CN1156460A

CN1156460A - Reconstructed hyman antibody against human interleukin-8

Info

Publication number: CN1156460A
Application number: CN 95194723
Authority: CN
Inventors: 松岛纲治; 松本义弘; 山田良树; 佐藤功; 土屋政幸; 山崎达美
Original assignee: Chugai Pharmaceutical Co Ltd
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 1994-07-13
Filing date: 1995-07-12
Publication date: 1997-08-06

Abstract

The present invention discloses a reshaped human antibody against human IL-8 comprising: (A) L chains each comprising: (1) a human L chain C region; and, (2) an L chain V region comprising a human L chain FR, and an L chain CDR of mouse monoclonal antibody against human IL-8; and, (B) H chains each comprising: (1) a human H chain C region; and, (2) an H chain V region comprising a human H chain FR, and an H chain CDR of mouse monoclonal antibody against human IL-8. Since the majority of this reshaped human antibody originates in human antibody and the CDR has low antigenicity, the reshaped human antibody of the present invention has low antigenicity to humans, and can therefore be expected to be useful in medical treatment.

Description

The reconstructed hyman antibody of anti-Ro 24-7472/000-8

Technical field:

The present invention relates to mouse monoclonal antibody complementarity-determining region (CDR) and variable region (the V district) of anti-Ro 24-7472/000-8 (IL-8), people/little mouse chimeric antibody and the reconstructed hyman antibody that relates to anti-people IL-8, wherein the complementarity-determining region of people's light chain (L chain) variable region and people's heavy chain (H chain) variable region are replaced by the mouse monoclonal antibody CDR of anti-people IL-8.And, the invention provides the DNA of coding above-mentioned antibody and its part.The invention still further relates to the carrier that contains above-mentioned DNA, more particularly, relate to expression vector and with described carrier host transformed.And, the invention provides the method for the reconstructed hyman antibody of producing anti-people IL-8 and the method for producing anti-people IL-8 chimeric antibody.

Background technology

Interleukin 8 (IL-8) is found in the monocyte cultures supernatant liquor that stimulates with lipopolysaccharides (LPS) and is a kind of chemotactic factor for neutrophil (MDNCF) of cells of monocytic origin or chemokine of neutrophil activating protein-1 (NAP-1) of also being called.IL-8 is produced by various kinds of cell, acts on polymorphonuclear leukocyte and lymphocyte and have along its concentration gradient to cause chemotactic activity.In addition, it not only induces the chemotaxis of neutrophil, but also activates the neutrophil function, and as degranulation, super-oxide discharges and promotion adheres to endotheliocyte.

In diseases associated with inflammation, more particularly, in respiratory tract disease, as the lung sac fibrosis, spontaneous lung fibrosis, the ailing syndromes of the respiratory tract of growing up, in sarcoidosis and the empyema and at dermatosis, as in the psoriasis and, in CrohnShi disease and the ulcerative colitis, on pathology, observe leukocyte infiltration in the inflammation site of these diseases at chronic rheumatoid arthritis.In addition, in patient's test sample of suffering from these diseases, detect IL-8, show that IL-8 may play central role in inflammation.(Mc Elvaney, people such as N.G., Journal of Clinical Investigation (J.Clin.Invest.), 90,1296-1301,1992; Lynch III, people such as J.P., Am.Rev.Respir.Dis., 145,1433-1439,1992; Donnelly, people such as S.C., Lancet, 341,643-647,1993; Car, people such as B.D., Am.J.Respir.Crit.Care Med., 149,655-659,1994; Antony, people such as V.B., Journal of Immunology (J.Immunol.), 151,7216-7223,1993; Takematsu, people such as H., Arch.Dermotol., 129,74-80,1993; Brennan, people such as F.M., European Journal of Immunology (Eur.J.Immunol.), 20,2141-2144,1990; IZZO, people such as R.S., Scand.J.Gastroenterol., 28,296-300,1993; IZZO, people such as R.S., Am.J.Gas-troenterol., 87,1447-1452,1992).

In personnel selection after IL-8 does the antigen immune mouse, Ko, people such as Y-C. have prepared in conjunction with people IL-8 and because this combines the bioactive mouse monoclonal antibody WS-4 that is had with people IL-8 in promptly with neutrophil in conjunction with suppressing people IL-8.Clearly illustrate that very the isotype of mouse monoclonal antibody WS-4 is by K-type L chain and C γ ¹-type H chain is formed (immunological method magazine, 149,227-235,1992).

A.5.12.14 the antibody example of the anti-people IL-8 of the known WS-4 of being different from comprises (Boylan, people such as A.M., Journal of Clinical Investigation, 89,1257-1267,1992), disclosed resisting-Pep-1 antibody and anti--Pep-3 antibody in international patent application no WO92-04372, and DM/C7 (Mulligan, M.S. wait people, Journal of Immunology, 150,5585-5595,1993).

Also find lung local asphyxia and heavy perfusion injury (people such as Sekido.N. through mouse monoclonal antibody WS-4 being administered to the experimental model that uses rabbit, nature, 365,654-657,1993), LPS inductive dermatitis (Harada, people such as A., international immunology (Inter-natl.Immunol.), 5,681-690,1993) and LPS-or interleukin 1 (IL-1) inductive sacroiliitis (Akahoshi, people such as T., lymphokinecyte factor research (Lymphokine Cyto Kine Res.), 13,113-116,1994) infiltration of middle inhibition neutrophil.

People IL-8 homologue is present in the rabbit and is called rabbit IL-8.Owing to having clearly illustrated that the combine (Harada of mouse monoclonal antibody WS-4 and rabbit IL-8 cross reaction and this antibody inhibition rabbit IL-8 with the rabbit neutrophil, A. wait the people, international immunology, 5,681-690,1993), these discoveries show: anti-people IL-8 antibody is useful in the therapeutical agent as the human inflammatory diseases of treatment.

The monoclonal antibody that produces in being different from human Mammals shows hyperimmunization originality (being also referred to as antigenicity) the mankind.Therefore, even mouse antibodies is administered to the mankind, owing to it is fallen by metabolism as allogenic material, so mouse antibodies is quite short in the transformation period of human body, thereby has stoped by well-verified desired result.And, to the mouse antibodies of using react and immune response that the human anti-mouse antibody that produces causes to patient be uncomfortable, also be dangerous, its example comprises serum disease or other anaphylaxis.Therefore, mouse antibodies can not often be administered to the mankind.

In order to address these problems, formed the method that produces humanization antibody.Mouse antibodies can be with two kinds of method human bodyizatioies.Simpler method relates to the production chimeric antibody, and wherein variable region (V district) from original mouse monoclonal antibody, constant region (C district) is from suitable human antibodies.Because the chimeric antibody of gained contains the mouse antibodies variable region in its complete form, so it has the specificity identical with original mouse antibodies, and expectation can conjugated antigen.

And in chimeric antibody, owing to compare a large amount of minimizings from non-human animal's protein sequence ratio with original mouse antibodies, therefore prediction is compared with original mouse antibodies and is had less immunogenicity.Although chimeric antibody combines with antigen better and has than reduced immunogenicity, still have with the mouse variable region that exists immunoreactive possibility (Lo Buglio, people such as A.F., Proc.Natl.Acad.Sci.USA, 86,4220-4224,1989) takes place.

Although the second method of humanization mouse antibodies is more complicated, mouse antibodies potential immunogenicity has sizable minimizing.In the method, have only complementarity-determining region (CDR) from the grafting of mouse antibodies variable region to the people variable region to produce reconstruct people variable region.Yet, for the CDR structure that makes reconstruct people variable region closer similar to original mouse antibodies structure, in some cases may be with the protein sequence part of supporting CDR framework region (FR) from the grafting of mouse antibodies variable region to the people variable region.

Then, these reconstruct people variable region is connected to human constant region.Those parts from the non-human protein sequence only partly are made up of CDR in the humanization antibody and very little FR.CDR is made up of the protein sequence of alterable height, and they do not show the kind specificity.Therefore, the reconstructed hyman antibody that contains mouse CDR should not have stronger immunogenicity than the natural human antibody that contains people CDR.

About other details of reconstructed hyman antibody referring to Riechmann, people such as L., nature, 332,323-327,1988; Verhoeyen, M, etc. the people, science, 239,1534-1536,1988; Kettleborough, people such as C.A, Protein Eng., 4,773-783,1991; Maeda, people such as H., human antibodies hybridoma, 2,124-134,1991; Gorman, people such as S.D., Proc.Nattl.Acad.Sci.USA, 88,4181-4185,1991; Tempest, people such as P.R., biotechnology, 9,266-271,1991; Co, people such as M.S., Proc.Natl.A-cad.Sci.USA, 88,2869-2873,1991; Carter, people such as P., Proc.Natl.A-cad.Sci.USA, 89,4285-4289,1992; Co, M.S waits the people, Journal of Immunology, 148,1149-1154,1992; And Sato, people such as K., cancer research, 53,851-856,1993.

Description of the invention

As mentioned above, although the expection reconstructed hyman antibody is useful to therapeutic purpose, still there is not understanding for the reconstructed hyman antibody of anti-people IL-8.And, there is not standard method can be common to any antibody to produce reconstructed hyman antibody.Therefore, producing the enough various designs in conjunction with active and/or the active reconstructed hyman antibody that neutralizes that show specific antigen is necessary (for example, Sato.K waits the people, cancer research, 53,851-856,1993).The invention provides the antibody of the anti-people IL-8 with reduced immunogenicity.

The invention provides the reconstructed hyman antibody of anti-people IL-8.The present invention also provides people/little mouse chimeric antibody useful in said reconstructed hyman antibody production method.And the present invention also provides the fragment of reconstructed hyman antibody.In addition, the invention provides production chimeric antibody and reconstructed hyman antibody and its segmental expression system.And the present invention also provides reconstructed hyman antibody and its segmental method of producing anti-people IL-8 chimeric antibody and its segmental method and producing anti-people IL-8.

More particularly, the invention provides:

(1) the L chain V district of the mouse monoclonal antibody of anti-people IL-8;

(2) the H chain V district of the mouse monoclonal antibody of anti-people IL-8.

And, the invention provides:

(1) comprises the L chain in the mouse monoclonal antibody L chain V district of people L chain C district and anti-people IL-8; With

(2) comprise the H chain in the mouse monoclonal antibody H chain V district of people H chain C district and anti-people IL-8.

And the present invention also provides the chimeric antibody of anti-people IL-8, comprises:

(1) respectively comprises the L chain in the mouse monoclonal antibody L chain V district of people L chain C district and anti-people IL-8; With

(2) respectively comprise the H chain in the mouse monoclonal antibody H chain V district of people H chain C district and anti-people IL-8.

And, the invention provides:

(1) the mouse monoclonal antibody WS-4L chain V district of anti-people IL-8; With

(2) the mouse monoclonal antibody WS-4H chain V district of anti-people IL-8.

And the present invention also provides:

(1) comprises the L chain in the mouse monoclonal antibody WS-4L chain V district of people L chain C district and anti-people IL-8; With

(2) comprise the H chain in the mouse monoclonal antibody WS-4H chain V district of people H chain C district and anti-people IL-8.

In addition, the invention provides the chimeric antibody of anti-people IL-8, comprise:

(1) respectively comprises the L chain in people L chain C district and anti-people IL-8 mouse monoclonal antibody WS-4L chain V district; With

(2) respectively comprise the H chain in people H chain C district and anti-people IL-8 mouse monoclonal antibody WS-4H chain V district.

And, the invention provides:

(1) CDR in the monoclonal antibody L chain V district of anti-people IL-8; With

(2) CDR in the monoclonal antibody H chain V district of anti-people IL-8.

And the present invention also provides:

(1) CDR in the mouse monoclonal antibody L chain V district of anti-people IL-8; With

(2) CDR in the mouse monoclonal antibody H chain V district of anti-people IL-8.

And the present invention also provides the people L chain V district of reconstruct of the antibody of anti-people IL-8, comprises:

(1) framework region (FR) in people L chain V district;

(2) CDR in the mouse monoclonal antibody L chain V district of anti-people IL-8;

And the people H chain V district of the reconstruct of the antibody of anti-people IL-8, comprise:

(1) people H chain V district FR; With

And, the invention provides the people L chain of reconstruct of the antibody of anti-people IL-8, comprise:

(1) people L chain C district; With

(2) comprise the L chain V district of the mouse monoclonal antibody L chain CDR of people L chain FR and anti-people IL-8;

And the people H chain of the reconstruct of the antibody of anti-people IL-8, comprise:

(1) people H chain C district; With

(2) comprise the H chain V district of H chain CDR of the mouse monoclonal antibody of people H chain FR and anti-people IL-8.

In addition, the present invention also provides people's antibody of the reconstruct of anti-people IL-8, comprises:

(A) each L chain comprises:

(1) people L chain C district; With

(2) comprise the L chain V district of the mouse monoclonal antibody L chain CDR of people L chain FR and anti-people IL-8; And

(B) each H chain comprises:

(1) people H chain C district; With

(2) comprise the H chain V district of the mouse monoclonal antibody H chain CDR of people H chain FR and anti-people IL-8.

More particularly, the invention provides:

(1) have the CDR in L chain V district of mouse monoclonal antibody WS-4 of the anti-people IL-8 of following sequence or its part:

CDR1：Arg?Ala?Ser?Glu?Ile?Ile?Tyr?Ser?Tyr?Leu?Ala

CDR2：Asn?Ala?Lys?Thr?Leu?Ala?Asp

CDR3：Gln?His?His?Phe?Gly?Phe?Pro?Arg?Thr

And:

(2) have the CDR in H chain V district of mouse monoclonal antibody WS-4 of the anti-people IL-8 of following sequence or its part:

CDR1：Asp?Tyr?Tyr?Leu?Ser

CDR2：Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?GluTyr?Ser?Ala?Ser?Val?Lys?Gly

CDR3：Glu?Asn?Tyr?Arg?Tyr?Asp?Val?Glu?Leu?Ala?Tyr

And, the invention provides the reconstruct people L chain V district of the antibody of anti-people IL-8, comprise:

(1) people L chain V district framework region (FR); With

(2) the mouse monoclonal antibody WS-4L chain V district CDR of anti-people IL-8; And:

The reconstruct people H chain V district of the antibody of anti-people IL-8 comprises:

(1) FR in people H chain V district; With

(2) CDR in the monoclonal antibody WS-4H chain V district of anti-people IL-8.

And, the invention provides the people L chain of the antibody reconstruct of anti-people IL-8, comprise:

(1) people L chain C district; With

(2) comprise the L chain V district of the mouse monoclonal antibody WS-4L chain CDR of people L chain FR and anti-people IL-8; And

The people H chain of the reconstruct of the antibody of anti-people IL-8 comprises:

(1) people H chain C district; With

(2) comprise the H chain V district of H chain CDR of the mouse monoclonal antibody WS-4 of people H chain FR and anti-people IL-8.

(A) L chain respectively comprises:

(1) people L chain C district; With

(2) comprise the L chain V district of the mouse monoclonal antibody WS-4L chain CDR of people L chain FR and anti-people IL-8; With

(B) H chain respectively comprises:

(1) people H chain C district; With

(2) comprise the H chain V district of the mouse monoclonal antibody WS-4H chain CDR of people H chain FR and anti-people IL-8.

The example of above mentioned people L chain FR comprises the FR with following amino acid sequences or its part:

FR1：Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?SerAla?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys

FR2：Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?LeuLeu?Ile?Tyr

FR3：Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?GlyThr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?IleAla?Thr?Tyr?Tyr?Cys

FR4:Phe Gly Gln Gly Thr Lys Val Glu Ile Lys or,

FR2：Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?LeuLeu?Ile?Tyr

FR3：Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?GlyThr?Asp?Tyr?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?IleAla?Thr?Tyr?Tyr?Cys

FR4：Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys

The example of people H chain FR above-mentioned comprises the FR with following amino acid sequences or its part:

FR1：Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?ValGln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?PheThr?Phe?Ser

FR2：Trp?Val?Arg?Gln?Ala?Gln?Gly?Lys?Gly?Leu?Glu?LeuVal?Gly

FR3：Arg?Leu?Thr?Ile?Ser?Arg?Glu?Asp?Ser?Lys?Asn?ThrLeu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Thr?Glu?Asp?Leu?Ala?ValTyr?Tyr?Cys?Ala?Arg

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；

FR2：Trp?Val?Arg?Gln?Ala?Gln?Gly?Lys?Gly?Leu?Glu?TrpVal?Gly

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；

FR2：Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?LeuVal?Gly

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；

FR2：Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?TrpVal?Gly

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；

FR2：Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?TrpVal?Gly

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；

FR2：Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Ala?Leu?Glu?TrpVal?Gly

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；

FR2：Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?TrpVal?Gly

FR3：Arg?Phe?Thr?Ile?Ser?Arg?Glu?Asp?Ser?Lys?Asn?ThrLeu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Thr?Glu?Asp?Leu?Ala?ValTyr?Tyr?Cys?Ala?Arg

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser；or，

FR2：Trp?Val?Arg?Gln?Ala?Gln?Gly?Lys?Gly?Leu?Glu?TrpVal?Gly

FR4：Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

In addition, the invention still further relates to the DNA that coding comprises peptide more than above-mentioned various antibody and its fragment.The invention still further relates to the carrier that contains above-mentioned DNA, its example is an expression vector.And, the invention provides with above-mentioned carrier host transformed.

And the present invention also provides chimeric antibody and its segmental method of producing anti-people IL-8 and has produced anti-people IL-8 reconstructed hyman antibody and its segmental method.

Brief description of the drawings

Fig. 1 represents to contain the expression vector HEF-VL-g κ and the HEF-VH-g γ 1 of people's elongation factor-1 α (HEF-1 α) promotor/enhanser, and they are useful in the expression of light chain of antibody of the present invention and H chain respectively.

Fig. 2 has shown for confirming the result of the ELISA that of the present invention chimeric WS-4 antibody (chL/chH) that secretion advances the COS cell culture medium and the binding ability of people IL-8 are carried out.

Fig. 3 is (RVHa) (RVLa) design of graphics of the DNA of the aminoacid sequence of (B) of (A) and reconstruct people WS-4 light chain of antibody V district's first kind " a " of coding reconstruct people WS-4 heavy chain of antibody V district's first kind of the present invention " a ".

Fig. 4 has shown respectively with the binding ability of the reconstruct people WS-4 light chain of antibody V district of the present invention (RVLa) of the chimeric WS-4 heavy chain of antibody V district (chH) of expressing in the COS cell and chimeric WS-4 light chain of antibody V district (chL) combination and H chain V district (RVHa) and people IL-8 and has secreted the ELISA result that the binding ability of the of the present invention chimeric WS-4 antibody (chL/chH) of COS cell culture medium compares.

Fig. 5 has shown that 8 classes of secreting in the COS cell culture medium contain the reconstruct people WS-4 antibody (RVLa/RVHa of RVLa of the present invention, RVLa/RVHb, RVLa/RVHc, RVLa/RVHd, RVLa/RVHe, RVLa/RVHf, RVLa/RVHg and RVLa/RVHh) advance the ELISA result that the binding ability of the of the present invention chimeric WS-4 antibody (chL/chH) in the COS cell culture medium compares with binding ability and the secretion of people IL-8.

Fig. 6 has shown that 8 classes that produce contain the reconstruct people WS-4 antibody (RVLb/RVHa of the second type RVLb of the present invention in COS cell culture supernatant liquid, RVLb/RVHb, RVLb/RVHc, RVLb/RVHd, RVLb/RVHe, RVLb/RVHf, RVLb/RVHg and RVLb/RVHh) advance the ELISA result that of the present invention chimeric WS-4 antibody (chL/chH) binding ability in the COS cell culture medium compares with people IL-8 binding ability and secretion.

Fig. 7 has shown reconstruct people WS-4 antibody RVLa/RVHg and the chimeric WS-4 antibody (chL/chH) of RVLb/RVHg and purifying of the present invention and the ELISA result that people IL-8 binding ability compares of purifying of the present invention.

Fig. 8 has shown that the reconstructed hyman antibody RVLa/RVHg of purifying more of the present invention and RVLb/RVHg suppress IL-8 combines inhibition mensuration with the ligand receptor of the ability of IL-8 receptors bind result with mouse WS-4 antibody of the present invention and chimeric WS-4 antibody (chL/chH).

Implement concrete mode of the present invention

The clone of the DNA in encoding murine V district

For the gene in the mouse monoclonal antibody V district of the anti-people IL-8 of clones coding, necessary The hybridization knurl of the mouse monoclonal antibody of the anti-people IL-8 of preparation generation is to obtain this gene. From After extracting mRNA in the hybridization knurl, according to known method mRNA is transformed into strand cD-NA then uses polymerase chain reaction (PCR) amplification target DNA to obtain this gene. Should The example of gene source is hybridization knurl WS-4, and it produces the mouse monoclonal of anti-people IL-8 Antibody is by productions such as Ko.Y.C.. Prepare the method for this hybridization knurl at the immunological method magazine 149,227-235 describes in 1992 and hereinafter as describing with reference to embodiment 1.

(1) extraction of total RNA:

For the target DNA in the mouse monoclonal antibody V district of the anti-people IL-8 of clones coding, can Through processing broken hybridization oncocyte with the thiocyanic acid guanidine and carrying out cesium chloride density gradient centrifugation and obtain Get total RNA (Chirgwin, the people such as J.M., biochemistry, 18,5294-5299,1979). And, also can use other used in clone gene method, as such as the vanadium compound Carrying out detergent-treatment and phenol under the condition that ribalgilase (RNase) inhibitor exists processes Method (Berger, the people such as S.L., biochemistry, 18,5143-5149,1979).

(2) cDNA is synthetic

Then, (a kind of poly (A) with being positioned at mRNA3 ' end is mutual through using oligomerization (dT) The oligonucleotide of mending) makes primer, to be included among total RNA that top mode obtains MRNA is template, processes the list that total RNA can obtain to be complementary to mRNA with reverse transcriptase Chain cDNA (Larrick, the people such as J.W., biochemistry, 7,934-938,1989). In addition, Simultaneously also can use at random primer. And, at needs at first in the situation of separating mRNA, Can be through total RNA being loaded to oligomerization (dT) in conjunction with mRNA poly (A) tail-fiber Finish on the plain post.

(3) through the DNA in polymerase chain reaction amplification coding V district

Then, use the specifically above-mentioned V of amplification coding district of polymerase chain reaction (PCR) CDNA. For the mouse monoclonal antibody K type L chain V district that increases, use 11 kinds SEQ ID NO:1 to 11 (the variable district of mouse K; MKV) Oligonucleolide primers shown in and At SEQ ID NO:12 (mouse K constant region, the Oligonucleolide primers branch shown in MKC) Not as 5 ' end primer and 3 ' end primer. Above-mentioned MKV primer and encoding murine K type L chain The dna sequence dna hybridization of guide's sequence, and above-mentioned MKC primer and encoding murine K type L The dna sequence dna hybridization in chain C district.

For the mouse monoclonal antibody H chain V district that increases, use respectively 12 kinds at SEQ ID NOs:13 to 24 (the variable district of murine heavy chain; MHV) Oligonucleolide primers shown in and Oligonucleolide primers (murine heavy chain constant region shown in the SEQ ID NO:25; MHC) divide Not as 5 ' end primer and 3 ' end primer. Above-mentioned MHV primer and encoding murine H chain guide The dna sequence dna hybridization of sequence, and above-mentioned MHC primer and encoding murine H chain C district Dna sequence dna hybridization.

And whole 5 ' end primers (MKV and MHV) contain is providing SaLI near 3 ' end The sequence GTCGAC of restriction enzyme site, and two 3 ' end primer (MKC and MHC) Contain at the nucleotide sequence CC-that the XmaI restriction enzyme site is provided near 5 ' end CGGG. These restriction enzyme sites are for the target dna fragment in two the V districts of will encoding Inferior clone advances clone's carrier separately. Also be present in coding two at these restriction enzyme sites In the situation of the target DNA sequence in individual V district, other restriction enzyme site should be used for inferior The clone advances clone's carrier separately.

(4) separation of the DNA in coding V district

Then, in order to obtain the dna fragmentation in encoding murine monoclonal antibody target V district, low On the fusing point agar sugar gel or through [PCR product purification reagent box (QIAGEN on post PCR purifying spin reagent box: QIAGEN); DNA purification kit (GENECLEAN II, BIO101)] separate and the purifying pcr amplification product. Through with restricted enzyme SaLI with The amplification product of XmaI processing purifying obtains encoding murine monoclonal antibody target V district Dna fragmentation.

And, through carrying with identical restricted enzyme SalI and the suitable clone of XmaI cutting Body (such as plasmid pUC19) also is connected to above-mentioned dna fragmentation enzymatic on this pUC19, can Acquisition contains the plasmid of the dna fragmentation in encoding murine monoclonal antibody target V district. According to appointing The meaning conventional method can be cloned the sequence of DNA and be measured, and an example is to use automatically Dna sequencing instrument (Applied Biosystems) order-checking. The clone of target DNA and sequence are measured In embodiment 1 and 2, describe in detail.

Complementarity-determining region (CDR)

The present invention also provides the height-V in the mouse monoclonal antibody V district of anti-people IL-8 District or complementarity-determining region (CDR). Two V districts of antibody L chain and H chain all form anti-Former in the site. These zones of L chain and H chain all have similar basic structure. This Article two, the V district of chain all contains four quite conservative framework regions of sequence, and these four framework regions with 3 high V districts or CDR connect (Kabat, the people such as E.A., " the protein order of immune target Row ", the healthy and human service department of the U.S., 1991)

The major part of above-mentioned four framework regions (FR) has the β-pleated sheet structure, 3 CDR shapes Cheng Huan. In some cases, CDR can form the part of β-pleated sheet structure. These three CDR maintains closely three-dimensional position and help CDR with three pairings of the utmost point with FR Form together antigen in conjunction with the site. The invention provides as the antibody composition of the mankindization useful CDR and their DNA of encoding. These CDR can be from Kabat, the people such as E.A. The experimental rules of " protein sequence of immune target " is through comparing V region sequence and known V The amino acid sequence in district determines that it is explained in detail and provides in embodiment 3.

Prepare chimeric antibody

Before the reconstructed hyman antibody V district of the anti-people IL-8 of design, must confirm what reality was used Whether CDR forms antigen binding domain. For this purpose prepares chimeric antibody. In order to prepare Chimeric antibody, the necessary DNA that makes up the chimeric antibody L chain of coding and H chain. Make up two kinds The basic skills of DNA relates to the mouse guide who observes in PCR clone's DNA The dna sequence dna separately of sequence and mouse V region sequence be present in mammalian cell The dna sequence dna in the encoding human C district in the expression vector connects.

Above-mentioned people's antibody C district can be any people L chain C district and any people H chain C district, closes In the L chain, example comprises people L chain C κ and C λ, and for the H chain, if IgG, the example bag Draw together C γ 1, C γ 2, C γ 3 or C γ 4 (EUison, the people such as J., DNA, 1,11-18 (1981), Takahashi, the people such as N., cell, 29,671-679 (1982), Krawinkel, the people such as U., Isosorbide-5-Nitrae 03-407 (1982)) or other type of the same race EMBO.J..

Two class expression vectors have been prepared, an expression vector for producing so-called chimeric antibody Contain in enhancers/promoters and express encoding murine L chain V district and the people who controls under district's control The DNA in L chain C district, an expression vector contains in the enhancers/promoters class expresses control Encoding murine H chain V district under the control in district and the DNA in people H chain C district. Then, use These two expression vectors transform the host's cell such as mammalian cell simultaneously, conversion thin Born of the same parents in body or in vitro culture to produce chimeric antigen (for example, WO91-16928).

As selection, the DNA in encoding murine L chain V district and people L chain C district and coding are little The DNA in mouse H chain V district and people H chain C district can import in the single expression vector, with said The carrier transformed host cell and then in external or body, cultivate to produce chimeric antibody.

Producing chimeric antibody from monoclonal antibody WS-4 describes among embodiment 4.

Use PCR clones coding mouse WS-4K-type L chain guide's sequence and V district CDNA and connection advance to contain the human gene group DNA's in encoding human L chain C κ district expression vector In. Equally, use PCR clones coding mouse WS-4 antibody H chain guide's sequence and The cDNA in V district and the expression that connects the human gene group DNA enter to contain encoding human C γ 1 district are carried In the body.

More particularly, use the PCR primer of specialized designs that suitable nucleotide sequence is led Enter encoding murine WS-4 antibody V district cDNA5 ' and 3 ' end in case (1) they be easy to by Insert expression vector and (2) suitable function (example of they performances in said expression vector As, through importing Kozak sequence of the present invention improve transcribe efficient).

Then, will use these primers anti-through the end code mouse WS-4 that pcr amplification obtains The DNA in body V district imports and has contained in the HEF expression vector (seeing Fig. 1) in required people C district. These carriers are suitable for the instantaneous or hereditary processing of stably express in various mammal cell line systems Antibody.

When testing the antigen-binding activity of the chimeric WS-4 antibody for preparing in this mode, card Real chimeric WS-4 antibody is in conjunction with the vigor (seeing Fig. 2) of people IL-8. Therefore, deducibility Clone correct mouse V district, and determined correct sequence.

The design of reconstruct people WS-4 antibody

Be transplanted to reconstructed hyman antibody on people's antibody in order to prepare mouse monoclonal antibody CDR, Need to the amino acid sequence of the mouse monoclonal antibody FR with CDR to be transplanted with CDR will transplant into the amino acid sequence of human monoclonal antibodies FR between have height Homology.

For reaching this purpose, through the amino acid sequence of smaller mouse monoclonal antibody FR with The amino acid sequence of people's antibody FR can be selected as design reconfiguration people WS-4 antibody V district The people V district on basis. More particularly, use hereditary analysis software GENETEX (soft Part development corporation, Ltd.) the V district of mouse WS-4 antibody L and H chain and in country relatively All known people V districts that find in biomedical research fund (NBRF) database.

With known people L chain V district relatively in, find mouse WS-4 antibody L chain The most similar (Watanabe, the people such as S., the Hoppe-Seyler ' s of V district and people's antibody HAV Z.Physiol.Chem., 351,1291-1295,1970), have 69.2% homology. On the other hand, with known people's antibody H chain V district relatively in, find WS-4 antibody H chain V district and people's antibody VDH26 the most similar (Buluwela, the people such as L., EMBOJ., 7,2003-2010,1988), have 71.4% homology.

In general, the homology of mouse V region amino acid sequence and people V region amino acid sequence Less than with the homology of mouse V region amino acid sequence. This shows mouse WS-4 antibody V District's incomplete similarity is in people V district, and the mankindization that show simultaneously mouse WS-4V district are to separate The best mode of patient's immunity originality problem of determining.

Further compared mouse WS-4 antibody V district and Kabat, the people such as E.A., (1991), the protein sequence of immune target, the 5th edition, the healthy and human service department of the U.S., The consensus sequence of the inferior base in the people V district that united states government printing office limits with comparison V district it Between FR. The result is displayed in Table 1.

Homology (%) between the FR of the people V district consensus sequence of table 1 mouse WS-4V district FR and each inferior base

FR in the A.L chain V district

HSGI HSGII HSGIII HSGIV

64.4 51.3 57.3 57.5

FR in the B.H chain V district

HSGI HSGII HSGIII

46.9 40.9 62.3

The FR in mouse WS-4 antibody L chain V district is similar in appearance to the people L chain V inferior basic I in district (HSGI) consensus sequence of FR has 64.4% homology. On the other hand, mouse WS The FR in-4 antibody H chain V districts is similar in appearance to the people H chain V inferior basic III's in district (HSGIII) Consensus sequence has 62.3% homology.

These results supported with known person antibody, belong to the people L chain V inferior basic I in district the people anti-Body HAU L chain V district and belong to people's antibody VDH26 H of the people H chain V inferior basic III in district The result that chain V compares in the district and obtains. For design reconfiguration people WS-4 antibody L chain V The district perhaps preferably uses the people L chain V district (HSGI) that belongs to inferior basic I, and in order to design heavily Structure people WS-4 antibody H chain V district preferably uses the people's antibody H chain that belongs to inferior basic III V district (HSGIII).

Compare with known person antibody L chain V district, mouse antibodies WS-4L chain V district Similar in appearance to people's antibody REI L chain V district, the member of the people L chain V inferior basic I in district. Cause This, the FR of REI is used for design reconfiguration people WS-4 antibody L chain V district. These with REI is among the people FR on basis, with original (Palm, the people such as W., Hoppe-Seylers Z, Physiol.Chem., 356,167-191,1975; With the people such as Epp.O., biochemistry 14,4943-4952,1975) the people REI of record compared 5 amino acid whose differences ( Position 39,71,104,105 and 107; See Table 2).

With kabat, the experience of E.A. etc. (1991) is the basis at the amino acid no shown in the table. Locate two amino acid whose changes and be present in rat CAMPATH-1H at 39 and 71 The change (Riechmann waits the people, 1988) that amino acid among the antibody L chain V district FR causes Identical. According to kabat, wait people (1991), in other 3 amino acid (positions of FR4 104,105 and 107) variation in is take the J district of other people KL chain as the basis and do not depart from the people Class.

Two class reconstruct people WS-4 antibody L chain V districts have been designed. At first kind RVLa In, FR and the REI that is present in the reconstruct people CAMPATH-1H antibody are basic FR (Riechmann waits the people, 1988) is identical, and CDR with at mouse WS-4 antibody L The CDR in chain V district is identical. Equations of The Second Kind RVLb is take RVLa as the basis, and its difference only exists In an amino acid in people FR371 position. Such as people such as Chothia.C., molecular biology Magazine, 196,901-917,1987 is defined, and residue 71 is L chain V district CDR1 rule The part of model structure

Amino acid expection in this position directly affects L chain V district CDR1 structure of rings, because of This thinks to antigen in conjunction with having obvious impact. At the people WS-4 of reconstruct antibody L chain Among the RVLb in V district, 71 phenylalanine changes over tyrosine. Table 2 has shown mouse WS-4 antibody L chain V district, be used for reconstruct people CAMPATH-1H antibody modification The people WS-4 antibody L chain V district of REI FR (Riechmann waits the people, 1988) and reconstruct Two types amino acid sequence separately.

Table 2

The design in the people WS-4L chain V district of reconstruct

1 2 3 4

12345678901234567890123 45678901234 567890123456789 WS-4L DIQMTQSPASLSASVGETVTITC RASEIIYSYLA WYQQKQGKSPQLLVY REI DIQMTQSPSSLSASVGDRVTITC WYQQKPGKAPKLLIY RVLa DIQMTQSPSSLSASVGDRVTITC RASEIIYSYLA WYQQKPGKAPKLLIY RVLb ----------------------- ----------- ---------------

FR1 CDR1 FR2

5 6 7 8 9

0123456 78901234567890123456789012345678 901234567 WS-4L NAKTLAD GVSSRFSGSGSGTQFSLRISSLQPEDFGSYYC QHHFGFPRT REI GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC RVLa NAKTLAD GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QHHFGFPRT RVLb ------- --------------Y----------------- ---------

CDR2 FR3 CDR3

10

8901234567 WS-4L FGGGTKLELK REI FGQGTKVEIK RVLa FGQGTKVEIK RVLb ----------

FR4 Annotate: the FR of REI the people CAMPATH-1H of reconstruct antibody (Riechmann, Deng the people, 1988) the middle discovery. The amino acid of 5 line in REI FR is to be different from the people The amino acid of REI amino acid sequence. Use single-letter coded representation amino acid. Amino acid no Consistent with the people's such as kabat definition.

FR in the mouse WS-4 antibody H chain V district is similar in appearance to belonging to inferior basic III's People H chain V district (table 1).

Compare the phase in mouse WS-4 antibody H chain V district with known people H chain V district Be similar to people's antibody VDH26 H chain V district, it is that people H chain V district is from FR1 to FR3 The member of inferior basic III (Buluwela, the people such as L., EMBO J., 7,2003-2010, 1988). For FR4, owing to do not report the FR4 sequence of VDH26, determine to use to belong to Amino acid sequence (Sanz, the people such as I., the immunology of the FR4 of people's antibody 4B4 of inferior basic III Magazine 142,883-887,1989). These people H chains V district is as design reconfiguration people WS The basis in-4 antibody H chain V districts.

Designed the H chain V district of 8 class reconstruct people WS-4 antibody. At all 8 types In, people FR1, FR2 and FR3 are with people's antibody VDH26 FR1, and FR2 and FR3 are base Plinth, and FR4 is take the FR4 of people's antibody 4B4 as the basis. Mouse CDR and mouse WS-4 The CDR in antibody H chain V district is identical.

Table 3 and table 4 have shown mouse WS-4 antibody H chain V district, people's antibody VDH26 Template FR1 is to FR3, the people WS-4 antibody H chain V of people's antibody 4B4 FR4 and reconstruct 8 types of amino acid sequences separately in district.

The design in the people WS-4 antibody H chain V district of table 3. reconstruct (table 4 is continuous)

1 2 3

123456789012345678901234567890 12345

WS-4H EVKLVESGGGLIQPGDSLRLSCVTSGFTFS DYYLS

VDH26 EVQLLESGGGLVQPGGSLRLSCAASGFTFS

RVHa～h EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYYLS

FR1 CDR1

4 5 6

67890123456789 012ABC3456789012345

WS-4H WVRQPPGKALEWVG LIRNKANGYTREYSASVKG

VDH26 WVRQAQGKGLELVG

RVHa WVRQAQGKGLFLVG LIRNKANGYTRFYSASVKG

RVHb -----------W-- -------------------

RVHc -----P-------- -------------------

RVHd -----P-----W-- -------------------

RVHe ----PP-----W-- -------------------

RVHf -----P--A--W-- -------------------

RVHg -----P-----W-- -------------------

RVHh -----------W-- -------------------

FR2 CDR2

The design (continued 3) in the people WS-4H chain V district of table 4. reconstruct

7 8 9 10

67890123456789012ABC345678901234 567890ABC12

WS-4H RFTISRDDSQSILYLQMNTLRGEDSATYYCAR ENYRYDVELAY

VDH26 RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR

RVHa RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR ENYRYDVELAY

RVHb -------------------------------- -----------

RVHc -------------------------------- -----------

RVHd -------------------------------- -----------

RVHe -------------------------------- -----------

RVHf -------------------------------- -----------

RVHg -F------------------------------ -----------

RVHh -F------------------------------ -----------

FR3 CDR3

11

34567890123

WS-4H WGQGTLVTVSA

4B4 WGQGTLVTVSS

RVHa～h WGQGTLVTVSS

FR4

Annotate: RVHa-h represents RVHa, RVHb, and RVHc, RVHd, RVHe, RVHf, RVHg and RVHh.

Use single-letter coded representation amino acid. The definition one of amino acid no and kabat etc. Cause.

The preparation of the DNA in coding reconstruct people WS-4 antibody V district

The preparation in reconstruct people WS-4 antibody V district is described in embodiment 5 in detail.

Composite coding reconstruct people WS-4 antibody L chain and H chain V district be the first kind separately DNA. Measure through sequence then and confirm reconstruct people's WS-4 antibody L chain and H chain V district class The correct amino acid sequence of global DNA sequence coding of " a " of type. Reconstruct people WS-4 The sequence of antibody L chain V district's type " a " shows in SEQ ID:62, and the people of reconstruct The sequence of WS-4 antibody H chain V district's type " a " shows in SEQ ID NO:38.

Use the disclosed PCR sudden change of slightly modified induce method (kammann, the people such as M., Nucleic acids research, 17,5404,1989) do the people that template prepares coding reconstruct with the first kind " a " The DNA of other type of WS-4 antibody V district. Such as the people WS-4 of front about reconstruct The design of antibody V district is described, another class of preparation coding reconstruct people WS-4 antibody L chain V district The DNA of type (type " b ") and coding reconstruct people WS-4 antibody H chain V district other 7 The DNA of type (type " b ", " c ", " d ", " e ", " f ", " g " and " h ").

These other types slightly change with the first kind in a series of amino acid sequences, In amino acid sequence these change through using the PCR sudden change method of inducing to make the order at DNA Slight change takes place in the row and obtain. The PCR design of primers becomes to import in dna sequence dna Required variation. After a series of PCR reactions, clone PCR products is then carried out order Row are measured with the change that confirms dna sequence dna and have been pressed designed generation. Reconstruct people WS The sequence of-4 antibody L chain V district's types " b " represents in SEQ ID NO:65, and reconstruct The type " b " in people WS-4 antibody H chain V district, " c ", " d ", " e ", " f ", " g's " and " h " Sequence is respectively at SEQ ID NO:41, expression in 44,45,48,51,54 and 55.

Measure all types of dna sequence dna in confirmation reconstruct people WS-4 antibody V district through sequence After, the inferior clone of DNA in coding reconstruct people WS-4 antibody V district advanced to have contained encoding human C In the mammalian cell expression vector of the DNA in district. That is, the reconstruct people WS-4 that will encode The DNA in antibody L chain V district is connected on the dna sequence dna in encoding human L chain C district, and The DNA in coding reconstruct people WS-4 antibody H chain V district is connected to encoding human C γ 1 district On the dna sequence dna.

Then, test reconstruct people L chain V district " a " or " b " type and H chain V district " a " arrive " h " All combinations of type and the combination of people IL-8. The result confirms to contain L chain " a " as shown in Figure 7 Two kinds of reconstructed hyman antibodies (RVLa/RVHg and RVLb/ of type or " b " type and H chain " g " type RVHg) ability in conjunction with people IL-8 is identical with chimeric WS-4 antibody.

Any expression system comprises mammalian cell, fungi such as zooblast or foundation Cell, the eukaryotic of yeast cells and thin such as the protokaryon of bacterium cell (such as Escherichia coli) Born of the same parents can be used for producing chimeric antibody or the reconstructed hyman antibody of anti-people IL-8 of the present invention. So And, preferably, chimeric antibody of the present invention or reconstruct antibody mammalian cell as Express in COS cell or the Chinese hamster ovary celI. In these examples, useful commonly used opening Mover is used in the mammalian cell and expresses. For example, preferred end user's giant cell virus Early stage (HCMV) promoter immediately. The example bag that contains the expression vector of HCMV promoter Draw together HCMV-VH-HC γ 1 and HCMV-VL-HC κ, and also comprise from The carrier of pSV2neo (International Patent Application Publication No. WO92-19759).

Can be used for of should using,, of the present invention heredity was expressed in mammalian cell that other opens The example of mover comprises such as reverse transcription virus, poliovirus, adenovirus and monkey disease poison The viral promotors of 40 (SV40) and such as human polypeptides chain elongation factor-1α (HEF-1 α) promoter that in mammalian cell, produces in. For example, using SV40 to start In the situation of son, pass through according to Mulligan, the people such as R.C. (nature, 277,108-114, 1979) method can be expressed or in the situation of using HEF-1 α promoter, pass through Press Mizu shima, the people's such as S. (nucleic acids research, 18,5322,1990) method can be shown Reach.

The concrete example of another of the promoter that the present invention is useful is HEF-1 α promoter. HEF-VH-g γ¹Be included in the table that contains this promoter with HEV-VL-g κ (Fig. 1) Reach in the carrier. From polyomavirus, adenovirus, SV40 or cow teats knurl virus (BPV) etc. Dna sequence dna can be used as the replicator point. And, for the something lost in the host's cell that increases Pass the copy number, can use aminoglycoside-3 '-phosphoric acid shifts acid, neomycin resistance gene, chest Glycosides kinases (TK) gene, Escherichia coli xanthine-guanine phosphoribosyl transferase (XG-PRT) gene or dihyrofolate reductase (dhfr) are as selecting mark.

Generally speaking, the present invention at first provides the mouse monoclonal antibody L of anti-people IL-8 The DNA in chain V district and H chain V district and the said L chain V district of encoding and the said H of coding The DNA in chain V district. These people at anti-people IL-8/little mouse chimeric antibody and reconstruct people are anti-Useful in the preparation of body. The example of monoclonal antibody is WS-4. L chain V district has all As in the amino acid sequence as shown in the SEQ ID NO:26, and H chain V district have such as Amino acid sequence shown in the SEQ ID NO:27. These amino acid sequences by such as Nucleotide sequence shown in the SEQ ID NO:26 and 27 is encoded respectively.

The of the present invention chimeric antibody of anti-people IL-8 comprises:

(1) people L chain C district and mouse L chain V district, and

(2) people H chain C district and mouse H chain V district.

Mouse L chain V district has been described in the front, mouse H chain V district and their DNA of encoding. Above-mentioned people L chain C district can be any people L chain C district, and its example comprises people C κ and C λ The district. Above-mentioned people H chain C district can be any people H chain C district, and its example comprises people C γ 1, C γ 2, C γ 3 or C γ 4 districts (Ellison, the people such as J., DNA, 1,11-18 (1981), Taka-Hashi, the people such as N., cell, 29,671-679 (1982) and Krawinkel, the people such as U., EMBO, J., Isosorbide-5-Nitrae 03-407 (1982)).

Prepare two class expression vectors for the production of chimeric antibody. Also be exactly to strengthen son/startup The subclass type express under the control of control district contain encoding murine L chain V district and people L chain C district it The expression vector of DNA and express the coding that contains under the control of control district in the enhancers/promoters class The expression vector of the DNA in mouse H chain V district and people H chain C district. Then show with these Reach simultaneously host's cell of transformed mammalian cell class of carrier, in external or body, cultivate and turn to The cell of changing is to produce chimeric antibody.

As selection, the DNA in encoding murine L chain V district and people L chain C district and the DNA in encoding murine H chain V district and people H chain C district can be imported in the single expression vector, use said carrier transformed host cell, then in external or these cell transformed of culturing in vivo to produce chimeric antibody.

The people WS-4 antibody of reconstruct of the present invention comprises:

(A) L chain respectively comprises:

(1) people L chain C district; With

(2) comprise the L chain V district of the mouse monoclonal antibody WS-4L chain CDR of people L chain FR and anti-people IL-8, and

(B) H chain respectively comprises:

(1) people H chain C district; With

In the preferred mode of the present invention, above-mentioned L chain CDR has the degree of the said aminoacid sequence that limits in table 5 in the aminoacid sequence shown in the SEQ ID NO:26; Above-mentioned H chain CDR has the said aminoacid sequence degree that limits in table 5 in the aminoacid sequence shown in the SEQ ID NO:27; Above-mentioned people L chain FR is from REI; Above-mentioned people H chain FR1, FR2 and FR3 are from VDH26, and FR4 is from 4B4; Above-mentioned people L chain C district is people C κ district; Above-mentioned people H chain C district is people C γ 1 district.In addition, above-mentioned people H chain C district can be people C γ 4 districts, but or the binding radioactivity isotropic substance replace above-mentioned people L chain C district and/or people H chain C district.

The partial amino-acid series that can preferably replace above-mentioned people FR has enough active reconstructed hyman antibody with preparation to specific antigens.

In the preferred mode of the present invention, L chain V district has at the aminoacid sequence shown in table 2RVLa or the RVLb, and H chain V district has RVHa in table 3 and 4, RVHb, RVHc, RVHd, RVHe, RVHf, the aminoacid sequence shown in RVHg or the RVHh.And the amino acid of position 41 should be proline(Pro) in H chain V district FR2, and the amino acid in said position 47 should be tryptophane, and/or the amino acid in said FR3 position 67 should be phenylalanine, and those have RVHb, RVHd, RVHe, RVHf, aminoacid sequence is more preferably shown in RVHg or the RVHh.Wherein RVHg exists most preferably as H chain V district.

Prepare two class expression vectors to produce reshaped antibody.That is to say, a kind ofly be contained in the expression vector that the enhancers/promoters class is expressed the DNA of the reconstruct people L chain that control region control coding down limits previously, and be contained in another expression vector that the enhancers/promoters class is expressed the DNA of the reconstruct people H chain that the coding of control region under controlling limit previously.Then, transform simultaneously such as the host cell of mammalian cell and external or cultivate transformant in vivo with these expression vectors to produce reconstructed hyman antibody.

As selection, the DNA of coding reconstruct people L chain and the DNA of coding reconstruct people H chain can be imported in the single expression vector, use said carrier transformed host cell, then external or cultivate these cell transformed in vivo with production purpose reconstructed hyman antibody.

According to such as the a-protein affinity chromatography, the ordinary method of ion-exchange chromatography or gel-filtration is separable and purifying produces by this way chimeric antibody or reconstructed hyman antibody.

The chimeric L chain of the present invention or the people L chain of reconstruct can be used for making up with the preparation complete antibody with the H chain.Similarly, chimeric H chain of the present invention or reconstruct people H chain can be used for making up with the preparation complete antibody with the L chain.

Mouse L chain V district, reconstruct people L chain V district, mouse H chain V district and reconstruct people H chain V district are in conjunction with the antigenic intrinsic zone with people IL-8 form.Think with they separately or with other proteinic fusion rotein form as medicine, useful during diagnostic aid etc.

And L chain V district CDR of the present invention and H chain V district CDR also are in conjunction with the antigenic intrinsic part with people IL-8 form.Think with they separately or with other proteinic fusion rotein form as medicine, useful during diagnostic aid etc.

The DNA in mouse L chain V of the present invention district of encoding is useful in the DNA of the DNA of the chimeric L chain of preparation coding or coding reconstruct people L chain.Similarly, the DNA in encoding murine H chain V district is useful when the DNA of the DNA of the chimeric H chain of preparation coding or the reconstruct people H chain of encoding.In addition, the DNA of code book invention L chain V district CDR is useful at the DNA of DNA that is used for preparation coding reconstruct people L chain V district or coding reconstruct people L chain.

Similarly, the encode DNA of H chain V district CDR of the present invention is useful at the DNA of the DNA that is used for preparation coding reconstruct people H chain V district and the reconstruct people H chain of encoding.And, reconstructed hyman antibody F (ab ') ₂, the strand Fv of the Fv of Fab or Fv or coupling connection H chain and L chain can produce and be used for above-described purpose (seeing, for example: Bird, people such as R.E., TIBTECH, 9,132-137,1991) in suitable host.

Strand Fv forms through the H chain V district and the L chain V district of the reconstructed hyman antibody of the anti-people IL-8 of connection.In this strand Fv, H chain V district is connected with connexon with L chain V district, and preferably connects (Huston, people such as J.S., Proc.Natl.Acad.Sci.USA, 85,5879-5883,1988) with the peptide connexon.

The H chain V district of this strand Fv and L chain V district can be one of above-mentioned reconstructed hyman antibody H chain and L chain V district.Its object lesson is included in SEQ ID NO:38, and 41,44,45,48, the H chain V district that aminoacid sequence described in 51 and 54 is formed and contain strand Fv (seeing WO88-01649) in the L chain V district of the composition of aminoacid sequence described in SEQ IDNO:62 or 65.

Preferably connect these zones with the peptide connexon.The example of the peptide connexon that uses comprises any any strand peptide of being made up of for example 12-19 residue (seeing WO88-09344).

Make template through the DNA that uses coding H chain or H chain V district and the DNA in above-mentioned reconstructed hyman antibody L chain of coding or L chain V district, the primer that uses the DNA two ends that the primer that limits two ends makes up restricted code polypeptide connexon to the DNA part and the process of these amino acid needed sequences of encoding through pcr amplification is to increasing to connect H and L chain obtain the to encode DNA of strand Fv respectively.

And, in case prepared the DNA of coding strand Fv, can obtain to contain the expression vector of this DNA according to conventional methods and with said expression vector host transformed.And, can obtain strand Fv according to a conventional method through using this host.

With antibody molecule relatively, strand Fv shows better tissue permeability, and expection is through being used for imaging and as the therapeutical agent that has with the reconstructed hyman antibody identity function with labelled with radioisotope.

ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent-antibody technique can be used for confirming the chimeric antibody of anti-IL-8 of the present invention, reconstructed hyman antibody and its F (ab ') ₂, Fab, the combination of Fv or strand Fv is active.For example, under the situation of the enzyme immunoassay of use and chimeric antibody and reconstructed hyman antibody, people IL-8 is added on the flat board that covers with anti-people IL-8 polyclonal antibody, add the chimeric antibody or the culture supernatants of reconstructed hyman antibody or the cell sample of purifying that produce anti-people IL-8, and add the suitable second antibody of using such as the enzyme labelling of alkaline phosphatase.Behind insulation and the washing flat board, add enzyme substrates, then measure absorbancy to estimate antigen-binding activity such as the p-nitrophenyl phosphoric acid ester.

With the chimeric antibody of common ligand receptor in conjunction with the anti-people IL-8 of inhibition evaluation of measuring, reconstructed hyman antibody and its F (ab ') ₂, Fab, Fv or strand Fv are to the IL-8 binding inhibition activity of IL-8 acceptor.For example, in order to measure behind the isolating neutrophil that centrifugal or alternate manner obtains from the blood that heparin is handled, the bonded of IL-8 acceptor suppresses on IL-8 and the neutrophil, prepares the cell suspension with suitable cell count that can be used for said determination.

With ¹²⁵Mark such as I etc. suitably contain the solution of IL-8, and with unlabelled IL-8 with the suitable concn preparation contain antibody of the present invention or its segmental solution mixes, then this mixture is added in the above-mentioned neutrophil suspension.After a period of time, separate neutrophil and be determined at mark activity on the neutrophil.

Conventional currently known methods, as at Grob, P.M. etc., journal of biological chemistry, 265,8311-8316, the method for describing in 1990 can be used for estimating antibody of the present invention or its fragment to the chemotactic inhibition of neutrophil.

Under the situation of using the chemotactic test kit that obtains with commercial form, dilute antibody of the present invention or its fragment with suitable medium after, IL-8 is added in the test kit, then add the antibody or the fragment of dilution.Subsequently, the neutrophil suspension of preparation is added in the test kit and makes leave standstill for some time.Because the neutrophil of migration can be through measuring the number of this neutrophil attached on the filter membrane that is placed in the test kit such as the usual way of dyeing or fluorescence antibody method.In addition, also can be through using microscopical microscopic evaluation or measuring through the automatic measurement of using machinery.

Behind the filtration sterilization that uses membrane filtration, the chimeric antibody of anti-people IL-8 of the present invention, reconstructed hyman antibody and its F (ab ') ₂, Fab, Fv or strand Fv fragment can be used as medicinal therapeutical agent preferably through parenteral, as through intravenous injection, intramuscular injection, peritoneal injection or subcutaneous injection or in tracheae are as through using the sprays medication.Although age and symptom according to patient change, and are 1-1000mg/ people at human normal dose, wherein can select the fractionated dose in 1-10mg/kg/ week.

For estimate its purifying in conjunction with active, can be through the ordinary method that is usually used in preparing physiologically active protein with chimeric antibody of the present invention, reconstructed hyman antibody and its F (ab ') ₂, Fab, Fv or strand Fv produced in fragments patent medicine therapeutical agent.For example, the goods that are used for injecting are by the refining chimeric antibody that is dissolved in such as the solvent of physiological saline or damping fluid, reconstructed hyman antibody or its F (ab ') ₂, Fab,, Fv or strand Fv fragment then add such as Tween 80, and gelatin or human serum albumin (HSA) are formed.As selection, but these goods also lyophilize with preceding dissolving and reprovision.Can be used for cryodesiccated carrier example and comprise that sugar-alcohol or sugar are such as the sweet dew alcohol and glucose.

Embodiment

Although hereinafter provide detailed explanation of the present invention by the embodiment that describes below, scope of the present invention is not subjected to the restriction of these embodiment.

Embodiment 1: the clone of the DNA in the mouse monoclonal antibody V district of the anti-people IL-8 that encodes

The DNA of the mouse monoclonal antibody variable region of the anti-people IL-8 of clones coding in the following manner.

1. the preparation of total RNA

Through modification in biological chemistry, 18,5294-5299, the Chirg-win described in 1979, people's such as J.M. cesium chloride density gradient centrifugation method prepares total RNA from hybridoma WS-4.

Just, complete homogenizing 1 * 10 in 25ml 4M guanidine thiocyanate (Flwka) ⁷Hybridoma WS-4 cell.Homogenate is taped against in the 5.7M caesium fluoride soln in the centrifuge tube, then in Beckman SW40 whizzer 20 ℃ with 31, centrifugal 14 hours precipitated rnas of 000rpm.

With 80% washing with alcohol RNA precipitation, be dissolved in 200 μ l then and contain the 20mM Tris-HCl of 10mM EDTA and 0.5%N-sodium lauryl sarcosinate (in (pH7.5).After adding proteolytic enzyme (Boehringer) was 0.5mg/ml to concentration, insulation gained mixture was 30 minutes in 37 ℃ of water-baths.With phenol and chloroform extraction mixture, use ethanol sedimentation RNA.Then, RNA precipitation is dissolved among the 10mM Tris-HCl (pH7.5) that 200 μ l contain 1mM EDTA.

2. the extraction of messenger RNA(mRNA) (mRNA)

In order to extract the mRNA of encoding murine monoclonal antibody WS-4H chain, use quick track mRNA separating kit Version 3.2 (Invitrogen) and extract poly (A)-positive mR-NA from total RNA that top step 1 obtains according to the described method of shop instruction.

3. strand cDNA's is synthetic

The synthesizing single-stranded cDNA of about 40ng mRNA that uses cDNA circulation test kit (Invitrogen) and described to specifications method from top step 2, to obtain.The cDNA that then product of gained is used for amplification coding mouse H chain V district.And, for the cDNA in amplification coding mouse L chain V district, from the synthesizing single-stranded cDNA of the above-mentioned total RNA of about 10 μ g.

4. through the gene of pcr amplification encoding antibody variable region

(1) amplification of the cDNA in encoding murine H chain V district

Be used as the PCR primer at MHV (murine heavy chain variable region) primer 1 to 12 shown in the SEQ ID NO:13 to 24 with at MHC (murine heavy chain constant region) primer (Jones, people such as S.T., biotechnology, 9,88-89,1991) shown in the SEQ ID NO:25.Obtain 100 μ l in the above in the step 3 and contain 10mM Tris-HCl (pH8.3), 50mM KCl, 0.1mM dNTP (dATP, dGTP, dCTP, dTTP), 1.5mM MgCl ₂0.001% (w/v) gelatin, the 5 archaeal dna polymerase AmpliTaq of unit (Perkin Elmer Cetus), one of MHV primer shown in the 0.25 μ M SEQ ID NO:13 to 24, the PCR solution of the MCH primer shown in the 75 μ M SEQID NO:25 and 1.5ml strand cDNA solution.Preparation PCR solution is used for each MHV primer 1-12.After covering this solution with 50 μ l mineral oil, with 94 ℃ of starting temperatures 3 minutes, then 94 ℃ 1 minute, the order of 55 ℃ of 1 minute and 72 ℃ of circulations of 1 minute heats.Repeat this heating cycle 30 times, at 72 ℃ of further insulation reaction mixtures.

(2) amplification of the cDNA in encoding murine L chain V district

Be used as the PCR primer at (the mouse K variable region) primer 1 to 11 of the MKV shown in the SEQ ID NO:1 to 11 with at (the mouse K constant region) primer of the MKC shown in the SEQ ID NO:12 (Jones, people such as S.T., biotechnology, 9,88-89,1991).

The 2.0 μ l strand cDNA that obtain with top step 3 are carried out the cDNA amplification, use and the described identical method of step 4 part (1) H chain V district's gene amplification in the above, different are to use the KMV primer mixture respectively 0.25 μ m and 3.0 μ m MCK primers increase.

5.PCR the purifying of product and fragmentation

Use 1.5% low melting-point agarose (Sigma) to separate by top described each dna fragmentation through agarose gel electrophoresis through the H of pcr amplification chain V district and L chain V district.Downcut the segmental agar sugar-tablet of L chain DNA of the H chain DNA fragment contain the about 450bp of length and the about 400bp of length respectively and, then add the isopyknic 20mM Tris-HCl (pH7.5) that contains 2mM ED-TA and 300mM NaCl 65 ℃ of fusings 5 minutes.

With phenol and this mixture of chloroform extraction, reclaim this dna fragmentation and be dissolved among the 10mM Tris-HCl (pH7.5) that contains 1mM EDTA through ethanol sedimentation.Then, use is dissolved in and contains 10mM MgCl ₂With the Restriction Enzyme XmaI (New England BioLabs) of 5 units among the 10mM Tris-HCl (pH7.9) of 1mM dithiothreitol (DTT) 37 ℃ of these fragments of digestion 3 hours.Subsequently, 37 ℃ of these dna fragmentations of digestion 2 hours, use 1.5% low melting-point agarose (sigma) with the Restriction Enzyme SalI (Takara shuzo) of 40 units through the separating obtained dna fragmentation of agarose gel electrophoresis.

Cutting-out contains the agar sugar-tablet of dna fragmentation and 65 ℃ of fusings 5 minutes, then adds the isopyknic 20mM Tris-HCl (pH7.5) that contains 2mM EDTA and 300mM NaCl.With phenol and this mixture of chloroform extraction, reclaim this dna fragmentation and be dissolved among the 10mM Tris-HCl (pH7.5) that contains 1mM EDTA through ethanol sedimentation.

Therefore, obtain respectively to contain encoding murine K type L chain V district gene dna fragmentation and contain the dna fragmentation of the gene in encoding murine H chain V district.Above-mentioned dna fragmentation all has the SalI attachment site at its 5 ' end, has the XmaI attachment site at its 3 ' end.

6. connect and conversion

The about 0.3 μ g of SalI-XmaI dna fragmentation of the gene that contains encoding murine K type L chain V district for preparing in top described mode with through using SalI, XmaI and escherichia coli alkaline phosphatase (BAP; Takara Shuzo) digests the about 0.1 μ g of pUC19 carrier (TakaraShuzo) the 16 ℃ of mixing in the buffering reaction mixture that contains 1 T4 of unit dna ligase (Gibco BRL) that prepare and also added additional damping fluid connection in 4 hours.

Then, the above-mentioned connection mixture of 5 μ l is added in the competent cell (GIBCO BRL) of 50 μ l bacillus coli DH 5 alphas, cell was left standstill on ice 30 minutes, 40 ℃ 1 minute and again at 1 minute on ice.Then, add 400 μ l2 * YT substratum (molecular cloning: laboratory manual, people such as Sambrook, press of cold spring harbor laboratory, 1989).37 ℃ the insulation 1 hour after, intestinal bacteria are applied to 2 * YT nutrient agar (molecular cloning: laboratory manual, Sambrook, Deng the people, press of cold spring harbor laboratory, 1989) on, this substratum contains 50 μ g/ml penbritins (Meiji Seika), follows 37 ℃ of overnight incubation to obtain the intestinal bacteria transformant.

Subsequently, with the selective marker of 50 μ gX-Gal (5-bromo-4-chloro-3-indyl-β-D-galactoside, Takara shuzo) as this moment.

Under 37 ℃, in the 10ml2 that contains 50 μ g/ml penbritins * YT substratum, cultivate this transformant and spend the night, use the method described in miniature test kit of qiagen plasmid (QIAGEN) and the by specification from this culture, to prepare plasmid DNA.

The plasmid called after pUC-WS4-VL that contains the gene in the mouse K-type L chain V district that in hybridoma WS-4, produces that coding obtains in this mode.

Except using JM109 as competent escherichia coli cell, according to top described identical method, contain the plasmid of coding from the preparation of SalI-Xmal dna fragmentation from the mouse H chain V district gene of hybridoma WS-4.The plasmid called after pUC-WS-4-VH of gained.

Determining of embodiment 2 dna nucleotide sequences

Use M13 primer RV and M13 primer M4 (all from Takara shuzo) as aligning primer, use automated DNA sequenator (Applied Biosystems, Inc.) and Taq DyeDeoxy Terminator cycle sequencing test kit (Applied Biosystems Inc.) and according to the method for producer's explanation determine the to encode nucleotide sequence of cDNA in above-mentioned plasmid zone.The nucleotides sequence that is included in the gene in the encoding murine WS-4 light chain of antibody V district among the plasmid pUC-WS-4-VL is listed among the SEQ ID NO:26 and shows.In addition, the nucleotides sequence that is included in the gene in the encoding murine WS-4 heavy chain of antibody V district among the plasmid pUC-WS4-VH is listed among the SEQ ID NO:27 and shows.

Embodiment 3:CDR determines

The basic structure in L and H chain V district has the common similarity, respectively has 4 framework regions that are called the hypervariable region connection of complementarity-determining region (CDR) by three.Although the aminoacid sequence of framework region is quite conservative, the aminoacid sequence mutability in CDR district high (ka-bat, people such as E.A., " protein sequence of immune target ", U.S. HHS, 1991).

According to this fact,, wait the amino acid sequence database of the antibody that the people prepares to determine that to study its homology CDR is as shown in table 5 through attempting mouse monoclonal antibody amino acid sequences and the Kabat of the anti-people IL-8 of coupling.

CDR plasmid sequence in table 5 mouse WS-4 light chain of antibody V district and the H chain V district is counted CDR1 CDR2 CDR3pUC-WS4-VL 26 24-34 50-56 89-97pUC-WS4-VH 27 31-35 50-68 101-111

Embodiment 4: the cDNA that confirms the clone expresses (preparation of chimeric WS-4 antibody)

Preparation of expression vectors

In order to prepare the carrier of expressing chimeric WS-4 antibody, the cDNA in encoding murine WS-4L chain and H chain V district clone pUC-WS4-VL and pUC-WS4-VH modify through PCR respectively.Then their are imported HEF expression vector (refer to foregoing, WO92-19759 and Fig. 1).

The reverse primer (SEQ ID NO:29) in reverse primer in L chain V district (SEQ ID NO:28) and H chain V district is hybridized with the DNA of coding V district leader starting point respectively and is designed to have Kozak common sequences (Kozak, M. wait the people, the molecular biology magazine, 196,947-950,1987) and the HindIII restriction site.The dna sequence dna hybridization of the forward primer (SEQ ID NO:31) in the forward primer (SEQID NO:30) in L chain V district and H chain V district and coding J chain end also is designed to be added with a donor splicing site sequence and BamHI restriction site.

100 μ l contain 20mM Tris-HCl (pH8.2), 10mM KCl, 6mM (NH4) ₂SO ₄, 1%Triton X-100,100mM dNTP, 1.5mM MgCl ₂, each primer 100pmol, the PCR reaction mixture of 100ng template DNA (pUC-VL or pUC-VH) and 2.5UAmpli Taq enzyme covers with 50 μ l mineral oil.94 ℃ of initial sex change after 3 minutes, repeat by 94 ℃ 1 minute, the heating cycle that 55 ℃ of 1 minute and 72 ℃ were formed in 1 minute 30 times then is incubated 10 minutes at last at 72 ℃.

Use 1.5% low melting-point agarose gel-purified PCR product, then with HindIII and BamHI digestion.L chain V district clone is advanced HEF expression vector HEF-VL-gk, and H chain V district clone is advanced HEF expression vector HEF-VH-g γ 1.After measuring dna sequence dna, contain the plasmid difference called after HEF-chWS4L-gk and the HEF-chWS4H-g γ 1 of dna fragmentation with correct dna sequence dna.

The COS cell is advanced in transfection

In order to observe the transient expression of chimeric WS-4 antibody, the above-mentioned expression vector of test in the COS cell.Use gene pulse instrument system (bioRad) HEF-chWS4L-gk and HEF-chWS-4H-g γ 1 transfection simultaneously to be advanced the COS cell through electroporation.Each DNA (10 μ g) added to be dissolved in contain 1 * 10 among the PBS ⁷In the 0.8ml aliquots containig of cell 1ml, then with 25 μ F electric capacity in the 1.5KV pulse.

After at room temperature experiencing decubation of 10 minutes, the cell suspension of electroporation is contained in the DMEM substratum of foetal calf serum of 5% no gamma globulin and is put in the tissue culture ware in 15ml.Be incubated after 96 hours, collect substratum, through the centrifugal cell debris that removes, then with having disk filter (Gelman Science) filtering supernatant in 0.45 μ m aperture.

ELISA

The ELISA flat board that is used to measure antigen combination and antibody concentration by following described preparation.Prepare the ELISA flat board of measuring antigen-binding activity by following mode.With 100 μ l goat Anti-Human IL-8 polyclonal antibody (R﹠amp; D system) concentration with 2 μ g/ml is dissolved in solid-phase layer damping fluid (0.1M sodium bicarbonate, 0.02% sodiumazide) in 96 each hole of hole flat board (Nunc), forms solid-phase layer also with 200 μ l dilution buffer liquid (50mM Tris-HCl (pH7.2), 1% bovine serum albumin (BSA), 1mM MgCl ₂, 0.15M NaCl, 0.05%Tween 20,0.02% sodiumazide) sealing after, add 100 μ l recombinant human IL-8 (Amersham) (5ng/ml).

The purification of samples of serial dilution chimeric antibody or express their COS cell culture supernatant liquid and add in each hole.Then, the anti-human IgG antibody of goat (TAGO) (1 μ g/ml) who adds 100 μ l alkali phosphatase enzyme marks.After insulation and the washing, add substrate solution (1mg/ml p-nitrophenyl phosphoric acid ester), then under 405nm, survey absorbancy.

In order to measure antibody concentration, after in dull and stereotyped each hole, 96 holes, forming solid-phase layer and sealing with the anti-human IgG antibody of goat (TAGO) of 100 μ l, 1 μ g/ml concentration, the purification of samples of serial dilution chimeric antibody or express their COS cell culture medium and be added in each hole.Then, the anti-human IgG antibody of goat (TAGO) (1 μ g/ml) who adds 100 μ l alkali phosphatase enzyme marks.After insulation and the washing, add substrate solution (1mg/ml p-nitrophenyl phosphoric acid ester) and under 405nm, survey absorbancy.

As a result, because chimeric antibody WS-4 demonstrates the specificity combination to IL-8, think that this chimeric antibody has the correct structure (see figure 2) in mouse monoclonal antibody WS-4V district.

And, intestinal bacteria with above-mentioned plasmid HEF-chWS4L-gk preserve with bacillus coli DH 5 alpha (HEF-chWS4L-gk), intestinal bacteria with above-mentioned plasmid HEF-chWS4H-g γ 1 preserve with e. coli jm109 (HEFOchWS4H-g γ 1), they are (the 1-1-3 Higashi of biotechnology Industrial Technology Research Institute that are kept at industrial science and technical body according to the requirement of budapest treaty respectively with the title of FERM BP-4739 and FEREBP-4740 on July 12nd, 1994, Tsukuba, Ibaraki, Japan).

Embodiment 5: the preparation of reconstruct people WS-4 antibody

The preparation in reconstruct people WS-4 heavy chain of antibody V district

Design the DNA in coding reconstruct people WS-4 heavy chain of antibody V district by following described mode.The global DNA in design coding reconstruct people WS-4 heavy chain of antibody V district makes the anti-VDH26 FR1 of the people that encodes respectively be connected with the dna sequence dna of encoding murine WS-4 heavy chain of antibody V district CDR to the known dna sequence of FR3 and people's antibody 4B4 FR4.

Then, HindIII recognition site/kozak common sequences and BamHI recognition site/donor splicing site sequence are added to 5 of this dna sequence dna ' and 3 ' side respectively, then import the HEF expression vector.To be divided into 4 oligonucleotide about equally with the dna sequence dna that this mode designs then, use the secondary structure of those oligonucleotide of these oligonucleotide assemblings of Computer Analysis possibility overslaugh subsequently.

These 4 oligonucleotide sequences show in SEQ ID NO:32 to 35.These oligonucleotide have the length of 113 to 143 bases, and adjacent oligonucleotide has the zone that overlaps each other by 20 based compositions.HF1 of these 4 oligonucleotide (SEQ ID NO:32) and HF3 (SEQ ID NO:34) have adopted dna sequence dna are arranged, and another HF2 (SEQID NO:33) and HF4 (SEQ ID NO:35) have antisense dna sequence.With synthetic these oligonucleotide of automatic dna synthesizer (applying biological system).

And the method for assembling these 4 oligonucleotide through PCR shows in Fig. 3.Merge HF1 and HF2 and HF3 and the HF4 of about each 100ng and add the PCR reaction mixture that has 98 μ l final volume and contain the 2.5Upfu archaeal dna polymerase.94 ℃ of beginning sex change after 3 minutes, with 2 circulations of this solution insulation, every circulation by 94 ℃ 2 minutes, 55 ℃ of 2 minutes and 72 ℃ of 2 minutes compositions.

After replacing the PCR reaction soln of half volume mutually, continue 2 circulations of insulation again.Add RVH5 ' primer (SEQ ID NO:36) of each 100pmol and RVH3 ' primer (SEQ ID NO:37) as behind the outside primer, cover the PCR reaction soln with 50 μ l mineral oil.94 ℃ of beginning sex change after 3 minutes, with 45 circulations of reaction soln insulation, every circulation be 94 ℃ 1 minute, 55 ℃ 1 minute 72 ℃ 1 minute, be incubated 10 minutes at 72 ℃ at last.

The dna fragmentation purifying on 1.5% low melting-point agarose gel that contains about 450 base pairs advances among the HEF expression vector HEF-VH-g γ 1 (Fig. 1) with HindIII and BamHI digestion and clone.After using EF-1 primer (SEQ ID NO:66) and HIP primer (SEQ ID NO:67) to measure dna sequence dna, contain the plasmid called after HEF-RVHa-g γ 1 of the dna fragmentation of the correct aminoacid sequence in coding H chain V district.Being contained in the aminoacid sequence in the H chain V district among this plasmid HEF-RVHa-g γ 1 and nucleotides sequence is listed among the SEQ ID NO:38 and shows.

Prepare reconstruct people WS-4 heavy chain of antibody V district " b " in the following manner, " c ", " d ", " e ", " f ", " g " and " h " is all types of.

Use mutagenic compound primer LTW1 (SEQ ID NO:39) and LTW2 (SEQ IDNO:40) (RVHb) through pcr amplification type " b ", the leucine that is designed to 47 is replaced by tryptophane, RVH5 ' (SEQ ID NO:36) and RVH3 ' (SEQ ID NO:37) be as the primer that limits two ends, plasmid HEF-RVHa-g γ 1 as template DNA to obtain plasmid HEF-RVHb-g γ 1.Being contained in the aminoacid sequence in the H chain V district among this plasmid HEF-RVHb-g γ 1 and nucleotides sequence is listed among the SEQ ID NO:41 and shows.

Use mutagenic compound primer QTP1 (SEQ ID NO:42) and QTP2 (SEQ IDNO:43) through pcr amplification type " c ", the L-glutamic acid that is designed at 41 is replaced by proline(Pro), and plasmid HEF-RVHa-g γ 1 makes template DNA to obtain plasmid HEF-RVHc-g γ 1.Being contained in the aminoacid sequence in the H chain V district among this plasmid HEF-RVHc-g γ 1 and nucleotides sequence is listed among the SEQ ID NO:44 and shows.

Use mutagenic compound primer QTP1 and QTP2 and plasmid HEF-RVHb-g γ 1 do template DNA through pcr amplification type " d " to obtain plasmid HEF-RVHd-g γ 1.Being contained in the aminoacid sequence in the H chain V district among this plasmid HEF-RVHd-g γ 1 and nucleotides sequence is listed among the SEQ ID NO:45 and shows.

Be designed to make template DNA amplification type " e " with acquisition plasmid HEF-RVHe-g γ 1 by the displaced mutagenic compound primer of proline(Pro) ATP1 (SEQ ID NO:46) and ATP2 (SEQ ID NO:47) and with plasmid HEF-RVHd-g γ 1 through using at 40 L-Ala.Being contained in the aminoacid sequence in the H chain V district among this plasmid HEF-RVHe-g γ 1 and nucleotides sequence is listed among the SEQ ID NO:48 and shows.

Use is designed to the mutagenic compound primer GTA1 (SEQ ID NO:49) that replaced by L-Ala at 44 glycine and GTA2 (SEQ ID NO:50) and makes template DNA amplification type " f " to obtain plasmid HEF-RVHf-g γ 1 with plasmid HEF-RVHd-g γ 1.Being contained in the aminoacid sequence in the H chain V district among this plasmid HEF-RVHf-g γ 1 and nucleotides sequence is listed among the SEQ ID NO:51 and shows.

Use is designed to mutagenic compound primer LTF1 that 67 leucine replaced by phenylalanine and makes template DNA amplification type " g " to obtain plasmid HEF-RVHg-g γ 1 with plasmid HEF-RVHd-g γ 1.Be contained in aminoacid sequence among this plasmid HEF-RVHg-g γ 1 and nucleotide sequence shown in SEQ ID NO:54.

Use mutagenic compound primer LTF1 and LTF2 and make template DNA amplification type " h " with plasmid HEF-RVHb-g γ 1.Being contained in the aminoacid sequence in this plasmid HEF-RVHh-g γ 1H chain V district and nucleotides sequence is listed among the SEQ ID NO:55 and shows.

The preparation in reconstruct people WS-4 light chain of antibody V district

The DNA in coding reconstruct people WS-4 light chain of antibody V district designs in the following manner.The complete DNA in design coding reconstruct people WS-4 light chain of antibody V district is so that the dna sequence dna of coding people antibody REI FR links to each other with the dna sequence dna of encoding murine WS-4 light chain of antibody V district CDR.

Then, HindIII recognition site/kozak common sequences and BamHI recognition site/donor splicing site sequence are added to respectively 5 of this dna sequence dna ' and 3 ' side make it can import the HEF expression vector.To be divided into 4 oligonucleotide about equally with the dna sequence dna that this mode designs then.May hinder the secondary structure of those oligonucleotide of these oligonucleotide assemblings subsequently with Computer Analysis.

4 oligonucleotide sequences show in SEQ ID NO:56 to 59.These oligonucleotide have the length of 106 to 124 bases, and adjacent oligonucleotide has the overlapping area by 19 to 23 based compositions.LF1 of these 4 oligonucleotide (SEQ ID NO:56) and LF3 (SEQ ID NO:58) have adopted dna sequence dna are arranged, and other LF2 (SEQID NO:57) and LF4 (SEQ ID NO:59) have antisense dna sequence.Use is used for above-mentioned HF1 to synthetic these oligonucleotide of the same procedure of HF4.

In order to assemble, the PCR mixture that contains 4 types each 100ng of Nucleotide and 50Ampli Taq at 94 ℃ of initial sex change 98 μ l is after 3 minutes, 2 circulations of this mixture insulation, and every circulation was by 94 ℃ of insulations 2 minutes.55 ℃ of 2 minutes and 72 ℃ were formed in 2 minutes.Behind the RVL5 ' primer (SEQ ID NO:60) and each 100pmol of RVL3 ' primer (SEQ ID NO:61) of adding as outside primer, cover the PCR reaction mixture with 50 μ l mineral oil.94 ℃ of initial sex change after 3 minutes, with 30 circulations of reaction mixture insulation, every circulation be 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ 1 minute then finally are incubated 10 minutes (see figure 3)s at 72 ℃.

The dna fragmentation that contains about 400 base pairs uses 1.5% low melting-point agarose gel-purified, advances HEF expression vector HEF-VL-g κ (Fig. 1) with HindIII and BamHI digestion and clone.Use EF-1 primer (SEQ ID NO:66) and KIP primer (SEQID NO:68) to measure dna sequence dna, contain the plasmid called after HEF-RVLa-g κ of the dna fragmentation of coding L chain V district correct nitrogen base acid sequence.Being contained in L chain V region amino acid sequence among this plasmid HEF-RVLa-g κ and nucleotides sequence is listed among the SEQ ID NO:62 and shows.

Use is designed to the mutagenic compound primers F TY1 (SEQ ID NO:63) and the FTY2 (SEQ ID NO:64) that are replaced by tyrosine at 71 phenylalanine, RVL5 ' (SEQ IDNO:60) and RVL3 ' (SEQ ID NO:61) be as the primer that limits its two ends, plasmid HEF-RVLa-g κ do template DNA through pcr amplification " b " type (RVLb) to obtain plasmid HEF-RVLb-g κ.The amino-acid sequence and the nucleotide sequence that are contained in the L chain V district among this plasmid HEF-RVLb-g κ show in SEQ ID NO:65.

In order to estimate the antigen-binding activity of each chain of reconstruct people WS-4 antibody, with foregoing expression vector HEF-RVLa-g κ and the mode of chimeric WS-4 heavy chain of antibody expression vector HEF-chWS4H-g γ 1 rotaring redyeing COS cell at first simultaneously about reconstruct people WS-4 light chain of antibody type " a ".By behind the collection substratum noted earlier, the antigen-binding activity of the antibody total amount of using the described method of ELISA part among the embodiment 4 in the above to measure to produce and the antibody of generation.These results show in Fig. 4.As shown in FIG. 4, confirm at the antibody of forming as the chimeric antibody (chL/chH) of positive control with by reconstruct L chain and chimeric H chain (RVLa/chH) at indistinction aspect the antigen-binding activity.

Simultaneously, combination for the expression vector HEF-chWS4L-g κ that estimates chimeric WS-4 light chain of antibody and reconstruct people WS-4 heavy chain of antibody type " a ", all simultaneously cotransfection advances the COS cell, the antibody total amount of using the described method of " ELISA " part among the embodiment 4 in the above to measure to produce and the antigen-binding activity of gained antibody.Do not confirm the antigen-binding activity (see figure 4) of this antibody (chL/RVHa).

As previously mentioned, because reconstruct people WS-4 light chain of antibody " a " type list reveals the antigen-binding activity that equates with chimeric WS-4 antibody, through carrying out all types of evaluation of all reconstruct H chains with all types of of reconstruct H chain and reconstruct people WS-4 light chain of antibody " a " type (RVLa) while rotaring redyeing COS cell.

The result has reconstruct H chain " b ", " d ", " e ", " f ", those antibody of " g " and " h " type show the antigen-binding activity that can compare with the chimeric WS-4 antibody (chL/chH) that is used as positive control, therefore show that this is combined to form the function antigen binding site of people's antibody.Yet, for the total amount of the antibody that produces, except type " g " (RVHg), all types of amounts that produce are lacked than chimeric WS-4 antibody (chL/chH).And, in antibody, do not observe the antigen-binding activity (see figure 5) with H chain type " c ".

According to these discoveries, inferring that the antibody with reconstruct people WS-4 light chain of antibody " a " type (RVLa) and reconstruct people WS-4 heavy chain of antibody " g " type forms again shows the functional antigen binding site of antigen-binding activity preferably, and after the COS cell was advanced in transfection simultaneously, the antibody amount of generation can be compared with chimeric WS-4 antibody (chL/chH).

Then, through carry out evaluation with reconstruct people WS-4 light chain of antibody " b " type (RVLb) and all types of while rotaring redyeing COS cells of H chain to reconstruct people WS-4 light chain of antibody " b " type (RVLb).The result shows, the antibody (RVLb/RVHg) that only has reconstruct people WS-4 heavy chain of antibody " g " type shows the antigen-binding activity that can compare with the chimeric WS-4 antibody (chL/chH) that is used as positive control, and infers that this is combined in formation functional antigen binding site in people's antibody.And, about the antibody total amount that produces, except type " g " (RVHg), the total amount that all types produces is lacked (see figure 6) than chimeric WS-4 antibody (chL/chH).

In above-mentioned evaluation, show two types that are comparable to the reconstructed hyman antibody (RVLa/RVHg and RVLb/RVHg) of chimeric WS-4 antibody (chL/chH) in conjunction with active and production degree of people IL-8 are used the a-protein column purification respectively, use among the embodiment 4 the described method exact evaluation of ELISA part subsequently in conjunction with activity.The result shows, chimeric WS-4 antibody (chL/chH), RVLa/RVHg antibody and RVLb/RVHg antibody all show the active (see figure 7) of combination of same degree.

According to these discoveries, infer and to have reconstruct people WS-4 light chain of antibody " a " class (RVLa) or " b " class (RVLb) and reconstruct people WS-4 heavy chain of antibody " g " type RVHg) antibody form the functional antigen binding site that shows better antigen-binding activity level again, and after the COS cell is advanced in transfection jointly, show the antibody producing level that is comparable to chimeric WS-4 antibody (chL/chH).

Estimated by reconstruct people WS-4 light chain of antibody " a " type (RVLa) and H chain " g " type (RVHg) or described L chain in conjunction with inhibition test through ligand receptor " b " reconstructed hyman antibody formed of type (RVLb) and described H chain " g " type (RVHg) is to the inhibition activity of IL-8 in conjunction with the IL-8 acceptor.

The blood sample that about 100ml is handled from normal subjects's heparin covers in the 15ml Mono-Poly separation solution (ICN Biomedicals) with the aliquots containig of 35ml, and according to the specification sheets that provides through centrifugation human neutrophils layer.After washing these cells with the RPMI-1640 substratum that contains 1%BSA, remove the red corpuscle of pollution with the 150mM ammonium chloride solution.After centrifugal, wash these cells and with 2 * 10 with the RPMI-1640 substratum that contains 1%BSA ⁷The concentration of individual cell/ml is resuspended.The neutrophil content of finding this cell suspension is 95% or more, and this is to use the result who measures behind the smear preparation of Cytospin (Shandon) preparation with Diff-Quik dyestuff (Green Cross) dyeing.

Centrifugal above-mentioned neutrophil suspension and with 2 * 10 ⁷The concentration of individual cell/ml is resuspended in the binding buffer liquid (D-PBS that contains 1%BSA and 0.1% sodiumazide).At this moment, add SK2 chimeric antibody (seeing international patent application no PCT/JP94/00859) with Fc part identical and its antigen people IL-6 and be respectively about 50 μ g/ml and about 40ng/ml to concentration with people's antibody of the present invention.In ice bath, be incubated 30 minutes for the Fc acceptor on the presaturation neutrophil.

Be mixed in preparation usefulness in the binding buffer liquid through concentration with each 4ng/ml ¹²⁵Radiolabeled IL-8 of I (74TBq/nmol.Amersham) and cold IL-8 (Amersham).Chimeric WS-4 antibody (chL/chH), reconstructed hyman antibody (RVLa/RVHg and RVLb/RVHg), negative control people antibody (PAESEL+LOREI) or positive control mouse WS-4 antibody use binding buffer liquid with 2000ng/ml and approximately progressively 2 times of dilutions of concentration between the 8ng/ml respectively.50 μ lIL-8 solution and 50 each antibody-solutions of μ l are incubated 30 minutes in ice bath.Then, add above-mentioned neutrophil suspension of 100 μ l and the further insulation of mixing in per 15 minutes 1 hour.After the insulation, cell suspension covers in the sucrose solution of 200 μ l20%, follows centrifugal and freezing.In order to measure the IL-8 that is attached on the cell, downcut cell precipitation and measure radioactivity with gamma counter (Aroka).These results show in Fig. 8.

Have reconstruct people WS-4 light chain of antibody " a " type (RVLa) and H chain " g " type (RVHg) or described L chain " b " type and described H chain " g " antibody of type clearly shows that IL-8 is had the binding inhibition activity that is comparable to chimeric antibody (chL/chH) with combining of IL-8 acceptor.

And, requirement according to budapest treaty, intestinal bacteria with above-mentioned plasmid HEF-RVLa-g κ are with bacillus coli DH 5 alpha (HEF-RVLa-g κ), the intestinal bacteria that contain plasmid HEF-RVHg-g γ 1 claim biotechnology Industrial Technology Research Institute (1-1-3 Higashi that FERM BP-4738 and FERM BP-4741 be stored in industrial science and technical body on July 12nd, 1994 with separately life with e. coli jm109 (HEF-RVHg-g γ 1), Tsukuba, Ibaraki, Japan).

Reference example 1: the preparation of hybridoma WS-4

Use will the choose BALB/C mice splenocyte of IL-8 immunity and murine myeloma cell P3 * 63-Ag8.653 of polyoxyethylene glycol to merge and prepare the hybridoma that produces anti-people IL-8 monoclonal antibody according to conventional methods.Screen (KO, people such as Y.C., immunological method magazine, 149,227-235,1992) by the standard use of setting up hybridoma WS-4 with the activity that combines of people IL-8.

Industrial applicibility:

The invention provides the reconstructed hyman antibody of anti-people IL-8, in this antibody, the CDR in people's antibody V district replaces with the mouse monoclonal antibody CDR of anti-people IL-8.Because mainly from human and have the inherent CDR of low antigenicity, therefore reconstructed hyman antibody of the present invention has the low antigenicity to the people to this reconstructed hyman antibody, so expect can be used for pharmacological agent.

Microorganism list by the requirement preservation of the 13rd of Patent Cooperation Treaty:

World preservation mechanism:

Title: the national bio-science of industrial science and technical body and human technical institute.

Address: Japanese 1-3 Higashi.1-chome, Tsukuba, Ibaraki.

Preserving number and preservation day:

(1) bacillus coli DH 5 alpha (HEF-RVLa-g κ)

Preserve number: FERM BP-4738

Preserve day: on July 12nd, 1994

(2) bacillus coli DH 5 alpha (HEF-chWS4L-g κ)

Preserve number: FERM BP-4739

Preserve day: on July 12nd, 1994

(3) e. coli jm109 (HEF-chWS4H-g γ 1)

Preserve number: FERM BP-4740

Preserve day: on July 12nd, 1994.(4) e. coli jm109 (HEF-RVHg-g γ 1)

Preserve number: FERM BP-4741

Preserve day: on July 12nd, 1994.

Sequence table SEQ ID NO:1 sequence length:40 sequence types:nucleic acid chain:strand topology:linear molecule type:synthetic DNA sequence title:MKV1 sequence A CTAGTCGAC ATGAAGTTGC CTGTTAGGCT GTTGGTGCTG 40SEQ ID NO:2 sequence length:39 sequence types:nucleic acid chain:strand topology:linear molecule type:synthetic DNA sequence title:MKV2 sequence A CTAGTCGAC ATGGAGWCAG ACACACTCCT GYTATGGGT 39SEQ ID NO:3 sequence length:40 sequence types:nucleic acid chain:strand topology:linear molecule type:synthetic DNA sequence title:MKV3; ACTAGTCGAC ATGAGTGTGC TCACTCAGGT CCTGGSGTTG 40SEQ ID NO：4：43：：：：DNA：MKV4ACTAGTCGAC ATGAGGRCCC CTGCTCAGWT TYTTGGMWTC TTG 43SEQ ID NO：5：40：：：：DNA：MKV5ACTAGTCGAC ATGGATTTWC AGGTGCAGAT TWTCAGCTTC 40SEQ ID NO：6：37：：：：DNA：MKV6ACTAGTCGAC ATGAGGTKCY YTGYTSAGYT YCTGRGG 37SEQ ID NO：7：41：：：：DNA：MKV7ACTAGTCGAC ATGGGCWTCA AGATGGAGTC ACAKWYYCWG G 41SEQ ID NO：8：41：：：：DNA：MKV8ACTAGTCGAC ATGTGGGGAY CTKTTTYCMM TTTTTCAATT G 41SEQ ID NO：9：35：：：：DNA：MKV9ACTAGTCGAC ATGGTRTCCW CASCTCAGTT CCTTG 35SEQ ID NO：10：37：：：：DNA：MKV10ACTAGTCGAC ATGTATATAT GTTTGTTGTC TATTTCT 37SEQ ID NO：11：38：：：：DNA：MKV11ACTAGTCGAC ATGGAAGCCC CAGCTCAGCT TCTCTTCC 38SEQ ID NO：12：27：：：：DNA：MKCGGATCCCGGG TGGATGGTGG GAAGATG 27SEQ ID NO：13：37：：：：DNA：MHV1ACTAGTCGAC ATGAAATGCA GCTGGGTCAT STTCTTC 37SEQ ID NO：14：36：：：：DNA：MHV2ACTAGTCGAC ATGGGATGGA GCTRTATCAT SYTCTT 36SEQ ID NO：15：37：：：：DNA：MHV3ACTAGTCGAC ATGAAGWTGT GGTTAAACTG GGTTTTT 37SEQ ID NO：16：35：：：：DNA：MHV4ACTAGTCGAC ATGRACTTTG GGYTCAGCTT GRTTT 35SEQ ID NO：17：40：：：：DNA：MHV5ACTAGTCGAC ATGGACTCCA GGCTCAATTT AGTTTTCCTT 40SEQ ID NO：18：37：：：：DNA：MHV6ACTAGTCGAC ATGGCTGTCY TRGSGCTRCT CTTCTGC 37SEQ ID NO：19：36：：：：DNA：MHV7ACTAGTCGAC ATGGRATGGA GCKGGRTCTT TMTCTT 36SEQ ID NO：20：33：：：：DNA：MHV8ACTAGTCGAC ATGAGAGTGC TGATTCTTTT GTG 33SEQ ID NO：21：40：：：：DNA：MHV9ACTAGTCGAC ATGGMTTGGG TGTGGAMCTT GCTATTCCTG 40SEQ ID NO：22：37：：：：DNA：MHV10ACTAGTCGAC ATGGGCAGAC TTACATTCTC ATTCCTG 37SEQ ID NO：23：38：：：：DNA：MHV11ACTAGTCGAC ATGGATTTTG GGCTGATTTT TTTTATTG 38SEQ ID NO：24：37：：：：DNA：MHV12ACTAGTCGAC ATGATGGTGT TAAGTCTTCT GTACCTG 37SEQ ID NO：25：28：：：：DNA：MHCGGATCCCGGG CCAGTGGATA GACAGATG 28SEQ ID NO：26：382：：：：cDNA：WS4VL：：pUC-WS4-VL：1..60

61..382 mature peptide sequence A TG AGT GTG CTC ACT CAG GTC CTG GGG TTG CTG CTG CTG TGG CTT ACA 48Met Ser Val Leu Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr-20-15-10-5GGT GCC AGA TGT GAC ATC CAG ATG ACT CAG TCT CCA GCC TCC CTA TCT 96Gly Ala Arg Cys Asp Ile Gln Mer Thr Gln Ser Pro Ala Ser Leu Ser

-1 1 5 10GCA?TCT?GTG?GGA?GAA?ACT?GTC?ACC?ATC?ACA?TGT?CGA?GCA?AGT?GAG?ATT 144Ala?Ser?Val?Gly?Glu?Thr?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Glu?Ile

15 20 25ATT?TAC?AGT?TAT?TTA?GCA?TGG?TAT?CAG?CAG?AAA?CAG?GGA?AAA?TCT?CCT 192Ile?Tyr?Ser?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Gln?Gly?Lys?Ser?Pro

30 35 40CAG?CTC?CTG?GTC?TAT?AAT?GCA?AAA?ACC?TTA?GCA?GAT?GGT?GTG?TCA?TCA 240Gln?Leu?Leu?Val?Tyr?Asn?Ala?Lys?Thr?Leu?Ala?Asp?Gly?Val?Ser?Ser?45 50 55 60AGG?TTC?AGT?GGC?AGT?GGA?TCA?GGC?ACA?CAG?TTT?TCT?CTG?CGG?ATC?AGC 288Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Gln?Phe?Ser?Leu?Arg?Ile?Ser

65 70 75AGC?CTG?CAG?CCT?GAA?GAT?TTT?GGG?AGT?TAT?TAC?TGT?CAA?CAT?CAT?TTT 336Ser?Leu?Gln?Pro?Glu?Asp?Phe?Gly?Ser?Tyr?Tyr?Cys?Gln?His?His?Phe

80 85 90GGT?TTT?CCT?CGG?ACG?TTC?GGT?GGA?GGC?ACC?AAG?CTG?GAA?CTC?AAA?C 382Gly?Phe?Pro?Arg?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Leu?Lys

95 100 105SEQ ID NO:27 sequence lengths: 424 sequence types: nucleic acid chain: double-stranded topology: thread-like molecule type: cDNA sequence title: WS4VH originates biological: mouse direct sources clone: pUC-WS4-VH feature: 1..57 signal peptide

58..424 mature peptide sequence A TG AAG TTG TGG TTA AAC TGG GTT TTT CTT GTG ACA CTT TTA AAT GGT 48Mer Lys Leu Trp Leu Asn Trp Val Phe Leu Val Thr Leu Leu Asn Gly-19-15-10-5ATC CAG TGT GAG GTG AAA CTG GTG GAG TCT GGA GGA GGC TTG ATA CAG 96Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Ile Gln

-1 1 5 10GCT?GGG?GAT?TCT?CTG?AGA?GTC?TCC?TGT?GTA?ACC?TCT?GGG?TTC?ACC?TTC 144Pro?Gly?Asp?Ser?Leu?Arg?Leu?Ser?Cys?Val?Thr?Ser?Gly?Phe?Thr?Phe

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAG?CCT?CCA?GGA?AAG?GCA?CTT 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Ala?Leu?30 35 40 45GAG?TGG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Trp?Val?Gly?Leu?lle?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

50 55 60TAC?AGT?GCA?TCT?GTG?AAG?GGT?CGG?TTC?ACC?ATC?TCC?AGA?GAT?GAT?TCC 288Tyr?Ser?Ala?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asp?Ser

65 70 75CAA?AGC?ATC?CTC?TAT?CTT?CAA?ATG?AAC?ACC?CTG?AGA?GGT?GAG?GAC?AGT 336Gln?Ser?Ile?Leu?Tyr?Leu?Gln?Met?Asn?Thr?Leu?Arg?Gly?Glu?Asp?Ser

80 85 90GCC?ACT?TAT?TAC?TGT?GCA?CGA?GAG?AAC?TAT?AGG?TAC?GAC?GTA?GAG?CTT 384Ala?Thr?Tyr?Tyr?Cys?Ala?Arg?Glu?Asn?Tyr?Arg?Tyr?Asp?Val?Glu?Leu

95 100 105GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT GCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala110 115 120SEQ ID NO：28：34：：：：DNA：chVLACAAAGCTTC CACCATGAGT GTGCTCACTC AGGT 34SEQ ID NO：29：37：：：：DNA：chVHGATAAGCTTC CACCATGAAG TTGTGGTTAA ACTGGGT 37SEQ ID NO：30：37：：：：DNA：chVLCTTGGATCCA CTCACGTTTG AGTTCCAGCT TGGTGCC 37SEQ ID NO：31：37：：：：DNA：chVHGTCGGATCCA CTCACCTGCA GAGACAGTGA CCAGAGT 37SEQ ID NO：32：137：：：：DNA：HF1TAAGCTTCCA CCATGGAGTT TGGGCTGAGC TGGGTTTTCC TTGTTGCTAT TTTAAAGGGT 60GTCCAGTGTG AAGTGCAGCT GTTGGAGTCT GGGGGAGGCT TGGTCCAGCC TGGGGGTTCT 120CTGAGACTCT CATGTGC 137SEQ ID NO：33：143：：：：DNA：HF2GCACTGTACT CTCTTGTGTA ACCATTGGCT TTGTTTCTAA TGAGACCCAC CAACTCTAGC 60CCTTTCCCTT GAGCTTGGCG GACCCAGCTC AGGTAGTAAT CACTGAAGGT GAATCCAGAG 120GCAGCACATG AGAGTCTCAG AGA 143SEQ ID NO：34：113：：：：DNA：HF3TACACAAGAG AGTACAGTGC ATCTGTGAAG GGCAGACTTA CCATCTCAAG AGAAGATTCA 60AAGAACACGC TGTATCTGCA AATGAGCAGC CTGAAAACCG AAGACTTGGC CGT 113SEQ ID NO：35：117：：：：DNA：HF4TCGGATCCAC TCACCTGAGG AGACGGTGAC CAGGGTTCCC TGGCCCCAGT AAGCAAGCTC 60TACGTCGTAG CGATAGTTCT CTCTAGCACA GTAATACACG GCCAAGTCTT CGGTTTT 117SEQ ID NO：36：37：：：：DNA：RVH5′GATAAGCTTC CACCATGGAG TTTGGGCTGA GCTGGGT 37SEQ ID NO：37：31：：：：DNA：RVH3′GTCGGATCCA CTCACCTGAG GAGACGGTGA C 31SEQ ID NO：38：424：：：：DNA：RVHa：：HEF-RVHa-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

-1 1 5 10CCT?GGG?GGT?TCT?CTG?AGA?CTC?TCA?TGT?GCT?GCC?TCT?GGA?TTC?ACC?TTC 144Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?GCT?CAA?GGG?AAA?GGG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Ala?Gln?Gly?Lys?Gly?Leu?30 35 40 45GAG?TTG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Leu?Val?Gly?Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

50 55 60TAG?AGT?GCA?TCT?GTG?AAG?GGC?AGA?CTT?ACC?ATC?TCA?AGA?GAA?GAT?TCA 288Tyr?Ser?Ala?Ser?Val?Lys?Gly?Arg?Leu?Thr?Ile?Ser?Arg?Glu?Asp?Ser

65 70 75AAG?AAC?ACG?CTG?TAT?CTG?CAA?ATG?AGC?AGC?CTG?AAA?ACC?GAA?GAC?TTG 336Lys?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Thr?Glu?Asp?Leu

80 85 90GCC?GTG?TAT?TAC?TGT?GCT?AGA?GAG?AAC?TAT?CGC?TAC?GAC?GTA?GAG?CTT 384Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Glu?Asn?Tyr?Arg?Tyr?Asp?Val?Glu?Leu

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：39：34：：：：DNA：LTW1GGCTAGAGTG GGTGGGTCTC ATTAGAAACA AAGC 34SEQ ID NO：40：36：：：：DNA：LTW2GAGACCCACC CACTCTAGCC CTTTCCCTTG AGCTTG 36SEQ ID NO：41：424：：：：DNA：RVHb：：HEF-RVHb-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?GCT?CAA?GGG?AAA?GGG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Ala?Gln?Gly?Lys?Gly?Leu?30 35 40 45GAG?TGG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Trp?Val?Gly?Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

50 55 60TAC?AGT?GCA?TCT?GTG?AAG?GGC?AGA?CTT?ACC?ATC?TCA?AGA?GAA?GAT?TCA 288Tyr?Ser?Ala?Ser?Val?Lys?Gly?Arg?Leu?Thr?Ile?Ser?Arg?Glu?Asp?Ser

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：42：32：：：：DNA：QTP1TGGGTCCGCC AAGCTCCAGG GAAAGGGCTA GA 32SEQ ID NO：43：32：：：：DNA：QTP2TCTAGCCCTT TCCCTGGAGC TTGGCGGACC CA 32SEQ ID NO：44：424：：：：DNA：RVHc：：HEF-RVHc-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?GCT?CCA?GGG?AAA?GGG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?30 35 40 45GAG?TTG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Leu?Val?Gly?Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：45：424：：：：DNA：RVHd：：HEF-RVHd-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?GCT?CCA?GGG?AAA?GGG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?30 35 40 45GAG?TGG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Trp?Val?Gly?Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：46：26：：：：DNA：ATP1TGGGTCCGCC AACCTCCAGG GAAAGG 26SEQ ID NO：47：26：：：：DNA：ATP2CCTTTCCCTG GAGGTTGGCG GACCCA 26SEQ ID NO：48：424：：：：DNA：RVHe：：HEF-RVHe-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?CCT?CCA?GGG?AAA?GGG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?30 35 40 45GAG?TGG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Trp?Val?Gly?Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：49：29：：：：DNA：GTA1CAAGCTCCAG GGAAAGCGCT AGAGTGGGT 29SEQ ID NO：50：29：：：：DNA：GTA2ACCCACTCTA GCGCTTTCCC TGGAGCTTG 29SEQ ID NO：51：424：：：：DNA：RVHf：：HEF-RVHf-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?GCT?CCA?GGG?AAA?GCG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Ala?Leu?30 35 40 45GAG?TGG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Trp?Val?Gly?Leu?Ile?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：52：23：：：：DNA：LTF1GTGAAGGGCA GATTTACCAT CTC 23SEQ ID NO：53：23：：：：DNA：LTF2GAGATGGTAA ATCTGCCCTT CAC 23SEQ ID N0：54：424：：：：DNA：RVHg：：HEF-RVHg-gγ1-19--1： 1-30：FR1 31-35：CDRl 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

15 20 25AGT?GAT?TAC?TAC?CTG?AGC?TGG?GTC?CGC?CAA?GCT?CCA?GGG?AAA?GGG?CTA 192Ser?Asp?Tyr?Tyr?Leu?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?30 35 40 45GAG?TGG?GTG?GGT?CTC?ATT?AGA?AAC?AAA?GCC?AAT?GGT?TAC?ACA?AGA?GAG 240Glu?Trp?Val?Gly?Leu?lle?Arg?Asn?Lys?Ala?Asn?Gly?Tyr?Thr?Arg?Glu

50 55 60TAC?AGT?GCA?TCT?GTG?AAG?GGC?AGA?TTT?ACC?ATC?TCA?AGA?GAA?GAT?TCA 288Tyr?Ser?Ala?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Glu?Asp?Ser

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：55：424：：：：DNA：RVHh：：HEF-RVHh-gγ1-19--1： 1-30：FR1 31-35：CDR1 36-49：FR2 50-68：CDR2 69-100：FR3 101-111：CDR3 112-122：FR4ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATT TTA AAG GGT 48Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly-19-15-10-5GTC CAG TGT GAA GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTC CAG 96Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

95 100 105GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G 424Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser110 115 120SEQ ID NO：56：124：：：：DNA：LF1TTGAAGCTTC CACCATGGGA TGGAGCTGTA TCATCCTCTT CTTGGTAGCA ACAGCTACAG 60GTGTCCACTC CGACATCCAG ATGACCCAGA GCCCAAGCAG CCTGAGCGCC AGCGTAGGTG 120ACAG 124SEQ ID NO：57：122：：：：DNA：LF2GCATTGTAGA TCAGCAGCTT TGGAGCCTTT CCTGGCTTCT GCTGGTACCA TGCTAAATAA 60CTGTAAATAA TCTCGCTTGC TCGACAGGTG ATGGTCACTC TGTCACCTAC GCTGGCGCTC 120AG 122SEQ ID NO：58：121：：：：DNA：LF3AGCTGCTGAT CTACAATGCA AAAACCTTAG CAGATGGAGT GCCAAGCAGA TTCAGCGGTA 60GCGGTAGCGG TACCGACTTC ACCTTCACCA TCAGCAGCCT CCAGCCAGAG GACATCGCTA 120C 121SEQ ID NO：59：106：：：：DNA：LF4GTAGGATCCA CTCACGTTTG ATTTCGACCT TGGTCCCTTG GCCGAACGTC CGAGGAAAAC 60CAAAATGATG TTGGCAGTAG TAGGTAGCGA TGTCCTCTGG CTGGAG 106SEQ ID NO：60：20：：：：DNA：RVL5′TTGAAGCTTC CACCATGGGA 20SEQ ID NO：61：20：：：：DNA：RVL3GTAGGATCCA CTCACGTTTG 20SEQ ID NO：62：379：：：：DNA：RVLa：：HEF-RVLa-gκ-19--1： 1-23：FR1 24-34：CDR1 35-49：FR2 50-56：CDR2 57-88：FR3 89-97：CDR3 98-107：FR4ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly-19-15-10-5GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

-1 1 5 10AGC?GTA?GGT?GAC?AGA?GTG?ACC?ATC?ACC?TGT?CGA?GCA?AGC?GAG?ATT?ATT 144Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Glu?Ile?Ile

15 20 25TAC?AGT?TAT?TTA?GCA?TGG?TAC?CAG?CAG?AAG?CCA?GGA?AAG?GCT?CCA?AAG 192Tyr?Ser?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?30 35 40 45CTG?CTG?ATC?TAC?AAT?GGA?AAA?ACC?TTA?GCA?GAT?GGA?GTG?CCA?AGC?AGA 240Leu?Leu?Ile?Tyr?Asn?Ala?Lys?Thr?Leu?Ala?Asp?Gly?Val?Pro?Ser?Arg

50 55 60TTC?AGC?GGT?AGC?GGT?AGC?GGT?ACC?GAC?TTC?ACC?TTC?ACC?ATC?AGC?AGC 288Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser

65 70 75GTC?CAG?CCA?GAG?GAC?ATC?GCT?ACC?TAC?TAC?TGC?CAA?CAT?CAT?TTT?GGT 336Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Mis?Phe?Gly

80 85 90TTT?CCT?CGG?ACG?TTC?GGC?CAA?GGG?ACC?AAG?GTC?GAA?ATC?AAA?C 379Phe?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys

95 100 105SEQ ID NO：63：38：：：：DNA：FTY1AGCGGTAGCG GTACCGACTA CACCTTCACC ATCAGCAG 38SEQ ID NO：64：38：：：：DNA：FTY2CTGCTGATGG TGAAGGTGTA GTCGGTACCG CTACCGCT 38SEQ ID NO：65：379：：：：DNA：RVLb：：HEF-RVLb-gκ-19--1： 1-23：FRl 24-34：CDRl 35-49：FR2 50-56：CDR2 57-88：FR3 89-97：CDR3 98-107：FR4ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly-19-15-10-5GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

15 20 25TAC?AGT?TAT?TTA?GCA?TGG?TAC?CAG?CAG?AAG?CCA?GGA?AAG?GCT?CCA?AAG 192Tyr?Ser?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?30 35 40 45CTG?CTG?ATC?TAC?AAT?GCA?AAA?ACC?TTA?GCA?GAT?GGA?GTG?CCA?AGC?AGA 240Leu?Leu?Ile?Tyr?Asn?Ala?Lys?Thr?Leu?Ala?Asp?Gly?Val?Pro?Ser?Arg

50 55 60TTC?AGC?GGT?AGC?GGT?AGC?GGT?ACC?GAC?TAC?ACC?TTC?ACC?ATC?AGC?AGC 288Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Thr?Phe?Thr?Ile?Ser?Ser

65 70 75CTC?CAG?CCA?GAG?GAC?ATC?GCT?ACC?TAC?TAC?TGC?CAA?CAT?CAT?TTT?GGT 336Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gln?His?His?Phe?Gly

95 100 105SEQ ID NO:66 sequence lengths: 18 sequence types: nucleic acid chain: strand topology: linear molecule type: synthetic DNA sequence title: EF1 sequence C AGACAGTGG TTCAAAGT 18SEQ ID NO:67 sequence length: 17 sequence types: nucleic acid chain: strand topology: linear molecule type: synthetic DNA sequence title: HIP sequence GCCCCAAAGC CAAGGTC 17SEQ ID NO:68 sequence length: 20 sequence types: nucleic acid chain: strand topology: linear molecule type: synthetic DNA sequence title: KIP sequence A ACTCAATGC TTTAGGCAAA 20

Claims

1. the mouse monoclonal antibody light chain of anti-Ro 24-7472/000-8 (IL-8) (L chain) variable region (V district).

2. the L chain V district of claim 1 has in aminoacid sequence or its part shown in the SEQ ID NO:26.

3. the mouse monoclonal antibody heavy chain of anti-people IL-8 (H chain) V district.

4. the H chain V district of claim 3 has in aminoacid sequence or its part shown in the SEQ ID NO:27.

5. the chimeric L chain that comprises the mouse monoclonal antibody L chain V district of people L chain constant region (C district) and anti-people IL-8.

6. the chimeric L chain of claim 5, wherein said mouse L chain V district has the aminoacid sequence shown in the SEQID NO:26 or its part.

7. the chimeric H chain that comprises the mouse monoclonal antibody H chain V district of people H chain C district and anti-people IL-8.

8. the chimeric H chain of claim 7, wherein said mouse H chain V district has the aminoacid sequence shown in the SEQ ID NO:27 or its part.

9. chimeric antibody comprises:

(1) each L chain comprises the mouse monoclonal antibody L chain V district of people L chain constant region (C district) and anti-people IL-8; With

(2) each H chain comprises the mouse monoclonal antibody H chain V district of people H chain C district and anti-people IL-8.

10. the chimeric antibody of claim 9, wherein said mouse L chain V district has the aminoacid sequence shown in the SEQ ID NO:26 or its part, and said mouse H chain V district has the aminoacid sequence shown in the SEQ ID NO:27 or its part.

11. the complementary determining region (CDR) in the mouse monoclonal antibody L chain V district of anti-people IL-8.

12. the CDR of claim 11 has following and in aminoacid sequence or its part shown in the SEQ ID NO:26:

CDR1：Arg?Ala?Ser/Glu?Ile?Ile/Tyr?Ser?Tyr/Leu?Ala/

CDR2：Asn?Ala?Lys/Thr?Leu?Ala/Asp

CDR3：Gln?His?His/Phe?Gly?Phe/Pro?Arg?Thr/

13. the CDR in the mouse monoclonal antibody H chain V district of anti-people IL-8.

14. the CDR of claim 13 has below and aminoacid sequence or its part shown in the SEQ ID NO:27.

CDR1：Asp?Tyr?Tyr/Leu?Ser.

CDR2：Leu?Ile?Arg/Asn?Lys?Ala/Asn?Gly?Tyr/Thr?Arg?Glu/TyrSer?Ala/Ser?Val?Lys/Gly

CDR3：Glu?Asn?Tyr/Arg?Tyr?Asp/Val?Glu?Leu/Ala?Tyr/

15. the reconstruct people L chain V district of the antibody of anti-people IL-8 comprises:

(1) framework region (FR) in people L chain V district; With

(2) CDR in the mouse monoclonal antibody L chain V district of anti-people IL-8.

16. the reconstruct people L chain V district of claim 15, wherein said CDR has in the aminoacid sequence shown in the claim 12 or its part.

17. the reconstruct people L chain V district of claim 15 and 16, wherein said FR is from people's antibody REI.

18. the reconstruct people L chain V district of claim 15, wherein said L chain V district has with the aminoacid sequence shown in RVLa in the table 2 or the RVLb or its part.

19. the reconstruct people H chain V district of the antibody of anti-people IL-8 comprises:

(1) FR in people H chain V district; With

20. the reconstruct people H chain V district of claim 19, wherein said CDR has in the aminoacid sequence shown in the claim 14 or its part.

21. the reconstruct people H chain V district in claim 19 and 20, wherein said FR1, FR2 and FR3 are from people's antibody VDH26, and said FR4 is from people's antibody 4B4.

22. the reconstruct people H chain V district in the claim 19, wherein said H chain V district have in table 3 and 4 with RVHa, RVHb, RNHc, RVHd, RVHe, RVHf, the aminoacid sequence shown in RVHg or the RVHh or its part.

23. the reconstructed hyman antibody L chain of anti-people IL-8 comprises:

(1) people L chain C district and

(2) L chain V district comprises the mouse monoclonal antibody L chain CDR of people L chain FR and anti-people IL-8.

24. the reconstructed hyman antibody L chain of claim 23, wherein said people L chain C district is people C κ district, and people L chain FR is from people's antibody REI, and described L chain CDR has the aminoacid sequence shown in the claim 12 or its part.

25. the reconstructed hyman antibody L chain of claim 23, wherein said L chain V district has in table 2 with the aminoacid sequence shown in RVLa or the RVLb or its part.

26. the reconstructed hyman antibody H chain of anti-people IL-8 comprises:

(1) people H chain C district; With

(2) H chain V district comprises the H chain CDR of the mouse monoclonal antibody of people H chain FR and anti-people IL-8.

27. the reconstructed hyman antibody H chain of claim 26, wherein said people H chain C district is C γ 1 district, said people H chain FR1, and FR2 and FR3 are from people's antibody VDH26, people H chain FR4 is from people's antibody 4B4, and said H chain CDR has in the aminoacid sequence shown in the claim 14 or its part.

28. the reconstructed hyman antibody H chain of claim 26, wherein said H chain V district has in table 3 and 4 with RVHa, RVHb, RVHc, RVHd, RVHe, RVHf, the aminoacid sequence that RVHg and RVHh represent or its part.

29. the reconstructed hyman antibody of anti-people IL-8 comprises:

(A) each L chain comprises:

(1) people L chain C district; With

(2) L chain V district comprises the mouse monoclonal antibody L chain CDR of people L chain FR and anti-people IL-8;

(B) the H chain comprises:

(1) people H chain C district; With

(2) H chain V district comprises the mouse monoclonal antibody H chain CDR of people H chain FR and anti-people IL-8.

30. the reconstructed hyman antibody of claim 29, wherein said L chain CDR has in the aminoacid sequence shown in the claim 12 or its part, and said H chain CDR has in the aminoacid sequence shown in the claim 14 or its part.

31. the reconstructed hyman antibody of claim 29, wherein said L chain CDR has in the aminoacid sequence shown in the claim 12 or its part, and said H chain CDR has in the aminoacid sequence shown in the claim 14 or its part; Said people L chain FR is from people's antibody REI; Said people H chain FR1, FR2 and FR3 are from people's antibody VDH26, and people H chain FR4 is from people's antibody 4B4, and said people L chain C district is people C κ district, and said people H chain C district is people C γ 1 district.

32. the reconstructed hyman antibody of claim 29, wherein said L chain CDR has the aminoacid sequence shown in the claim 12 or its part, and said H chain CDR has the aminoacid sequence shown in the claim 14 or its part; Said people L chain FR is from people's antibody REI; Said people H chain FR1, FR2 and FR3 are from people's antibody VDH26, and people H chain FR4 is from people's antibody 4B4, and said people L chain C district is people C κ district, and said people H chain C district is C γ 4.

33. the reconstructed hyman antibody of claim 29, wherein said L chain V district has in table 2 with the aminoacid sequence shown in RVLa or the RVLb or its part.

34. the reconstructed hyman antibody of claim 29, wherein said H chain V district has in table 3 and 4 with RVHa, RVHb, RVHc, RVHd, RVHe, RVHf, the aminoacid sequence that RVHg or RVHh represent or its part.

The DNA of the chimeric L chain of the antibody of anti-people IL-8 35. encode, contain:

(1) people L chain C district; With

(2) the mouse monoclonal antibody L chain V district of anti-people IL-8.

36. the DNA in the claim 35, aminoacid sequence or its part shown in the wherein said L chain V district coding SEQ IDNO:26.

37. the DNA in the claim 35, the DNA in the said L chain V district of wherein encoding has in nucleotide sequence or its part shown in the SEQ ID NO:26.

The DNA of the chimeric H chain of the antibody of anti-people IL-8 38. encode, contain:

(1) people H chain C district and

(2) the mouse monoclonal antibody H chain V district of anti-people IL-8.

39. the DNA of claim 38, wherein said H chain V district has in the aminoacid sequence shown in the SEQ IDNO:27 or its part.

40. the DNA of claim 38, wherein said H chain V district has in the nucleotide sequence shown in the SEQ IDNO:27 or its part.

The DNA in the mouse monoclonal antibody L chain V district of anti-people IL-8 41. encode.

42. the DNA of claim 41, wherein said L chain V district is coded in the aminoacid sequence shown in the SEQ IDNO:26 or its part.

43. the DNA of claim 41, the DNA in the said L chain V district of wherein encoding has in nucleotide sequence or its part shown in the SEQ ID NO:26.

The DNA in the mouse monoclonal antibody H chain V district of anti-people IL-8 44. encode.

45. the DNA of claim 44, wherein said H chain V district is coded in the aminoacid sequence shown in the SEQ IDNO:27 or its part.

46. the DNA of claim 44, the DNA in the said H chain V district of wherein encoding has in nucleotide sequence or its part shown in the SEQ ID NO:27.

The DNA of the mouse monoclonal antibody L chain V district CDR of anti-people IL-8 47. encode.

48. the DNA of the coding CDR of claim 47, aminoacid sequence or its part shown in the wherein said CDR coding claim 12.

49. the DNA of the coding CDR of claim 47, wherein said CDR have nucleotide sequence or its part shown in the SEQ ID NO:26.

The DNA of the mouse monoclonal antibody H chain V district CDR of anti-people IL-8 50. encode.

51. the DNA of the coding CDR of claim 50, wherein said CDR has the aminoacid sequence shown in the claim 14 or its part.

52. the DNA of the coding CDR of claim 50, wherein said CDR has the nucleotide sequence shown in the SEQ ID NO:27 or its part.

The DNA in the reconstruct people L chain V district of the antibody of anti-people IL-8 53. encode comprises:

(1) FR in people L chain V district; With

54. the DNA in the coding reconstruct people L chain V district of claim 53, wherein said CDR has the aminoacid sequence shown in the claim 12 or its part.

55. the DNA in claim 53 and 54 coding reconstruct people L chain V districts, wherein said FR is from people's antibody REI.

56. the DNA of claim 53, aminoacid sequence or its part represented with RVLa or RVLb in the wherein said L chain V district coding schedule 2.

57. the DNA of claim 53 has in nucleotide sequence or its part shown in SEQ ID NO:62 or the SEQ IDNO:65.

The DNA in the reconstruct people H chain V district of the antibody of anti-people IL-8 58. encode comprises:

(1) FR in people H chain V district; With

59. the DNA in the coding reconstruct people H chain V district of claim 58, wherein said CDR has in the aminoacid sequence shown in the claim 14 or its part.

60. the DNA in the coding reconstruct people H chain V district of claim 58 and 59, wherein said FR1, FR2 and FR3 are from people's antibody VDH26, and FR4 is from people's antibody 4B4.

61. the DNA in the coding reconstruct people H chain V district of claim 58, wherein said H chain V district are coded in table 3 and 4 with RVHa, RVHb, RVHc, RVHd, RVHe, RVHf, the aminoacid sequence that RVHg or RVHh represent or its part.

62. the DNA of claim 48 has the NO:38 at SEQ ID, the nucleotide sequence or its part that show in 41,44,45,48,51,54 or 55.

The DNA of the reconstruct people L chain of the antibody of anti-people IL-8 63. encode comprises:

(1) people L chain C district; With

(2) L chain V district comprises the mouse monoclonal antibody CDR of people FR and anti-people IL-8.

64. the DNA of claim 63, aminoacid sequence or its part represented with RVLa or RVLb in the wherein said L chain V district coding schedule 2.

65. the DNA of claim 63, wherein said L chain V district has in nucleotide sequence or its part shown in SEQ IDNO:62 or the SEQ ID NO:65.

66. the DNA of claim 63,64 and 65, wherein said people L chain C district are people L chain C κ districts.

The DNA of the reconstruct people H chain of the antibody of anti-people IL-8 67. encode comprises:

(1) people H chain C district; With

(2) H chain V district comprises the mouse monoclonal antibody CDR of people FR and anti-people IL-8.

68. the DNA of the coding reconstruct people H chain of claim 67, wherein said H chain V district are coded in table 3 and 4 with RVHa, RVHb, RVHc, RVHd, RVHe, RVHf, the aminoacid sequence that RVHg or RVHh represent or its part.

69. the DNA of claim 67, wherein said H chain V district has the IDNO:38 at SEQ, 41,44,45,48, and the nucleotide sequence shown in 51,54 or 55 or its part.

70. the DNA of claim 67,68 or 69, wherein said people H chain C district are people H chain C γ 1 districts.

71. the DNA in the claim 67,68 or 69, wherein said people H chain C district are people H chain C γ 4 districts.

72. contain claim 35, the carrier of the DNA of any one in 36,37,38,39,40,63,64,65,66,67,68,69,70 and 71.

73. with the described carrier transformed host cells of claim 72.

74. method of producing the chimeric antibody of anti-people IL-8, comprise to cultivate and require 35 with containing right, the expression vector of any one described DNA and the expression vector that is contained in any one described DNA in the claim 38,39 and 40 transformed host cells and reclaim the step of target antibody simultaneously in 36 and 37.

75. method of producing the chimeric antibody of anti-people IL-8, comprise to cultivate and require 35 with containing right, in 36 and 37 any one described DNA and in claim 38,39 and 40 any one described DNA the expression vector transformed host cells and reclaim the step of this target antibody.

76. method of producing the reconstructed hyman antibody of anti-people IL-8, comprise: cultivate with containing right and require 63,64, the expression vector of any one described DNA and contain right and require 67 in 65 and 66,68, the expression vector of any one described DNA transformed host cells and reclaim the step of this target antibody simultaneously in 69,70 and 71.

77. a method of producing the reconstructed hyman antibody of anti-people IL-8 comprises and cultivates with containing right requirement 63,64, any one described DNA and claim 67 in 65 and 66, the expression vector transformed host cells of any one described DNA and reclaim the step of this target antibody in 68,69,70 and 71.