Reconstructed human antibodies to human interleukin-8

 

(57) Abstract:

The invention relates to a reconstructed human antibodies to human IL-8. A large part of the reconstructed antibody is a human antibody, a CDR has low antigenicity for human. Reconstructed human antibody is encoded by a DNA sequence contained in the expression vector. Reconstructed human antibodies of this invention have low antigenicity for people and therefore they can be used for therapy in medicine. 11 C. and 4 h.p. f-crystals, 8 ill., 5 table.

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8. In addition, this invention is a DNA which encodes wisewoman, and more specifically, the expression vector and the cell host transformed by the specified vector. In addition, this invention is a process for obtaining a reconstructed human antibodies to IL-8, as well as to the process of obtaining chimeric antibodies to human IL-8.

Interleukin-8 (IL-8) was opened in the culture supernatant of monocytes stimulated by lipopolysaccharide (LPS), and is a chemokine that is known as produced by monocytes factor of chemotaxis of neutrophils (PMPIN) or activated neutrophils protein-1 (at-1). IL-8 is produced by various cells, acts on neutrophilic granulocytes and lymphocytes and possesses activity that causes chemotaxis along the gradient of its concentration. In addition, it not only induces chemotaxis of neutrophils, but also activates the function of neutrophils, such as degranulation, secretion of peroxides and increased adhesion to endothelial cells.

In inflammatory diseases, and more specifically, for respiratory diseases such as pulmonary fibrosis, idiopathic pulmonary fibrosis, respiratory distress syndrome of adults, sarcoidosis and empyema, and also in skin diseases such as psoriasis, and XP is nocito at the site of inflammation in these diseases. In addition, IL-8 detected in samples from patients with these diseases, suggesting that IL-8 may play a Central role in inflammation (McElvaney, N. G. et al., J. Clin. Invest., 90, 1296-1301, 1992; Lynch III, J. P. et al., Am. Rev. Respir. Dis., 145, 1433-1439, 1992; Donnelly, S. C. et al., Lancet, 341, 643-647, 1993; Car, B. D. et al., Am. J. Respir. Crit. Care Md., 149, 655-659, 1994; Antony, V. C. et al., J. Immunol. , 151, 7216-7223, 1993; Takematsu, H. et al., Arch. Denmatol., 129, 74-80, 1993; Brennan, F. M. et al., Eur. J. Immunol., 20, 2141- 2144, 1990; Izzo, R. S. et al., Scand. J. Gastroenterol., 28, 296-300, 1993; Izzo, R. S. et al., Am. J. Gastroenterol., 87, 1447-1452, 1992).

After immunization of mice with human IL-8 as an antigen To Y-C., et al. got a mouse monoclonal antibody WS-4 that bind to human IL-8 and inhibit the binding of human IL-8 by neutrophils as a result of this binding, and it will neutralize the biological activity possessed by IL-8. It was clearly shown that the isotope mouse monoclonal antibody WS-4 consist of K-type L chain and C1-type H chain (J. Immunol. Methods, 149, 227-235, 1992).

Well-known examples of antibodies to human IL-8, in addition to WS-4, include A. 5.12.14 (Boylan, A. M. et al., J. Clin. Invest., 89, 1257-1267, 1992), antibody anti-PEP-1 and antibodies anti-PEP-3 disclosed in international patent applications N WO 92-04372 and DM/C7 (Mulligan, M. S. et al., J. Immunol., 150, 5585-5595, 1993).

the use of rabbits, the infiltration of neutrophils is inhibited in ischemic and reperfusion of the lungs (Sekido n, et al., Nature, 365, 654-657, 1993), LPS induced dermatitis (Harada , A., et al., Internatl. Immunol., 5, 681-690, 1993) and induced by LPS or interleukin-1 (IL-1) arthritis (Akahoshi T., et al., Lymphokine Res Cytoline., 13, 113-116, 1994).

Homolog of human IL-8 exists in rabbit called rabbit IL-8. As was clearly shown that mouse monoclonal antibody WS-4 cross-react with rabbit IL-8, and that the antibodies inhibit the binding of rabbit IL-8 rabbit neutrophils (Harada, A., et al., Internatl. Immunol., 5, 681-690, 1993), these data suggest that antibodies to human IL-8 should be useful as a therapeutic agent for the treatment of inflammatory diseases in humans.

Monoclonal antibodies derived from other mammals, and not of man, show a high degree of immunogenicity (also called antigenicity) in humans. For this reason, even if mouse antibodies are introduced to people, due to the fact that they are metabolized as a foreign substance, the half-life of murine antibodies in humans is relatively short, and thus prevents a corresponding manifestation of unwanted antibodies, cause immune response, which ncomfortable and dangerous for the patient, examples of which include serum sickness and other allergic reactions. For this reason, mouse antibodies cannot often enter people.

To solve these problems, we developed a process for obtaining humanized antibodies. Mouse antibodies can be humanized in two ways. A simpler method involves the production of chimeric antibodies in which the variable region (V region) is from the original murine monoclonal antibody and a constant region (C region) is of a suitable human antibody. Because of the resulting chimeric antibody containing the variable region of mouse antibody in its full form, they have specificity identical to the specificity of the original murine antibody, and it can be expected that they will bind the antigen.

In addition, as the proportion of protein sequences derived from another animal, not human, chimeric antibodies significantly reduced compared to the original mouse antibody, predicts that they have lower immunogenicity compared to the original mouse antibody. Although chimeric who here appears immune response to murine variable region (LoBuglio, A. F., et al., Proc. Natl. Acad. Sci. USA, 86, 4220-4224, 1989).

Although the second method of humanization of murine antibodies is more complex latent immunogenicity of murine antibodies is reduced significantly. In this method only the hypervariable region (CDR) is transplanted from variable regions of a mouse antibody into a human variable region to create a reconstructed human variable regions. However, in order to bring the structure of the CDR reconstructed human variable regions to the structure of the original mouse antibody, there are circumstances in which it may be necessary to transplant part of the protein sequence of the structural region (FR), carrier CDR of the variable region of mouse antibody to a human variable region.

These are then reconstructed human variable regions linked to human constant region. These parts are derived from nonhuman protein sequences are humanized antibody only from CDR and a very small part of CO. The CDR consists of hypervariable protein sequences, and they do not show species specificity. For this reason, the reconstructed chelovecheskaya human antibodies, containing human CDR.

Additional details regarding the reconstructed human antibodies can be found by contacting L. Riechmann, et al., Nature, 332, 323-327, 1988; Verhoeyen, M., et al., 239, 1534-1536, 1988; Kettleborough, C. A. et al. Protein Eng. , 4, 773-783, 1991; Maeda, H. et al., Hum. Antibodies Hybridomas, 2, 124-134, 1991; Gorman, S. D. et al., Proc. Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest, P. R. et al., Bio/Technology, 9, 266-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA, 88, 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154, 1992; and, Sato, K. et al., Cancer Res. , 53, 851-856, 1993.

As stated above, although the reconstructed human antibodies, as predicted, will be applicable for the purpose of therapy is unknown reconstructed human antibody to human IL-8. In addition, there is no standard processes that can be universally applied to arbitrary antibody to obtain a reconstructed human antibodies. Thus, require different tricks in order to create a reconstructed human antibodies that exhibit sufficient binding activity and/or neutralizing activity against a specific antigen (e.g., Sato K., et al., Cancer Res., 53, 851-856, 1933). This invention is antibody to human IL-8 with no antibody to human IL-8. The invention also is a human/murine chimeric antibodies, which are applicable in the process of production of these human antibodies. In addition, this invention also represents the reconstructed fragment of a human antibody. Additionally, this invention represents an expression system for the production of chimeric antibodies and reconstructed human antibodies and their fragments. In addition, the invention is also the process of obtaining chimeric antibodies to human IL-8 and their fragments, as well as the process of obtaining a reconstructed human antibodies to human IL-8 and their fragments.

More specifically, this invention is:

1) V region of the L chain of the mouse monoclonal antibody to human IL-8 and

2) the V region of the H chain of the mouse monoclonal antibody to human IL-8.

In addition, this invention is:

1) an L chain containing region of a human L chain and the V region of the L chain of the mouse monoclonal antibodies to IL-8 and

2) H chain containing region of a human H chain and the V region of the H chain of the mouse monoclonal antibody to human IL-8.

In addition, this from the traveler includes a human With the region of the L chain and the V region of the L chain of the mouse monoclonal antibody to human IL-8 and

2) H circuits, each of which contains a region of a human H chain and the V region of the H chain of the mouse monoclonal antibody to human IL-8.

In addition, this invention is:

1) V region of the L chain of the mouse monoclonal antibody WS-4 human IL-8 and

2) the V region of the H chain of the mouse monoclonal antibody WS-4 human IL-8.

In addition, this invention also is:

1) an L chain containing region of a human L chain and the V region of the L chain of the mouse monoclonal antibody WS-4 human IL-8 and

2) H chain containing region of a human H chain and the V region of the H chain of the mouse monoclonal antibody WS-4 human IL-8.

Additionally, the invention is a chimeric antibody to human IL-8, consisting of:

1) L chains, each of which includes a region of a human L chain and the V region of the L chain of the mouse monoclonal antibody WS-4 human IL-8 and

2) H circuits, and each includes a region of a human H chain and the V region of the H chain of the mouse monoclonal antibody WS-4 human IL-8.

In addition, this invention is:

1) CDR V region of the L chain of a monoclonal antibody to human IL-8 and
also present:

1) CDR V region of the L chain of the mouse monoclonal antibody to human IL-8 and

2) CDR V region of the H chain of the mouse monoclonal antibody to human IL-8.

In addition, this invention also is reconstructed V region of a human L chain of the antibody to human IL-8, consisting of:

1) structural regions (FR) V region of a human L chain and

2) CDR V region of the L chain of the mouse monoclonal antibody to human IL-8;

and also reconstructed as V region of the H chain of the antibody to the human IL-8, consisting of:

1) FR V region of H chain and

2) CDR V region of the H chain of the mouse monoclonal antibody to human IL-8.

In addition, this invention is a reconstructed human L chain of the antibody to human IL-8, consisting of:

1) With the human L chain and

2) the V region of L chain consisting of FR of a human L chain and the CDR of the L chain of the mouse monoclonal antibody to human IL-8;

as well as the reconstructed H chain of the antibody to human IL-8, consisting of:

1) C region of a human H chain and

2) the V region of H chain consisting of the FR of a human H chain and the CDR of the H chain of the mouse monoclonal antibody to human IL-8.


(A) L chains, each includes:

1) the human With the region of the L chain and

2) the V region of L chain containing the FR of a human L chain and the CDR of the L chain of the mouse monoclonal antibody to human IL-8;

and

(B) H circuits, and each includes:

1) the human With the region of H chain and

2) the V region of H chain containing the FR of a human H chain and the CDR of the H chain of the mouse monoclonal antibody to human IL-8.

More specifically, this invention is:

1) CDR V region of the L chain of the mouse monoclonal antibody WS-4 human IL-8 having the following sequences or part of them:

CDR1: Arg Ala Ser Glu Ile Ile Ser Tight Tight Leu Ala

CDR2: Asn Ala Lys Thr Leu Ala Asp

CDR3: Gin His His Phe Gly Phe Pro Arg Thr

and

2) CDR V region of the H chain of the mouse monoclonal antibody WS-4 human IL-8 having the following sequences or part of them:

CDR1: Asp Tight Tight Leu Ser

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

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

In addition, this invention is reconstructed V region of the L chain of the antibody to human IL-8 comprising:

1) structural sections (FR) V region of the L chain and

2) CDR V region of the L chain of the mouse monoclonal antibody WS-4 to the, stoaway from:

1) FR V region of the H chain; and

2) CDR V region of the H chain of a monoclonal antibody WS-4 human IL-8.

In addition, this invention is a reconstructed human L chain of the antibody to human IL-8, consisting of:

1) the human With the region of L chain and

2) the V region of L chain consisting of FR of a human L chain and the CDR of the L chain of the mouse monoclonal antibody WS-4 human IL-8;

and reconstructed human H chain of the antibody to human IL-8, consisting of:

1) human With a region of H chain and

2) the V region of H chain including FR human H chain and the CDR of the H chain of a monoclonal antibody WS-4 human IL-8.

Moreover, this invention also is reconstructed human antibody to human IL-8, consisting of

(A) L chains, each includes:

1) With the area of human L chain and

2) the V region of L chain consisting of FR of a human L chain and the CDR of the L chain of the mouse monoclonal antibody WS-4 human IL-8, and

(B) H circuits, and each includes:

1) With the region of H chain and

2) the V region of H chain consisting of the FR of a human H chain and the CDR of the H chain of the mouse monoclonal antibody WS-4 human IL-8.

Examples of the above-mentioned FR human H chain include those which have the amino acid sequence of B or part, provided at the end of the description.

Additionally, this invention also relates to DNA that encodes the polypeptide, are included in the above various antibodies, and fragments. This invention also relates to a vector, which contains the above-mentioned DNA, an example of which is the expression vector. In addition, the invention is a cell host, which transforms the above vector.

In addition, the invention is also the process of obtaining chimeric antibodies to human IL-8 and its fragments, as well as the process of obtaining a reconstructed human antibodies to human IL-8 and their fragments.

Fig. 1 shows the expression vectors HEF-VL-DK and HEF-VH-g1 containing the promoter/enhancer of the human elongation factor 1(HEF-1), which is applicable for the expression of L chain and H chain, respectively, of the antibodies of the present invention.

Fig. 2 is a graph showing the results of ELISA for confirmation of the ability of binding to human IL

Fig. 3 is a chart design DNA, which encodes the amino acid sequence of each of the first version "a" (RVHa) V region of the H chain of the reconstructed human antibody WS-4 of the present invention (a) and the first version "a" (RVLa) V region of L chain reconstructed human antibody WS-4 (B).

Fig. 4 is a graph showing the results of ELISA compared the ability of binding to human IL-8 V region of the L chain (RVLa) and the V region of the H chain (RVHa) reconstructed human antibody WS-4 of this invention in combination with, respectively, the V region of the H chain chimeric antibody WS-4 (chH) and the V region of the L chain chimeric antibody WS-4 (chL), expressed in COS cells, with the ability of binding chimeric antibody WS-4 (chL/chH) of the present invention, secreted into the culture medium of COS cells.

Fig. 5 is a graph showing the results of ELISA compared the ability of binding to human IL-8 of 8 types of reconstructed human antibody WS-4, containing RVLa of the present invention (RVLa/RVHa, RVLa/RVHb, RVLa/RVHc, RVLa/RVHd, RVLa/RVHe, RVLa/RVHf, RVLa/RVHg and RVLa/RVHh), secreted into the culture medium of COS cells.

Fig. 6 is a graph showing the results of ELISA compared the ability of casinogo invention (RVLb/RVHa, RVLb/RVHb, RVLb/RVRc, RVLb/RVHe, RVLb/RVHf, RVLb/RVHg and RVLb/RVHh) produced in the culture supernatant of COS cells with the ability of binding chimeric antibody WS-4 (chL/chH) of the present invention, secreted into the culture medium of COS cells.

Fig. 7 is a graph showing the results of ELISA compared the ability of binding to human IL-8 treated reconstructed human antibody WS-4 RVLa/RVHg and RVLb/RVHg of the present invention and purified chimeric antibody WS-4 (chL/chH) this invention.

Fig. 8 is a graph showing the results of the research suppress the binding of the ligand to the receptor as compared to the ability to inhibit the binding of IL-8 receptors for IL-8 treated reconstructed human antibodies RVLa/RVHg and RVLb/RVHg of the present invention with the ability to inhibit binding of the mouse antibody WS-4 chimeric antibody WS-4 (chL/chH) this invention.

Cloning of DNA encoding murine V region

To clone the gene that encodes the V region of mouse monoclonal antibodies to human IL-8, you must obtain hybridoma, which produces a mouse monoclonal antibody to human IL-8, to obtain such a gene. After extracting mRNA from gebremariam polymerase chain reaction (PCR) for gene. An example of a source of this gene is hybridoma WS-4, which produces a mouse monoclonal antibody to human IL-8 received by Y. C., et al. The process of obtaining this hybridoma described in J. Immunol. Methods, 149, 227-235, 1992, and further described as reference example 1.

1) Extract all DNA

For cloning of target DNA, which encodes the V region of mouse monoclonal antibodies to IL-8, all RNA can be obtained by destroying cells hybridoma by processing the guanidine thiocyanate and conducting centrifugation in density gradient based on cesium chloride (Chirgwin J. M., et al. , Biochemistry, 18, 5294-5299, 1979). In addition, you can also use other methods that are used in the cloning of genes, such as those which are used for processing the detergent and processing of phenol in the presence of an inhibitor of the ribonuclease (RNase), such as vanadium complex (Berger, S. L., et al., Biochemistry, 18, 5143-5149, 1979).

2) Synthesis of cDNA

Then you can get single-stranded cDNA complementary to mRNA by treatment of total RNA reverse transcriptase using oligo(dt) oligonucleotide, complementary to the poly(A) tail, is located on the 3' end of the mRNA, as the primer and the mRNA contained in useone also at the same time to use a random primer. In addition, when it is desirable to first isolate mRNA, this can be done by applying total RNA on a column of oligo(dt)-cellulose, which is associated poly(A) tail of mRNA.

3) Amplification of the DNA encoding the V region using polymerase chain reaction.

Then cDNA, which encodes the above region-specific amplificates using polymerase chain reaction (PCR). For amplification of the V region of the L chain of (K) - type mouse monoclonal antibodies are 11 types of oligonucleotide primers, shown in the LAST ID NN 1-11 (mouse Kappa variable; ΜV) and oligonucleotide primer shown in TH ID N 12 (murine Kappa constant; ICC) as the 5' end of the primer and the 3' end of the primer, respectively. The above-mentioned primers ΜV hybridize with the DNA sequence, which encodes the murine leader sequence L chain Kappa-type.

For amplification of the V region of the H chain of the mouse monoclonal antibodies used 12 types oligonucleotide primers shown in TH ID NN 13-24 (mouse heavy variable; MTV) and oligonucleotide primer, shown in the LAST ID No. 25 (mouse heavy const; MTC), as the 5' end of the primer and 3' Coraya encodes mouse With the region of H chain.

In addition, all 5' end primers (ΜV and MTV) contain the sequence GTCGAC, which creates a cleavage site of restriction enzyme SalI about 3' end, whereas both the 3' end of the primer (ICC and ITC) contain the nucleotide sequence CCCGGG, which creates a cleavage site of restriction enzyme XmaI near the 5' end. These sites cleavage with restriction enzymes are used to sublimirovanny target DNA fragments that encode both V region into appropriate cloning vectors. When these cleavage sites of restriction enzymes are also present in the target DNA sequence, which encodes both the V region, should be used other cleavage sites of restriction enzymes to sublimirovanny into appropriate cloning vectors.

4) Isolation of DNA encoding the V region

Then, to obtain a DNA fragment that encodes the target V region of mouse monoclonal antibodies, the products of PCR amplification separated and purified in agarose gel with a low melting point or column [set for purification of PCR products (QIAGEN PCR Purification Spin Kit, QIAGEN); the kit for DNA purification (GENECLEAN II, BIO 101). The DNA fragment that encodes celevuyu restriction enzymes SalI and XmaI.

Further, with cleavage of a suitable cloning vector, such as plasmid pUC19 with the same restriction enzymes, SalI and XmaI, and enzymatic binding of the above-mentioned DNA fragment from pUC19 this get a plasmid that contains the DNA fragment that encodes the target V region of mouse monoclonal antibodies. Sequencing of the cloned DNA can be performed by the method, an example of which is the use of automated DNA sequencing machine (Applied Biosystems). Cloning and sequencing of the target DNA are described in detail in examples 1 and 2.

Hypervariable sites (CDR)

This invention also represents a hypervariable area or complementarity determining region (CDR) of the variable regions of murine monoclonal antibodies to human IL-8. V region of L chain and H chain of these antibodies form the antigen binding site. These areas on the L chains and H chains have a similar basic structure. V region of both circuits contain four structural region in which the sequence is relatively conservative, and these four structural areas are connected by three hypervariable sites or CDRs (Kabat E. the PEX structural regions (FR) are-plate structure, and three CDRs form loops. CDR may in some cases form part of the vane structure. Three CDR supported three-dimensional in extremely close positions using FR and participate in the formation of the antigen binding site together with three paired CDR. This invention is a CDR that is applicable as components of humanized antibodies, and the DNA that encodes them. These CDRs can be identified by experimental rules Kabat E. A. et al., "Sequences of Proteins of Immunological Interest", by comparing the sequence of the V region with known amino acid sequences of the V region, a detailed explanation of what is presented in exercise 3.

Obtaining chimeric antibodies

Before creating a reconstructed human V region antibody to human IL-8, you must confirm form really used CDR antigen binding region. With this purpose were obtained chimeric antibodies. To obtain chimeric antibodies, it is necessary to construct a DNA, which encodes the L chain and H chain chimeric antibody. The basic method of constructing both DNA involves the binding of the corresponding DNA sequences of the mouse leader sequence, poray encodes the human region, already present in the expression vector for mammalian cells.

Above With human antibodies may be any regions of the L chain and all regions of the H chain, and as for the L chain, examples include SC or human L chain, whereas, as for the H chain, if it is IgG, examples include C1, C2, C3 or C4 (J. Ellison et al., DNA, 1, 11-18 (1981), N. Takahashi Et al., Cell, 29, 671-679 (1982), Krawinkel, U. Et al., EMBO J., 1, 403-407 (1982)) or other isotypes.

Received two types of expression vectors for the production of chimeric antibodies, namely the expression vector that contains DNA encoding the V region of mouse L chain and a human L chain under control enhancer/promotor region of the regulation of expression, and the expression vector that contains DNA, which encodes the V region of the mouse H chain and a human H chain under the control of the management expression enhancer/promoter type. The cells are then-owners, such as mammalian cells, simultaneously transformed both expressing vectors, and transformed cells were cultured either in vitro or in vivo for the production of chimeric antigen (for example, WO91-16928).

Alternatively, the DNA that encodes the V region of the L chain of the mouse and the area L C is the result of the expression vector, cell host transformed with the use of this vector and then cultured either in vitro or in vivo for the production of chimeric antibodies.

Obtaining chimeric antibodies of monoclonal antibody WS-4 is described in exercise 4.

cDNA, which encodes a leader sequence circuit to the type of mouse WS-4 and V region, is cloned using PCR and connects with the expression vector that contains the DNA of the human genome, which encodes the IC region of a human L chain. Similarly, cDNA, which encodes the leader sequence of the H chain and the V region of the mouse antibody WS-4, is cloned using PCR and associated with the expression vector that contains the DNA of the human genome, which encodes human C1 region.

More specifically, suitable nucleotide sequences are introduced at the 5' and 3' ends of the cDNA, which encodes the V regions of mouse antibody WS-4 using specially designed primers for PCR, so that (1) they can be easily inserted into the expression vector and (2) they will accordingly operate in the specified expression vector (for example, the efficiency of transcription is enhanced by the introduction sequence, the Goat is the amplification by PCR using these primers, is introduced into the expression vector HEF (see Fig. 1), which already contains the desired human C region. These vectors are suitable for temporary or permanent expression of genetically-engineered antibodies in different systems of mammalian cells.

When tested for binding activity to the antigen chimeric antibody WS-4, thus obtained, was demonstrated activity of the antibody WS-4 in respect of binding to human IL-8 (see Fig. 2). Thus, it was concluded that there was cloned faithful murine V region and determined the correct sequence.

Creating a reconstructed human antibody WS-4

To obtain a reconstructed human antibodies in which CDR of mouse monoclonal antibody grafted into a human antibody, it is desirable to have a high degree of homology between the amino acid sequences of FR of murine monoclonal antibody with CDR subject transplantation, and amino acid sequences of FR of the human monoclonal antibodies, which should be transplanted these CDR.

With this purpose, the human V region to serve as a basis for constructing V regions reconstruing monoclonal antibodies with the amino acid sequence of FR of the human antibody. More specifically, the V region of the L and H chains of the murine antibody WS-4 compared with all known human V regions found in the database of the National Fund for biomedical research (National Biomedical Research Foundation, NBRF) using genetic analytical software, GENETEX (Software Development Co., Ltd.).

When compared with known V region of the human L chains was found that the V region of the L chain of the mouse antibody WS-4 most closely similar to human antibodies HAU (S. Watanabe et al., Hoppe-Seyler's Z. Physiol. Chem. , 351, 1291-1295, 1970), which has homology equal to 69,2%. On the other hand when compared with known V regions of the H chain of a human antibody, it was found that the V region of the H chain of the antibody WS-4 most closely similar to the V region of human antibody VDH26 (L. Buluwela et al., EMBO J. 7, 2003-2010, 1988), which has homology equal to 71.4 per cent.

Basically homology of amino acid sequences of murine V regions with amino acid sequences of human V regions less than homology with the amino acid sequences of murine V regions. This shows that V region of a mouse antibody WS-4 is not completely similar to the human V region, and at the same time indicates that the humanization V the region of murine antibody WS-4, in addition, compared with the consensus sequence of subgroup human V region according to Kabat E. A., et al., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health Services, U. S. Government Printing Office, in order to compare FR V region. These results are shown in table 1.

FR V region of the L chain of the mouse antibody WS-4 most closely resembles the consensus sequence of FR of the V region of a human L chain subgroup I (HSGI) with homology equal to 64.4%. On the other hand FR V region of the H chain of the mouse antibody WS-4 most closely resembles the consensus sequence of the V region of a human H chain subgroup III (HSGIII) with homology equal to 62.3 percent.

These results confirm the results obtained from the comparison with the known human antibody V region of the L chain of a human antibody HAU, belonging to subgroup I V regions of a human L chain and the V region of the H chain of a human antibody VDH26 belonging to subgroup III V regions of H chain. To create the V region of L chain reconstructed human antibody WS-4, it is probably best to use the V region of a human L chain belonging to subgroup I (HSGI), then how to create the V region of H chain reconstructed human antibody WS-4, may be better sun is to the V region of the L chain known human antibody V region of the L chain of the mouse monoclonal antibody WS-4 most closely similar to the V region of the L chain of a human antibody REI, representative subgroups 1 V regions of the L chain. Thus, used FR from REI when creating the V region of L chain reconstructed human antibody WS-4. In these human FR based on the REI there are differences in five amino acids (positions 39, 71, 104, 105, and 107; see table 2) compared with the sequence of the human REI, documentary shown in the literature (W. Palm et al., Hoppe-Seyler's Z. Physiol. Chem., 356, 167-191, 1975; Epp, O. et al., Biochemistry, 14, 4943-4952, 1975).

The numbers of amino acids shown in the table on the basis of experience Kabat E. A. et al. (1991). Changing two amino acids at positions 39 and 71 were those changes that are caused by the amino acids present in the FRV region of the L chain of the rat antibody CAMPATH-IH (Riechmann et al., 1988). By Kabat et al. (1991) changes in the other three amino acids in FR4 (position 104, 105, and 107) based on the J region from other human kL circuits and do not differ from those of men.

It was created two versions of the V region of L chain reconstructed human antibody WS-4. In the first embodiment RVLa FR were identical to the FR-based REI, present in reconstructed human antibody CAMPATH-IH (Riechmann et al., 1988), whereas the CDR were identical CDR V region of the L chain of the mouse antibody WS-4. The second option, RVLb, based on Rl., 196, 901-917, 1987, the residue 71 is part of the canonical structure CDRI V region of L chain.

Predicted amino acid in this position directly affects the structure of the loop CDRI V region of the L chain, and for this reason it is believed that it has a significant effect on binding to the antigen. In the V region of L chain RVLb reconstructed human antibody WS-4 phenylalanine at position 71 is replaced by tyrosine. Table 2 shows the corresponding amino acid sequence of V region of the L chain of the mouse antibody WS-4, FR-modified REI for use in reconstructed human antibody CAMPATH-1H (Riechmann, et al., 1988) and two versions of the V region of L chain reconstructed human antibody WS-4.

FR V region of the H chain of the mouse antibody WS-4 most closely similar to the V region of a human H chain, belonging to subgroup I (table 1).

When compared with known V region of the human H chain and the V region of the H chain of the mouse antibody WS-4 most closely similar to the V region of the H chain of a human antibody VDH26, representative subgroup III V regions of human H chains, from FR1 to FR3 (L. Buluwela et al., EMBO J. 7, 2003-2010, 1988). In relation to FR4, since the sequence of FR4 VDH26 not reported, it was decided to use , 142, 883-887, 1989). These V region of a human H chain was used as the basis for creating the V region of H chain reconstructed antibody WS-4.

It was created eight versions of the V region of H chain reconstructed human antibody WS-4. In all eight variants of the human FR1, FR2 and FR3 are based on FR1, FR2 and FR3 of a human antibody VDH26, while FR4 based on FR4 of the human antibody 4B4. Mouse CDRs were identical CDR V region of the H chain of the mouse antibody WS-4.

In tables 3 and 4 shows the corresponding amino acid sequence of V region of the H chain of the mouse antibody WS-4, matrix from FR1 through FR3 of a human antibody VDH26, FR4 of the human antibody 4B4 and 8 variants of the V region of H chain reconstructed human antibody WS-4.

Amino acids are identified using single letter code. The numbers of amino acids are in accordance with the definition of Kabat et al.

Obtaining DNA encoding the V region of the reconstructed human antibody WS-4

Getting the V region of the reconstructed human antibody WS-4 is described in detail in example 5.

Was synthesized DNA, which encodes the corresponding first variants of the V regions of the L chain and H chain of the reconstructed human antibodies is avannah human antibody WS-4 encodes the correct amino acid sequence by sequence determination. Sequence variant "a" of the V region of L chain reconstructed human antibody WS-4 shown in TH ID N 62, and the sequence variant "a" of the V region of H chain reconstructed human antibody WS-4 shown in TH ID N 38.

DNA that encode other variants of the V region of the reconstructed human antibody WS-4 were produced using minor modifications openly described method of induction PCR mutation (M. Kamman et al., Nucleic Acids Res., 17, 5404, 1989) with the first option "a" as a matrix. As previously described in relation to the creation of the V region of the reconstructed human antibody WS-4, were obtained DNA that encodes one additional variant of the V region of L chain reconstructed human antibody WS-4 (option "b"), and the DNA that encodes seven additional variants of the V region of H chain reconstructed human antibody WS-4 (options "b", "C", "d", "e", "f", "g" and "h").

These additional options contained light changes in a series of amino acid sequences from the first option, and these changes in amino acid sequences were achieved through the implementation of minor changes in the DNA sequence in the application of indukti DNA. After a series of PCR reactions, the PCR product was cloned, followed by sequencing to confirm that there have been changes in DNA sequence, which was expected. Sequence variant "b" V region of L chain reconstructed human antibody WS-4 shown in TH ID N 65, and the sequence variants "b", "C", "d", "e", "f", "g" and "h" V region of H chain reconstructed human antibody WS-4 shown in the LAST NN ID 41, 44, 45, 48, 51, 54 and 55, respectively.

After confirming the DNA sequences of different variants of the V region of the reconstructed human antibody WS-4 by determining the DNA sequence, which encodes the V region of the reconstructed human antibody WS-4, was subcloned into the expression vectors for mammalian cells that already contain DNA which encodes a human With the area. Namely DNA, which encodes the V region of L chain reconstructed human antibody WS-4, was associated with the DNA sequence that encodes the region of the L chain and the DNA that encodes the V region of H chain reconstructed human antibody WS-4, was associated with the DNA sequence which encodes human C1 region.

Then all areas of the H chain were tested for binding to human IL-8. As a result, as shown in Fig. 7, both the reconstructed antibodies containing "a" or "b" and "g" H chain (RVLa/RVHg and RVLb/RVHg) demonstrated the ability to bind to human IL-8 to the same extent as chimeric antibody WS-4.

Any expression system, including eukaryotic cells, such as cells of animals or certain mammalian cells, fungi cells, yeast cells and prokaryotic cells such as bacterial cells (e.g., Escherichia coli), can be used for the production of chimeric antibodies or reconstructed human antibodies to human IL-8 of the present invention. Preferably, however, to chimeric antibodies or reconstructed antibodies of the present invention expressibility in mammalian cells such as COS cells or CHO cells. In these cases, for expression in mammalian cells can be used suitable commonly used promoter. For example, it is preferable to use immediate-early promoter of human cytomegalovirus (HCMV). Examples of expression vectors that contain the HCMV promoter include HCMV-VH-HC1 and HCMV-VL-NSC, and this includes those that are produced from pSV2neo (International Patent Application Publication No. WO92-19759 which can be used in this invention, you need to use, include the promoters of viruses, such as retroviruses, virus polyoma, adenovirus and simian virus 40 (SV40), promoters derived from mammalian cells such as human factor-1 elongation of polypeptide chain (HEF-1). For example, in the case of SV40 promoter expression can be produced by the following method Mulligan R. C. et al. (Nature, 277, p.108-114, 1979), or in the case of promoter HEF-1, the expression can be made according to the following method Mizushima, S. et al. (Nucleic Acids Res., 18, 5322, 1990).

Another specific example of a promoter suitable for this invention is the promoter HEF-1. HEF-VH-g1 and HEF-VL-DC (Fig. 1) contained in the expression vector comprising the promoter. DNA sequence derived from virus polyoma, adenovirus, SV40 or bovine human virus (BPV), etc. can be used as replicating points. In addition, in order to amplify the number of genetic copies in the cells of the host, you can use the aminoglycoside-3'-phosphotransferase, neo-resistant gene, a gene timedancing (TS), gene xanthine-guaninephosphoribosyltransferase (XGPRT) or dihydrotetrazolo (dhfr) as a selective marker.

In conclusion, this invention, in which also DNA, which encodes the specified V region of the L chain and the DNA that encodes the specified V region of the H chain. They are applicable when receiving a human/mouse chimeric antibodies and reconstructed human antibodies to human IL-8. An example of a monoclonal antibody is WS-4. V region of the L chain has the amino acid sequence shown, for example, in TH ID N 26 and the V region of the H chain has the amino acid sequence shown, for example, in TH ID N 27. These amino acid sequences encoded by the nucleotide sequences shown in, for example, PET ID NN 26 and 27, respectively.

Chimeric antibody to human IL-8 of the present invention consist of:

1) With the human L chain and the V region of mouse L chain; and

2) With human L chain and the V region of the mouse H chain.

V region of mouse L chain V region of the mouse H chain and the DNA that encodes these areas, as described earlier. Above the area of human L chain may be any human With a region of L chain, examples of which include human SC and area. Above the region of H chain can be any human With a region of H chain can be any human With the region of H chain, inkel U., et al., EMBO J., 1, 403-407 (1982)).

For the production of chimeric antibodies obtained two types of expression vectors. Namely, the expression vector that contains DNA, which encodes the V region of mouse L chain and the human With the region of L chain under the control of the management expression enhancer/promoter type, and an expression vector that contains DNA, which encodes the murine V region of H chain and the human With the region of H chain under the control of the management expression enhancer/promoter type. The cells are then-owners, as mammalian cells, simultaneously transformed with these expression vectors, and transformed cells are cultured either in vitro or in vivo with obtaining chimeric antibodies.

Or DNA, which encodes the murine V region of the L chain and the human With the region of the L chain and the DNA that encodes the murine V region of H chain and the human With the region of the H chain, can be introduced into a single expression vector, cell host transformed with this vector, and these transformed cells are then cultured either in vitro or in vivo to obtain chimeric antibodies.

Reconstructed human antibody WS-4 of the present invention consists of:

A) L capocelli L chain and the CDR of the L chain of the mouse monoclonal antibody WS-4 human IL-8, and

In) H circuits, and each consists of:

1) human With a region of H chain and

2) the V region of H chain consisting of the FR of a human H chain and the CDR of the H chain of the mouse monoclonal antibody WS-4 human IL-8.

In the preferred implementation method of this invention the above-mentioned CDR of the L chain are the amino acid sequence shown in TH ID N 26, and within the specified amino acid sequence defined in table 5; the above CDR of the H chain are the amino acid sequence shown in TH ID N 27, and within the specified amino acid sequence defined in table 5; the above FR of a human L chain originate from REI; the above FR1, FR2 and FR3 of a human H chain derived from VDH26, and FR4 derived from 4B4; With the above region of the L chain is a human Ck region; the above-mentioned human With the region of H chain is the human C1 region. Besides the above-mentioned human With the region of H chain may be human C4 region or a radioisotope can be connected instead of the above-mentioned human With the region of L chain and/or human With the field H chain.

It is preferable to replace part of the amino acid which possess activity against a specific antigen.

In the preferred method of this invention V region of the L chain has the amino acid sequence shown as RVLa or RVLb in table 2, and the V region of the H chain has the amino acid sequence shown as RVHa, RVHb, RVHc, RVHd, RVHe, RVHf, RVHg or RVHh in tables 3 and 4. In addition, the amino acid at position 41 in FR V region of H chain must be a Proline, the amino acid at the specified position 47 should be tryptophan and/or the amino acid at position 67 of the specified FR3 should be phenylalanine, and these antibodies having the amino acid sequence shown as RVHb, RVHd, RVHe, RVHf, RVHg or RVHh, is more preferable. Those in which there is RVHg as the V region of H chain preferred.

For the production of reconstructed antibodies obtained two types of expression vectors. Namely, the resulting expression vector, which contains the DNA, which encodes the previously described reconstructed human L chain under the control of the management expression enhancer/promoter type, as well as another expression vector that contains DNA, which encodes the previously described reconstructed human H chain under the control of the management expression enhancer/promoter type. Then, the transformed cells are cultured or in vitro, or in vivo to obtain a reconstructed human antibodies.

Or DNA that encodes a reconstructed human L chain, and DNA which encodes a reconstructed human H chain, are inserted into a single expression vector, cell host transformed with this vector, and these transformed cells are then cultured either in vitro or in vivo to obtain the reconstructed target antibodies.

Chimeric antibodies or reconstructed human antibodies thus obtained can be isolated and purified by conventional methods, such as affinity chromatography with protein A, ion-exchange chromatography or gelfiltration.

Chimeric L chain or reconstructed human L chain of the present invention can be used for a complete antibodies by combining with H chain. Similarly, chimeric H chain or reconstructed human H chain of the present invention can be used for a complete antibodies by combining with L chain.

Murine V region of the L chain, reconstructed human V region of L chain murine V region of H chain and reconstructed human V region of H chain Aut be used as pharmaceutical preparations diagnostic tools, etc. by themselves or in the form of a fused protein with other proteins.

In addition, CDR V region of the L chain and the CDR of the V region of the H chain of the present invention are also inherently parts that bind antigen in the form of human IL-8. Believe that they will be used as pharmaceuticals, diagnostics, etc. by themselves or in the form of a fused protein with other proteins.

DNA which encodes the murine V region of the L chain of the present invention, applicable to obtain a DNA which encodes a chimeric L chain, or a DNA which encodes a reconstructed human L chain. Similarly, a DNA, which encodes the V region of the mouse H chain applicable to obtain a DNA which encodes a chimeric H chain, or the DNA that encodes the reconstructed human H chain. Moreover, DNA which encodes a CDR of the V region of the L chain of the present invention, applicable to obtain DNA that encodes a reconstructed human V region of the L chain, or a DNA which encodes a reconstructed human L chain.

Similarly, DNA which encodes a CDR of the V region of the H chain of the present invention, applicable for obtaining DNA, which codero H chain. In addition, a reconstructed human antibody F(ab')2, Fab or Fv, or single chain Fv, which is associated with Fv H chain and L chain may be produced by suitable cell host and used for the purposes described above (see, for example. Bird, R. E. et al., TIBTECH, 9, 132-137, 1991).

Single chain Fv is formed by linking the V region of H chain and the V region of L chain reconstructed human antibody to human IL-8. This single chain Fv V region of H chain and the V region of the L chain are linked through the linker, and preferably, by using a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883, 1988).

V region of H chain and the V region of the L chain of this single chain Fv may be one of the above V regions of H chain and L chain of the reconstructed human antibodies. Specific examples of this include the V region of H chain formed by the amino acid sequences described in the LAST NN ID 38, 41, 44, 45, 48, 51 and 54, and single chain Fv containing the V region of L chain, made up of the amino acid sequences described in TH ID NN 62 or 65 (see WO88-01649).

These V region, preferably related peptide linker. Examples of peptide linkers that are used for this purpose include the LNC, which encodes a single chain Fv obtained by using a DNA, which encodes the H chain or the V region of H chain and the DNA that encodes the L chain or the V region of the L chain of the above reconstructed human antibodies, as matrix, the amplification of the DNA which encodes the amino acid sequence, which is desirable, using primerno pair that defines both ends using PCR, and amplification by connecting primerno pair, which determines DNA which encodes a polypeptide linker together with both of these ends, so that respectively bind H and L chains.

Moreover, if the resulting DNA that encode single chain Fv, an expression vector that contains them, together with the host-cell, which is transformed by the specified expression vector can be obtained by conventional methods. In addition, a single chain Fv can be obtained by conventional methods using the host cell.

Compared with molecules of antibody single chain Fv are the best penetration into the fabric, and it is assumed that they will be used when rendering using radioisotope labels, and as a therapeutic agent having a function of Oia, chimeric antibodies, reconstructed human antibodies and F(ab')2, Fab, Fv or single chain Fv against IL-8 of the present invention can be used in ELISA (enzyme linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay-ELISA), RIA (radioimmunoassay, RIA) or method with fluorescent antibodies. For example, in the case of application of enzyme immunoassay with chimeric antibodies and reconstructed human antibodies to human IL-8 add to card coated with polyclonal antibody to human IL-8, add the culture supernatant or the purified sample of cells that produce chimeric antibodies or reconstructed human antibody to human IL-8, and add the appropriate second antibody which is labeled with an enzyme such as alkaline phosphatase. After incubation and washing Board type substrate of the enzyme, such as p-nitrophenylphosphate, with subsequent absorption measurement to evaluate the activity of binding antigen.

Activity by inhibition of binding to receptors of IL-8 chimeric antibody, a reconstructed human antibodies and F(ab')2, Fab, Fv or single chain Fv to human IL-8, estimated by the usual definition of suppression of light is neutrophils, after separation of neutrophils obtained from heparinized blood by centrifugation or other means, receive a suspension of cells with the corresponding number of cells that can be used in the aforementioned study.

The solution containing IL-8, respectively labeled125I and the like, and unlabeled IL-8 is mixed with a solution containing antibodies of the present invention or fragments thereof, prepared in the appropriate concentration, and then adding this mixture to the above suspension of neutrophils. After a certain period of time, the neutrophils are separated, and is determined by the activity of the label on the neutrophils.

To assess the inhibition of chemotaxis of neutrophils antibodies or fragments of this invention can use conventional known methods such as a method described in Grob, P. M. et al., J. Biol. Chem., 265, 8311-8316, 1990.

In the case of commercially available cameras for chemotaxis after dilution of antibodies or their fragments of the present invention in a suitable medium cultivation in camera type IL-8 and then adding the dilution of the antibodies or fragments. Then in the camera add the prepared suspension nauts on the filter, placed in the chamber, the number of neutrophils can be determined by conventional methods, such as staining or methods with fluorescent antibodies. In addition, quantification can be performed using the evaluation under a microscope or by using automatic measurement using any device.

After sterilization by filtration using a membrane filter of a chimeric antibody, a reconstructed human antibodies and their fragments F(ab')2, Fab, Fv or Fv, single chain to human IL-8 of the present invention can be introduced in the form of pharmaceutical therapeutic compound, preferably parenterally, by, for example, intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection, or intratrahealno, using, for example, an inhaler. Although varying depending on the age and symptoms of the patient, normal dosage in humans is 1-1000 mg/body, which can be selected division at doses equal to 1-10 mg/kg/week.

After assessing their cleansing binding activity of the chimeric antibody, a reconstructed human antibody to human IL-8 and their fragments F (the use of methods, usually used for the production of drugs physiologically active proteins. For example, the drug for injection consists of purified chimeric antibody, a reconstructed human antibodies to human IL-8 or their fragments F(ab')2, Fab, FV or FV, single chain, dissolved in a solvent such as physiological saline or a buffer, with the addition of antiobsessional tools, such as tween 80, gelatin or human serum albumin (CSA). Or this drug can also be liofilizirovannam for dilution and preparation before use. Examples of media that can be used for lyophilization include sugar alcohols or sugars such as mannitol and glucose.

Although the subsequent is a detailed explanation of the present invention through its implementation, described below, the scope of the present invention is not limited to these examples.

Example 1: Cloning of the DNA encoding the V region of mouse monoclonal antibodies to human IL-8

The DNA that encodes the variable region of murine monoclonal antibodies to human IL-8, cloned by the method described below.

1. Getting all PHK

All PHK gwin J. M., et al., described in Biochemistry, 18, 5294-5299, 1979.

Namely 1 107cells hybridoma WS-4 fully homogenized in 25 ml of 4 b guanidine thiocyanate (Fluka). The homogenate was layered onto a 5.7 M solution of caesium chloride in a centrifuge tube, followed by the deposition of PHK by centrifugation for 14 hours at 20oC when 31000 rpm rotor Beckman SW40.

Sediment PHK were washed in 80% ethanol and then dissolved in 200 μl of 20 mm solution of Tris-HCl (pH 7.5) containing 10 mm EDTA and 0.5% N-laurylsarcosine sodium. After adding proteinase (Boeringer) to a concentration of 0.5 mg/ml, the resulting mixture was incubated in a water bath at 37oC for 30 minutes. The mixture was extracted with phenol and chloroform, and PHK precipitated with ethanol. Then the precipitate PHK was dissolved in 200 ál of 10 mm solution of Tris-HCl (pH 7.5) containing 1 mm EDTA.

2. Extraction of messenger RNA (mRNA)

To be extracted mRNA encoding the H chain of the mouse monoclonal antibody WS-4, a poly(A)-positive mRNA was extracted from total RNA obtained in stage 1 above, using the dial to highlight the Fast Track mRNA, variant 3,2 (Invitrogen) and following the procedure described in the manufacturer's instructions.

3. Synthesis of single-stranded cDNA.

The single-stranded chancadora, described in the instructions. The resulting product is then used for amplification of cDNA, which encodes the murine V region of the H chain. In addition, in order to amplify cDNA, which encodes the murine V region of the L chain, was synthesized single-stranded cDNA from about 10 μg of the above total RNA.

4. Amplification of the gene encoding the variable region of the antibody, using PCR

1) Amplification of cDNA that encodes a murine V region of H chain

Primers MHv(mouse heavy variable) from 1 to 12 shown in TH ID NN: from 13 to 24 and primer MHC (mouse heavy constant), shown in TH ID N: 25 (Jones S. T., et al., Bio/Tichnology, 9, 88-89, 1991) was used for PCR primers. 100 μl of PCR solution containing 10 mm Tris-HCl (pH 8,3), 50 mm KCl, 0.1 mm dNTP (dATP, DSTF, DCTP, dTTP), 1.5 mm MgCl2, of 0.001% (wt/vol) gelatin, 5 units of DNA polymerase AmpliTaq (Perkin Elmer Cetus), 0.25 mm of one MHvprimers shown in TH ID NN: 13 to 24, 75 μm MCH primer shown in TH ID N: 25, and 1.5 μl of a solution of single-stranded cDNA obtained in stage 3 above. The PCR solutions were prepared for each of the primers MHV1-12. After coating each solution 50 μl of mineral oil was heated in a period on the order of 3 minutes at an initial temperature, ti is the next repetition of this cycle of heating 30 times the reaction mixture was further incubated for 10 minutes at 72oC.

2) Amplification of cDNA that encodes a murine V region of L chain

Primers MKv(mouse Kappa variable 1 to 11 shown in TH ID NN: 1 to 11 and primer MKC (mouse Kappa constant), shown in TH ID N: 12 (Jones S. T. et al., Bio/Technology, 9, 88-89, 1991) as primers in PCR.

Amplification of cDNA was made from 2 μl of single-stranded cDNA obtained in stage 3 above, using the same method described for amplification of the gene of the V region of H chain in stage 4, part (1) above, except that the amplification was performed using 0.25 μm of each of the mixtures of primers MKv and 3.0 μm primer GMT.

5. Clearing and fragmentation of PCR product

The appropriate DNA fragments of the V region of H chain and the V region of L chain, amplificatoare by PCR, as described above, were separated using agarose gel electrophoresis using 1.5% agarose (Sigma) with low melting point. Pieces of agarose containing the DNA fragment of the H chain length of about 450 and a DNA fragment of the L chain length of about 400 on carved separately and melted for 5 minutes at 65oC followed by the addition of equal volume of 20 mm Tris-HCl (pH 7.5) containing 2 mm EDTU and 300 mm NaCl.

This mixture was extracted with phenol and hdtu. Then the fragments were digested for 3 hours at 37oC using 5 units Xmal restriction enzyme (New England BioLabs) in 10 mm Tris-HCl (pH of 7.9) containing 10 mm MgCL2and 1 mm dithiothreitol. Then the DNA fragments were digested for 2 hours at 37oC using 40 units of restriction enzyme SalI (Takara Shuzo), and the resulting DNA fragments were separated using agarose gel electrophoresis using 1.5% agarose (Sigma) with low melting point.

Pieces of agarose containing the DNA fragments were cut out and melted for 5 minutes at 65owith the subsequent addition of an equal volume of 20 mm Tris-HCl (pH 7.5) containing 2 mm EDTA and 300 mm NaCl. This mixture was then extracted with phenol and chloroform, the DNA fragments were isolated by precipitation with ethanol and dissolved in 10 mm Tris-HCl (pH 7.5) containing EDTU.

Thus, accordingly, received a DNA fragment containing a gene that encodes murine V region of L chain-type, and the DNA fragment containing the gene that encodes murine V region of the H chain. The above-mentioned DNA fragments, both have customers join SalI at the 5' end and attach XmaI at their 3' end.

6. The clutch and transformation

About 0.3 μg of a DNA fragment SalI-XmaI containing about 0.1 µg of the vector pUC19 (Takara Shuzo), prepared by cleavage with SalI, XmaI and alkaline phosphatase of Escherichia coli (VAR; Takara Shuzo) for 4 hours at 16oC in the buffer of the reaction mixture containing 1 unit of DNA T4 ligase (Gibco BRL), and was added to the supplied buffer for binding.

Then the above mixture for binding were added to 50 μl of competent cells of E. Coli DH5 (GIBCO BRL), after which the cells were left to stand for 30 minutes on ice, for 1 minute at 42oC and again on ice for 1 minute. Then add 400 ál of 2 x YT medium (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989). After incubation for 1 hour at 37oC E. Coli was divided into 2 x agarized medium YT (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) containing 50 μg/ml ampicillin (Meiji Seika), followed by incubation overnight at 37oC to obtain transformant E. Coli.

Then at this time used X-Gal (5-bromo-4-chloro-3-indolyl--D-galactoside, Takara Shuzo) as a marker for selection.

This transformant was incubated over night at 37oC in 10 ml 2 x environment YT containing 50 μg/ml ampicillin, and the culture was obtained plasmid DNA using a kit QIAGEN Plasmid Mini Kit (QIAGEN) and following the procedure described in inst hybridoma WS-4, thus obtained, was named pUC-WS4-VL.

A plasmid containing the gene that encodes murine V region of H chain originating from hybridoma WS-4, were obtained from DNA fragments SalI-XmaI, following the same method described above except using JM109 competent cells of E. coli. The obtained plasmid was named pUC-WS4-VH.

Example 2: Determination of nucleotide sequence of DNA

The nucleotide sequence of the coding region of cDNA in the above-mentioned plasmid was determined using M13 primer RV and M13 primer M4 (both from Takara Shuzo) as primers sequences, automated DNA sequencing machine (Applied Biosystems Inc.) and a set of Taq Dye Deoxy Tertinator Cycle Sequencing Kit (Applied Biosystems Inc.) and following the Protocol described by the manufacturer. The nucleotide sequence of the gene that encodes the V region of the L chain of the mouse monoclonal antibody WS-4, contained in the plasmid pUC-WS4-VL shown in TH ID N: 26. In addition, the nucleotide sequence of the gene that encodes the V region of the H chain of the mouse monoclonal antibody contained in the plasmid pUC-WS4-VH shown in TH ID N: 27.

Example 3: Determination of CDR

The basic structure of the V regions of the L and H chains have mutual affinity, and each has four Karka the (CDR). Although the amino acid sequence of framework region is relatively well preserved, the variability of the amino acid sequences of the CDR regions is extremely high (Kabat E. A. et al., "Sequences of Proteins of Immunological Interest", US Dept. Of Health and Human Services, 1991).

Based on this fact were defined CDR, which are shown in table 5, by examining their homology when trying to find the amino acid sequence of the variable regions of murine monoclonal antibodies to human IL-8 using a database of amino acid sequences of the antibodies obtained Kabat et al.

Example 4: Confirmation of expression of the cloned cDNA (obtaining chimeric WS-4 antibody)

Obtaining the expression vector of

To get a vector that expresses a chimeric WS-4 antibody, clone cDNA pUC-WS4-VL and pUC-WS4-VH that encode the V region of L chain and H chain of the mouse WS-4, respectively, were modified by PCR. Then they were introduced into the expression vector HEF (see previously described, W92-19759 and Fig. 1)

Reverse primer (PEFC ID N: 28) for the V region of the L chain and the reverse primer (PEFC ID N: 29) for the V region of H chain were respectively hybridized with the DNA that encodes the start of the leader sequence -950 V, 1987) and the restriction site HindIII. Direct primer (PEFC ID N: 30) for the V region of the L chain and the direct primer (PEFC ID N: 31) for the V region of H chain hybridized with a DNA sequence which encodes a terminal J chain, and designed to add splicing donor sequence and restriction site BamHI.

100 μl PCR reaction mixture containing 20 mm Tris-HCl (pH 8,2), 10 mm KCl, 6 mm (NH4)2SO4, 1% Triton X-100, 100 μm dNTP, 1.5 mm MgCl2100 pmole each primer, 100 ng DNA templates (Fig-VL or pUC-VH) and 2.5 U of enzyme AmpliTaq, was covered with 50 μl of mineral oil. After initial denaturation for 3 minutes at 94oC cycle heating, consisting of 1 minute at 94oC, 1 minute at 55oC and 1 minute at 72oC, was repeated 30 times, followed the final incubation for 10 minutes at 72oC.

The PCR product was purified using a 1.5% agarose gel with a low melting point, followed by cleavage with HindIII and BamHI. Gene V region of the L chain was cloned in the expression vector HEF, HEF-VL-DK, whereas gene V region of the H chain was cloned into the HEF expression vector HEF-VH-g1. After determining the DNA sequences of the plasmid containing the DNA fragment having the correct posledovatelnostyu expression of chimeric antibody WS-4, the above expression vectors were tested in COS cells. HEF-chWS4L-DK and HEF-chW4H-g1 at the same time transfusional in COS cells by electroporation using the Gene Pulser (Biorad). Each DNA (10 μg) was added to 0.8 ml of the sample containing 1107cells/ml in phosphate-buffered saline (FBFR) and then subjected to pulsations at 1.5 kV with a capacity of 25 μf.

After being left to stand for a period of time equal to 10 minutes, at room temperature, elektrooborudovanie cells suspended in 15 ml of cultivation media DMEM (GIBCO) containing 5% fetal bovine serum, exempt from-globulin, placed in a Cup for tissue culture. After incubation for 96 hours the culture medium was collected, remaining cells were removed by centrifugation and the supernatant was then filtered using disc filter having a pore diameter of equal to 0.45 μm (Gelman Science).

ELISA

Board for ELISA for the quantitative determination of the binding of antigen and antibody concentrations were prepared as described below. Board for ELISA to determine the binding activity of the antigen were prepared as follows. After the formation of the solid layer in each cell 96-cell Board (Nunc) with 100 μl of goat powney 2 µg/ml (0.1 M sodium bicarbonate, 0,02% sodium azide) and blocked with 200 μl of buffer for dilution (50 mm Tris-HCl (pH of 7.2), 1% bovine serum albumin (BSA), 1 mm MgCl2, 0.15 M NaCl, 0.05% tween-20 and 0.02% sodium azide) was added to 100 μl of recombinant human IL-8 (Amersham) (5 ng/ml).

The purified sample chimeric antibody or the culture supernatant of COS cells that expressed them, were serially diluted and added to each cell. Then added 100 μl of labeled alkaline phosphatase goat to human IgG antibody (TAGO) (1 μg/ml). After incubation and washing was added to the substrate solution (1 mg/ml p-nitrophenylphosphate) followed by measuring the absorption at 405 nm.

For measuring the concentration of antibody after formation of a hard layer in the cells in the 96-cell Board with 100 μl of goat-human IgG antibody (TAGO) with a concentration of 1 μg/ml and blocked, the purified sample of chimeric antibodies or culture medium of COS cells that expressed them, were serially diluted and added to each cell. Then added 100 μl of labeled alkaline phosphatase goat to human IgG antibody (TAGO) (1 μg/ml). After incubation and washing was added to the substrate solution (1 mg/ml p-nitrophenylphosphate) and measured the absorbance at 405 nm.

Furthermore, E. coli having the above-mentioned plasmid HEF-chWS4L-DK, placed in storage as Escherichia coli DH5 (HEF-chWS4L-DK), a Escherichia coli having the above-mentioned plasmid HEF-chWS4H-g1, placed in storage as Escherichia coli JM109 (HEF-chWS4H-g1) in the Industrial technology research Institute of bioengineering of the Agency of industrial science and technology (Bioengineering Industrial Technology Research Institute of the Agency of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, lbaraki, Japan)) July 12, 1994 under the appropriate heading FERM BP-4739 and FERM BP-4740 in accordance with the provisions of the Budapest Convention.

Example 5: Receiving a reconstructed human antibody WS-4

Getting the V region of H chain reconstructed human antibody WS-4

DNA which encodes the V region of H chain reconstructed human antibody WS-4 was constructed by the method described below. Full DNA, which encodes the V region of H chain reconstructed human antibodies, was designed so that a known DNA sequence, which respectively encode from FR1 through FR3 of a human antibody VDH26 and FR4 of the human antibody 4B4, associated with the military was added to the site HindIII recognition/consensus Kozak sequence and the site of the BamHI recognition/splicing donor sequence at the 5' and 3' ends of the DNA sequence with the subsequent introduction of the expression vector HEF. The DNA sequence, is designed in such a way, then was divided into four approximately equal oligonucleotide, then the secondary structure of these nucleotides, for which there was a possibility of interference with the Assembly of these oligonucleotides was determined using the computer. Four oligonucleotide sequences shown in TH ID NN: 32-35. These oligonucleotides have a length equal to 113-143 grounds, and adjacent oligonucleotides have a region of overlap, typically consisting of 20 bases. HF1 (PEFC ID NN: 32) and HF3 (PEFC ID NN: 34) of these four oligonucleotides have a sense DNA sequence, whereas the other HF2 (PEFC ID N: 33) and HF4 (PEFC ID N: 35) have antisense DNA sequence. These oligonucleotides were synthesized using an automatic DNA synthesizer (Applied Biosystem).

In addition, the method of Assembly of these four nucleotides by PCR is illustrated in Fig. 3. Approximately 100 ng of each HF1 and HF2, and HF3 and HF4 were combined and added to a PCR reaction mixture with a final volume equal to 98 ál containing 2.5 U Pfu DNA polymerase. After initial denaturation for 3 minutes at 94oC solutions were incubated for 2 cycles, where each cycle consisted of inkseine half of the volume of the reaction solution of PCR incubation was continued for an additional two cycles. After adding 100 pmole each primer RVH5' (LETTER ID N: 36) and primer RVH3' (LETTER ID N: 37) as external primers, the reaction solution of PCR were covered with 50 µl of mineral oil. After initial denaturation for 3 minutes at 94oC the reaction solutions were incubated for 45 cycles of 1 minute at 94oC, 1 minute at 55oC and 1 minute at 72oC followed by incubation for 10 minutes at 72oC.

The DNA fragment containing approximately 450 base pairs was purified by 1.5% agarose gel with a low melting point, was digested with HindIII and BamHI and cloned into the HEF expression vector HEF-VH-g1 (Fig. 1). After determining the DNA sequence using primer EF-1 (PEFC ID N: 66) and primer HIP (TH ID NN: 67) plasmid, which contained a DNA fragment that encodes the correct amino acid sequence of V region of the H chain, called HEF-RVHa-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHa-g1, shown in TH ID N: 38.

Each option "b", "C", "d", "e", "f", "g" and "h" V region of H chain reconstructed human antibody WS-4 was obtained by a method described below.

Option "b" is private so that leucine at position 47 was replaced by tryptophan, RVH5' (LETTER ID N: 36) and RVH3' (LETTER ID N: 37) as primers that define both ends, and the plasmid HEF-RVHa-g1 as a matrix DNA to obtain plasmid HEF-RVHb-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHb g1 shown in TH ID N: 41.

Option "C" amplified by PCR using mutagenic primers QTP1 (PEFC ID N: 42) and QTP2 (PEFC ID N: 43), designed so that the glutamic acid at position 41 was replaced by Proline, and the plasmid HEF-RVHa-g1 as a matrix DNA to obtain plasmid HEF-RVHc-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHc-g1, shown in TH ID N: 44.

Option "d" amplified by PCR using mutagenic primers QTP1 and QTP2 and plasmid HEF-RVHb-g1 as a matrix DNA to obtain plasmid HEF-RVHd-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHd-g1, shown in TH ID N: 45.

Option "e" amplified by using mutagenic primers ATR (PEFC ID N: 46) and ATR (PEFC ID N: 47), score obtain plasmid HEF-RVHe-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHe-g1, shown in TH ID N: 48.

Option "f" amplified using the mutagenic primers GTA1 (PEFC ID N: 49) and GTA2 (PEFC ID N: 50), designed so that the glycine at position 44 was replaced by alanine, and plasmid HEF-RVHd-g1 as a matrix DNA to obtain plasmid HEF-RVHf-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHf-g1, shown in TH ID N: 51.

Option "g" amplified using the mutagenic primers LTF1 (PEFC ID N: 52) and LTF2 (PEFC ID N: 53), are designed so that the leucine at position 67 was replaced by phenylalanine, and plasmid HEF-RVHd-g1 as a matrix DNA to obtain plasmid HEF-RVHg-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHg-g1, shown in TH ID N: 54.

Option "h" amplified using the mutagenic primers LTF1 and LTF2 and plasmid HEF-RVHb-g1 as a matrix DNA to obtain plasmid HEF-RVHh-g1. Amino acid sequence and nucleotide sequence of the V region of H chain enclosed in this plasmid HEF-RVHh-
DNA which encodes the V region of L chain reconstructed human antibody WS-4 was created by the method described below. Full DNA, which encodes the V region of L chain reconstructed human antibody WS-4 was created so that the DNA sequence, which encodes the FR of the human antibody REI, binds to the DNA sequence which encodes a CDR of the V region of the L chain of the mouse antibody WS-4.

Then the website recognition HindIII/consensus Kozak sequence and the site of the BamHI recognition/splicing donor sequence was added, respectively, at the 5' and 3' ends of this DNA sequence, so as to allow its introduction into the expression vector HEF. The DNA sequence generated in such a way, then is divided into four approximately equal nucleotide, then the secondary structure of these nucleotides, for which there is a risk of interference with the Assembly of these nucleotides were analyzed using the computer.

Four oligonucleotide sequences shown in TH ID N: 56-59. These oligonucleotides have a length equal to 106-124 grounds adjacent oligonucleotides have a region of overlap, usually consisting of 19-23 bases. LF1 (PEFC ID N: 56) and LF3 (PEFC ID N: 58) of e is L ID N: 59) have antisense sequence. These oligonucleotides were synthesized by the same method as applied to the above-mentioned HF1-HF4.

For assemblies, after initial denaturation 98 μl PCR mixture containing 100 ng of each of the four types of nucleotides and 5 U Ampli Taq, for 3 minutes at 94oC, the mixture is incubated for 2 cycles, where each cycle consisted of incubation for 2 minutes at 94oC, 2 minutes at 55oC and 2 minutes at 72oC. After adding 100 µl of each: primer RVL5' (LETTER ID N: 60) and primer RVL3' (LETTER ID N: 61) as external primers, PCR reaction mixture was covered with 50 μl of mineral oil. After initial denaturation for 3 minutes at 94oC, the reaction solution was incubated for 30 cycles of 1 minute at 94oC, 1 minute at 55oC and 1 minute at 72oC, followed by incubation in the end for 10 minutes at 72oC (see Fig. 3).

The DNA fragment consisting of about 400 base pairs was purified using a 1.5% agarose gel with a low melting point, were digested HindIII and BamHI and cloned into the HEF expression vector HEF-VL-DC (Fig. 1). After determining the DNA sequence using primers EF-1

(LAST ID N: 66) and primer KIP (PEFC ID N: 68), a plasmid, which is named La HEF-RVLa-DK. Amino acid sequence and nucleotide sequence of the V region of L chain enclosed in this plasmid HEF-RVLa-DK, shown in TH ID N: 62.

Option "b" amplified by PCR using mutagenic primers FTY1 (PEFC ID N: 63) and FTY2 (PEFC ID N: 64), is designed so that the phenylalanine at position 71 was replaced by tyrosine, RVL5' (LETTER ID N: 60) and RVL3' (LETTER ID N: 61) as primers that define both ends, and the plasmid HEF-RVLa-DK as a matrix DNA to obtain plasmid HEF-RVLb-DK. Amino acid sequence and nucleotide sequence of the V region of L chain enclosed in this plasmid HEF-RVHb-DK, shown in TH ID N: 65.

To assess the binding activity of the antigen each circuit reconstructed human antibody WS-4, COS cells first at the same time transfusional in the manner described previously in relation to the expression vector HEF-RVLa-DK for option "a" L chain reconstructed human antibody WS-4 and the expression vector HEF-chWS4H-g1 for H chain chimeric antibody WS-4. After selection of culture medium, as described above, was determined by the number of produced antibodies and activity by binding to antigen for antibodies produced using the method described in railo, not that there were differences in activity by binding to the antigen between chimeric antibody (chL/chH), used as positive control, and antibodies, consisting of reconstructed L-chain, and chimeric H chain (RVLa/chH).

At the same time, to evaluate the combination of the expression vector HEF-chWS4L-DK for L chain chimeric antibody WS-4 and option "and" H chain reconstructed human antibody WS-4, were both at the same time cotransfection in COS cells, and the obtained antibodies was determined by the number of produced antibodies and activity by binding to the antigen, using the method described in the section "ELISA" in the above example 4. Activity by binding to the antigen was not observed in these antibody (chL/RVHa) (see Fig. 4).

As described previously, since the option "a" L chain reconstructed human antibody WS-4 was active in binding to the antigen, is equal to the activity of the L chain chimeric antibody WS-4, assessment of each option, all reconstructed H chains was performed by simultaneous transliterowany COS cells each option reconstructed H chain and option "a" L chain reconstructed human antibody WS-4 (RVLa).

The result was such that these antibodies with variations who want to make a chimeric antibody WS-4 (chL/chH), used as a positive control, showing thus that this combination provides a functional binding site of the antigen from human antibodies. However, as the number of produced antibodies, all options were producirovanie in a smaller amount than that of the chimeric antibody WS-4 (chL/chH), except for the option "g" (RVHg). In addition, the binding activity of the antigen was not observed in antibodies with variant "with" H chain (see Fig. 5).

Based on these data it was concluded that antibodies with variant "a" L chain reconstructed human antibody WS-4 (RVLa) and "g" H chain reconstructed human antibody WS-4, again form a functional binding site of the antigen, which shows favorable activity by binding to the antigen, and that the amount of produced antibodies is comparable to the number of chimeric antibody WS-4 (chL/chH) after simultaneous transfection in COS cells.

Then estimate variant "b" L chain reconstructed human antibodies (RVLb) was produced by simultaneous transliterowany COS cells each option H chain version "b" L chain reconstructed human antibody WS-4 (RVLb). The result showed that only antimania antigen, comparable to the activity of the chimeric antibody WS-4 (chL/chH), used as positive control, and concluded that this combination forms a functional binding site of the antigen in human antibodies. In addition, as the number of produced antibodies, all options were producirovanie less than the chimeric antibody WS-4 (chL/chH), except for the option "g" (RVHg) (see Fig. 6).

When the above evaluation two types of reconstructed human antibodies (RVLa/RVHg and RVLb/RVHg), which was active for binding to human IL-8, and the degree of product comparable to the activity and production of chimeric antibody (chL/chH), respectively, were purified using a column with protein A, and then evaluated the activity on binding, accurately applying the method described in the section on ELISA in example 4. The result showed that the chimeric antibody (chL/chH), antibodies RVLa/RVHg and antibodies RVLb/RVHg, all showed the same degree of activity on binding (see Fig. 7).

Based on these data it was concluded that antibodies having either option "a" (RVLa), or option "b" (RVLb) L chain reconstructed human antibody WS-4 and g (RVHg) H chain reconstructed human entitely the level of production of antibodies comparable to the activity and level of production, which Express the chimeric antibody WS-4 (chL/chH), after simultaneous transfection in COS cells.

Inhibitory activity against the binding of IL-8 IL-8 receptors in reconstructed human antibodies comprising variant "a" (RVLa) H chain and the option "g" (RVHg) H chain reconstructed human antibody WS-4, or option "b" (RVLb) of the L chain and the option "g" (RVHg) of the H chain was evaluated using a study on the inhibition of binding of ligand to receptor.

Approximately 100 ml sample of heparinized blood from normal subjects were layered in 35 ml samples of 15 ml mono-poly-separating solution (ICN Biomedicals), and a layer of human neutrophils were isolated by centrifugation on the provided instructions. After washing these cells medium RPMI-1640 containing 1% BSA, contaminating erythrocytes were removed using a 150 mm solution of ammonium chloride. After centrifugation the cells were washed with medium RPMI-1640 containing 1% BSA, and resuspendable to a concentration of 2 to 107cells/ml Was found that the content of neutrophils in this cell suspension was 95% or more, according to the results of the quantitative determination after staining smears of samples obtained from ispolnali and resuspendable to a concentration of 2 to 107cells/ml with buffer for binding (D-FBFR containing 1% BSA and 0.1% sodium azide). At this time added to the chimeric SK2 antibody having an Fc portion that is identical to a part of the human antibodies of the present invention (see International patent application N PCT/JP 94/00859) and the antigen to him, human IL-6, up to concentrations of approximately 50 mg/ml and about 40 ng/ml, respectively, and incubated for 30 minutes in an ice bath to pre-saturation of Fc receptors on neutrophils.

IL-8 labeled with a radioactive label125I (74 SBC/mmol, Amersham) and its IL-8 (Amersham) was prepared by mixing buffer binding at concentrations of 4 ng/ml, each. Chimeric antibody WS-4 (chL/chH), reconstructed human antibodies (RVLa/RVHg and RVLb/RVHg), human antibody negative control (PAESEL + LOREI) or mouse antibody WS-4 positive control, respectively, were diluted with buffer to bind to the concentration of 2000 ng/ml to about 8 ng/ml, serially 2-multiple dilutions. 50 μl of a solution of IL-8 and 50 μl of a solution of each antibody were incubated for 30 minutes in an ice bath. Then added 100 μl of the above suspension of neutrophils and incubation continued additionally for 1 hour at PE is blowing by centrifugation and freezing. In order to quantify IL-8 associated with the cell sediment was cut off and the radioactivity was measured using a gamma counter (Aroka). These results are shown in Fig. 8.

Clearly shown that antibodies with variant "a" of the L chain (RVLa) and "g" H chain (RVHg) reconstructed human antibody WS-4, or option "b" specified L-chain and the option "g" specified H chain, have inhibitory binding activity comparable to the activity of the chimeric antibody (chL/chH) in respect of binding IL-8 IL-8 receptors.

Furthermore, E. coli having the above-mentioned plasmid HEF-RVLa-DK, was deposited as Escherichia coli DH5 (HEF-RVLa-DK), and Escherichia coli containing the plasmid HEF-RVHg-g1 was deposited as Escherichia coli JM109 (HEF-RVHg-g1) in the research Institute of industrial bioengineering technology of Agency of industrial science and technology (Bioengeneering Industrial Technology Research Institute of Agency of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, lbaraki, Japan) July 12, 1994, under the respective names of FERM BP-4738 and FERM BP-4741 under the provisions of the Budapest Convention.

Reference example 1: Getting hybridoma WS-4

Hybridoma, which produces monoclonal antibody to human IL-8, was obtained by liani the m methods using polyethylene glycol. The selection was made using the activity of binding human IL-8 as a criterion to confirm hybridoma WS-4 (Ko Y. C., et al., J. Immunol. Methods, 149, 227-235, 1992).

This invention represents a reconstructed human antibody to human IL-8, and in these antibodies, CDR V region of human antibody is replaced with CDR of mouse monoclonal antibodies to human IL-8. Since most of this reconstructed human antibody has a human origin, and the CDR is inherently has low antigenicity, reconstructed human antibodies of this invention have low antigenicity for people, and for this reason it can be expected that they will be applied in medicine for treatment.

The list of microorganisms, deposited according to the provisions of article 13 bis of the patent cooperation Treaty from the International community repositories:

Name: national Institute of biological Sciences and the Agency for technology related to human industrial science and technology National Institute of Bioscience and Human Technology Agency of Industrial science and Technology

Address: 1-3, Higashi 1-chome, Taukuba, lbaruki, Japan

The registration number of the store and the date of receipt in storage:

1) EscHEF-chWS4L-DK)

The number of storage: FERM BP-4739

Date: July 12, 1994

3) Escherichia coli JM109 (HEF-chWS4H-g1)

The number of storage: FERM BP-4740

Date: July 12, 1994

4) Escherichia coli JM109 (HEF-RVHg-g1)

The number of storage: FERM BP-4741

Date: July 12, 1994

1. Reconstructed human V region of L chain antibodies RVLa or RVLb to human IL-8, consisting of a series of alternating frame region (FR) of a human V region of the L chain and the CDR of the V region of L chain mouse monoclonal antibodies to human IL-8: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and containing the amino acid sequence presented in table.2.

2. Reconstructed human V region of L chain under item 1, with the specified FR comes from a human antibody REI.

3. Reconstructed human V region of the H chain of the antibody RVLa, RVLb, RVLc, RVLd, RVLe, RVLf, RVLg or RVLh to human IL-8, consisting of a series of alternating frame region (FR) of a human V region of H chain and the CDR of the V region of the H chain of the mouse monoclonal antibody to human IL-8: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and containing the amino acid sequence presented in table.3 and 4.

4. Reconstructed human V region of H chain under item 3, being the l 4B4.

5. L chain reconstructed human antibody to human IL-8, consisting of: 1) human C region of the L chain, representing the human Ctoregion, and 2) the V region of L chain under item 1.

6. H chain reconstructed human antibody to human IL-8, consisting of: 1) human C region of the H chain, representing the human C1or C4region, and 2) the V region of H chain under item 3.

7. Reconstructed human antibody to human IL-8 comprising: 1) an L chain under item 5 and 2) an H chain under item 6.

8. DNA encoding the reconstructed human V region of the L chain of the antibody to human IL-8 p. 1 and containing the nucleotide sequence or a portion thereof, presented in TH ID N 62 or N 65.

9. DNA encoding the reconstructed human V region of the H chain of the antibody to human IL-8 under item 3 and containing the nucleotide sequence or part of it, presented in the LAST NN ID 38, 41, 44, 45, 48, 51, 54 or 55.

10. DNA encoding the reconstructed human L chain of the antibody to human IL-8 under item 5 and containing the nucleotide sequence or a portion thereof, presented in TH ID N 62 or N 65.

11. DNA encoding realnosti or part thereof, presented in TH ID NN 38, 41, 44, 45, 48, 51, 54 or 55.

12. A vector containing the DNA, under item 10.

13. A vector containing the DNA, under item 11.

14. Method for the production of a reconstructed human antibodies to human IL-8 comprising the stage of culturing host cells transformed simultaneously expression vector containing the DNA, which is presented in paragraph 10, and the expression vector containing the DNA, which is presented in paragraph 11, and selection of the target antibodies.

15. Method for the production of a reconstructed human antibodies to human IL-8 comprising the stage of culturing host cells transformed by the expression vector containing the DNA, which is presented in paragraph 10, or DNA, which is presented in paragraph 11, and selection of the target antibodies.

 

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