CN101454343B

CN101454343B - Modified humanised anti-interleukin-18 antibodies

Info

Publication number: CN101454343B
Application number: CN200780018859.3A
Authority: CN
Inventors: J·H·艾利斯; V·格尔马谢夫斯基; P·A·汉布林; I·柯比
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2006-05-25
Filing date: 2007-05-23
Publication date: 2013-07-03
Anticipated expiration: 2027-05-23
Also published as: GB0610438D0; UA96595C2; CN101454343A

Abstract

The present invention discloses humanised anti-IL-18 antibodies, methods of manufacture and methods of treatment with said antibodies. Further disclosed are screening methods using for example surface plasmon resonance to identify antibodies with therapeutic potential.

Description

Humanized anti--IF1 8 antibody of modifying

1. invention field

The present invention relates generally to the immunoglobulin (Ig) field, for example antibody, particularly humanized antibody can be used for treating and diagnosing the indication that is mediated by human interferon-18.

2. background of invention

Human interferon-18 (IL-18) is the cytokine (people such as Ushio, J.Immunol.156:4274,1996) that 193 amino acid whose precursor protein matter forms as the lifeless matter activity are synthesized.By for example caspase-1 or caspase-4 cutting precursor protein, discharge 156 amino acid whose mature proteins (people such as Gu, Science, 275:206,1997; People such as Ghayur, Nature386:619,1997), show biologic activity, comprise secondary stimulus T cell proliferation, strengthen cytotoxicity, inducing T cell and the NK cells produce IFN-γ of NK cell and strengthen I type t helper cell (Th1) differentiation (people such as Okamura, Nature 378:88,1995; People such as Ushio, J.Immunol.156:4274,1996; People such as Micallef, Eur.J.Immunol.26:1647,1996; People such as Kohno, J.Immunol.158:1541,1997; People such as Zhang, Infect.Immunol.65:3594,1997; People such as Robinson, Immunity 7:571,1997).In addition, IL-18 still is the effective inductor of the short inflammatory mediator of person monocytic cell, and these media comprise IL-8, tumor necrosis factor-alpha (TNF-α) and prostaglandin E2 (PGE2) (Ushio, people such as S., J.Immunol.156:4274-4279,1996; Puren, people such as A.J., J.Clin.Invest.10:711-721,1997; People such as Podolin, J.Immunol.submitted, 1999).

Through differentiating, clone's IL-1 receptor associated protein(RAP) (IL-1Rrp) people such as (, J.Biol.Chem.271:3967,1996) Parnet was the subunit (Kd=18nM) people such as (, J.Biol.Chem.272:25737,1997) Torigoe of IL-18 acceptor in the past.Second subunit of IL-18 shows the homology with the additional albumen of IL-1 acceptor, called after AcPL (gaining the name in additional albumen-sample).IL-1Rrp and AcPL are the NF-κ B that induces of IL-18 and JNK activation necessary people such as (, J.Biol.Chem.273:29445,1998) Born.Except NF-κ B and JNK, the IL-18 signal is also by IL-1 receptor-associated kinase (IRAK), p56lck (LCK) and mitogen activated protein kinase (MAPK) work (people such as Micallef, Eur.J.Immunol.26:1647,1996; People such as Matsumoto, Biophys Biochem.Res.Comm.234:454,1997; People such as Tsuji-Takayama, Biochem.Biophys.Res.Comm.237:126,1997).

TH1 cells produce pro-inflammatory cytokine is IFN-γ, IL-2 and TNF-β (people such as Mosmann for example, J.Immunol.136:2348,1986), relate to mediation various autoimmune disease, comprise multiple sclerosis (MS), rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel (IBD) and psoriasis (Mosmann and Sad, Immunol.Today 17:138,1996).Therefore, expection TH1-promote property cytokine for example the antagonist of IL-18 will suppress disease progression.IL-18 monoclonal antibody specific (mAb) can be used as antagonist.

Verified IL-18 is in the developing effect of autoimmune disease.Correspondingly, verified before disease takes place, significantly increase in the pancreas that is expressed in non-obese diabetes (NOD) mouse of IL-18 and the spleen people such as (, J.Clin.Invest.99:469,1997) Rothe.Similarly, shown in the synovia of patient with rheumatoid arthritis the level of IL-18 significantly raise (people such as Kawashima, Arthritis and Rheumatism 39:598,1996).In addition, verified IL-18 has increased the clinical severity of mouse experiment allergic encephalomyelitis (EAE).In addition, also verified in female Louis rat, neutralization is anti--and rat IL-18 antiserum(antisera) prevented development people such as (, J.Immunol.161:6368,1998) Wildbaum of EAE.Therefore, IL-18 is the new autoimmune desirable target for the treatment of of development.

People such as Taniguchi, J.Immunol.Methods 206:107 have described the anti-human il-18 monoclonal antibody (mAb) of seven kinds of mouse and six kinds of rats, and it is in conjunction with four kinds of different antigen sites.The IFN-γ that a kind of mouse mAb (#125-2H) and six kinds of rat mAb suppress the KG-1 cell that IL-18 induces produces, in the rat mAb performance and active low 10 times of the neutralization of specific activity #125-2H.Western blotting detect to confirm, three kinds of mouse mAb, but do not have among the rat mAb a kind of, with membrane-bound human il-18 kickback.In addition, also described the enzyme linked immunological absorption that utilizes #125-2H and rat mAb to detect human il-18 and detected (ELISA).The limit that this ELISA detects is 10pg/ml.

European patent application EP 0 712 931 discloses two kinds of mouse anti human IL-18mAb, H1 (IgG1) and H2 (IgM).Western blotting detects and confirms, two kinds of mAb react with membrane-bound human il-18, and do not react with membrane-bound people IL-12.In immune affinity chromatographic experiment rules, used H1 to come the purifying human il-18, and be used in and measure human il-18 among the ELISA.H2 is used to measure human il-18 in the radioimmunoassay detection.

In and IL-18 antibody can be potentially be used for alleviating autoimmune disease and people's related symptoms.Therefore, this area needs the IL-18 antagonist of high-affinity, the neutralizing monoclonal antibody of antihuman interleukin 18 for example, and it can reduce differentiation and the propagation of Th1 cell, thereby reduces autoimmune disease and related symptoms.

All reference of mentioning in this specification are clear and definite and complete by reference being incorporated into herein all.

3. summary of the invention

According to the present invention, humanized anti-il-1 8 antibody are provided, comprise heavy chain and light chain with following complementary determining region (CDR):

CDRH1：SEQ.I.D.NO：1

CDRH2：SEQ.I.D.NO：2

CDRH3：SEQ.I.D.NO：3

CDRL1：SEQ.I.D.NO：4

CDRL2：SEQ.I.D.NO：5

CDRL3：SEQ.I.D.NO：6

CDRH1：SEQ.I.D.NO：1

CDRH2：SEQ.I.D.NO：2

CDRH3：SEQ.I.D.NO：3

CDRL1：SEQ.I.D.NO：4

CDRL2：SEQ.I.D.NO：5

CDRL3：SEQ.I.D.NO：6

Wherein, the 71st of described light chain the residue corresponding residue of finding in the donor antibody of CDR of being derived has replaced.

It will be apparent to those skilled in the art that, physics source that term " is derived " and not only is intended to define described material, but also defined material and be not that to be derived from the material of reference source structurally consistent.Therefore, " from the donor antibody framework of the CDR that derives, find " corresponding residue must be from the donor antibody framework purifying.Similarly, " derive from donor antibody " that CDR neither be from the donor antibody purifying.

Unless prompting in addition, CDR and framework region (FR) and amino acid counting are all deferred to people such as Kabat " Sequences of immunological interest ", the Kabat that proposes among NIH definition.

In another aspect of this invention, providing humanized resists-il-1 8 antibody, it comprises the CDR that derives from donor antibody that is transplanted on people's acceptor framework region, described anti--interleukin 18 antibody comprises the CDR with sequence of display in SEQ ID NO:1,2,3,4,5 and 6, wherein said anti--residue found in the corresponding position in the 71st residue of the light chain of interleukin 18 antibody and the donor antibody framework is identical.

In another aspect of this invention, provide humanized anti--il-1 8 antibody, it comprises the CDR with sequence of display in SEQ ID NO:1,2,3,4,5 and 6, described antibody comprises tyrosine the 71st of light chain.

In another aspect of this invention, providing humanized resists-il-1 8 antibody, comprise the heavy chain of the CDR that has in SEQ ID NO:1,2 and 3 display and have the light chain of the CDR of display in SEQ ID NO:4,5 and 6, the CDR of wherein said light chain derives from donor antibody, and described donor antibody has tyrosine the 71st of donor antibody light chain.

In another aspect of this invention, providing humanized resists-il-1 8 antibody, comprise the CDR from donor antibody, and the 71st at the light chain of described humanized antibody has tyrosine, and wherein said donor antibody is that (that is, described humanized antibody comprises the CDR identical with 2C10 for 2C10 or its framework variant, but different framework, referring to US patent 6,706,487).

In another aspect of this invention, provide humanized and resisted-il-1 8 antibody, comprised:

(a) heavy chain, its have contain be transplanted on people's heavy chain acceptor framework in SEQ ID NO:1,2 and 3 display sequence CDR and

(b) light chain, it has and contains the CDR of sequence of display in SEQ ID NO:4,5 and 6 that is transplanted on people's light chain acceptor framework, wherein said people's light chain acceptor framework comprises the framework region of deriving from SEQ IDNO:38, and wherein the 71st of SEQ ID NO:38 the is tyrosine.

(a) heavy chain, its have allow the CDR be combined with the human il-18 specificity and

(b) light chain, it has the acceptor framework, and has the CDR that contains the sequence of display in SEQ ID NO:4,5 and 6, and has tyrosine residues at the 71st.

The CDR of described light chain is preferably located in the position in the acceptor framework, and these positions are corresponding to the position of the sequence that displays in SEQ ID NO:4,5 and 6 in the sequence that displays in SEQ ID NO:35.Described light chain and/or heavy chain preferably in human patients right and wrong immunogenic.

(a) heavy chain, its comprise the sequence that has in SEQ ID NO:1,2 and 3 display CDR and

(b) light chain, it comprises the CDR that is transplanted to the sequence that displays on people's light chain acceptor framework in SEQ ID NO:4,5 and 6, the described light chain acceptor framework region of wherein said humanized resisting-il-1 8 antibody is derived from the variant of the sequence that displays in SEQ ID NO:38, wherein said variant comprises tyrosine at the 71st, and described variant comprises with the sequence that displays in SEQ ID NO:38 and has 75% or the sequence of higher identity.Preferably, described variant comprises with the sequence that displays in SEQ ID NO:38 80% or higher for example 81%, 82%, 83%, 84% identity, more preferably, 85% or higher, 86%, 87%, 88%, 89% identity for example, even more preferably, 90% or higher, 91%, 92%, 93%, 94% identity for example, more preferably, 95% or higher, 96%, 97%, 98%, 99% identity for example.

In another aspect of this invention, provide humanized and resisted-il-1 8 antibody, this antibody comprises:

(a) CDR of sequence of display among SEQ ID NO:1,2,3,4,5 and 6 that derives from donor antibody, described donor antibody contains tyrosine the 71st of donor antibody;

(b) people's acceptor framework, described acceptor framework contains phenylalanine the 71st of people's light chain;

Wherein said anti--interleukin 18 antibody contains tyrosine the 71st of light chain.

(a) CDR of display among SEQ ID NO:1,2,3,4,5 and 6 that derives from donor antibody, described donor antibody contains die aromatischen Aminosaeuren the 71st of donor antibody light chain;

(b) people's acceptor framework, described acceptor framework the 71st of light chain acceptor framework contain with (a) part in the dissimilar die aromatischen Aminosaeuren of die aromatischen Aminosaeuren;

Wherein said resisting-interleukin 18 antibody comprises the light chain of deriving from antibody of (a) part, and it contains die aromatischen Aminosaeuren at the 71st.

In another aspect of this invention, provide humanized anti--il-1 8 antibody, when at 37 ℃ with surface plasma body resonant vibration (for example, Biacore ^TM, preferably use Biacore ^TM3000 instruments and the condition that provides among the 7.4.1 hereinafter) when measuring, described antibody is at the equilibrium constant that has 300pM in conjunction with human il-18.

In another aspect of this invention, provide humanized anti--il-1 8 antibody, described antibody is included in the CDR of display among the SEQ ID NO:1,2,3,4,5 and 6, and when at 37 ℃ with surface plasma body resonant vibration (for example, Biacore ^TM, preferably use Biacore ^TM3000 instruments and the condition that provides among the 7.4.1 hereinafter) when measuring, described antibody is for the equilibrium constant that has 300pM in conjunction with human il-18.

Preferably, the equilibrium constant of relevant antibodies human il-18 at 37 ℃ with surface plasma body resonant vibration (for example, Biacore ^TM, preferably use Biacore ^TM3000 instruments and the condition that provides among the 7.4.1 hereinafter) be less than 90pM when measuring.More preferably, the equilibrium constant is 70pM or still less, more preferably is 65pM, 60pM, 55pM or 50pM or still less.

In another aspect of this invention, provide humanized anti--il-1 8 antibody, described antibody at 37 ℃ with surface plasma body resonant vibration (for example, Biacore ^TM, preferably use Biacore ^TMT100 instrument and the condition that provides among the 7.4.2 hereinafter) when measuring, show 0.00021/s or higher dissociation constant or dissociation rate (kd) for human il-18.

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on the heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:37, one or more residues of the 27th, 28,29,93,39,40,36,71,89,91 of wherein said heavy chain are identical with the corresponding residue in the donor antibody heavy chain;

(b) comprise the light chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on the light chain acceptor framework that displays in SEQ ID NO:4,5 and 6, described light chain acceptor framework comprises the framework region that the sequence that displays is derived from SEQ ID NO:38, the 71st of wherein said light chain identical with corresponding residue in the donor antibody light chain with the 45th, 83,84,85 optional one or more residues.

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on people's heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region that the display sequence is derived from SEQ ID NO:37, and the 27th, 28,29,93 residue of wherein said heavy chain is identical with the corresponding residue in the donor antibody heavy chain;

(b) comprise the light chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on the light chain acceptor framework that displays in SEQ ID NO:4,5 and 6, described light chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:38, the 71st residue of the light chain of wherein said resisting-il-1 8 antibody is identical with the corresponding residue in the donor antibody light chain.

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on people's heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:37, the 27th, 28,29,39,40,93 residue of wherein said heavy chain is identical with the corresponding residue in the donor antibody heavy chain;

(b) comprise the light chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on the light chain acceptor framework that displays in SEQ ID NO:4,5 and 6, described light chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:38, the 71st residue of wherein said light chain is identical with the corresponding residue in the donor antibody light chain.

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on people's heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:37, the 27th, 28,29,36,39,40,71,89,91,93 residue of wherein said heavy chain is identical with the corresponding residue in the donor antibody heavy chain;

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on people's heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:37, the 27th, 28,29,93 residue of wherein said heavy chain is identical with the corresponding residue in the donor antibody heavy chain;

(b) comprise the light chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on the light chain acceptor framework that displays in SEQ ID NO:4,5 and 6, described light chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:38, the 71st, 45,83,84,85 residue of wherein said light chain is identical with the corresponding residue in the donor antibody light chain.

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on people's heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:37, the 27th, 28,29,93,39,40 residue of wherein said heavy chain is identical with the corresponding residue in the donor antibody heavy chain;

(a) comprise the heavy chain of the CDR that derives from donor antibody, described CDR has the sequence of being transplanted on people's heavy chain acceptor framework that displays in SEQ ID NO:1,2 and 3, described heavy chain acceptor framework comprises the framework region of deriving from the sequence that displays among SEQ ID NO:37, the 27th, 28,29,93,39,40,36,71,89,91 residue of wherein said heavy chain is identical with the corresponding residue in the donor antibody heavy chain;

In another aspect of this invention, provide the humanization that comprises heavy chain and light chain to resist-il-1 8 antibody, wherein said antibody in the time of 25 ℃ and the dissociation rate (kd) of the human il-18 of combination and described antibody in the time of 37 ℃ and the ratio between the dissociation rate (kd) of the human il-18 of combination be 1:5 or still less, wherein said antibody comprises CDR and people's acceptor framework of deriving from donor antibody, and the 71st residue of the light chain of wherein said people's acceptor framework is replaced by the corresponding residue from donor antibody.Preferably use Biacore ^TMT100 instrument and the condition that provides among the 7.4.2 are hereinafter measured dissociation rate.

In another aspect of this invention, provide humanization to resist-il-1 8 antibody the light chain that it comprises the heavy chain that is selected from SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 and is selected from SEQ IDNO:13, SEQ ID NO:29.

In another aspect of this invention, provide humanization to resist-il-1 8 antibody, it comprises the heavy chain of SEQID NO:9 and the light chain of SEQ ID NO:13, the perhaps light chain of the heavy chain of SEQ ID NO:9 and SEQ ID NO:29.

In another aspect of this invention, provide humanization to resist-il-1 8 antibody, it comprises the heavy chain of SEQID NO:17 and the light chain of SEQ ID NO:13, the perhaps light chain of the heavy chain of SEQ ID NO:17 and SEQ ID NO:29.

In another aspect of this invention, provide humanization to resist-il-1 8 antibody, it comprises the heavy chain of SEQID NO:21 and the light chain of SEQ ID NO:13, the perhaps light chain of the heavy chain of SEQ ID NO:21 and SEQ ID NO:29.

In another aspect of this invention, provide and comprised herein anti--il-1 8 antibody of describing and the pharmaceutical composition of pharmaceutically acceptable carrier.

In another aspect of this invention, provide the method for screening the antibody (particularly suppressing interactional antibody between part and the acceptor, for example anti--il-1 8 antibody) that is used for the treatment of purposes, described method comprises:

(a) binding affinity of measuring the antigen of antibody antagonist specificity combination at 30 to 45 ℃ (preferred 37 ℃) (for example uses: Biacore ^TMSurface plasma body resonant vibration);

(b) binding affinity of measuring the antigen of antibody antagonist specificity combination at 20 to 25 ℃ (preferred 25 ℃) (for example uses: Biacore ^TMSurface plasma body resonant vibration);

(c) if avidity (a) greater than the avidity of (b), preferably, if avidity (a) is bigger 2 times or more than the avidity of (b), more preferably, 4 times or more, selects described antibody to be used for the treatment of purposes.

In another aspect of this invention, provide the method for the antibody (particularly suppressing interactional antibody between part and the acceptor, for example anti--il-1 8 antibody) of selecting to be used for the treatment of purposes, described method comprises:

(a) (use for example: Biacore when 30 to 45 ℃ (preferred 37 ℃), measure dissociation rate between antibody and the antigen that antibodies specific is combined ^TMSurface plasma body resonant vibration);

(b) (use for example: Biacore measure dissociation rate between antibody and the antigen that antibodies specific is combined at 20 to 25 ℃ (preferred 25 ℃) ^TMSurface plasma body resonant vibration);

(c) if dissociation rate (a) is lower than the dissociation rate of (b), then select described antibody to be used for the treatment of purposes.

Term " anti--il-1 8 antibody " mean when referring to antibody of the present invention this type of can in and the antibody of the biologic activity of human interleukin-18.It does not get rid of this antibody-like also can in and the situation of the biologic activity of non-human primates (for example rhesus monkey and/or stump-tailed macaque) il-1 8.

Brief description of drawings

Also further describe invention with reference to the accompanying drawings, wherein:

Fig. 1 has shown the influence of temperature to the association rate (ka) of H1L1 and H1L2;

Fig. 2 has shown the influence of temperature to dissociation rate (kd);

Fig. 3 has shown the influence of temperature to the equilibrium constant (KD);

Fig. 4 A-4C has shown the representative data from an experiment of the EC50 value shown in the generation table 7;

Fig. 5 has shown that the humanization variant of four kinds of selections is in conjunction with the EC50 value of human il-18;

Fig. 6 has shown that the humanization variant of selection is in conjunction with the EC50 value of human il-18;

Fig. 7 has shown the combination of H1L2 and human il-18 when having 50% synovia;

Fig. 8 has shown in KG1 detects, the restraining effect that the IFN-γ that IL-18 is excited produces;

Fig. 9 A and 9B have shown respectively in 10% and 25% autoserum, the restraining effect that the IFN-γ that in human PBMC S donor LPS is excited produces;

Figure 10 has shown that 2C10 is in conjunction with the hIL18 that is caught by hIL18BP;

Figure 11 has shown that 9 kinds of humanized variants suppress the ability that discharges IFN-γ in the KG1 cell that IL-18 excites;

Figure 12 has shown the restraining effect of the IFN-γ production that H1 variant and 2C10 excite IL-18 in the KG1 cell;

Figure 13 has shown the IC50 data of H1 variant when getting 95% fiducial interval;

Figure 14 has shown the restraining effect of the IFN-γ production that in the KG1 cell IL-18 is excited;

Figure 15 has shown the restraining effect of the IFN-γ production that in the KG1 cell rhesus monkey IL-18 is excited;

Figure 16 has shown that the human il-18 that uses chimeric 2C10 is in conjunction with the result of ELISA;

Figure 17 has shown that the rhesus monkey IL-18 that uses chimeric 2C10 is in conjunction with the result of ELISA;

Figure 18 A and 18B shown and used H1L2 and 2C10 respectively, in conjunction with the result in conjunction with ELISA of the IL-19BP of human il-18 combination.

4. humanized antibody

Utilize complete non-human antibody to treat human diseases or obstacle, itself just has the potential immunogenicity problem that has generally been confirmed at present, particularly based on the repeat administration of antibody.That is, patient's immune system is identified as inhuman complete antibody the dissident and initiates neutralization reaction.Except developing completely people's antibody (referring to above), develop various technology for many years and overcome these problems, these technology relate generally to reduce the non-human aminoacid sequence composition in the complete antibody, but keep the relative simplification that obtains the non-human antibody from inoculation animal (for example mouse, rat or rabbit).Put it briefly, existing two kinds of methods can reach this purpose.First method is chimeric antibody, generally comprises the variable region of the non-human (for example: rodents is mouse for example) that merges with human constant region.Because the antigen binding site of antibody is positioned at the variable region, chimeric antibody has still kept it to the binding affinity of antigen, but has obtained the effector function of human constant region, therefore can bring into play the effector function of above describing.The general using recombinant DNA method prepares chimeric antibody.Separate encoding antibody DNA (for example: cDNA), with ordinary method order-checking (for example using the oligonucleotide probe that can be combined specifically with the H of code book invention antibody and the gene of L chain (the above-mentioned SEQ ID NO:1 that for example encodes, 2,3,4,5 and 6 DNA)).With the common source of hybridoma as this type of DNA.Express chimeric vector if desired, the cDNA that separates the complete ripe variable region of coding light chain and heavy chain, to be inserted in the suitable expression vector in its frame, in addition, also to contain generally be that the suitable constant region for immunoglobulin of human origin and signal sequence, terminator codon, promotor, terminator and other obtain the required element of antibody expression to described carrier.Then, with the examples of such carriers transfection to as then can not produce without transfection and to obtain the synthetic of antibody in the host cell (for example: E.Coli, COS cell, Chinese hamster ovary celI or myeloma cell) of immunoglobulin (Ig).H and the L constant region that can replace corresponding non-human (for example mouse) by the encoding sequence of personnel selection L and H chain are come DNA is modified, referring to Morrison, and PNAS81,6851 (1984).

Second method relates to the preparation humanized antibody, wherein, has reduced the non-human component of antibody by the humanization variable region.Two kinds of humanization technology commonly used are arranged.First kind is to transplant by CDR to realize humanization.CDR forms ring at the N-terminal near antibody, and the support that provides at framework region forms the surface.The antigen-binding specificity of antibody mainly is to be determined by the topology on this CDR surface and chemical feature.These features be again conversely by the relative distribution of the conformation of each CDR, CDR and comprise CDR the residue side chain character and distribute to determine.Only be transplanted on people's framework region (acceptor framework) and the constant region by the CDR with non-human (for example mouse) antibody (donor antibody), just can reduce immunogenicity greatly (referring to people such as Jones, (1986) people such as Nature321:522-525 and Verhoeyen M, (1988) Science 239:1534-1536).But the antigen binding characteristic that the CDR transplanting may not can obtain to keep fully itself, people often find: if recover main antigen-binding affinity, just need to keep some framework residues (being sometimes referred to as " reverse mutation ") in the donor antibody (referring to, people such as Queen C, (1989) PNAS 86,10,029-10,033, Co, people such as M, (1991) Nature 351,501-502).In the case, for people's framework region (FR) is provided, can from database, select the people V district with non-human donor antibody sequence homology the highest (general 60% or higher).Can from people's consensus sequence or each one antibody, select people FR.In case of necessity, the Key residues of donor antibody can be replaced in people's acceptor framework region, thereby keep the CDR conformation.The computer model of antibody can be used for assisting to identify residue important on these structures, referring to W099/48523.

Alternatively, can also realize humanization by the process of " finishing (veneering) ".The heavy chain immunoglobulin of people and mouse uniqueness and the statistical analysis of variable region of light chain disclose, in the antibody of people and mouse, the accurate model of exposed residue is different, most single surface locations to seldom several different residues have strong preference (referring to, people such as Padlan E.A., (1991) Mol.Immunol.28, people such as 489-498 and Pedersen J.T., (1994) J.Mol.Bio.235; 959-973).Therefore, by replacing exposed residues different with the residue of in people's antibody, finding usually in the framework region, just may reduce the immunogenicity of inhuman Fv.Because the antigenicity of protein may be relevant with its surperficial accessibility, replacing surface residue may be enough to make human immune system " to ignore " the mouse variable region (also referring to people such as Mark G E., (1994) the 113rd phase of Handbook of ExperimentalPharmacology: The pharmacology of monoclonal Antibodies, Springer-Verlag, the 105-134 page or leaf).This humanization process is called as " finishingization ", does not disturb and supports residue because only having changed the surface of antibody.Other optional method comprises method and the Humaneering that proposes among the WO04/006955 ^TM(Kalobios) method, its utilize approach on the expression system production sequence of bacterium human antibody of planting system (Alfenito-MAdvancing Protein Therapeutics, January 2007, San Diego, Califomia).In addition, present humanization method relates on the basis of the structural similarity in people CDR district and donor mice antibody CDR district, but not based on other zone of antibody homology of framework region for example, selects people's acceptor framework.This method is also referred to as Superhumanization ^TM(Evogenix Inc.; People such as Hwang, (2005) Methods 36:35-42).

Therefore, the present invention relates to the above humanized antibody of the 3rd joint proposition.This antibody-like preferably contains IgG isotype (for example IgG1 or IgG4)

In alternate embodiment, can allow the humanized antibody of above the 3rd joint proposition and non-human constant region (" anti-phase chimeric ") merge, for example non-human primates, rat, mouse or rabbit.

Certainly it will be apparent to those skilled in the art that to be that the acceptor framework of display has constituted the immunoglobulin amino acid of being encoded by VH and V kappa gene respectively in SEQ ID NO:37 and 38.So, they have comprised the CDR of framework region and receptor antibody simultaneously.The donor CDR that is used among the SEQ ID NO:1,2,3,4,5 and 6 display replaces receptor antibody CDR and with the sequence that obtains and suitable frame 4 sequences (for example SEQ ID NO:39 and SEQ ID NO:40 display sequence) combination, thereby (for example produce complete immune globulin variable region, SEQ ID NO:11 and SEQ ID NO:15 display), belong to those skilled in the art's ability category fully.

4.1 other modification

Think the Fc district of antibody and the effector function of the interaction mediate antibody between each Fc acceptor (Fc γ R), comprise fixing, the cytophagy of cytotoxicity (ADCC), complement of antibody dependent and antibody the transformation period/elimination.Can carry out various modifications to the Fc district of antibody of the present invention according to desirable effector characteristic.For example describe in detail among EP 0 629 240 B1 and EP 0 307 434 B2, carry out specific sudden change in the Fc district, make the antibody of original cracking performance become non-cracking performance; Perhaps can in antibody, mix and remedy the receptors bind epi-position, improve its serum half-life (seeing US5739277).5 kinds of human Fc gamma receptors of identification are Fc γ R (I), Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and newborn FcRn at present.People such as Shields, (2001) J.Biol.Chem276 6591-6604) has confirmed that one group of public IgG1 residue participates in and the combination of all Fc γ R, Fc γ RII and Fc γ RIII also utilize this to organize some different loci outside the public residue simultaneously.In the time of will organizing the IgG1 residue and change into L-Ala with next, reduce the combination with all Fc γ R: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239.Described residue all is positioned at the CH2 district of IgG, and bunch collection is near the hinge that connects CH1 and CH2.Fc γ R1 only utilizes public IgG1 residue group to carry out combination, and Fc γ RII and Fc γ RIII also interact with some unique residues except public residue group.The change of some residues only weaken with the combination (for example Arg-292) of Fc γ RII or with the combination (for example Glu-293) of Fc γ RIII.Some variants show the combination with the improvement of Fc γ RII or Fc γ RIII, but do not influence and the combination of other acceptors (for example, Ser-267Ala improves the combination with Fc γ RII, but does not influence the combination with Fc γ RIII).Other variant then shows the combination with the improvement of Fc γ RII or Fc γ RIII, but with other acceptors in conjunction with descend (for example, Ser-298Ala has improved the combination with Fc γ RIII, but with Fc γ RII in conjunction with descending).For Fc γ RIIIa, the L-Ala that combines at Ser-298, Glu-333 and Lys-334 place in conjunction with best IgG1 variant replaces.Think that neonatal Fc Rn acceptor has participated in cleaning antibody and inter-organization transcytosis (referring to, people such as Junghans R.P (1997) Immunol.Res 16.29-57 and Ghetie, (2000) Annu.Rev.Immunol.18,739-766).Determine to comprise with human IgG1's residue of people FcRn direct interaction: Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435.Therefore, the present invention relates to have the antibody of the invention that the residue of each above-detailed changes, described change can modify the transformation period/removings and/or change effector function (for example ADCC and/or complement cracking).

Other modifications comprise the glycosylation variant of antibody of the present invention.The glycosylation antagonist function of known conservative site antibody at antibody constant region particularly has deep effect to for example above-described effector function, referring to for example: people such as Boyd, (1996), MoI.Immunol.32,1311-1318.Also consider treatment of the present invention with the glycosylation variant of antibody or its Fab, wherein increase, replace, delete or modified one or more carbohydrate part.Introduce l-asparagine-X-Serine or l-asparagine-x-Threonine motif, produced the attached potential site of enzymatic that is used for the carbohydrate part, therefore can be used for handling the antibody glycosylation.People such as Raju, (2001) Biochemistry 40 is among the 8868-8876, utilize β-1,4-galactosyltransferase and/or α-2, and the 3-sialytransferase has improved the terminal sialylated of TNFR-IgG immunoadhesin by semi-lactosi glycosylation and/or sialylated more again.Thinking increases the terminal sialylated transformation period that can prolong immunoglobulin (Ig).The same with most glycoprotein, antibody normally produces with the form of sugared type mixture.At eukaryotic cell, when particularly producing antibody in mammalian cell, this mixture is especially obvious.Develop several different methods and prepared the sugared type of appointment, referring to people such as Zhang, Science (2004), people such as 303,371, Sears, Science, (2001) 291,2344, people such as Wacker, (2002) Science, 298 1790, people such as Davis, (2002) Chem.Rev.102,579, people such as Hang, (2001) Acc.Chem.Res 34,727.Therefore, the present invention has considered to relate to multiple therapeutic as described herein (mono-clonal) antibody (can be IgG isotype, for example IgG1), described antibody comprise specified quantity (for example 7 or below, for example 5 or below, for example 2 or 1) described antibody or the sugared type of Fab.

Other embodiment of the present invention comprises with the treatment of the present invention of non-albumen polymer (for example polyoxyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene (polyoxyalkylene)) coupling with antibody or its Fab.Making protein and PEG coupling is prior art, the antigenicity and the immunogenicity that can be used for improving the protein transformation period, reduce protein.The purposes that the PEG that has adopted complete antibody and Fab ' fragment to study different molecular weight and form (linear or branch) modifies is referring to people such as Koumenis I.L., (2000) Int.J.Pharmaceut.198:83-95.

5. preparation method

Can in genetically modified organism, produce antibody of the present invention, these genetically modified organisms for example are that goat is (referring to people such as Pollock, (1999), J.Immunol.Methods 231:147-157), chicken (referring to Morrow KJJ (2000) Genet.Eng.News 20:1-55), mouse (see people such as Pollock, the same) or plant (see Doran PM, (2000) Curr.Opinion Biotechnol.11,199-204, Ma JK-C (1998), Nat.Med.4; 601-606, people such as Baez J, BioPharm (2000) 13:50-54, people such as Stoger E; (2000) Plant MoI.Biol.42:583-590).Also can produce antibody by chemosynthesis.But antibody of the present invention normally utilizes reconstitution cell culture technique preparation well known to those skilled in the art.The polynucleotide that separate encoding antibody are inserted into replicable vector (for example plasmid) and are used for further cloning (amplification) or expressing at host cell.An effectively expressing system is NADPH-linked glutamate synthase system (for example Lonza Biologics sells), especially (sees below) when host cell is CHO or NSO.Utilize traditional program (for example oligonucleotide probe) can be easily the polynucleotide of encoding antibody to be separated and check order.Available carrier comprises plasmid, virus, phage, transposon, minichromosomes, and wherein plasmid is representational embodiment.In general, examples of such carriers also comprises signal sequence, replication orgin, one or more marker gene, enhancer element, promotor and the transcription termination sequence that operably is connected with light chain and/or heavy chain polynucleotide, thereby is conducive to express.The polynucleotide of coding light chain and heavy chain can be inserted isolated vectors, and while or priority introducing (for example: by conversion, transfection, electroporation or transduction) same host cell, perhaps if desired, can before above-mentioned transduction, heavy chain be inserted identical carrier with light chain.

It will be apparent for a person skilled in the art that because the redundancy of genetic code the alternative polynucleotide sequence of the disclosed polynucleotide polypeptide of the present invention of also can encoding in the literary composition.

5.1 signal sequence

Can produce antibody of the present invention with the fusion rotein form that has the allos signal sequence, described signal sequence has the specificity cleavage site at the N-terminal of mature protein.Host cell can be identified and the processing signal sequence.For prokaryotic host cell, signal sequence can be alkaline phosphatase, penicillinase or heat-staple enterotoxin 1 I leader sequence.For the yeast secretion, signal sequence can be yeast invertase leader sequence, alpha factor leader sequence or acid phosphatase leader sequence (seeing as W090/13646).In the mammal cell line system, obtainable is viral secretory leader sequence (for example hsv gD signal) and native immunoglobulin signal sequence (for example people's Ig heavy chain).Usually, the polynucleotide of signal sequence and code book invention antibody are connected in the same reading frame.

5.2 replication orgin

Replication orgin well known in the art contains pBR322, the various virus replication starting points that are fit to most saccharomycetic 2 μ plasmids and are fit to most mammalian cells, for example SV40, polyomavirus, adenovirus, VSV or the BPV that is fit to most gram negative bacteriums.Usually, the mammalian expression vector of integration does not need the replication orgin composition, unless need breed by carrier in E.Coli.But SV40 ori contains early promoter, so can be used.

5.3 selective marker

Typically select the such protein of genes encoding: (a) give the resistance to microbiotic (for example penbritin, Xin Meisu, methotrexate or tsiklomitsin) or other toxin, perhaps (b) complementary auxotroph or provide the nutrition that does not have in the complex medium, perhaps (c) both combinations.The principle of selecting can relate to stops growing the host cell that does not contain carrier.Those have successfully transformed the cell of code book invention treatment with the gene of antibody, owing to for example drug resistance that the selective marker of sending is jointly given is survived.An example is the DHFR-selective system, and wherein, transformant is (for example referring to, Page and Sydenham 1991Biotechnology 9:64-68) that produces from the host strain of DHFR feminine gender.In this system, DHFR gene and antibody polynucleotide sequence of the present invention are sent jointly, select the DHFR positive cell by removing Nucleotide then.If desired, can also use the inhibitor methotrexate of DHFR to select the transformant of DHFR gene amplification.By the DHFR gene being operably connected to antibody coding sequence of the present invention or its functional derivatives, the amplification of DHFR gene can obtain the common amplification of needed target antibody sequence.Chinese hamster ovary celI is particularly suitable for this DHFR/ methotrexate and selects, it also is generally known in the art using the amplification of DHFR system and selecting the method for host cell, referring to people such as Kaufman R.J., J.MoI.Biol. (1982) 159,601-621, about summary, referring to Werner RG, NoeW, Kopp K, Schluter M, ＂ Appropriate mammalian expression systems forbiopharmaceuticals ＂, Arzneimittel-Forschung.48 (8): 870-80,1998Aug.Other example is NADPH-linked glutamate synthase expression system (Lonza Biologics).The selection gene that is suitable for using in yeast is the trpl gene, referring to people such as Stinchcomb, and Nature282,38,1979.

5.4 promotor

Be operatively coupled on for the DNA/ polynucleotide of the suitable promotor of expressing antibody of the present invention with encoding antibody.The promotor that is used for prokaryotic hosts comprises phoA promotor, β-Nei Xiananmei and lactose promoter systems, alkaline phosphatase, tryptophane and hybrid promoter, for example Tac.Be suitable in yeast cell, carrying out expression promoter and comprise glycerol 3-phosphate kinases or other glycolytic ferments, for example Hydratase, phosphoenolpyruvate, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, G-6-P isomerase, glycerol 3-phosphate mutase and glucokinase.Derivable Yeast promoter comprises the enzyme of alcoholdehydrogenase 2, homotype cytochrome C, acid phosphatase, metallothionein(MT) and responsible nitrogen metabolism or maltose/galactose utilization.

Be used for comprising the rna plymerase ii promotor in mammal cell line system expression promoter, comprise viral promotors, for example polyomavirus, fowlpox virus and adenovirus (for example adenovirus 2), bovine papilloma virus, avian sarcomata virus, cytomegalovirus (particularly immediate early gene promotor), retrovirus, hepatitis B virus, Actin muscle, Rous sarcoma virus (RSV) promotor and early stage or late period simian virus 40; And non-viral promotors EF-1 α (Mizushima and Nagata Nucleic Acids Res 1,990 18 (17): 5322) for example.The selection of promotor can based on the consistency of the host cell of be used for expressing.

5.5 enhancer element

When suitable (for example be used for when higher eucaryotic cells is expressed), can use with promoter element and operably be connected in enhancer element in the carrier mutually.Suitable Mammals enhancer sequence comprises the enhancer element from sphaeroprotein, elastoser, white protein, fetoprotein and Regular Insulin.Alternatively, can use the enhancer element from eukaryotic cell virus, for example SV40 enhanser, the sub-enhanser of cytomegalovirus early promoter, polyomavirus enhanser, baculovirus enhanser or mouse IgG2a seat (referring to W004/009823).General this type of enhanser is positioned at the position of promotor upstream at carrier, but also can be positioned at any position, for example: in the downstream of non-translational region or polyadenylic acid signal.The selection of enhanser and place can based on the consistency of the host cell that is used for expressing.

5.6 poly-adenosine/termination

In eukaryotic system, the poly-adenosine signal operationally is connected with the polynucleotide of code book invention antibody.This type of signal generally is positioned at 3 ' end of open reading frame.In mammlian system, nonrestrictive signal example from tethelin, elongation factor-1 α and virus (for example: SV40) the long terminal repetition signal of deriving of gene or retrovirus comprises.In Yeast system, poly-adenosine/termination signal comprises the signal of deriving from phosphoglycerokinase (PGK) and ethanol dehydrogenase 1 (ADH) gene.In prokaryotic system, generally do not need the poly-adenosine signal, replace and general use shorter and terminator sequence more clearly.The selection of poly-adenosine/termination signal can based on the consistency of the host cell of be used for expressing.

5.7 be used for improving other method/element of output

Except foregoing, can also use further feature to improve output, comprise chromosome reconstitution element, intron and the modification of host cell specificity codon.The codon that can adapt to host cell has a preference for to modify the codon usage of antibody of the present invention, thus improve transcribe and/or product production (for example: people such as Hoekema A, Mol Cell Biol 1,987 7 (8): 2914-24).The selection of codon can based on the consistency of the host cell of be used for expressing.

5.8 host cell

The proper host cell that is used for the carrier of clone or expression code book invention antibody is protokaryon, yeast or higher eucaryotic cells.Suitable prokaryotic cell prokaryocyte comprises eubacterium, enterobacteriaceae for example, and (for example (for example: ATCC 31446 for E.coli as Escherichia; 31537; 27325)), enterobacter, erwinia, Klebsiella, proteus, salmonella such as Salmonella typhimurium (Salmonella typhimurium), serratia such as serratia marcescens (Serratiamarcescans) and Shigella, and bacillus such as subtilis (B.subtilis) and Bacillus licheniformis (B.licheniformis) (referring to, DD 266 710), false pseudomonas bacillus such as pseudomonas aeruginosa (P.aeruginosa) and streptomycete.Yeast host cell have yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (schizosaccharomyces pombe), kluyveromyces (Kluyveromyces) (for example: ATCC 16045,12424,24178,56500), Ye Shi yeast (yarrowia) (EP402226), pichia pastoris phaff (Pichia pastoris) (EP183070; Also referring to people such as Peng, J.Biotechnol.108 (2004) 185-192), candidiasis (Candida), Trichodermareesei (Trichoderma reesia) (EP 244 234), Penicillium notatum (Penicillin), curved neck mould (Tolypocladium) and Aspergillus (Aspergillus), for example: Aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Though the present invention considers eucaryon and yeast host cell especially, usually, host cell of the present invention is vertebrate cells.Suitable vertebrate host cell comprises mammalian cell, for example: COS-1 (ATCC No.CRL 1650), COS-7 (ATCC CRL 1651), the human embryonic kidney cell is 293, baby hamster kidney cell (BHK) (ATCC CRL.1632), BHK570 (ATCCNO:CRL 10314), 293 (ATCC NO.CRL 1573), Chinese hamster ovary cell CHO (CHO-K1 for example, ATCC NO:CCL 61), DHFR-CHO clone (DG44 for example, referring to people such as Urlaub, (1986) the same), particularly the Chinese hamster ovary celI of those suitable suspension culture is, mouse sustenticular cell (sertoli cell), monkey-kidney cells, African green monkey kidney cell (ATCCCRL-1587), the HELA cell, Madin-Darby canine kidney(cell line) (ATCC CCL 34), human pneumonocyte (ATCC CCL75), Hep G2 and myelomatosis or lymphoma cell are as NSO (referring to US 5807715), Sp2/0, Y0.

Therefore, one embodiment of the invention provides the host cell of stable conversion, and it comprises coding the treatment described herein heavy chain of antibody and/or the carrier of light chain.General this type of host cell comprises encode first carrier of light chain and second carrier of encoding heavy chain.

This type of host cell be can also further transform or debug, thereby quality, function and/or the output of antibody of the present invention modified.Non-limiting instance comprises enzyme and the protein folding companion who expresses specific modification (for example glycosylation).

5.9 cell culture processes

Can cultivate by any method known to those skilled in the art with the carrier transformed host cells of code book invention treatment with antibody.Host cell can be cultivated at the rotation angle bottle, shake bottle, roll in bottle or the tubular fibre system, but for scale operation, preferred agitator tank reactor or bag reactor (for example Wave Biotech, Somerset, New Jersey USA) carry out suspension culture.General using for example decollator, baffle plate or the low shear agitation wing is transformed into the adaptation ventilation with stirred pot.For bubble tower and airlift reactor, can directly ventilate with the air or oxygen bubble.When host cell is cultivated in serum free medium, preferably in substratum, replenish cytoprotective (for example Pluronic F-68), the protection cell is avoided the damage that venting process causes.According to the characteristics of host cell, anchorage-dependent cell system can use microcarrier as growth matrix, perhaps makes cell adapted suspension culture (common situation).Host cell, the particularly cultivation of vertebrate host cell, can use multiple modes of operation, for example fed-batch, repeated batch are cultivated (referring to people such as Drapeau, (1994) cytotechnology 15:103-109), are continued single batch of cultivation or perfusion cultivation.Though can in the substratum that contains serum (as foetal calf serum (FCS)), cultivate the mammalian host cell of recombinant conversion, but, preferably at synthetic serum free medium (for example people such as Keen is disclosed among (1995) Cytotechnology 17:153-163) or commercially available substratum (for example ProCHO-CDM or UltraCHO ^TM(Cambrex NJ, USA)) middle this type of host cell of cultivating, if desired, can also replenish necessary energy source such as glucose and the synthetically grown factor such as Recombulin.The serum-free culture of host cell requires these cell adaptedly to grow under the condition of serum-free.A kind of adaptive method is to cultivate this type of host cell in containing the substratum of serum, repeatedly change 80% substratum into serum free medium then, make host cell study adapt to serum-free condition (referring to people such as for example Scharfenberg K, (1995) in Animal Cell technology:Developments towards the 21st century (people such as Beuvery E.C. writes), the 619-623 page or leaf, Kluwer Academic publishers).

Can utilize various technology from substratum, to reclaim and purifying secreted antibody of the present invention in substratum, thereby the degree of purification that is fit to required purposes is provided.For example, treatment of the present invention is used for the treatment of the common requirement of purposes of human patients with antibody, compares with the substratum that contains therapeutic antibodies, has at least 95% purity when measuring with reductibility SDS-PAGE, more common 98% or 99% the purity that requires.In first example, utilize the cell relic in the centrifugal removal substratum usually, utilize for example micro-filtration, ultrafiltration and/or Depth Filtration to carry out the clarification step of supernatant liquor subsequently.Alternatively, can under not centrifugal condition, utilize micro-filtration, ultrafiltration or Depth Filtration results antibody.Also can use multiple other technologies, for example: dialysis, gel electrophoresis and chromatographic technique (for example hydroxyapatite chromatography (HA), affinity chromatography (randomly relating to affinity labelling system such as poly Histidine) and/or hydrophobic interaction chromatography (HIC is referring to US5429746).In one embodiment, after a plurality of clarification steps, utilizing albumin A or G affinity chromatography to catch antibody of the present invention, is further chromatographic step such as ion-exchange and/or HA chromatography, negatively charged ion or cationic exchange, size exclusion chromatography and ammonium sulfate precipitation subsequently.Usually, also use the various steps (for example carrying out nanofiltration etc. with the DV-20 filter membrane) of removing virus.Through these steps, (normally monoclonal) goods of purifying are provided, it comprises the antibody of the present invention of 10mg/ml at least or more (for example 100mg/ml or more), thereby has constituted an embodiment of invention.Can produce 100mg/ml or higher concentration by ultracentrifugation.This based article that is fit to is substantially devoid of the antibody of the present invention of aggregated forms.

The bacterium system is particularly suitable for the expressing antibodies fragment.These fragments are positioned at born of the same parents or matter between week.According to method known to those skilled in the art, can extract matter albumen between insoluble week, and be folded into active protein again, referring to people such as Sanchez, (1999) J.Biotechnol.72, people such as 13-20 and Cupit PM, (1999) Lett Appl Microbiol, 29,273-277.

6. pharmaceutical composition

Above-described purifying antibody preparation of the present invention (particularly mono-clonal preparation) can be incorporated in the pharmaceutical composition, is used for the treatment of human diseases and disorder that above-outlined is crossed.Usually this based composition also comprise pharmaceutically acceptable (namely, inertia) carrier, described carrier is that acceptable pharmacy practice is known and require, referring to for example: Remingtons PharmaceuticalScience, the 16th edition, (1980) Mack publishing company.The example of examples of such carriers comprises the sterilization carrier that is buffered to pH5-8 with suitable damping fluid, for example: salt solution, Ringer's solution or glucose solution.Injection (for example: in intravenously, intraperitoneal, intracutaneous, subcutaneous, intramuscular or portal vein) or the pharmaceutical composition of lasting infusion do not contain the visible particle thing suitably, and can comprise 0.1ng-100mg antibody, usually 5mg-25mg antibody.The method for preparing this class pharmaceutical composition is well known in the art.In one embodiment, the per unit dosage form of pharmaceutical composition contains 0.1ng-100mg treatment antibody of the present invention, randomly also has operation instruction.Can freeze-drying (lyophilize) pharmaceutical composition of the present invention, be used for before administration, rebuilding according to well known by persons skilled in the art or apparent method.When invention embodiment comprises the IgG1 isotype of antibody of the present invention, can in pharmaceutical composition, add the copper intercalating agent, as Citrate trianion (for example Trisodium Citrate) or EDTA or Histidine, thus the palliating degradation degree of this isotype antibody of reduction copper mediation, referring to EP0612251.

Generally rule of thumb determine effective dose and the treatment plan of antibody administration of the present invention, depend on disease or the obstacle of patient's age, body weight and healthy state and treatment.These influence factors are in doctor in charge's consideration.People such as for example Smith, (1977) Antibodies in humandiagnosis and therapy, Raven Press can find the guidance to the selection suitable dose among the New York, but generally between 1mg to 1000mg.In one embodiment, treatment suffers from the antibody of the present invention (or its Fab) that the dosage of human patients of RA is (for example, between 50mg to 200mg) weekly or about the subcutaneous 100mg of giving of per two weeks.Composition of the present invention can also be used for preventative purposes.

According to the disease for the treatment of and obstacle, can the pharmaceutical composition of the antibody of the present invention for the treatment of significant quantity and the another kind of medicament use simultaneously, respectively or successively of significant quantity will be comprised, described medicament for example is: anti-inflammatory agent such as NSAID, methotrexate, Bucillamine (bucillamine), sodium thioacetate, perhaps one or more anti-TNF alpha treatments, for example: Enbrel ^TM(etanercept), Remicade ^TM(infliximab), Humira ^TM(adalimumab) and/or CDP870.Antibody of the present invention can be united use with the anti-TNF alpha receptor antibody of significant quantity, referring to people such as Davis MW, (2000) Ann Rheum Dis 59 (Suppl 1): 41-43.In another embodiment, antibody of the present invention can be united use with the reagent at following material of significant quantity: IL-1/IL-1R (Kineret for example ^TM), CTLA4-1g, IL-6 be (referring to people such as Choy, 54), IL-8, IL-15, VEGF, IL-17, IL-18 be (referring to people such as Taylor (2002) Ann.Rheum.Dis61 (suppl 1):, (2001) Curr.Opin.Immunol.13:611-616), anti--ICAM and/or anti-CD 4 antibodies, at the member's (as MMP-1,2,3 and/or 13) of MMP family reagent.The antibody of invention can also be united use with the reagent of extracing the known cell that relates at inflammatory process, and for example CD20 positive B cell uses Mabthera ^TM(Rituximab).The treatment of other and antibodies of the present invention comprises that also vasculogenesis suppresses therapy, for example: the antagonist of integrin alpha V β 3, Kringle1-5 are (referring to people such as Sumariwalla P, (2003), Arthritis Res Ther5:R32-R39), solubility Flt-1 is (referring to people such as Miotla, (2000) Lab.Invest.80:1195-1205), anti--COX-2 reagent or anti--OSM reagent (as anti--OSM antibody), referring to WO2005/095457, its complete content is integrated into herein by reference, and can be consulted by the reader.By convention, the present invention also should consider such pharmaceutical composition, and it comprises antibody of the present invention or its Fab and other medicine, and the test kit that randomly constitutes with operation instruction.These are combined in treatment joint disease/illness perhaps is useful especially in the rheumatoid arthritis for example.

7. clinical application

Antibody of the present invention can be used for treating the disease of IL-18 mediation, for example autoimmune disease.What mention especially is multiple sclerosis, arthritis disease such as rheumatoid arthritis, type i diabetes, inflammatory bowel (IBD) and psoriasis.Therefore, the present invention also comprises the method for the treatment of the human patients of suffering from the disease (for example multiple sclerosis, rheumatoid arthritis, type i diabetes, IBD, psoriasis) of replying the hIL-18 neutralizing effect, described method comprises the antibody of the present invention that gives described patient treatment significant quantity, particularly has the heavy chain of the sequence that displays and the antibody of the light chain of the sequence that displays in SEQID NO:13 in SEQ ID NO:9.

Also provide antibody of the present invention to be used for the treatment of purposes in the medicine of a kind of (or multiple) above-mentioned diseases in production.

Table A

The description of protein or polynucleotide (PN)	Recognition sequence symbol (SEQ ID NO :)
		CDRH1	1
CDRH2	2
		CDRH3	3
CDRL1	4
		CDRL2	5
CDRL3	6
		Human il-18	7
Human il-18 PN	8
		H1 heavy chain (variable region+constant region)	9
H1 heavy chain (PN)	10
		The H1 variable region	11
H1 variable region (PN)	12
		L2 light chain (variable region+constant region)	13
L2 light chain (PN)	14
		The L2 variable region	15
L2 variable region (PN)	16
		H2 heavy chain (variable region+constant region)	17

H2 heavy chain (PN)	18
		The H2 variable region	19
H2 variable region (PN)	20
		H3 heavy chain (variable region+constant region)	21
H3 heavy chain (PN)	22
		The H3 variable region	23
H3 variable region (PN)	24
		L1 light chain (variable region+constant region)	25
L1 light chain (PN)	26
		The L1 variable region	27
L1 variable region (PN)	28
		L3 light chain (variable region+constant region)	29
L3 light chain (PN)	30
		The L3 variable region	31
L3 variable region (PN)	32
		2c10 rat-human IgG1's mosaic	33
2c10 rat-human IgG1's mosaic (PN)	34
		2c10 rat-people C κ mosaic	35
2c10 rat-people C κ mosaic (PN)	36
		Heavy chain acceptor framework	37
Light chain acceptor framework	38
		Add the JH6 aminoacid sequence of SEQ ID NO:37 to	39
Add the J κ aminoacid sequence of SEQ ID NO:38 to	40

7. illustrate

The following example has exemplified all respects of the present invention.All general clones, connection and other recombinant DNA technology are all according to people such as Maniatis, Molecular cloning (A laboratorymanual), Cold Spring Harbor Laboratory, or people such as Sambrook, MolecularCloning (A laboratory manual), the general instruction among the Cold Spring Harbor Laboratory is implemented.Carrier system used herein and other molecular biology method all are disclosed among the WO2005/095457, and its complete content is integrated into herein by reference, and the reader can consult specially.

7.1 clone hybridization knurl variable region

Parental generation rat antibody 2C10 displays in US patent 6706487.The reader can consult this document specially.On the basis in above-mentioned disclosed rat V district, connect human IgG1 or κ C district, design chimeric antibody 2C10c.Introduce general immunoglobulin (Ig) signal sequence and translation initiation codon ATG at heavy chain and light chain construct.Design Hind III and BsiWI restriction endonuclease sites come frame to go out the VL structural domain, and allow it is cloned in the mammalian expression vector that contains people C κ district (SEQID NO:36).Design Hind III and SpeI restriction endonuclease sites come frame to go out the VH structural domain, and allow it is cloned in the mammalian expression vector that contains people γ 1C district (SEQID NO:34).This causes framework 4 (Kabat residue 107 and 108) and disclosed sequence in the 2C10Vh district that 2 amino acid whose changes are arranged, as shown in the SEQ IDNO:33.

Use overlapping oligonucleotide to make up entire coded sequence by PCR, and be cloned in the expression vector of above-outlined.After the sequence checking, chimeric antibody expression in Chinese hamster ovary celI.Come the antibody that purifying is produced from cell culture supernatant by affinity chromatography on rProtein A agarose.At external chimeric antibody in conjunction with assessment 2C10 in detecting, confirm that itself and parental generation rat 2C10 have comparable tiring.By in ELISA, measuring the EC50 value (Figure 16 and 17) in conjunction with people or rhesus monkey IL-18, or in the KG-1 biological detection, measure the restraining effect (referring to Figure 15) to IFN-γ release, realize above-mentioned assessment.

7.2 humanization

7.2.1 the humanization strategy of light chain

For the variable sequence of light chain of 2C10 rat, selection is acceptor framework (F_IGV1D-12-1, SEQ IDNO:38) with the ethnic group that the variable sequence of light chain of rat 2C10 has 64% identity (comprising CDR).Planting is to be combined with suitable FR4 in computer simulation in the V district, and in the case, J district κ 2 minigenes (Kabat Vol.II) are based on sequence similarity (SEQ ID NO:40).Possible influence based on sequence comparison and antagonist function has produced three-type-person source variant.Construct L1 is that rat CDR (using the Kabat definition) is grafted directly on above selected people's acceptor framework.Construct L2 has the extra reverse mutation in a place of the 71st residue on the basis of L1.Construct L3 has the extra reverse mutation in 4 places at the 45th, 83,84 and 85 residue place on the basis of L2.Referring to table 1.

Table 1: the general introduction of the humanization VL variant of generation

Construct	Acceptor/template framework	Be positioned at the reverse mutation (Kabat) at aa# place	The reverse mutation sum	People's acceptor framework	Original rat sequence
						L1	F_IGV1D-12-1/J2SEQ.I.D.NO：38	------	Do not have	----	------
L2	L1	71	1	F	Y
						L3	L2	45838485	5	KFAT	QEGD

7.2.2 the humanization countermeasure of heavy chain

For 2C10 rat variable heavy chain sequence, selection is acceptor framework (Fp_IGHV1-f_2, SEQ IDNO:37) with the ethnic group that rat 2C10 variable heavy chain sequence has 59% identity (comprising CDR).Planting is to be combined with suitable FR4 in computer simulation in the V district, and in the case, JH6 minigene (Kabat Vol.II) is based on sequence similarity (SEQ ID NO:39).Produced three-type-person source variant based on this framework.H1 is the graft of rat CDR (using the Kabat definition), has 4 extra reverse mutations at the 27th, 28,29 and 93 residue place.This allows the rare aminoacid sequence in CDR1 upstream at adjacent parental generation (that is, donor) antibody, and it can component part CDR (referring to the definition of Cnothia).H2 has the extra reverse mutation in 2 places of the 39th and 40 residue on the basis of H1.H3 has the extra reverse mutation in other 4 places at the 36th, 71,89 and 91 residue place on the basis of H2.Referring to table 2.

Table 2: the general introduction of the humanization Vh variant of generation

Construct	Acceptor/template framework	Be positioned at the reverse mutation (Kabat) at aa# place	The reverse mutation sum	People's acceptor framework	Original rat sequence
						H1	Fp_IGHV1-f_2SEQ.I.D.NO：37	27282993	4	YTLA	EIST
H2	H1	3940	6	QA	RR
						H3	H2	36718991	10	WEVY	FATF

7.3 the humanization of 2C10C

Rebuild humanized V district by pcr amplification and overlapping oligonucleotide.The human normal immunoglobulin signal sequence that primer comprises be used to the restriction enzyme site of being cloned into mammalian expression vector and is used for secreting.Use Hind III and SpeI that humanized V district is cloned in the mammalian expression vector that contains people γ 1 constant region as H1, H2 and H3, use Hind III and BsiWI to be cloned in the mammalian expression vector that contains people κ constant region as L1, L2 and L3.It has produced the human IgG isotype of humanization heavy chain variant and the people κ isotype of humanization light chain variant.

7.3.1 express the combination of humanized heavy chain and light chain antibody

With transfection CHOK1 cell instantaneously in quadruplicate.Detect the antibody concentration of supernatant liquor, then by comparing 2C10 rat-people's mosaic, be used for external in conjunction with detecting.

Implement the extensive transient expression of all 9 kinds of variants as follows: each culturing bottle, in 8ml substratum (OptiMEM/glutamax/5% FBS), mix 51.4 μ g light chain plasmids and 8.6 μ g heavy chain plasmids and 240 μ g transfection liquid (at WO2006/053783, this liquid has been described among the embodiment 13, it is incorporated into herein by reference in full), and this mixture is applied on the CHOK1 cell that almost converges of two bottles of T175 culturing bottles, under typical conditions of tissue culture, kept 72 hours.Also in polyclone Chinese hamster ovary celI system, express described antibody with the mg magnitude, use and shake bottle and use FPLC and albumin A to carry out purifying.

7.4 it is external in conjunction with detecting

7.4.1 Biacore analyzes

Utilize albumin A to catch HBS-EP damping fluid (Biacore ^TM) in antibody, at Biacore ^TMCarry out the Biacore of humanization 2C10 antibody on 3000 instruments ^TMDynamic analysis.In brief, by the primary amine coupling albumin A is fixed on the CM5 chip, the experiment rules of utilizing the manufacturer to recommend reach the about 2000-4000 resonance units of density (RU ' s).Then, allow humanized antibody through the albumin A surface, the level of catching is about 200-500RU ' s, and behind after a while stable, with concentration the antibody surface by catch of IL18 (people's or rhesus monkey) to determine, the induction that obtains combination is composed.Utilize acid elution requirement regeneration, remove the antibody of catching fully from the albumin A surface, and can significantly not reduce the binding capacity on surface.All curves all double with reference to the buffering injection liquid to replace IL18, utilize the global pervasive parameter in BiaEval 4.1 to fit data to the 1:1 combination model.Dissociation rate grading experiment also is to utilize identical albumin A catching method to set up, but has only used single IL18 concentration (10nM).Also use the combination model fitting data identical with dynamic analysis simultaneously, because the report dissociation rate uses a kind of analyte concentration, this value is more suitable for for ordering rather than provides definite kinetic measurement, as the method that will select which kind of antibody further to be studied.

Initial result indication in the time of 25 ℃, all constructs have the people IL18 binding affinity similar to rat 2C10 parental generation antibody.Yet when when carrying out dissociation rate evaluation experiment for 37 ℃, the L1 construct is poorer than L2 and L3 performance, the visible dissociation rate that increases (table 3a and 3b).

Table 3a: the people is anti--kinetic parameter that the Biacore of IL18 antibody analyzes, detect in the time of 25 ℃.

Antibody	ka	Kd	KD(pM)
				2C10	2.55e6(7e4)	3.5e-5(4.2e-6)	13.9(2.2)
H1L1	1.4e6	4.7e-5	33.2
				H1L2	1.3e6(1.4e5)	3.85e-5(1.5e-5)	30.3(8.7)
H1L3	1.25e6(2.1e4)	2.8e-5(5.7e-6)	22.5(7.2)
				H2L1	1.03e6(1.0e5)	3.35e-5(1.1e-5)	33.5(13.4)
H2L2	1.4e6(1.4e5)	2.8e-5(0.0)	20.1(1.8)

H2L3	1.15e6(7e5)	2.8e-5(2.8e-6)	23.8(0.7)
				H3L1	2.5e6(4.2e5)	4.7e-5(9.9e-6)	19.4(7.6)
H3L2	2.6e6(2.8e5)	4.3e-5(3.6e-6)	16.5(2.7)
				H3L3	1.7e6(4.2e5)	4.0e-5(5.7e-6)	24.2(9.3)

Data: twice result of experiment (standard deviation).

The dissociation rate grading of the people IL18 of anti--IL18 antibodies that table 3b:Biacore detects the humanization of catching with albumin A detects in the time of 37 ℃.

Antibody	Kd
		2C10	7.01e-5
H1L1	1.62e-4
		H1L2	4.81e-5
H1L3	5.54e-5
		H2L1	9.93e-5
H2L2	4.15e-5
		H2L3	4.62e-5
H3L1	1.3e-4
		H3L2	8.22e-5
H3L3	7.01e-5

Data a: result of experiment.

Not only performance is poor in the time of 37 ℃, and the L1 construct is in conjunction with the avidity of rhesus monkey IL18 25 ℃ the time not good (showing 4a) too.Based on these observationss, 37 ℃ of combinations that study selected antibody and people and rhesus monkey IL-18 in great detail.The data of the human il-18 that shows among the table 4b are six independent mean values of measuring (and standard deviation).The data presentation of rhesus monkey IL-18 mean value and the standard deviation of twice use of H1L2 and H1L3, the data of H3L2 and H3L3 are then from single experiment.The higher relatively standard deviation of these data may be to carry out this result of experiment in the time of 37 ℃.

The relatively poor relatively fact of L1 construct performance is surprising, considers that difference between L1 and the L2 construct only is to replace with phenylalanine the reverse mutation of the 71st tyrosine of light chain.Tyrosine and phenylalanine all are die aromatischen Aminosaeurens, therefore when 37 ℃ (but not 25 ℃), at Biacore ^TMObserving in the skeleton construction this trickle change in the system, to produce tangible result (for binding affinity) be beyong contemplation.

Table 4a:Biacore detects the kinetic parameter of the rhesus monkey IL-18 of being combined with the humanized antibody construct, detects in the time of 25 ℃.

Antibody	ka	Kd	KD(pM)
				2C10	1.2E6	6.6E-5	54.7
H1L1	4.3E5	1.6E-4	380
				H1L2	4.3E5	4.7E-5	108
H1L3	5.8E5	6.4E-5	109
				H2L1	2.9E5	1.8E-4	627
H2L2	5.3E5	5.5E-5	104
				H2L3	4.7E5	8.8E-5	189
H3L1	9.1E5	1.4E-4	149
				H3L2	1.1E6	6.6E-5	59.6
H3L3	1.0E6	7.0E-5	69.1

Data a: result of experiment.

Table 4b:Biacore detects the kinetic parameter of the rhesus monkey IL-18 of being combined with the humanized antibody construct, detects in the time of 37 ℃.

Antibody/IL18	ka	Kd	KD(pM)
				H1L2 people IL18 rhesus monkey IL18	7.75e5(2.9e4)1.01e6(9.2e5)	1.38e-4(1.7e-5)1.40e-4(2.1e-5)	197(66.3)139(8.5)
H1L3 people IL18 rhesus monkey IL18	7.12e5(2.5e4)1.08e6(2.2e5)	1.18e-4(1.9e-5)1.86e-4(6.1e-5)	188(81)170(21.9)
				H3L2 people IL18 rhesus monkey IL18	1.52e6(4.9e5)1.85e6	1.45e-4(2.2e-5)1.19e-4	105(39.6)64.3
H3L3 people IL18 rhesus monkey IL18	1.49e6(4.5e5)1.79e6	1.52e-4(1.7e-5)1.35e-4	110(36.1)75.6

Select variant HIL1, H1L2 and H1L3 to be used for the further analysis of joint 7.4.2 down.

7.4.2 Biacore analyzes the T100 data

Use T100Biacore ^TMInstrument has further characterized some variant antibody.Owing to used the inline degassing instrument that can reduce the influence of damping fluid under the high temperature, this instrument is comparing Biacore aspect at high temperature stable of sensitivity, temperature control and baseline ^TM3000 more have superiority.It also provides the software that strengthens, for example automated data analysis.

Method is identical with the method that above-mentioned 7.4.1 joint uses basically; Albumin A is fixed on the CM5 chip by the density of primary amine coupling with 2000-6000RU ' s.At HBS-EP (Biacore ^TM) middle operation.Catch the density of anti-IL-18 antibody between 100-500RUs, allow human il-18 catch the surface with the concentration of 16-0.0625nM by this, and the double-basis criterion is used 0nM concentration (that is, only injecting the damping fluid of capture antibody).Behind the per injection IL18, regenerate by soft acid elutriant (10mM glycine, twice of pH1.5 injection).This regeneration step from the albumin A surface removal therefore the antibody of catching (and removed all in conjunction with the IL18 on it).Regeneration can significantly not change the ability at the later antibody peak of albumin A surface bonding, allows the generation of another time capturing events.Use the built-in analysis software of T100 instrument, the binding curve that utilizes the analysis of 1:1 combination model to obtain.Shown in move under the temperature.

Combination at 15 ℃, 20 ℃, 25 ℃, 32 ℃ and 37 ℃ of following analyses and H1L1 and H1L2

Under different temperature, utilize aforesaid method to experimentize.Fig. 1 has shown the influence of temperature to association rate (ka), and Fig. 2 has shown the influence to dissociation rate (kd) simultaneously, and Fig. 3 has shown the influence to the equilibrium constant (KD).Table 5 is described in detail for drawing the kinetics value that these figure use.

Table 5: from the kinetic parameter of temperature experiment acquisition

Temperature ℃	Antibody	Ka	Kd	KD(pM)
					15	H1L1	1.49e6	2.98e-5	20.1
	H1L2	2.07e6	2.07e-5	10.0
					20	H1L1	2.02e6	4.77e-5	23.7
	H1L2	2.84e6	2.53e-5	8.9
					25	H1L1	2.52e6	7.82e-5	31.1

H1L2	3.37e6	4.22e-5	12.5
				32	H1L1	3.48e6	1.64e-4	47.2
H1L2	4.51e6	8.57e-5	18.9
				37	H1L1	6.30e6	3.36e-4	53.3
H1L2	5.89e6	1.31e-4	22.3

Data are from single experiment.

Data presentation the test two kinds of antibody association rate the test temperature range in similar, H1L2 generally has association rate faster, before 37 ℃ end value, this moment H1L1 have association rate faster.Yet dissociation rate demonstrates bigger difference, and two kinds of antibody have similar dissociation rate in the time of 15 ℃, 20 ℃, 25 ℃, but begins to deviate from when 32 ℃ and 37 ℃, observes H1L1 dissociation rate faster.Overall balance constant (it is the function of kd/ka) has reflected that these change, and the difference between prompting H1L1 and the H1L2 is mainly aspect the antibody/IL18 complex body by dissociation rate (kd) definition stable.

Combination at 25 ℃ and 37 ℃ of following analyses and H1L1, H1L2, H1L3 and chimeric 2C10

Experimentize by above-mentioned.Table 6 is described the kinetic parameter of acquisition in detail.Data presentation, according to the combination by equilibrium constant KD definition, when 25 ℃ and 37 ℃, H1L2 is than the better antibody of H1L1, still, and the kinetic parameter demonstration, in the time of 25 ℃, H1L2 has than the better association rate of H1L1 (ka).And in the time of 37 ℃, the position has been reversed, and the better combination of indication observed H1L2 in the time of 37 ℃ is because the reason of dissociation rate (kd) indicates the sudden change that makes L2 be different from L1 to give the stability that the IL18-antibody complex body under higher temperature increases.

Table 6: under 25 ℃ and 37 ℃, the kinetics of being combined with people IL18 with H1L1, H1L2, H1L3 and chimeric 2C10

The people IL18 of people IL18 in the time of 25 ℃ in the time of 37 ℃

Antibody

Ka

Kd

KD(pM)

Antibody

Ka

Kd

KD(pM)

H1L1(n＝4)

2.49e6(4.41e5)

7.94e-5(1.02e-5)

33.1(9.3)

H1L1(n＝6)

5.64e6(2.42e6)

4.58e-4(1.02e-4)

94.3(39.6)

H1L2(n＝4)

2.88e6(7.39e5)

4.32e-5(9.75e-6)

16.3(7.0)

H1L2(n＝6)

4.86e6(1.88e6)

1.98e-4(5.20e-5)

46.0(18.8)

H1L3(n＝3)

2.36e6(9.89e5)

4.53e-5(7.46e-6)

22.0(9.9)

H1L3(n＝3)

6.43e6(6.10e6)

2.11e-4(4.84e-5)

64.8(57.3)

Chimeric 2C10 (n=3)

6.88e6(3.22e6)

5.22-5(8.94e-5)

9.1(4.7)

Chimeric 2C10 (n=2)

2.55e7(1.25e7)

5.62-4(1.97e-4)

22.9(3.5)

Data are the mean value of a plurality of independent data set, have shown mean value and standard deviation, and standard deviation is positioned at bracket.Also comprised in this data sheet obtain the analysis under five differing tempss, in the value of H1L1 and the H1L2 of 25 ℃ and 37 ℃ operations.

7.4.3 IL18 in conjunction with ELISA in assessment 2C10 humanization variant

Utilize the antibody purification of different batches, carried out the ELISA of at least 6 all 9 kinds of humanization variants.Fig. 4 A-4C has shown the representative data of testing from once, and described experiment is the experiment of the generation EC50 value rank shown in the table 7.Use the 16D10 (non-neutral mouse monoclonal antibody) of 2.5 μ g/ml that human il-18 is fixed on the Nunc Maxisorp 96 hole flat boards, catch the recombinant human IL-18 of 5ng/ml.With difference anti--the IL-18 humanized antibody adds in the various diluents.Utilize anti-human IgG Fc specificity peroxidase conjugated thing (Sigma A0170) to detect the humanized antibody of combination.

The EC50 value (with [μ g/ml] expression) of the increase of table 7:2C10 humanization variant

	2C10c	H3L2	H3L3	H1L1	H1L2	H1L3	H2L2	H3L1	H2L3	H2L1
											EC50 ^*	0.007	0.008	0.009	0.010	0.011	0.011	0.012	0.013	0.016	0.021

All standard errors are all between 0.001 and 0.002.

The ability of all variants all shows very near the 2C10 mosaic, and the prompter source turns into only having produced Disability seldom.Though the ordering that the EC50 value that the several times by these detections repeat to produce has produced variant really,, only ELISA can not produce the clearly difference (referring to table 7 and Fig. 4 A-4C) between these variants.Utilize Biacore ^TM(referring to 7.4.1 and 7.4.2) obtained some differences of variant, and it causes utilizing in the independent experiment that repeats of people and rhesus monkey IL18 4 checked variants more to approach (table 8, Fig. 5 [people] and 6[rhesus monkey]) at some.

The EC50 value of six independent repeated experiments that 8:4 selected humanization variant of table is combined with human il-18

	Experiment 1/1	Experiment 1/2	Experiment 2/1	Experiment 2/2	Experiment 3/1	Experiment 3/2	Average	SE
									2C10c	0.015	0.016	0.013	0.011	0.020	0.020	0.0158	0.004
H1L2	0.029	0.030	0.021	0.025	0.024	0.027	0.0260	0.003
									H1L3	0.027	0.025	0.029	0.028	0.029	0.027	0.0275	0.002
H3L2	0.032	0.030	0.026	0.018	0.025	0.022	0.0255	0.005
									H3L3	0.035	0.028	0.018	0.021	0.025	0.025	0.0253	0.006

7.4.4 IL-18 in conjunction with ELISA in assessment 2C10 humanization H1 variant

With three-type-person source H1 variant: H1L1, H1L2 and H1L3 carry out ELISA, when room temperature and 37 ℃, assess its in human serum and lock solution (PBS 0.05%TWEEN and 1%BSA (w/v)) with the combination of human il-18.The humanized antibody variant is fixed on the Nunc Maxisorp 96 hole flat boards with 2.5 μ g/ml.When room temperature and 37 ℃, in human serum and lock solution, carry out catching the recombinant human IL-18 of 5ng/ml.Add anti-IL-18 mouse monoclonal antibody 16D10.Utilize anti-mouse κ peroxidase conjugated thing (Serotec MCA 1291P) to detect the mouse antibodies of combination.Shown the typical EC50 value that produces from data in the table 9.

Table 9: under room temperature and 37 ℃, the EC50 value of 2C10 humanization H1 variant

	EC50(ng/ml)	Standard error
			Incubated at room
When having serum
			The 2C10 mosaic	7.296	0.358
H1L1	10.189	0.512
			H1L2	9.791	0.471
H1L3	8.989	0.411
			In the sealing damping fluid
The 2C10 mosaic	3.814	0.068
			H1L1	3.315	0.136
H1L2	3.552	0.079
			H1L3	3.790	0.133
Hatch at 37 ℃
			When having serum
The 2C10 mosaic	10.140	1.254
			H1L1	12.069	0.740
H1L2	9.791	0.471
			H1L3	11.438	1.861
In the sealing damping fluid
			The 2C10 mosaic	3.794	0.114
H1L1	3.430	0.104
			H1L2	3.404	0.145
H1L3	3.334	0.222

Carry out the temperature of human il-18 integrating step by change, from room temperature to 37 ℃, the ability of three-type-person source H1 variant is unaffected in this detects.When having antibody in the human serum, observe lower binding signal.

7.4.5 the stability of assessment humanization H1 variant antibody in the time of 37 ℃

Measure three-type-person source H1 variant: H1L1, H1L2 and H1L3,14 days stability in storage by a definite date in the human serum under 37 ℃ and the phosphate buffered saline (PBS).With antibody dilution to 50 μ g/ml, 37

℃ hatch

0,1,4,6,8 and 14 day time after, detect stability by IL-18 in conjunction with ELISA., in conjunction with ELISA 16D10 (non-neutral mouse monoclonal antibody) is fixed on the Nunc Maxisorp 96 hole flat boards for IL-18, catches the recombinant human IL-18 of 5ng/ml.Add anti--IL-18 humanized antibody 37 ℃ of different time points of hatching time-histories.Utilize Anti-Human IgG Fc specificity peroxidase conjugated quality testing to survey the humanized antibody of combination.

Based on the ability that described antibody under this detecting pattern is combined with human il-18, prolongation is exposed to the binding ability that did not influence humanization H1 antibody variants under 37 ℃ of temperature in 0,1,4,6,8 and 14 day.

7.5 the combination of H1L2 and people IL18 when having synovia

The recombinant human IL18 of 500ng/ml to 0ng/ml scope is incorporated in 50% people's synovia, implements ELISA.Then the IL-18 that contains in this solution is used for the hole with the Maxisorp96 hole flat board (Nunc) of H1L2 antibody sandwich.Then, by biotinylated anti-IL-18 antibody (D045-6, MBL) and streptavidin-HRP detects the IL-18 of combination.Recombinant human IL18 in titration 50% synovia (SF) produced with the titration damping fluid in the almost identical curve of recombinant human IL18, only have slight skew in half maximum value.Referring to Fig. 7.Even this has confirmed to exist 50% people SF, antibody also has the ability in conjunction with hIL-18, and described condition has closer been simulated the combining environmental that antibody will run in the therapeutic scene.

7.5.1 when having IL18 in conjunction with albumen (IL18bp) and the combination of IL18

Use BiacoreTM technology (BiacoreTM3000) and ELISA to determine whether H1L2 still can be in conjunction with people IL18 when having people IL18bp.IL18bp has the high-affinity with IL18, as the natural inhibitor of IL-18 function and work (Fig. 8, Fig. 9 A and B, table 10 and table 11).

Biacore detects

In brief, by the primary amine coupling density of albumin A with about 4000 resonance units (RU ' s) is fixed on the CM5 chip surface.Then, be that the reorganization Fc-IL18 of 3 μ g/ml is in conjunction with albumen (R﹠amp with concentration; D Systems) with 10 μ l/ minutes flow velocity by 1 minute; Cause catching the IL-18 of about 1400RU ' s in conjunction with albumen.Then, with concentration be the IL18 of 30nM with 10 μ l/ minutes flow velocity by the IL18 that catches in conjunction with protein surface 5 minutes.After this, with concentration be the rat 2C10 parental generation antibody of 10nM with 30 μ l/ minutes flow velocity by IL18 in conjunction with albumen/IL18 surface 3 minutes.IL18 is last will to be disturbed site with the IL18 binding protein interactions by the epi-position of 2C10 antibody recognition, then thereafter should invisible any binding signal.Figure 10 has confirmed 2C10 in conjunction with the hIL-18 that has been caught by hIL-18BP, and the binding site of indication 2C10 and hIL-18BP is nonoverlapping.

The IFN γ secretion of table 10:IL-18BP rheumatoid arthritis (RA) synovial cell's influence

Sample, the IL-18BP state	Contrast	IL-12	IL-18	IL-12+IL-18
					RA1
-	ND	0.63	ND	1.63
					+	ND	0.29	ND	0.13
RA2
					-	ND	0.85	ND	0.70
+	ND	0.10	ND	0.10
					RA3
-	ND	1.06	ND	1.62
					+	ND	0.32	ND	0.40

ELISA detects

Humanization H1L2 or 2C10 rat MAb and the combination that is combined in the people IL18 that catches on the IL18BP, recombinant human IL18bp-Fc fusion rotein (R﹠amp among the described ELISA in directly in conjunction with ELISA, have been tested; D Systems#119-BP) is coated on the Nunc Maxisorp flat board with 0.5 μ g/ml.Recombinant human IL18 (interior reagent) is added in the sealing damping fluid (containing 1%w/v BSA) with 100ng/ml.Add concentration range at the antibody purification of 0.5ng/ml to 1 μ g/ml.With the specificity HRP conjugate (Sigma) of Anti-Human κ light chain or the antibody of anti--rat IgG HRP detection combination.Figure 18 A and B for example understand the result who obtains.

7.6 vitro bioassay

7.6.1 the humanization construct in and KG-1 clone IL18 in stress IFN-γ discharging Active

The neutralization activity at the antibody of IL-18 has been measured specifically in this detection, and discharges based on the IFN-γ in the KG-1 cell of IL-18-mediation.KG-1 (ATCC#CCL-246) is people's myelomonocyte system of the functional IL-18 acceptor of constitutive expression, thereby replys exogenous IL-18 and excite.

Measured the inhibition ability (table 12 and accompanying drawing 11) that IFN-γ that whole nine kinds of humanization variants excite people IL18 in the KG-1 cell discharges.

Table 12: utilize all nine kinds of humanization variants in the KG-1 biological detection in and the IC50 value of recombinant human IL18

Antibody	IC50
		H1L1	0.071
H1L2	0.033
		H1L3	0.027
H2L1	0.145
		H2L2	0.054
H2L3	0.046
		H3L1	0.027
H3L2	0.035
		H3L3	0.034
2C10(1)	0.042
		2C10(2)	0.039

Also implemented repeated experiments at least 6 times for 4 kinds of preferred humanization variants, described preferred variant is at Biacore ^TMShow the best avidity with the people who recombinates and rhesus monkey IL18 in the detection.Fig. 8 for example understands the representative result of four kinds of preferred humanization variants and H1L1, and table 13 has been summarized the result of these detections, and the material from the cell-derived same protein of CHOe1a batch is all used in above-mentioned detection.

Table 13: use 4 or the KG-1 biological detection of 5 kind of selected humanization variant in and the IC50 value of recombinant human IL18

	Mean value 2C10IC50	(H1L1)	(H1L2)	(H1L3)	(H3L2)	(H3L3)	IL-18bp
								Experiment
1	0.046	Do not detect	0.062	0.064	0.064	0.057	Do not detect
								Experiment 2	There is not match	Do not detect	There is not match	There is not match	There is not match	There is not match	Do not detect
Test 3 flat boards 1	0.074	Do not detect	0.109	0.144	0.090	0.091	Do not detect
								Test 3 flat boards 2	0.075	Do not detect	0.173	0.156	0.128	0.115	Do not detect
Experiment 4	0.017	0.091	0.017	0.044	There is not match	0.016	There is not match
								Experiment 5 (EC80 of estimation)	0.075	0.757	0.122	0.085	0.072	0.056	0.054
Experiment 5 (EC50 of estimation)	0.044	0.180	0.047	0.046	0.039	0.038	0.034
								Experiment 6 (EC80 of estimation)	There is not match	0.078	0.023	0.021	There is not match	0.013	0.007
Experiment 6 (EC50 of estimation)	0.020	0.069	0.019	0.019	0.015	0.016	0.01

Under the situation that compares H1L1 and other humanization variant, H1L1 shows other four kinds of humanization constructs and lower the tiring of 2C10 parental generation MAb than test.

Also implement further analysis with four kinds of preferred monoclonal antibodies, compared the inhibition ability that the KG-1 cell that people IL18 is excited discharges IFN-γ: 2C10 and the humanization variant (H1L1, H1L2 and H1L3) of deriving from 2C10.In 96 hole flat boards, implement the KG-1 biological assay, by at 37 ℃ and 5%CO ₂In hatch the antibody (from 2 μ g/ml to 7.8ng/ml, doubling dilution) of specificity IL18 of the recombinant human IL-18 of 50ng/ml and different concns or negative isotype contrast (Synagis, anti-RSV antibodies), every hole adds 3.10 then ⁵The KG-1 cell.The most dull and the most stereotyped at 37 ℃, 5%CO ₂In hatched 20-24 hour.The results supernatant liquor utilizes people IFN γ ELISA test kit (the Biosource AHC4432 that is purchased; AHC4539) measure IFN γ output.

Three kinds of experiments have been implemented.The inhibition normalization method as a result that the IFN γ that will excite IL-18 according to negative control produces.The target of statistical study is to obtain after replying adjustment at any Synagis, to the IC50 estimated value of every kind of mAb of every kind of experiment.Analyze the IC50 estimated value then statistically, produce the comprehensive estimated value of IC50 of every kind of mAb with 95% fiducial interval (that is statistics likelihood scope).Final use Deng Naiteshi check (Dunnett ' s test) every kind of humanization variant and 2C10 are looked back comparison.

Figure 12 for example understands representational experiment.Table 14 and Figure 13 have shown under 95% fiducial interval the comprehensive estimated value to the IC50 of every kind of monoclonal antibody, and change with the % of rat 2C10 under p value and fiducial interval.

Table 14: the IC50 value of the neutralizing effect that the IFN γ that IL18 excites in the KG-1 biological assay produces

Monoclonal antibody	Average IC50 μ g/ml (95%CI)	% changes vs2C10 (95%CI)	Dunnett p value
				2C10	0.095(0.071，0.127)	-	-
H1L1	0.352(0.264，0.470)	269.4(117.2，528.2)	0.0001
				H1L2	0.154(0.112，0.211)	61.5(-7.6，182.0)	0.0968
H1L3	0.164(0.121，0.221)	71.6(-0.4，195.8)	0.0519

Tiring of H1L1 is markedly inferior to 2C10 (p＜0.001) statistically.Other humanization variant then is not different from 2C10 significantly, though relatively being positioned on the boundary of H1L3 and 2C10.

7.6.2 the H1L2 activity that IFN-γ discharges in the human PBMC that neutralization excites

Excite human PBMC from three donors with recombinant human IL18 and anti-CD 3 antibodies, and add the dilution gradient of four kinds of selected humanized antibody variants studying.Each donor has comprised parental generation 2C10 antibody and IL18bp as a comparison.For two in three donors, IL18 and anti--exciting of CD3 are failed, and do not detect any IFN-γ.In the donor of remainder, the result difference during lower concentration is very big, but by add various anti--IL18 antibody, comprise the humanization variant, the inhibition fully that the IFN-γ that can realize IL18 is induced produces.Referring to Figure 14.

Also the human PBMC who excites with LPS experimentizes, and the described generation that also causes IL18 and the relevant IFN γ of exciting discharges.LPS induces IFN γ to produce in the mode of concentration dependent, adds 2C10 parental generation monoclonal antibody with the fixed concentration of 1 μ g/ml and has suppressed this excitation fully, and indicating described effect is the IL18 mediation, and can in and endogenous IL18 (not display data).In whole blood, also can confirm this effect, though still dose-dependently of the retarding effect of 2C10, but more not obvious (data not shown).Fig. 9 for example understands 3 independent donors and the experimental result that suppresses with parental generation rat mono-clonal 2C10, H1L2 and IL18bp.Donor 1 and 3 has produced similar result, and 2 couples of LPS of donor excite and do not show IFN γ and discharge (not shown).When existence has been added〉during 10% or 25% the human serum of 1 μ g/ml antibody or IL18bp, the IFN γ that can suppress the IL18 mediation fully discharges.At 10ng/ml and abovely just can observe restraining effect, table 11 has shown this inhibiting IC50 value when having 10% or 25% human serum.

Table 11: when having human serum, the IC50 value that the IFN γ that utilizes 2C10, H1L2 and IL18bp inhibition LPS-to excite discharges, described release is caused by endogenous IL18 neutralizing effect

	IL18bp	H1L2	2C10
				Donor
1 10% serum	0.024	0.102	0.023
				Donor 1 25% serum	0.064	0.113	0.069
Donor 3 10% serum	0.032	0.042	0.033
				Donor 3 25% serum	0.046	0.108	0.086

7.6.3 the general introduction of being combined with the lineal homologue of IL18

Antibody and combination from the IL18 of other species have been utilized ELISA and Biacore and KG-1 cell biological to measure to have checked.This at first utilizes parental generation 2C10 and the rhesus monkey/stump-tailed macaque of chimeric 2C10c to carry out, but adopts some humanization variants to repeat.Utilize Biacore and ELISA to test the combination of parental generation 2C10 and pig, mouse and rat IL18, tested the combination of humanization variant and rhesus monkey/stump-tailed macaque and the dog IL18 of 4 kinds of the bests.With rhesus monkey/stump-tailed macaque IL18 and three kinds of monoclonal antibodies, 2C10 mosaic and representational humanization variant H3L3, implement the KG-1 biological assay.Consider the similarity of all humanization variants, it also may represent other variant of generation, comprises that H1L2 is (referring to table 15, Fig. 8).

The general introduction of the lineal homologue combination of the mixture of table 15:2C10 parental generation rat MAb, the chimeric 2C10c of rat-people and selected 4 kinds of humanization variants

	Rhesus monkey/stump-tailed macaque IL18	Dog IL18	Mouse ﹠ rat IL18	Pig IL18
					2C10 rat MAb	ELISA(+)KG-1(+)	ELISA(-)	ND	ND
2C10 mosaic (rat/people)	ELISA(+)KG-1(+)Biacore ^TM(+)	ELISA(-)Biacore ^TM(-)	ELISA(-)Biacore ^TM(-)	ELISA(-)Biacore ^TM(-)
					Humanization (H1L2, H1L3, H3L1, H3L3)	ELISA (+) KG-1 (+) has only tested H3L3Biacore ^TM(+)	ELISA(-)	ELISA(-)	ELISA(-)

ND: do not determine; +: detectable combination

7.7IL18 the circular dichroism of antibody and thermally denature research

Utilize circular dichroism (CD) to study the change of secondary structure of IL18 antibody as the function of temperature, particularly from 25 ℃ to 37 ℃.Carry out thermally denature research of these same antibody and determine their thermostability and their melting temperature(Tm) (Tm).

CD method: with 0.5nm step pitch and 1nm bandwidth scanning 180nm-280nm, obtain CD spectrum with Applied Photophysics Chirascan spectrograph.The acquisition time of each point/be 5 seconds.Sample is diluted in PBS～0.2mg/ml, and is placed in the 1mm optical path length cell.Gather the spectrum of every kind of protein with the thermostatic bath that is set in 4 ℃, 25 ℃ and 37 ℃.Determine the actual temperature of sample by being placed on probe in the cell interior liquid, and described actual temperature design temperature～3 ℃ of scopes in.

Tm method: in the 1:1000Sypro orange of phosphate buffered saline(PBS) (PBS), all proteins is diluted to 0.2mg/ml.Utilize Bioneer Exicycler instrument at 620nm (exciting at 490nm) in per 0.5 ℃ of interval measurement fluorescent emission, sample temperature tiltedly is increased to 95 ℃ from 10 ℃, each temperature spot is waited for 10 seconds.Utilize Grafit that the sex change fitting of a curve is melted thermoisopleth to standard.

As expection, compare the shape from the CD spectrum of all four kinds of antibody, the structure that has shown them is the height βZhe Die, and has essentially identical structure.CD has disclosed, and in 4 ℃-37 ℃ temperature range, the secondary structure of three kinds of antibody (H1L1, H1L2, H1L3) does not show any significant change.But in this temperature range, 2C10 antibody chimeric body structurally shows slight minimizing really.This with following table 16 in the thermostability trend that provides be consistent.

The thermally denature of table 16:IL18 antibody

Antibody	H1L1	H1L2	H1L3	Mosaic
					Tm	73℃	70℃	67℃	65℃

Tm=sex change/melting temperature(Tm)

H1L1, H1L2 and H1L3 also are stable significantly under the condition far above 37 ℃, under body temperature without any the signal of sex change.Therefore, they unlikely give any otherness advantage in the difference on the thermostability under normal body temperature and envrionment temperature.

Claims

1. humanized resisting-il-1 8 antibody, it comprises variable region of heavy chain and the variable region of light chain shown in SEQ ID NO:15 shown in SEQ ID NO:11.

Claim 1 described humanized anti--il-1 8 antibody, it comprises heavy chain and the light chain shown in SEQ ID NO:13 shown in SEQ IDNO:9.

3. comprise according to anti--il-1 8 antibody of claim 1 or claim 2 and the pharmaceutical composition of pharmaceutically acceptable carrier, wherein said pharmaceutical composition is used for the treatment of autoimmune disease.

The described antibody of claim 1 or claim 2 for the preparation of the treatment autoimmune disease medicine in purposes.

5. the described purposes of claim 4, wherein said autoimmune disease is selected from multiple sclerosis, arthritis disease, type i diabetes, inflammatory bowel and psoriasis.

6. the described purposes of claim 5, wherein said autoimmune disease is rheumatoid arthritis.

7. the polynucleotide of the coding defined heavy chain of antibody of claim 2 and light chain of antibody.

8. the polynucleotide of the coding defined antibody heavy chain variable region of claim 1 and antibody chain variable region.

9. the carrier of the coding defined heavy chain of antibody of claim 2 and light chain of antibody.

10. the carrier of the coding defined antibody heavy chain variable region of claim 1 and antibody chain variable region.

11. comprise the host cell of the stable conversion of claim 9 or 10 described carriers.

12. for the production of the method according to the antibody of claim 1 or claim 2, described method is included in and allows to express under the condition of described antibody, cultivates that carrier with the polynucleotide that comprise encoding said antibody transforms or the host cell of transfection.