's turn to do Definition clearly eliminates the humanized antibody comprising non-human antigen-binding residues.
" people shares frame " is such frame, is represented in the selection of human immunoglobulin(HIg) VL or VH Frame sequence most often The amino acid residue seen.In general, subgroup of the selection from variable domain sequence of human immunoglobulin(HIg) VL or VH sequence.It is logical Often, the subgroup of sequence is such as Kabat, Sequences of Proteins of Immunological Interest, the 5th Version, NIH publish 91-3242, Bethesda MD (1991), roll up the subgroup in 1-3.In one embodiment, for VL, institute Stating subgroup is the subgroup κ I in such as above Kabat.In one embodiment, for VH, the subgroup is such as the above Kabat Such subgroup III in.
" humanization " antibody refers to a kind of chimeric antibody, it includes the amino acid residue from inhuman HVR and comes from people FR Amino acid residue.In certain embodiments, humanized antibody will at least one be (and typical comprising substantially all of Ground, two) variable domains, wherein all or substantially all HVR (for example, CDR) corresponds to the HVR of non-human antibody, and And all or substantially all FR corresponds to the FR of human antibody.Humanized antibody optionally may include from human antibody At least part of antibody constant region." humanization form " of antibody, for example, non-human antibody, refers to and carried out humanization Antibody.
Term " hypervariable region " or " HVR " as used in this article refer to that the change of sequence height (" determine by complementation in constant region for immunoglobulin sequence Determine area " or " CDR ") and/or formed structure determination ring (" hypervariable loop ") and/or containing with antigen contact residue (" antigen touching Point ") each region.In general, antibody includes three (L1, L2, L3) in three (H1, H2, the H3) and VL in six HVR:VH. Exemplary HVR herein includes: that (a) is appeared in amino acid residue 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), the hypervariable loop (Chothia, J.Mol.Biol.196:901-917 (1987)) at 53-55 (H2) and 96-101 (H3)； (b) it appears in amino acid residue 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95- CDR (Kabat etc., Sequences of Proteins of Immunological Interest, 5th at 102 (H3) Ed.Public Health Service, NIH, Bethesda, MD (1991))；(c) amino acid residue 27c-36 is appeared in (L1), the antigen contact at 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum etc., J.Mol.Biol.262:732-745 (1996))；And (d) (a), (b) and/or combination (c), including HVR amino acid residue 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49- 65 (H2), 93-102 (H3) and 94-102 (H3).
Unless otherwise noted, herein, other residues (such as such as, FR residue) in HVR residue and variable domains It is numbered according to above Kabat etc..
" immunoconjugates " are the antibody with the heterologous molecule conjugation of one or more including but not limited to cytotoxic agents.
" individual " or " subject " are mammals.Mammal includes but is not limited to domestic animal (such as such as, ox, silk floss Sheep, cat, dog and horse), primate (for example, people and non-human primate such as monkey), rabbit and rodent (for example, Mouse and rat).In certain embodiments, individual or subject are people.
" separation " antibody is the antibody separated with the component of its natural surroundings.In some embodiments, antibody It is purified to the purity greater than 95% or 99%, such as by such as electrophoresis (such as such as, SDS-PAGE, isoelectric focusing (IEF), capillary Electrophoresis tube) or chromatograph what (for example, ion exchange or reversed-phase HPLC) method determined.For the method for assessing antibody purity Summary, see, e.g., Flatman etc., J.Chromatogr.B 848:79-87 (2007).
" separation " nucleic acid refers to the nucleic acid molecules separated with the component of its natural surroundings.Isolated nucleic acid includes this The nucleic acid molecules of sample, the nucleic acid molecules are comprised in the cell for usually containing the nucleic acid molecules, but the nucleic acid point Son is present in outside chromosome or is present at the chromosome location different from its native chromosomal sites.
" the isolated nucleic acid of the anti-C5 antibody of coding " refers to one or more encoding antibody heavies and light chain (or its segment) Nucleic acid molecules, including in single carrier or such nucleic acid molecules in separated carrier, and be present in host cell One or more positions such nucleic acid molecules.
Term " monoclonal antibody " as used in this article refers to the antibody of the group obtained from substantially homologous antibody, That is, the individual antibody including the group is identical and/or in conjunction with identical epitope, in addition to possible variant antibodies, example Such as, it is generated containing naturally occurring mutation or in the preparation process of monoclonal antibody formulation, such variant is generally present in a small amount.It is right Than in polyclonal antibody preparations (generally including the different antibodies for different determinants (epitope)), monoclonal antibody formulation Each monoclonal antibody be for the single determinant on antigen.Therefore, the property of attribute " monoclonal " instruction antibody is to obtain From substantially homologous antibody population, and it is not intended as requiring to prepare the antibody by any specific method.For example, according to The monoclonal antibody that the present invention uses can be prepared by multiple technologies, including but not limited to hybridoma method, recombinant DNA method, Phage display, and using the method for the transgenic animals containing all or part of human immunoglobulin gene's seats, originally The such method of described in the text and other be used to prepare the illustrative methods of monoclonal antibody.
" exposed antibody " refers to the antibody not being conjugated with heterologous moiety (for example, cytotoxic moieties) or radioactive label.It is naked Antibody can reside in pharmaceutical preparation.
" natural antibody " refers to the naturally occurring immunoglobulin molecules with various structures.For example, native IgG antibodies It is about 150, the heterologous four glycan albumen of 000 dalton is identical by the two identical light chains and two that pass through disulfide bonding Heavy chain composition.From N-terminal to C-terminal, each heavy chain has variable region (VH), is also referred to as variable heavy chain domain or weight chain variable Structural domain is three constant domains (CH1, CH2 and CH3) later.Similarly, from N-terminal to C-terminal, each light chain has variable region (VL), variable light chain domain or light variable domains are also referred to as, are chain constant (CL) structural domain later.Antibody Amino acid sequence of the light chain based on its constant domain one of can be assigned to two types, referred to as κ (kappa) and λ (lambda)。
Term " package insert " is for referring to the operation instruction being generally comprised in the commercial packing for the treatment of product, containing related In the idicatio of such treatment product, purposes, dosage, administration, combination treatment, contraindication and/or use warning.
" percentage (%) amino acid sequence identity " relative to reference polypeptide sequence is defined as carrying out to sequence It compares and introduces gap (gap) if necessary to realize maximum percentage sequence identity, without recognizing any conservative substitution For be sequence identity a part after, amino acid identical with the amino acid residue in reference polypeptide sequence is residual in candidate sequence The percentage of base.In order to determine that the comparison of percentage amino acid sequence identity can be with a variety of in technology in the art Mode is realized, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the suitable parameters for aligned sequences, including in the sequence compared Realize high specific to required any algorithm in overall length.The author that ALIGN-2 sequence compares computer program is Genentech, Inc., and source code is filed in U.S. Copyright Office together with user file, Washington D.C., and 20559, It is registered with S. Copyright registration number TXU510087.However, For the purpose of this paper, comparing computer program using sequence ALIGN-2 generates % amino acid sequence identity value.The public can from Genentech, Inc., South San Francisco, California obtains ALIGN-2 program or described program can be from compilation of source code.ALIGN-2 program should be compiled as For UNIX operating system, including digital UNIX V4.0D.All sequences compare parameter by ALIGN-2 program setting and are not required to Change.
In the case where ALIGN-2 is used for amino acid sequence relatively, amino acid sequence A and or relatively given amino are given (it can alternatively be expressed as given amino acid sequence A and or relatively given amino to the % amino acid sequence identity of acid sequence B Acid sequence B has or comprising specific % amino acid sequence identity) calculate it is as follows: 100 multiplied by score X/Y；Wherein X is in A and B Program compare in by the amino acid residue that alignment programs ALIGN-2 scoring is identical match number, and Y is in B The sum of amino acid residue.It is to be understood that A is to B when the length of amino acid sequence A is not equal to the length of amino acid sequence B % amino acid sequence identity will be equal to B to the % amino acid sequence identity of A.Unless otherwise expressly stated, herein All % amino acid sequence identity values used are all using ALIGN-2 computer program to obtain as described in the preceding paragraph.
Term " pharmaceutical preparation " refers to such preparation, the life for the active constituent that the form having allows to be included in Object activity is effective, and its be free of to the subject that will be administered the preparation have unacceptable toxicity other Component.
" pharmaceutical carrier " refers to the ingredient in pharmaceutical preparation in addition to the active ingredient (s, is avirulent to subject.Medicine It include but is not limited to buffer, excipient, stabilizer or preservative with carrier.
Term " C5 " as used in this article includes coming from any vertebrate origin, including mammal such as primate Any natural C5 of (for example, people and monkey) and rodent (for example, mouse and rat).Unless otherwise noted, term " C5 " refers to People's C5 egg with amino acid sequence shown in SEQ ID NO:39 and containing β chain-ordering shown in SEQ ID NO:40 It is white.The term includes any type of C5 of " overall length " unprocessed C5 and the processing in cell.The term also wraps The variant of naturally occurring C5 is included, for example, splice variant or allelic variant.The amino acid sequence of example people C5 is shown in SEQ ID In NO:39 (" wild type " or " WT " C5).The amino acid sequence of the exemplary β chain of people C5 is shown in SEQ ID NO:40.People The exemplary MG1 of the β chain of C5, the amino acid sequence of MG2 and MG1-MG2 structural domain are respectively displayed on SEQ ID NO:41,42 Hes In 43.The amino acid sequence of exemplary machin and mouse C5 are respectively displayed in SEQ ID NO:44 and 105.SEQ ID NO: 39,40,43,44 and 105 amino acid residue 1-19 corresponds to signal sequence, is removed simultaneously during the processing in cell And it therefore can not find in corresponding exemplary amino acid sequence.
As used in this article, " treat (treatment) " (and its phraseological variant such as " is treated (treat) " or " is controlled Treat (treating) ") refer to the clinical intervention for attempting to change the natural process of treated individual, and can in order to prevent or It is carried out during clinical disease course.The ideal effect for the treatment of includes but is not limited to prevent disease from occurring or recurring, and mitigates symptom, eliminates Any direct or indirect pathological examination of disease, prevents from shifting, and reduces the rate of progression of disease, improves or mitigate disease shape State, and eliminate or improve prognosis.In some embodiments, antibody of the invention be used to postpone the development of disease or be used for Slow down the progress of disease.
Term " variable region " or " variable domains " refer to the structure for participating in heavy chain of antibody or light chain that antibody antigen combines Domain.The heavy chain and light variable domains (respectively VH and VL) of natural antibody usually have similar structure, wherein each structure Domain includes four conservative framework regions (FR) and three hypervariable regions (HVR).(see, e.g., Kindt etc., Kuby Immunology, 6thEd., W.H.Freeman&Co., page 91 (2007)).Single VH or VL structural domain can be enough to give antigen Binding specificity.In addition, in conjunction with specific antigen antibody can use respectively VH from the antibody with the antigen binding or VL structural domain screens the library of complementation VL or VH structural domain to separate.See, e.g., Portolano etc., J.Immunol.150: 880-887(1993)；Clarkson etc., Nature352:624-628 (1991).
As used in this article, term " carrier " is the nucleic acid point for referring to make another coupled nucleic acid reproduction Son.The term include the carrier of the nucleic acid structure as self-replacation and be integrated to introduce have its host cell genome In carrier.Certain carriers can instruct the expression that the nucleic acid being connected can be operated with it.Such carrier is referred to herein as " table Up to carrier ".
II. composition and method
In one aspect, the present invention is based partially on anti-C5 antibody and application thereof.In certain embodiments, combination is provided The antibody of C5.Antibody of the invention, which can be used for for example diagnosing or treating, is related to the complement-mediated of excessive or uncontrolled C5 activation Disease or illness.
A. exemplary anti-C5 antibody
In one aspect, the present invention provides the isolated antibody for combining C5.In certain embodiments, of the invention anti- Epitope in the β chain of C5 antibody combination C5.In certain embodiments, the MG1-MG2 structure of the β chain of anti-C5 antibody combination C5 Epitope in domain.In certain embodiments, anti-C5 antibody combines in the segment being made of the amino acid 1 9-180 of the β chain of C5 Epitope.In certain embodiments, the MG1 structural domain (amino acid of SEQ ID NO:40 of the β chain of anti-C5 antibody combination C5 20-124 (SEQ ID NO:41)) in epitope.In certain embodiments, anti-C5 antibody combines β chain (the SEQ ID by C5 NO:40 the epitope in the segment of amino acid 33-124 composition).In another embodiment, the antibody do not combine than by The short segment of the segment of the amino acid 33-124 composition of the β chain of C5, for example, the amino acid of the β chain (SEQ ID NO:40) by C5 The segment of 45-124,52-124,33-111,33-108 or 45-111 composition.
On the other hand, the present invention provides anti-C5 antibody, and the antibody shows the binding property of pH dependence.Such as this It is used herein, statement " combination of pH dependence " indicate antibody " acid pH show and C5 combination and its in neutral pH Combination compared to reduce " (for the disclosure, two kinds of expression can be used convertibly).For example, " the combination with pH dependence Property " antibody include under neutral ph in conjunction with C5 affinity than higher antibody at acidic.In certain embodiments In, antibody of the invention combine under neutral ph C5 affinity be at least the 2 of acid pH, 3,5,10,15,20,25,30, It is 35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000,10000 times or more high.Some In embodiment, affinity ratio of the antibody at pH7.4 in conjunction with C5 is higher in pH5.8.In a further embodiment, institute State affinity of the antibody at pH7.4 in conjunction with C5 be at least the 2 of pH5.8,3,5,10,15,20,25,30,35,40,45,50, It is 55,60,65,70,75,80,85,90,95,100,200,400,1000,10000 times or more high.
For the disclosure, antibody indicates " affinity " of C5 with the KD of antibody.The KD of antibody refers to that antibody-antigene is mutual The equilibrium dissociation constant of effect.Antibody combines the KD value of its antigen bigger, weaker to the binding affinity of the specific antigen. Therefore, as used in this article, state " higher than at acidic in the affinity of neutral pH " (or equivalent statements " pH dependence Property combination ") refer to acid pH antibody combination C5 KD be higher than neutral pH antibody combination C5 KD.For example, in the present invention Context in, if being at least 2 times of height in the KD of neutral pH antibody combination C5 in the KD of acid pH antibody combination C5, then it is assumed that Antibody combines the affinity of C5 higher than at acidic under neutral ph.Therefore, the present invention includes such antibody, described anti- Body is in the KD at least 2 that the KD of acid pH combination C5 is that the antibody combines C5 under neutral ph, and 3,5,10,15,20,25,30, It is 35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000,10000 times or more high.Another In a embodiment, the antibody can be 10 in the KD value of neutral pH-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M with Under.In another embodiment, the antibody can be 10 in the KD value of acid pH-9M, 10-8M, 10-7M, 10-6M or more.
In a further embodiment, if being the KD in pH7.4 antibody combination C5 in the KD of pH5.8 antibody combination C5 At least 2 times of height, then it is assumed that antibody combines the affinity of C5 higher than at acidic under neutral ph.In some embodiments In, the antibody provided is at least the 3,5,10,15 of the KD that the antibody combines C5 at pH7.4 in the KD of pH5.8 combination C5, 20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000,10000 times or more It is high.In another embodiment, the antibody can be 10 in the KD value of pH7.4-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M or less.In another embodiment, the antibody can be 10 in the KD value of pH5.8-9M, 10-8M, 10-7M, 10-6M Or it is bigger.
Antibody can also be expressed as the kd of antibody to the binding property of specific antigen.The kd of antibody refers to antibody about specific The dissociation rate constant of antigen and with the inverse of unit second (that is, sec-1) indicate.The increase instruction antibody of kd value and its antigen Combination it is weaker.Present invention accordingly comprises such antibody, the antibody acid pH combination C5 kd value ratio neutral pH more It is high.The present invention includes such antibody, and the antibody is that the antibody combines C5 under neutral ph in the kd of acid pH combination C5 Kd at least 2,3,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100, It is 200,400,1000,10000 times or more high.In another embodiment, the antibody can be 10 in the kd value of neutral pH- 21/s, 10-31/s, 10-41/s, 10-51/s, 10-61/s or less.In another embodiment, kd of the antibody in acid pH Value can be 10-31/s, 10-21/s, 10-11/s or more.The invention also includes such antibody, the antibody is combined in pH5.8 The kd value ratio of C5 is higher in pH7.4.The present invention includes such antibody, and the antibody is described anti-in the kd of pH5.8 combination C5 Body combines at least the 3,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80 of the kd of C5 at pH7.4, It is 85,90,95,100,200,400,1000,10000 times or more high.In another embodiment, the antibody is pH7.4's Kd value can be 10-21/s, 10-31/s, 10-41/s, 10-51/s, 10-61/s or less.In another embodiment, described anti- Body can be 10 in the kd value of pH5.8-31/s, 10-21/s, 10-11/s or more.
In some cases, " reduce compared with its combination in neutral pH in acid pH with the combination of C5 " and be represented as resisting Body the KD value of acid pH combination C5 combined under neutral ph with antibody the ratio between KD value of C5 (or vice versa).For example, for The present invention, if antibody shows 2 or more acidity/neutrality KD ratio, it may be considered that the antibody is shown is " in acid pH and C5 Reduce in conjunction with compared with its combination in neutral pH ".In certain exemplary implementation schemes, the pH5.8/ of antibody of the invention PH7.4KD ratio is 2 or more.In certain exemplary implementation schemes, acidity/neutrality KD ratio of antibody of the invention can be 2,3, 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000, 10000 or more.In another embodiment, the antibody can be 10 in the KD value of neutral pH-7M, 10-8M, 10-9M, 10- 10M, 10-11M, 10-12M or less.In another embodiment, the antibody can be 10 in the KD value of acid pH-9M, 10-8M, 10-7M, 10-6M or more.In the case where in addition, for the present invention, if antibody shows 2 or more pH5.8/pH7.4KD Than it may be considered that the antibody shows " reducing compared with its combination in neutral pH in acid pH with the combination of C5 ".At certain In a little exemplary implementation schemes, the pH5.8/pH7.4KD ratio of antibody of the invention can be 3,5,10,15,20,25,30,35, 40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000,10000 or more.In another embodiment party In case, the antibody can be 10 in the KD value of pH7.4-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M or less.Another In a embodiment, the antibody can be 10 in the KD value of pH5.8-9M, 10-8M, 10-7M, 10-6M or more.
In some cases, " reduce compared with its combination in neutral pH in acid pH with the combination of C5 " and be represented as resisting Body the kd value of acid pH combination C5 combined under neutral ph with antibody the ratio between kd value of C5 (or vice versa).For example, for The present invention, if antibody shows 2 or more acidity/neutrality kd ratio, it may be considered that the antibody is shown is " in acid pH and C5 Reduce in conjunction with compared with its combination in neutral pH ".In certain exemplary implementation schemes, the pH5.8/ of antibody of the invention PH7.4kd ratio is 2 or more.In certain exemplary implementation schemes, acidity/neutrality kd ratio of antibody of the invention can be 2,3, 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000, 10000 or more.In other exemplary implementation scheme, the pH5.8/pH7.4kd ratio of antibody of the invention can be 2,3,5, 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,400,1000,10000 More than.In another embodiment, the antibody can be 10 in the kd value of neutral pH-21/s, 10-31/s, 10-41/s, 10- 51/s, 10-61/s or less.In another embodiment, antibody can be 10 in the kd value of pH 7.4-21/s, 10-31/s, 10- 41/s, 10-51/s, 10-61/s or less.In another embodiment, the antibody can be 10 in the kd value of acid pH-31/ S, 10-21/s, 10-11/s or more.In another embodiment, the antibody can be 10 in the kd value of pH5.8-31/s, 10- 21/s, 10-11/s or more.
As used in this article, statement " acid pH " refers to 4.0 to 6.5 pH.Stating " acid pH " includes 4.0,4.1, 4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0, Any of 6.1,6.2,6.3,6.4 and 6.5 pH value.In particular aspect, " acid pH " is 5.8.
As used in this article, statement " neutral pH " refers to the pH of 6.7 to about 10.0.Stating " neutral pH " includes 6.7, 6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6, Any of 8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9 and 10.0 pH value.In spy Further aspect, " neutral pH " are 7.4.
As indicated herein, the biosensor based on surface plasma resonance is can be used come really in KD value and kd value It is fixed to be interacted with characterizing antibody-antigene.(see, e.g., embodiment 3 herein).KD value and kd value can at 25 DEG C or 37 DEG C of determinations.
In certain embodiments, anti-C5 antibody of the invention combine by MG1 structural domain in the β chain of C5 (SEQ ID NO: 41) epitope formed.In certain embodiments, in the β chain (SEQ ID NO:40) of anti-C5 antibody combination C5 of the invention Epitope, the epitope include the segment that at least one is selected from the group being made up of: amino acid 47-57,70-76 and 107- 110.In certain embodiments, the table in the segment of the β chain (SEQ ID NO:40) of anti-C5 antibody combination C5 of the invention Position, the epitope include the amino acid that at least one is selected from the group being made up of: Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, LVs109, and His110.In certain embodiments, in the segment of the β chain (SEQ ID NO:40) of anti-C5 antibody combination C5 of the invention Epitope, the epitope include the amino acid that at least one is selected from the group being made up of: Glu48, Asp51, His70, His72, Lys109 and His110.In certain embodiments, anti-C5 antibody of the invention itself and open country compared with the combination of C5 mutant The combination of raw type C5 weakens, wherein the C5 mutant has at least one ammonia at the position selected from the group being made up of Base acid displacement: Glu48, Asp51, His72 and Lys109.In another embodiment, anti-C5 antibody and C5 of the invention The pH- dependence of mutant is combined to be weakened compared in conjunction with its pH- dependence with wild type C5, wherein the C5 mutant has At least one is in the amino acid replacement at the position of group being made up of: His70, His72 and His110.Another In a embodiment, in C5 mutant, the amino acid at the position selected from Glu48, Asp51 and Lys109 is replaced as the third ammonia Acid, and the amino acid at the position selected from His70, His72 and His110 is replaced as tyrosine.
In certain embodiments, anti-C5 antibody of the invention and the antibody competition comprising VH selected from the following and VL couples In conjunction with C5:(a) VL of the VH and SEQ ID NO:11 of SEQ ID NO:1；(b) VH the and SEQ ID NO:26 of SEQ ID NO:22 VL；(c) VL of the VH and SEQ ID NO:25 of SEQ ID NO:21；(d) VH the and SEQ ID NO:15 of SEQ ID NO:5 VL；(e) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；(f) VH the and SEQ ID NO:16 of SEQ ID NO:6 VL；(g) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；(h) VL of the VH and SEQ ID NO:13 of SEQ ID NO:3； (i) VL of the VH and SEQ ID NO:19 of SEQ ID NO:9；(j) VL of the VH and SEQ ID NO:17 of SEQ ID NO:7；(k) The VL of the VH and SEQ ID NO:18 of SEQ ID NO:8；(l) VL of the VH and SEQ ID NO:27 of SEQ ID NO:23；(m) The VL of the VH and SEQ ID NO:20 of SEQ ID NO:10.
In certain embodiments, anti-C5 antibody combination C5 of the invention and the amino acid of SEQ ID NO:39 is contacted Asp51(D51).In a further embodiment, anti-C5 antibody combination C5 of the invention and the ammonia of SEQ ID NO:39 is contacted Base acid Lys109 (K109).In another embodiment, anti-C5 antibody combination C5 of the invention and SEQ ID NO is contacted: 39 amino acid Asp51 (D51) and amino acid Lys109 (K109).
In certain embodiments, anti-C5 antibody of the invention compared with the combination of C5 mutant its with wild type C5's In conjunction with decrease, wherein the C5 mutant is replaced with the Glu48Ala (E48A) of SEQ ID NO:39.In another embodiment party In case, the pH- dependence knot of anti-C5 antibody of the invention itself and wild type C5 compared with the pH- dependence of C5 mutant combines It closes and weakens, wherein the C5 mutant is replaced with the Glu48Ala (E48A) of SEQ ID NO:39.
In another embodiment, anti-C5 antibody combines the C5 egg being made of the amino acid sequence of SEQ ID NO:39 It is white, but the C5 albumen being made of the amino acid sequence of the SEQ ID NO:39 replaced with H72Y is not combined, wherein the C5 Albumen and with H72Y displacement C5 albumen prepare and screen under the same conditions.In another embodiment, anti-C5 antibody The C5 albumen being made of the amino acid sequence of SEQ ID NO:39 is combined at pH7.4, but not combining at pH7.4 has The C5 albumen of H72Y displacement.
It is not only restricted to specific theory, it is anticipated that when an amino acid residue on C5 is replaced as another ammonia The combination of anti-C5 antibody and C5 weaken (or almost losing) when base acid, it means that the amino acid residue on C5 is for anti- Interaction between C5 antibody and C5 is crucial, and the antibody can identify on C5 around the amino acid residue Epitope.
It has been found that one group contends with one other or can show that pH- is relied in conjunction with the anti-C5 antibody of same epitope in the present invention Property binding characteristic.In amino acid, the histidine that pKa value is about 6.0 to 6.5 can have difference between neutral and acid pH Proton dissociation state.Therefore, the histidine residues on C5 can be with pH- dependence phase of the power-assisted between anti-C5 antibody and C5 Interaction.It is not only restricted to specific theory, it is anticipated that anti-C5 antibody can identify near the histidine residues on C5 Conformational structure, the structure can be changed according to pH.The expection can be consistent with experimental result described below: when the group on C5 When histidine residue is replaced as another amino acid, the pH- dependence of anti-C5 antibody weakens (or almost losing) (that is, in neutrality PH, the anti-C5 antibody with pH- dependence binding characteristic is with the Histidine mutagenesis of C5 in conjunction with the affinity similar with wild type C5 Body, and in acid pH, identical antibody is with the Histidine mutagenesis body of affinity combination C5 more higher than wild type C5).
In certain embodiments, anti-C5 antibody of the invention combines the C5 from more than a kind of species.In other reality It applies in scheme, anti-C5 antibody combines the C5 from people and non-human animal.In a further embodiment, anti-C5 antibody is in conjunction with next From the C5 of people and monkey (for example, machin, macaque, marmoset, chimpanzee or baboon).
In one aspect, the present invention provides the anti-C5 antibody for inhibiting the activation of C5.In certain embodiments, it provides anti- C5 antibody prevents C5 from being cut to C5a and C5b, therefore prevents to generate anaphylatoxin activity relevant to C5a, and hinder The only assembling of C5b-9 membrane attack complex (MAC) relevant to C5b.In certain embodiments, anti-C5 antibody is provided, is hindered Conversion of the open close C5 for crossing C5 convertase to C5a and C5b.In certain embodiments, anti-C5 antibody is provided, C5 is prevented to turn Change enzyme close to the cleavage site on C5.In certain embodiments, anti-C5 antibody is provided, is blocked as caused by the activation of C5 Hemolytic activity.In a further embodiment, anti-C5 antibody of the invention inhibits C5 via classical pathway and/or alternative pathway Activation.
In one aspect, the present invention provides anti-C5 antibody, inhibits the activation of C5 variant.C5 variant refers to due to heredity Variation is as being mutated, the genetic variation of C5 caused by polymorphism or equipotential variation.Heredity variation may include one or more nucleosides The missing of acid, displacement or insertion.C5 variant may be embodied in the one or more heredity variation in C5.In certain embodiments In, C5 variant has biological activity similar with wild type C5.Such C5 variant may include at least one and be selected from by following The change of the group of composition: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D.Herein, For example, R885H indicates such heredity variation, wherein the arginine at position 885 is by histidine.In certain embodiments In, anti-C5 antibody of the invention inhibits wild type C5 to be selected from at least one by V145I, R449G, V802I, R885H, The activation of both C5 variants of group of R928Q, D966Y, S1310N and E1437D composition.
In one aspect, the present invention provides anti-C5 antibody, it includes at least one, two, three, four, five or six A hypervariable region selected from the following (HVR): (a) HVR-H1, the HVR-H1 include the ammonia of any of SEQ ID NO:45-54 Base acid sequence；(b) HVR-H2, the HVR-H2 include the amino acid sequence of any of SEQ ID NO:55-64；(c)HVR- H3, the HVR-H3 include the amino acid sequence of any of SEQ ID NO:65-74；(d) HVR-L1, the HVR-L1 include The amino acid sequence of any of SEQ ID NO:75-84；(e) HVR-L2, the HVR-L2 include in SEQ ID NO:85-94 The amino acid sequence of any one；(f) HVR-L3, the HVR-L3 include the amino acid of any of SEQ ID NO:95-104 Sequence.
In one aspect, the present invention provides antibody, and the antibody includes at least one, and at least two, or all three choosings From VH HVR sequence below: (a) HVR-H1, the HVR-H1 include the amino acid sequence of any of SEQ ID NO:45-54 Column；(b) HVR-H2, the HVR-H2 include the amino acid sequence of any of SEQ ID NO:55-64；(c) HVR-H3, institute State the amino acid sequence that HVR-H3 includes any of SEQ ID NO:65-74.In one embodiment, the antibody includes HVR-H3, the HVR-H3 include the amino acid sequence of any of SEQ ID NO:65-74.In another embodiment, The antibody includes HVR-H3 and HVR-L3, and the HVR-H3 includes the amino acid sequence of any of SEQ ID NO:65-74, The HVR-L3 includes the amino acid sequence of any of SEQ ID NO:95-104.In another embodiment, described anti- Body includes HVR-H3, HVR-L3 and HVR-H2, and the HVR-H3 includes the amino acid sequence of any of SEQ ID NO:65-74 Column, the HVR-L3 include the amino acid sequence of any of SEQ ID NO:95-104, and the HVR-H2 includes SEQ ID The amino acid sequence of any of NO:55-64.In another embodiment, the antibody includes (a) HVR-H1, described HVR-H1 includes the amino acid sequence of any of SEQ ID NO:45-54；(b) HVR-H2, the HVR-H2 include SEQ ID The amino acid sequence of any of NO:55-64；(c) HVR-H3, the HVR-H3 include any in SEQ ID NO:65-74 A amino acid sequence.
On the other hand, the present invention provides antibody, and the antibody includes at least one, and at least two, or all three VL HVR sequence selected from the following: (a) HVR-L1, the HVR-L1 include the amino acid of any of SEQ ID NO:75-84 Sequence；(b) HVR-L2, the HVR-L2 include the amino acid sequence of any of SEQ ID NO:85-94；(c) HVR-L3, The HVR-L3 includes the amino acid sequence of any of SEQ ID NO:95-104.In one embodiment, the antibody Comprising (a) HVR-L1, the HVR-L1 includes the amino acid sequence of any of SEQ ID NO:75-84；(b) HVR-L2, institute State the amino acid sequence that HVR-L2 includes any of SEQ ID NO:85-94；(c) HVR-L3, the HVR-L3 include SEQ The amino acid sequence of any of ID NO:95-104.
On the other hand, antibody of the invention includes (a) VH structural domain, and the VH structural domain includes at least one, until It is two few, or all three VH HVR sequences selected from the following: (i) HVR-H1, the HVR-H1 include SEQ ID NO:45-54 Any of amino acid sequence, (ii) HVR-H2, the HVR-H2 include the amino acid of any of SEQ ID NO:55-64 Sequence, and (iii) HVR-H3, the HVR-H3 include the amino acid sequence of any of SEQ ID NO:65-74；And (b) VL structural domain, the VL structural domain include at least one, and at least two, or all three VL HVR sequences selected from the following: (i) HVR-L1, the HVR-L1 include the amino acid sequence of any of SEQ ID NO:75-84, (ii) HVR-L2, the HVR- L2 includes the amino acid sequence of any of SEQ ID NO:85-94, and (c) HVR-L3, the HVR-L3 include SEQ ID The amino acid sequence of any of NO:95-104.
On the other hand, the present invention provides antibody, and the antibody includes: (a) HVR-H1, and the HVR-H1 includes SEQ The amino acid sequence of any of ID NO:45-54；(b) HVR-H2, the HVR-H2 include any in SEQ ID NO:55-64 A amino acid sequence；(c) HVR-H3, the HVR-H3 include the amino acid sequence of any of SEQ ID NO:65-74； (d) HVR-L1, the HVR-L1 include the amino acid sequence of any of SEQ ID NO:75-84；(e) HVR-L2, it is described HVR-L2 includes the amino acid sequence of any of SEQ ID NO:85-94；(f) HVR-L3, the HVR-L3 include SEQ The amino acid sequence of any of ID NO:95-104.
In one aspect, the present invention provides anti-C5 antibody, and the anti-C5 antibody includes at least one, and two, three, four A, five or six HVR:(a selected from the following) HVR-H1, the HVR-H1 includes SEQ ID NO:45, in 54,117,126 The amino acid sequence of any one；(b) HVR-H2, the HVR-H2 include SEQ ID NO:55, any in 64,118-120,127 A amino acid sequence；(c) HVR-H3, the HVR-H3 include SEQ ID NO:65, any of 74,121,128 amino Acid sequence；(d) HVR-L1, the HVR-L1 include SEQ ID NO:75, any of 84,122,129 amino acid sequence； (e) HVR-L2, the HVR-L2 include SEQ ID NO:85,94,123-124, any of 130 amino acid sequence；(f) HVR-L3, the HVR-L3 include SEQ ID NO:95, any of 104,125,131 amino acid sequence.
In one aspect, the present invention provides antibody, and the antibody includes at least one, and at least two, or all three choosings From VH HVR sequence below: (a) HVR-H1, the HVR-H1 include SEQ ID NO:45, any of 54,117,126 Amino acid sequence；(b) HVR-H2, the HVR-H2 include SEQ ID NO:55,64,118-120, any of 127 amino Acid sequence；(c) HVR-H3, the HVR-H3 include SEQ ID NO:65, any of 74,121,128 amino acid sequence. In one embodiment, the antibody includes HVR-H3, and the HVR-H3 includes SEQ ID NO:65, is appointed in 74,121,128 One amino acid sequence.In another embodiment, the antibody includes HVR-H3 and HVR-L3, and the HVR-H3 includes SEQ ID NO:65, any of 74,121,128 amino acid sequence, the HVR-L3 include SEQ ID NO:95,104, Any of 125,131 amino acid sequence.In another embodiment, the antibody include HVR-H3, HVR-L3 and HVR-H2, the HVR-H3 include SEQ ID NO:65, any of 74,121,128 amino acid sequence, the HVR-L3 packet The NO:95 of ID containing SEQ, any of 104,125,131 amino acid sequence, the HVR-H2 include SEQ ID NO:55,64, 118-120, any of 127 amino acid sequence.In another embodiment, the antibody includes (a) HVR-H1, described HVR-H1 includes SEQ ID NO:45, any of 54,117,126 amino acid sequence；(b) HVR-H2, the HVR-H2 packet The NO:55 of ID containing SEQ, 64,118-120, any of 127 amino acid sequence；(c) HVR-H3, the HVR-H3 include SEQ ID NO:65, any of 74,121,128 amino acid sequence.
On the other hand, the present invention provides antibody, and the antibody includes at least one, and at least two, or all three VL HVR sequence selected from the following: (a) HVR-L1, the HVR-L1 include SEQ ID NO:75, any of 84,122,129 Amino acid sequence；(b) HVR-L2, the HVR-L2 include SEQ ID NO:85,94,123-124, any of 130 ammonia Base acid sequence；(c) HVR-L3, the HVR-L3 include SEQ ID NO:95, any of 104,125,131 amino acid sequence Column.In one embodiment, the antibody include (a) HVR-L1, the HVR-L1 include SEQ ID NO:75,84,122, Any of 129 amino acid sequence；(b) HVR-L2, the HVR-L2 include SEQ ID NO:85, in 94,123-124,130 The amino acid sequence of any one；(c) HVR-L3, the HVR-L3 include SEQ ID NO:95, any of 104,125,131 Amino acid sequence.
On the other hand, antibody of the invention includes (a) VH structural domain, and the VH structural domain includes at least one, until It is two few, or all three VH HVR sequences selected from the following: (i) HVR-H1, the HVR-H1 include SEQ ID NO:45, Any of 54,117,126 amino acid sequence, (ii) HVR-H2, the HVR-H2 include SEQ ID NO:55,64,118- Any of 120,127 amino acid sequence, and (iii) HVR-H3, the HVR-H3 include SEQ ID NO:65,74,121, Any of 128 amino acid sequence；(b) VL structural domain, the VL structural domain include at least one, and at least two, or it is complete Three, portion VL HVR sequence selected from the following: (i) HVR-L1, the HVR-L1 include SEQ ID NO:75, in 84,122,129 The amino acid sequence of any one, (ii) HVR-L2, the HVR-L2 include SEQ ID NO:85, any in 94,123-124,130 A amino acid sequence, and (c) HVR-L3, the HVR-L3 include SEQ ID NO:95, any of 104,125,131 ammonia Base acid sequence.
On the other hand, the present invention provides antibody, and the antibody includes (a) HVR-H1, and the HVR-H1 includes SEQ ID NO:45, any of 54,117,126 amino acid sequence；(b) HVR-H2, the HVR-H2 include SEQ ID NO:55, 64,118-120, any of 127 amino acid sequence；(c) HVR-H3, the HVR-H3 include SEQ ID NO:65,74, Any of 121,128 amino acid sequence；(d) HVR-L1, the HVR-L1 include SEQ ID NO:75, in 84,122,129 The amino acid sequence of any one；(e) HVR-L2, the HVR-L2 include SEQ ID NO:85, any in 94,123-124,130 A amino acid sequence；(f) HVR-L3, the HVR-L3 include SEQ ID NO:95, any of 104,125,131 ammonia Base acid sequence.
In certain embodiments, any one or more amino acid in anti-C5 antibody as provided above are following HVR is replaced at position: (a) in HVR-H1 (SEQ ID NO:45), at position 5 and 6；(b) in HVR-H2 (SEQ ID NO:55 in), at position 1,3,9,11,13 and 15；(c) in HVR-H3 (SEQ ID NO:65), in 2,5,6,12 He of position At 13；(d) in HVR-L1 (SEQ ID NO:75), at position 1,5,7 and 9；(e) at HVR-L2 (SEQ ID NO:85) In, at position 4,5 and 6；(f) in HVR-L3 (SEQ ID NO:95), at position 2,4 and 12.
In certain embodiments, the displacement is conservative substitution, as described herein.In certain embodiments, with Any one or more in lower displacement can carry out in any combination: (a) in HVR-H1 (SEQ ID NO:45), M5V or C6A；(b) in HVR-H2 (SEQ ID NO:55), C1A or G, Y3F, T9D or E, Y11K or Q, S13D or E or A15V；(c) In HVR-H3 (SEQ ID NO:65), G2A, V5Q or D, T6Y, Y12H or L13Y；(d) at HVR-L1 (SEQ ID NO:75) In, Q1R, N5Q or G, G7S, D9K or S；(e) in HVR-L2 (SEQ ID NO:85), K4T or E, L5T or A6H, A6E, or A6Q；(f) in HVR-L3 (SEQ ID NO:95), C2S, C2N or C2T, F4K；Or A12T or A12H.
The all possible combination replaced above respectively by HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and The consensus sequence SEQ ID NO:126 of HVR-L3,127,128,129,130 and 131 cover.
In any embodiments above, anti-C5 antibody is humanization.In one embodiment, anti-C5 antibody packet HVR in embodiment containing any of the above, and also include receptor people frame, for example, human immunoglobulin(HIg) frame or people are shared Frame.In another embodiment, anti-C5 antibody includes the HVR in any of the above embodiment, and also comprising VH or VL, the VH or VL include FR sequence, wherein the FR sequence is as follows.For heavy-chain variable domains, FR1 includes SEQ ID The amino acid sequence of any of NO:132-134, FR2 include the amino acid sequence of any of SEQ ID NO:135-136, FR3 includes the amino acid sequence of any of SEQ ID NO:137-139, and FR4 includes any of SEQ ID NO:140-141 Amino acid sequence.For light variable domains, FR1 includes the amino acid sequence of any of SEQ ID NO:142-143, FR2 includes the amino acid sequence of any of SEQ ID NO:144-145, and FR3 includes any of SEQ ID NO:146-147 Amino acid sequence, FR4 include SEQ ID NO:148 amino acid sequence.
On the other hand, anti-C5 antibody includes to have extremely with the amino acid sequence of any of SEQ ID NO:1-10 The heavy chain of few 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity can Structure changes domain (VH) sequence.In certain embodiments, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, The VH sequence of 97%, 98% or 99% identity includes displacement (for example, conservative substitution) relative to reference sequence, is inserted into, or is lacked It loses, but includes that the anti-C5 antibody of the sequence keeps combining the ability of C5.In certain embodiments, in SEQ ID NO: In any of 1-10, amounts to 1 to 10 amino acid and be replaced, be inserted into and/or lack.In certain embodiments, it replaces, Insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, anti-C5 antibody includes SEQ ID NO:1-10 Any of in VH sequence, the posttranslational modification including the sequence.In special embodiment, VH is comprising one, two A or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes the amino acid of any of SEQ ID NO:45-54 Sequence, the amino acid sequence of (b) HVR-H2, the HVR-H2 comprising any of SEQ ID NO:55-64, and (c) HVR-H3, The HVR-H3 includes the amino acid sequence of any of SEQ ID NO:65-74.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes any of with SEQ ID NO:11-20 Amino acid sequence have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% The light variable domains (VL) of sequence identity.In certain embodiments, have at least 90%, 91%, 92%, 93%, The VL sequence of 94%, 95%, 96%, 97%, 98% or 99% identity includes displacement (for example, conservative relative to reference sequence Displacement), insertion, or missing, but include that the anti-C5 antibody of the sequence keeps combining the ability of C5.In certain embodiments In, in any of SEQ ID NO:11-20, amounts to 1 to 10 amino acid and be replaced, be inserted into and/or lack.Certain In embodiment, the displacement, insertion, or lack in (that is, in FR) region of the generation outside HVR.Optionally, anti-C5 is anti- Body includes the VL sequence in any of SEQ ID NO:11-20, the posttranslational modification including the sequence.Special real Apply in scheme, VL includes one, two or three HVR:(a selected from the following) HVR-L1, the HVR-L1 includes SEQ ID The amino acid sequence of any of NO:75-84；(b) HVR-L2, the HVR-L2 include any of SEQ ID NO:85-94 Amino acid sequence；(c) HVR-L3, the HVR-L3 include the amino acid sequence of any of SEQ ID NO:95-104.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes any in embodiment provided above VL in any of VH and embodiment provided above in a.In one embodiment, the antibody wraps respectively VH and VL sequence in any of any of NO:1-10 of ID containing SEQ and SEQ ID NO:11-20, including it is described The posttranslational modification of sequence.
On the other hand, anti-C5 antibody includes the amino acid sequence with SEQ ID NO:10, any of 106-110 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Heavy-chain variable domains (VH) sequence.In certain embodiments, have at least 90%, 91%, 92%, 93%, 94%, The VH sequence of 95%, 96%, 97%, 98% or 99% identity includes displacement (for example, conservative set relative to reference sequence Change), insertion or missing, but include that the anti-C5 antibody of the sequence keeps combining the ability of C5.In certain embodiments, In SEQ ID NO:10,106-110 it is any in 1 to 10 amino acid of total be replaced, be inserted into and/or lack.In certain realities It applies in scheme, the displacement, is inserted into, or missing occurs in (that is, in FR) region outside HVR.Optionally, anti-C5 antibody Comprising the VH sequence in any in SEQ ID NO:10,106-110, the posttranslational modification including the sequence.Special real Apply in scheme, VH includes one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes SEQ ID NO:45, any of 54,117,126 amino acid sequence, (b) HVR-H2, the HVR-H2 include SEQ ID NO:55,64, 118-120, any of 127 amino acid sequence, and (c) HVR-H3, the HVR-H3 include SEQ ID NO:65,74, Any of 121,128 amino acid sequence.
On the other hand, anti-C5 antibody includes VH sequence, is appointed in the VH sequence and SEQ ID NO:10,106-110 One amino acid sequence have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.In certain embodiments, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, The VH sequence of 97%, 98% or 99% identity includes displacement (for example, conservative substitution), insertion or scarce relative to reference sequence It loses, but includes that the anti-C5 antibody of the sequence keeps combining the ability of C5.In certain embodiments, SEQ ID NO:10, In 106-110 it is any in 1 to 10 amino acid of total be replaced, be inserted into and/or lack.In certain embodiments, described Displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, anti-C5 antibody includes SEQ ID NO: In 10,106-110 it is any in VH sequence, the posttranslational modification including the sequence.In special embodiment, VH includes One, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes SEQ ID NO:45, and 54,117,126 Any of amino acid sequence, (b) HVR-H2, the HVR-H2 include SEQ ID NO:55, are appointed in 64,118-120,127 One amino acid sequence, and (c) HVR-H3, the HVR-H3 include SEQ ID NO:65, and any of 74,121,128 Amino acid sequence.
On the other hand, anti-C5 antibody includes VH sequence, and the VH sequence and amino acid sequence SEQ ID NO:10 have There is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.? In certain embodiments, VH sequence is amino acid sequence SEQ ID NO:10.In certain embodiments, have at least 90%, The VH sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence packet Containing displacement (for example, conservative substitution), insertion or missing, but include that the anti-C5 antibody of the sequence keeps combining the ability of C5. In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:10 is replaced, and is inserted into and/or is lacked.Certain In embodiment, the displacement, insertion, or lack in (that is, in FR) region of the generation outside HVR.Optionally, anti-C5 is anti- Body includes the VH sequence in SEQ ID NO:10, the posttranslational modification including the sequence.In special embodiment, VH packet Containing one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes amino acid sequence SEQ ID NO: 54, (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:64, and (c) HVR-H3, the HVR-H3 include Amino acid sequence SEQ ID NO:74.
On the other hand, anti-C5 antibody includes VH sequence, the VH sequence and amino acid sequence SEQ ID NO:106 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, VH sequence is amino acid sequence SEQ ID NO:106.In certain embodiments, have at least The VH sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:106 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VH sequence in SEQ ID NO:106, the posttranslational modification including the sequence.In special embodiment party In case, VH includes one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes amino acid sequence SEQ ID NO:117, (b) HVR-H2, the HVR-H2 includes amino acid sequence SEQ ID NO:118, and (c) HVR-H3, described HVR-H3 includes amino acid sequence SEQ ID NO:121.
On the other hand, anti-C5 antibody includes VH sequence, the VH sequence and amino acid sequence SEQ ID NO:107 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, VH sequence is amino acid sequence SEQ ID NO:107.In certain embodiments, have at least The VH sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:107 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VH sequence in SEQ ID NO:107, the posttranslational modification including the sequence.In special embodiment party In case, VH includes one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes amino acid sequence SEQ ID NO:117 (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:119, and (c) HVR-H3, the HVR- H3 includes amino acid sequence SEQ ID NO:121.
On the other hand, anti-C5 antibody includes VH sequence, the VH sequence and amino acid sequence SEQ ID NO:108 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, VH sequence is amino acid sequence SEQ ID NO:108.In certain embodiments, have at least The VH sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:108 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VH sequence in SEQ ID NO:108, the posttranslational modification including the sequence.In special embodiment party In case, VH includes one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes amino acid sequence SEQ ID NO:117 (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:118, and (c) HVR-H3, the HVR- H3 includes amino acid sequence SEQ ID NO:121.
On the other hand, anti-C5 antibody includes VH sequence, the VH sequence and amino acid sequence SEQ ID NO:109 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, VH sequence is amino acid sequence SEQ ID NO:109.In certain embodiments, have at least The VH sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:109 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VH sequence in SEQ ID NO:109, the posttranslational modification including the sequence.In special embodiment party In case, VH includes one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes amino acid sequence SEQ ID NO:117 (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:118, and (c) HVR-H3, the HVR- H3 includes amino acid sequence SEQ ID NO:121.
On the other hand, anti-C5 antibody includes VH sequence, the VH sequence and amino acid sequence SEQ ID NO:110 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, VH sequence is amino acid sequence SEQ ID NO:110.In certain embodiments, have at least The VH sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:110 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VH sequence in SEQ ID NO:110, the posttranslational modification including the sequence.In special embodiment party In case, VH includes one, two or three HVR:(a selected from the following) HVR-H1, the HVR-H1 includes amino acid sequence SEQ ID NO:117 (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:120, and (c) HVR-H3, the HVR- H3 includes amino acid sequence SEQ ID NO:121.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes to appoint with SEQ ID NO:20,111-113 One amino acid sequence have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or The light variable domains (VL) of 100% sequence identity.In certain embodiments, have at least 90%, 91%, 92%, The VL sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity includes displacement (example relative to reference sequence Such as, conservative substitution), insertion or missing, but include that the anti-C5 antibody of the sequence keeps combining the ability of C5.In certain realities Apply in scheme, it is any in SEQ ID NO:20,111-113 in 1 to 10 amino acid of total be replaced, be inserted into and/or lack. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VL sequence in any in SEQ ID NO:20,111-113, the posttranslational modification including the sequence.? In special embodiment, VL includes one, two or three HVR (a) HVR-L1 selected from the following, the HVR-L1 include SEQ ID NO:75, any of 84,122,129 amino acid sequence；(b) HVR-L2, the HVR-L2 include SEQ ID NO:85,94,123-124, any of 130 amino acid sequence；(c) HVR-L3, the HVR-L3 include SEQ ID NO: Any of 95,104,125,131 amino acid sequence.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes to have with amino acid sequence SEQ ID NO:20 There is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity VL.In certain embodiments, VL sequence is amino acid sequence SEQ ID NO:20.In certain embodiments, have at least The VL sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:20 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VL sequence in SEQ ID NO:20, the posttranslational modification including the sequence.In special embodiment In, VL includes one, two or three HVR (a) HVR-L1 selected from the following, the HVR-L1 include amino acid sequence SEQ ID NO:84；(b) HVR-L2, the HVR-L2 include amino acid sequence SEQ ID NO:94；(c) HVR-L3, the HVR-L3 packet The ID of SEQ containing amino acid sequence NO:104.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes and amino acid sequence SEQ ID NO:111 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity VL.In certain embodiments, VL sequence is amino acid sequence SEQ ID NO:111.In certain embodiments, have at least The VL sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:111 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VL sequence in SEQ ID NO:111, the posttranslational modification including the sequence.In special embodiment party In case, VL includes one, two or three HVR (a) HVR-L1 selected from the following, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 include amino acid sequence SEQ ID NO:123；(c) HVR-L3, it is described HVR-L3 includes amino acid sequence SEQ ID NO:125.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes and amino acid sequence SEQ ID NO:112 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity VL.In certain embodiments, VL sequence is amino acid sequence SEQ ID NO:112.In certain embodiments, have at least The VL sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:112 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VL sequence in SEQ ID NO:112, the posttranslational modification including the sequence.In special embodiment party In case, VL includes one, two or three HVR (a) HVR-L1 selected from the following, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 include amino acid sequence SEQ ID NO:123；(c) HVR-L3, it is described HVR-L3 includes amino acid sequence SEQ ID NO:125.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes and amino acid sequence SEQ ID NO:113 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity VL.In certain embodiments, VL sequence is amino acid sequence SEQ ID NO:113.In certain embodiments, have at least The VL sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is relative to reference sequence Arranging includes displacement (for example, conservative substitution), insertion or missing, but includes the anti-C5 antibody holding of the sequence in conjunction with C5 Ability.In certain embodiments, 1 to 10 amino acid of total in SEQ ID NO:113 is replaced, and is inserted into and/or is lacked. In certain embodiments, the displacement, insertion, or missing occur in (that is, in FR) region outside HVR.Optionally, Anti- C5 antibody includes the VL sequence in SEQ ID NO:113, the posttranslational modification including the sequence.In special embodiment party In case, VL includes one, two or three HVR (a) HVR-L1 selected from the following, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 include amino acid sequence SEQ ID NO:124；(c) HVR-L3, it is described HVR-L3 includes amino acid sequence SEQ ID NO:125.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes in any embodiment provided above VL in VH and any embodiment provided above.In one embodiment, the antibody separately includes SEQ ID NO: In 10,106-110 it is any in any and SEQ ID NO:20,111-113 in VH and VL sequence, the translation including the sequence After modify.In one embodiment, the antibody includes the VH sequence of SEQ ID NO:10 and the VL sequence of SEQ ID NO:20 Column.In one embodiment, the antibody includes the VH sequence of SEQ ID NO:106 and the VL sequence of SEQ ID NO:111. In another embodiment, the antibody includes the VH sequence of SEQ ID NO:107 and the VL sequence of SEQ ID NO:111. In a further embodiment, the antibody includes the VH sequence of SEQ ID NO:108 and the VL sequence of SEQ ID NO:111. In another embodiment, the antibody includes the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO:111. In another embodiment, the antibody includes the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO:112. In another embodiment, the antibody includes the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO:113. In another embodiment, the antibody includes the VH sequence of SEQ ID NO:110 and the VL sequence of SEQ ID NO:113.
In one aspect, anti-C5 antibody is provided, wherein the antibody includes to include VH sequence below: (a) HVR-H1, The HVR-H1 includes amino acid sequence SEQ ID NO:54, and (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:64, and (c) HVR-H3, the HVR-H3 includes amino acid sequence SEQ ID NO:74, and includes VL sequence below: (a) HVR-L1, the HVR-L1 include amino acid sequence SEQ ID NO:84；(b) HVR-L2, the HVR-L2 include amino acid sequence Arrange SEQ ID NO:94；(c) HVR-L3, the HVR-L3 include amino acid sequence SEQ ID NO:104.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes to include VH sequence below: (a) HVR- H1, the HVR-H1 include amino acid sequence SEQ ID NO:117, and (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:118, and (c) HVR-H3, the HVR-H3 include amino acid sequence SEQ ID NO:121, and comprising below VL sequence: (a) HVR-L1, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 packet The ID of SEQ containing amino acid sequence NO:123；(c) HVR-L3, the HVR-L3 include amino acid sequence SEQ ID NO:125.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes to include VH sequence below: (a) HVR- H1, the HVR-H1 include amino acid sequence SEQ ID NO:117, and (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:119, and (c) HVR-H3, the HVR-H3 include amino acid sequence SEQ ID NO:121, and comprising below VL sequence: (a) HVR-L1, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 packet The ID of SEQ containing amino acid sequence NO:123；(c) HVR-L3, the HVR-L3 include amino acid sequence SEQ ID NO:125.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes to include VH sequence below: (a) HVR- H1, the HVR-H1 include amino acid sequence SEQ ID NO:117, and (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:118, and (c) HVR-H3, the HVR-H3 include amino acid sequence SEQ ID NO:121, and comprising below VL sequence: (a) HVR-L1, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 packet The ID of SEQ containing amino acid sequence NO:124；(c) HVR-L3, the HVR-L3 include amino acid sequence SEQ ID NO:125.
On the other hand, anti-C5 antibody is provided, wherein the antibody includes to include VH sequence below: (a) HVR- H1, the HVR-H1 include amino acid sequence SEQ ID NO:117, and (b) HVR-H2, the HVR-H2 include amino acid sequence SEQ ID NO:120, and (c) HVR-H3, the HVR-H3 include amino acid sequence SEQ ID NO:121, and comprising below VL sequence: (a) HVR-L1, the HVR-L1 include amino acid sequence SEQ ID NO:122；(b) HVR-L2, the HVR-L2 packet The ID of SEQ containing amino acid sequence NO:124；(c) HVR-L3, the HVR-L3 include amino acid sequence SEQ ID NO:125.
In certain embodiments, anti-C5 antibody of the invention include any embodiment provided above in VH and Include SEQ ID NO:33, the heavy chain constant region of any of 34,35,114,115 and 116 amino acid sequence.In certain realities It applies in scheme, anti-C5 antibody of the invention includes VL in any embodiment provided above and comprising SEQ ID NO:36, The constant region of light chain of any of 37 and 38 amino acid sequence.
On the other hand, the present invention provides antibody, and the antibody is identical in conjunction with anti-C5 antibody provided herein Epitope.For example, in certain embodiments, providing such antibody, the antibody is identical in conjunction with antibody described in table 2 Epitope.As proving following working examples, all anti-C5 antibody described in table 2 are all grouped into the identical of C5 In epitope frame and show pH- dependence binding characteristic.
In a further aspect, the present invention provides antibody, the antibody identical epitope in conjunction with antibody provided herein. In a further aspect, the present invention provides antibody, the antibody identical epitope in conjunction with antibody described in table 7 or 8.Certain In embodiment, antibody is provided, the antibody combines the piece being made of the amino acid 33-124 of the β chain (SEQ ID NO:40) of C5 Epitope in section.In certain embodiments, antibody, the table in the β chain (SEQ ID NO:40) of the antibody combination C5 are provided Position, the epitope include the segment that at least one is selected from the group being made up of: amino acid 47-57,70-76 and 107-110. In certain embodiments, antibody, the epitope in the segment of the β chain (SEQ ID NO:40) of the antibody combination C5, institute are provided Stating epitope includes the amino acid that at least one is selected from the group being made up of: Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109, and His110.In another embodiment, the epitope of anti-C5 antibody of the invention is comformational epitope.
It in another aspect of the invention, is monoclonal antibody according to the anti-C5 antibody of any embodiments above, including Chimeric, humanization or human antibody.In one embodiment, anti-C5 antibody is antibody fragment, for example, Fv, Fab, Fab ', ScFv, double antibody or F (ab ')2Segment.In another embodiment, the antibody is full length antibody, for example, completely IgG1 or IgG4 antibody or other antibody isotypes or isotype being defined herein.
On the other hand, can be appointed in conjunction with described in following 1-7 section according to the anti-C5 antibody of any embodiments above It anticipates feature (either individually or in combination).
1. affinity of antibody
In certain embodiments, the dissociation constant (Kd) that antibody provided herein has are as follows: 1 μM or less, 100nM with Under, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less or 0.001nM or less be (for example, 10-8M is hereinafter, for example, 10- 8M to 10-13M, for example, 10-9M to 10-13M)。
In one embodiment, Kd measures (RIA) measurement by radiolabeled antigen binding.In an embodiment party In case, RIA is carried out using the Fab form and its antigen of purpose antibody.For example, Fab passes through the solution binding affinity of antigen Following manner measurement: there are the titration series of unlabelled antigen Cmin (125I) antigen marked is flat Weigh Fab, then with the antigen of anti-Fab antibody coated plate capture combination (see, e.g., Chen etc., J.Mol.Biol.293:865-881 (1999)).In order to determine determination condition, by MICROTITER (registered trademark) porous plate (Thermo Scientific) is used in the anti-Fab antibody (Cappel of capture of 5 μ g/ml in 50mM sodium carbonate (pH9.6) Labs) overnight coating, and 2% (w/v) fetal calf serum albumin being subsequently used in PBS (about 23 DEG C) of room temperature closing two to Five hours.In non-adsorbent plate (Nunc#269620), by 100pM or 26pM [125I]-antigen is continuous dilute with purpose Fab's Release liquid mixing (for example, with Presta etc., anti-VEGF antibodies in Cancer Res.57:4593-4599 (1997), Fab-12 Assessment it is consistent).Then purpose Fabb is incubated overnight；However, longer time (for example, about 65 hours) can be continued by being incubated for To guarantee to realize balance.Thereafter, mixture is transferred to capture plate and is used to be incubated at room temperature (for example, continuing one hour).Then It removes solution and 0.1% polysorbate20 (TWEEN-20 (registered trademark)) that plate is used in PBS washs eight times.When flat When plate has been dried, the scintillator (MICROSCINT-20 in 150 holes μ l/ is addedTM；Packard), and by plate exist TOPCOUNTTMDozens of minutes are counted on γ calculating instrument (Packard).Selection causes each less than or equal to 20% maximum combined The concentration of Fab is used for competitive binding assay.
According to another embodiment, Kd uses the measurement measurement of BIACORE (registered trademark) surface plasma resonance.Example Such as, it is carried out with~10 response units (RU) using BIACORE (registered trademark)-at 25 DEG C using immobilized antigen CM5 chip The measurement of 2000 or BIACORE (registered trademark) -3000 (BIACORE (registered trademark), Inc., Piscataway, NJ).One In a embodiment, according to the operation instruction of supplier, by carboxymethylated dextran biosensor chip (CM5, BIACORE (registered trademark), Inc.) use N- ethyl-N '-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- HOSu NHS (NHS) activation.Antigen is diluted to 5 μ g/ml (~0.2 μM) with 10mM sodium acetate pH4.8, later with 5 μ L/ minutes flow velocitys inject about 10 response units (RU) to realize coupling protein.After injections of antigens, 1M ethanol amine is injected To close unreacted radical.It is continuous dilute by twice of Fab with about 25 l/ minutes flow velocitys of μ at 25 DEG C for kinetic measurement Releasing liquid, (0.78nM to 500nM) is injected into 0.05% polysorbate20 (TWEEN-20TM) surfactant PBS (PBST) in.Use simple one-to-one (one-to-one) Langmuir (Langmuir) binding model (BIACORE (registered trademark) Evaluation Software version 3 .2) by simultaneously fitting Combination and dissociation influence chart come calculations incorporated rate (kon) reconciliation From rate (koff).As koff/konThe ratio between calculated equilibrium dissociation constant (Kd).See, e.g., Chen etc., J.Mol.Biol.293:865-881 (1999).If the association rate by the measurement measurement of the above surface plasma resonance is more than 106M-1s-1, then association rate can be determined in the following manner: such as in spectrometer such as equipped with cut-off equipment (stop-flow) Spectrophotometer (Aviv Instruments) or 8000- series SLM-AMINCO with teeter chamberTMSpectrophotometer (ThermoSpectronic) it is measured in, using measurement in the case where there is the antigen for increasing concentration, at 25 DEG C, in PBS, The fluorescent quenching technology of the fluorescent emission intensity of the anti-antigen-antibody of 20nM (Fab form) in pH 7.2 increasedd or decreased (swashs Hair=295nm；Transmitting=340nm, 16nm band logical).
2. antibody fragment
In certain embodiments, antibody provided herein is antibody fragment.Antibody fragment includes but is not limited to Fab, Fab ', Fab '-SH, F (ab ')2, Fv and scFv segment and other segments described below.For the comprehensive of specific antibodies segment It states, referring to the Nat.Med.9:129-134 such as Hudson (2003).For the summary of scFv segment, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore Editor, (Springer-Verlag, New York), pp.269-315 (1994)；It sees also, WO 93/16185；And the U.S. The patent No. 5,571,894 and 5,587,458.For comprising salvage receptor binding epitope residue and with increased Half-life in vivo Fab and F (ab ')2The discussion of segment, referring to U.S. Patent number 5,869,046.
Double antibody is tool there are two the antibody fragment of antigen binding site, be can be divalent or bispecific.Ginseng See, for example, EP 404,097；WO 1993/01161；Hudson etc., Nat.Med.9:129-134 (2003)；And Hollinger Deng Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993).Hudson etc., Nat.Med.9:129-134 (2003) In also illustrate ternary antibody and quaternary antibody.
Single domain antibody is such antibody fragment, it includes all or part of heavy-chain variable domains of antibody or All or part of light variable domains.In certain embodiments, single domain antibody is people's single domain antibody (Domantis, Inc., Waltham, MA；See, e.g., U.S. Patent number 6,248,516.
Antibody fragment can be prepared by multiple technologies, and the technology includes but is not limited to that the proteolysis of complete antibody disappears Change and pass through the preparation of recombinant host cell (for example, Escherichia coli or bacteriophage), as described herein.
3. chimeric and humanized antibody
In certain embodiments, antibody provided herein is chimeric antibody.Certain chimeric antibodies are described in for example U.S. Patent number 4,816,567；With Morrison etc., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)) In.In an example, chimeric antibody includes that (for example, deriving from mouse, rat, hamster, rabbit or inhuman spirit are long for non-human variable domains The variable region of class animal such as monkey) and human constant region.In additional examples, chimeric antibody is " type conversion " antibody, wherein class Type or subclass change via the type of parental antibody or subclass.Chimeric antibody includes its antigen-binding fragment.
In certain embodiments, chimeric antibody is humanized antibody.Typically, by non-human antibody's humanization to reduce pair The immunogenicity of people, while retaining the specificity and affinity of parent non-human antibody.In general, humanized antibody includes one or more A variable domains, wherein HVR, for example, CDR (or part thereof) from non-human antibody and FR (or part thereof) derive from people Antibody sequence.Humanized antibody optionally also includes at least part of human constant region.In some embodiments, humanization is anti- Some FR residues in body are replaced as the corresponding residue from non-human antibody (for example, antibody that HVR residue is derived from), example Such as, to restore or improve antibody specificity or affinity.
Humanized antibody and preparation method thereof is summarized in such as Almagro and Fransson, Front.Biosci.13: It in 1619-1633 (2008), and is further described in such as Riechmann, Nature 332:323-329 (1988)； Queen etc., Proc.Nat ' lAcad.Sci.USA86:10029-10033 (1989)；U.S. Patent number 5,821,337,7,527, 791,6,982,321 and 7,087,409；Kashmiri etc., Methods 36:25-34 (2005) (describe specificity and determine area (SDR) it transplants)；Padlan, Mol.Immunol.28:489-498 (1991) (describe " surface reconstruction (resurfacing)″)；Dall ' Acqua etc., Methods 36:43-60 (2005) (describe " FR reorganization (shuffling)″)；With Osbourn etc., Methods 36:61-68 (2005) and Klimka etc., Br.J.Cancer, 83: In 252-260 (2000) (describing " M8003 line " method for FR reorganization).
The people's framework region that can be used for humanization includes but is not limited to: using " best match (best-fit) " method choice Framework region (see, e.g., the J.Immunol.151:2296 such as Sims (1993))；From the light chain with specific subgroup or again The framework region of the consensus sequence of the human antibody of chain variable region is (see, e.g., Carter etc., Proc.Natl.Acad.Sci.USA 89:4285 (1992)；With the J.Immunol.151:2623 (1993) such as Presta)；People's maturation (body mutation) framework region or life Growing is framework region (see, e.g., Almagro and Fransson, Front.Biosci.13:1619-1633 (2008))；With Derived from FR library screening framework region (see, e.g., Baca etc., J.Biol.Chem.272:10678-10684 (1997) and Rosok etc., J.Biol.Chem.271:22611-22618 (1996)).
4. human antibody
In certain embodiments, antibody provided herein is human antibody.Human antibody can be used as is generally known in the art Multiple technologies preparation.Human antibody is described generically in van Dijk and van de Winkel, Curr.Opin.Pharma.5:368-74 (2001) and Lonberg, Curr.Opin.Immunol.20:450-459 (2008) In.
Human antibody can be by being modified to production complete human antibody or complete antibody with people variable region with sound The transgenic animals for answering antigen to attack apply immunogene to prepare.Such animal typically contains all or part of people and ball is immunized Protein gene seat, substitutes endogenous immunoglobulin genes seat or it is present in outside chromosome or by random integration to moving In the chromosome of object.In such transgenic mice, endogenous immunoglobulin genes seat has usually been deactivated.For by turning base Because animal obtains the summary of the method for human antibody, referring to Lonberg, Nat.Biotech.23:1117-1125 (2005).Also join See, for example, U.S. Patent number 6,075,181 and 6,150,584, which depict XENOMOUSETMTechnology；U.S. Patent number 5, 770,429, which depict HUMAB (registered trademark) technologies；U.S. Patent number 7,041,870, which depict K-M MOUSE (notes Volume trade mark) technology and U.S. Patent Application Publication No. US 2007/0061900, which depict VELOCIMOUSE (registered trademarks) Technology).Can be further embellished from the people variable region of complete antibody that thus kind animal generates, for example, by with difference Human constant region combination.
Human antibody can also be prepared by the method based on hybridoma.Be used to prepare human monoclonal antibodies human myeloma and Mouse-human heteromyeloma's cell line has been described.(see, e.g., Kozbor J.Immunol., 133:3001 (1984)； Brodeur etc., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Dekker, Inc., New York, 1987)；With Boerner etc., J.Immunol.147:86 (1991).).Via people's B- cell The human antibody of hybridoma technology preparation is also described in Li et al., Proc.Natl.Acad.Sci.USA 103:3557-3562 (2006) in.Other method is included in such as U.S. Patent number 7,189,826 and (describes and prepare Dan Ke by hybridoma cell line Grand human IgM antibody) and (2006) Ni, Xiandai Mianyixue 26 (4): 265-268 (describing people-people's hybridoma) in retouch Those of state.People's hybridoma technology (Trioma technology) is also described in Vollmers, Histology and (2005) Histopathology 20 (3): 927-937 and Vollmers, Methods and Findings in In (2005) Experimental and Clinical Pharmacology 27 (3): 185-191.
Human antibody can also be produced by separating Fv clone's variable domain sequence selected from source of people phage display library It is raw.Then such variable domain sequence can be combined with required people's constant domain.It is described below for by antibody library Select the technology of human antibody.
5. deriving from the antibody in library
Antibody of the invention can have the combinatorial libraries of required one or more active antibody to separate by screening. For example, a variety of methods are used to generate phage display library and for the antibody with required binding property as is generally known in the art Screen the library.Such method is summarized in such as Hoogenboom etc., Methods in Molecular Biology In 178:1-37 (O ' Brien etc., ed., Human Press, Totowa, NJ, 2001) and it is further described in for example McCafferty etc., Nature 348:552-554；Clackson etc., Nature 352:624-628 (1991)；Marks etc., J.Mol.Biol.222:581-597 (1992)；Marks, Meth.Mol.Biol., 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003)；Sidhu etc., J.Mol.Biol.338 (2): 299-310 (2004)；Lee etc., J.Mol.Biol.340 (5): 1073-1093 (2004)；Fellouse, Proc.Natl.Acad.Sci.USA 101 (34): 12467-12472(2004)；In Lee etc., J.Immunol.Methods284 (1-2): 119-132 (2004).
In some bacteriophages methods of exhibiting, VH and VL gene pool by polymerase chain reaction (PCR) respectively clone and It is recombinated at random in phage library, then can be directed to the library in conjunction with described in the phage selection of antigen, such as Winter, Described in Ann.Rev.Immunol.12:433-455 (1994).Antibody fragment is typically shown as scFv by bacteriophage (scFv) segment or Fab segment.Library from the source being immunized provides high-affinity antibody without structure to immunogene Build hybridoma.It is alternatively possible to clone natural library (naive) (for example, from people) to a variety of non-self antigens and itself The antibody that antigen provides single source is any immune without carrying out, such as Griffiths, EMBO J, 12:725-734 (1993) described.Naive libraries are prepared finally, can also be synthesized by the following: the V- gene that white spirit cell clone is not reset Section, and reset using the PCR primer containing random sequence with encoding the area Gao Bian CDR3 and realizing in vitro, such as Hoogenboom, J.Mol.Biol.227:381-388 (1992) are described.The patent disclosure packet of human antibody phage library is described It includes, such as: U.S. Patent number 5,750,373 and U.S. Publication No 2005/0079574,2005/0119455,2005/ 0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.
Herein, the antibody or antibody fragment for being isolated from human antibody library are considered as human antibody or human antibody segment.
6. multi-specificity antibody
In certain embodiments, antibody provided herein is multi-specificity antibody, for example, bispecific antibody.It is more Specific antibody is such monoclonal antibody, has binding specificity at least two different sites.In certain implementations In scheme, a binding specificity is for C5 and the other is for another antigen.In certain embodiments, double special Property antibody can in conjunction with C5 two different epitopes.Bispecific antibody can be also used for for cytotoxic agent being positioned at expression The cell of C5.Bispecific antibody can be used as full length antibody or antibody fragment is produced.
The technology for being used to prepare multi-specificity antibody includes but is not limited to two immunoglobulins with not homospecificity The recombinant co-expression (referring to Milstein and Cuello, Nature305:537 (1983)) of heavy chain-light chain pair, WO 93/ 08829 and Traunecker etc., EMBO are J.10:3655 (1991)), and " protrusion-hole (knob-in-hole) " engineered (see, e.g., U.S. Patent number 5,731,168).Multi-specificity antibody can also be used to prepare antibody Fc-by engineered The electrostatic guiding role of heterodimeric molecule prepares (WO2009/089004A1)；It is crosslinked more than two antibody or segment (ginseng See, for example, U.S. Patent number 4,676,980 and Brennan etc., Science, 229:81 (1985))；Using leucine zipper with Prepare bispecific antibody (see, e.g., Kostelny etc., J.Immunol.148 (5): 1547-1553 (1992))；Using " Double antibody " technology be used to prepare bispecific antibody fragment (see, e.g., Hollinger etc., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993))；With use scFv (scFv) dimer (referring to example Such as, Gruber etc., J.Immunol.152:5368 (1994))；And three-specific antibody is prepared, such as such as Tutt, Described in J.Immunol.147:60 (1991).
It herein further include that there are three the modified antibody of above functions antigen binding site, including " octopus antibody " for tool (see, e.g., US 2006/0025576).
Antibody or segment herein further includes " double action FAb " or " DAF ", and it includes combine C5 and another kind not The antigen binding site of same antigen (for example, see US2008/0069820).
7. antibody variants
In certain embodiments, consider the amino acid sequence variation of antibody provided herein.For example, it is desirable that mentioning The binding affinity and/or other biological property of high antibody.The amino acid sequence variation of antibody can be by described in coding The nucleotide sequence of antibody introduces suitable modification or is prepared by peptide synthesis.Such modification includes, for example, from antibody amino groups The missing of acid sequence, and/or the insertion into antibody amino acids sequence and/or the residue in displacement antibody amino acids sequence.It can It to be lacked, is inserted into and displaced any combination is to obtain final construct, on condition that needed for final construct has Feature, for example, antigen-combination.
A. it replaces, insertion and deletion mutants
In certain embodiments, the antibody variants with one or more amino acid replacements are provided.Replace the mesh of mutagenesis Site include HVR and FR.Conservative substitution is shown under the title of " the preferred displacement " in table 1.More change is provided at It is under the title of " exemplary permutation " in table 1 and such as described further below in relation to amino acid side chain classification.Amino acid replacement Can be introduced into purpose antibody and carry out needed for activity (for example, keep/improve antigen binding, reduced immunogenicity or The ADCC or CDC of raising) screening product in.
[table 1]
Initial residue Exemplary permutation It is preferred that replacing
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gin；His；Asp, Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Lcu；Val；Met；Ala；Phc；Nor-leucine Leu
Leu(L) Nor-leucine；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；Nor-leucine Leu
Amino acid can be grouped according to shared side chain properties are as follows: (1) hydrophobicity: nor-leucine, Met, Ala, Val, Leu, Ile；(2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln；(3) acid: Asp, Glu；(4) alkaline: His, Lys, Arg；(5) residue of chain orientation: Gly, Pro is influenced；(6) armaticity: Trp, Tyr, Phe.
The member that non-conservative displacement needs the member by one of these groups to change another group into.
A kind of one or more hypervariable regions of the displacement variant including displacement parental antibody (for example, humanization or human antibody) are residual Base.In general, certain biological property (examples will be had relative to parental antibody by being selected for the resulting variant further studied Such as, increased affinity, reduced immunogenicity) change (for example, improve) and/or by be kept substantially parental antibody certain A little biological properties.Exemplary permutation variant is affinity maturation antibody, can be for example, using the parent based on phage display With power mature technology (as described in this article those) conventional preparation.In short, by one or more HVR residue mutations and will become Body antibody display is on bacteriophage and screens specific biological activity (for example, binding affinity).
Changing (for example, displacement) can carry out in HVR, for example, to improve affinity of antibody.Such change can be It is carried out in HVR " hot spot ", the HVR " hot spot " is compiled i.e. during body cell maturation with the codon that high-frequency is mutated The residue (see, e.g., Chowdhury, Methods Mol.Biol.207:179-196 (2008)) of code, and/or contact are anti- Former residue, tests the binding affinity of resulting variant VH or VL.Reselection is carried out by building second level library and from it Affinity maturation has been described in such as Hoogenboom, Methods in Molecular Biology178:1-37 (O ' Brien etc., ed., Human Press, Totowa, NJ, (2001)) in.In some embodiments of affinity maturation, pass through Diversity is introduced selection and used by any one of a variety of methods (for example, fallibility PCR, chain reorganization or oligonucleotides directed mutagenesis) In mature variable gene.Then second level library is generated.Then it is any with required affinity to identify to screen the library Antibody variants.Introducing multifarious another method includes HVR orientation method, wherein several HVR residues are (for example, 4-6 simultaneously A residue) it is randomized.Can be for example using alanine scanning mutagenesis or modeling, the HVR that specific identification participates in antigen binding is residual Base.Particularly, CDR-H3 and CDR-L3 are usually targeted.
In certain embodiments, it replaces, insertion or missing can occur in one or more HVR, so long as Change the ability for not being substantially reduced antibodies bind antigen.For example, not being substantially reduced the conservative change of binding affinity (for example, originally Conservative substitution described in the text) it can be carried out in HVR.Such change can contact the outer of the residue of antigen for example in HVR Portion.In certain embodiments of variant VH and the VL sequence of above-mentioned offer, each HVR is unchanged, or contains and be no more than one, Two or three amino acid replacements.
Can be used for identify can be targeted for mutagenesis antibody residue or region method be referred to as " alanine scanning lure Become ", such as Cunningham, Science 244:1081-1085 (1989) is described.In the method, a residue or one group of mesh Residue (for example, electrically charged residue such as arg, asp, his, lys and glu) is marked to be accredited and by neutral or electronegative amino acid (for example, alanine or polyalanine) substitutes the interaction to determine whether influence antibody and antigen.It can be to initially setting It changes at the amino acid position of display function sensibility and introduces further displacement.Alternatively, or additionally, Ag-Ab is compound The crystal structure of object is to determine the contact point between antibody and antigen.Such contact residues and adjacent residues can be targeted or by It excludes as displaced candidate.Variant can be screened to determine if the property for having required.
Amino acid sequence insertion include length be residue to the aminoterminal for the polypeptide for containing 100 or more residues and/or Insertion in the sequence of c-terminus fusion and single or multiple amino acid residues.The example of end insertion includes having the end N- first The antibody of sulphur methionyl residues.Other insertion variants of antibody molecule include that the N-terminal of antibody or C-terminal partly decline with antibody serum is increased The enzyme (for example, for ADEPT) or peptide fusion of phase.
B. glycosylation variants
In certain embodiments, antibody provided herein is changed to increased or decrease the journey that antibody is glycosylated Degree.Glycosylation site is added to antibody or it is allow to lack glycosylation site by changing amino acid sequence so that generating or removing One or more glycosylation sites are gone easy to accomplish.
When antibody includes the area Fc, thus it is possible to vary carbohydrate connected to it.The day generated by mammalian cell Right antibody typically comprises branch, (biantennary) oligosaccharides of bifurcation, and the oligosaccharides is usually connected to by N- connection The Asn297 of the CH2 structural domain in the area Fc.See, e.g., Wright etc., TIBTECH 15:26-32 (1997).Oligosaccharides can wrap Include multiple kinds of carbohydrate, for example, mannose, N-acetyl-glucosamine (GlcNAc), galactolipin and sialic acid, and with bifurcation Oligosaccharide structure " stem " in the connected fucose of GlcNAc.In some embodiments, it can carry out in antibody of the present invention Oligosaccharides modification to generate the antibody variants with specific improved property.
In one embodiment, antibody variants are provided, has and lacks the rock algae being connected with the area Fc (direct or indirect) The carbohydrate structure of sugar.For example, in such antibody fucose amount can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%.The amount of fucose is (such as compound relative to all sugar structures being connected with Asn 297 by calculating Object, heterocomplex and high mannose structures) summation Asn297 at sugar chain in the average magnitude of fucose determine, such as pass through The measurement of MALDI-TOF mass spectrography, as described in WO2008/077546, such as.Asn297 refers to positioned at the position 297 in the area Fc The asparagicacid residue of (Eu of the area Fc residue is numbered) nearby；However, due to sequence variations small in antibody, Asn297 can also be with At the upstream of position 297 or about +/- 3 amino acid in downstream, that is, between position 294 and 300.Such fucosido Changing variant can have the ADCC function of raising.See, e.g., U.S. Patent Publication number US 2003/0157108 (Presta, L.)；US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).It is related to " removing fucosylation " or " fucose lacks " the disclosed example variant of antibody includes: US 2003/0157108 to swaged；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704； US 2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586； WO 2005/035778；WO 2005/053742；WO 2002/031140；Okazaki etc., J.Mol.Biol.336:1239- 1249(2004)；Yamane-Ohnuki etc., Biotech.Bioeng.87:614 (2004).It is anti-that fucosylation can be prepared The example of the cell line of body include albumen fucosylation deficiency Lecl3CHO cell (Ripka etc., Arch.Biochem.Biophys.249:533-545 (1986)；US 2003/0157108, Presta, L；And WO2004/ 056312, Adams etc., especially embodiment 11), and the cell line knocked out, such as α -1,6- fucosyl transferase gene, FUT8, the Chinese hamster ovary celI of knockout is (see, e.g., Yamane-Ohnuki etc., Biotech.Bioeng.87:614 (2004)； Kanda etc., Biotechnol.Bioeng.94 (4): 680-688 (2006)；And WO2003/085107).
The antibody variants for having the oligosaccharides being bisected also are provided, for example, the bifurcation being wherein connected with antibody Fc district Oligosaccharides is halved by GlcNAc.Such antibody variants can have the ADCC function of reduced fucosylation and/or raising.This The example of kind antibody variants is described in such as WO 2003/011878 (Jean-Mairet)；U.S. Patent number 6,602,684 (Umana etc.)；In US 2005/0123546 (Umana etc.).Also providing in the connected oligosaccharides in the area Yu Fc has at least one The antibody variants of galactose residue.Such antibody variants can have the CDC function of raising.Such antibody variants are described in example Such as WO 1997/30087 (Patel)；WO 1998/58964 (Raju, S.)；In WO 1999/22764 (Raju, S.).
C.Fc region variants
In certain embodiments, one or more amino acid modifications can be introduced into the Fc of antibody provided herein Thus Qu Zhong generates Fc region variants.It includes amino acid modification that Fc region variants, which may be embodied in one or more amino acid positions, The people Fc region sequence (for example, human IgG1, IgG2, the area IgG3 or IgG4 Fc) of (for example, displacement).
In certain embodiments, the present invention considers such antibody variants, with some but not all effector function Can, this makes that it is important antibody Half-life in vivo and certain effector functions (such as complement and ADCC) are inessential or harmful Application be ideal candidate.The measurement of external and/or in vivo cytotoxicity can be carried out to confirm CDC and/or ADCC activity Reduction/elimination.For example, Fc receptor (FcR) binding assay can be carried out with guarantee antibody lack Fc γ R combine (therefore may Lack ADCC activity), but it is to maintain FcRn binding ability.The main cell of ADCC is mediated, NK cell only expresses Fc γ RIII, And monocytes Fc γ RI, Fc γ RII and Fc γ RIII.On hematopoietic cell FcR expression be summarized in Ravetch and In table 3 on Kinet, Annu.Rev.Immunol.9:457-492 (1991) page 464.ADCC for purpose of appraisals molecule The non-limiting example of active external test is described in U.S. Patent number 5,500,362 (see, e.g., Hellstrom etc. Proc.Nat ' l Acad.Sci.USA 83:7059-7063 (1986)) and Hellstrom etc., Proc.Nat ' l Acad.Sci.USA 82:1499-1502 (1985)；U.S. Patent number 5,821,337 (referring to Bruggemann etc., J.Exp.Med.166:1351-1361 (1987)) in.It is alternatively possible to using on-radiation measuring method (see, e.g., being used for The ACTI of flow cytometryTMNon-radioactive cell toxicity test (CellTechnology, Inc.Mountain View, CA)；With (registered trademark) the non-radioactive cell toxicity test of CytoTox 96 (Promega, Madison, WI)).It can be used for such measurement Effector cell include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cell.Alternatively, or additionally, Ke Yi In vivo, such as in the animal model as described in the Proc.Nat ' l Acad.Sci.USA95:652-656 such as Clynes (1998) The ADCC activity of middle purpose of appraisals molecule.C1q binding assay can also be carried out to confirm therefore antibody cannot be in conjunction with C1q and lacking Few CDC activity.See, e.g., C1q the and C3c combination ELISA in WO2006/029879 and WO2005/100402.In order to comment Estimate complement activation, can carry out CDC measurement (see, e.g., Gazzano-Santoro etc., J.Immunol.Methods 202: 163(1996)；Cragg etc., Blood101:1045-1052 (2003)；With Cragg etc., Blood 103:2738-2743 (2004)).Also method progress FcRn as known in the art can be used to combine with internal removing/half-life period measurement (referring to example Such as, Petkova etc., Int ' l.Immunol.18 (12): 1759-1769 (2006)).
Antibody with reduced effector function includes having 238,265,269,270,297,327 He of the area Fc residue One or more of 329 displaced antibody (U.S. Patent number 6,737,056).Such Fc mutant is included in amino acid position Setting at the two or more in 265,269,270,297 and 327 has displaced Fc mutant, including so-called residue 265 and 297 It replaces to " DANA " the Fc mutant (U.S. Patent number 7,332,581) of alanine.
Describe certain antibody variants with combination improve or reduction and FcR.(see, e.g., United States Patent (USP) Numbers 6,737,056；WO 2004/056312 and Shields etc., J.Biol.Chem.9 (2): 6591-6604 (2001).)
In certain embodiments, antibody variants include the area Fc, and the area Fc has one or more ammonia for improving ADCC Base acid displacement, for example, the displacement at the position 298,333 and/or 334 (EU of residue is numbered) in the area Fc.
In some embodiments, be changed in the area Fc, it is described change cause change (that is, improve or reduce ) C1q is combined and/or complement-dependent cytotoxicity (CDC), for example, such as U.S. Patent number 6,194,551, WO1999/ Described in 51642 and Idusogie etc., J.Immunol.164:4178-4184 (2000).
Such antibody is described in US2005/0014934 (Hinton etc.), with increased half-life period and raising Be responsible for by Maternal immunoglobulin G transfer to fetus neonatal Fc receptor (FcRn) (J.Immunol.117:587 (1976) and Kim etc., J.Immunol.24:249 (1994)) combination.The antibody includes the area Fc with one or more of displacements, described Displacement improves the combination in the area Fc and FcRn.Such Fc variant includes that at the following one or more area Fc residue with displacement It is a little: 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380, 382,413,424 or 434, for example, the displacement (U.S. Patent number 7,371,826) of the area Fc residue 434.
Referring also to Duncan, Nature 322:738-40 (1988)；U.S. Patent number 5,648,260；U.S. Patent number 5,624,821；And it is related to the WO 1994/29351 of other examples of Fc region variants.
D. cysteine engineering reform antibody variant
In certain embodiments, it may be desirable to cysteine engineering reform antibody is prepared, for example, " thioMAb ", Wherein one or more residues of antibody are replaced as cysteine residues.In special embodiment, displaced residue goes out At the access site of present antibody.By the way that the residue is replaced as cysteine, thus reactive thiol group group is placed in anti- At the access site of body and it can be used for antibody conjugate to other parts, such as drug moiety or linker-drug part, thus Immunoconjugates are generated, as further described herein.In certain embodiments, any one or more in following residue It can be replaced as cysteine: the V205 (Kabat number) of light chain；The A118 (EU number) of heavy chain；With the area heavy chain Fc S400 (EU number).Cysteine engineering reform antibody can be generated as described in such as U.S. Patent number 7,521,541.
E. antibody derivatives
In certain embodiments, antibody provided herein can be further embellished to containing as is generally known in the art And readily available other non-protein part.Part suitable for antibody derivatization includes but is not limited to water-soluble poly Close object.The non-limiting example of water-soluble polymer includes but is not limited to polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, Carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1,3- dioxolane, poly- 1,3,6- tri- dislike Alkane, ethylene/copolymer-maleic anhydride, polyaminoacid (homopolymer or random copolymer) and glucan or poly- (n- vinyl pyrrole Alkanone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, oxyethylated polyols (for example, Glycerol), polyvinyl alcohol and its mixture.Methoxy PEG-propionaldehyde can be in preparation advantageous due to its stability in water 's.Polymer can have any molecular weight, and can be branch or non-branch.The number for the polymer being connected with antibody Mesh can change, and if more than one polymer of connection, can be identical or different molecule.Generally, due to derivative The quantity and/or type of the polymer of change can based on determination considered below, including but not limited to, improved antibody it is specific Whether property or function, antibody derivatives can be used for the treatment, etc. under qualifications.
In another embodiment, the antibody that can be selectively heated and being exposed to radiation and non-protein are provided Partial conjugate.In one embodiment, non-protein part be carbon nanotube (Kam etc., Proc.Natl.Acad.Sci.USA 102:11600-11605 (2005)).The radiation can have any wavelength, and wrap Such wavelength is included but is not limited to, the wavelength does not damage normal cell, but it is non-that non-protein part is heated to neighbouring antibody- The killed temperature of the cell of protein part.
B. recombination method and composition
Recombination method and composition preparation can be used in antibody, for example, such as U.S. Patent number 4, described in 816,567.? In one embodiment, the nucleic acid of separation, code book anti-C5 antibody described in the text are provided.Such nucleic acid can encode packet The amino acid sequence of the VL containing antibody and/or amino acid sequence (for example, light chain and/or heavy chain of antibody) comprising antibody VH.? In another embodiment, one or more carriers (for example, expression vector) comprising such nucleic acid are provided.In another implementation In scheme, the host cell comprising such nucleic acid is provided.In such embodiment, host cell includes (for example, turning Change has): (1) carrier, the carrier include nucleic acid, and the nucleic acid encode includes the amino acid sequence of antibody VL and includes antibody VH Amino acid sequence, or (2) include coding comprising antibody VL amino acid sequence nucleic acid first vector, and include coding packet The Second support of the nucleic acid of the amino acid sequence of the VH containing antibody.In one embodiment, host cell is eukaryon, for example, Chinese hamster ovary (CHO) cell or lymphocyte (for example, Y0, NS0, Sp20 cell).In one embodiment, system is provided The method of standby anti-C5 antibody, wherein the method includes cultivating to include under conditions of being suitable for expressing antibody as described above The host cell of the nucleic acid of encoding said antibody, and optionally antibody is recycled from host cell (or host cell culture medium).
In order to be prepared by recombinant anti-C5 antibody, such as the nucleic acid of encoding antibody as described above is separated and is inserted into one kind Or it for the further clone in host cell and/or is expressed in variety carrier.Conventional method appearance can be used in such nucleic acid It changes places and separation and (for example, using the oligonucleotide probe for the gene for capableing of specific bond encoding antibody heavy and light chain) is sequenced.
The host cell of carrier suitable for cloning or expressing encoding antibody includes that protokaryon or eukaryon described herein are thin Born of the same parents.For example, antibody can be prepared in bacterium, especially when not needing glycosylation and Fc effector function.For antibody piece Section and expression of the polypeptide in bacterium, see, e.g., U.S. Patent number 5,648,237,5,789,199 and 5,840,523.(also Referring to, Charlton, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which depict in expression in escherichia coli antibody fragment).After expression, antibody can To be separated in solvable fraction and can be further purified from bacterial cell thickener.
Other than prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also for the carrier of encoding antibody Suitable clone or expressive host, including glycosylation approach have partially or completely people's sugar by " humanization " so as to cause generation The fungi of the antibody of base mode and yeast strainss.Referring to Gerngross, Nat.Biotech.22:1409-1414 (2004), And Li et al., Nat.Biotech.24:210-215 (2006).
Host cell suitable for expressing glycosylated antibodies also derives from multicellular organism, and (invertebrate and vertebra are dynamic Object).The example of no vertebrate cell includes plant and insect cell.A variety of baculoviral strains have been identified, it can be with insect Cell is used together, especially for transfecting Spodopterafrugiperda (Spodoptera frugiperda) cell.
Plant cell cultures are also used as host.See, e.g., U.S. Patent number 5,959,177,6,040, 498,6,420,548,7,125,978 and 6,417,429 (which depict for producing antibody in transgenic plants PLANTIBODIESTMTechnology).
Vertebrate cells are also used as host.For example, the mammal cell line for the culture that is suitable for suspending can be with It is useful.Other examples of useful mammalian host cell line are the monkey kidney CV1 cell line of SV40 (COS-7) conversion； Human embryonic kidney cell (293 or such as such as Graham, 293 cells described in J.Gen Virol.36:59 (1977))；Children Hamster kidney cell (BHK)；Mouse sertoli cell (TM4 cell, such as such as Mather, Biol.Reprod.23:243-251 (1980) described in)；MK cells (CVl)；African green monkey kidney cell (VERO-76)；Human cervical carcinoma cell (HELA)；Dog kidney is thin Born of the same parents (MDCK；Buffalo hepatocytes (BRL 3A)；Human pneumonocyte (W138)；Human liver cell (Hep G2)；Mouse mammary tumor (MMT 060562)；TRI cell described in such as such as Mather, AnnalsN.Y.Acad.Sci.383:44-68 (1982)；MRC 5 cells；With FS4 cell.Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cell, including DHFR-Chinese hamster ovary celI (Urlaub etc., Proc.Natl.Acad.Sci.USA 77:4216 (1980))；Such as with myeloma cell line Y0, NS0 and Sp2/0.For being suitable for the summary of certain mammalian host cell lines of antibody producing, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).
It is more to prepare in animal that related antigen and adjuvant are preferably injected by (ip) in multiple subcutaneous (sc) or peritonaeum Clonal antibody.It might be useful that using difunctional or derivatization reagent, for example, maleimide yl benzoic acid succinimide Ester (is conjugated) by cysteine residues, and n-hydroxysuccinimide base (passes through lysine residue), glutaraldehyde, succinic anhydride, SOCl2Or R1N=C=NR, wherein R and R1Be different alkyl, by related antigen in the species to be immunized have exempt from The albumen of epidemic focus is (for example, keyhole limpet hemocyanin (keyhole limpet hemocyanin), serum albumin, bovine thyroid Globulin or soybean trypsin inhibitor) conjugation.
By the way that such as 100 μ g or 5 μ g albumen or conjugate (respectively for rabbit or mouse) and the Freund of 3 volumes are helped completely Agent merges and solution is carried out intracutaneous injection in multiple sites, makes animal (usually non-human mammal) for antigen, is immunized Immunogenic conjugate or derivative are immunized.After one month, by the subcutaneous injection at multiple sites, helped completely used in Freund The peptide or conjugate of 1/5 to 1/10 original amount in agent carry out booster immunization to animal.After 7 to 14 days, to animal bloodletting and Measure the antibody titer of serum.Booster immunization is carried out to animal until valency platform.Preferably, with from different albumen conjugation and/ Or the conjugate for the same antigen being conjugated by different crosslinking agents carries out booster immunization to animal.Conjugate can also be used as egg White fusions are prepared in recombinant cell culture thing.In addition, aggregating agent such as alum is suitable for enhancing immune response.
Antibody population of the monoclonal antibody obtained from substantially homogeneous, that is, constitute the individual antibody of the group in addition to can It can existing possible naturally occurring mutation and/or posttranslational modification (for example, isomerization, amidation) be in addition on a small quantity identical 's.Therefore, antibody characteristic of attribute " monoclonal " instruction not as the mixture of discrete antibody.
For example, hybridoma method preparation can be used in monoclonal antibody, the method is first by Kohler etc., Nature 256 (5517): 495-497 (1975) description.In hybridoma method, mouse or other suitable host animal (such as hamster) quilts It is immune to cause to generate or can generate and the lymph for the antibody in conjunction with immune protein-specific as above Cell.Alternatively, lymphocyte can be immunized in vitro.
Immunoreagent will typically comprise antigen protein or it merges variant.In general, if necessary to the cell in people source, then Using peripheral blood lymphocytes (PBL), or if necessary to non-human mammalian sources, then splenocyte or lymph node cells are used. Then lymphocyte is merged with the cell line of immortality using suitable fusion reagent (such as polyethylene glycol), to form hybridoma Cell (Goding, Monoclonal Antibodies:Principles and Practice, Academic Press (1986), pp.59-103).
The myeloma in the mammalian cell that immortal cell line usually converts, especially rodent, ox and people source is thin Born of the same parents.In general, using rat or mouse myeloma cell line.Therefore the hybridoma of preparation is inoculated in suitable culture medium And cultivate, the culture medium preferably contains one or more objects for inhibiting nonfused Parent Myeloma Cell growth or survival Matter.For example, if Parent Myeloma Cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), It typically will include then hypoxanthine, aminopterin and thymidine (HAT culture medium) for the culture medium of hybridoma, and be to prevent The substance of HGPRT deficient cells growth.
Preferred immortal myeloma cell is those cells, the cell effective integration, supports that selected antibody is raw The high-caliber production antibody of cytotostatic is produced, and sensitive to culture medium such as HAT culture medium.These, it is preferred to rat bone marrow tumour Cell line such as derives from and is available from Salk Institute Cell Distribution Center, San Diego, Those of MOPC-21 and MPC-11 mouse tumor of California USA, and be available from American Type Culture preservation The SP-2 cell of the heart (American Type Culture Collection, Manassas, Virginia USA) is (and its derivative Object, for example, X63-Ag8-653).Human myeloma and mouse-human heteromyeloma's cell line are also been described as producing people's list Clonal antibody (J such as Kozbor Immunol.133 (6): 3001-3005 (1984)；Brodeur etc., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp.51-63).
Production for the monoclonal antibody for antigen, the culture medium of measurement culture hybridoma.Preferably, by miscellaneous The binding specificity of the monoclonal antibody of oncocyte production is handed over by immunoprecipitation or is exempted from by external binding assay, such as radioactivity Epidemic disease measures (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA) to determine.Such technology and measurement are as known in the art.Example Such as, binding affinity can pass through Munson, Anal.Biochem.107 (1): 220-239 (1980) Scatchard analysis It determines.
There is required specificity identifying to generate, after the hybridoma of affinity and/or active antibody, Ke Longke To be subcloned by limiting dilution assay and cultivate (Goding, ibid) by standard method.Culture medium suitable for this purpose Including for example, D-MEM or RPMI-1640 culture medium.In addition, to can be used as tumour internal in mammals for hybridoma Culture.
Such as by conventional immune globulins purification process, for example, albumin A-Sepharose, hydroxylapatite chromatography, gel Electrophoresis, dialysis or affinity chromatography will suitably be separated by the monoclonal antibody and culture medium of subclone secretion, ascites or serum.
Antibody can be prepared by the way that suitable host animal is immunized for antigen.In one embodiment, antigen is Polypeptide comprising overall length C5.In one embodiment, antigen is the polypeptide of the β chain (SEQ ID NO:40) comprising C5.One In a embodiment, antigen is the polypeptide of the MG1-MG2 structural domain (SEQ ID NO:43) of the β chain comprising C5.Implement at one In scheme, antigen is the polypeptide of the MG1 structural domain (SEQ ID NO:41) of the β chain comprising C5.In one embodiment, antigen It is the polypeptide in the region of the amino acid at 19 to 180 place of position comprising the β chain corresponding to C5.In one embodiment, antigen It is the polypeptide in the region of the amino acid at 33 to 124 place of position comprising the β chain corresponding to C5.In one embodiment, antigen It is to be selected from the more of the segment of the amino acid 47-57,70-76 and 107-110 of the β chain (SEQ ID NO:40) of C5 comprising at least one Peptide.In one embodiment, antigen is such polypeptide, and the polypeptide includes the segment of the β chain of C5, and the segment includes extremely Few amino acid selected from the group being made up of: Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109 and His110.Implement at one In scheme, antigen is such polypeptide, and the polypeptide includes the segment of the β chain of C5, the segment include at least one be selected from by The amino acid of group consisting of: Glu48, Asp51, His70, His72, Lys109 and His110.It is also included in the present invention In be by for the antigen-immunized animal preparation antibody.The antibody can " exemplary anti-C5 be anti-in conjunction with above Body " described in any feature (either individually or in combination).
C. it measures
It can be identified by many measure known in the art, screen anti-C5 antibody provided herein or characterize its object Reason/chemical property and/or biological activity.
1. binding assay and other measurements
In one aspect, for example, by known method such as ELISA, Western blotting, BIACORE (registered trademark) etc. comes Test the antigen-binding activity of antibody of the invention.
On the other hand, competition assay can be used for identifying the C5 in conjunction with anti-C5 antibody competition described herein Antibody.In certain embodiments, in the presence of such competition antibody excess, (for example, reduction) reference antibody and C5 are blocked Combination reach at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or higher.In some cases, in conjunction with being suppressed at least 80%, 85%, 90%, 95% or higher.In certain implementations In scheme, epitope (for example, linear or comformational epitope) and anti-C5 antibody (example described herein that such competition antibody combines Such as, anti-C5 antibody described in table 2) combine epitope it is identical.Epitope mapping for combining antibody it is detailed exemplary Method is provided in Morris " Epitope Mapping Protocols, " in Methods in Molecular Biology In vol.66 (Humana Press, Totowa, NJ) (1996).
In exemplary competition assay, fixed C5 is incubated in the solution, the solution includes the (ginseng of the first label Than) antibody and the second unlabelled antibody, antibody the combination C5 of first label, test the second unlabelled antibody and The ability of first antibody competitive binding C5.Secondary antibody can reside in doma supernatant.As control, by fixed C5 It is incubated in the antibody comprising the first label but the solution not comprising the second unlabelled antibody.First antibody and C5 is being allowed to tie After being incubated under conditions of conjunction, excessive unbonded antibody is removed, and measure the amount of the label in conjunction with fixed C5.If phase For control sample, in the test sample, the amount of the label in conjunction with fixed C5 is substantially reduced, then this instruction secondary antibody with First antibody competitive binding C5.Referring to, Harlow and Lane, Antibodies:A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1988).
In another exemplary competition assay, analyzed using BIACORE (registered trademark) to determine the anti-C5 antibody of test The ability of C5 in conjunction with the anti-C5 antibody competition of second (reference).BIACORE (registered trademark) is being operated according to the recommendation of manufacturer The other side of instrument (for example, BIACORE (registered trademark) 3000), using standard technique as known in the art by C5 egg White capture generates the surface of C5 coating on CM5BIACORE (registered trademark) chip.Typically, 200-800 is responded The C5 of unit is coupled to chip, and (amount is easy to provide the test antibody that can measure horizontal combination and can easily be used Concentration saturation).It will assess and be combined with the two kinds of antibody (that is, test antibody and reference antibody) for the ability of contending with one other with 1: 1 Site molar ratio is blended in suitable buffer to prepare and test mixture.When calculating concentration based on binding site, test Or the molecular weight of reference antibody is assumed the number by the total molecular weight of corresponding antibodies divided by C5 binding site on antibody.It is each anti- Concentration of the body (that is, test antibody and reference antibody) in test mixture should be sufficiently high to be easy to make binding site for catching Obtain the antibody saturation on the C5 molecule on BIACORE (registered trademark) chip.Test and reference antibody in mixture In identical molar concentration (based on combine), typically 1.00 to 1.5 μM (being based on binding site).It also prepares individually only Containing test antibody and the solution for containing only reference antibody.Test antibody and reference antibody in these solution should be mixed with test In identical buffer in object and it is in identical concentration and condition.Mix the test containing test antibody and reference antibody Object passes through and records the total amount of combination on BIACORE (registered trademark) chip that C5 is coated.Then chip is handled to remove In conjunction with test or reference antibody without destroying C5 in conjunction with chip.Typically, this by with 30mM HCl by chip processing It carries out within 60 seconds.Pass through the solution of individual test antibody on the surface that C5 is coated and records the amount of combination.Again Processing chip is to remove the antibody of all combinations without destroying the C5 in conjunction with chip.Then make the solution of individual reference antibody Pass through and record the amount of combination on the surface of C5 coating.Next the maximum of the mixture of test antibody and reference antibody is calculated Theory combines, and is the summation of each antibody (i.e. test and reference) the individually combination when passing through on the surface C5.If practical The combination of the mixture of record is less than the theoretical maximum, then test antibody and reference antibody contend with one other in conjunction with C5.Therefore, lead to Often, competition test anti-C5 antibody be it is such, in the above BIACORE (registered trademark) closing measurement combine C5 so that During measurement and in the presence of reference anti-C5 antibody, the combination for being combined into test antibody and reference antibody of record is most Broad theory combines 80% to 0.1% (for example, 80% > to 4%) of (as defined above), specially maximum theoretical binding 75% to 0.1% (for example, 75% to 4%), and more specifically maximum theoretical binding 70% to 0.1% (for example, 70% to 4%).
In certain embodiments, anti-C5 antibody of the invention and the VH comprising being selected from antibody CFA0341 and CFA0330 With VL couples of antibody competition combination C5.In some embodiments, anti-C5 antibody C5 in conjunction with antibody competition selected from the following: CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675 and CFA0672.In some embodiments, Anti- C5 antibody and antibody CFA0329 competitive binding C5.In some embodiments, anti-C5 antibody and antibody CFA0666 are competed In conjunction with C5.
In certain embodiments, anti-C5 antibody of the invention and the VH comprising antibody CFA0305 or 305LO5 and VL pairs Antibody competition combination C5.
In a further embodiment, anti-C5 antibody combines the affinity of C5 higher than at acidic under neutral ph. In certain embodiments, the anti-C5 antibody of the invention C5 in conjunction with the antibody competition comprising VH selected from the following and VL couples: CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675 and CFA0672.In some embodiments, Anti- C5 antibody and antibody CFA0666 competitive binding C5.In a further embodiment, anti-C5 antibody combines C5 at pH7.4 Affinity ratio it is higher in pH5.8.
In a further embodiment, anti-C5 antibody combines the affinity of C5 higher than at acidic under neutral ph. In certain embodiments, anti-C5 antibody of the invention and the VH comprising antibody CFA0305 or 305LO5 and VL pairs of antibody are competing It strives and combines C5.In a further embodiment, affinity ratio of the anti-C5 antibody at pH7.4 in conjunction with C5 is higher in pH5.8.
In certain embodiments, anti-C5 antibody of the invention and the antibody competition comprising VH selected from the following and VL couples In conjunction with VH the and SEQ ID NO:25 of the VL or SEQ ID NO:21 of the VH and SEQ ID NO:26 of C5:SEQ ID NO:22 VL.In some embodiments, anti-C5 antibody C5:(a in conjunction with the antibody competition comprising VH selected from the following and VL couples) SEQ The VL of the VH and SEQ ID NO:15 of ID NO:5；(b) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；(c)SEQ ID The VL of the VH and SEQ ID NO:16 of NO:6；(d) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；(e) SEQ ID NO: The VL of 3 VH and SEQ ID NO:13；(f) VL of the VH and SEQ ID NO:11 of SEQ ID NO:1；(g) SEQ ID NO:9 The VL of VH and SEQ ID NO:19；(h) VL of the VH and SEQ ID NO:17 of SEQ ID NO:7；(i) SEQ ID NO:8 The VL of VH and SEQ ID NO:18.In some embodiments, anti-C5 antibody and VH the and SEQ ID comprising SEQ ID NO:23 The antibody competition combination C5 of the VL of NO:27.In some embodiments, anti-C5 antibody and the VH comprising SEQ ID NO:7 and The antibody competition combination C5 of the VL of SEQ ID NO:17.
In certain embodiments, anti-C5 antibody of the invention and the antibody competition comprising VH selected from the following and VL couples In conjunction with C5:(a) VL of the VH and SEQ ID NO:11 of SEQ ID NO:1；(b) VH the and SEQ ID NO:26 of SEQ ID NO:22 VL；(c) VL of the VH and SEQ ID NO:25 of SEQ ID NO:21；(d) VH the and SEQ ID NO:15 of SEQ ID NO:5 VL；(e) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；(f) VL of the VH and SEQ ID NO:16 of SEQ ID NO:6； (g) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；(h) VL of the VH and SEQ ID NO:13 of SEQ ID NO:3；(i) The VL of the VH and SEQ ID NO:19 of SEQ ID NO:9；(j) VL of the VH and SEQ ID NO:17 of SEQ ID NO:7；(k)SEQ The VL of the VH and SEQ ID NO:18 of ID NO:8；(1) VL of the VH and SEQ ID NO:27 of SEQ ID NO:23；(m) SEQ The VL of the VH and SEQ ID NO:20 of ID NO:10.
In certain embodiments, anti-C5 antibody of the invention and the antibody competition comprising VH selected from the following and VL couples In conjunction with C5:(a) VL of the VH and SEQ ID NO:26 of SEQ ID NO:22；(b) VH the and SEQ ID NO of SEQ ID NO:21: 25 VL；(c) VL of the VH and SEQ ID NO:15 of SEQ ID NO:5；(d) VH the and SEQ ID NO:14 of SEQ ID NO:4 VL；(e) VL of the VH and SEQ ID NO:16 of SEQ ID NO:6；(f) VH the and SEQ ID NO:12 of SEQ ID NO:2 VL；(g) VL of the VH and SEQ ID NO:13 of SEQ ID NO:3；(h) VL of the VH and SEQ ID NO:19 of SEQ ID NO:9； (i) VL of the VH and SEQ ID NO:17 of SEQ ID NO:7；(j) VL of the VH and SEQ ID NO:18 of SEQ ID NO:8；(k) The VL of the VH and SEQ ID NO:27 of SEQ ID NO:23.
In certain embodiments, anti-C5 antibody of the invention and VH the and SEQ ID comprising being selected from SEQ ID NO:1 The VH of the VL of the VH and SEQ ID NO:20 of the VL or SEQ ID NO:10 of NO:11 and VL couples of antibody competition combination C5.
In a further embodiment, anti-C5 antibody combines the affinity of C5 higher than at acidic under neutral ph. In certain embodiments, anti-C5 antibody combine under neutral ph the affinity of C5 than it is higher at acidic and with comprising VH selected from the following and VL couples of antibody competition combination C5:(a) SEQ ID NO:1 VH and SEQ ID NO:11 VL；(b) The VL of the VH and SEQ ID NO:15 of SEQ ID NO:5；(c) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；(d)SEQ The VL of the VH and SEQ ID NO:16 of ID NO:6；(e) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；(f)SEQ ID The VL of the VH and SEQ ID NO:13 of NO:3；(g) VL of the VH and SEQ ID NO:19 of SEQ ID NO:9；(h) SEQ ID NO: The VL of 7 VH and SEQ ID NO:17；(i) VL of the VH and SEQ ID NO:18 of SEQ ID NO:8；(j) SEQ ID NO: The VL of 10 VH and SEQ ID NO:20.In a further embodiment, anti-C5 antibody combines the affinity of C5 at pH7.4 It is higher than in pH5.8.
In some embodiments, anti-C5 antibody combine under neutral ph the affinity of C5 than at acidic more it is high simultaneously And the C5:(a in conjunction with the antibody competition comprising VH selected from the following and VL couples) SEQ ID NO:5 VH and SEQ ID NO:15 VL；(b) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；(c) VL of the VH and SEQ ID NO:16 of SEQ ID NO:6； (d) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；(e) VL of the VH and SEQ ID NO:13 of SEQ ID NO:3；(f) The VL of the VH and SEQ ID NO:11 of SEQ ID NO:1；(g) VL of the VH and SEQ ID NO:19 of SEQ ID NO:9；(h)SEQ The VL of the VH and SEQ ID NO:17 of ID NO:7；(i) VL of the VH and SEQ ID NO:18 of SEQ ID NO:8.Other In embodiment, affinity ratio of the anti-C5 antibody at pH7.4 in conjunction with C5 is higher in pH5.8.
In some embodiments, anti-C5 antibody combine under neutral ph the affinity of C5 than at acidic more it is high simultaneously And the VH and SEQ ID NO with VL the or SEQ ID NO:10 comprising VH the and SEQ ID NO:11 selected from SEQ ID NO:1: The VH of 20 VL and VL couples of antibody competition combination C5.In a further embodiment, anti-C5 antibody combines C5 at pH7.4 Affinity ratio it is higher in pH5.8.
In certain embodiments, it can determine whether anti-C5 antibody of the invention combines defined epitope as follows: will be on C5 Amino acid (in addition to alanine) be replaced as the C5 point mutation body of alanine and expressed in 293 cells, and via ELISA, Western blot or BIACORE (registered trademark) test the combination of anti-C5 antibody and C5 mutant；Wherein anti-C5 antibody and C5 are prominent The combination of variant is obviously reduced or eliminates indicate to include described on C5 in conjunction with anti-C5 antibody with the combination of wild type C5 relative to it The epitope of amino acid.In certain embodiments, the upper amino acid that be replaced as alanine of C5 is selected from the group being made up of: Glu48, Asp51, His70, His72, Lys109 and the His110 of the β chain (SEQ ID NO:40) of C5.In other embodiment party In case, the upper amino acid that be replaced as alanine of C5 is the Asp51 or Lys109 of the β chain (SEQ ID NO:40) of C5.
In another embodiment, it can determine whether the anti-C5 antibody with pH- dependence binding characteristic is tied as follows It closes defined epitope: the C5 point mutation body that the histidine residues on C5 are replaced as another amino acid (for example, tyrosine) is existed It is expressed in 293 cells, and tests anti-C5 antibody and C5 mutation via ELISA, western blot or BIACORE (registered trademark) The combination of body；Wherein anti-C5 antibody acid pH and wild type C5 combination relative to its acid pH and C5 mutant knot Conjunction is obviously reduced, and indicates the epitope on anti-C5 antibody combination C5 comprising the histidine residues.In a further embodiment, Anti- C5 antibody and wild type C5 do not subtract significantly relative to itself and C5 mutant in the combination of neutral pH in the combination of neutral pH It is weak.In certain embodiments, the upper histidine residues that be replaced as another amino acid of C5 are selected from the group being made up of: His70, His72 and the His110 of the β chain (SEQ ID NO:40) of C5.In another embodiment, histidine residues His70 is replaced as tyrosine.
2. determination of activity
In one aspect, measurement is provided for identifying the anti-C5 antibody with biological activity.Biological activity can be with Including for example, inhibiting the activation of C5, C5 being prevented to be formed C5a and C5b by cutting, preventing C5 convertase close to the cutting on C5 Site blocks hemolytic activity as caused by the activation of C5 etc..It also provides with described internal and/or In vitro biological activity anti- Body.
In certain embodiments, antibody of the invention is tested for the biological activity.
In certain embodiments, for example, by Isenman etc., institute in J Immunol.124 (1): 326-331 (1980) The method stated determines whether test antibody inhibits C5 to be cut into C5a and C5b.In another embodiment, this passes through use It is determined in the method (for example, ELISA or western blot) of the C5a and/or C5b albumen of specific detection cutting.When existing The amount of the cleaved products (that is, C5a and/or C5b) of C5 is detected in the case where test antibody (or after contacting with test antibody) It reduces, then test antibody is accredited as being the antibody for being able to suppress C5 cutting.In certain embodiments, the concentration of C5a and/or Physiologically active can by method, for example, chemotactic assay, RIA or ELISA come determine (see, for example, Ward and Zvaifler J.Clin.Invest.50 (3): 606-616 (1971)).
In certain embodiments, pass through the method (example for detecting the protein-interacting between C5 convertase and C5 Such as, ELISA or BIACORE (registered trademark)) determine whether test antibody prevents C5 convertase close to C5.When in the presence of test When the decrease that interacts in the case where antibody (or after contacting with test antibody), test antibody is accredited as being that can prevent C5 Antibody of the invertase close to C5.
In certain embodiments, C5 activity can be used as its cytolytic ability in subject's body fluid function and It is measured.The cytolytic ability of C5 or its decrease can be measured by method as known in the art, for example, conventional haemolysis is surveyed It is fixed, such as Kabat and Mayer (editor), Experimental Immunochemistry, second edition, 135-240, Springfield, IL, CC Thomas (1961), the haemolysis measurement of 135-139 pages of description or the conventional deformation of the measurement, Chicken red blood cell haemolysis method described in such as such as Hillmen, N.Engl.J.Med.350 (6): 552-559 (2004).? In certain embodiments, quantify C5 activity or the inhibition to it using CH50eq measurement.CH50eq measurement is for measuring blood The method of total classical complement activity in clear.The test is lysis measurement, and immune body sensitized red blood cell is used to mend as classics The activator of body approach, and determined needed for generating 50% lysis (CH50) using the different diluent of test serum Amount.Percent hemolysis can be determined for example using spectrophotometer.CH50eq measurement is provided to final complement complex (TCC) The indirect measurement formed, because TCC itself is directly responsible for the haemolysis of measurement.Inhibit C5 activation that working Examples also can be used In provide and exemplary method detection and/or measurement.Using the measurement of these or other suitable type, can filter out can press down The candidate antibodies of C5 activation processed.In certain embodiments, the C5 activation is inhibited to include: and negative control under similar conditions Effect is compared, and C5 activation in the assay reduces at least 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% or more. In some embodiments, refer to and C5 activation inhibited to reach at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more.
D. immunoconjugates
The present invention also provides immunoconjugates, the immunoconjugates include anti-C5 antibody herein, and the antibody is sewed Together in one or more cytotoxic agents such as chemotherapeutics or drug, growth inhibitor, toxin is (bacterium, true for example, proteotoxin Bacterium, the enzyme activity toxin of plant or animal origin or its segment) or radioactive isotope.
In one embodiment, immunoconjugates are antibody-drug conjugates (ADC), and wherein antibody conjugate is in one kind Or a variety of drugs, including but not limited to maytansine class compound is (referring to U.S. Patent number 5,208,020,5,416,064 and Europe 0 425 235 B1 of patent EP)；Auristatin such as monomethyl auristatin drug moiety DE and DF (MMAE and MMAF) (referring to U.S. Patent number 5,635,483 and 5,780,588 and 7,498,298)；Dolastatin (dolastatin)；Add benefit Vehicle mycin (calicheamicin) or derivatives thereof (referring to U.S. Patent number 5,712,374,5,714,586,5,739,116, 5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296；Hinman etc., Cancer Res.53: 3336-3342(1993)；With Lode etc., Cancer Res.58:2925-2928 (1998))；Anthracycline antibiotic (anthracycline) such as daunomycin (daunomycin) or Doxorubicin (doxorubicin) (referring to Kratz, Current Med.Chem.13:477-523 (2006)；Jeffrey etc., Bioorganic&Med.Chem.Letters 16: 358-362(2006)；Torgov etc., Bioconj.Chem.16:717-721 (2005)；Nagy etc., Proc.Natl.Acad.Sci.USA 97:829-834 (2000)；Dubowchik etc., Bioorg.&Med.Chem.Letters 12:1529-1532 (2002)；King etc., J.Med.Chem.45:4336-4343 (2002)；With U.S. Patent number 6,630, 579)；Methotrexate (MTX) (methotrexate)；Eldisine (vindesine)；Taxane (taxane) such as docetaxel (docetaxel), taxol (paclitaxel), La Luotasai (larotaxel), tesetaxel (tesetaxel) and it is difficult to understand he It matches (ortataxel)；Crescent toxin (trichothecene)；And CC1065.
In another embodiment, immunoconjugates include antibody described herein, and the antibody conjugate is in enzyme activity Property toxin or its segment, including but not limited to diphtheria A chain, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (comes from copper Green pseudomonad (Pseudomonas aeruginosa)), ricin A chain, abrin A chain, modeccin A chain, α-sarcine, tung oil tree (Aleurites fordii) albumen, caryophyllin (dianthin) albumen, dyers' grapes (Phytolaca Americana) albumen (PAPI, PAPII and PAP-S), balsam pear (momordica charantia) inhibitor, manioca poison egg It is white, crotin, Saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, morphine (mitogellin), restrictocin, phenomycin, enomycin and unit cell Mei Xi race toxin (tricothecene).
In another embodiment, immunoconjugates include antibody as described herein, and the antibody conjugate is in putting Penetrating property atom is to form radioactivity conjugate.A variety of radioactive isotopes can be used for preparing radioactivity conjugate.Example includes At211, I131, I125, y90, Re186, Re188, Sm153, Bi212, P32, Pb212With the radioactive isotope of Lu.When radioactivity conjugate When for detecting, it may include radioactive atom and being used for for scintigraphy research, such as tc99m or I123 or spin labeling (also referred to as magnetic resonance imaging, mri) is imaged in nuclear magnetic resonance (NMR), and such as iodo- 123 (again), iodine -131, indium -111 are fluoro- 19, carbon -13, nitrogen -15, oxygen -17, gadolinium, manganese or iron.
A variety of bifunctional protein coupling agent preparations can be used in the conjugate of antibody and cytotoxic agent, and the coupling agent is such as N- succinimido -3- (2- pyridyl group two is thio) propionic ester (SPDP), succinimido -4- (N- dimaleoyl imino Methyl) hexamethylene -1- formic acid esters (SMCC), iminothiolane hydrochloride (IT), dual-function derivative (such as diformazan of imines base ester Base adipic acid HCl), active ester (such as two succinimidyl suberates), aldehyde (such as glutaraldehyde), diazido compound is (such as Two (to azidobenzoyl) hexamethylene diamines), two-diazonium radical derivatives (such as two-(to diazo benzoyl)-ethylenediamines), Diisocyanate (such as toluene 2,6- diisocyanate) and two-active fluorine compounds (fluoro- 2, the 4- dinitrobenzene of such as 1,5- bis-). For example, ricin immunotoxin can such as Vitetta, described in Science 238:1098 (1987) prepare.Carbon-14- The different sulphur cyanato- benzyl -3- methyl diethylene-triamine pentaacetic acid (MX-DTPA) of the 1- of label is for radionuclide to be conjugated To the Exemplary chelators of antibody.Referring to WO94/11026.Connector can be " cleavable connector ", and power-assisted is thin in cell The release of cytotoxic drugs.It is, for example, possible to use the unstable connector of acid, peptidase-sensitive connector, photo-labile connector, dimethyl Connector or connector (Chari etc., Cancer Res.52:127-131 (1992) containing disulfide bond；U.S. Patent number 5,208, 020)。
Immunoconjugates or ADC herein take explicitly into account, but not limited to this conjugation that kind is prepared using crosslinking agent reagent Object, the crosslinking agent include, but are not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio- SMCC and thio-SMPB and SVSB (succinimido-(4- vinyl sulfone) benzoic ether) is commercially available (for example, coming From Pierce Biotechnology, Inc., Rockford, IL., U.S.A).
E. the method and composition for diagnosing and detecting
In certain embodiments, any anti-C5 antibody provided herein can be used in C5 in detection biological sample Presence.As used in this article, term " detection " includes quantitative detection or qualitative detection.In certain embodiments, biological The product of imitating include cell or tissue, such as serum, whole blood, blood plasma, biopsy samples, tissue sample, cell suspension, saliva, phlegm, oral cavity Liquid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary gland secretion, lymph, urine, sweat, tear, gastric juice, synovia, peritonaeum Liquid, ocular lens fluid and mucus.
In one embodiment, it provides for diagnosing or the anti-C5 antibody of detection method.On the other hand, it provides Detect C5 method present in biological sample.In certain embodiments, the method includes make biological sample with such as Anti- C5 antibody described herein contacts under conditions of allowing anti-C5 antibody combination C5, and detect in anti-C5 antibody and Whether compound is formed between C5.Such method can be method in vitro or in vivo.In one embodiment, anti-C5 is resisted Body is used to select to be suitable for use with the subject of anti-C5 Antybody therapy, for example, wherein C5 is the biological marker for selecting patient Object.
In another embodiment, present invention offer, which is selected as, is suitable for the treatment comprising anti-C5 antibody of the invention Method, disease with complement-mediated or be related to excessive or uncontrolled C5 activation illness individual method.Certain In embodiment, which comprises (a) detection changes from the heredity of the C5 of individual, and (b) when from described When detecting heredity variation in the C5 of body, select described individual as being suitable for the therapy comprising anti-C5 antibody of the invention.? In another embodiment, the present invention provides method of the selection for the therapy of individual, and the individual suffers from the disease of complement-mediated Disease is related to the illness of excessive or uncontrolled C5 activation.In certain embodiments, which comprises (a) detection comes Derived from the heredity variation of the C5 of individual, and (b) when detecting heredity variation in the C5 from the individual, selection includes The therapy of anti-C5 antibody of the invention is used for the individual.
In another embodiment, the present invention provides the method for the treatment of individual, and the individual suffers from the disease of complement-mediated Disease is related to the illness of excessive or uncontrolled C5 activation.In certain embodiments, which comprises (a) detection comes Heredity variation derived from the C5 of individual selects described (b) when detecting heredity variation in the C5 from the individual Body applies anti-C5 antibody of the invention to the individual as being suitable for the therapy comprising anti-C5 antibody of the invention, and (c).
In another embodiment, the present invention is provided to treat the anti-C5 antibody of the invention of individual, the individual Disease with complement-mediated is related to the illness of excessive or uncontrolled C5 activation.In certain embodiments, when next When detecting heredity variation in the C5 of the individual, the individual is treated using anti-C5 antibody of the invention.
In another embodiment, the present invention provides the in vitro use of the heredity variation of C5, is used to be selected as suitable Together in the therapy comprising anti-C5 antibody of the invention, disease with complement-mediated or be related to excessive or uncontrolled C5 The individual of the illness of activation.In certain embodiments, when detecting heredity variation in the C5 from the individual, choosing The individual is selected as being suitable for the therapy.In another embodiment, the present invention provides the body of the heredity variation of C5 Outer purposes, be used for select for individual therapy, it is described individual with complement-mediated disease or be related to it is excessive or not by The illness of the C5 activation of control.In certain embodiments, when detecting heredity variation in the C5 from the individual, choosing The therapy comprising anti-C5 antibody of the invention is selected for the individual.
It is reported that in C5 have heredity variation some patients show go on business to the treatment comprising existing anti-C5 antibody The reaction (Nishimura et al., N.Engl.J.Med.370:632-639 (2014)) of method.It is recommended that by such patient with comprising The therapy of anti-C5 antibody of the invention is treated, because such antibody all has the activation of C5 variant and wild type C5 There is inhibitory activity, as proved in following embodiment.
The heredity that can be detected in C5 by using method well known in the prior art is changed.Such method may include Sequencing, PCR, RT-PCR and the method based on hybridization, such as southern trace or northern trace, but not limited to this.C5 Variant may include at least one heredity variation.Heredity variation can be selected from the group being made up of: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D.Here, such as R885H means the arginine quilt at position 885 The heredity variation of histidine.In certain embodiments, C5 variant has bioactivity similar with wild type C5.
The Exemplary conditions that antibody diagnosis of the invention can be used include: rheumatoid arthritis (RA)；Systemic red Yabbi sore (SLE)；Lupus nephritis；Ischemical reperfusion injury (IRI)；Asthma；Paroxysmal nocturnal hemoglobinuria (PNH)；It is molten Hemorrhagic uremia syndrome (HUS) (for example, Atypical Hemolytic Uremic Syndrome (aHUS))；Dense sediment disease (DDD)； Neuromyelitis optica (NMO)；Multifocal motor neuropathy (MMN)；Multiple sclerosis (MS)；Systemic sclerosis；Macular degeneration (example Such as, age-related macular degeneration (AMD))；Haemolysis, liver enzyme increases and low platelet (HELLP) syndrome；Thrombotic blood is small Plate reduction property purpura (TTP)；Spontaneous abortion；Epidermolysis bullosa；Recidivity miscarriage；Pre-eclampsia (pre- eclampsia)；Traumatic brain injury；Myasthenia gravis；Cold coagulation disease；Sjogren syndrome (Sjogren ' s syndrome)；Dermatomyositis；Bullous pemphigoid；Phototoxic reaction；Shiga toxin Escherichia coli correlation haemolytic uraemic is comprehensive Simulator sickness (Shiga toxin E.coli-related hemolytic uremic syndrome)；Typical or infectious hemolytic Uremia syndrome (typical or infectious hemolytic uremic syndrome (tHUS))；C3 glomerulus Ephritis；Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis；Body fluid and vasotransplantation rejection；Acute antibodies are situated between The repulsion (AMR) led；Graft function obstacle；Myocardial infarction；Allograft；Septicemia；Coronary artery disease；Heredity Angioedema；Dermatomyositis；Graves disease (Graves ' disease)；Atherosclerosis；Alzheimer disease (Alzheimer ' s disease, AD)；Huntington disease (Huntington ' s disease)；Gram refined Er Shi disease (Creutzfeld-Jacob disease)；Parkinson's disease (Parkinson ' s disease)；Cancer；Wound；Septic shock (septic shock)；Spinal cord injury；Uveitis；Diabetic eye diseases；Retinopathy of prematurity；Glomerulonephritis；Film property kidney It is scorching；IgANP；Adult respiratory distress syndrome (ARDS) (ARDS)；Chronic obstructive pulmonary disease (COPD)；Cystic fibrosis； Hemolytic anemia；Paroxysmal cold hemoglobinuria；Anaphylactic shock；Allergy；Osteoporosis；Osteoarthritis；Bridge this first shape Adenositis (Hashimoto ' s thyroiditis)；Type-1 diabetes mellitus；Psoriasis；Pemphigus；Autoimmune hemolytic anemia (AIHA)；Idiopathic Thrombocytopenic Purpura (ITP)；Goodpasture's syndrome (Goodpasture syndrome)；Moral Goss disease (Degos disease)；Antiphospholipid syndrome (APS)；Catastrophic APS (catastrophic APS, CAPS))；The heart Vascular diseases；Myocarditis；Cerebrovascular disorder；Peripheral artery disease；Renal vascular diseases；Mesenterium/intestines vascular diseases；Vasculitis； Anaphy lactoid purpura nephritis (Henoch-Schonlein purpura nephritis)；Aortic arch syndrom (Takayasu ' s disease)；Dilated cardiomyopathy；Diabetic angiopathy；Kawasaki disease (Kawasaki ' s disease) (arteritis)；Vein gas Body embolism (VGE), the restenosis after bracket placement；Turnery art (rotational atherectomy)；Membranous nephropathy；Ji Lan- Barre syndrome (Guillain-Barre syndrome, GBS)；Fisher's syndrome (Fisher syndrome)；Antigen lures The arthritis led；Synovial membrane inflammation；Virus infection；Bacterium infection；Fungal infection；With by myocardial infarction, cardiopulmonary bypass and blood Damage caused by dialysis.
In certain embodiments, the anti-C5 antibody of label is provided.Mark the label that including but not limited to directly detects or Partially (such as fluorescence, chromophore, electron density, chemiluminescence and radioactive label), and the part such as enzyme that detects indirectly or match Body, for example, passing through enzyme reaction or interaction of molecules.Exemplary indicia includes but is not limited to radioactive isotope32P,14C,125I,3H, and131I, fluorogen such as Rare Earth Chelate or fluorescein and its derivative, rhodamine and its derivative, dansyl, umbrella shape Ketone, luciferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent number 4,737,456), luciferin, 2, 3- dihydro phthalazine diketone, horseradish peroxidase (HRP), alkaline phosphatase, beta galactosidase, glucoamylase, lysozyme, Carbohydrate oxidase, for example, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase And xanthine oxidase, it is coupled with using the enzyme of hydrogen peroxide with oxidation dye precursors such as HRP, lactoperoxidase or micro- Peroxidase, biotin/avidin, spin labeling, bacteriophage labels, stable free radical etc..
F. pharmaceutical preparation
The pharmaceutical preparation of anti-C5 antibody as described herein pass through the antibody of the purity that will have required degree with One or more optional pharmaceutical carrier (Remington ' s Pharmaceutical Sciences the 16th edition, Osol, A.Ed. (1980)) mix and be prepared to the form of lyophilized preparation or aqueous solution.Pharmaceutical carrier under the dosage and concentration used for Recipient is usually avirulent, and includes, but are not limited to: buffer such as phosphate, citrate and other organic acids； Antioxidant, including ascorbic acid and methionine；Preservative (such as stearyl dimethyl benzyl ammonium chloride；Chlorination pregnancy is double Ammonium；Benzalkonium chloride；Benzethonium chloride；Phenol, butyl or benzyl alcohol；Alkyl paraben such as nipagin or to hydroxyl Propyl benzoate；Catechol；Resorcinol；Cyclohexanol；3- amylalcohol；And metacresol)；Low molecular weight (less than about 10 residues) Polypeptide；Albumen, such as serum albumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid Such as glycine, glutamine, aspartic acid, histidine, arginine or lysine；Monosaccharide, disaccharides and other carbohydrate, packet Include glucose, mannose or dextrin；Chelating agent such as EDTA；Sugar such as sucrose, mannitol, trehalose or sorbierite；At salt contend with from Son such as sodium；Metal composite (for example, Zn- albumen composition)；And/or nonionic surfactant such as polyethylene glycol (PEG).This Exemplary pharmaceutical carrier in text further includes interstitial drug dispersing agent such as soluble neutral reactive transparent matter acid enzyme glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.).Certain exemplary sHASEGP and application method, including rHuPH20, are described in beauty In state's publication No. 2005/0260186 and 2006/0104968.In one aspect, by sHASEGP and one or more other sugar Amine dextranase such as chondroitinase combines.
The antibody preparation of exemplary freeze-drying is described in U.S. Patent number 6,267,958.Aqueous antibody preparation includes beauty Described in state's patent No. 6,171,586 and WO 2006/044908 those, the preparation of the latter includes histidine-acetate buffer Liquid.
When needed, preparation herein can also contain have more than a kind of activity that the specific adaptations for treatment are demonstrate,proved at Point, it is therefore preferred to have those of complementary activity not adversely affected between each other.Such active constituent is suitably to purpose Purposes, which is effectively measured, to be present in combination.
Active constituent can be encapsulated in for example by condensation technique or by interfacial polymerization such as hydroxylmethyl cellulose respectively In element or the microcapsules of gelatin-microcapsule and poly- (methyl methacrylate) microcapsules preparation, in colloid drug delivery systems (example Such as, liposome, albumin microspheres, microemulsion, nanoparticle and Nano capsule) in or in thick lotion.Such technology is disclosed in In Remington ' s Pharmaceutical Sciences the 16th edition, Osol, A.Ed. (1980).
Extended release preparation can be prepared.The suitable example of extended release preparation includes the solid hydrophobic polymerization containing antibody The form of the semipermeability matrix of object, the matrix is physical items, for example, film or microcapsules.
The preparation that can be used for applying in vivo is usually sterile.It is sterile can for example by via aseptic filter membrane filter and Easily realize.
G. Treatment and composition for
Any anti-C5 antibody provided herein may be used in treatment method.
In one aspect, the anti-C5 antibody as drug is provided.In a further aspect, it provides for treating complement-mediated Disease or be related to excessive or uncontrolled C5 activation illness anti-C5 antibody.In certain embodiments, it provides and is used for The anti-C5 antibody for the treatment of method.In certain embodiments, the present invention provides anti-C5 antibody, is used to treat with complement Jie The method of the individual of the disease led or the illness for being related to excessive or uncontrolled C5 activation, the method includes applying to individual A effective amount of anti-C5 antibody.In such embodiment, the method also includes a effective amount of at least to individual application A kind of other therapeutic agent.People is preferably according to " individual " of any embodiments above.
When antigen is soluble protein, the combination of antibody and its antigen can lead to Increased Plasma Half-life of the antigen in blood plasma (that is, the removing of reduced antigen from blood plasma), because half-life period of the antibody in blood plasma itself is longer and serves as the load of antigen Body.This is because antigen-antibody complex by FcRn via in cell endosomal recycling (Roopenian, Nat.Rev.Immunol.7 (9): 715-725 (2007)).It is contemplated, however, that antibody (its of the binding property with pH dependence It is discharged into acid endosomal compartment in conjunction with its antigen by antigen after entering cell in neutral extracellular environment) in antigen Neutralize and remove counterpart that aspect is combined in such a way that pH is not dependent relative to it have preferable property (Igawa etc., Nat.Biotech..28 (11): 1203-1207 (2010)；Devanaboyina etc., mAbs5 (6): 851-859 (2013)；WO 2009/125825)。
In another embodiment, the present invention is provided to enhance the anti-C5 antibody of the removing of C5 from blood plasma.Certain In embodiment, the present invention is provided to enhance C5 in individual from the anti-C5 antibody in the method for the removing of blood plasma, the side Method includes applying a effective amount of anti-C5 antibody to individual to enhance removing of the C5 from blood plasma.In one embodiment, with do not have There is the anti-C5 antibody of the routine of pH dependence binding characteristic to compare, anti-C5 antibody enhances removing of the C5 from blood plasma.According to it is any with " individual " of upper embodiment is preferably people.
In another embodiment, the present invention is provided to inhibit the anti-C5 antibody of accumulation of the C5 in blood plasma.At certain In a little embodiments, the present invention is provided to inhibit the anti-C5 antibody in the method for accumulation of the C5 in blood plasma, institute in individual The method of stating includes that accumulation of a effective amount of anti-C5 antibody to inhibit C5 in blood plasma is applied to individual.In one embodiment, Accumulation of the C5 in blood plasma is to form the result of antigen-antibody complex.In another embodiment, it is relied on without pH Property binding characteristic the anti-C5 antibody of routine compare, anti-C5 antibody inhibits accumulation of the C5 in blood plasma.According to any above implementation " individual " of scheme is preferably people.
Anti- C5 antibody of the invention can inhibit the activation of C5.In another embodiment, the present invention is provided to press down The anti-C5 antibody of the activation of C5 processed.In certain embodiments, the present invention is provided to inhibit the side of the activation of C5 in individual Anti- C5 antibody in method, the method includes applying a effective amount of anti-C5 antibody to individual to inhibit the activation of C5.At one In embodiment, inhibit the cytotoxicity of C5 mediation by inhibiting the activation of C5.According to the " a of any embodiments above Body " is preferably people.
On the other hand, the present invention provides purposes of the anti-C5 antibody in preparation or compounding pharmaceutical.In an embodiment party In case, the drug is used to treat the disease of complement-mediated or is related to the illness of excessive or uncontrolled C5 activation.Another In a embodiment, the drug is used to treat the disease of complement-mediated or is related to the illness of excessive or uncontrolled C5 activation Method, the method includes to the disease with complement-mediated or being related to for the illness that excessive or uncontrolled C5 is activated Body applies a effective amount of drug.In such embodiment, the method also includes a effective amount of extremely to individual application A kind of few other therapeutic agent.People is preferably according to " individual " of any embodiments above.
In another embodiment, the drug is for enhancing removing of the C5 from blood plasma.In another embodiment, The drug is used to enhance in individual C5 from the method for the removing of blood plasma, and the method includes a effective amount of to individual application Drug is to enhance removing of the C5 from blood plasma.In one embodiment, with do not have pH dependence binding characteristic the anti-C5 of routine Antibody is compared, and anti-C5 antibody enhances removing of the C5 from blood plasma.It can be people according to " individual " of any embodiments above.
In another embodiment, the drug is for inhibiting accumulation of the C5 in blood plasma.In another embodiment In, in method of the drug for inhibiting accumulation of the C5 in blood plasma in individual, the method includes having to individual application The drug of effect amount is to inhibit accumulation of the C5 in blood plasma.In one embodiment, accumulation of the C5 in blood plasma is to form antigen- The result of antibody complex.In another embodiment, with do not have pH dependence binding characteristic the anti-C5 antibody phase of routine Than anti-C5 antibody inhibits accumulation of the C5 in blood plasma.It can be people according to " individual " of any embodiments above.
Anti- C5 antibody of the invention can inhibit the activation of C5.In another embodiment, the drug is for inhibiting The activation of C5.In another embodiment, in the method for activation of the drug for inhibiting C5 in individual, the method Including applying a effective amount of drug to individual to inhibit the activation of C5.In one embodiment, by inhibit C5 activation come The cytotoxicity for inhibiting C5 to mediate.It can be people according to " individual " of any embodiments above.
In terms of another, the present invention is provided to treat the disease of complement-mediated or be related to excessive or uncontrolled The method of the illness of C5 activation.In one embodiment, the method includes to the complement-mediated disease or relate to And the individual of the illness of excessive or uncontrolled C5 activation applies a effective amount of anti-C5 antibody.In such embodiment party In case, the method also includes applying a effective amount of at least one other therapeutic agent to individual.According to any above embodiment party Case " individual " it can be people.
On the other hand, the present invention is provided to enhance the method for the removing of C5 from blood plasma in individual.In a reality It applies in scheme, the method includes to apply a effective amount of anti-C5 antibody to the individual to enhance removing of the C5 from blood plasma.One In a embodiment, compared with the anti-C5 antibody of routine for not having pH dependence binding characteristic, anti-C5 antibody enhances C5 from blood The removing of slurry.In one embodiment, " individual " is people.
On the other hand, the present invention is provided to inhibit the method for accumulation of the C5 in blood plasma in individual.At one In embodiment, the method includes to apply a effective amount of anti-C5 antibody to the individual to inhibit accumulation of the C5 in blood plasma. In one embodiment, accumulation of the C5 in blood plasma is to form the result of antigen-antibody complex.In another embodiment In, compared with the anti-C5 antibody of routine for not having pH dependence binding characteristic, anti-C5 antibody inhibits accumulation of the C5 in blood plasma. In one embodiment, " individual " is people.
Anti- C5 antibody of the invention can inhibit the activation of C5.On the other hand, the present invention is provided in individual Inhibit the method for the activation of C5.In one embodiment, the method includes and applies a effective amount of anti-C5 to the individual to resist Body is to inhibit the activation of C5.In one embodiment, the cytotoxicity for inhibiting C5 to mediate by inhibiting the activation of C5.One In a embodiment, " individual " is people.
On the other hand, the present invention is provided for example for any above-mentioned treatment method comprising provided herein any The pharmaceutical preparation of anti-C5 antibody.In one embodiment, the pharmaceutical preparation is anti-comprising any anti-C5 provided herein Body and pharmaceutical carrier.In another embodiment, the pharmaceutical preparation includes any anti-C5 antibody provided herein and extremely A kind of few other therapeutic agent.
On the other hand, the pharmaceutical preparation is used to treat the disease of complement-mediated or is related to excessive or uncontrolled The illness of C5 activation.In another embodiment, the pharmaceutical preparation is for enhancing removing of the C5 from blood plasma.Implement at one In scheme, compared with the anti-C5 antibody of routine for not having pH dependence binding characteristic, anti-C5 antibody enhances C5 from the clear of blood plasma It removes.In another embodiment, the pharmaceutical preparation is for inhibiting accumulation of the C5 in blood plasma.In one embodiment, Accumulation of the C5 in blood plasma is to form the result of antigen-antibody complex.In another embodiment, it is relied on without pH Property binding characteristic the anti-C5 antibody of routine compare, anti-C5 antibody inhibits accumulation of the C5 in blood plasma.Anti- C5 antibody of the invention It can inhibit the activation of C5.In another embodiment, the pharmaceutical preparation is used to inhibit the activation of C5.In an embodiment party In case, inhibit the cytotoxicity of C5 mediation by inhibiting the activation of C5.In another embodiment, by the pharmaceutical preparation It is applied to the disease with complement-mediated or is related to the individual of excessive or uncontrolled C5 activation.According to any above embodiment party " individual " of case is preferably people.
In one aspect, individual has wild type C5.On the other hand, individual has C5 variant.In certain embodiment party In case, C5 variant has bioactivity similar with wild type C5.Such C5 variant may include selected from being made up of At least one variation of group: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D.Here, for example R885H means that the arginine at position 885 is changed by the heredity of histidine.
On the other hand, the present invention is provided to prepare the method for drug or pharmaceutical preparation, the method includes inciting somebody to action this Any anti-C5 antibody provided in text is mixed with pharmaceutical carrier, for example, to be used for any above-mentioned treatment method.Implement at one In scheme, the method for being used to prepare drug or pharmaceutical preparation further includes adding at least one other therapeutic agent to drug or drug Preparation.
In certain embodiments, the disease or illness for being related to the complement-mediated of excessive or uncontrolled C5 activation are selected from The group being made up of: rheumatoid arthritis (RA)；Systemic loupus erythematosus (SLE)；Lupus nephritis；Ischemia-reperfusion damage Hurt (IRI)；Asthma；Paroxysmal nocturnal hemoglobinuria (PNH)；Hemolytic uremic syndrome (HUS) is (for example, atypia is molten Hemorrhagic uremia syndrome (aHUS))；Dense sediment disease (DDD)；Neuromyelitis optica (NMO)；Multifocal motor neuropathy (MMN)；Multiple sclerosis (MS)；Systemic sclerosis；Macular degeneration (for example, age-related macular degeneration (AMD))；Haemolysis, Liver enzyme increases and low platelet (HELLP) syndrome；Thrombotic thrombocytopenic purpura (TTP)；Spontaneous abortion；Bleb Property epidermidolysis；Recidivity miscarriage；Pre-eclampsia；Traumatic brain injury；Myasthenia gravis；Cold coagulation disease；Sjogren is comprehensive Sign；Dermatomyositis；Bullous pemphigoid；Phototoxic reaction；Shiga toxin Escherichia coli correlation hemolytic uremic syndrome；Allusion quotation Type or infectious hemolytic uremic syndrome (tHUS)；C3 glomerulonephritis；Anti-neutrophil cytoplasmic antibody (ANCA) phase Closing property vasculitis；Body fluid and vasotransplantation rejection；The repulsion (AMR) that acute antibodies mediate；Graft function obstacle；Cardiac muscle Infraction；Allograft；Septicemia；Coronary artery disease；Hereditary angioedema；Dermatomyositis；Graves disease；Artery Atherosis；Alzheimer disease (AD)；Huntington disease；Gram refined Er Shi disease；Parkinson's disease；Cancer；Wound；Septic shock； Spinal cord injury；Uveitis；Diabetic eye diseases；Retinopathy of prematurity；Glomerulonephritis；Membraneous nephritis；Immunoglobulin A kidney Disease；Adult respiratory distress syndrome (ARDS) (ARDS)；Chronic obstructive pulmonary disease (COPD)；Cystic fibrosis；Hemolytic anemia；Paroxysmal Cold hemoglobinuria；Anaphylactic shock；Allergy；Osteoporosis；Osteoarthritis；Hashimoto thyroiditis；Type-1 diabetes mellitus；Silver Bits disease；Pemphigus；Autoimmune hemolytic anemia (AIHA)；Idiopathic Thrombocytopenic Purpura (ITP)；Goodpasture Syndrome；Degos' disease；Antiphospholipid syndrome (APS)；Catastrophic APS (CAPS)；Cardiovascular disease；Myocarditis；Cerebrovascular barrier Hinder；Peripheral artery disease；Renal vascular diseases；Mesenterium/intestines vascular diseases；Vasculitis；Anaphy lactoid purpura nephritis；Aortic arch syndrom； Dilated cardiomyopathy；Diabetic angiopathy；Kawasaki disease (arteritis)；Venous gas embolism (VGE), it is narrow again after bracket placement It is narrow；Turnery art；Membranous nephropathy；Guillain-Barre&1& syndrome (GBS)；Fisher's syndrome；The arthritis of antigen induction；Synovitis Disease；Virus infection；Bacterium infection；Fungal infection；It is damaged with as caused by myocardial infarction, cardiopulmonary bypass and haemodialysis.
In certain embodiments, the disease of complement-mediated or illness are eye disease or illness.In another embodiment party In case, the eye disorders are macular degenerations.In another embodiment, the macular degeneration is AMD.In another implementation In scheme, the AMD is the AMD of dry form.
In certain embodiments, the disease of complement-mediated or illness are PNH.
In certain embodiments, the disease of complement-mediated or illness are myocardial infarctions.
In certain embodiments, the disease of complement-mediated or illness are RA.
In certain embodiments, the disease of complement-mediated or illness are osteoporosis or osteoarthritis.
In certain embodiments, the disease of complement-mediated or illness are inflammation.
In certain embodiments, the disease of complement-mediated or illness are cancers.
Antibody of the invention can be used separately for treating or be used to treat in combination with other reagents.For example, of the invention Antibody can be co-administered at least one other therapeutic agent.
Above-mentioned such combined therapy includes that (the above therapeutic agent of two of them is comprised in same or separated combined administration In preparation) and separate administration, in such situation, the application of antibody of the invention can occur in other therapeutic agent or reagent Application before, meanwhile, and/or later.In one embodiment, the application of anti-C5 antibody and applying for other therapeutic agent With occurring within about one month each other, or within about one week, two weeks or three weeks, or at about one day, two days, three days, four days, Within five days or six days.
Antibody (and any other therapeutic agent) of the invention can be applied by any suitable means, including stomach Outer application, intrapulmonary application and intranasal administration, and, if local treatment needs, application in lesion.Parenteral infusions include flesh Application in meat, intravenous to apply, intra-arterial is applied, application or subcutaneous administration in peritonaeum.Medication can be by any suitable Approach, for example, such as intravenous or subcutaneous injection, it is of short duration or long-term for being partially dependent upon application by injection.Herein It is middle to consider a variety of therapeutic regimens, including but not limited to single administration or the multiple applications at multiple time points, inject application and arteries and veins Punching injection.
Antibody of the invention can be prepared with the consistent mode of good medical practice, medication and application.Consider in the context Factor include treatment disease specific, the specific mammal for the treatment of, the clinical condition of individual patient, the cause of disease, drug delivery Other factors known to site, medication, administration time arrangement and healthcare practitioners.The antibody does not need, but optionally Ground, it is formulated together with one or more medicaments currently used for prevention or therapeutic purpose disease.Other such medicaments it is effective Amount depends on the type and other factors discussed above of the amount of antibody present in preparation, disease or treatment.These are usually It to be used with identical dosage described herein and administration route, or is about the 1 to 99% of dosage described herein, or with warp Test/clinic is determined as suitable any dosage and any approach.
In order to prevent or treat disease, the suitable dosage of antibody of the invention (when be used alone or with it is one or more its When his other therapeutic agent is applied in combination) will depend on disease to be treated type, the type of antibody, the severity of disease and The course of disease, the application of antibody are the treatments before in order to prevent purpose or for therapeutic purposes, the clinical history of patient and to antibody Reaction and attending physician judgement.Antibody is suitably applied to patient disposably or in a series of treatments.According to disease The type and severity, the antibody of about 1 μ g/kg to 15mg/kg (for example, 0.1mg/kg-10mg/kg) of disease can be for applying To the initial candidate dosage of patient, whether, for example, by one or many separated applications, or by being continuously injected into.One A typical daily dose can be about 1 μ g/kg to 100mg/kg or more, this depends on above-mentioned factor.For several Repetitive administration in it or longer time usually can be with continued treatment until disease symptoms needed for occurring according to condition Suppress.One exemplary dose of antibody is about 0.05mg/kg to about 10mg/kg.Therefore, can by one or more dosage, About 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) it is applied to patient.Such dosage can between It has a rest application, for example, once a week or once every three weeks (for example, making patient receive about two to about 20, or for example, about The antibody of six dosage).Initial higher load dosage can be applied, one or more lower dosage are followed by.The treatment Process can easily pass through routine techniques and measurement monitoring.
It is to be understood that any of above preparation or treatment method can be using immunoconjugates of the invention (to replace Anti- C5 antibody or other than anti-C5 antibody) carry out.
H. product
In another aspect of the invention, product is provided, it includes can be used for treating, prevents and/or diagnose above-mentioned disease Material.The product includes container and label or package insert on container or in conjunction with container.Suitable container includes, For example, bottle, bottle, syringe, IV solution bag etc..The container can be made of multiple material (such as glass or plastics).Institute It states container and contains composition, the composition is individual or with for treatment, and prevention and/or diagnosis illness are effectively another Combination of compositions, and the container can have sterile access interface (such as the container can be have can subcutaneously be infused Penetrate the intravenous solution bag or bottle of the plug that needle punctures).At least one of composition activating agent is antibody of the invention.Mark Note or package insert indication composition are used to treat selected illness.In addition, the product, which may include (a), is wherein loaded with group The first container of object is closed, wherein the composition includes antibody of the invention；(b) be wherein loaded with composition second is held Device, wherein the composition also includes cytotoxicity or other therapeutic agents.Product in the embodiment of the invention can be with Comprising package insert, the package insert indication composition can be used for treating particular condition.Alternatively, or additionally, described Product can also include second (or third) container, and the container includes acceptable buffer, such as injection bacteriostatic water (BWFI), phosphorus Acid buffering salt water, Ringer's solution and dextrose solution.Its other materials that can also be needed comprising business or user, including other Buffer, dilution, filler, needle and syringe.
It is to be understood that any of above product can include immunoconjugates of the invention to substitute anti-C5 antibody, or For person other than anti-C5 antibody, any of above product can also include immunoconjugates of the invention.
Embodiment
It is the embodiment of method and composition of the invention below.It should be understood that, it is known that generality provided above is retouched It states, it is possible to implement various other embodiments.
Embodiment 1
Prepare C5
1.1. expression and purification of recombinant human and machin C5
Use FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) transient expression recombined human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID NO:39).The bodies such as the conditioned medium use of people C5 will be expressed Long-pending milliQ water dilution, is then applied to Q-sepharose FF or Q-sepharose HP anion-exchange column (GE Healthcare, Uppsala, Sweden), NaCl gradient elution is used later.The fraction for collecting the C5 containing people, then respectively by salinity 80mM NaCl and pH6.4 are adjusted to pH.By resulting sample administration in SP-sepharose HP cation exchange column (GE Healthcare, Uppsala, Sweden) and with NaCl gradient elution.The fraction of the C5 containing people is collected and passes through CHT ceramics hydroxyl Apatite column (Bio-Rad Laboratories, Hercules, CA, USA).Then people's C5 eluate is applied to Superdex 200 solvent resistant columns (GE healthcare, Uppsala, Sweden).The fraction of the C5 containing people is collected and is stored in -150 DEG C.
Recombination machin C5 (NCBI GenBank accession number: XP_ is carried out in a manner of identical with people's counterpart 005580972, SEQ ID NO:44) expression and purifying.
1.2. from plasma purification machin C5 (cynoC5)
Plasma sample from machin is applied to SSL7- agarose (Invivogen, San Diego, CA, USA), It is eluted later with 100mM sodium acetate pH3.5.Fraction containing cynoC5 is neutralized immediately and with peptide M agarose (Invivogen, San Diego, CA, USA) pass through albumin A HP column (GE healthcare, Uppsala, Sweden) together.Then grade will be flowed through It gives and is added on Superdex200 solvent resistant column (GE healthcare, Uppsala, Sweden).Fraction containing cynoC5 is converged Collect and is stored in -80 DEG C.
Embodiment 2
Generate anti-C5 antibody
2.1. antibody screening
Anti- C5 antibody is prepared, selects and measured as follows:
By 12-16 week old NZW rabbit employment C5 and/or monkey C5 intradermal immunization (50-100 μ g/ dosage/rabbit).2 months when Between in by the dosage repeat 4-5 times.Latter week is finally immunized, collects spleen and blood from immune rabbit.Antigen-specific b-is thin The antigen dyeing of born of the same parents' label, is sorted with FCM cell sorter (FACS aria III, BD), and with cells/well The EL4 cell (European Collection of Cell Cultures) and dilution 20 of density and 25,000 cells/wells Times the rabbit T- cell conditioned medium of activation bed board and is cultivated 7-12 days in 96- orifice plate together.By EL4 cell silk Rimocidin C (Sigma, catalog number (Cat.No.) M4287) is handled 2 hours and is washed 3 times in advance.By in-the M containing phytolectin (Roche, 1 1082132-001 of catalog number (Cat.No.)), phorbol 12-myristate 13-acetate (Sigma, catalog number (Cat.No.) P1585) and Rabbit thymocyte is cultivated in the RPMI-1640 of 2%FBS to prepare the rabbit T- cell conditioned medium of activation.After incubation, it collects B- cell culture supernatant is for further analysis and freezes cell precipitation (pellet).
ELISA measurement is used for the specificity of antibody in test b-cell culture supernatant.By the 50nM strepto- in PBS Avidin (GeneScript, catalog number (Cat.No.) Z02043) is coated on the hole 384- MAXISorp (Nunc, catalog number (Cat.No.) 164688), in room Temperature was up to 1 hour.Then the plate Blocking One (Nacalai Tesque, catalog number (Cat.No.) 03953-95) for diluting 5 times is sealed It closes.People or monkey C5 are marked with NHS-PEG4- biotin (PIERCE, catalog number (Cat.No.) 21329) and are added closed ELISA plate, It is incubated for 1 hour and washs.B- cell culture supernatant is added to ELISA plate, is incubated for 1 hour and washs.It is anti-by goat Rabbit igg-horseradish peroxidase (BETHYL, catalog number (Cat.No.) A120-111P) and the ABTS added later (KPL, catalog number (Cat.No.) 50-66- 06) combination is detected.
It is used to assess antibody for ELISA measurement to combine the pH- dependence of C5.The mountain of 1 μ g/ml will be diluted to PBS (-) The hole 384- MAXISorp (Nunc, catalog number (Cat.No.) 164688) is added in goat-anti-rabbit igg-Fc (BETHYL, catalog number (Cat.No.) A120-111A), Incubation at room temperature 1 hour, and closed with 5 times of dilution of Blocking One (Nacalai Tesque, catalog number (Cat.No.) 03953-95).? After incubation, by Plate wash and B- cell culture supernatant is added.Plate is incubated for 1 hour, washing, and the life of 500pM is added The people or monkey C5 of object element are simultaneously incubated for 1 hour.After incubation, by Plate wash and with pH7.4MES buffer (20mM MES, 150mM NaCl and 1.2mM CaCl2) or pH5.8MES buffer (20mM MES, 150mM NaCl and 1mM EDTA) in room temperature It is incubated for 1 hour.After incubation, pass through Streptavidin-horseradish peroxidase conjugate (Thermo Scientific, catalogue Number 21132) ABTS (KPL, catalog number (Cat.No.) 50-66-06) and being added later detects the combination of biotinylated C5.
By Octet RED384 system (Pall Life Sciences) be used to assess antibody to the affinity of C5 and pH- according to Property is relied to combine.The antibody secreted in B- cell culture supernatant is loaded to albumin A biosensor tips (Pall Life Sciences it) and is immersed in 50nM people or the monkey C5 in pH7.4MES buffer to analyze binding kinetics.? Dissociation is analyzed in pH7.4MES buffer and pH5.8MES buffer.
Screening amounts to 41,439 B- cell lines and combines to the affinity and pH- dependence of people or monkey C5 and select 677 A cell line and it is appointed as CFA0001-0677.Use ZR-96 Quick-RNA kit (ZYMO RESEARCH, catalog number (Cat.No.) R1053 the RNA of selected cell line) is purified by the cell precipitation frozen.By the encoding antibody heavy variable region in selected cell line DNA by reverse transcription PCR expand and with coding F760G4 (SEQ ID NO:33) or F939G4 (SEQ ID NO:34) weight The DNA of chain constant region is recombinated.By the DNA of encoding antibody light variable region by reverse transcription PCR expand and with coding k0MTC light chain The DNA of constant region (SEQ ID NO:36) is recombinated.The anti-C5 antibody of existing humanization is separately synthesized according to library pearl monoclonal antibody (EcuH- G2G4, SEQ ID NO:29 and EcuL-k0, SEQ ID NO:30) heavy chain and light chain gene.VH (EcuH, SEQ ID will be encoded NO:31 the DNA of 4 CH of human IgG (F760G4, SEQ ID NO:33) of DNA and coding modification) are merged with frame, and will encode VL The DNA of (EcuL, SEQ ID NO:32) is merged with the DNA of coding k0 constant region of light chain (SEQ ID NO:37) with frame.It will also be each The coded sequence of fusion is cloned into expression vector.By antibody in FreeStyleTMExpression in 293-F cell (Invitrogen) And it purifies from culture supernatant with evaluation function activity.The inhibition of test complement activity is measured by using liposome dissolving to comment The neutralization activity for estimating antibody, as described in embodiment 5.1.
2.2. simultaneously by the epitope frame of sandwich ELISA
Select that there is high-affinity, the anti-C5 antibody of pH dependence or neutralization activity is for further analyzing.Using sandwich The antibody of selection is grouped into identical or overlapping epitope the different epitope frame in conjunction with C5 albumen by ELISA measurement.It will not The capture antibody of label is diluted to 1 μ g/ml with PBS (-) and the hole 384- MAXISorp plate (Nunc, catalog number (Cat.No.) is added 164688).Blocking One (Nacalai Tesque, catalogue by plate being incubated at room temperature 1 hour and with diluting 5 times Number 03953-95) closing.Plate is incubated for 1 hour, washing, and the people C5 of 2nM is added and is incubated for 1 hour.After incubation, it will put down Plate washs and the detection antibody (1 μ g/mL, by NHS-PEG4- biotin biotinylation) of label is added.After 1 hour is incubated for, pass through It Streptavidin-horseradish peroxidase conjugate (Thermo Scientific, catalog number (Cat.No.) 21132) and is added later ABTS (KPL, catalog number (Cat.No.) 50-66-06) detects the combination of biotinylated antibody.
All anti-C5 antibody are used as both capturing antibody and detecting antibody, and match comprehensively.As shown in fig. 1, phase The antibody mutually competed is classified into 7 epitope frames: CFA0668, CFA0334 and CFA0319 are grouped into epitope A, CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330 and CFA0318 are grouped into epitope B, CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 are divided Group is grouped into epitope D, CFA0329 is grouped into epitope E, CFA0359 into epitope C according to library pearl monoclonal antibody and CFA0322 It is grouped into epitope F with CFA0217, and CFA0579, CFA0328 and CFA0272 are grouped into epitope G.Fig. 1 is shown The epitope frame of some anti-C5 chimeric antibodies is simultaneously.VH the and VL sequence for the anti-C5 antibody being grouped into epitope C is listed in Table 2.
[table 2]
The anti-C5 antibody being grouped into epitope C
2.3. humanization and optimization
Humanization is carried out to reduce the possible immunogenicity of the antibody to the variable region of some anti-C5 antibody.It uses The complementary determining region (CDR) of anti-C5 rabbit antibody is transplanted to by conventional CDR transplantation method (Nature 321:522-525 (1986)) On homologous human antibody frame (FR).The gene of composite coding humanization VH and VL and by its respectively with 4 CH of the human IgG of modification (SG402, SEQ ID NO:35) and people CL (SK1, SEQ ID NO:38) combination, and each combined sequence is cloned into expression and is carried In body.
Several mutation and mutation combination are detected to identify the mutation of some binding properties in the leading antibody of raising and dash forward Become combination.Then multiple mutation are introduced into humanization variable region to enhance binding affinity or the reduction in neutral pH to C5 In acid pH to the binding affinity of C5.Thus a kind of variant of optimization is generated by CFA0305,305LO5 (VH, SEQ ID NO: 10；VL, SEQ ID NO:20；HVR-H1, SEQ ID NO:54；HVR-H2, SEQ ID NO:64；HVR-H3, SEQ ID NO: 74；HVR-L1, SEQ ID NO:84；HVR-L2, SEQ ID NO:94；And HVR-L3, SEQ ID NO:104).
By antibody with being expressed in the HEK293 cell of heavy chain and the mixture cotransfection of light chain expression vector and pass through egg White A purifying.
Embodiment 3
The combination of anti-C5 antibody characterizes
3.1. the expression and purifying of recombinant antibodies
It is recombinated using FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) transient expression anti- Body.The purifying of the conditioned medium of expression antibody certainly is carried out using albumin A using conventional method.If it is required, then further into Row gel filtration.
3.2, assess pH dependence
BIACORE (registered trademark) T200 instrument (GE Healthcare) is used at 37 DEG C, in pH7.4 and pH5.8, is commented Anti- C5 antibody is estimated to the kinetic parameter of recombined human C5.Using amine coupling kit (GE Healthcare) according to GE The setting that Healthcare recommends, is fixed on CM4 sensor core on piece for ProA/G (Pierce).Antibody and analyte are diluted To corresponding running buffer ACES pH7.4 and pH5.8 (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05% Tween20,0.005%NaN3) in.By ProA/G by each antibody capture on the sensor surface.Antibody capture is horizontal typical Ground is 60-90 response units (RU).Then, recombined human C5 is injected with the concentration of 10 and 20nM or 20 and 40nM, dissociated later. Using 25mM NaOH by surface regeneration.By utilizing 1: 1 binding model, using BIACORE (registered trademark) T200Evaluation software, version 2 .0 (GE Healthcare) are fitted influence chart to determine the dynamics under the conditions of two pH Parameter.The influence chart of all antibody is shown in Figures 2 A and 2 B.The association rate (ka) of antibody, dissociation rate (kd) and combination parent It is listed in Table 3 with power (KD).In addition to CFA0330 (VH, SEQ ID NO:21 and VL, SEQ ID NO:25) and CFA0341 (VH, SEQ ID NO:22 and VL, SEQ ID NO:26) except the dissociation rate that shows in pH 5.8 of all antibody all than in pH7.4 Relatively faster.
[table 3]
Kinetic parameter of anti-C5 antibody under the conditions of pH7.4 and pH5.8
3.3. cross reactivity inspection
In order to observe the cross reactivity of anti-C5 antibody on human C5 (hC5) and machin C5 (cynoC5), carry out BIACORE (registered trademark) dynamic analysis.Measurement setting with described in embodiment 3.2, will recombination cynoC5 with 2,10 Hes The concentration of 50nM is injected.Kinetic parameter is determined by being fitted with identical data described in embodiment 3.2.PH7.4's Binding kinetics and affinity are listed in Table 4.The kinetic parameter for hC5 provided in table 4 is the result of embodiment 3.2. All anti-C5 antibody other than CFA0672 all show comparable KD to hC5 and cynoC5.KD of the CFA0672 to cynoC5 It is weak to 8 times of the KD of hC5.
[table 4]
In the anti-C5 antibody of pH7.4 to the binding kinetics and affinity of hC5 and cynoC5
Embodiment 4
The epitope mapping of anti-C5 antibody
4.1. the combination of peptide derived from anti-C5 MAb and C5 beta chain
The combination of peptide derived from anti-C5 monoclonal antibody (MAb) and C5 beta chain is detected in western blot analysis.It will be with The C5 peptide of GST- label (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49) fusion: 19-180, 161-340,321-500 and the 481-660 expression in Escherichia coli (DH5 α, TOYOBO, DNA-903).With 1mM isopropyl ss- D-1- Thiogalactopyranoside (IPTG) harvests E. coli SampLes after being incubated for 5 hours at 37 DEG C, and with 20000x g centrifugation 1min is to be precipitated.Precipitating is suspended with Sample Buffer solution (2ME+) (Wako, 191-13272), and is used for western blot Analysis.The expression (Fig. 3) of each peptide is confirmed using anti-GST antibody (Abcam, ab9085).Arrow instruction fusion has the C5 peptide of GST (46-49kDa).Anti- C5MAb:CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675, in conjunction with the 19-180 (Fig. 3) of C5.
4.2. the expression and purifying of the MG1-MG2 structural domain (1-225) of people C5
Use FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) transient expression people C5 β- The recombination MG1-MG2 structural domain (SEQ ID NO:43) of chain.1/2 volume of conditioned medium of MG1-MG2 structural domain will be expressed The dilution of milliQ water, be applied to later Q-sepharose FF anion-exchange column (GE healthcare, Uppsala, it is auspicious Allusion quotation).By from anion-exchange column flow through fraction be adjusted to pH 5.0 and be applied to SP-sepharose HP cation hand over Change column (GE healthcare, Uppsala, Sweden) and with NaCl gradient elution.Structural domain containing MG1-MG2 is collected from eluent Fraction and then pass through with 1x PBS balance 75 solvent resistant column of Superdex (GE healthcare, Uppsala, Sweden).Then collect the fraction of the structural domain containing MG1-MG2 and be stored in -80 DEG C.
4.3. the ability in conjunction with MG1-MG2 structural domain
It is set using measurement identical with described in embodiment 3.2 to measure anti-C5 antibody to the knot of MG1-MG2 structural domain Conjunction ability, the difference is that only being measured under the conditions of pH7.4.By MG1-MG2 structural domain with the concentration of 20nM and 40nM Injection.As shown in Figure 4, in addition to all showing increased association reaction according to all antibody other than the pearl monoclonal antibody-F760G4 of library, this table These bright antibody are MG1-MG2 bonding agents.It is not shown as known α-link mixture according to library pearl monoclonal antibody-F760G4 and MG1- The combination of MG2 structural domain.
4.4. the combination of peptide derived from anti-C5MAb and C5MG1-MG2 structural domain
The combination of peptide derived from anti-C5MAb and MG1-MG2 structural domain is tested in western blot analysis.It will be marked with GST- Sign the C5 peptide of fusion: 33-124,45-124,52-124,33-111,33-108 and 45-111 (SEQ ID NO:40) are in large intestine It is expressed in bacillus.Harvest E. coli SampLes after being incubated for 5 hours with 1mM IPTG at 37 DEG C, and with 20000x g be centrifuged 1min with It is precipitated.Precipitating is suspended with Sample Buffer solution (2ME+), and is used for western blot analysis.Confirmed using anti-GST antibody The expression (Fig. 5 A) of peptide derived from C5.Peptide (Fig. 5 B) of the CFA0305 only in conjunction with 33-124.CFA0305 combination recombined human C5 (rhC5) beta chain (about 70kDa) is used as control.Fig. 5 C outlines reacting for anti-C5MAb and peptide derived from C5.
4.5. the combination of anti-C5MAb and C5 mutant
Because predicting three amino acid residues in C5 beta chain: E48, D51 and K109 by crystal structure analysis, C5 is participated in Combination between anti-C5-MAb, so testing the combination of anti-C5MAb and people's C5 point mutation body in western blot analysis.It is logical Fat transfection is crossed to express E48, any of D51 and K109 the C5 point mutation body for being replaced as alanine in FS293 cell.Rouge 5 days harvest culture mediums after transfection are used for Western blotting later.SDS-PAGE is carried out under the reducing conditions.As the result is shown in Fig. 6 In.According to α-chain of C5 and three C5 point mutation body of library pearl monoclonal antibody combination wild type (WT), and CFA0305 combines WT C5's strongly Beta chain, and E48A C5 mutant beta chain combination it is weak, and do not combine the beta chain of D51A and K109A C5 mutant, show This 3 amino acid residues participate in antibody/antigen interaction.Table 5 provide anti-C5MAb (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675) western blot analysis general introduction.It is anti- C5MAb is grouped into identical epitope C, but the binding pattern between antibody is slightly different, shows C5 for anti-C5MAb's Bond area is located adjacent one another without identical.
[table 5]
The general introduction of anti-C5 MAb and C5 mutant reacted
4.6. BIACORE (registered trademark) binding analysis of anti-C5 antibody and C5 mutant
In order to test whether residue E48, G51 and K109 actually participate in antibody/antigen interaction, BIACORE is carried out (registered trademark) binding analysis.Three kinds of C5 mutant: E48A, G51A and K109A are prepared, as described in embodiment 4.5.Preparation contains There is the culture supernatant sample for the mutant C5 being overexpressed in FS293 cell, wherein mutant C5 is 40 μ g/ml.For BIACORE (registered trademark) binding analysis, by sample with BIACORE (registered trademark) running buffer (ACES pH7.4, 10mg/ml BSA, 1mg/ml Sensor Chip CM 5) the final sample concentration of 10x to mutant C5 is diluted for 4 μ g/ml.
BIACORE (registered trademark) T200 instrument (GE Healthcare) is utilized at 37 DEG C, using retouching in embodiment 3.2 The determination condition stated assesses the interaction of three kinds of C5 mutant Yu anti-C5 antibody.10mg/ml BSA, 1mg/ml carboxylic first will be contained 7.4 buffer of ACES pH of base glucan is used as running buffer.Pass through Monoclonal mouse Anti-Human IgG, Fc fragments specific Antibody (GE Healthcare) will capture on different flow chambers according to library pearl monoclonal antibody-F760G4 and 305LO5.1 quilt of flow chamber As reference surfaces.With the concentration of 4 μ g/ml by wild type and mutant C5 protein injection on the sensor surface with capture Antibody interaction.At the end of each analysis circulation, by sensor surface 3M MgCl2Regeneration.Utilize Bia Evaluation Software, version 2 .0 (GE Healthcare) analyze result.Reference flow chamber (flow chamber 1) and blank running buffer are injected Curve from have capture antibody flow chamber curve in subtract.
As shown in Figure 7, compared to wild type C5, three kinds of C5 mutant can be combined with similar binding characteristic according to library Pearl monoclonal antibody.For 305LO5, three kinds of mutant all show the lower association reaction to 305LO5 compared with wild type C5.D51A Weaken the combination of 305LO5 and C5 to baseline level with K109A mutant.
4.7.C5 the identification of the His residue of pH dependence interaction of the upper power-assisted between anti-C5 antibody and C5
Crystal structure analysis discloses 3 histidine residues on people C5 and is located at antibody/antigen interface.It is known that there is typical case About 6.0 pKa histidine residues power-assisted in pH dependence protein-protein-interacting (Igawa etc., Biochim (2014) Biophys Acta 1844 (11): 1943-1950).In order to which which the His residue studied on antibody/antigen interface helps PH dependence interaction of the power between anti-C5 antibody and C5, carries out BIACORE (registered trademark) binding analysis.Following system It is standby to be mutated three kinds of people C5 mutant of (H70Y, H72Y and H110Y) with single His and there are double His to be mutated (H70Y+ H110Y H70, any of H72 and H110 mutant): are replaced as to the single His mutant of tyrosine by fat transfection It is expressed in FS293 cell with H70 and the H110 double His mutant for being all replaced as tyrosine.C5His mutant and 305LO5 The antigen binding property of (the anti-C5 antibody of pH- dependence) is measured by improved BIACORE (registered trademark) and is determined, is such as implemented Described in example 4.6.In short, the other dissociation stage in pH5.8 is integrated into BIACORE (registered trademark) measurement, And then after the dissociation stage of pH7.4, thus between antibody and antigen of the assessment from the compound formed in pH7.4 The dissociation of pH- dependence.PH5.8 dissociation rate by using Scrubber2.0 (BioLogic Software) curve matching Software processing and fitting data determine.
As shown in Figure 8, it does not influence in the mono- His mutation of the C5 of H70 or H110 and double His mutation (H70+H110) in neutrality The combination of pH C5 and 305LO5.Meanwhile it being mutated in single His of H72 and showing that C5 is significant impaired in conjunction with 305LO5.C5His is prominent Variant and C5-wt albumen are shown in table 6 in the dissociation rate of pH5.8.As shown in table 6, in the C5 antigen of test, PH5.8, C5-wt show the most fast dissociation from 305LO5.Compared with C5-wt, single His of H70 be mutated show in pH5.8 it is several Twice of slow dissociation rate and single His of H110 mutation lead to slightly slow dissociation rate in pH5.8.In H70 and H110 Double His mutation cause to pH- dependence combine larger impact, wherein the dissociation rate in pH5.8 is almost the three of C5-wt It is slow again.
[table 6]
The pH5.8 dissociation rate value of C5 His mutant combination 305LO5
Embodiment 5
The inhibitory activity that anti-C5 antibody activates C5
5.1. anti-C5 MAb inhibits the liposome dissolving of complement activation
The inhibition to complement activity of anti-C5 MAb is tested by liposome dissolving measurement.By normal person's blood of 30 μ L (6.7%) (Biopredic, SER018) is mixed in 96- orifice plate with the diluted MAb of 20 μ L and is incubated on shaking table at 25 DEG C clearly Educate 30min.The liposome being sensitized with the antibody (Autokit CH50, Wako, 995-40801) for dinitrophenyl is shifted It is placed on shaking table into each hole and at 25 DEG C by plate up to 2min.Each hole is added in the substrate solution (Autokit CH50) of 50 μ L And 2min is mixed in 25 DEG C of oscillations.Final mixture is incubated for 40 minutes at 37 DEG C, measures mixture later 340nm's OD.The percentage of liposome dissolving is defined as 100x [(ODMAb-ODSerum and liposome background)]/[(ODWithout MAb-ODSerum and liposome background)].Figure 9A shows anti-C5 Mab:CFA0305,0307,0366,0501,0538,0599,0666,0672 and 0675, inhibit liposome Dissolution.Two kinds of non-TCP friendly flow antibody: CFA0330 and 0341, also inhibition lysis (Fig. 9 B).
5.2. anti-C5MAb inhibits C5a to generate
The C5a during liposome dissolving for testing anti-C5MAb is generated to confirm that anti-C5 MAb inhibits C5 to be cut into C5a and C5b.Quantify the supernatant measured from liposome dissolving using C5a ELISA kit (R&D systems, DY2037) In C5a it is horizontal.All MAb inhibit the C5a in supernatant to generate (Figure 10 A and 10B) in a manner of dose-dependent.
5.3. anti-C5 MAb inhibits the haemolysis of complement activation
The inhibition of the classical complement activity of anti-C5MAb is tested in haemolysis measurement.By chicken red blood cell (cRBC) (Innovative research, IC05-0810) uses MgCl containing 0.5mM2With 0.15mM CaCl2(GVB++) gelatin/bar ratio The salt water (Boston BioProducts, IBB-300X) of appropriate buffering washs, later with the anti-chicken RBC antibody of 1 μ g/ml (Rockland 103-4139) is sensitized 15 minutes at 4 DEG C.Then cell washed with GVB++ and with 5x107A cell/ml is outstanding It floats in identical buffer.In individual round bottom 96- hole micrometering test plate (panel), by the normal human serum (20%) of 50 μ L (Biopredic, SER019) is mixed with the diluted Mab of 50 μ L and is incubated for 30 minutes on shaking table at 37 DEG C.Then by 60 μ L Sensitization cRBC suspension be added the hole containing serum in, and by antibody mixture 37 DEG C be incubated for 30 minutes.After incubation, will Plate is centrifuged 2 minutes with 1000x g at 4 DEG C.Supernatant (100 μ L) is transferred in the hole of the flat hole 96- micrometering test plate (panel) to be used for OD, reference wavelength 630nm of the measurement in 415nm.The percentage of haemolysis is defined as 100x [(ODMAb-ODSerum and cRBC)]/ [(ODWithout MAb-ODSerum and cRBC background)].Figure 11 shows anti-C5 Mab:CFA0305 and 305LO5, inhibits the haemolysis of cRBC.
5.4. anti-C5MAb inhibits alternative com-plement pathway
Measure for the haemolysis of alternative pathway to measure similar mode with classical pathway haemolysis.It will be collected from Xin Xi The blood of blue white rabbit (InVivos) and the Alsever solution (Sigma, A3551) of same volume mix, and mixture is used as Rabbit RBC (rRBC).By rRBC with being supplemented with 2mM MgCl2It washs and with the GVB of 10mM EGTA with 7x108A cell/ml suspends In identical buffer.In round bottom 96- hole micrometering test plate (panel), by the normal human serum (25%) of 40 μ L (Biopredic, SER019 it) mixes with the diluted Mab of 40 μ L and is incubated for 30 minutes on shaking table at 37 DEG C.Then by the rRBC suspension of 20 μ L It is added into the hole containing serum, and antibody mixture is incubated for 60 minutes at 37 DEG C.After incubation, by plate with 1000x g 4 DEG C centrifugation 2 minutes.Supernatant (70 μ L) is transferred in the hole on the flat hole 96- micrometering test plate (panel) with for measuring the OD in 415nm, Reference wavelength is 630nm.Figure 12 shows anti-C5 Mab:CFA0305 and CFA0672, inhibits the haemolysis of rRBC, indicates that these are anti- Body inhibits alternative com-plement pathway.
Embodiment 6
Anti- C5 monoclonal antibody and pharmacokinetic of the people C5 in mouse
6.1. the internal test of C57BL/6 mouse is used
Individual people C5 or people C5 and Anti-Human C5 antibody are being applied to C57BL/6 mouse (In Vivos or Biological Resource Centre, Singapore) after, the body of evaluator C5 (Calbiochem) and Anti-Human's C5 antibody Internal dynamics.By people C5 solution (0.01mg/ml) or C5 containing someone and Anti-Human C5 antibody (respectively, 0.01mg/ml and 2mg/ml(CFA0305-F760G4、CFA0307-F760G4、CFA0366-F760G4、CFA0501-F760G4、CFA0538- F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 and CFA0675-F760G4) or 0.2mg/ Ml (CFA0330-F760G4 and CFA0341-F760G4)) the solution of mixture tail is administered to the single dose of 10ml/kg In vein.In this case, Anti-Human C5 antibody is present in excess relative to people C5, and therefore, it is considered that almost everyone C5 is in conjunction with antibody.5 minutes after application, 7 hours, 1 day, 2 days, 3 days and 7 days collection blood.Immediately by the blood of collection 10 minutes are centrifuged with separated plasma at 4 DEG C with 14,000rpm.Isolated blood plasma is stored in -80 DEG C of refrigerators until measuring.Make Anti-Human's C5 antibody be CFA0305-F760G4, CFA0307-F760G4 as described above, CFA0330-F760G4, CFA0341-F760G4、CFA0366-F760G4、CFA0501-F760G4、CFA0538-F760G4、CFA0599-F760G4、 CFA0666-F760G4, CFA0672-F760G4 and CFA0675-F760G4.
6.2. pass through electrochemical luminescence (ECL) measurement measurement total plasma concentration of people C5
The total concentration of people C5 in mice plasma is measured by ECL.
There are in the case where CFA0330-F760G4, CFA0341-F760G4 or individual people C5 in plasma sample, make Using the following method.Anti-Human C5 antibody (Santa Cruz) is assigned to 96 hole bare board (Meso Scale of MULTI-ARRAY Discovery it is stood overnight on) and at 4 DEG C, to prepare the plate for being fixed with Anti-Human C5.Prepare calibration curve sample and with 1 μ The injection of antibodies (CFA0330-F760G4 or CFA0341-F760G4) of g/ml dilutes 100 times or more of mice plasma sample, and And it is incubated for 30 minutes at 37 DEG C.Then, sample is assigned on the plate for being fixed with Anti-Human C5, and keeps it quiet in room temperature It sets 1 hour.Then, Anti-Human's IgG antibody (Meso Scale Discovery) of addition SULFO-TAG label is with anti-in room temperature It answers one hour, and washs.It immediately distributes Read buffer T (x4) (Meso Scale Discovery) and uses Sector Imager2400 (Meso Scale Discovery) is measured.
There are CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501- in plasma sample F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 or CFA0675- In the case where F760G4, following methods are used.By Anti-Human's C5 antibody (CFA0329-F939G4；VH, SEQ ID NO:23, and VL, SEQ ID NO:27) it is assigned on 96 hole bare board of MULTI-ARRAY (Meso Scale Discovery) and is stood at 4 DEG C Overnight, to prepare the plate for being fixed with Anti-Human C5.It prepares calibration curve sample and dilutes 100 times with acid solution (pH 5.5) Above mice plasma sample, and it is incubated for 30 minutes at 37 DEG C.Then, sample is assigned to and is fixed with Anti-Human C5's On plate, and it is being stored at room temperature it 1 hour.Then, Anti-Human's C5 antibody (CFA0300- of addition SULFO-TAG label F939G4；VH, SEQ ID NO:24 and VL, SEQ ID NO:28) in room temperature reaction one hour, and wash.Immediately It distributes Read buffer T (x4) (Meso Scale Discovery) and uses (the Meso Scale of Sector Imager 2400 Discovery it) measures.
Response based on calibration curve calculates people C5 using analysis software SOFTmax PRO (Molecular Devices) Concentration.The time-histories of blood plasma people's C5 concentration is shown in Figure 13 after the intravenous application measured by this method.Data are plotted as and 5 The remaining percentage that blood plasma people's C5 concentration is compared when minute.
6.3. measurement Anti-Human C5 antibody plasma concentration is measured by ECL
The concentration of Anti-Human's C5 antibody in mice plasma is measured by ECL.By Anti-Human IgG (γ-chain specificity) F (ab ') 2 It is naked that antibody fragment (Sigma) or Anti-Human IgG κ chain antibody (Antibody Solutions) are assigned to 96 hole MULTI-ARRAY It is stood overnight on plate (Meso Scale Discovery) and at 4 DEG C, to prepare the plate for being fixed with Anti-Human IgG.Preparation correction Curve sample and the mice plasma sample for diluting 100 times or more.Then, sample is assigned to the plate for being fixed with Anti-Human IgG On, and it is being stored at room temperature it 1 hour.Then, add biotinylated Anti-Human's IgG antibody or SULFO-TAG label it is anti- Human IgG Fc antibody (Southernbiotech) is washed with reacting at room temperature one hour.Then, only when use biotin When Anti-Human's IgG antibody of change, just add SULFO-TAG label Streptavidin (Meso Scale Discovery) with Room temperature reaction one hour, and wash.It immediately distributes Read buffer T (x4) (Meso Scale Discovery) and makes It is measured with Sector Imager 2400 (Meso Scale Discovery).Response based on calibration curve, using point It analyses software SOFTmax PRO (Molecular Devices) and calculates Anti-Human C5 antibody concentration.The vein measured by this method The time-histories of blood plasma Anti-Human C5 antibody concentration is shown in Figure 14 after interior application.Data are plotted as and blood plasma Anti-Human C5 at 5 minutes The remaining percentage that antibody concentration is compared.
6.4.pH dependence Anti-Human C5 antibody combines the effect removed to internal people C5
Internal test pH dependence Anti-Human's C5 antibody (CFA0305-F760G4, CFA0307-F760G4, CFA0366- F760G4、CFA0501-F760G4、CFA0538-F760G4、CFA0599-F760G4、CFA0666-F760G4、CFA0672- F760G4 and CFA0675-F760G4) and non-TCP friendly flow Anti-Human C5 antibody (CFA0330-F760G4 and CFA0341- ), and more resulting blood plasma Anti-Human C5 antibody concentration and blood plasma people's C5 concentration F760G4.As shown in figure 14, antibody exposure is It is comparable.Meanwhile compared to non-TCP friendly flow Anti-Human's C5 antibody, the people C5's being administered simultaneously with pH dependence Anti-Human's C5 antibody Removing is to accelerate (Figure 13).
Embodiment 7
Optimize anti-C5 monoclonal antibody (305 variant)
By the variable region of several optimizations for being mutated and being introduced into anti-C5 antibody 305LO5 further to improve its property, and Generate variable region 305LO15,305LO16,305LO18,305LO19,305LO20,305LO22 and the 305LO23 of optimization.305 The amino acid sequence of the VH and VL of variant are listed in respectively in table 7 and 8.By the human IgG1 of the gene of encoding humanized VH and modification CH variant SG115 (SEQ ID NO:114), and the human IgG 4CH variant SG422 (SEQ ID NO:115) or SG429 of modification (SEQ ID NO:116) combination.It combines the gene of encoding humanized VL and people CL (SK1, SEQ ID NO:38).It is respectively synthesized Encoding humanized anti-C5 antibody BNJ441 (BNJ441H, SEQ ID NO:149；BNJ441L, SEQ ID NO:150) heavy chain It is respectively cloned into expression vector with light chain gene and by it.
By antibody with expressing in the HEK293 cell of heavy chain and the combination cotransfection of light chain expression vector, and pass through albumen A purifying.
[table 7]
The VH amino acid sequence of 305 variants
[table 8]
The VL amino acid sequence of 305 variants
Embodiment 8
The combination of anti-C5 antibody (305 variant) characterizes
37 DEG C using BIACORE (registered trademark) T200 instrument (GE Healthcare) at three kinds under different conditions Anti- C5 antibody is assessed to the kinetic parameter of recombined human C5；(1) all in pH7.4, (2) association and dissociation all exists association and dissociation PH5.8, and (3) are incorporated in pH7.4 and dissociate in pH5.8.Using amine coupling kit (GE Healthcare) according to GE ProA/G (Pierce) is fixed to CM1 sensor core on piece by the setting that Healthcare recommends.By the anti-of condition (1) and (3) Body and analyte are diluted in ACES pH7.4 buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05%Tween 20,0.005%NaN3) in and by the antibody of condition (2) and analyte be diluted in ACES pH5.8 buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05%Tween 20,0.005%NaN3) in.Each antibody capture is being passed by ProMG In sensor surfaces.Antibody capture level is typically 60-90 response units (RU).Then, with 3 to 27nM or 13.3 to 120nM (being prepared by three times serial dilution) injects recombined human C5, dissociates later.Surface 25mM NaOH is regenerated.Condition (1) passed through with the kinetic parameter of (2) by the dissociation rate for being fitted influence chart determination and condition (3) with 1: 1 binding model Influence chart is fitted with 1: 1 dissociation for MCK model to determine, wherein using BIACORE (registered trademark) T200Evaluation software, version 2 .0 (GE Healthcare).The pH dependence of all antibody be shown as condition (2) and (1) the ratio between dissociation rate.
Association rate (ka), dissociation rate (kd), binding affinity (KD) and pH dependence are listed in Table 9.All antibody It is all about 20 times than fast and its pH dependence in pH7.4 in the dissociation rate that pH 5.8 is shown.
[table 9]
Kinetic parameter of anti-C5 antibody variants under the conditions of pH7.4 and pH5.8
Combination of the anti-C5 antibody (BNJ441, according to library pearl monoclonal antibody and 305 variants) to recombined human C5 at pH7.4 and pH5.8 Affinity is determined at 37 DEG C using BIACORE (registered trademark) T200 instrument (GE Healthcare), to assess pH to antigen knot The effect of conjunction.The setting recommended using amine coupling kit (GE Healthcare) according to manufacturer, by goat anti-human IgG (Fc) polyclonal antibody (KPL#01-10-20) is fixed to CM4 sensor core on piece.Antibody and analyte are diluted in containing 20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05%Tween 20 and 0.005%NaN3ACES pH7.4 buffer or In ACES pH5.8 buffer.Using anti-Fc method by antibody capture on the sensor surface, capture horizontal is typically 50- 80 response units (RU).It is begun through from 27nM (for pH7.4 determination condition) or 135nM (for 5.8 determination condition of pH) Three times serial dilution preparation and reorganization people C5.Surface 20mM HCl, 0.01%Tween 20 are regenerated.It uses 2.0 software of BiaEvaluation (GE Healthcare) is fitted by data processing and with 1: 1 binding model.
BNJ441 at pH7.4 and pH5.8, according to library pearl monoclonal antibody and 305 variants to the binding affinity of recombined human C5 (KD) it is shown in table 10.The ratio between (in the KD of pH 5.8) that 305 variants are shown/(in KD of pH 7.4) is almost 800, is aobvious Show (in the KD of pH 5.8)/the ratio between (in the KD of pH 7.4) be only 93 BNJ441 8 times of height.
[table 10]
Embodiment 9
The inhibitory activity that anti-C5 antibody (305 variant) activates C5
9.1. anti-C5MAb inhibits the liposome dissolving of complement activation
The inhibition of the complement activity of anti-C5MAb is tested by liposome dissolving measurement.By the normal human serum of 30 μ L (6.7%) (Biopredic, SER019) is mixed in 96- orifice plate with the diluted MAb of 20 μ L and is incubated on shaking table in room temperature 30min.The liposome solutions being sensitized with the antibody (Autokit CH50, Wako, 995-40801) for dinitrophenyl are turned It moves on in each hole and places 2min on shaking table at 37 DEG C.The substrate solution (Autokit CH50) of 50 μ L is added to each hole simultaneously 2min is mixed in 37 DEG C of oscillations.Final mixture is incubated for 40 minutes at 37 DEG C, measures the OD in 340nm later.Liposome The percentage of dissolution is defined as 100x [(ODMAb-ODSerum and liposome background)]/[(ODWithout MAb-ODSerum and liposome background)].Figure 15 shows anti- C5 Mab:305LO15-SG422,305LO16-SG422,305LO18-SG422,305LO19-SG422,305LO20-SG422 And 305LO20-SG115, inhibit liposome dissolving.Two kinds of antibody with Fc variant: 305LO15-SG115 and 305LO23- SG429 also shows and inhibits liposome dissolving (Figure 16).
Test the inhibition of the recombined human C5 (SEQ ID NO:39) of anti-C5MAb.By the human serum for lacking C5 of 10 μ L (Sigma, C1163) is mixed and in shaking table with the recombinant C 5 (0.1 μ g/mL) of the diluted MAb of 20 μ L and 20 μ L in 96- orifice plate On 37 DEG C be incubated for 1 hour.Liposome (Autokit CH50) is transferred in each hole and places 2min on shaking table at 37 DEG C. The substrate solution (Autokit CH50) of 50 μ L is added to each hole and mixes 2min in 37 DEG C of oscillations.Final mixture is existed 37 DEG C are incubated for 180 minutes, measure the OD in 340nm later.The percentage of liposome dissolving is defined above.Figure 17 is shown Anti- C5 Mab:305LO22-SG115,305LO22-SG422,305LO23-SG115 and 305LO23-SG422 inhibit liposome Dissolution.
9.2. anti-C5MAb inhibits C5a to generate
The C5a during liposome dissolving for testing anti-C5MAb is generated to confirm that anti-C5 MAb inhibits C5 to be cut into C5a and C5b.Quantify to measure from liposome dissolving using C5a ELISA kit (R&D systems, DY2037) upper C5a in clear is horizontal.All MAb inhibit the C5a in supernatant to generate (Figure 18 and 19) in a manner of dose-dependent.
9.3. the complement activity in machin blood plasma is measured
The inhibition of the complement activity of anti-C5MAb is tested in machin blood plasma.Monkey will be applied in anti-C5 MAb (20mg/kg), and periodically plasma sample is collected until the 56th day.By chicken red blood cell (cRBC) (Innovative Research, IC05-0810) use MgCl containing 0.5mM2With 0.15mM CaCl2(GVB++) gelatin/barbital buffering salt water (Boston BioProducts, IBB-300X) washing, later with anti-chicken RBC antibody (the Rockland 103- of 1 μ g/ml 4139) it is sensitized 15 minutes at 4 DEG C.Then cell washed with GVB++ and with 1x108A cell/ml is suspended in identical buffering In liquid.In individual round bottom 96- hole micrometering test plate (panel), monkey blood plasma and the cRBC of sensitization are incubated for 20 minutes at 37 DEG C.It is being incubated for Afterwards, plate is centrifuged 2 minutes with 1000xg at 4 DEG C.Supernatant is transferred in the hole on the flat hole 96- micrometering test plate (panel) to be used to survey Measure the OD, reference wavelength 630nm in 415nm.Percent hemolysis is defined as 100x [(ODAfter application-ODBlood plasma and cRBC background)]/ [(ODBefore application-ODBlood plasma and cRBC background)].Figure 20 shows anti-C5 Mab:305LO15-SG422,305LO15-SG115,305LO16- SG422,305LO18-SG422,305LO19-SG422,305LO20-SG422,305LO20-SG115 and 305LO23-SG115, Inhibit the complement activity in blood plasma.
9.4. anti-C5 MAb inhibits the biological activity of C5 variant
The recombined human C5 variant of anti-C5 MAb: V145I, R449G, V802I, R885H, R928Q, D966Y is tested, The inhibition of S1310N and E1437D.It is reported that with the R885H PNH patient's display difference being mutated to according to library pearl monoclonal antibody in C5 Reaction (see for example, Nishimura etc., New Engl.J.Med.370:632-639 (2014)).By each individual's C5 variant table Next research is used for up in FS293 cell, and by supernatant.By the human serum (Sigma, C1163) for lacking C5 of 10 μ L It is mixed simultaneously in 96- orifice plate with the cell culture medium containing 5 variant of recombinant C (2-3 μ g/mL) of the diluted MAb of 20 μ L and 20 μ L It is incubated for 0.5 hour on shaking table at 37 DEG C.Liposome (Autokit CH50) is transferred in each hole and at 37 DEG C on shaking table Place 2min.The substrate solution (Autokit CH50) of 50 μ L is added to each hole and mixes 2min in 37 DEG C of oscillations.It will be final Mixture 37 DEG C be incubated for 90 minutes, measure the OD in 340nm later.The percentage of liposome dissolving is as defined above.Figure The 21 anti-C5 MAb (according to library pearl monoclonal antibody) of display do not inhibit R885H C5 variant, but inhibit other variants of test.Figure 22 is shown Anti- C5 MAb (305 variant) inhibits the C5 variant of all tests.
9.5. anti-C5 MAb inhibits the liposome dissolving of complement activation
The inhibition of the complement activity of anti-C5MAb is tested by liposome dissolving measurement.By the normal human serum of 30 μ L (6.7%) (Biopredic, SER019) is mixed in 96- orifice plate with the diluted MAb of 20 μ L and is incubated on shaking table in room temperature 30min.The liposome solutions being sensitized with the antibody (Autokit CH50, Wako, 995-40801) for dinitrophenyl are turned It moves on in each hole and places 2min on shaking table at 25 DEG C.The substrate solution (Autokit CH50) of 50 μ L is added in each hole And 2min is mixed in 25 DEG C of oscillations.Final mixture is incubated for 45 minutes at 37 DEG C, measures the OD in 340nm later.Lipid The percentage inhibition of body dissolution is defined as 100x [(ODMAb-ODSerum and liposome background)]/[(ODWithout MAb-ODSerum and liposome background)].Figure 23 is aobvious Show anti-C5MAb, BNJ441 and 305 variants inhibit liposome dissolving, and the inhibitory activity of 305 variants is better than BNJ441.
Embodiment 10
Pharmacokinetic of the anti-C5 monoclonal antibody (305 variant) in machin
10.1. the internal test of machin is used
Anti-Human C5 is applied in machin (Shin Nippon Biomedical Laboratories, Ltd., Japan) After antibody, the In vivo kinetics of Anti-Human C5 antibody are assessed.It was transfused by 30 minutes, by the solution (2.5mg/ of Anti-Human's C5 antibody Ml it) is administered in the cephalic vein of forearm with the single dose of about 8ml/kg.Before administration and application after 5 minutes, 7 hours, 1 It, 2 days, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days collection blood.By the blood of collection immediately with 1,700xg is centrifuged 10 minutes at 4 DEG C with separated plasma.Isolated blood plasma is stored in -70 DEG C or less refrigerators until measuring.Such as Preparation Anti-Human C5 antibody described in embodiment 7.
10.2. the measurement total plasma concentration of machin C5 is measured by ELISA
The total concentration of machin C5 in machin blood plasma is measured by ELISA.By Anti-Human C5 antibody (using in embodiment 2 The internal antibodies that the method for description generates) it is assigned to Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International it is stood overnight on) and at 4 DEG C, to prepare the plate for being fixed with anti-machin C5.Prepare calibration curve sample The machin plasma sample of product and 20000 times of the injection of antibodies dilution with 0.4 μ g/ml, and it is incubated for 60 minutes at 37 DEG C. Then, sample is assigned on the plate for being fixed with anti-machin C5, and is being stored at room temperature it 1 hour.Then, HRP is added Anti-Human's IgG antibody (SouthernBiotech) of label is washed with reacting at room temperature 30 minutes.Then, ABTS is added ELISA HRP substrate (KPL).By microplate reader under the wavelength of 405nm measuring signal.Response based on calibration curve uses It analyzes software SOFTmax PRO (Molecular Devices) and calculates machin C5 concentration.It is measured by this method intravenous The time-histories of blood plasma machin C5 concentration is shown in Figure 24 after application.Data are plotted as and apply preceding blood plasma machin C5 concentration phase The remaining percentage of ratio.Compared with non-TCP friendly flow Anti-Human's C5 antibody, pH dependence Anti-Human C5 antibody (305LO15-SG422, 305LO15-SG115、305LO16-SG422、305LO18-SG422、305LO19-SG422、305LO20-SG422、 305LO20-SG115,305LO22-SG422,305LO23-SG422 and 305LO23-SG115) show the lower product of plasma C 5 It is poly-.
10.3. measurement Anti-Human C5 antibody plasma concentration is measured by ELISA
The concentration of Anti-Human's C5 antibody in machin blood plasma is measured by ELISA.By Anti-Human's IgG κ chain antibody (Antibody Solutions) is assigned to Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) It above and at 4 DEG C stands overnight, to prepare the plate for being fixed with Anti-Human IgG.Prepare 100 times of calibration curve sample and dilution or more Machin plasma sample.Then, sample is assigned on the plate for being fixed with Anti-Human IgG, and makes it to be stored at room temperature 1 small When.Then, Anti-Human's IgG antibody (SouthernBiotech) of addition HRP label is to react at room temperature 30 minutes, and washes It washs.Then, ABTS ELISA HRP substrate (KPL) is added.By microplate reader under the wavelength of 405nm measuring signal.Based on school The response of positive curve calculates Anti-Human C5 antibody concentration using analysis software SOFTmax PRO (Molecular Devices).It is logical The time-histories for crossing blood plasma Anti-Human's C5 antibody concentration after the intravenous application of this method measurement is shown in Figure 25.With non-TCP friendly flow Anti-Human's C5 antibody is compared, pH dependence Anti-Human's C5 antibody (305LO15-SG422,305LO15-SG115,305LO16- SG422、305LO18-SG422、305LO19-SG422、305LO20-SG422、305LO20-SG115、305LO22-SG422、 305LO23-SG422 and 305LO23-SG115) show longer half-life period.
Embodiment 11
The X ray analysis of crystal structure of 305 variant Fab and people's C5-MG1 domain complex
11.1. the expression and purifying of the MG1 structural domain (20-124) of people C5
Using pGEX-4T-1 carrier (GE healthcare), will melt via the connector of blood coagulation enzyme cleavable and GST- label The MG1 structural domain (the amino acid residue 20-124 of SEQ ID NO:39) (GST-MG1) of conjunction is in Escherichia coli strain Expression in BL21DE3pLysS (Promega).It is lured at 25 DEG C with 0.1mM isopropyl ss-D-1- Thiogalactopyranoside (IPTG) Protein expression is led up to 5 hours.Bacterium cell pellet is mixed with lysonase (Merck) and adequate proteins enzyme inhibitor is supplemented with The Bugbuster (Merck) of object (Roche) is cracked, and uses GSTrap column (GE healthcare) saying according to the producer later The bright purifying carried out from solvable fraction to GST-MG1.GST label is cut off with fibrin ferment (Sigma), and resulting MG1 is tied Structure domain is further purified with 75 solvent resistant column of Superdex (GE healthcare).The fraction of the structural domain containing MG1 is collected And it is stored in -80 DEG C.
11.2. the Fab segment of 305 variants is prepared
Fab segment from 305 one of the variant of optimization is prepared by a conventional method, using utilizing papain The limited digestion of (Roche Diagnostics, catalog number (Cat.No.) 1047825), be loaded into later albumin A column (MabSlect SuRe, GE Healthcare) on to remove Fc segment, and be loaded into cation exchange column (HiTrap SP HP, GE Healthcare) and on solvent resistant column (Superdex200 16/60, GE Healthcare).By the fraction of the segment containing Fab Collect and is stored in -80 DEG C.
11.3. 305 variant Fab and people's C5-MG1 domain complex are prepared
The recombined human C5-MG1 structural domain of purifying is mixed with 305 variant Fab segments of purifying with 1:1 molar ratio.It will answer It closes object and passes through gel filtration chromatography (Superdex200 10/300 increases, GE Healthcare) purifying, use 25mM The column of HEPES pH 7.5,100mM NaCl balance.
11.4. it crystallizes
The compound of purifying is concentrated into about 10mg/mL, and passes through sitting drop vapor phase grafting (drop vapor at 4 DEG C Diffusion method) and kind of a crystallization is combined to be crystallized.Mother liquor removes water magnesium formate, 15.0%w/v polyethylene glycol by 0.2M 3350 are constituted.This successfully generates flat crystal in several days.Crystal is immersed in 0.2M and removes water magnesium formate, 25.0%w/v is poly- In the solution of ethylene glycol 3350 and 20% glycerol.
11.5. data collection and structure determination
X ray diffracting data is measured in SPring-8 by BL32XU.During measurement, crystal is placed in always -178 DEG C To keep freezing state in nitrogen stream, and collected always using the MX-225HS CCD detector (RAYONIX) being connected with bunch 180 X-ray diffraction images are counted, while once by 1.0 degree of Crystal Rotation.Using Xia2 program (J.Appl.Cryst.43: 186-190 (2010), XDS software package (Acta.Cryst.D66:125-132 (2010)) and Scala (Acta.Cryst.D62: 72-82 (2006)) carry out the determinations of cell parameters, the indexing of point diffraction and the diffraction data obtained from diffraction image plus Work, and resolution ratio is finally obtained up to 2.11 angstroms of diffracted intensity data.Crystallographic data statistics is shown in table 11.
[table 11]
X-ray data is collected and refine statistics
a；Rmerge=∑ hk/ ∑ j | Ij (hkl)-< I (hkl)>|/∑ hk/ Σ j | Ij (hkl) |, wherein Ij (hkl) and<I (hkl) > it is the intensity of measurement j and the mean intensity of the reflection with index hkl respectively.
b；The R factor=∑ hkl | Fcacl(hkl)|-|Fobs(hkl)|/∑hkl|Fobs(hkl) |, wherein FobsAnd FcalcRespectively It is the mechanism factor amplitude of observation and calculating.
c；RfreeIt is calculated using 5% reflection of random picking.
Structure is determined using program Phaser (J.Appl.Cryst.40:658-674 (2007)) by molecular replacement.Fab The human IgG 4Fab crystal structure (PDB coding: 1BBJ) of structural domain searched model and derive from announcement, and the lookup of MG1 structural domain People C5 crystal structure (PDB coding: 3CU7, Nat.Immunol.9:753-760 (2008)) of the model from announcement.Model utilizes Coot program (Acta Cryst.D66:486-501 (2010)) build and using program Refmac5 (Acta Cryst.D67: 355-367 (2011)) carry out refine.The crystallography reliability factor (R) of 25-2.11 angstroms of diffracted intensity data is 20.42%, and Free R value is 26.44%.Structure refinement statistics is shown in table 11.
11.6.305 the overall structure of variant Fab and C5-MG1 domain complex
From 305 optimization variant Fab segment (" 305Fab ") with 1: 1 ratio combination people C5-MG1 structural domain (" MG1 "), and the asymmetric unit of crystal structure includes two compounds, molecule 1 and 2, as shown in Figure 26 A.Using all Molecule 1 and 2 can be aligned by the C alpha atom position in residue well with 0.717 angstrom of RMSD, as shown in Figure 26 B.It begs for below The figure of opinion is prepared using molecule 1.
In Figure 27 A and 27B, the epitope of the contact zone 305Fab is respectively in MG1 amino acid sequence and in crystal structure Mapping.The epitope includes containing one or more be located in 4.5 angstroms of distances of arbitrary portion away from 305Fab in crystal structure The amino acid residue of the MG1 of a atom.In addition, the epitope in 3.0 angstroms is highlighted in Figure 27 A.
11.7.E48 the interaction of D51 and K109
As described in embodiment 4.5 and 4.6, pass through western blot and BIACORE (registered trademark) binding analysis, test bag Include the combination of the anti-C5MAb and three kinds of people's C5 point mutation bodies E48A, D51A and K109A of 305 antibody series.Although 305 variants are strong Strong combination WT C5, but they only weaker combine E48A C5 mutant and do not combine D51A and K109A mutant. The crystal structure of 305Fab and MG1 compound discloses three amino acid E48, D51 and K109 all away from 3.0 angstroms of 305Fab In distance, several hydrogen bonds are formed with Fab, as depicted in fig. 28 a.According to more detailed research, the K109 residue of MG1 is embedded in In the ditch formed in the interface of the heavy chain of Fab and pass through three with H-CDR3_G97, H-CDR3_Y100 and H-CDR3_T100b A hydrogen bond and (Figure 28 D) is closely interacted with Fab with the salt bridge of H-CDR3_D95.D51 is located at MG1's and 305Fab Two hydrogen bonds are formed between heavy chain and with H-CDR1_Ser32 and H-CDR2_Ser54 to filling space (Figure 28 C).These Instruction, the K109 and D51 of C5 are crucial residues for the combination of 305 antibody series.On the other hand, E48 is positioned next to Surface and only with Fab formed a hydrogen bond, show its to antibody combine contribution be less than K109 and E51 contribution (Figure 28 B). The western blot of these relationships and people C5 mutant and BIACORE (registered trademark) binding analysis result (embodiment 4.5 and 4.6) consistent.Supplement prompt: the residue numbering of Fab amino acid is based on Kabat numbering plan.(Kabat etc., Sequences Of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md., 1991)
11.8. the interaction of H70, H72 and the H110 of people C5 and 305 antibody series
Crystal structure analysis discloses, three on people C5 histidine residues, that is, H70, H72 and H110 are comprised in In the epitope of 305 variant Fab, as shown in Figure 27 A and Figure 29 A.BIACORE (registered trademark) binding analysis is carried out to study this A little histidine residues are to pH dependence protein-protein-interacting contribution between people C5 and 305 variant Fab, wherein using People C5 mutant H70Y, H72Y, H110Y and H70Y+H110Y (embodiment 4.7).H72Y causes 305 variant Fab in conjunction with C5 It completely loses.The C5 residue is located in the pocket formed by the CDR2 ring of 305 Fab heavy chains and the ring (L73, S74 and E76) of MG1 And the space is tightly packed, as shown in Figure 29 C.In addition, the H72 residue of C5 and H-CDR2_Y58 form hydrogen bond.It is expected that H72Y mutation will not be tolerated, because of the side chain that the volume for accommodating tyrosine without enough spaces is bigger.With H-CDR2_ The hydrogen bond of Y58 also cannot keep.Contribution about H70 and H110 to pH dependence, H70Y and H110Y mutation cause in pH 5.8 The dissociation of lower 305 variant Fab from C5 is slower.The T53 of H70 and MG1 forms intramolecular hydrogen bond, it is believed that works as the H70 of C5 in pH 5.8 Protonation when causing conformational change in the corresponding portion of the interaction interface of MG1, the hydrogen bond is destroyed (Figure 29 B). For H110, the protonation of the C5 residue is expected will to cause electrical charge rejection to 305Fab, adjacent histidine residues H-CDR3_ The repulsion (Figure 29 D) can be enhanced in the protonation of H100c.
Although in order to be clearly understood that by illustrating and embodiment describes some details of foregoing invention, specification and Embodiment is not construed as limiting the scope of the invention.The disclosure of all patents referred to herein and scientific literature passes through Reference completely clearly combines.

Claims (29)

1. combining the antibody of C5, it is used to treat the disease or illness of complement-mediated, the disease or illness are related to the excessive of C5 Or uncontrolled activation, wherein the antibody is under neutral ph than the β chain of higher affinity combination C5 at acidic Interior epitope.
2. combining the antibody of C5, it is used to enhance removing of the C5 from blood plasma, wherein the antibody is under neutral ph than in acidity Epitope under pH in the β chain of higher affinity combination C5.
3. combine the antibody of C5 in preparation for treating the purposes in the disease of complement-mediated or the drug of illness, the disease or Illness is related to the excessive or uncontrolled activation of C5, wherein the antibody is under neutral ph than higher parent at acidic With the epitope in the β chain of power combination C5.
4. combine the antibody of C5 in preparation for enhancing C5 from the purposes in the drug of the removing of blood plasma, wherein the antibody with Than the epitope in the β chain of higher affinity combination C5 at acidic under neutral pH.
5. the method for the individual of disease or illness of the treatment with complement-mediated, the disease or illness are related to the excessive of C5 or not Controlled activation, the method includes applying the antibody of a effective amount of combination C5 to the individual, wherein the antibody with Than the epitope in the β chain of higher affinity combination C5 at acidic under neutral pH.
The method of removing of the C5 from blood plasma during 6. enhancing is individual comprising apply the antibody of a effective amount of combination C5 to the individual To enhance removing of the C5 from blood plasma, wherein the antibody is under neutral ph than higher affinity combination C5's at acidic Epitope in β chain.
7. the antibody used of any one of claims 1 to 6, purposes or method, wherein the antibody has selected from by following The feature of the group of composition:
(a) antibody is contacted with the amino acid D51 and K109 of C5 (SEQ ID NO:39)；
(b) antibody is greater than the antibody to the affinity of C5 (SEQ ID No:39) and sets to the E48A by SEQ ID NO:39 Change the affinity of the C5 mutant of composition；With
(c) antibody combines the C5 albumen that forms of amino acid sequence by SEQ ID NO:40 in pH7.4, but pH7.4 not The C5 albumen formed in conjunction with the amino acid sequence by the SEQ ID NO:39 replaced with H72Y.
8. the antibody used of any one of claims 1 to 6, purposes or method, wherein the antibody with comprising selected from following VH and VL pairing antibody competition combination C5:
(a) VL of the VH and SEQ ID NO:11 of SEQ ID NO:1；
(b) VL of the VH and SEQ ID NO:15 of SEQ ID NO:5；
(c) VL of the VH and SEQ ID NO:14 of SEQ ID NO:4；
(d) VL of the VH and SEQ ID NO:16 of SEQ ID NO:6；
(e) VL of the VH and SEQ ID NO:12 of SEQ ID NO:2；
(f) VL of the VH and SEQ ID NO:13 of SEQ ID NO:3；
(g) VL of the VH and SEQ ID NO:19 of SEQ ID NO:9；
(h) VL of the VH and SEQ ID NO:17 of SEQ ID NO:7；
(i) VL of the VH and SEQ ID NO:18 of SEQ ID NO:8；With
(j) VL of the VH and SEQ ID NO:20 of SEQ ID NO:10.
9. the antibody used of any one of claims 1 to 8, purposes or method, wherein the β chain of the antibody combination C5 Epitope in MG1-MG2 structural domain.
10. the antibody used of any one of claims 1 to 9, purposes or method, wherein the antibody combines the β chain by C5 Epitope in the segment of the amino acid 33-124 composition of (SEQ ID NO:40).
11. the antibody used of any one of claims 1 to 10, purposes or method, wherein the β chain of the antibody combination C5 Epitope in (SEQ ID NO:40), the epitope include in the group for selecting free amino acid 47-57,70-76 and 107-110 to form At least one segment.
12. the antibody used of any one of claims 1 to 11, purposes or method, wherein the β chain of the antibody combination C5 Epitope in the segment of (SEQ ID NO:40), the epitope include selected from by Glu48, Asp51, His70, His72, Lys109 With at least one amino acid in the group of His110 composition.
13. the antibody used of any one of claims 1 to 12, purposes or method, wherein in the antibody and table 2,7 and 8 The antibody combines identical epitope.
14. the antibody used of any one of claims 1 to 13, purposes or method, wherein the antibody inhibits the activation of C5.
15. the antibody used of any one of claims 1 to 14, purposes or method, wherein the antibody inhibits C5 variant The activation of R885H.
16. the antibody used of any one of claims 1 to 15, purposes or method, wherein the antibody is monoclonal antibody.
17. the antibody used of any one of claims 1 to 16, purposes or method, wherein the antibody is human antibody, source of people Change antibody or chimeric antibody.
18. the antibody used of any one of claims 1 to 17, purposes or method, wherein the antibody is in conjunction with C5 Antibody fragment.
19. the antibody used of any one of claims 1 to 18, purposes or method, wherein the antibody includes (a) HVR- H3, it includes amino acid sequence DX1GYX2X3PTHAMX4X5, wherein X1It is G or A, X2It is V, Q or D, X3It is T or Y, X4It is Y or H, X5It is L or Y (SEQ ID NO:128), (b) HVR-L3, it includes amino acid sequence QX1TX2VGSSYGNX3, wherein X1It is S, C, N Or T, X2It is F or K, X3It is A, T or H (SEQ ID NO:131), and (c) HVR-H2, it includes amino acid sequences X1IX2TGSGAX3YX4AX5WX6KG, wherein X1It is C, A or G, X2It is Y or F, X3It is T, D or E, X4It is Y, K or Q, X5It is S, D or E, X6It is A or V (SEQ ID NO:127).
20. the antibody used of any one of claims 1 to 18, purposes or method, wherein the antibody includes (a) HVR- H1, it includes amino acid sequence SSYYX1X2, wherein X1It is M or V, X2It is C or A (SEQ ID NO:126), (b) HVR-H2, Include amino acid sequence X1IX2TGSGAX3YX4AX5WX6KG, wherein X1It is C, A or G, X2It is Y or F, X3It is T, D or E, X4It is Y, K Or Q, X5It is S, D or E, X6It is A or V (SEQ ID NO:127), and (c) HVR-H3, it includes amino acid sequences DX1GYX2X3PTHAMX4X5, wherein X1It is G or A, X2It is V, Q or D, X3It is T or Y, X4It is Y or H, X5Be L or Y (SEQ ID NO: 128)。
21. the antibody of claim 20 used, purposes or method, wherein the antibody further includes (a) HVR-L1, packet X containing amino acid sequence1ASQX2IX3SX4LA, wherein X1It is Q or R, X2It is N, Q or G, X3It is G or S, X4It is D, K or S (SEQ ID NO:129)；(b) HVR-L2, it includes amino acid sequence GASX1X2X3S, wherein X1It is K, E or T, X2 is L or T, X3It is A, H, E Or Q (SEQ ID NO:130)；(c) HVR-L3, it includes amino acid sequence QX1TX2VGSSYGNX3, wherein X1S, C, N or T, X2It is F or K, X3It is A, T or H (SEQ ID NO:131).
22. the antibody used of any one of claims 1 to 18, purposes or method, wherein the antibody includes (a) HVR- L1, it includes amino acid sequence X1ASQX2IX3SX4LA, wherein X1It is Q or R, X2It is N, Q or G, X3It is G or S, X4It is D, K or S (SEQ ID NO:129)；(b) HVR-L2, it includes amino acid sequence GASX1X2X3S, wherein X1It is K, E or T, X2It is L or T, X3 It is A, H, E or Q (SEQ ID NO:130)；(c) HVR-L3, it includes amino acid sequence QX1TX2VGSSYGNX3, wherein X1It is S, C, N or T, X2It is F or K, X3It is A, T or H (SEQ ID NO:131).
23. the antibody of claim 20 used, purposes or method, wherein the antibody further includes: weight chain variable structure Domain frame FR1, it includes the amino acid sequences of any of SEQ ID NO:132-134；Heavy-chain variable domains frame FR2, It includes the amino acid sequences of any of SEQ ID NO:135-136；Heavy-chain variable domains frame FR3, it includes SEQ The amino acid sequence of any of ID NO:137-139；With heavy-chain variable domains frame FR4, it includes SEQ ID NO: The amino acid sequence of any of 140-141.
24. the antibody of claim 22 used, purposes or method, wherein the antibody further includes: light chain variable domain Domain frame FR1, it includes the amino acid sequences of any of SEQ ID NO:142-143；Light variable domains frame FR2, It includes the amino acid sequences of any of SEQ ID NO:144-145；Light variable domains frame FR3, it includes SEQ The amino acid sequence of any of ID NO:146-147；With light variable domains frame FR4, it includes SEQ ID NO:148 Amino acid sequence.
25. the antibody used of any one of claims 1 to 18, purposes or method, wherein the antibody includes: (a) and SEQ The amino acid sequence of ID NO:10, any of 106-110 have the VH sequence of at least 95% sequence identity；(b) and SEQ The amino acid sequence of ID NO:20, any of 111-113 have the VL sequence of at least 95% sequence identity；Or (c) basis (a) VH sequence in and according to the VL sequence in (b).
26. the antibody of claim 25 used, purposes or method, wherein the antibody includes SEQ ID NO:10,106-110 Any of VH sequence.
27. the antibody of claim 25 used, purposes or method, wherein the antibody includes SEQ ID NO:20,111-113 Any of VL sequence.
28. the antibody of claim 26 or 27 used, purposes or method, wherein the antibody includes SEQ ID NO:10, The VH sequence of any of 106-110 and the VL sequence of SEQ ID NO:20, any of 111-113.
29. the antibody of any one of claim 1 to 28 used, purposes or method, wherein the antibody be overall length IgG1 or IgG4 antibody.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021218953A1 (en) * 2020-04-28 2021-11-04 上海普铭生物科技有限公司 Antibody against human complement protein (c5) and application thereof

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104761637B (en) 2006-03-31 2021-10-15 中外制药株式会社 Method for regulating antibody hemodynamics
EP3127921A1 (en) 2007-09-26 2017-02-08 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substition in cdr
TWI564021B (en) 2008-04-11 2017-01-01 Chugai Pharmaceutical Co Ltd Repeated binding of antigen to antigen binding molecules
TWI761912B (en) 2010-11-30 2022-04-21 日商中外製藥股份有限公司 Antibody having calcium-dependent antigen-binding ability
TW201809008A (en) 2014-12-19 2018-03-16 日商中外製藥股份有限公司 Anti-C5 antibodies and methods of use
IL309975A (en) 2016-06-14 2024-03-01 Regeneron Pharma Anti-c5 antibodies and uses thereof
TWI789369B (en) * 2016-06-17 2023-01-11 日商中外製藥股份有限公司 Anti-c5 antibodies and methods of use
WO2018025982A1 (en) 2016-08-05 2018-02-08 中外製薬株式会社 Composition for prophylaxis or treatment of il-8 related diseases
EP3692064A1 (en) * 2017-10-04 2020-08-12 Alexion Pharmaceuticals, Inc. Dosage and administration of anti-c5 antibodies for treatment of patients with membranoproliferative glomerulonephritis
CA3083113A1 (en) 2017-12-13 2019-06-20 Regeneron Pharmaceuticals, Inc. Anti-c5 antibody combinations and uses thereof
SG11202101853PA (en) * 2018-09-06 2021-03-30 Univ Pennsylvania Humanized anti-c5 antibodies and uses thereof
CN114072174A (en) * 2019-04-24 2022-02-18 宾夕法尼亚大学理事会 Dual-function humanized anti-C5 antibody and factor H fusion protein and uses thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995029697A1 (en) * 1994-05-02 1995-11-09 Alexion Pharmaceuticals, Inc. Methods and compositions for the treatment of glomerulonephritis and other inflammatory diseases
WO2010015608A1 (en) * 2008-08-05 2010-02-11 Novartis Ag Compositions and methods for antibodies targeting complement protein c5
US20110111406A1 (en) * 2008-04-11 2011-05-12 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding to two or more antigen molecules repeatedly
WO2011111007A2 (en) * 2010-03-11 2011-09-15 Rinat Neuroscience Corporation ANTIBODIES WITH pH DEPENDENT ANTIGEN BINDING
WO2014160958A1 (en) * 2013-03-29 2014-10-02 Alexion Pharmaceuticals, Inc. Compositions and methods for increasing the serum half-life of a therapeutic agent targeting complement c5
WO2015134894A1 (en) * 2014-03-07 2015-09-11 Alexion Pharmaceuticals, Inc. Anti-c5 antibodies having improved pharmacokinetics
US20160068592A1 (en) * 2013-01-31 2016-03-10 Seoul National University R&Db Foundation C5 antibody and method for preventing and treating complement-related diseases
WO2016098356A1 (en) * 2014-12-19 2016-06-23 Chugai Seiyaku Kabushiki Kaisha Anti-c5 antibodies and methods of use

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0230985Y2 (en) 1985-07-08 1990-08-21
JP4643139B2 (en) 2001-06-12 2011-03-02 テレフオンアクチーボラゲット エル エム エリクソン（パブル） Synchronization in the UMTS Terrestrial Radio Access Network (UTRAN)
WO2003015819A1 (en) 2001-08-17 2003-02-27 Tanox, Inc. Complement pathway inhibitors binding to c5 and c5a without preventing formation of c5b
JP4333939B2 (en) 2001-08-27 2009-09-16 ロード・コーポレーション Trolley wire suspension system
DE10253331A1 (en) 2002-11-14 2004-06-03 Symrise Gmbh & Co. Kg Use of trans-pellitori as a flavoring
US7267849B2 (en) 2004-03-02 2007-09-11 Nitto Denko Corporation Compensator for liquid crystal display
KR20080069889A (en) 2007-03-20 2008-07-29 (주)화도 Pe pipe
EA200901211A1 (en) * 2007-03-22 2010-04-30 Новартис Аг ANTIGENS OF C5 PROTEIN AND THEIR APPLICATION
WO2008126921A1 (en) 2007-04-11 2008-10-23 Sekisui Chemical Co., Ltd. Method for production of crosslinked polyvinyl acetal resin, and crosslinked polyvinyl acetal resin
KR20100054403A (en) 2008-11-14 2010-05-25 삼성전자주식회사 Online composition apparatus and method for writing by multiple participants
TW201206466A (en) * 2010-03-11 2012-02-16 Rinat Neuroscience Corp Antibodies with pH dependent antigen binding
KR20110111007A (en) 2010-04-02 2011-10-10 이화여자대학교 산학협력단 Octahydro-binaphthol derivatives for l/d optical conversion and optical resolution
KR20110122011A (en) 2010-05-03 2011-11-09 황보철종 System for administrating combine store point and method therefor
AR092098A1 (en) * 2012-08-13 2015-03-25 Regeneron Pharma ANTI-PCSK9 ANTIBODIES WITH PHASE DEPENDENT UNION CHARACTERISTICS
SG11201610782YA (en) * 2015-12-18 2017-07-28 Chugai Pharmaceutical Co Ltd Anti-c5 antibodies and methods of use
TWI789369B (en) * 2016-06-17 2023-01-11 日商中外製藥股份有限公司 Anti-c5 antibodies and methods of use

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995029697A1 (en) * 1994-05-02 1995-11-09 Alexion Pharmaceuticals, Inc. Methods and compositions for the treatment of glomerulonephritis and other inflammatory diseases
US20110111406A1 (en) * 2008-04-11 2011-05-12 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding to two or more antigen molecules repeatedly
WO2010015608A1 (en) * 2008-08-05 2010-02-11 Novartis Ag Compositions and methods for antibodies targeting complement protein c5
WO2011111007A2 (en) * 2010-03-11 2011-09-15 Rinat Neuroscience Corporation ANTIBODIES WITH pH DEPENDENT ANTIGEN BINDING
US20160068592A1 (en) * 2013-01-31 2016-03-10 Seoul National University R&Db Foundation C5 antibody and method for preventing and treating complement-related diseases
WO2014160958A1 (en) * 2013-03-29 2014-10-02 Alexion Pharmaceuticals, Inc. Compositions and methods for increasing the serum half-life of a therapeutic agent targeting complement c5
WO2015134894A1 (en) * 2014-03-07 2015-09-11 Alexion Pharmaceuticals, Inc. Anti-c5 antibodies having improved pharmacokinetics
WO2016098356A1 (en) * 2014-12-19 2016-06-23 Chugai Seiyaku Kabushiki Kaisha Anti-c5 antibodies and methods of use

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021218953A1 (en) * 2020-04-28 2021-11-04 上海普铭生物科技有限公司 Antibody against human complement protein (c5) and application thereof

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