CN101248088A

CN101248088A - Anit-il-23 antibiodies

Info

Publication number: CN101248088A
Application number: CNA2006800310995A
Authority: CN
Inventors: C·B·白德尔; S·W·布赖特; C·D·迪金森; K·K·基克利; D·M·马奎斯; A·P·瓦塞罗特
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2005-08-25
Filing date: 2006-08-23
Publication date: 2008-08-20

Abstract

The present invention encompasses isolated antibodies, or antigen-binding portions thereof, that specifically bind to the p19 subunit of IL-23. These antibodies, or antigen-binding portions thereof, are high affinity, neutralizing antibodies useful for the treatment of autoimmune disease.

Description

Anti-il-23 antibodies

Invention field

The invention belongs to medical field, specifically belong to anti-interleukin-22 3 (IL-23) monoclonal antibody field.The present invention relates more specifically to be used for the treatment of the neutrality anti-il-23 monoclonal antibody of autoimmune disease.

Background of invention

It is believed that IL-23 is for the vital a kind of cytokine of the activation of inducing the required a large amount of inflammatory cells of chronic inflammatory diseases.IL-23 has the IL-12 of being different from but complementary function with it, and IL-12 is the heterodimer cytokine of 70KDa, is made up of covalently bound p40 and p35 subunit.IL-23 is made of the p40 subunit identical with IL-12, but this subunit and p19 subunit covalency pairing people " immunology summary " (Immunological Reviews) 202:96-105 such as (, 2004) Langrish.

In addition, IL-23 plays an important role by promoting activating T cell secretion pro-inflammatory cytokine IL-17 to involve in the memory/t cell response that causes a disease.There is increasing proof to show that high-caliber IL-17 and autoimmune disease, inflammatory disease are relevant, comprise rheumatoid arthritis, psoriatic and multiple sclerosis (people such as Aggarwal, J.Biol.Chem 278 (3): 1910-1914,2003).Therefore, the neutrality antibody of IL-23 will suppress secretion and the short scorching effect of IL-17, and the IL-17 of terminal is for the effect of inflammatory disease.

Though the existing in the past report of human IL-2's 3 antibody, high-affinity neutrality human IL-2 3 antibody of discerning the specificity epitope on the p19 subunit do not have open as yet.Undoubtedly, in the physics that need improve aspect the treatment autoimmune disease.

Correspondingly, need specificity to seal the high-affinity neutrality antibody of the short scorching effect of IL-17 thus in conjunction with the IL-23p19 subunit, it can be used as therapeutical agent.Why need the high-affinity antibody of specificity in conjunction with the IL-23p19 subunit, reason is that also described antibody can carry out subcutaneous administration but not intravenously is used to the patient.Also need suppress in the assay method with low IC at IL-23 ₅₀The value specificity has the anti-il-23 antibodies of minimum dose therapeutically effective in conjunction with the antibody of IL-23p19 subunit with generation.The present invention has satisfied these demands, and relevant advantage is provided, and therefore the useful therapeutics of autoimmune disease is provided.

Summary of the invention

One embodiment of the invention are antibody or its antigen-binding portion thereof, and its specificity is in conjunction with the IL-23p19 subunit, in and the IL-23 activity, K _DValue is lower than about 160pM, IC ₅₀Value is lower than about 20pM.

Another embodiment is antibody or its antigen-binding portion thereof, and it is in conjunction with the IL-23p19 subunit that is positioned at aminoacid sequence amino-acid residue 129-159 shown in the SEQ ID NO:60.

Another embodiment of the present invention is antibody or its antigen-binding portion thereof, comprise the variable region of heavy chain shown in the SEQ ID NO:52, variable region of light chain shown in the SEQ ID NO:57, the constant region of light chain shown in CH shown in the SEQ ID NO:62 and the SEQ ID NO:63.

Another embodiment of the present invention comprises antibody or its antigen-binding portion thereof, its specificity is in conjunction with the IL-23p19 subunit, the variable region of heavy chain that comprises contains the aminoacid sequence that is selected from SEQ ID NO:44-55, and variable region of light chain contains the aminoacid sequence that is selected from SEQ ID NO:56-59.

Another embodiment of the present invention is antibody or its antigen-binding portion thereof, its specificity is in conjunction with the IL-23p19 subunit, the variable region of light chain that comprises contains the aminoacid sequence shown in the SEQ ID NO:57, and variable region of heavy chain contains and is selected from SEQ ID NO:48,49 or 52 aminoacid sequence.

In another embodiment, the LCVR of anti-il-23 monoclonal antibody of the present invention contains 1,2 or 3 peptide that is selected from following group respectively at LCDR1, LCDR2 and LCDR3 place: have (a) SEQ IDNO:26-35; (b) SEQ ID NO:37-38 and (c) peptide of sequence shown in the SEQ ID NO:40 (that is, for the antibody that contains described 3 peptides, a peptide is selected from (a), and a peptide is selected from (b), and a peptide is selected from (c)).

In another embodiment, the HCVR of anti-il-23 monoclonal antibody of the present invention contains 1,2 or 3 peptide that is selected from following group respectively at HCDR1, HCDR2 and HCDR3 place: have (a) SEQ IDNO:1-4; (b) SEQ ID NO:5-11 and (c) peptide of sequence shown in the SEQ ID NO:13-21 (that is, for the antibody that contains described 3 peptides, a peptide is selected from (a), and a peptide is selected from (b), and a peptide is selected from (c)).

The present invention also provides the anti-il-23 monoclonal antibody, and it contains 6 peptides that are selected from following group respectively at LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 place: have (a) SEQID NO:26-35; (b) SEQ ID NO:37-38; (c) SEQ ID NO:40; (d) SEQ ID NO:1-4; (e) SEQ ID NO:5-11 and (f) peptide of sequence shown in the SEQ ID NO:13-21 (promptly from (a-f), respectively selecting a peptide).

The present invention also provides the anti-il-23 monoclonal antibody, contain 6 peptides: be respectively peptide at LCDR1, LCDR2 and LCDR3 place, and be respectively peptide with sequence shown in the SEQ ID NO:22,23 and 24 at HCDR1, HCDR2 and HCDR3 place with sequence shown in the SEQ ID NO:41,42,43.

Another embodiment of the present invention provides humanized anti-il-23 monoclonal antibody and antigen-binding portion thereof thereof, it is in conjunction with the specificity epitope that comprises IL-23p19 subunit amino-acid residue 129-159 (SEQ ID NO:60 residue 129-159), and the antagonism or the interior biological activity of at least a external or body with IL-23 or its part correlation that neutralizes.

In another embodiment, in as embodiment 1 described external mouse boosting cell assay method, the IC of antibody of the present invention ₅₀Value is less than or equal to about 100pM, 75pM, 50pM, 25pM, 20pM, 15pM, 13pM, 10pM, 8pM, 5pM, 2pM or 1pM.The IC of preferred antibody of the present invention ₅₀Value is less than or equal to about 20pM.

In another embodiment, antibody of the present invention is characterised in that the strong binding affinity (K to human IL-2 3p19 subunit _D), promptly be lower than about 160pM, 100pM, 75pM, 50pM or 25pM.The K of preferred antibody of the present invention _DValue is lower than about 100pM.And the feature of invention antibody also is from the dissociation rate (k of human IL-2 3p19 subunit _Off) be lower than 4 * 10 ^-4s ^-1

Another embodiment of the present invention comprises the isolated antibody of arbitrary above-mentioned embodiment, and wherein said antibody is full length antibody, complete antibody, chimeric antibody, Fab fragment, F (ab ') basically ₂Fragment or strand Fv fragment.Isolated antibody or its antigen-binding portion thereof of preferred arbitrary above-mentioned embodiment are humanized antibody.

The present invention includes isolating nucleic acid, the antibody that its polynucleotide encoding that comprises is described herein and claimed.The present invention also comprises with containing these polynucleotide and expressing the carrier transformed host cells of described herein and claimed antibody.

The present invention includes the method for treatment autoimmune disease, comprise to the experimenter and use the described herein and claimed antibody of significant quantity.Preferred autoimmune disease of being treated is a multiple sclerosis.

At last, the present invention includes antibody and the experimenter of needs is treated purposes in the medicine of autoimmune disease in preparation.

Detailed Description Of The Invention

The invention provides the aminoacid sequence of isolating people's antibody or its antigen-binding portion thereof, described antibody or its antigen-binding portion thereof specificity be in conjunction with the p19 subunit of IL-23, and can be used for treating autoimmune disease.

For the present invention is more readily understood, at first some term is defined.

Human IL-2 3 p19 subunit is 189 amino acid whose polypeptide, contains 21 amino acid whose leader sequences [SEQ ID NO:60].

As used herein, term " antibody " is intended to expression and comprises the immunoglobulin molecules that 4 polypeptide chains promptly pass through disulfide linkage two weight (H) chains connected to one another and two light (L) chains.Every heavy chain comprises variable region of heavy chain (being abbreviated as HCVR or VH in the literary composition) and CH (being abbreviated as HCCR or CH in the literary composition).CH comprises 3 structural domain: CH1, CH2 and CH3.Every light chain comprises variable region of light chain (being abbreviated as LCVR or VL in the literary composition) and constant region of light chain (being abbreviated as LCCR in the literary composition).Constant region of light chain comprises 1 domain C L.

HCVR and LCVR district can further be subdivided into the hypervariable region, are called complementary determining region (" CDR "), and the more conservative zone that interspersing therebetween is called framework region (" FR ").Each HCVR and LCVR comprise 3 CDR and 4 FR, arrange in the following order from the N-terminal to the C-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.3 of heavy chain CDR are called " HCDR1, HCDR2 and HCDR3 " in the literary composition, and 3 CDR of light chain are called " LCDR1, LCDR2 and LCDR3 ".CDR contains and the interactional most of residue of antigen formation specificity.The well-known Kabat numbering convention of the numbering of cdr amino acid residue and basis on location in HCVR and the LCVR district.

Light chain is divided into κ chain and λ chain.Heavy chain is divided into γ, μ, α, δ, ε, and antibody isotype is defined as IgG (subclass IgG respectively ₁, IgG ₂, IgG ₃And IgG ₄), IgM, IgA, IgD and IgE.In light chain and heavy chain inside, variable region and constant region link together by about 12 or more a plurality of amino acid whose " J " district, and wherein heavy chain also comprises about 3 or more a plurality of amino acid whose " D " district.

As used herein, " antigen-binding portion thereof " of term antibody is meant one or more antibody fragments that kept the ability of specificity conjugated antigen (as the p19 subunit of IL-23).Verified, the fragment of full length antibody can be carried out the antigen combined function of antibody.The example of the binding fragment that " antigen-binding portion thereof " of term antibody contained comprises (i) Fab fragment, promptly comprises the unit price fragment of VL, VH, CL and CH1 structural domain; (ii) F (ab ') ₂Fragment promptly comprises segmental pair of valency fragment of two Fab, and described Fab fragment is by the disulfide bridge connects of hinge area; The Fd fragment that (iii) comprises VH and CH1 structural domain; The Fv fragment that (iv) comprises the VL and the VH structural domain of single antibody arm; (v) dAb fragment, it comprises the VH structural domain; (vi) independent complementary determining region (CDR).In addition, although segmental two structural domains of Fv are VL with VH is by different genes encodings, they can utilize recombination method to link together by synthetic linker, make them be prepared as the wall scroll protein chain, and wherein the pairing of VL and VH district forms monovalent molecule (being called strand Fv (scFv)).Such single-chain antibody also is intended to be included in " antigen-binding portion thereof " of term antibody.Also comprise other forms of single-chain antibody such as binary antibody (diabody).Binary antibody is two valency bi-specific antibodies, wherein VH and VL structural domain are expressed on the wall scroll polypeptide chain, but the joint that uses is too short, match thereby can't make between these two structural domains on the same chain, force the complementary structure territory pairing on described structural domain and other chains thus, and produce two antigen binding sites.

The CDR in the antigen binding domain territory of antibody of the present invention is the mouse source fully or basically, randomly changes some amino-acid residue, for example, replaces (referring to for example table 1 and 2) with different amino-acid residues, to optimize the specified property of antibody, as K _D, k _Off, IC ₅₀In other embodiments, the antigen binding domain territory of IL-23 antibody of the present invention can derive from other inhuman species, includes but not limited to: rabbit, rat or hamster.Alternatively, the antigen binding domain territory can derive from the human sequence.

Term " monoclonal antibody " is not limited to the antibody by hybridoma technology production as used herein." monoclonal antibody " is meant the antibody that derives from single copy or clone, comprises for example eucaryon, protokaryon or phage clone, rather than refers to its production method." monoclonal antibody " can be complete antibody (comprising all or total length Fc district), basically complete antibody, the part or the fragment that perhaps comprise the antibody of antigen-binding portion thereof, for example Fab fragment, Fab ' fragment or the F of mouse antibodies or chimeric, humanization or people's antibody (ab ') ₂Fragment.

Aminoacid sequence or retracing sequence that term " humanized antibody " expression antibody moiety or whole origin come from people's antibody reproductive tract constitute, and are prepared by the sequence that changes non-human antibody's (preferred mouse monoclonal antibody) complementary determining region (CDR).The framework region of variable region is by corresponding people's framework region (or its sizable part or major part, promptly at least about 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%, according to the Kabat numbering) replace, and by the nucleic acid sequence information or its reorganization or the mutant form coding that are present in people's reproductive tract immunoglobulin (Ig) zone, and whether in people's cell, produce regardless of described antibody.

The CDR of humanized antibody can change from the CDR of its inhuman parental antibody of originating or optimize, to produce the performance of expectation, as specificity, avidity and/or preferential combination.Compare with parent CDR, can have aminoacid replacement, interpolation and/or disappearance through the CDR that changes or optimize, preferably in six CDR structural domains, add up to about 1,2,3,4,5,6,7,8,9 or 10 such aminoacid replacement, interpolation and/or disappearance.For example, the cdr amino acid position among the SEQ ID NO:44-59 is through altered position by CDR shown in the Mab3A8.Alternatively, mouse antibodies Mab3A8 can be the parental antibody that is used for comparison antibody CDR of the present invention.Such as discussed herein, the antibody with regard to humanized antibody is not limited to full length antibody, and can comprise fragment and single stranded form.

As used herein, term " recombinant human antibody " is intended to comprise by recombinant means preparation, expression, establishment or isolating everyone antibody, the antibody that for example utilizes transfection to express to the recombinant expression vector of host cell, by the combination people antibody library isolated antibody of reorganization, by the genetically modified animal of human immunoglobulin gene (as mouse) isolated antibody; Perhaps by comprising with any other means preparation, expression, establishment or the isolated antibody of human immunoglobulin gene's montage to other dna sequence dnas.Such recombinant human antibody has variable region and the constant region that derives from people's reproductive tract immunoglobulin sequences.But, in certain embodiments, such recombinant human antibody is carried out vitro mutagenesis, thereby the aminoacid sequence in described recombinant antibodies VH and VL district is such sequence, though it derives from people's reproductive tract VH and VL sequence and relevant with it, may not be present in natively in people's antibody reproductive tract storehouse in vivo.

As used herein, " isolated antibody " is intended to represent not contain the antibody (being substantially free of specificity in conjunction with other antigenic antibody in conjunction with the isolated antibody of human IL-2 3p19 subunit as specificity) of other antibody with different antigen-specifiies.But, specificity has cross reactivity in conjunction with isolated antibody and other antigen of human IL-2 3p19 subunit as the IL-23 molecule from other species.And isolated antibody can be substantially free of other cell materials and/or chemical.

As used herein, " neutrality antibody " is intended to represent such antibody, itself and the biological activity that causes suppressing human IL-2 3 that combines of human IL-2 3p19 subunit.

As measuring among mouse boosting cell bioassay method [embodiment 1] or the human IL-2 3 and in the assay method [embodiment 2],, can assess this human IL-2's 3 bioactive restraining effect by measuring the bioactive one or more indexs of IL-23.

" variant " antibody refers to such molecule in the text, owing in the parental antibody sequence, add, lack and/or replace one or more amino-acid residues, and on aminoacid sequence, be different from " parent " antibody aminoacid sequence.In preferred embodiments, variant antibody comprises at least one aminoacid addition, disappearance and/or replacement in the parental antibody CDR district (for example, 1 to about 10, preferred 2,3,4,5,6,7 or 8).Identity with regard to the variant antibody sequence or homology are defined as in the text through aligned sequences and if the necessary room of introducing is with after obtaining maximal sequence identity per-cent, the amino-acid residue per-cent identical with the parental antibody residue in the variant antibody sequence.Variant antibody keeps the ability that combines with parental antibody institute bonded antigen or preferred epi-position, and preferably has at least one performance that is better than parental antibody or biological activity.For example, compare with parental antibody, variant antibody preferably has stronger binding affinity, lower dissociation rate, lower IC ₅₀Or enhanced suppresses the bioactive ability of antigen.The variant antibody of special concern in the literary composition is such antibody, and when comparing with parental antibody, its performance or biological activity strengthen about at least 2 times, preferably about at least 5 times, 10 times or 20 times.

" parent " antibody in the literary composition contains the aminoacid sequence that is useful on preparation variant antibody.Parental antibody can have the framework sequence in mouse source, but preferred framework sequence all or basically is that the people originates.Parental antibody can be mouse, chimeric, humanization or people's antibody.

The antibodies human IL-2 3p19 subunit of " specificity combination ", but debond human IL-2 3p40 subunit.Specificity can show some cross reactivities with the IL-23 from other species in conjunction with human IL-2 3 antibody.

Term " epi-position " is meant the part of molecule, and it can be by antibody recognition, and is one or more antigen binding domains territory institute combination of antibody.Epi-position is made of chemically active surface molecular group such as amino acid or sugared side chain usually, and has specific Three Dimensions Structure and specific charge characteristic." inhibition epi-position " and/or " neutrality epi-position " is intended to represent such epi-position, under the situation of complete antigen molecule, when being specific to the antibodies of described epi-position, cause body interior or external or contain the loss of bioactivity or the reduction of molecule described in the organism of described molecule.As used herein, term " antigenic epitopes " is defined as the part of polypeptide, antibody capable specificity combination with it, as known in the art arbitrary method measure like that, Chang Gui immunoassay for example." non-linear epi-position " or " conformational epitope " comprise discontinuous polypeptide (or amino acid) in the antigen protein, are specific to the antibody combination with it of described epi-position.

Term " k as used herein _On" be intended to represent binding constant or association rate constant, or the specific rate of reaction of forward reaction or mixture formation reaction, linear module: M ^-1Sec ^-1

As used herein, term " k _Off" be intended to represent dissociation constant or dissociation rate constant, or antibody is from the specific rate of reaction of antibody/antigen complex dissociation, linear module: sec ^-1

As used herein, term " K _D" be intended to represent the dissociation constant of specific antibodies-AI.Its calculation formula is as follows:

k _off/k _on＝K _D

Antibody of the present invention is high-affinity antibody, generally shows low k _OffValue.Be the open purpose meter of the present invention, term " high-affinity " expression 1.6 * 10 ^-10M is to about 4.5 * 10 ^-11The avidity of M or K _DValue.

Term " is tired " and is bioactive linear module, and is expressed as IC ₅₀, or in embodiment 1 described bioassay method being and mouse boosting cell on the required antibody effective concentration of 50%IL-23 biological activity.

As used herein, term " nucleic acid molecule " is intended to comprise dna molecular and RNA molecule.Nucleic acid molecule can be strand or double-stranded, but is preferably double-stranded DNA.

As used herein, with regard to coding in conjunction with the term " isolated nucleic acid molecule " with regard to the nucleic acid of human IL-2 3 antibody or antibody moiety (as VH, VL, CDR3), be intended to represent such nucleic acid molecule, wherein the nucleotide sequence of encoding said antibody or antibody moiety does not contain coding in conjunction with being different from antigenic antibody of the human IL-2 3 or other nucleotide sequences of antibody moiety, and wherein said other sequences can the nucleic acid of natural side joint in the human gene group DNA.Thereby for example, the isolating nucleic acid of the present invention in the anti-human IL-2 3 antibody VH districts of encoding does not contain coding in conjunction with other sequences that are different from antigenic other VH districts of human IL-2 3.

As used herein, term " carrier " is intended to represent to transport the nucleic acid molecule of other nucleic acid that are attached thereto.One class carrier is " plasmid ", is meant cyclic double-stranded DNA ring, wherein can be connected into additional DNA section.Another kind of carrier is a virus vector, wherein can be connected into additional DNA section in viral genome.

As used herein, term " recombinant host cell " (or only being " host cell ") is intended to represent to the cell of wherein having introduced recombinant expression vector.

Monoclonal antibody 3A8 is mouse monoclonal antibody (MAb3A8), and its specificity is in conjunction with the epi-position on the human IL-2 3p19 subunit.MAb3A8 is produced the more antibody of high-affinity by humanization and optimization, have among the strong IL-23 and active, and high special is in the p19 of IL-23 subunit but not the p40 subunit.

The preferred antibody of the present invention or its antigen-binding portion thereof reveal high-affinity (low K to the identical table bit table of MAb3A8 _DBe worth), and binding affinity is better than among the MAb3A8 viewed.The bonding properties of definition antibody of the present invention is the variable region of antibody fully, more specifically is the CDR district of antibody.

Be when antibody is reduced the possibility that it causes immunne response in human patients the time as injection of therapeutic agent from the primary impellent of other species humanized antibodies.The human sequence who adopts in the humanized antibody is many more, and immunogenic risk is low more.In addition, the humanized antibody of injection has longer circulating half-life than the non-human antibody of injection usually.In addition, if expectancy effect thing function, because described effector partly is the people, humanized antibody can more preferably interact with other parts of human immune system.Can change sequence preference heavy chain as herein described and light chain district, and biological property that can remarkably influenced antibody.This is for not influencing CDR in antibody constant region and the variable region in conjunction with especially correct for the part of IL-23 ability.

In addition, such as discussed herein, people's framework variable region and variant thereof can be used for the present invention.But, no matter selected framework how, if focus is to reduce the immunogenicity risk, then makes the varied number with respect to choosing framework minimize.

Humanized antibody of the present invention can comprise or derive from people's reproductive tract light chain framework.In specific embodiments, described light chain reproductive tract sequence is selected from people VK sequence, includes but not limited to: A1, A10, A11, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4 and O8.In certain embodiments, this light chain people reproductive tract framework is selected from V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4 and V5-6.

In other embodiments, humanized antibody of the present invention can comprise or derive from people's reproductive tract heavy chain framework.In specific embodiments, this heavy chain people reproductive tract framework is selected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1 and VH7-81.

In specific embodiments, variable region of light chain and/or variable region of heavy chain comprise framework region or at least the part frame district (as, contain 2 or territory, 3 subprovinces, as FR2 and FR3).In certain embodiments, FRL1, FRL2, FRL3 or FRL4 are the people fully at least.In other embodiments, FRH1, FRH2, FRH3 or FRH4 are the people fully at least.In some embodiment, at least FRL1, FRL2, FRL3 or FRL4 be reproductive tract sequence (as people's reproductive tract) or comprise as described in people's consensus sequence of specific frame.In other embodiments, at least FRH1, FRH2, FRH3 or FRH4 be reproductive tract sequence (as people's reproductive tract) or comprise as described in people's consensus sequence of specific frame.In preferred embodiments, all framework region behaviour framework region.

The preferred people's CH of humanized antibody of the present invention aminoacid sequence comprises IgG1 constant region IgG1[SEQ ID NO:61] or IgG4 constant region [SEQ ID NO:62].The preferred people's constant region of light chain of humanized antibody of the present invention aminoacid sequence is κ chain constant region [SEQ ID NO:63].

In addition, preferred people's variable region of heavy chain framework is VH1-24[SEQ ID NO:64], and preferred people's variable region of light chain framework is A17[SEQ ID NO:65].SEQ ID NO:64 and 65 representatives have people's reproductive tract sequence of natural CDR.Added the people J district together with optimum matching, to form complete variable region from the CDR of MAb3A8.Should be understood that except other people heavy chain or constant region of light chain and the variable region framework sequence those mentioned above, also consider to be used for the present invention and be well known in the art.

The present invention includes antibody or its antigen-binding portion thereof, it is in conjunction with the specificity epitope on the IL-23p19 subunit, and in and the IL-23 activity.Thereby, utilize CDR as herein described and heavy chain and variable region of light chain to prepare full length antibody and functional fragment and analogue, described fragment and analogue are kept the combination of proteins avidity that is specific to the CDR of IL-23p19 subunit at employing.

The binding affinity of MAb3A8 utilizes surface plasmon resonance (BIAcore ^TM) determine.In these experiments, BIAcore ^TMA-protein on the chip or anti-Fc antibody is with the low density capture antibody, and makes part flow through chip.The increase of improving quality in the measured chip surface.This analytical procedure can be determined association rate and the speed of dissociating in real time, to obtain relevant bonded avidity (K _D).The K of MAb3A8 _DBe worth about 300pM (picomole).The K of humanized antibody of the present invention _DBe worth about 45 to about 160pM; About 45 to about 150pM; About 45 to about 100pM; About 50 to about 95pM; About 60 to about 85pM; And about 70 to about 80pM.The K of preferred humanized antibody of the present invention _DValue is lower than about 100pM.

In antibody of the present invention or its antigen-binding portion thereof and the biological activity of IL-23.Utilize two kinds of assay methods to test in MAb3A8 and the preferred antibody of the present invention and the active ability of IL-23 [referring to embodiment 1 and 2].

The invention still further relates to the recombinant DNA of encoding antibody, specificity is in conjunction with human IL-2 3p19 subunit when described antibody expression.The such antibody of preferred described dna encoding comprises one or more heavy chains of the present invention and light chain CDR[table 1 and 2 when it is expressed].

Table 1CDR sequence-variable region of heavy chain (HCDR)

FAb	HCDR1	HCDR2	HCDR3
FAb	HCDR1	HCDR2	HCDR3	3A8	GKTFTSYGIN (SEQ ID NO：1)	YIYIGNGYTESNEKFKG (SEQ ID NO：5)	IGAYYGNFDY (SEQ ID NO：12)

FAb	HCDR1	HCDR2	HCDR3
FAb	HCDR1	HCDR2	HCDR3	1	GKTFFSYGIN (SEQ ID NO：2)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
2	GKTFWSYGIN (SEQ ID NO.3)	YIYIGGGYTEPNPKYKG (SEQ ID NO.6)	IGGYYGNFTD (SEQ ID NO：13)	1	GKTFFSYGIN (SEQ ID NO：2)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
2	GKTFWSYGIN (SEQ ID NO.3)	YIYIGGGYTEPNPKYKG (SEQ ID NO.6)	IGGYYGNFTD (SEQ ID NO：13)	3	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
4	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO.6)	IGGYYGNFTD (SEQ ID NO：13)	3	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
4	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO.6)	IGGYYGNFTD (SEQ ID NO：13)	5	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
6	GKTFYSYGIN (SEQ ID NO.4)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	5	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
6	GKTFYSYGIN (SEQ ID NO.4)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	7	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
8	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	7	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
8	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	9	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
10	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)	9	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
10	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)	11	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
12	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	11	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
12	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	13	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFDD (SEQ ID NO.14)
14	GKTFWSYGIN	YIYIGNGYTEPNPKYKG	IGGYYGNFTD	13	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFDD (SEQ ID NO.14)

FAb	HCDR1	HCDR2	HCDR3
FAb	HCDR1	HCDR2	HCDR3		(SEQ ID NO：3)	(SEQ ID NO：7)	(SEQ ID NO：13)
15	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IG GYYGNFTD (SEQ ID NO：13)		(SEQ ID NO：3)	(SEQ ID NO：7)	(SEQ ID NO：13)
15	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IG GYYGNFTD (SEQ ID NO：13)	16	GKTFTSYGIN (SEQ ID NO：1)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
17	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)	16	GKTFTSYGIN (SEQ ID NO：1)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
17	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)	18	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)
19	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)	18	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)
19	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)	20	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)
21	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	20	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)
21	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	22	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)
23	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	22	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)
23	GKTFFSYGIN (SEQ ID NO：2)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	24	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
25	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	24	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
25	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)	26	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)
27	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFDD (SEQ ID NO.14)	26	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFTD (SEQ ID NO：13)

FAb	HCDR1	HCDR2	HCDR3
FAb	HCDR1	HCDR2	HCDR3	28	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
29	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFDD (SEQ ID NO.14)	28	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFTD (SEQ ID NO：13)
29	GKTFWSYGIN (SEQ ID NO：3)	YIYIGGGYTEPNPKYKG (SEQ ID NO：6)	IGGYYGNFDD (SEQ ID NO.14)	30	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFHD (SEQ ID NO.15)
31	GKTFWSYGIN (SEQ ID NO：3)	YIYIGT GYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFAD (SEQ ID NO：16)	30	GKTFWSYGIN (SEQ ID NO：3)	YIYIGNGYTEPNPKYKG (SEQ ID NO：7)	IGGYYGNFHD (SEQ ID NO.15)
31	GKTFWSYGIN (SEQ ID NO：3)	YIYIGT GYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFAD (SEQ ID NO：16)	32	GKTFWSYGIN (SEQ ID NO：3)	YIYIGSGYTEPNPKYKG (SEQ ID NO：9)	IGGYYGNFHD (SEQ ID NO：15)
33	GKTFWSYGIN (SEQ ID NO：3)	YIYIGTGYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFKD (SEQ ID NO：17)	32	GKTFWSYGIN (SEQ ID NO：3)	YIYIGSGYTEPNPKYKG (SEQ ID NO：9)	IGGYYGNFHD (SEQ ID NO：15)
33	GKTFWSYGIN (SEQ ID NO：3)	YIYIGTGYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFKD (SEQ ID NO：17)	34	GKTFWSYGIN (SEQ ID NO：3)	YIYIGTGYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFDH (SEQ ID NO：18)
35	GKTFWSYGIN (SEQ ID NO：3)	YIYIGTGYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFDQ (SEQ ID NO：19)	34	GKTFWSYGIN (SEQ ID NO：3)	YIYIGTGYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFDH (SEQ ID NO：18)
35	GKTFWSYGIN (SEQ ID NO：3)	YIYIGTGYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFDQ (SEQ ID NO：19)	36	GKTFWSYGIN (SEQ ID NO：3)	YIYIGRGYTEPNPKYKG (SEQ ID NO：10)	IGGYYGNFQD (SEQ ID NO：20)
37	GKTFWSYGIN (SEQ ID NO：3)	YIYIGYGYTEPNPKYKG (SEQ ID NO：11)	IGGYYGNFED (SEQ ID NO：21)	36	GKTFWSYGIN (SEQ ID NO：3)	YIYIGRGYTEPNPKYKG (SEQ ID NO：10)	IGGYYGNFQD (SEQ ID NO：20)
37	GKTFWSYGIN (SEQ ID NO：3)	YIYIGYGYTEPNPKYKG (SEQ ID NO：11)	IGGYYGNFED (SEQ ID NO：21)	38	GKTFWSYGIN (SEQ ID NO：3)	YIYIGT GYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFED (SEQ ID NO：21)
Consensus sequence ^＊	GKTFX ₁SYGIN (SEQ ID NO：22)	YIYIGX ₂GYTEX ₃NX ₄KX ₅KG (SEQ ID NO：23)	IGX ₆YYGNFX ₇X ₈ (SEQ ID NO：24)	38	GKTFWSYGIN (SEQ ID NO：3)	YIYIGT GYTEPNPKYKG (SEQ ID NO：8)	IGGYYGNFED (SEQ ID NO：21)

^*X ₁Be T, F, W or Y; X ₂Be N, T, S, R, Y or G; X ₃Be S or P; X ₄Be E or P; X ₅Be F or Y; X ₆Be A or G; X ₇Be D, H, A, K, Q, E or T; And X ₈Be Y, H, Q or D.

Table 2CDR sequence-variable region of light chain (LCVR)

FAb	LCDR1	LCDR2	LCDR3
FAb	LCDR1	LCDR2	LCDR3	MAb3A8	KSSQSLLDSDGKTYLN (SEQ ID NO：25)	LVSKLDS (SEQ ID NO：36)	WQGTHFPLT (SEQ ID NO：39)
1	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	MAb3A8	KSSQSLLDSDGKTYLN (SEQ ID NO：25)	LVSKLDS (SEQ ID NO：36)	WQGTHFPLT (SEQ ID NO：39)
1	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	2	QSSQSLLISGGKTYLN (SEQ ID NO：27)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
3	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	2	QSSQSLLISGGKTYLN (SEQ ID NO：27)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
3	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	4	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
5	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	4	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
5	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	6	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
7	QSSQSLLISGGKTYLN (SEQ ID NO：27)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	6	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
7	QSSQSLLISGGKTYLN (SEQ ID NO：27)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	8	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40
9	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	8	QSSQSLLISGGNTYLN (SEQ ID NO：26)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40
9	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	10	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
11	RSSQSLLISGGKTYLN (SEQ ID NO：29)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	10	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
11	RSSQSLLISGGKTYLN (SEQ ID NO：29)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	12	KSSQSLLISGGNTYLN	LVSKLDQ	WQGTYFPLT

EAb	LCDR1	LCDR2	LCDR3
EAb	LCDR1	LCDR2	LCDR3		(SEQ ID NO：30)	(SEQ ID NO：37)	(SEQ ID NO：40)
13	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)		(SEQ ID NO：30)	(SEQ ID NO：37)	(SEQ ID NO：40)
13	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	14	QSSQSLLISGGKTYLN (SEQ ID NO：27)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
15	KSSQ SLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	14	QSSQSLLISGGKTYLN (SEQ ID NO：27)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
15	KSSQ SLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	16	RSSQSLLISGGPTYLN (SEQ ID NO：32)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
17	RSSQSLLISGGPTYLN (SEQ ID NO：32)	KVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	16	RSSQSLLISGGPTYLN (SEQ ID NO：32)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
17	RSSQSLLISGGPTYLN (SEQ ID NO：32)	KVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	18	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
19	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	18	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
19	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	20	KSSQSLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
21	KSSQSLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	20	KSSQSLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
21	KSSQSLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	22	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
23	QSSQSLLISGGKTYLN (SEQ ID NO：27)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	22	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
23	QSSQSLLISGGKTYLN (SEQ ID NO：27)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	24	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
25	QSSQSLLISGGPTYLN (SEQ ID NO：33)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	24	KSSQSLLISGGKTYLN (SEQ ID NO：31)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)

FAb	LCDR1	LCDR2	LCDR3
FAb	LCDR1	LCDR2	LCDR3	26	KSSQSLLISGGPTYLN (SEQ ID NO：34)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
27	KSSQSLLISGGNTYLN (SEQ ID NO：35)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	26	KSSQSLLISGGPTYLN (SEQ ID NO：34)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)
27	KSSQSLLISGGNTYLN (SEQ ID NO：35)	KVSKLDQ (SEQ ID NO：38)	WQGTYFPLT (SEQ ID NO：40)	28	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
29	KSSQSLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	28	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
29	KSSQSLLISGGKTYLN (SEQ ID NO：31)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	30	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
31	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	30	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
31	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	32	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
33	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	32	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
33	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	34	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
35	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	34	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
35	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	36	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
37	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	36	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
37	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)	38	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)
Consensus sequence ^＊＊	X ₉SSQSLLX ₁₀SX ₁₁GX ₁₂TYLN	X ₁₃VSKLDX ₁₄	WQGTX ₁₅FPLT	38	RSSQSLLISGGNTYLN (SEQ ID NO：28)	LVSKLDQ (SEQ ID NO：37)	WQGTYFPLT (SEQ ID NO：40)

FAb	LCDR1	LCDR2	LCDR3
FAb	LCDR1	LCDR2	LCDR3		(SEQ ID NO：41)	(SEQ ID NO：42)	(SEQ ID NO：43)

^*X ₉Be K, Q or R; X ₁₀Be D or I; X ₁₁Be D or G; X ₁₂Be K, N or P; X ₁₃Be L or K; X ₁₄Be S or Q; And X ₁₅Be H or Y.

The present invention includes such antibody, it comprises the light chain CDR consensus sequence shown in the SEQ ID NO:41,42 and 43, and the heavy chain CDR consensus sequence shown in the SEQ ID NO:22,23 and 24.The exemplary weight chain variable region amino acid sequence of preferred antibody of the present invention that utilizes VH framework VH1-24 is shown in SEQ ID NO:44-55.The exemplary light chain variable region amino acid sequence of preferred antibody of the present invention that utilizes VL framework A17 is shown in SEQ ID NO:56-59.SEQ ID NO:58 and 59 amino-acid residue 41 change over the mouse residue that is positioned at this site.And exemplary antibody representative of the present invention is as follows: the constant region of light chain shown in CH shown in the SEQ ID NO:62 and the SEQ ID NO:63.

Thereby exemplary antibody of the present invention is selected from down group:

(a) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:47, the SEQ ID NO:63 and the SEQ ID NO:62;

(b) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:48, the SEQ ID NO:63 and the SEQ ID NO:62;

(c) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:49, the SEQ ID NO:63 and the SEQ ID NO:62;

(d) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:51, the SEQ ID NO:63 and the SEQ ID NO:62;

(e) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:52, the SEQ ID NO:63 and the SEQ ID NO:62;

(f) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:53, the SEQ ID NO:63 and the SEQ ID NO:62; With

(g) has the antibody of the HCCR shown in LCCR shown in the HCVR shown in the LCVR shown in the SEQ ID NO:57, the SEQ ID NO:55, the SEQ ID NO:63 and the SEQ ID NO:62.

Neutrality antibody of the present invention is an aminoacid sequence and realizing by arranging suitable nucleotide sequence and express these sequences in suitable clone, producing suitable antibody gene sequence.The nucleotide sequence of expectation can utilize the mutafacient system based on codon to produce.Such method allows codon position place any desired in oligonucleotide to produce the amino-acid residue of any and all frequencies.This can comprise any 20 amino acid whose replacements of desired location place completely random.Alternatively, can implement this method, thereby obtain specific amino acid on the expectation site in amino acid chain such as the new CDR sequence of the present invention.Substantially, can easily obtain to express the suitable nucleotide sequence of any desired aminoacid sequence, and utilize such method can reproduce new CDR sequence of the present invention.This makes us can synthesize polypeptide such as the antibody with any desired aminoacid sequence.For example, might determine the aminoacid sequence of any desired structural domain of selected antibody at present, and randomly prepare wherein one or more amino acid and be replaced by other and expect amino acid whose homology chain, so that the analogue of a large amount of replacements to be provided.

When using such method, should understand because the degeneracy of genetic code, synthesize the redundant phenomenon of codon that to introduce particular amino acid residue on the relevant regulations specific position with part degenerate oligonucleotide synthetic method such as random oligonucleotide, the method of even now can be used for providing might aminoacid sequence main collection, and screening is as the best-of-breed functionality of antibody structure or be used for other purposes.Alternatively, such antibody sequence can chemosynthesis or is produced in other modes well known to those skilled in the art.

According to invention disclosed herein, can produce the enhanced high-titer antibody by the one or more new CDR sequences disclosed herein of combination in the wall scroll polypeptide structure, wherein the verified enhanced that causes independently of each CDR is tired or biological activity.By this way, a plurality of amino acid combined sequence can be advanced among the identical or different CDR of an antibody, be had the bioactive antibody of aspiration level with generation.The effect that described amino acid coding has can make the k of described antibody _OffReduce, preferably follow the increase of affinity of antibody.Higher tiring can be passed through lower k _OffValue realizes, even avidity keeps identical or reduces to a certain extent.Such antibody is preferably produced by synthetic required polypeptide chain in suitable engineering cell, has mixed the suitable nucleotide sequence that coding contains the required polypeptide chain of the CDR section that changes in the wherein said cell.As non-limiting instance, can adopt so new CDR sequence, and utilize among splenocyte bioassay method as herein described or the human IL-2 3 and with scheme the antibody that is produced is tired or bioactivity screening, antibody shows high-affinity to specific antigen structure such as human IL-2 3p19 subunit in these methods.

In contrast, it must be understood that not to be that the interior all sites of different antibodies structural domain sequence all is equal to, because the replacement of any type may be useful on the specific site, also may be deleterious.In addition, the aminoacid replacement of some type with regard to avidity may be or plus or minus equally on some site.For example, on given position, may need not to attempt all possible hydrophobic amino acid.Also may be that any hydrophobic amino acid can.On the other hand, significantly swing may provided to acidity on the locating point or basic aminoacids aspect the avidity of measuring.

Such as has already been described, K _DPass through k _OnWith k _OffThe ratio of constant is measured.For example, 3.1 * 10 ⁷(M ^-1s ^-1) k _OnWith 0.9 * 10 ^-4(s ^-1) k _OffCombination will obtain 2.9 * 10 ^-12The K of M _DThereby avidity can be by improving k _OnOr reduction k _OffImproved.Correspondingly, antibody k of the present invention _OffReduction produce more efficiently therapeutical agent possibly.

According to aforementioned content as can be known, antibody of the present invention is the high-affinity monoclonal antibody.But, only can to derive from the meaning of clone or individual cells type at it be monoclonal for such antibody.But, this does not mean and refers to that it is confined to specific source.Such antibody can easily be produced in the cell that does not produce antibody usually such as CHO, NSO or COS cell.In addition, such antibody can particularly be produced in Mammals or even bacterium and the vegetable cell at the other types cell, and the cell that genetic modification is such is to express and assembling forms the polypeptide light chain and the heavy chain of antibody product.In addition, can the such chain of chemosynthesis, but because they are specific to given antigenic determinant, under the use spirit of this term, will constitute " mono-clonal " antibody.Thereby as used herein, the term monoclonal antibody more is intended to represent the specificity and the purity of antibody molecule, and is not only the mechanism that is used to produce described antibody.

As used herein equally, term is tired and is intended to describe when being used for the therapeutic interest purposes, and the antibody effect is for the dependency of such antibody concentration.Thereby, the biological activity of expression with regard to given antigen of tiring.As non-limiting instance, by measuring tiring of anti-il-23 antibodies or biological activity or biological effect with scheme among splenocyte bioassay method described herein or the human IL-2 3.The relative potency called after IC of the antibody of producing according to the inventive method ₅₀, the about 1pM of scope is to about 100pM, about 1pM to about 50pM usually; About 1pM is to about 25pM.Preferred IC ₅₀About 25pM, 20pM, 15pM, 13pM, 10pM, 8pM, 5pM, 2pM or 1pM.The IC of antibody of the present invention most preferably ₅₀About 20pM.

In contrast, antibody only is k to antigenic avidity _OnWith k _OffThe mathematical measure of ratio.In addition, the K of the antibody of producing according to the inventive method _DCommon scope about 2 * 10 ^-10M is to about 25 * 10 ^-12M preferably is lower than about 160pM, 100pM, 75pM, 50pM or 25pM.The K of antibody of the present invention most preferably _DBe lower than about 100pM.

In one embodiment, antibody of the present invention or its antigen-binding portion thereof contain preferred human constant region of Mammals and variable region usually, heavy chain and light chain framework region and heavy chain and light chain CDR are contained in described variable region, wherein heavy chain and light chain framework region have the peculiar sequence of the preferred people's antibody of Mammals antibody, and wherein the CDR sequence is similar to the CDR of the preferred mouse antibodies of some species that is different from the people.

In one embodiment of the invention, utilize K _DThe anti-human IL-2 3 neutrality antibody Fab fragments that value is lower than about 160pM strengthen tires, and with k _OffValue is brought down below about 1 * 10 ^-3s ^-1, preferably be lower than about 5 * 10 ^-4s ^-1, more preferably less than about 1 * 10 ^-4s ^-1The amino acid that exists among the segmental CDR of such Fab is shown in table 1 and 2.

In specific embodiment, the present invention relates to K _DValue is lower than the isolated antibody of about 160pM, wherein k _OffValue is lower than about 1 * 10 ^-3s ^-1, preferably be lower than about 5 * 10 ^-4s ^-1, and most preferably be lower than about 1 * 10 ^-4s ^-1(comprising its all combinations).Thereby, the ripe human IL-2 3p19 of the preferred antibodies of the present invention subunit, K _DBe worth about 160pM or lower, k _OffRate constant 1 * 10 ^-3s ^-1Or lower, and in and human IL-2's 3 activity.

The DNA of code book invention antibody generally includes the expression regulation polynucleotide sequence that effectively is connected in antibody coding sequence, and this comprises natural bonded or allogenic promoter region.The preferred expression regulating and controlling sequence for can transform or the carrier of transfection eukaryotic host cell in the eukaryotic promoter system, but also can use the regulating and controlling sequence of prokaryotic hosts.In case carrier has been incorporated in the proper host cell system, then be suitable for expressing under the condition of nucleotide sequence, and if desired, under the condition that is suitable for reclaiming with purifying light chain, heavy chain, light/heavy chain homodimer or complete antibody, binding fragment or other immunoglobulin (Ig) forms, breed host cell.

The nucleotide sequence of the present invention that can finally express expectation antibody can utilize multiple any technology of knowing in the technology to be made of multiple different polynucleotide (genome or cDNA, RNA, synthetic oligonucleotide etc.) and component (as V, J, D and C district).Connecting suitable genome and composition sequence is the production method of using always, but also can use the cDNA sequence.

The human constant region dna sequence dna can separate from the various human cell according to the method for knowing, but preferably separates from the B cell of immortalization.The suitable derived cell of polynucleotide sequence and be used for immunoglobulin expression and the excretory host cell can obtain from many sources well-known in the art.

As described herein, except the specifically described antibody of this paper, can design other " homologous basically " modified antibodies and utilize multiple recombinant DNA technology well known to those skilled in the art to produce.For example, framework region can replace because of several amino acid, terminal and middle interpolation is different with native sequences with disappearance or the like.And, can utilize the basis of multiple different people's framework region alone or in combination as Humanized immunoglobulin of the present invention.Usually, genetic modification can easily be realized by the multiple technology of knowing such as site-directed mutagenesis.

Alternatively, can produce the polypeptide fragment of a part that only comprises main antibody structure, described fragment has one or more immune globulin activities (as the complement fixation(CF) activity).These polypeptide fragments can be produced by proteolysis cutting complete antibody by method well-known in the art, or terminator codon is inserted in the site that utilizes side-directed mutagenesis to expect in carrier, for example after CH1 producing the Fab fragment, or hinge area after with generation F (ab ') ₂Fragment.Single-chain antibody can be by producing with DNA joint connection VL and VH.

As discussed previously, after polynucleotide sequence effectively being connected in (promptly arranging) expression regulation sequence, will in the host, express to guarantee its functionating.These expression vectors are reproducible in host organisms usually, perhaps as episome, perhaps as the integrated part of host chromosome DNA.Generally speaking, expression vector contains selective marker, as tsiklomitsin, Xin Meisu and Tetrahydrofolate dehydrogenase, allow to detect those cells that transformed with the expectation dna sequence dna.

Intestinal bacteria (E.coli) are particularly useful for cloning the prokaryotic hosts of polynucleotide of the present invention.Other microorganism host that are suitable for using comprise bacillus, as subtilis (Bacillus subtilus), and other enterobacteriaceaes (enterobacteriaceae), as salmonella (Salmonella), serratia (Serratia) and multiple Rhodopseudomonas (Pseudomonas) species.In these prokaryotic hosts, people also can prepare expression vector, and described carrier contains the expression regulation sequence compatible with host cell (as replication orgin) usually.In addition, can there be any multiple promotor of knowing, as lactose promoter systems, tryptophane (trp) promoter systems, β-Nei Xiananmei promoter systems or from the promoter systems of lambda particles phage.The common regulating and expressing of promotor randomly has operon sequence, and has ribosome bind site sequence or the like, with initial and finish and transcribe and translate.

Except bacterium, other microorganisms such as yeast also can be used for expressing.Pichia pastoris phaff (Pichiapastoris) is preferred host, together with suitable carriers, has the expression regulation sequence of expectation, as promotor, comprises glycerol 3-phosphate acid kinase or other glycolytic ferments, and replication orgin, terminator sequence or the like.

The mammalian tissues cell culture also can be used for expressing and producing polypeptide of the present invention.In fact preferred eukaryotic cell, because the many proper host cell systems that can secrete complete immunoglobulin (Ig) have been developed in this area, and comprise B cell, human embryonic kidney cell line or the hybridoma of Chinese hamster ovary celI system, multiple COS clone, HeLa cell, myeloma cell line, conversion.Preferred cell is CHO and myeloma cell line such as SP2/0 and NS0.

The expression vector that is used for these cells can comprise expression regulation sequence, as replication orgin, promotor, enhanser and necessary machining information site, and as ribosome bind site, RNA splice site, polyadenylation site, and the Transcription Termination subsequence.Preferred expression regulation sequence has the promotor that derives from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc.Preferred polyadenylation site comprises the sequence that derives from SV40 and Trobest.

The carrier that contains polynucleotide of interest sequence (as heavy chain and light chain encoding sequence and expression regulation sequence) can advance host cell by the method transfection of knowing, and this becomes according to the type of cell host.For example, the calcium chloride transfection is generally used for prokaryotic cell prokaryocyte, and calcium phosphate is handled or electroporation can be used for other cell hosts.

Antibody is once expression, can be according to the standard method purifying, comprise ammonium sulfate precipitation, ion-exchange, affine (as a-protein), anti-phase, hydrophobic interaction column chromatography, gel electrophoresis or the like.For medicinal application, preferred purity is at least about the pure basically immunoglobulin (Ig) of 90-95%, most preferably 98-99% or higher purity.Polypeptide one is purified, can be partial purification or homogeneous as required, then can use in the treatment or in the prevention as teaching herein.

Antibody of the present invention can as described hereinly be used for the treatment of, prevent, diagnose and study application.Antibody of the present invention can be used for diagnosing with human IL-2 3 expresses relevant disorder or disease.In a similar manner, antibody of the present invention can be used for measuring, and is just testing the experimenter's of its IL-23 associated conditions IL-23 level with monitoring.Research is used and to be comprised antibody of the present invention and the mark method with IL-23 in test sample such as people's body fluid or the cell or tissue extract of utilizing.Antibody of the present invention can be modified or not modified use, but and gives mark by covalently or non-covalently adhering to the test section.But the test section can be any part that can directly or indirectly produce detectable signal.

Be used to measure the multiple ordinary method of IL-23, comprise for example ELISA, RIA and FACS, for known in this field, and for diagnosing the IL-23 expression level to change or providing the foundation unusually.Normal or standard expression values utilizes any technology well known in the art to establish, for example, and by being suitable for forming antigen with for example antibody: mix under the condition of antibody complex by the sample that will contain the IL-23 polypeptide.Antibody is directly or indirectly by detectable material mark, to promote combination or the not detection of binding antibody.Suitable detectable substance comprises plurality of enzymes, prothetic group, fluorescent substance, luminophore and radioactive substance.

For simplicity, antibody of the present invention can be provided as test kit, i.e. the packaged combination of predetermined amount reagent, and have the specification sheets of implementing diagnostic assay.Under the situation of antibody by enzyme labelling, test kit will comprise substrate and the cofactor (as the substrate that can detect chromophoric group or fluorophore precursor is provided) that enzyme is required.In addition, can comprise other additives, as stablizer, buffer reagent (as sealing damping fluid or lysis buffer) or the like.The relative quantity of plurality of reagents can change significantly, so that the fully preferred reagent solution concentration of measuring sensitivity to be provided.Especially, reagent can be provided as powder form, and normally lyophilisate comprises vehicle, and the reagent solution of proper concn will be provided when dissolving.

The invention still further relates to the people's of the inflammation disorder for the treatment of experience IL-23 mediation method, comprise that the patient to needs uses the antibody that is specific to human IL-2 3p19 subunit of significant quantity.Antibodies of the present invention and in and IL-23.The disorder of multiple IL-23 mediation comprises multiple sclerosis, rheumatoid arthritis (RA), graft versus host disease (GVH disease), psoriatic, Crohn's disease (Crohn ' s disease), other inflammatory bowels and cancer.The IL-23 antibody that preferred the present invention is contained is used for the treatment of recurrence remission form multiple sclerosis, the multiple sclerosis of most common form.

Antibody of the present invention or its antigen-binding portion thereof can be the forms of composition, and described composition comprises the antibody of the present invention that is suspended in pharmaceutically acceptable diluent or the vehicle.These pharmaceutical compositions can be used for the treatment of autoimmune disease by realization well known in the art, preferably any means of the general objects purposes of multiple sclerosis are used.Preferred route of administration is a parenteral, and it is defined as such method of application in the text: comprise (intrasternal), subcutaneous and intra-articular injection and infusion in intravenously, intramuscular, intraperitoneal, the breastbone.More preferably route of administration is subcutaneous and uses once in a week.Application dosage will depend on age, health and the body weight of acceptor, the type of synchronous therapeutic [if any], the character of therapeutic frequency and desired effects.

Composition in the scope of the invention comprises the composition that all are such, and wherein the amount of antibody or its antigen-binding portion thereof realizes treating the desired medical science effect of multiple sclerosis effectively.Although individual demand may be different because of the patient, within the limit of power of determining to fall into the clinicist with ordinary skill of all components optimum range significant quantity.

The designer drug compositions to be making it being suitable for selected method of application, and uses pharmaceutically useful vehicle such as buffer reagent, tensio-active agent, sanitas, solubilizing agent, isotonic agent, stablizer, carrier or the like in appropriate circumstances.That is known as the practitioner is such, " Lei Shi pharmaceutical science " ( Remington ' s Pharmaceutical Sciences), Mack Publishing Co., Easton PA, latest edition is incorporated this paper into as a reference, and the general introduction of relevant compounding process is provided.

The concentration of IL-23 antibody by weight can be from being low to moderate about 0.1% to height to 15 or 20% in the preparation, and mainly select based on liquid volume, viscosity, stability etc. according to selected concrete method of application.Preferred IL-23 antibody concentration scope is generally 1 to about 100mg/mL.Preferred 10 to about 50mg/mL.

Preparation can comprise damping fluid.Preferred buffer is citrate buffer solution or phosphoric acid buffer or its combination.Usually the pH of preparation is about 4 to about 8.Preferred pH is about 5 to about 7.5.The pH that can select preparation is with balance antibody stability (chemistry with physics), and makes the patient comfortable when using.Preparation also can comprise salt such as NaCl.In addition, preparation can comprise washing agent, to prevent to assemble and help to keep stability.

Preparation can carry out sterile filtration after the preparation preparation, or otherwise makes it can accept on microbiology.Can add sanitas such as meta-cresol or phenol or its mixture, to prevent microorganism growth and pollution.

The exemplary composition that is used for intravenous infusion can have liquid volume such as the aseptic Ringer solution up to 250mL, and 1-100mg/mL or higher antibody concentration.Therapeutical agent of the present invention can be freezing or freeze-drying store, and with preceding reconstruct in suitable sterile carrier.Freeze-drying and reconstruct can cause the antibody activity loss (as for the routine immunization sphaeroprotein, IgM antibody often has bigger loss of activity than IgG antibody) of varying degree.May need to adjust dosage by way of compensation.

Although as if preceding method is the most convenient and be suitable for administration of protein such as humanized antibody most, other application techniques such as transdermal administration and Orally administered through suitable transformation also can adopt, as long as the design appropriate formulation.In addition, may expect to utilize biological degradable membrane and matrix or miniature osmotic pump or based on the delivery system of dextran bead, alginate or collagen and adopt the sustained release preparation.Generally speaking, exist and can be used for using the preparation of antibody of the present invention, and can be selected according to multiple option.

Can utilize the typical dosage level of standard clinical techniques optimization, and this will depend on method of application and patient's situation.Usually, dosage range will be that the 10 μ g/kg/ months are to the 10mg/kg/ month.

On the other hand, consider to utilize antibody of the present invention or its antigen-binding portion thereof to be used for the treatment of autoimmune disease as medicine.

Again on the other hand, also provide and contained the goods that are used for the treatment of or prevent the material of above-mentioned disorder or illness.Described goods comprise container and mark.For example, suitable containers comprises bottle, bottle, syringe and test tube.Container can be made of a variety of materials, as glass or plastics.Container fills the antibody compositions of the present invention of effective prevention or treatment disorder or illness, and can have sterile access port (for example, container can be the bottle of intravenous solution bag or band stopper, described stopper can by the subcutaneous injection needle-penetration).Active agents in the composition is an anti-il-23 antibodies of the present invention.On the container or mark attached thereto show that described composition is used for the treatment of selected illness.Described goods can further comprise second container, and it contains pharmaceutically acceptable damping fluid such as phosphate-buffered saline, Ringer solution and dextran solution.Described goods can further comprise commerce or other desired materials of user perspective, the package insert (package insert) that comprises other buffers, thinner, filter, pin, syringe and have description of use.

The present invention illustrates by following embodiment, is not to be intended to limit by any way.

Embodiment 1

IL-23 suppresses to measure

Based on the ability that IL-23 stimulates mouse boosting cell secretion IL-17, utilize the mouse boosting cell assay method to detect the active inhibition of IL-23.With antibody of the present invention and MAb3A8 and commercially available mouse monoclonal antibody MAB1290 (peace enlightening biotechnology (R﹠amp; D Systems)) compare.

General planning is as follows: take out spleen from 1BALB/c or C57BL/6 mouse, results splenocyte, and preparation single cell suspension.Splenocyte is with complete RPMI (containing 1% non-essential amino acid, 1% Sodium.alpha.-ketopropionate, 2.5mM HEPES, 1%L-L-glutamic acid, 0.00035%2-mercaptoethanol, 1% penicillin/streptomycin, 10% heat-inactivated serum FCS and 50ng/mL human IL-2 (peace enlightening biotechnology)) washing and resuspended.Then splenocyte is inoculated in 96 well culture plates according to 500,000 cells/well, volume 100 μ l.

The human IL-2 3 (peace enlightening biotechnology) of concentration 10pM is carried out preincubate with test antibody 3 times of serial dilutions, scope 0.001-54 μ g/mL.In other assay plate in 37 ℃, 5%CO ₂In hatch 90 minutes by a definite date.

Each sample with 100 μ l preincubates is added in the culture plate that contains splenocyte then, and in 37 ℃, 5%CO ₂In hatched 48-72 hour.Utilize mouse IL-23 sandwich ELISA test kit (M1700, peace enlightening biotechnology) to measure the amount of IL-23 in each culture supernatants.IC ₅₀Be and mouse boosting cell on the required antibody amount of 50%IL-23 biological activity.

Table 3 has shown the MAb3A8 that compares with MAB1290 in the mouse boosting cell assay method (Murine Splenocyte Assay) and the IC of antibody of the present invention ₅₀As an example Shuo Ming antibody MAbQF20 and MAbQF37 significantly in and IL-23 to the stimulation of mouse boosting cell secretion IL-17, IC ₅₀Value is lower than 20pM.

Table 3: the IC that measures in the mouse boosting cell assay method ₅₀

Sample	SEQ ID NO HCVR/LCVR	SEQ ID NO HCCR/LCCR	IC ₅₀pM
Sample	SEQ ID NO HCVR/LCVR	SEQ ID NO HCCR/LCCR	IC ₅₀pM	MAb3A8			4
MAB1290			2800	MAb3A8			4
MAB1290			2800	MAbQF20	48/57	62/63	2
MAbQF37	52/57	62/63	2	MAbQF20	48/57	62/63	2

Embodiment 2

Human IL-2 3 neutralization

Human IL-2 3 is expelled in the mouse with IL-2, can stimulates splenocyte to produce mouse IL-17.Block this IL-17 hormesis at the neutrality antibody of IL-23p19 subunit, this is read by the IL-17 output of (ex vivo) in the first external back body in mouse model in as lower body and is confirmed.

Add preceding 22 hours of human IL-2 3 (hIL-23) stimulation C57BL/6 mouse with mouse IL-2 (mIL-2), by injection mIL-2 sensitized mice.Stimulating with mIL-2 and hIL-23 preceding 2 hours, to the humanized antibody of injected in mice high-affinity of the present invention or (IgG4) control antibodies of isotype coupling.

Following group is set then:

0 time point, only intraperitoneal (i.p.) injection mIL-2 (5 μ g) or mIL-2 (5 μ g) add human IL-2 3 (10 μ g)

Located in 7 hours, only peritoneal injection mIL-2 (10 μ g) or mIL-2 (10 μ g) add human IL-2 3 (10 μ g)

Located in 23 hours, only peritoneal injection mIL-2 (5 μ g) or mIL-2 (5 μ g) add human IL-2 3 (10 μ g)

Located in 30 hours, and put to death mouse, take out spleen, results splenocyte, and preparation single cell suspension.With complete RPMI (containing 1% non-essential amino acid, 1% Sodium.alpha.-ketopropionate, 2.5mM HEPES, 1%L-L-glutamic acid, 0.00035%2-mercaptoethanol, 1% penicillin/streptomycin, 10% heat-inactivated foetal calf serum) washing splenocyte, and according to 200,000 cell/200 μ l/ holes are inoculated in 96 well culture plates that wrap quilt with the anti-mouse CD3e of hamster antibody (5 μ g/ml PBS solution, 4 ℃ are spent the night) in advance.Then with 96 orifice plates in 37 ℃, 5%CO ₂In hatched 48-72 hour

Utilize mouse IL-17 sandwich ELISA test kit (M1700, peace enlightening biotechnology) to measure the amount of IL-17 in each culture supernatants.As shown in table 4, among MAbQF20 and the MAbQF37 and IL-23, and significantly block the IL-17 hormesis.

Table 4

Antibody/stimulation	IL-17 level (pg/mL) experiment 1	IL-17 level (pg/mL) experiment 2
Antibody/stimulation	IL-17 level (pg/mL) experiment 1	IL-17 level (pg/mL) experiment 2	Isotype contrast/IL-2/IL-23	4100	4500
MAbQF20/IL-2/IL-23	1600	1500	Isotype contrast/IL-2/IL-23	4100	4500
MAbQF20/IL-2/IL-23	1600	1500	MAbQF37/IL-2/IL-23	1600	1700

Embodiment 3

Binding affinity

Utilize the BIAcore measuring system to detect the avidity (table 3) of MAb3A8 and antibody of the present invention.BIAcore ^TMBe the automatic biological sensing system of measuring interaction of molecules people (1991) " immunological method magazine " (J.Immunol. Methods) 145:229-240 such as () Karlsson.In these experiments, antibody is captured BIAcore with low density ^TMOn the surface of chip.Utilize the active amino of ethyl dimethylaminopropyl carbodiimide (EDC) that a-protein is coupled to carboxymethyl (CM5) BIAcore ^TMThe flow cell of sensor chip.A-protein is diluted in the sodium-acetate buffer of pH 4.5, and utilize EDC to be fixed to the flow cell of CM5 chip, produce 1000 response units.Unreacted site is sealed with thanomin.Use flow velocity 60 μ l/min.Each circulation injection 10 μ l, 2 μ g/mL antibody-solutions of the present invention, then the human IL-2 3 that reduces gradually of injection concentration (as 1500,750,375,188,94,47,23.5,12 and 0pm), thereby carry out a plurality of combinations/wash-out circulation.Glycine hydrochloride with pH 1.5 carries out wash-out.Utilize BIAevaluation ^TMThe analytic dynamics data.

K with MAb3A8 _DValue and antibody of the present invention and commercially available anti-IL23 neutralizing monoclonal antibody MAb1290 compare (table 5).The result shows as an example the affinity constant of the antibody MAbQF20 of explanation and MAbQF37 than the low 2-6 of MAb3A8 times, the K of these antibody _DValue is well below about 160pM.

Table 5. anti-il-23 antibodies is to human IL-2 3 bonding properties

Measure by BIAcore (HBS-EP damping fluid, pH 7.4 is in 25 ℃)

ANTIBODY	k _on(M ^-1s ^-1)	k _off(s ^-1)	K _D(pM)
ANTIBODY	k _on(M ^-1s ^-1)	k _off(s ^-1)	K _D(pM)	MAb3A8	3.0×10 ⁵	9.12×10 ^-5	300
MAb1290 ^＊	3.68×10 ⁴	7.68×10 ^-4	21,400	MAb3A8	3.0×10 ⁵	9.12×10 ^-5	300
MAb1290 ^＊	3.68×10 ⁴	7.68×10 ^-4	21,400	MAbQF20	1.55×10 ⁶	1.15×10 ^-4	74
MAbQF37	1.55×10 ⁶	1.53×10 ^-4	98	MAbQF20	1.55×10 ⁶	1.15×10 ^-4	74

^*Anti-human IL-2's 3 monoclonal antibodies, mouse (peace enlightening biotechnology)

Embodiment 4

The IL-23 epitope mapping

The epi-position of the IL-23p19 subunit that humanized antibody of the present invention is discerned is included among the amino-acid residue 129-159 of SEQ IDNO:60.Detect epi-position by aminocompound deuterium exchange mass spectrum (DXMS).

DXMS can be used for detecting the protein-protein interaction between IL-23p19 subunit and the humanized antibody of the present invention.The Fab fragment of utilizing MAb3A8 is as the pattern binding fragment.Identify a plurality of epi-positions with this method, the residue 129-159 except SEQ ID NO:60 also comprises residue 129-152,151-159 and 134-152, and wherein all residue positions are all based on SEQ ID NO:60.

Utilize the standard ELISA competition assay to determine that humanized antibody of the present invention and MAb3A8 are in conjunction with identical epi-position.Candidate's humanized antibody is measured in the mode of concentration dependent with biotinylated MAb3A8.The competition of concentration dependent has shown that the ripe IL-23 of the antibodies of vying each other goes up identical epi-position.Thereby, humanized antibody of the present invention in conjunction with the IL-23p19 subunit epi-position of above being identified and in and the IL-23 activity, can be used for thus treating with IL-23 and express relevant inflammation disorder, as autoimmune disease, preferred multiple sclerosis.

Embodiment 5

The EAE model of multiple sclerosis

EAE is the cell-mediated central nervous system of CD4+T (CNS) demyelination, as being people MS model.The mechanism of causing a disease of EAE development comprises T cells with antigenic specificity activation and Th1 differentiation, then is T cell and macrophages infiltration CNS.IL-23 stimulates T emiocytosis IL-17, and IL-17 has facilitated the pathology of multiple sclerosis (MS).The IL-23 that the level that identified in the MS patients serum raises, and the microarray analysis of human patients MS pathology has shown that IL-17 increases (people's " natural medical science " such as Lock is 8:500-508 (Nat.Med.), 2002).The monocyte (MNC) of expressing IL-17mRNA in many MS blood samples of patients and the celiolymph significantly raises, and compare with the catabasis, detect blood MNC (people's " multiple sclerosis " (Multiple Sclerosis) such as Matusevicius of the expression IL-17mRNA of higher quantity in the MS clinical deterioration rates phase, 5:1-1-104,1999).

Embodiment described herein has shown the effect of anti-il-23 antibodies in recurrence remission form EAE model, wherein anti-mouse IL-23 antibody suppressor T cell secretion IL-17, thus reduce the EAE score of enlivening in the EAE model.For inducing an illness, with the 100 μ l complete Freund's adjuvants (CFA) that contain 50 μ g PLP 139-151 and 200 μ g mycobacterium tuberculosis (Mycobacterium tuberculosis) H37RA the SJL/J mouse in 8-9 age in week was carried out subcutaneous injection in two places of flank at the 0th day.On first same day catabasis (23-26 days), at random mouse is divided into 3 treatment groups: (i) carrier (PBS), (ii) IgG ₁The isotype control antibodies, 10mg/kg and (iii) mouse anti mouse IL-23 mono-clonal IgG ₁Antibody, 10mg/kg.Since first same day catabasis, animal is carried out the 0.2ml peritoneal injection, weekly then, 6 weeks by a definite date.

From the 7th day up to the 60th day the paralysis level of mouse is marked.Last is handled one week of back and is put to death mouse.The clinical symptom of disease was in development in about the 12nd day to the 15th day, and the disease peak period appears at about the 17th day to the 22nd day.Independently individual animals is carried out subjective scoring by at least two score keepers that do not understand the treatment stack features according to clinical CNS disease severity.0 is divided into normally; 0.5 divide: the unable or spasm of afterbody far-end; 1 minute: whole afterbody was unable; 1.5 divide: the unable and hind leg weakness of afterbody; 2.0 divide: acroparalysis after the one-sided part; 2.5 divide: bilateral part back acroparalysis; 3.0 divide: the whole back of bilateral acroparalysis; 3.5 divide: acroparalysis before whole hind leg and the one-sided part; 4.0 divide: forelimb and hind leg are all paralysed; 5.0 divide: dying or dead.Compare with the isotype control group, IL-23 Antybody therapy group disease score is significantly lower.

Take out the spleen of each treatment group mouse, and prepare the single cell suspension of splenocyte respectively.Splenocyte was cultivated 2 days down in the existence of PLP (be used to induce the peptide of EAE outbreak, thereby the formerly external intravital experiment in back being provided with down immune stimulatory cell mass again).After 2 days, measure the concentration of 22 kinds of cytokine/chemokines in the culture supernatants and (but use woods company limited (Linco, stdn test kit Inc.)).The IL-17 concentration of isotype contrast MAb treatment group culture supernatants is about 450pg/ml, and the IL-17 concentration of anti-il-23 MAb treatment group culture supernatants is about 175pg/ml.Thereby in anti-il-23 MAb treatment group, observe the remarkable reduction (p＜0.002) of mouse IL-17 concentration.

Sequence table

＜110〉Eli Lilly Company

＜120〉anti-il-23 antibodies

<130>X17070

<150>60/711336

<151>2005-08-25

<150>60/772355

<151>2006-02-10

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115

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115

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＜213〉homo sapiens

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50 55 60

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65 70 75 80

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85 90 95

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<212>PRT

＜213〉homo sapiens

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1 5 10 15

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100 105 110

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＜213〉homo sapiens

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Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr

1 5 10 15

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20 25 30

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35 40 45

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50 55 60

Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln

65 70 75 80

Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly

85 90 95

Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu

100 105 110

Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln Leu His Ala Ser Leu

115 120 125

Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr

130 135 140

Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu

145 150 155 160

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

165 170 175

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

180 185

<210>61

<211>329

<212>PRT

＜213〉homo sapiens

<400>61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Glu Trp Glu Thr Trp Arg Arg

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210>62

<211>329

<212>PRT

＜213〉homo sapiens

<400>62

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210>63

<211>107

<212>PRT

＜213〉homo sapiens

<400>63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>64

<211>119

<212>PRT

＜213〉homo sapiens

<400>64

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

1 5 10 15

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

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Thr Ile Gly Ala Tyr Tyr Gly Asn Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210>65

<211>112

<212>PRT

＜213〉homo sapiens

<400>65

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Thr His Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Claims

1. antibody or its antigen-binding portion thereof, its specificity be in conjunction with the IL-23p19 subunit, in and the IL-23 activity, K _DBe worth about 45pM to about 160pM, IC ₅₀Be worth about 1pM to about 20pM.

2. the antibody of claim 1 or its antigen-binding portion thereof, it is in conjunction with the IL-23p19 subunit that is positioned at aminoacid sequence amino-acid residue 129-159 shown in the SEQ ID NO:60.

3. each antibody or its antigen-binding portion thereof among the claim 1-2 are humanized antibody.

4. each antibody or its antigen-binding portion thereof among the claim 1-3, the variable region of light chain that comprises the variable region of heavy chain that is selected from SEQ IDNO:44-55 and be selected from SEQ ID NO:56-59.

5. each antibody or its antigen-binding portion thereof among the claim 1-4, the heavy chain of wherein said antibody have and are selected from following constant region: human IgG ₁, IgG ₂, IgG ₃And IgG ₄CH.

6. antibody or its antigen-binding portion thereof comprise the variable region of light chain shown in variable region of heavy chain shown in the SEQ ID NO:52 and the SEQ ID NO:57.

7. the antibody of claim 6, the heavy chain of wherein said antibody has the constant region shown in the SEQ ID NO:62, and the light chain of described antibody has the constant region shown in the SEQ ID NO:63.

8. pharmaceutical composition comprises:

(a) according to each antibody or its antigen-binding portion thereof in the claim 1 to 7; With

(b) pharmaceutically acceptable carrier.

9. the patient of needs is recurred the method for remission form multiple sclerosis therapy, comprise antibody or its antigen-binding portion thereof of using in the claim 1 to 7 of significant quantity each to described patient.

10. according to each antibody or its antigen-binding portion thereof in the claim 1 to 7, as medicine.

11. each antibody or its antigen-binding portion thereof purposes in the medicine of preparation treatment recurrence remission form multiple sclerosis patients in the claim 1 to 7.