HK1024261B - Reconstituted human anti-hm 1.24 antibody - Google Patents

Description

reconstituted human anti-HM 1.24 antibody

Technical Field

The present invention relates to a reconstructed human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody, genes encoding the same, a method for producing the same, and uses of the same. The reconstructed human antibodies and chimeric antibodies of the invention are useful as pharmaceutical agents, for example, for myeloma.

Background

Human B cells undergo various differentiation processes classified according to the kind of expressed surface antigen, and finally develop into antibody-producing plasma cells. In the final stage of their differentiation, on the one hand, B cells acquire the capacity to produce cytoplasmic immunoglobulins, and on the other hand B cell-associated antigens such as cell surface immunoglobulins, HLA-DR, CD20, Fc receptors, complement C3 receptors, etc. disappear (Ling, n.r. et al, type III leukocytes (1986) p 320, Oxford, UK, Oxford).

At present, there have been reports of the recognition of antigens on plasma cell membranes by monoclonal antibodies such as anti-PCA-1 (Anderson, K.C. et al, J. Immunol (1983)130, 1132), anti-PC-1 (Anderson, K.C. et al, J. Immunol (1983)132, 3172), anti-MM 4(Tong, A.W. et al, blood (1987)69, 238), etc. However, monoclonal antibodies against CD38 are still used for the detection of plasma cells and myeloma cells (Epstein, J. et al, New England journal of medicine, (1990)322, 664, Terstappen, L.W.M. et al, blood (1990)76, 1739, Leo, R. et al, Ann. Hensch (1992)64, 132, shimazaki, C. et al, American journal of hematology (1992)39, 159, Hata, H. et al, blood (1993)81, 3357, Harada, H. et al, blood (1993)81, 2658, Billadeau, D. et al, journal of Experimental medicine (1993)178, 1023).

However, the anti-CD 38 monoclonal antibody is an antigen associated with T cell activation rather than B cell differentiation and is expressed on a variety of cells including B cells. Furthermore, although CD38 is not expressed on some lymphoplasmacytoid cells (lymphoplasmacytoids), it is expressed in large amounts on hematopoietic precursor cells. Therefore, the anti-CD 38 monoclonal antibody is considered unsuitable for studying the differentiation and maturation of human B cells or for treating plasma cell diseases.

Goto, T et al have reported that mouse anti-HM 1.24 antibody was recognized to specifically express an antigen with a molecular weight of 29 to 33Kda on a B cell line (blood (1994)84, 1922-. It is considered that an antigen recognized by the anti-HM 1.24 monoclonal antibody is associated with terminal differentiation of B cells (Goto, T. et al, J.Clin Immunol (1992)16, 688-691), and that administration of the anti-HM 1.24 antibody to a mouse transplanted plasmacytoma resulted in specific accumulation of the antibody at the tumor site (shuji Ozaki et al, conference set of 19 th Japanese society for myeloma research, general lecture 3), and therefore it is suggested that the radioisotope-labeled anti-HM 1.24 antibody can be used for diagnosis of tumor sites, missile therapy such as radioimmunotherapy, and the like.

Furthermore, the aforementioned journal of blood describes the complement dependent cytotoxic activity of the anti-HM 1.24 antibody against the human myeloma cell line RPMI 8226.

Myeloma is a neoplastic disease characterized by the accumulation of monoclonal plasma cells (myeloma cells) in the bone marrow. Myeloma is a disease in which terminally differentiated B cells that produce and secrete immunoglobulin or plasma cells increase primarily monoclonally in the bone marrow, and thus monoclonal immunoglobulin or its constituents, L chains or H chains, are detectable in serum (Masaakikosaka et al, Nippon Rinsho (1995)53, 91-99).

Conventional chemotherapeutic agents have been used to treat myeloma, but no effective therapeutic agents have been found that can cause reduction of myeloma and prolong the survival of myeloma patients. Therefore, there has been a long-standing emergence of drugs for myeloma which have therapeutic effects.

For example, a mouse antibody administered to a human may be metabolized as a foreign substance, resulting in a relatively short half-life of the mouse antibody in the human and thus failing to exert its intended effect.

To solve the above problems, methods for reducing the immunogenicity of non-human antibodies such as murine monoclonal antibodies have been developed. As such an example, there is a method for producing a chimeric antibody in which the variable region (V region) of the antibody is derived from a mouse and the constant region (C region) thereof is derived from an appropriate human antibody.

Since the chimeric antibody thus obtained comprises the variable regions of the original mouse antibody in an intact form, it is expected to have the same binding specificity to an antigen as the original human murine antibody. In addition, the ratio of amino acid sequences of non-human origin in the chimeric antibody is greatly reduced, and therefore the antibody is expected to have low immunogenicity as compared with the original mouse antibody. Chimeric antibodies bind to antigens in the same manner as the original mouse monoclonal antibodies and, although less immunogenic, may include an immune response against the mouse variable region (LoBuglio, A.F., et al, Proc. Natl. Acad. Sci. USA 86, 4220-.

Although the second approach to reducing the immunogenicity of mouse antibodies is much more complex, the potential immunogenicity of mouse antibodies can be further significantly reduced. In this method, only Complementarity Determining Regions (CDRs) of a mouse antibody variable region are grafted to a human antibody variable region to prepare a "reconstructed" human antibody variable region.

However, in order to make the structure of the CDRs of the reconstructed human antibody variable region as similar as possible to that of the original mouse antibody, a part of the amino acid sequence of the Framework Region (FR) supporting the CDRs may be grafted from the variable region of the mouse antibody to the variable region of the human antibody, if necessary. This portion of the V region of the humanized reconstituted human antibody is then ligated to the constant region of the human antibody. Finally, the portions derived from the amino acid sequence of non-human origin in the reconstructed humanized antibody are portions of the CDR and FR. CDRs are composed of highly variable amino acid sequences that do not exhibit species-specific sequences. Thus, a humanized antibody carrying mouse CDRs should not be more immunogenic than a natural human antibody with human antibody CDRs.

For humanized antibodies see Riechmann, L. et al, Nature, 332, 323-327, 1988; verhoeye, M. et al, science 239, 1534-1536, 1988; kettleborough, C.A., et al, protein engineering, 4, 773-; meada, H.et al, human antibodies and hybridomas, 2, 124-; groman, 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, 1869-; carter, p. et al, proceedings of the national academy of sciences USA, 89, 4285-; co, M.S. et al, J. immunology, 148, 1149-1154, 1992; and Sato, K. et al, cancer research, 53, 851-.

Queen et al (International application publication No. WO90-07861) describe a method for producing humanized antibodies, anti-IL-2 receptor antibodies, anti-Tac. However, it is difficult to fully humanize all antibodies even according to the method described in WO 90-07861. Thus, WO90-07861 does not describe a general method for humanising antibodies, but only describes a method for humanising an anti-Tac antibody which is one of the anti-IL-2 receptor antibodies. Furthermore, even if the method of WO90-07861 is completely followed, it is difficult to prepare a humanized antibody having exactly the same activity as the original mouse antibody.

Generally, the CDR/FR amino acid sequences of each antibody are different. Thus, the determination of amino acid residues to be substituted for the construction of humanized antibodies and the screening of amino acid residues for substitution of the above amino acid residues will vary with the particular antibody. Therefore, the method of preparing humanized antibodies as described in WO90-07861 cannot be applied to humanization of all antibodies.

Queen et al, Proc. Natl.Acad.Sci., USA, (1989)86, 10029-10033 have similar disclosures to WO 90-07861. This document describes that the humanized antibody produced according to the method described in WO90-07861 gives only one third of the activity of the original mouse antibody. In other words, this indicates that the method of WO90-07861 by itself is not capable of producing fully humanized antibodies with the same activity as the original mouse antibody.

Co et al, cancer research (1996)56, 1118-1125, were published by the Queen et al research group mentioned above. This document describes that even the method for producing a humanized antibody according to WO90-07861 cannot construct a humanized antibody having the same activity as the original mouse antibody. Thus, not only does the fact indicate that the WO90-07861 method by itself does not produce fully humanized antibodies having the same activity as the original murine antibody, but also that the method of constructing humanized antibodies as described in WO90-07861 cannot be applied to humanization of all antibodies.

Ohtomo et al, molecular immunology (1995)32, 407-. This document discloses that the amino acid residues used for humanization of anti-Tac antibodies in WO90-07861 are not related to activity and methods such as those described in WO90-07861 cannot be used.

Kettleborough et al, protein engineering (1991)4, 773-783 disclose the construction of several humanized antibodies from mouse antibodies by substitution of amino acid residues. However, there are many proposals for humanising anti-Tac antibodies which require alternative amino acid residues, such as those described in WO 90-07861.

The aforementioned document indicates that the method for producing a humanized antibody described in WO90-07861 is a technique applicable only to the anti-Tac antibody described therein and does not produce the same activity as the original mouse antibody even using this technique.

The original mouse antibodies described in these documents have amino acid sequences different from the anti-Tac antibody described in WO 90-07861. Thus, the method for constructing a humanized antibody that can be used for the anti-Tac antibody cannot be used for other antibodies. Similarly, since the mouse anti-HM 1.24 antibody of the present invention has an amino acid sequence different from that of the anti-Tac antibody, a method of constructing a humanized antibody of the anti-Tac antibody cannot be used. Furthermore, the successfully constructed humanized antibody of the present invention has an amino acid sequence which is different from that of the humanized anti-Tac antibody described in WO 90-07861. This fact indicates that the same method cannot be used for humanization of antibodies having different CDR-FR sequences

Thus, even if the original mouse antibody used for humanization is known, the identity of the CDR-FR sequences of an active humanized antibody can be confirmed after repeated experiments. WO90-07861 does not mention the FR sequences bound in the humanized antibody constructed according to the present invention and the fact that an active humanized antibody is obtained from binding to FR, less CDR sequences.

As previously mentioned, it is expected that the humanized antibody may be used for therapeutic purposes, but humanized anti-HM 1.24 antibodies are unknown or not mentioned. Furthermore, there is no standard method for humanized antibody production that can be universally applied to any antibody, and various inventions for constructing humanized antibodies exhibiting sufficient binding activity, binding inhibition activity and neutralizing activity are required (e.g., sato, k. et al, cancer research, 53, 851-.

Disclosure of the invention

The present invention provides a reconstituted antibody of anti-HM 1.24 antibody. The invention also provides human/mouse chimeric antibodies useful in methods of constructing such reconstructed antibodies. The invention further provides fragments of the reconstructed antibodies. In addition, the invention provides expression systems for the production of chimeric antibodies, reconstructed antibodies and fragments thereof. The present invention further provides methods for producing chimeric antibodies against HM1.24 antibody and fragments thereof and reconstituted antibodies against HM1.24 antibody and fragments thereof.

More specifically, the present invention provides methods for specifically recognizing a polypeptide having the sequence of SEQ ID No: 103, or a chimeric antibody and a reconstructed antibody of the polypeptides of the amino acid sequences listed in 103. The cDNA encoding the polypeptide was inserted between XbaI cleavage sites of the pUC19 vector, and prepared as plasmid pRS38-pUC 19. Escherichia coli containing plasmid pRS38-pUC19 was stored as Escherichia coli DH5 alpha (pRS38-pUC19) at 5.10.1993 at national institute of bioscience and human technology, Industrial science and technology agency, MITI (Higashi l-Chome 1-3, Tsukuba City, Ibalaki prediction, Japan) and was deposited under the accession number FERM BP-4434 (see Japanese unexamined patent publication (Kokai) No. 7-196694) under the terms of the Budapest treaty.

As an embodiment of such a chimeric antibody or a reconstituted antibody, a chimeric anti-HM 1.24 antibody or a reconstituted human anti-HM 1.24 antibody is mentioned. A detailed description of the chimeric anti-HM 1.24 antibody or the reconstituted human anti-HM 1.24 antibody will be given below.

Accordingly, the present invention also provides a chimeric L chain comprising a human light (L) chain constant region (C region) and an L chain variable (V) region of anti-HM 1.24 antibody, and a chimeric H chain comprising a human heavy (H) chain constant region and a heavy (H) chain V region of anti-HM 1.24 antibody.

The present invention further provides a chimeric antibody comprising:

(1) an L chain comprising a human L chain C region and an L chain V region of anti-HM 1.24 antibody; and

(2) an H chain comprising the C region of a human H chain and the V region of an H chain of anti-HM 1.24 antibody.

The invention further includes a reconstituted human L chain V region of anti-HM 1.24 antibody comprising: (1) framework Regions (FR) of human L chain V region and (2) Complementarity Determining Regions (CDR) of L chain V region of anti-HM 1.24 antibody; and a reconstructed human H chain V region of the anti-HM 1.24 antibody comprising: (1) FR of human H chain V region and (2) CDR of H chain V region of anti-HM 1.24 antibody.

The invention further provides a reconstituted human L chain of anti-HM 1.24 antibody comprising: (1) a human L chain C region and (2) an L chain V region comprising a human L chain FR and an anti-HM 1.24 antibody L chain CDR; and a reconstituted human H chain of anti-HM 1.24 antibody comprising: (1) a C region of a human H chain, and (2) an H chain V region comprising human H chain FR and H chain CDR of anti-HM 1.24 antibody.

The invention further provides a reconstituted human antibody against the HM1.24 antibody, comprising:

(A) an L chain comprising (1) a human L chain C region and (2) an L chain V region comprising the FR of a human L chain and the L chain CDR of an anti-HM 1.24 antibody; and

(B) an H chain comprising (1) a human H chain C region and (2) an H chain V region comprising the FR of a human H chain and the CDR of an H chain of an anti-HM 1.24 antibody.

The present invention further provides a DNA encoding the L chain V region of anti-HM 1.24 antibody and a DNA encoding the H chain V region of anti-HM 1.24 antibody.

The present invention further provides a DNA encoding a chimeric L chain comprising (1) a human L chain C region and (2) an L chain V region of anti-HM 1.24 antibody; and a DNA encoding a chimeric H chain comprising (1) the C region of human H chain and (2) the H chain V region of anti-HM 1.24 antibody.

The present invention further provides a reconstituted human L chain V region of anti-HM 1.24 antibody comprising: (1) the FR of human L chain V region and (2) the CDR of L chain V region of anti-HM 1.24 antibody; and a DNA encoding a reconstructed human H chain V region of the anti-HM 1.24 antibody, which V region comprises (1) the FR of the human H chain V region and (2) the CDR of the H chain V region of the anti-HM 1.24 antibody.

The present invention further provides a DNA encoding a reconstructed human L chain of anti-HM 1.24 antibody, comprising: (1) a human L chain C region and (2) an L chain V region comprising the FR of a human L chain and the CDR of an anti-HM 1.24 antibody L chain; and a DNA encoding a reconstructed human H chain of the anti-HM 1.24 antibody, comprising: (1) a human H chain C region and (2) an H chain V region comprising the FR of a human H chain and the CDR of an anti-HM 1.24 antibody H chain.

The present invention further provides a vector comprising any of the plurality of DNAs described above.

The present invention further provides a host cell transformed with the above-described vector.

The present invention also provides a method for producing a chimeric antibody against HM1.24 antibody, which method comprises culturing a host cell transformed with an expression vector containing a DNA encoding the chimeric L chain and an expression vector containing a DNA encoding the H chain and recovering the desired antibody.

The present invention further provides a method for producing a reconstructed human antibody against HM1.24 antibody, which method comprises culturing a host cell transformed with an expression vector containing a DNA encoding said reconstructed human L chain and an expression vector containing a DNA encoding said reconstructed human H chain and recovering the desired antibody.

The invention further provides pharmaceutical compositions comprising the chimeric or reconstructed human antibodies, particularly therapeutic agents for myeloma.

The invention further provides a polypeptide containing the amino acid sequence which specifically recognizes the polypeptide with the sequence shown in SEQ ID No: 103, and a pharmaceutical composition containing a chimeric antibody of a polypeptide having an amino acid sequence shown in SEQ ID No: 103 as shown in the table, and a pharmaceutical composition containing as an active ingredient a reconstructed human antibody of a polypeptide having an amino acid sequence shown in the table 103. As a pharmaceutical composition, a therapeutic agent for myeloma is specifically provided.

Brief Description of Drawings

FIG. 1 shows that the fluorescence intensity of the chimeric anti-HM 1.24 antibody and that of the mouse anti-HM 1.24 antibody in the FCM assay using the human myeloma cell line KPMM2 changed in a similar manner to the control antibody.

FIG. 2 shows that chimeric anti-HM 1.24 antibody similar to mouse anti-HM 1.24 antibody inhibits biotinylated mouse anti-HM 1.24 antibody from binding to WISH cells in a dose-dependent manner in a cell ELISA using WISH cells.

FIG. 3 shows that control human IgG1 or mouse anti-HM 1.24 antibody was not cytotoxic and that chimeric anti-HM 1.24 antibody showed increased cytotoxicity to RPMI8226 cells with increased E/T ratios.

FIG. 4 is a schematic representation of a method for reconstructing the L chain of human anti-HM 1.24 antibody constructed by CDR-grafting in a PCR manner.

FIG. 5 is a diagram illustrating a method of assembling oligonucleotides RVH1, RVH2, RVH3 and RVH4 by PCR in the preparation of a reconstituted human anti-HM 1.24 antibody H chain.

FIG. 6 is a diagram illustrating a method of constructing H chain V region of human-mouse hybrid anti-HM 1.24 antibody by PCR.

FIG. 7 is a diagram illustrating a method of constructing H chain V region of mouse and human hybrid anti-HM 1.24 antibody by the PCR method.

FIG. 8 shows that L chain a type of the reconstituted human anti-HM 1.24 antibody has the same antigen-binding activity as that of the chimeric anti-HM 1.24 antibody. -1 and-2 represent different lot numbers.

FIG. 9 shows the antigen binding activity of the reconstituted human anti-HM 1.24 antibody, in which type a of the L chain binds to type a, b, f or H of the H chain, and chimeric anti-HM 1.24 antibody.

FIG. 10 shows the binding activity of the reconstituted human anti-HM 1.24 antibody, in which type b of the L chain binds to type a, b, f or H of the H chain, and the chimeric anti-HM 1.24 antibody.

FIG. 11 shows the binding inhibition activity of the reconstituted human anti-HM 1.24 antibody, in which type a of the L chain binds to type a, b, f or H of the H chain, and the chimeric anti-HM 1.24 antibody.

FIG. 12 shows the binding inhibition activity of the reconstituted human anti-HM 1.24 antibody, in which type b of the L chain binds to type a, b, f or H of the H chain, and the chimeric anti-HM 1.24 antibody.

FIG. 13 shows the a, b, c and d forms of the H chain of reconstituted human anti-HM 1.24 antibody, and the antigen-binding activity of the chimeric anti-HM 1.24 antibody.

FIG. 14 shows the a and e forms of the H chain of reconstituted human anti-HM 1.24 antibody, and the antigen-binding activity of chimeric anti-HM 1.24 antibody. -1 and-2 represent different lot numbers.

FIG. 15 shows the binding inhibition activity of the a, c, p and r forms of the H chain of the reconstituted human anti-HM 1.24 antibody, and the chimeric anti-HM 1.24 antibody.

FIG. 16 shows the antigen binding activity of human-murine anti-HM 1.24 antibody, murine-human anti-HM 1.24 antibody, and chimeric anti-HM 1.24 antibody.

FIG. 17 shows the antigen binding activity of the a, b, c or f forms of the H chain of the reconstituted human anti-HM 1.24 antibody, and the chimeric anti-HM 1.24 antibody.

FIG. 18 shows the antigen binding activity of the a, g forms of the H chain of reconstituted human anti-HM 1.24 antibody and that of the chimeric anti-HM 1.24 antibody.

FIG. 19 shows the binding inhibition activity of the a and g forms of the H chain of reconstituted human anti-HM 1.24 antibody and chimeric anti-HM 1.24 antibody.

FIG. 20 shows the antigen binding activity of the H and I forms of the H chain of reconstituted human anti-HM 1.24 antibody and the chimeric anti-HM 1.24 antibody.

FIG. 21 shows the antigen binding activity of the f, H and j forms of the H chain of reconstituted human anti-HM 1.24 antibody and that of the chimeric anti-HM 1.24 antibody.

FIG. 22 shows the binding inhibition activity of the H and I forms of the H chain of reconstituted human anti-HM 1.24 antibody and that of the chimeric anti-HM 1.24 antibody.

FIG. 23 shows the binding inhibition activity of the f, H and j forms of the H chain of reconstituted human anti-HM 1.24 antibody and chimeric anti-HM 1.24 antibody.

FIG. 24 shows the antigen binding activity of the H, k, l, m, n and o forms of the reconstructed human anti-HM 1.24 antibody H chain and the chimeric anti-HM 1.24 antibody.

FIG. 25 shows the antigen binding activity of the a, H, p and q forms of the H chain of reconstituted human anti-HM 1.24 antibody and chimeric anti-HM 1.24 antibody.

FIG. 26 shows the inhibitory activity of H chain H, k, l, m, n and o forms of reconstructed human anti-HM 1.24 antibody and binding of chimeric human anti-HM 1.24 antibody to WISH cells.

FIG. 27 shows the binding inhibition activity of the a, H, p and q types of the H chain of reconstituted human anti-HM 1.24 antibody and chimeric anti-HM 1.24 antibody.

FIG. 28 shows the antigen binding activity of the a, c, p and r forms of the H chain of reconstituted human anti-HM 1.24 antibody and chimeric anti-HM 1.24 antibody.

FIG. 29 shows that the S form of the reconstituted human anti-HM 1.24 antibody has the same antigen-binding activity as the r form of the reconstituted human anti-HM 1.24 antibody.

FIG. 30 shows that the type of reconstituted human anti-HM 1.24 antibody has the same binding inhibition activity as that of the type r of reconstituted human anti-HM 1.24 antibody.

FIG. 31 shows that purified reconstituted human anti-HM 1.24 antibody has the same antigen-binding activity as chimeric anti-HM 1.24 antibody.

FIG. 32 shows that purified reconstituted human anti-HM 1.24 antibody has the same binding inhibition activity as that of chimeric anti-HM 1.24 antibody.

FIG. 33 shows that administration of chimeric anti-HM 1.24 antibody results in an increase in survival compared to administration of control human IgG1 in human myeloma cell-transplanted mice.

FIG. 34 shows that when cells from healthy human peripheral blood were used as effector cells, control human IgG1 was non-cytotoxic to KPMM2 cells and mouse anti-HM 1.24 antibody was also weakly cytotoxic, whereas reconstituted human anti-HM 1.24 antibody was strongly cytotoxic to KPMM2 cells.

FIG. 35 shows that when peripheral blood cells from healthy humans were used as effector cells, the control human IgG1 was non-cytotoxic and the mouse anti-HM 1.24 antibody was also weakly cytotoxic to ARH-77 cells, whereas the reconstituted human anti-HM 1.24 antibody was strongly cytotoxic to ARH-77 cells.

FIG. 36 shows that when bone marrow cells from SCID mice were used as effector cells, control human IgG1 was non-cytotoxic to KPMM2 cells, while the cytotoxicity of reconstituted human anti-HM 1.24 antibody to KPMM2 increased with increasing antibody concentration.

FIG. 37 shows that in human myeloma cell-transplanted mice, serum IgG levels were increased after administration of control human IgG1 compared to levels before administration, while administration of reconstituted human anti-HM 1.24 antibody inhibited the increase in serum human IgG levels.

FIG. 38 shows that administration of reconstituted human anti-HM 1.24 antibody prolongs survival in human myeloma cell-transplanted mice compared to administration of control human IgG 1.

FIG. 39 is a graph showing increased serum human IgG levels in mice transplanted with human myeloma cells after administration of melphalan and control human IgG1, as compared to before administration.

FIG. 40 shows that administration of reconstituted human anti-HM 1.24 antibody prolongs survival compared to administration of melphalan or control human IgG1 in human bone myeloma cell-transplanted mice.

Method for carrying out the invention

1. Construction of chimeric antibodies

(1) Cloning of DNA encoding V region of mouse anti-HM 1.24 monoclonal antibody

Preparation of mRNA

Total RNA is prepared from the harvested myeloma by a known method such as the guanidine ultracentrifugation method (Chirgwin, J.M., et al, biochemistry (1979), 18, 5294-. In addition, mRNA can be prepared without a total RNA extraction step using the QuickPrep mRNA purification kit (produced by Pharmacia).

Preparation and amplification of cDNA

cDNA for the V region of the L chain and H chain is synthesized from the mRNA obtained in the above-mentioned mRNA preparation method using reverse transcriptase. cDNA for the L chain V region was synthesized using AMV reverse transcriptase first strand cDNA Synthesis kit. The synthesized cDNA is amplified by hybridizing appropriate primers to the leader sequence and C region of the antibody gene (for example, MKV primer contains the base sequences shown by SEQ ID Nos. 29 to 39, and MKC primer contains the base sequence shown by SEQ ID No. 40).

The cDNA for the H chain V region was synthesized and amplified using the 5 '-AmpliFINDER RACE kit (CLONTECH) by PCR (polymerase chain reaction) using the 5' -RACE method (Frohman, M.A., et al, Proc. Natl. Acad. Sci. USA, 85, 8998-. The Ampli FINDER Anchor was ligated to the end of the above-mentioned synthesized cDNA 5' and used as a primer for amplifying the H chain V region, a primer hybridizing with an Anchor primer (SEQ ID No: 77) and a mouse H chain (e.g., MHC22 primer containing the base sequence shown in SEQ ID No: 42) constant region (Cr region) was used.

DNA purification and determination of base sequence thereof

The PCR product is subjected to agarose gel electrophoresis by a known method to separate a DNA fragment of interest, and the DNA is recovered and purified, and then ligated into a vector DNA.

The DNA was purified using a commercial kit (e.g., GENECLEAN II; BIO 101). The DNA fragment can be preserved with a known vector DNA (e.g., pUC19, Bluescript, etc.).

The above DNA and the DNA vector are ligated by using a known ligation reaction kit (produced by Takara Shuzo) to obtain a recombinant vector. After selection of ampicillin-resistant clones and preparation of vector DNA according to known methods (J.Sambrook et al, "molecular cloning", Cold spring harbor laboratory Press 1989), the resulting recombinant vector was introduced into E.coli JM 109. After digesting the above vector DNA with a restriction enzyme, the base sequence of the objective DNA is determined by a known method (e.g., dideoxy method) (J.Sambrook et al, "molecular cloning", Cold spring harbor laboratory Press, 1989). According to the present invention, an automatic sequencing system (DNA sequencer 373A; manufactured by ABI Co., Ltd.) can be used.

Complementarity determining region

The H chain V region and the L chain V region form an antigen binding site, and both have similar characteristics in the entire structure. Thus, each of the four Framework Regions (FR) is linked to three hypervariable regions, the Complementarity Determining Regions (CDRs). The amino acid sequence of the FR is well conserved and the variation of the amino acid sequence of the CDR region is extremely high (Kabat, E.A., et al, "sequences of immune related proteins", U.S. department of health and human services, 1983).

The majority of the four FRs described above have a beta-sheet structure resulting in three CDRs forming loops. CDRs may sometimes form part of the beta sheet structure. The three CDRs are in close spatial proximity to each other and form the antigen binding sites of the three CDRs with the mating regions.

Based on these facts, the amino acid sequence of the variable region of mouse anti-HM 1.24 antibody was compared with the amino acid sequence database of antibody prepared by Kabat et al ("sequence of immune-related protein", in the US health and human services, 1983) to observe homology and find CDR regions.

(2) Construction of expression vector for chimeric antibody

Once the DNAs encoding the L chain and H chain V regions of the mouse monoclonal antibody have been cloned, a chimeric anti-HM 1.24 antibody can be obtained by ligating these mouse V regions with a DNA encoding a human antibody constant region and then expressing the same.

The basic method for constructing chimeric antibody includes connecting the mouse leader sequence and V region sequence in the cloned cDNA to the human anti-C region encoding sequence in the mammal cell expression vector.

The human antibody C region may be any of the H chain C region and any of the L chain C region. For example, C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4 of the human H chain, or C.lambda.or C.kappa.of the L chain.

Two tables were constructed to generate chimeric antibodies in the vector: these are an expression vector comprising DNA encoding mouse L chain V region and human L chain C region under the control of an expression regulatory region such as an enhancer/promoter system, and an expression vector comprising DNA encoding mouse H chain V region and human H chain C region under the control of an expression regulatory region such as an enhancer/promoter system. Then, host cells such as mammalian cells are co-transformed with these expression vectors, and the transformed cells are cultured in vitro or in vivo to produce chimeric antibodies (e.g., WO 91-16928).

Alternatively, DNAs encoding the mouse leader sequence and mouse L chain V region and human L chain C region in the cloned cDNA and DNAs encoding the mouse leader sequence and mouse H chain V region and human H chain C region are introduced into a single expression vector (see International application publication No. WO94-11523), and a host cell is transformed with the vector. The transformed host cells are then cultured in vitro or in vivo to produce the chimeric antibody of interest.

1) Construction of chimeric H chain

Expression vectors suitable for chimeric antibody H chains can be obtained by introducing cDNA encoding mouse H chain V regions into appropriate expression vectors containing genomic CDN or cDNA encoding human antibody H chain C regions. H chain C regions mentioned are, for example, Cr1, Cr2, Cr2 or Cr 4.

Construction of expression vector for chimeric H chain containing Cr genomic DNA

An expression vector containing a genomic DNA in which Cr1 is an H chain C region, such as HFE-PMh-g.gamma.1 (International publication No. WO92/19759) or DHFR-. DELTA.E-RVh-PM 1f (International publication No. WO92/19759), may be used.

An appropriate base sequence can be introduced by a PCR method so as to insert cDNA encoding mouse H chain V region into these expression vectors. These appropriate base sequences can be introduced by PCR methods using PCR primers, one designed to have an appropriate restriction enzyme recognition sequence at its 5 'end and a Kozak conserved sequence immediately before the start codon, and the other designed to have an appropriate restriction enzyme recognition site and splice donor site at its 3' end where the primary transcript of genomic DNA is appropriately spliced to mRNA.

The cDNA encoding the mouse H chain V region thus constructed is treated with an appropriate restriction enzyme and inserted into the above expression vector to construct a chimeric H chain expression vector containing C.gamma.1 DNA.

construction of expression vector for cDNA chimeric H chain

An expression vector containing cDNA whose C.gamma.1 is an H chain C region can be constructed by first preparing mRNA from CHO cells into which a genomic DNA expression vector DHFR-. DELTA.E-RVh-PM 1f (International application publication No. WO92/19759) encoding the H chain V region of a humanized PM1 antibody and the H chain C region of a human antibody Cr1(N.Takahashi et al, cell 29, 671-679, 1982) and an expression vector RV1-PM1a (International application publication No. WO92/19759) encoding the L chain V region of a humanized PM1 antibody and the K chain C region of a human antibody L chain have been integrated; the cDNA containing the humanized PM1 antibody H chain V region and human antibody H chain C region Cr1 was cloned by RT-PCR method and ligated to an appropriate expression vector of animal cells by an appropriate restriction endonuclease site.

An appropriate base sequence was introduced by PCR so as to directly link the cDNA encoding the mouse H chain V region with Cr1 encoding the human anti-H chain C region. For example, these appropriate base sequences can be introduced by PCR method using PCR primers, one designed to have an appropriate restriction enzyme recognition sequence at its 5 'end and Kozak conserved sequence immediately before the start codon, and the other designed to have an appropriate restriction enzyme recognition sequence for the direct ligation of H chain C region Cr1 at its 3' end.

An expression vector containing a cDNA chimeric H chain can be constructed by treating the cDNA encoding the mouse H chain V region thus constructed with an appropriate restriction enzyme, ligating it to the above cDNA containing the H chain C region C.gamma.1, and inserting it into an expression vector such as pCOS1 or pCHO 1.

2) Construction of L chain of chimeric antibody

An expression vector for a chimeric antibody L chain can be obtained by ligating cDNA encoding a mouse L chain V region with genomic DNA or cDNA encoding a human antibody L chain C region, and then introducing the same into an appropriate expression vector. The L chain C region is, for example, a kappa chain or a lambda chain.

Construction of expression vector for chimeric L chain kappa chain cDNA

An appropriate base sequence can be introduced by a PCR method to construct an expression vector containing cDNA encoding mouse L chain V region. For example, these appropriate base sequences can be introduced by PCR methods using PCR primers, one designed to have a recognition sequence for an appropriate restriction enzyme and a Kozak consensus sequence at its 5 'end and the other designed to have a recognition sequence for an appropriate restriction enzyme at its 3' end.

A human L chain C region K chain linked to a mouse L chain V region can be constructed, for example, from HEF-PM1K-gk containing genomic DNA (see International application publication No. WO 92/19759). A method for constructing an expression vector for L chain kappa chain of cDNA chimeric antibody comprises incorporating a restriction enzyme recognition sequence into the 5 '-end or 3' -end of DNA encoding K chain of L chain C region by PCR method, ligating the mouse L chain V region thus constructed and L chain C region kappa chain, and inserting it into an expression vector such as pCOS1 or pCHO 1.

2. Construction of reconstructed human antibodies

(1) Design of the V region of the reconstructed human anti-HM 1.24 antibody

It is necessary to have high homology between the FR of the mouse monoclonal antibody and the FR of the human antibody in order to construct a reconstructed human antibody in which the CDRs of the mouse monoclonal antibody have been grafted onto a human antibody. Thus, the V regions of the L chain and H chain of mouse anti-HM 1.24 antibody were compared with all known structurally clear antibody V regions using a protein database.

The L chain V region of mouse anti-HM 1.24 antibody is most similar to the subgroup IV of the L chain V region of human antibody (HSGIV), with a homology of 66.4%. On the other hand, it has homology of 56.9%, 55.8% and 61.5% with HSGI, HSGII and HSGIII, respectively.

The L chain V region of the mouse anti-HM 1.24 antibody has 67.0% homology with the human antibody REIL chain V region in the human antibody L chain V region subgroup I, as compared with the known human antibody L chain V region. Thus, the FR of REI was used as a starting material for constructing the L chain V region of the reconstructed human anti-HM 1.24 antibody.

The type a of L chain V region of the reconstructed human anti-HM 1.24 antibody was designed. In this type, the FR of the human antibody and the REI-based FR of the reconstructed human CMPATH-1H antibody are made identical (see Riekchmann, L. et al, Nature, 322, 21-25, (1988), the L chain V region model a of the reconstructed human PM-1 antibody described in International application publication No. WO92-19759 contains FR), and the CDR in the mouse CDR and the CDR in the L chain V region of the mouse anti-HM 1.24 antibody are made identical.

The H chain V region of mouse anti-HM 1.24 antibody is most similar to the consensus sequence of the H chain V region of human antibody (HSGI), with a homology of 54.7%. On the other hand, it has 34.6% and 48.1% homology with HSGII and HSGIII, respectively. When the H chain V region of the mouse anti-HM 1.24 antibody is compared with the H chain V region of a known human antibody, FR1 to FR3 are most similar to the chain V region of the human antibody HG3H in human H chain V region subgroup I (Recavi, G. et al, Proc. Natl. Acad. Sci. USA, 80, 855) with a homology of 67.3%.

Therefore, the FR of the human antibody HG3 is used as a starting material for constructing the H chain V region of the reconstructed human anti-HM 1.24 antibody.

However, since the amino acid sequence of FR4 of human antibody HG3 is not disclosed, the amino acid sequence of FR4 of human antibody JH6 (Ravetch, J.V, etc., cell, 27, 583-Bu 591) having the highest homology with the FR4 of mouse anti-HM 1.24 antibody is used as FR 4. FR4 of JR6 has the same amino acid sequence as FR4 of the H chain of mouse anti-HM 1.24 antibody, except that it differs by only one amino acid.

In the first model a of the reconstructed H chain V region of human anti-HM 1.24 antibody, VFRs 1 to FR3 were made identical to FR1 to FR3 of human antibody HG3, except that the 30 th positions of human FR1 and the 71 th positions of human FR3 were identical to the amino acids of mouse anti-HM 1.24 antibody, and the CDRs were identical to those of the H chain V region of mouse anti-HM 1.24 antibody.

(2) Construction of L chain V region of reconstructed human anti-HM 1.24 antibody

The L chain of the reconstructed human anti-HM 1.24 antibody was constructed by the PCR method for CDR-grafting. This method is illustrated in fig. 4. The reconstructed human anti-HM 1.24 antibody (type a) containing the FRs from the human antibody RE1 was constructed using 8 PCR primers. The outer primers A (SEQ ID No: 47) and H (SEQ ID No: 48) were designed to hybridize to the DNA sequence of the HEF vector HEF-VL-gk.

The CDR-grafting primers L1S (SEQ ID No: 49), L2S (SEQ ID No: 50) and L3S (SEQ ID No: 51) have sense DNA sequences. The CDR-grafting primers L1A (SEQ ID No: 52), L2A (SEQ ID No: 53) and L3A (SEQ ID No: 54) have antisense DNA sequences, each having a complementary DNA sequence (20 to 23bp) to the 5' -terminal DNA sequence of primers L1S, L2S and L3S, respectively.

Four reactions A-L1A, L1S-L2A, L2SA-L3A, and L3S-H were performed in the first stage of the PCR reaction and each PCR product was purified. The four PCR products of the first PCR can be assembled with each other according to their respective complementarity (see WO 92-19759). Then, the full-length DNA encoding the L chain V region of the reconstructed human anti-HM 1.24 antibody is amplified by adding the outer primers A and H (second PCR). In the above PCR, the plasmid HEF-RVL-M21a (see International application publication No. WO95-14041) encoding a human ONS-M21 antibody L chain V region model a reconstructed on the basis of FR of human antibody REI source was used as a template.

In the first stage of PCR, a template DNA and each primer are used.

The PCR products A-L1A (215bp), L1S-L2A (98bp), L2S-L3A (140bp) and L3S-H (151bp) were purified on a 1.5% low melting agarose gel and assembled in a second PCR. In the second PCR, the first PCR product and each of the outer primers (A and H) were used.

The 516bp DNA fragment from the second PCR was purified on a 1.5% low melting agarose gel, digested with BamHI and HindIII, and the resulting DNA fragment was cloned into the HEF expression vector HEF-VL-gk. The plasmid containing the DNA fragment containing the correct amino acid sequence of the L chain V region of the reconstructed human anti-HM 1.24 antibody was designated as the plasmid HEF-RVLa-AHM-gk after its DNA sequence was determined. The amino acid sequence and the base sequence of the L chain V region contained in the plasmid HEF-RVLa-AHM-gk are shown in SEQ ID No: 9.

the reconstructed type b of the L chain V region of human anti-HM 1.24 antibody can be constructed by PCR for mutagenesis. Mutagen primers FTY-1(SEQ ID No: 55) and FTY-2(SEQ ID No: 56) were designed to mutate phenylalanine at position 71 to tyrosine.

After amplifying the above primers using the plasmid HEF-RVLa-AHM-gk as template, the final product was purified. The DNA fragment obtained by digestion with BamHI and HindIII was cloned into the HEF expression vector HEF-VL-gk to obtain the plasmid HEF-RVLb-AHM-gk. The amino acid sequence and the base sequence of the L chain V region contained in the plasmid HEF-RVLb-AHM-gk are shown in SEQ ID NO: 10.

(3) construction of the reconstructed H chain V region of human anti-HM 1.24 antibody

3-1 reconstruction of the a to e forms of the H chain V region of human anti-HM 1.24 antibody

The DNA encoding the H chain V region of the reconstructed human anti-HM 1.24 antibody was designed as follows. By linking the DNA sequences encoding FRs1 to 3 of the human antibody HG3 and FR4 of the human antibody JH6 with the DNA sequence encoding the CDRs of the H chain V region of the mouse anti-HM 1.24 antibody, the full-length DNA encoding the H chain V region of the reconstituted human anti-HM 1.24 antibody can be obtained.

Then, HindIII recognition site/KOZAK consensus sequence and BamHI recognition site/splice donor sequence were added to the 5 'end and 3' end of this DNA sequence, respectively, to allow insertion into the HEF expression vector.

The DNA sequence thus designed is divided into four oligonucleotides, which are then analyzed in silico for the secondary structure of oligonucleotides that could potentially prevent the assembly of these oligonucleotides.

SEQ ID No: 57 to 60 lists four oligonucleotide sequences of RVH1 to RVH 4. These oligonucleotides are 119 to 144 bases in length and have an overlap of 25 to 26 bp. Of these oligonucleotides, RVH2(SEQ ID NO: 58) and RVH4(SEQ ID NO: 60) contain sense DNA sequences and RVH1(SEQ ID NO: 57) and RVH3(SEQ ID NO: 59) contain antisense DNA sequences. The method of assembling these four oligonucleotides by PCR is shown in FIG. 5.

PCR was performed using four oligonucleotides and RHP1(SEQ ID No: 60) and RHP2(SEQ ID No: 62) as the outer primers.

The amplified 438bpDNA fragment was purified, digested with HindIII and BamHI and cloned into the HEF expression vector HEF-VH-g.gamma.1. The plasmid containing the DNA fragment encoding the correct amino acid sequence of the H chain V region was named HEF-RVH α -AHM-g γ 1 after its base sequence was determined. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHa-AHM-g gamma 1 are shown in SEQ ID No: shown at 11.

Types b, c, d and e of the H chain V region of the reconstructed human anti-HM 1.24 antibody were constructed as follows. In constructing forms b to e of the H chain V region of each of the reconstituted human anti-HM 1.24 antibodies, a three-dimensional structural model of the V region of mouse anti-HM 1.24 antibody is constructed in order to predict alternative amino acid positions in the antibody molecule.

The plasmid HEF-RVHb-AHM-g gamma 1 is obtained by amplifying b type by a PCR method by using mutagen primers BS (sequence 63) and BA (SEQ ID No: 64) and the plasmid HEF-RVHa-AHM-g gamma 1 which are designed to mutate arginine at the 66 th position into lysine as templates. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHb-AHM-g gamma 1 are shown in SEQ ID NO: shown at 13.

The model d was amplified by PCR using the mutagen primers DS (SEQ ID No: 68) and DA (SEQ ID No: 68) designed to mutate arginine at position 66 to lysine and threonine at position 73 to lysine and using the plasmid HEF-RVHa-AHM-g γ 1 as template DNA to obtain the plasmid HEF-RVHd-AHM-g γ 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHd-AHM-g gamma 1 are shown in SEQ ID No: as shown at 14.

The plasmid HEF-RVHa-AHM-g gamma 1 is obtained by amplifying the model e by using mutagen primers ES (sequence 69) and EA (SEQ ID No: 70) which are designed to mutate alanine mutated at the valine position 67 and methionine to leucine at the methionine position 69 and taking the plasmid HEF-RVHa-AHM-g gamma 1 as template DNA. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHe-AHM-g gamma 1 are shown in SEQ ID No: shown at 15.

Construction of 3-2H chain hybrid V region

By constructing H chain hybrid V regions, it is possible to investigate which of the humanized antibody V region FRs is associated with the binding activity and the binding inhibition activity. Of the two V regions constructed, the amino acid sequences of FR1 and FR2 of one V region were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from model a of the H chain V region of reconstituted human anti-HM 1.24 antibody (mouse human hybrid anti-HM 1.24 antibody), and the amino acid sequences of the other FR1 and FR2 were derived from model a of the H chain V region of reconstituted human anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from mouse anti-HM 1.24 antibody (human mouse hybrid anti-HM 1.24 antibody). The amino acid sequences of the CDR regions were derived from mouse anti-HM 1.24 antibody.

Two H-chain hybrid V regions can be constructed by PCR methods. This method is illustrated in fig. 6 and 7. Four primers were used to construct two H-strand hybrid V regions. The outer primers a (SEQ ID No: 71) and h (SEQ ID No: 72) were designed to hybridize to the DNA sequence of the HEF expression vector HEF-VH-g γ 1. Construction primer HYS for H-strand hybridization (SEQ ID No: 73) was designed to contain a sense DNA sequence and H-strand hybridization primer HYA (SEQ ID No: 74) was designed to contain an antisense DNA sequence so that these DNA sequences were complementary to each other.

In the first stage of PCR, PCR was carried out using the plasmid HEF-1.24H-g γ 1 as a template, the outer primer a and the H chain hybrid primer HYA, and PCR using the plasmid HEF-RVHa-AHM-g γ 1 as a template, the H chain hybrid primer HYA and the outer primer H to construct H chain hybrid V region, in which the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from the reconstructed human anti-HM 1.24 antibody H chain V region model a, and each PCR product was purified.

The two PCR products of the first PCR can be assembled on the basis of their own complementarity (see International application publication No. WO 92-19759). The full length DNA encoding the H chain hybrid V region, in which the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from the reconstructed human anti-HM 1.24 antibody H chain V region type a, was then amplified in a second PCR stage by the addition of outer primers a and H.

In the first stage of PCR, PCR using the plasmid HEF-RVHa-AHM-g gamma 1 as a template and the outer primer a and the H chain hybrid primer H gamma A and PCR using the plasmid HEF-1.4H-g gamma 1 as a template and the H chain hybrid primer H gamma S and the outer primer H were carried out to construct an H chain hybrid V region in which the amino acid sequences of FR1 and FR2 were derived from type a of the reconstructed H chain V region of human anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from mouse anti-HM 1.24 antibody and to purify each PCR product.

The two PCR products of the first PCR are assembled on the basis of their own complementarity (see International application publication No. WO 92-19759). The full length DNA encoding the H chain hybrid V region, in which the amino acid sequences of FR1 and FR2 were derived from type a of the reconstructed H chain V region of human anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from mouse anti-HM 1.24 antibody, was then amplified in a second PCR stage by the addition of outer primers a and H.

First PCR, purification of PCR products, assembly, second PCR and cloning into HEF expression vector HEF-VH-g.gamma.1 were carried out according to the procedure described in "construction of L chain V region of reconstituted human anti-HM 1.24 antibody" in example 9. The plasmid containing the DNA fragment encoding the correct amino acid sequence of the H chain hybrid V region was designated HEF-MH-RVH-AHM-g.gamma.1 after determination of the DNA sequence, in which the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from type a of the reconstructed human anti-HM 1.24 antibody H chain V region.

The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-MH-RVH-AHM-g gamma 1 are shown in SEQ ID No: shown at 75. The DNA fragment plasmid containing the correct amino acid sequence encoding the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 were derived from the reconstructed human anti-HM 1.24 antibody type a and the amino acid sequences of FR3 and FR4 were derived from the mouse anti-HM 1.24 antibody H chain V region may also be designated as HEF-HM-RVH-AHM-g.gamma.1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-HM-RVH-AHM-g gamma 1 are shown in SEQ ID No: as shown at 76.

3-3 construction of reconstructed human anti-HM 1.24 antibody H chain V region f to s type

Each type f, g, H, i, j, k, l, m, n, o, p, q, r, and s of the H chain V region of the reconstructed human anti-HM 1.24 antibody was constructed as follows. In constructing the f to s forms of the H chain V region of the reconstructed human anti-HM 1.24 antibody, a three-dimensional structure model of the V region of the mouse anti-HM 1.24 antibody should be constructed as described above in order to predict the positions of the substituted amino acid residues in the antibody molecule.

The plasmid HEF-RVHf-AHM-g gamma 1 was obtained by amplifying the f-type by PCR method using mutagen primers FS (SEQ ID No: 79) and FA (SEQ ID No: 78) designed to mutate threonine at position 75 to serine and valine at position 78 to alanine and using the plasmid HEF-RVHe-AHM-g gamma 1 as template DNA. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHf-AHM-g gamma 1 are shown in SEQ ID No: shown at 16.

The g-form was amplified with the plasmid HEF-RVHa-AHM-g.gamma.1 master template DNA using mutagen primers GS (SEQ ID No: 80) and GA (SEQ ID No: 81) designed to mutate alanine at position 40 to arginine to give the plasmid HEF-RVHg-AHM-g.gamma.1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHg-AHM-g gamma 1 are shown as SEQ ID No: shown at 17.

And amplifying the h type by using mutagen primers FS and FA and taking the plasmid HEF-RVHb-AHM-g gamma 1 as a template DNA to obtain the plasmid HEF-RVHh-AHM-g gamma 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHh-AHM-g gamma 1 are shown in SEQ ID No: 18, respectively.

The type I was amplified using mutagen primers IS (SEQ ID No: 83) and IA (SEQ ID No: 83) designed to mutate arginine at position 83 to alanine and serine at position 84 to phenylalanine and using plasmid HEF-RVHh-AHM-g γ 1 as template DNA to obtain plasmid HEF-RVHi-AHM-g γ 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHi-AHM-g gamma 1 are shown in SEQ ID No: 19, respectively.

Mutagen primers JS (SEQ ID No: 84) and JA (SEQ ID No: 85) for mutating arginine at position 66 into lysine are used, plasmid HEF-RVHf-AHM-g gamma 1 is used as template DNA, and j type is amplified to obtain plasmid HEF-RVHj-AHM-g gamma 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHj-AHM-g gamma 1 are shown in SEQ ID No: shown at 20.

The plasmid HEF-RVHk-AHM-g gamma 1 is obtained by amplifying the k type by using mutagen primers KS (SEQ ID NO: 86) and KA (SEQ ID NO: 87) which are designed to mutate the 81 th glutamic acid into glutamine and using the plasmid HEF-RVHh-AHM-g gamma 1 as a template DNA. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHk-AHM-g gamma 1 are shown in SEQ ID No: shown at 21.

Mutagen primers LS (SEQ ID No: 88) and LA (SEQ ID No: 89) designed to mutate glutamic acid at position 81 into glutamic acid and serine at position 82B into isoleucine are used, plasmid HEF-RVHh-AHM-g gamma 1 is used as template DNA, and type 1 is amplified to obtain plasmid HEF-RVH1-AHM-g gamma 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVH1-AHM-g gamma 1 are shown in SEQ ID No: 22, respectively.

Mutagen primers MS (SEQ ID No: 90) and MA (SEQ ID No: 91) designed to mutate 81 th glutamic acid into glutamic acid, 82b th serine into isoleucine and 87 th threonine into transhistidine are used, and plasmid HEF-RVHh-AHM-g gamma 1 is used as template DNA to amplify m type to obtain plasmid HEF-RVHm-AHM-g gamma 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHm-AHM-g gamma 1 are shown in SEQ ID No: shown at 23.

The plasmid HEF-RVHh-AHM-g gamma 1 is obtained by amplifying n type by using mutagen primers NS (SEQ ID No: 92) and NA (SEQ ID No: 93) which are designed to mutate serine at position 82B into isoleucine and taking the plasmid HEF-RVHh-AHM-g gamma 1 as template DNA. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHn-AHM-g gamma 1 are shown in SEQ ID No: as shown at 24.

The O type was amplified using mutagen primers OS (SEQ ID NO: 94) and OA (SEQ ID NO: 95) designed to mutate threonine 87 to serine and plasmid HEF-RVHh-AHM-g γ 1 as template DNA to obtain plasmid HEF-RVHo-AHM-g γ 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHo-AHM-g gamma 1 are shown in SEQ ID No: shown at 25.

The P type was amplified by PCR using mutagen primers PS (SEQ ID NO: 96) and PA (SEQ ID NO: 97) designed to mutate the 78 th valine to alanine and plasmid HEF-RVHa-AHM-g γ 1 as template DNA to obtain plasmid HEF-RVHp-AHM-g γ 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHp-AHM-g gamma 1 are shown in SEQ ID No: shown at 26.

A Q type was amplified by PCR method using mutagen primers QS (SEQ ID NO: 98) and QA (SEQ ID NO: 99) designed to mutate threonine 75 to serine and using plasmid HEF-RVHa-AHM-g γ 1 as template DNA to obtain plasmid HEF-RVHq-AHM-g γ 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHq-AHM-g gamma 1 are shown in SEQ ID No: as shown at 27.

The plasmid HEF-RVHp-AHM-g gamma 1 is obtained by amplifying the r type by a PCR method by using mutagen primers CS (SEQ ID No: 65) and CA (SEQ ID No: 66) and taking the plasmid HEF-RVHp-AHM-g gamma 1 as template DNA. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-RVHr-AHM-g gamma 1 are shown in SEQ ID No: shown at 28.

The S type was amplified using mutagen primers SS (SEQ ID NO: 100) and SA (SEQ ID NO: 101) designed to mutate methionine 69 to isoleucine and plasmid HEF-RVHr-AHM-g γ 1 as template DNA to obtain plasmid HEF-RVHs-AHM-g γ 1. The amino acid sequence and the base sequence of the H chain V region contained in the plasmid HEF-HVHs-AHM-gGamma 1 are shown in SEQ ID NO: 102, respectively.

Table 1 shows the amino acid sequences of the constructed L chain V regions, and tables 2 to 4 show the amino acid sequences of the H chain V regions.

TABLE 1 amino acid sequence of L chain V region

FR1 CDR1 FR2

1 2 3 4

12345678901234567890123 45678901234 567890123456789

AHM DIVMTQSHKFMSTSVGDRVSITC KASQDVNTAVA WYQQKPGQSPKLLIY

HuSG I DIQMTQSPSSLSASVGDRVTITC WYQQKPGKAPKLLIY

REI DIQMTQSPSSLSASVGDRVTITC WYQQKPGKAPKLLIY

RVLa ----------------------- ----------- ---------------

RVLb ----------------------- ----------- ---------------

CDR2 FR3

5 6 7 8

0123456 78901234567890123456789012345678

AHM SASNRYT GVPDRITGSGSGTDFTFTISSVQAEDLALYYC

HuSG I GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

REI GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC

RVLa ------- --------------------------------

RVLb ------- --------------Y-----------------

CDR3 FR4

9 10

901234567 8901234567

AHM QQHYSTPFT FGSGTKLEIK

HuSG I FGQGTKVEIK

REI FGQGTKVEIK

RVLa --------- ----------

RVLb --------- ----------

TABLE 2 amino acid sequence of H chain V region (1)

FR1 CDR1 FR2

1 2 3 4

123456789012345678901234567890 12345 67890123456789

AHM QVQLQQSGAELARPGASVKLSCKASGYTFT PYWMQ WVKQRPGQGLEWIG

HuSGI EVQLVQSGADVKKPGXSVXVSCKASGYTFS WVRQAPGXGLDWVG

HG3 QVQLVQSGAEVKKPGASVKVSCKASGYTFN WVRQAPGQGLEWMG

RVHa -----------------------------T ----- --------------

RVHb -----------------------------T ----- --------------

RVHc -----------------------------T ----- --------------

RVHd -----------------------------T ----- --------------

RVHe -----------------------------T ----- --------------

RVHf -----------------------------T ----- --------------

RVHg -----------------------------T ----- ----R---------

RVHh -----------------------------T ----- --------------

RVHi -----------------------------T ----- --------------

RVHj -----------------------------T ----- --------------

RVHk -----------------------------T ----- --------------

RVHl -----------------------------T ----- --------------

RVHm -----------------------------T ----- --------------

RVHn -----------------------------T ----- --------------

RVHo -----------------------------T ----- --------------

RVHp -----------------------------T ----- --------------

RVHq -----------------------------T ----- --------------

RVHr -----------------------------T ----- --------------

RVHs -----------------------------T ----- --------------

TABLE 3H chain V region amino acid sequence (2)

CDR2 FR3

5 6 7 8 9

012A3456789012345 67890123456789012A8C345678901234

AHM SIFPCDGDTRYSQKFKG KATLTADKSSSTAYMQLSILAFEDSAVYYCAR

HuSG I RVTXTXDXSXNTAYMELSSLRSEDTAVYYCAR

HG3 RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR

RVHa ----------------- -----A--------------------------

RVHb ----------------- K----A--------------------------

RVHc ----------------- -----A-K------------------------

RVHd ----------------- K----A-K------------------------

RVHe ----------------- -A-L-A--------------------------

RVHf ----------------- -A-L-A---S--A-------------------

RVHg ----------------- -----A--------------------------

RVHh ----------------- K----A---S--A-------------------

RVHi ----------------- K----A---S--A-------AF----------

RVHj ----------------- KA-L-A---S--A-------------------

RVHk ----------------- K----A---S--A--Q----------------

RVHl ----------------- K----A---S--A--Q--I-------------

RVHm ----------------- K----A---S--A--Q--I-----S-------

RVHn ----------------- K----A---S--A-----I-------------

RVHo ----------------- K----A---S--A-----------S-------

RVHp ----------------- -----A------A-------------------

RVHq ----------------- -----A---S----------------------

RVHr ----------------- -----A-K----A-------------------

RVHs ----------------- ---I-A-K----A-------------------

TABLE 4 amino acid sequence of H chain V region (3)

CDR3 FR4

10 11

57890ABJK12 34567890123

AHM GLRRGGYYFDY WGQGTTLTVSS

HuSG I WGQGTLVTVSS

JH6 WGQGTTVTVSS

RVHa ----------- ------------

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

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

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

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

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

RVHg ----------- ------------

RVHh ----------- ------------

RVHi ----------- ------------

RVHj ----------- ------------

RVHk ----------- ------------

RVHl ----------- ------------

RVHm ----------- ------------

RVHn ----------- ------------

RVHo ----------- ------------

RVHp ----------- ------------

RVHq ----------- ------------

RVHr ----------- ------------

RVHs ----------- ------------

3. Synthesis of chimeric and reconstructed human antibodies

To synthesize a chimeric antibody or to reconstruct a human antibody, two expression vectors are constructed for each antibody, one comprising DNAs encoding a mouse H chain V region and a human H chain C region under the control of an expression regulatory region such as an enhancer/promoter system and DNAs encoding a mouse L chain V region and a human L chain C region under the control of an expression regulatory region such as an enhancer/promoter system, or one comprising DNAs encoding a humanized H chain V region and a human H chain C region under the control of an expression regulatory region such as an enhancer/promoter system and DNAs encoding a humanized L chain V region and a human L chain C region under the control of an expression regulatory region such as an enhancer/promoter system.

Subsequently, host cells such as mammalian cells are co-transformed with these vectors, and the transformed cells are cultured in vitro or in vivo to produce chimeric antibodies or reconstructed human antibodies (for example, international application publication No. WO 91-16928). In addition, antibody genes can be introduced into mammals such as goats to produce transgenic animals from which chimeric or reconstructed human antibodies can be obtained from their milk.

The H chain V region and H chain C region, L chain V region and L chain C region may also be linked to a single vector to transform an appropriate host cell and then produce an antibody. DNAs encoding a mouse leader sequence and H chain V region and human H chain C region in the cloned cDNA, and DNAs encoding a mouse leader sequence and L chain V region and human L chain C region are introduced into a single expression vector to express the chimeric antibody (see International application publication No. WO 94-11523).

The DNA encoding the humanized H chain V region and the human H chain C region, and the DNA encoding the humanized L chain V region and the human L chain C region are introduced into a single expression vector to express the reconstructed human antibody (see International application publication No. WO 94-11523). Host cells are transformed with the vectors and these transformed host cells are cultured in vivo or in vitro to produce the chimeric or reconstructed human antibodies of interest.

A transformant transformed by the above-described method with a gene encoding a desired chimeric antibody or a reconstructed human antibody can be cultured, and the produced chimeric antibody or reconstructed human antibody can be isolated from the inside or outside of the cell and purified into a homogeneous antibody.

The target protein, chimeric antibody or reconstructed human antibody of the present invention is isolated and purified by using an affinity column. This column uses protein A, such as HyperD, POROS, Sepharose F, etc. Alternatively, conventional methods for separating and purifying proteins may be used and this method is not limited in any way. For example, chimeric antibodies or reconstructed human antibodies can also be isolated and purified by various chromatographic binding methods, ultrafiltration, salting out, dialysis, and the like, as appropriate.

Any expression method may be used to produce the chimeric anti-HM 1.24 antibody or the reconstituted human anti-HM 1.24 antibody of the present invention, including, for example, eukaryotic cells such as animal cells, established mammalian cell line systems, insect cell systems, fungal cell systems, and yeast cell systems, and prokaryotic cells such as bacterial cells of E.coli. The chimeric antibody or the reconstructed human antibody of the present invention is preferably expressed in COS cells, CHO cells, HeLa cells, Vero cells, myeloma cells or BHK cells.

In this case, a common promoter suitable for expression in mammalian cells can be used. For example, the human cytomegalovirus early (HCMV) promoter is preferably used. Examples of such expression vectors containing HCMV promoter include HCMV-VH-HC γ 1, HCMV-VL-HC κ and the like and vectors derived from pSV2neo (International application publication No. WO 92-19759).

In addition, as a promoter for gene expression of the mammalian cell in the present invention, a viral promoter such as retrovirus, polyoma virus, adenovirus, simian virus 40(SV40), etc., and a promoter derived from a mammalian cell such as human polypeptide chain elongation factor 1d (HEF-12) can be used. For example, when SV40 promoter is used, expression can be easily carried out by the method of Mullingan et al (Nature 277, 108 (1979)), and when HEF-12 promoter is used, the method of Mizushima, S. et al (nucleic acid research, 18, 5322, 1990) is used.

Promoters derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), etc. can be used as a replication source, and expression vectors include Aminoglycoside Phosphotransferase (APH) gene, Thymidine Kinase (TK) gene, E.coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (DHRF) gene, etc. as a selection marker to amplify gene copy number in a host cell system.

4. Binding inhibition Activity of chimeric antibody or reconstructed human antibody

(1) Determination of antibody concentration

The concentration of purified antibody is determined by ELISA or absorbance measurement methods.

ELISA plates for determining antibody concentration were prepared as follows. Each well of a 96-well ELISA plate (e.g., Maxisorp, manufactured by NUNC) was immobilized with 100 μ g of goat anti-human IgG antibody at a concentration of 1. mu.g per ml.

With 100. mu.g per ml of dilution buffer (e.g.50 mM Tris-HCl, mM MgCl)₂，0.15M Nacl，0.05％Tween20，0.02％Nan₃1% fetal Bovine Serum Albumin (BSA), pH8.1), serial dilutions of culture supernatants of, e.g., COS cells or CHO cells expressing chimeric, hybrid or reconstituted human antibodies, or purified chimeric, hybrid or reconstituted human antibodies are added to each well. Then, 100. mu.l of alkaline phosphatase conjugated to goat anti-human IgG antibody was added, 1mg/ml of a substrate solution (Sigma 104, p-nitrophenol phosphate, manufactured by SIGMA) was added, and its absorbance at 405nm was measured using a microplate reader (Bio Rad). Human IgClk (produced by BInding Site) can be used as a standard for concentration determination. The concentration of purified antibody was determined by measuring the absorbance of the antibody at 280nm and calculating as 1mg per ml of 1.35 OD.

(2) Binding Activity

Binding activity was determined by cell ELISA with human amniotic cell line WISH (ATCC CCL 25). Cell ELISA was prepared as follows. WISH cells at an appropriate concentration in PRMI1640 medium supplemented with 10% calf serum were added to a 96-well plate, cultured overnight, and washed twice with PBS (-), followed by fixation with 0.1% glutaraldehyde (produced by Nakalai-teque).

After blocking, 100. mu.l of a continuous buffer such as COS cells or CHO cell culture supernatant, which can express the chimeric anti-HM 1.24 antibody, hybrid anti-HM 1.24 antibody or reconstituted human anti-HM 1.24 antibody, or purified chimeric anti-HM 1.24 antibody, hybrid anti-HM 1.24 antibody or reconstituted human anti-HM 1.24 antibody was added to each well, followed by the addition of peroxidase-labeled rabbit anti-HM 1.24 antibody (produced by DAKO).

After 1 hour of incubation at room temperature, the substrate solution was added and incubated again. The reaction was then stopped with 50. mu.l of 6N sulfuric acid, and its absorbance at 490nm was measured with a microplate reader model 3550 (manufactured by Bio-Rad).

(3) Determination of binding inhibition Activity

The binding inhibition activity of biotinylated murine anti-HM 1.24 antibody was determined by cell ELISA using the human amniotic membrane cell line WISH (ATCC CCL 25). cell-ELISA plates were prepared according to (2) above. WISH cells at an appropriate concentration in PRMI1640 medium supplemented with 10% calf serum were added to a 96-well plate, cultured overnight, and washed twice with PBS (-) and fixed with 0.1% glutaraldehyde (produced by Nakalai tesque).

After blocking, 50. mu.l of serial dilutions of COS cell or CHO cell culture supernatants, or purified chimeric anti-HM 1.24 antibody, hybrid anti-HM 1.24 antibody or reconstituted human anti-HM 1.24 antibody, which may express chimeric anti-HM 1.24 antibody, hybrid anti-HM 1.24 antibody or reconstituted human anti-HM 1.24 antibody, were added to each well, along with 50. mu.l of 2 mg per ml of biotinylated murine anti-HM 1.24 antibody, followed by incubation at room temperature for 2 hours and, after rinsing, peroxidase-labeled streptavidin was added.

After incubation at room temperature for 1 hour and washing, the substrate solution was added and incubated. Subsequently, the reaction was stopped with 50. mu.l of 6N sulfuric acid, and then absorbance at 490nm was measured by means of microplate reading rake transfer model 3550 manufactured by Bio-Rad).

Measurement of ADCC Activity

ADCC activity of the chimeric antibody or the reconstructed human antibody of the present invention was determined as follows. First, monocytes are separated from human peripheral blood or bone marrow by a density centrifugation method and prepared into effector cells. Human myeloma cells were prepared as target cells by labeling RPMI8226 cells (ATCC CCL155) with C γ. Then, a chimeric antibody or a reconstructed human antibody to be tested for ADCC activity is added to the labeled target cells and cultured, and then effector cells are added to the cells in an appropriate ratio and cultured.

After incubation, the supernatant was taken and the radioactivity was measured by an r counter. The maximum released radioactivity can now be determined with 1% NP-40. Cytotoxicity (%) was calculated as (A-C)/(B-C). times.100, where A is the radioactivity released in the presence of antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released by the culture fluid in the absence of antibody (cpm).

When ADCC activity or CDC activity is expected in the antibody C region, human C.gamma.1 or human C.gamma.3 can be used as the antibody C region. Furthermore, by adding a part of the amino acids in the C region of the modified or modified form, high ADCC activity or CDC activity can be induced.

For example, there are IgG-like polymerization by amino acid substitution (Smith, R.I.F. and Morrison, S.L., BIO/TECHNOLOGY (1994)12, 683-688), IgM-like polymerization by amino acid additive (Smith, R.I.F. et al, J. Immunol (1995)154, 2226-2236), sequential linked expression of genes encoding the L chain (Shuford, W. et al, science (1991)252, 724-727), IgG dimerization by amino acid substitution (Caron, P.C. et al, J. Immunol. 1992)176, 1191-1195, shop, B.J. immunology (1992)148, 2918-2922), IgG dimerization by chemical modification (Wolff, E.A. et al, cancer research (1993)53, 2560, 2565), and antibody hinge region induction effect by amino acid substitution (Nordher. 2379, European Ed. 19921, 2379). These changes can be accomplished by oligo site directed mutagenesis with primers, addition of base sequences with restriction endonuclease cleavage sites, and induction of chemical modifications of covalent bonds.

In vivo diagnosis of myeloma

The chimeric anti-HM 1.24 antibody or the reconstituted human anti-HM 1.24 antibody of the present invention can be used for in vivo diagnosis of myeloma by linking with a labeling compound such as a radioisotope.

Furthermore, fragments of chimeric anti-HM 1.24 antibody or reconstituted human anti-HM 1.24 antibody linked to a labelled compound such as a radioisotope may be used for in vivo diagnosis of myeloma, for example Fab, F (ab') 2, Fv, or single chain Fv (scFv), in which Fv or Fv of H and L chains are linked by a suitable linker.

These antibody fragments can be specifically obtained by constructing genes encoding these antibody fragments, introducing them into an expression vector, and then expressing them in an appropriate host cell, or digesting the chimeric anti-HM 1.24 antibody with an appropriate enzyme or reconstructing the human anti-HM 1.24 antibody.

The above-mentioned in vivo diagnostic method for myeloma can be administered systemically in a parenteral manner.

Pharmaceutical composition and therapeutic agent for myeloma

The antibody was administered to an animal transplanted with myeloma cells and the anti-tumor effect was evaluated to confirm the therapeutic effect of the chimeric anti-HM 1.24 antibody or humanized anti-HM 1.24 antibody of the present invention.

Preferred myeloma cells to be transplanted into an animal are, for example, KPMM2 (Japanese unexamined patent publication (Kokai) No. 7-236474), RPMI8226(ATCC CCL155), ARH-77(ATCC CRL1621) and S6B45(Suzuki, H., et al, European journal of immunology (1992)22, 1989-1993). As the animal to which the cell is transplanted, an animal having reduced or deleted immune function is preferable, and examples thereof include a nude mouse, a SCID mouse, a brown mouse and a nude rat.

Furthermore, the evaluated antitumor effect was confirmed by the change in the amount of human immunoglobulin in serum, measurement of tumor volume and/or weight, change in the weight of human Benth-Jones protein in urine, animal survival and the like.

The myeloma pharmaceutical composition or therapeutic agent comprising the chimeric anti-HM 1.24 antibody or the reconstituted human anti-HM 1.24 antibody of the present invention as an active ingredient may be systemically or topically administered parenterally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection or subcutaneous injection can be selected and an appropriate dosage regimen is selected according to the age of the patient and medical conditions.

An effective dosage range is from 0.01 mg to 1000 mg per kg body weight per dose. Alternatively the dose is 5mg per kg body weight, preferably 50mg to 100 mg per kg body weight.

The myeloma pharmaceutical composition or therapeutic agent comprising as an active ingredient the reconstituted anti-HM 1.24 antibody or reconstituted human anti-HM 1.24 antibody of the present invention may contain a pharmaceutically acceptable carrier or additive, depending on the route of administration.

Examples of such carriers and additives are water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, Human Serum Albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, and the like. The additives used are selected from, but not limited to, the additives mentioned above or combinations thereof.

Examples

The present invention is described more specifically below.

Example 1 cloning of cDNA encoding the V region of mouse anti-HM 1.24 antibody

1. Isolation of messenger RNA (mRNA)

Using the Rapid mRNA isolation kit of version 3.2 (manufactured by Invitrogen corporation), mouse anti-HM 1.24 antibody was produced from 2X 10 according to the instructions attached thereto⁸mRNA was isolated from hybridoma cells.

2. The genes encoding the antibody variable regions were amplified by PCR.

PCR was performed using an amplification thermal cycler (produced by Perkin Elmer Cetus).

2-1, amplification and enzyme digestion of gene encoding mouse L chain V region

Single-stranded cDNA was synthesized from the isolated mRNA using AMV reverse transcriptase cDNA first strand synthesis kit (produced by Life Science) and used for PCR. For the primers for PCR, primers hybridizing to the mouse Kappa-type L chain leader sequence as shown in SEQ ID No: MKV (mouse Kappa variable) primers from 29 to 39 (Jones, S.T. et al, Bio/technology, 9, 88-89, (1991)).

Containing 10mM Tris-HCl (pH8.3), 50mM KCl, 0.1mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5mM MgCl₂5 units of DNA polymerase Ampli Taq (produced by Perkinelmer Cetus), 0.25mM shown in SEQ ID No: MKV primer in 29 to 39, 3mM shown in SEQ ID No: mu.l of the PCR solution of MKC primer in 40 and 100ng of single-stranded cDNA was covered with 50. mu.l of mineral oil, then heated at an initial temperature of 94 ℃ for 3 minutes, and then circulated in the order of 94 ℃ for 1 minute, 35 ℃ for 1 minute, and 72 ℃ for 1 minute. After repeating this cycle 30 times, the reaction mixture was incubated at 72 ℃ for 10 minutes. The amplified DNA fragment was purified by using a low melting point agarose gel (manufactured by Sigma), and digested with XmaI (manufactured by New England Biolabs) and SalI (manufactured by Takara Shuzo) at 37 ℃.

2-2 amplification and restriction of cDNA encoding mouse H chain V region

The gene encoding the V region of the H chain of mice was amplified using the 5' -RACE method (rapid amplification of cDNA ends; Frohman, M.A., et al, Proc. Natl. Acad. Sci. USA, 85, 8998-. After cDNA was synthesized using primer P1(SEQ ID No: 41) which specifically hybridizes to the constant region of mouse IgG2a, primer MHC2a (SEQ ID No: 42) and anchor primer (SEQ ID No: 77) which specifically hybridizes to the constant region of mouse IgG2a and which were attached to the kit were used by 5' -AmpliFINDER cDNA terminal rapid amplification kit (manufactured by CLONTECH). cDNA encoding mouse H chain V region was amplified. The amplified DNA fragment was purified with a low melting point agarose gel (manufactured by Sigma) and digested with EcoRI (manufactured by Takara Shuzo) and XmaI (manufactured by New England BioLabs) at 37 ℃.

3. Ligation and transformation

By adding 50mM Tris-HCl (pH7.6), 10mM Mgcl₂10mM dithioreitol, 1mM ATP, 50mg/ml polyethylene glycol (8000) and 1 unit of T4 DNA ligase (manufactured by Gibco-BRL) were reacted at 16 ℃ for 2.5 hours, and the DNA fragment containing the V region gene encoding mouse Kappa type L chain prepared as described above was ligated with the PUC vector digested with SalI and XmaI. Similarly, the pUC19 vector containing the DNA fragment encoding the mouse H chain V region gene and digested with EcoRI and XmaI was reacted at 16 ℃ and ligated for 3 hours.

Then 10. mu.l of the above ligation mixture was added to 50. mu.l of competent cells of E.coli DH52, followed by 30 minutes on ice, 1 minute at 42 ℃ and 1 minute on ice. Next, 400. mu.l of 2XYT medium (molecular cloning: A laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press, (1989)) was added thereto, cultured at 37 ℃ for 1 hour, and then E.coli was plated on zXYT agar medium containing 50. mu.g/ml ampicillin (molecular cloning: a laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press, (1989)) and cultured overnight at 37 ℃ to obtain E.coli transformants.

The transformant was cultured overnight at 37 ℃ in 10ml of 2XYT medium containing 50. mu.g/ml ampicillin, and then plasmid DNA was prepared from this culture using the alkaline lysis method (molecular cloning: A laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press, (1989)).

The plasmid thus obtained containing the gene encoding mouse Kappa-type L chain V region from the hybridoma producing the anti-HM 1.24 antibody was designated as pUCHHMVL 9. The plasmid obtained by the above method and containing the mouse H chain V region-encoding gene derived from the hybridoma producing the anti-HM 1.24 antibody was designated as pUCHVHR 16.

Example 2 determination of DNA base sequence

The base sequence of the cDNA coding region in the above plasmid was determined by the method described in the manufacturer's instructions using an automatic DNA sequencer (manufactured by Applied Biosystem Inc.) and a Taq dye dideoxy terminator cycle sequencing kit (manufactured by Applied Biosystem Inc.).

The base sequence of the gene encoding mouse anti-HM 1.24 antibody L chain V region contained in plasmid pUCHMLVL 9 is shown in SEQ ID No: 1. the base sequence of the gene encoding the H chain V region of mouse anti-HM 1.24 antibody contained in the plasmid pUCHVHR 16 is shown in SEQ ID No: 2.

example 3 determination of CDRs

The overall structures of the L and H chain V regions share similarities with each other, with the four framework regions being connected by three highly variable regions, the Complementarity Determining Regions (CDRs). The amino acid sequence within the framework is relatively well conserved, but the variation in amino acid sequence is extremely high (Kabat, e.a., et al, "sequences of proteins of interest for immunization", US dept.health and Human Services, 1983).

Based on these facts, the amino acid sequence of the variable region of the anti-HM 1.24 antibody was compared with the amino acid sequences of antibodies in the database to investigate homology, and CDR regions were determined as shown in Table 5.

TABLE 5

Plasmids	Serial number	CDR(1)	CDR(2)	CDR(3)
					pUCHMV19	3-5	24-34	50-56	89-97
pUCHMVHR16	6-8	31-35	50-66	99-109

Example 4 determination of expression of cloned cDNA (construction of chimeric anti-HM 1.24 antibody)

1. Construction of expression vectors

To construct an expression vector expressing the chimeric anti-HM 1.24 antibody, cDNA clones pUCHHMVL 9 and pUCHHMVHR 16 encoding the L chain and H chain V regions of mouse anti-HM 1.24 antibody, respectively, were modified by PCR method and then introduced into the HEF expression vector (International application publication No. WO 92-19759).

The reverse primer ONS-L722S (SEQ ID No: 43) corresponding to the L chain V region and the reverse primer VHR16S (SEQ ID No: 44) corresponding to the H chain V region were designed so that they could hybridize to the DNA encoding the beginning of each V region leader sequence and they had Kozak conserved sequences (Kozak, M. et al, J. mol. biol., 196, 947-950, (1987)) and recognition sites for Hind III restriction enzymes. The forward primer VL9A (SEQ ID NO: 45) corresponding to the L chain V region and the forward primer VHR16A (SEQ ID NO: 46) corresponding to the H chain V region were designed so that they could hybridize to the DNA sequence encoding the termini of the splice region and they had a splice donor sequence and a recognition site for a BamHI restriction enzyme.

A100. mu.l PCR reaction mixture containing 10mM Tris-HCl (pH8.3), 50mM KCl, 0.1mM dNTPs, 1.5mM MgCl2, 100pmol of each primer, 100ng template DNA (pUCHVL 9 or pUCHVHR 16) and 5 units Ampli Taq enzyme was covered with 50. mu.l mineral oil, heated after 94% initial denaturation for 30 cycles at 94 ℃ for 1 minute, 55 ℃ for 1 minute and 72 ℃ for 1 minute, respectively, and finally incubated at 72 ℃ for 10 minutes.

The PCR product was purified on a 1.5% low melting agarose gel and digested with HindIII and BamHI, followed by cloning into HEF-VL-g.kappa.for L chain V region and HEF-VL-g.gamma.1 for H chain V region. After the DNA sequence was determined, the plasmids containing the DNA fragment containing the correct DNA sequence were designated HEF-1.24L-g.kappa.and HEF-1.24H-g.gamma.1, respectively.

To prepare restriction fragments, the regions encoding the respective variable regions were excised from the above-mentioned plasmids HEF-1.34L-gk and HEF-1.24H-g.gamma.1 using HindIII and BamHI restriction enzymes, and the fragments were inserted into HindIII and BamHI sites of a plasmid vector pUC19, which were designated pUC 19-1.24L-g.kappa.and pUC 19-1.24H-g.gamma.1, respectively.

Coli containing plasmids pUC19-1.24L-gk and pUC-1.24H-g γ 1 were designated E.coli DH5 α (pUC19-1.24L-gk) and E.coli DH5 α (pUC19-1.24H-g γ 1), respectively, and were stored under the terms of the Budapest treaty at 29.8.1996 under international conventions at the national institute of Life sciences and human technology, Industrial science and technology agency, MITI (Higashi 1-chome 1-3, Tsukuba city, Ibalakiprefecture, Japan) and were given the accession numbers FERM-5646 and FERM BP-5644, respectively.

2. Transfection into COS-7 cells

In order to observe transient expression of the chimeric anti-HM 1.24 antibody, the above-described expression vector was examined in COS-7 (American type culture Collection CRL-1651) cells. HEF-1.24L-g κ and HEF-1.24H-g γ 1 were co-transformed into COS-7 cells by electroporation using a gene pulser (manufactured by BioRad). Each DNA aliquot (10. mu.g) was added to 0.8ml of PBS containing 1X 10⁷Cells/ml COS-7 cells were then pulsed with 1500V and 25UF capacitance.

After a 10-minute recovery period at room temperature, the electroporated cells were added to 30ml of DMEM medium (produced by GIBCO) containing 10% gamma globulin-free fetal bovine serum. In CO₂After culturing in incubator BNA120D (produced by TABAI) for 72 hours, culture supernatant was collected, centrifuged to remove cell debris, and then usedIn the following experiments.

FCM analysis

The antigen-binding activity of the chimeric anti-HM 1.24 antibody was analyzed by FCM (flow cytometry) using KPMM2 cells. 4.7X 10⁵After KPMM2 cells (Japanese unexamined patent publication (Kokai) No. 7-236475) were washed with PBS (-), 50. mu.l of COS-7 cell culture capable of producing the above-described chimeric anti-HM 1.24 antibody and 50. mu.l of FACS buffer (PBS (-) -containing 2% fetal bovine serum and 0.1% sodium azide) were added, or 5. mu.l of 500. mu.g/ml purified mouse anti-HM 1.24 antibody and 95. mu.l of FACS buffer were added, and incubated on ice for 1 hour.

As a control, 50. mu.l of 2. mu.g/ml chimeric SK2 (International application publication No. WO94-28159) and 50. mu.l of FACS buffer were added. Or 5. mu.l of 500. mu.g/ml purified mouse IgG2aR (UPC10) (produced by CAPPEL) was added instead of the purified mouse anti-HM 1.24 antibody and 95. mu.l of FACS buffer, and similarly incubated. After washing with FACS buffer, 100. mu.l of 25. mu.g/ml FITC-conjugated goat anti-human antibody (GAH) (manufactured by CAPPEL) or 10. mu.g/ml FITC-conjugated goat anti-mouse antibody (GAM) (manufactured by Becton Dickinson) was added and incubated on ice for 30 minutes. After washing with FACS buffer, the cells were suspended in 1ml of FACS buffer, and the fluorescence intensity of each cell was measured by FACScan (produced by Becton Dickinson).

As shown in fig. 1, it was revealed that the chimeric anti-HM 1.24 antibody and KPMM2 cells were bound because the peak of fluorescence intensity in the cells in which the chimeric anti-HM 1.24 antibody was introduced was shifted rightward compared to the control, similarly to the case where mouse anti-HM 1.24 antibody was added. This confirmed that the cloned cDNA encodes the variable region of mouse anti-HM 1.24 antibody.

Example 5 establishment of CHO cell line capable of stably producing chimeric anti-HM 1.24 antibody

1. Construction of expression vector for chimeric H chain

After digesting the above plasmid HEF-1.24H-g.gamma.1 with the restriction enzymes PvuI and BamHI, each 2.8kbp fragment containing the EF1 promoter and DNA encoding the H chain V region of mouse anti-HM 1.24 antibody was purified on a 1.5% low melting point agarose gel. The above DNA fragment was then inserted into a fragment of about 6kbp prepared by digesting an expression vector DHFR-DELTA E-Rvh-PM1f (see International application publication No. WO92/19759) for a human H chain expression vector, which comprises a DHFR gene and a gene encoding a human H chain constant region, with PvuI and BamHI to construct an expression vector DHFR-DELTA E-HEF-1.24H-ggamma 1 of a chimeric anti-HM 1.24 antibody H chain.

2. Gene transfer into CHO cells

To establish a stable synthesis system for chimeric anti-HM 1.24 antibody, the genes of the above-described expression vectors HEF-1.24-gk and DHFR-. DELTA.E-HEF-1.24H-g.gamma.1 were linearized by PvuI digestion, and simultaneously introduced into CHO cells DXB11 (given by the Council of medical research Council) by electroporation under conditions similar to those described above (transfection of COS-7 cells described above).

3. MTX-induced Gene amplification

Among CHO cells into which genes were introduced, only those into which both L chain and H chain expression vectors were introduced survived in the nucleoside-free α -MEM culture broth (produced by GIBCO-BRL) to which 500. mu.g/ml of G418 (produced by GIBCO-BRL) and 10% of fetal bovine serum were added, and thus they were selected. Next, 10nm MTX (produced by Sigma) was added to the above culture solution. Among the expanded clones, those capable of synthesizing the chimeric anti-HM 1.24 antibody in large amounts were selected. Clone #8-13, which synthesized chimeric antibody with a synthesis efficiency of about 20. mu.g/ml, was thus obtained and was designated as a chimeric anti-HM 1.24 antibody-synthesizing cell line.

Example 6 construction of chimeric anti-HM 1.24 antibody

The chimeric anti-HM 1.24 antibody was constructed by the following method. The chimeric anti-HM 1.24 antibody-synthesized CHO cells were cultured for 30 days in Iscove's modified Dulbeccoo's medium containing 5% gamma-globulin-free newborn bovine serum (GIBCO-BRL) by a high-density cell culture apparatus Verax system 20 (produced by CELLEX BIOSCIENCE Inc.).

On days 13, 20, 23, 26 and 30 after the start of the culture, the culture solution was recovered using a pressure filter apparatus SARTOBRAN (manufactured by Sartorius), followed by affinity purification using a bulk antibody collection system, an Afiprep system (manufactured by Nippon Gaishi) and a super protein A column (bed volume: 100ml, manufactured by Nippon Gaishi), and PBS as an adsorption/washing buffer and 0.1M sodium citrate buffer (pH3) as an elution buffer, chimeric anti-HM 1.24 antibody according to the attached description. The eluted fractions were immediately adjusted to pH7.4 by adding 1M Tris-HCl (pH 8.0). The antibody concentration was determined by absorbance at 280nm and calculated with 1. mu.g/ml corresponding to 1.35 OD.

Example 7 Activity assay of chimeric anti-HM 1.24 antibody

The chimeric anti-HM 1.24 antibody was evaluated by the following binding inhibition activity.

1. Determination of binding inhibition Activity

1-1 construction of biotinylated anti-HM 1.24 antibody

After diluting the mouse anti-HM 1.24 antibody to 4mg/ml with 0.1M bicarbonate buffer, 4. mu.l of 50mg/ml biotin-N-hydroxysuccinamide (manufactured by EY LABS InC.) was added and reacted at room temperature for 3 hours. Thereafter, 1.5ml of a 0.2M glycine solution was added thereto, and the reaction was terminated by incubating at room temperature for 30 minutes, followed by collecting the biotinylated IgG fraction with a PD-10 column (produced by Pharmacia Biotech).

1-2 measurement of binding inhibition Activity

The binding inhibitory activity of biotinylated mouse anti-HM 1.24 antibody was measured by cell ELISA using human amniotic membrane cell line WISH cells (American type culture Collection CCL 25). Cell ELISA plates were prepared as follows. To a 96-well plate, 4X 10 cells cultured in PRMI1640 medium supplemented with 10% fetal bovine serum were added⁵Cells/ml, cultured overnight, washed twice with PBS (-), and fixed with 0.1% glutaraldehyde (produced by Nakalai tesque).

After blocking, 50. mu.l of a serial dilution of either chimeric anti-HM 1.24 antibody or mouse anti-HM 1.24 antibody obtained by affinity purification was added to each well, and 50. mu.l of 2. mu.g/ml biotinylated mouse anti-HM 1.24 antibody was simultaneously added, incubated at room temperature for 2 hours, and then peroxidase-labeled streptavidin (manufactured by DAKO) was added. After incubation for 1 hour at room temperature, rinsing was performed and a substrate solution was added. After the reaction was terminated by adding 50. mu.l of 6N sulfuric acid, absorbance at 490nm was measured by using a microplate reader 3550 type (manufactured by Bio-Rad).

The results shown in Table 2 indicate that the chimeric anti-HM 1.24 antibody and the mouse anti-HM 1.24 antibody have the same binding inhibition activity against biotinylated mouse anti-HM 1.24 antibody. This indicates that the chimeric antibody and the mouse anti-HM 1.24 antibody have the same V region.

Example 8 measurement of ADCC Activity of chimeric anti-HM 1.24 antibody

ADCC (antibody dependent cellular cytotoxicity) activity was determined according to current methods of immunology, chapter 7, human immunology studies, compiled by the method set forth by John E, Cokigan et al, John Wiley & Sons, inc.

1. Preparation of Effector cells

Monocytes were isolated from peripheral blood or bone marrow of healthy subjects and multiple myeloma patients by density centrifugation. Thus, an equal amount of PBS (-) was added to peripheral blood and bone marrow of healthy subjects and multiple myeloma patients, which were centrifuged at 400g for 30 minutes in the upper layer of ficoll (produced by Pharmacia) -Conrey (produced by Daiichi Pharmaceutical Co. Ltd.) (specific gravity, 1.077). The monocytes were collected, washed twice with RPMI1640 (manufactured by Sigma) supplemented with 10% fetal bovine serum (manufactured by Witaker), and the cells were diluted to 5X 10 with the same culture medium⁶/ml。

2. Preparation of target cells

A human myeloma cell line RPML8226 (American type culture Collection CCL155) was radiolabeled by culturing in RPMI1640 supplemented with 10% fetal bovine serum (produced by Witaker) and 0.1mci 51Cr sodium chromate RPMI1640 at 37 ℃ for 60 minutes. After radiolabelling, cells were washed three times with HanKS Balanced Salt Solution (HBSS) and the concentration was adjusted to 2 × 10⁵/ml。

ADCC assay

To a 96-well U-shaped plate (manufactured by Corning), 50. mu.l of 2X 10 was added⁵Target cells/ml, 1. mu.g/ml affinity-purified chimeric anti-HM 1.24 antibody and mouse anti-HM 1.24 antibody, or control human IgG (manufactured by Serotec), were reacted at 4 ℃ for 1 minute.

Then, when the ratio (E: T) of effector cells (E) to target cells (T) was set to 0: 1, 5: 1, 20: 1, or 50: 1, 100. mu.l of 5X 10 cells was added thereto⁶Effector cells/ml in CO₂The culture was carried out in an incubator for 4 hours.

100. mu.l of the supernatant was taken out, and radioactivity released into the culture supernatant was measured by a counter (ARC361, manufactured by Aloka). To determine maximum radioactivity, 1% NP-40 was used. Cytotoxicity (%) was calculated as (A-C)/(B-C). times.100, where A is the radioactivity released in the presence of antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released by the culture broth in the absence of antibody alone (cpm).

As shown in FIG. 3, cytotoxicity increased with increasing E: T ratio when chimeric anti-HM 1.24 antibody was added, as compared to control IgG1, indicating ADCC activity of this chimeric anti-HM 1.24 antibody. Furthermore, since cytotoxicity was observed even when mouse anti-HM 1.24 antibody was added, it was suggested that the Fc portion of human antibody is required in order to obtain ADCC activity when the effector cells are human-derived cells.

Example 9 construction of reconstituted human anti-HM 1.24 antibody

1. Design of reconstructed human anti-HM 1.24 antibody V region

In order to construct a reconstructed human antibody in which CDRs of a mouse monoclonal antibody have been grafted to a human antibody, it is preferable that there be a high homology between FRs of the mouse antibody and FRs of the human antibody. Thus, the L chain and H chain V regions of mouse anti-HM 1.24 antibody were compared with the V regions of all known antibodies whose structures are clear using a protein database.

The L chain V region of mouse anti-HM 1.24 antibody is most similar to the conserved sequence of human L chain V region subgroup IV (HSGIV), with 66.4% homology. On the other hand, they have 56.9%, 55.8% and 61.5% homology with HSGI, HSGII and HSGIII, respectively.

When the L chain V region of the mouse anti-HM 1.24 antibody is compared with the L chain V region of a known human antibody, it shows 67.0% homology with the human L chain V region REI, i.e., one of the human L chain V region subgroups I. The FR of FRI thus serves as a starting material for the construction of the L chain V region of the reconstituted human anti-HM 1.24 antibody.

The type a of L chain V region of the reconstructed human anti-HM 1.24 antibody was designed. In this type, the REI-based FRs occurring in the human FR and in the reconstructed human CAMPATH-1H antibody (see Riechmann, L., et al, Nature, 322, 21-25, (1988) International application No. WO92-19759, FR contained in type a of the reconstructed human PM-1L chain V region) are identical, and the mouse CDR is identical to the CDR in the L chain V region of the mouse anti-HM 1.24 antibody.

The H chain V region of mouse anti-HM 1.24 antibody is most similar to the conserved sequence of HSGI of human H chain V region. Has 54.7 percent of homology. On the other hand, it showed 34.6% and 48.1% homology with HSGII and HSGIII, respectively. FR1 and FR3 are most similar to the human antibody HG3H chain V region, one of the human H chain V region subgroups I (Recavi, G.et al., Proc. Natl. Acad. Sci. USA, 80, 855) when the mouse anti-HM 1.24 antibody H chain V region is compared with the known human antibody H chain V region, and have a homology of 67.3%.

Therefore, the FR of the human antibody HG3 serves as a starting material for the construction of the H chain V region of the reconstructed human anti-HM 1.24 antibody. However, since the amino acid sequence of FR4 of human HG3 has not been described, the amino acid sequence of human antibody JH6 FR4 having the highest homology with the H chain FR4 of mouse anti-HM 1.24 antibody was used (ravatch, J.V. et al, cell, 27, 583-591). FR4 of JH6 and FR4 of H chain of mouse anti-HM 1.24 antibody have the same amino acid sequence except for one amino acid.

In the first type a form of the reconstructed H chain V region of human anti-HM 1.24 antibody, FR1 to FR3 are identical to FR1 to FR3 of human FR3 and the CDRs are identical to those of the H chain V region of mouse anti-HM 1.24 antibody, except that the amino acid at position 30 of human FR1 and the amino acid at position 71 of human FR3 are identical to those of mouse anti-HM 1.24 antibody.

2. Construction of L chain V region of reconstructed human anti-HM 1.24 antibody

L chain of human anti-HM 1.24 antibody was reconstructed by CDR-grafting construction in the PCR method. The process is shown in figure 4. 8 PCR primers were used to construct a reconstituted human anti-HM 1.24 antibody (type a) with the derived human antibody REI FR. The outer primers A (SEQ ID No: 47) and H (SEQ ID No: 48) were designed to hybridize to the DNA sequence of the expression vector HEF-VL-g κ.

The CDR-grafting primers L1S (SEQ ID No: 49), L2S (SEQ ID No: 50) and L3S (SEQ ID No: 51) have sense DNA sequences. CDR-grafting primers L1A (SEQ ID No: 52), L2A (SEQ ID No: 53) and L3A (SEQ ID No: 54) have antisense DNA sequences each having a DNA sequence (20 to 23bp) complementary to the DNA sequence at the 5' -end of primers L1S, L2S and L3S 5, respectively.

In the first stage of PCR, four reactions A-L1A, L1S-L2A, L2S-L3A and L3S-H were performed to purify each PCR product. The four products of the first stage PCR can be assembled with one another by their own complementation (see International application publication No. WO 92-19759). Next, the full-length DNA encoding the L chain V region of the reconstructed human anti-HM 1.24 antibody was amplified by adding the outer primers A and H (second PCR). In the above PCR, the plasmid HEF-RVL-M21a (see International application publication No. WO95-14041) encoding the human ONS-M21 antibody L chain V region type a reconstructed based on the FR derived from the human antibody REI was used as a template.

In the first stage of PCR, a PCR solution containing 10mM Tris-HCl (pH8.3), 50mM KCl, 0.1mM dNTP, 1.5mM MgCl was used₂100ng template DNA, 100pmol each primer and 5U Ampli Taq. Each PCR tube was covered with 50. mu.mineral oil. Heating at 94 ℃ first denatured, followed by a reaction cycle of 94 ℃ for 1 minute, 55 ℃ for 1 minute, and 72 ℃ for 1 minute, and then incubation at 72 ℃ for 10 minutes.

The PCR products A-L1A (215bp), L1S-L2A (98bp), L2S-L3A (140bp) and L3S-H were purified on a 1.5% low melting agarose gel and assembled in a second PCR. In a second PCR, a 98. mu.l PCR mixture containing 1. mu.g of each first stage PCR product and 5U Ampli Taq was incubated at 94 ℃ for 2 minutes, 55 ℃ for 2 minutes and 72 ℃ for two minutes for 2 reaction cycles, followed by the addition of 100pmol of each outer primer (A and H). The PCR tube was covered with 50. mu.l of mineral oil, and 30 cycles of PCR reaction were performed under the same conditions as above.

The 516bp DNA fragment was purified from the second PCR using a 1.5% low melting agarose gel, then digested with BamHI and HindIII, and the DNA fragment thus obtained was cloned into the HEF expression vector HEF-VL-g κ. After the DNA sequence was determined, a plasmid containing a DNA fragment of the correct amino acid sequence of the L chain V region of the reconstructed human anti-HM 1.24 antibody was designated as the plasmid HEF-RVLa-AHM-g κ. The amino acid sequence and base sequence of the L chain V region contained in the plasmid HEF-RVLa-AHM-g κ are shown in SEQ ID No: 9.

the b-form of the L chain V region of the reconstructed human anti-HM 1.24 antibody was constructed by mutagenesis using PCR. Mutagenesis primers FTY-1(SEQ ID No: 55) and FTY-2(SEQ ID No: 56) were designed so that phenylalanine at position 71 was mutated to tyrosine.

After amplifying the above primers using the plasmid HEF-RVLa-AHM-g kappa as a template, the final product was purified and digested with BamHI and HindIII. The obtained DNA fragment was cloned into HEF expression vector HEF-VL-g κ to obtain plasmid HEF-RVLb-AHM-g κ. The amino acid sequence and base sequence of the L chain V region contained in plasmid HEF-RVLb-AHM-gk are shown in SEQ ID No: 10.

3. construction of the reconstructed H chain V region of human anti-HM 1.24 antibody

3-1 construction of the reconstructed human anti-HM 1.24 antibody H chain V region a to e forms

The DNA encoding the H chain V region of the reconstructed human anti-HM 1.24 antibody was designed as follows. A full length DNA encoding the H chain V region of the reconstituted human anti-HM 1.24 antibody is designed by ligating DNA sequences encoding the FR1 to 3 of the human antibody HG3 and the FR4 of the human antibody JH6 to DNA sequences encoding the CDRs of the H chain V region of the mouse anti-HM 1.24 antibody.

Next, HindIII recognition site/KOZAK conserved sequence and BamHI recognition site/splice donor sequence were added to the 5 '-end and 3' -end of this DNA sequence, respectively, to allow insertion into the HEF expression vector.

The DNA sequence thus designed was divided into four oligonucleotides. It is then possible that oligonucleotides which prevent assembly of these oligonucleotides will be subjected to computer analysis of secondary structure. The sequences of the four oligonucleotides RVH1 to RVH4 are shown in SEQ ID nos: 57 to 60. These oligonucleotides have a length of 119 to 144 bases and an alternating region of 25 to 26 bp. Of these oligonucleotides, RVH2(SEQ ID No: 58) and RVH4(SEQ ID No: 60) have a sense DNA sequence and RVH1(SEQ ID No: 57) and RVH3(SEQ ID No: 59) have an antisense DNA sequence. The method of assembling these four oligonucleotides by PCR is shown in the figure (see FIG. 5).

A PCR mix (98. mu.l) containing 100ng of each oligonucleotide and 5U of Ampli Taq was first denatured by heating at 94 ℃ for 2 minutes and subjected to two cycles of incubation at 94 ℃ for 2 minutes, 55 ℃ for 2 minutes and 72 ℃ for 2 minutes. After addition of 100pmol of RHP1(SEQ ID No: 61) and RHP2(SEQ ID No: 62) as outer primers, the PCR tubes were covered with 50. mu.l of mineral oil. Followed by denaturation by first heating at 94 ℃ for 1 minute, followed by 30 cycles at 94 ℃ for 1 minute, 55 ℃ for 1 minute, 72 ℃ for 1 minute, and incubation at 72 ℃ for 10 minutes.

The 438bp DNA fragment was purified on a 1.5% low melting agarose gel, digested with HindIII and BamHI, and cloned into the HEF expression vector HEF-VH-g γ 1. After the nucleotide sequence was determined, the plasmid containing the DNA fragment encoding the correct V region amino acid sequence of H chain was designated HEF-RVHa-AHM-g.gamma.1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHa-AHM-g gamma 1 are shown in SEQ ID No: 11.

each of types b, c, d, and e of the H chain V region of the human anti-HM 1.24 antibody reconstructed was constructed as follows.

Mutagenesis primers BS (SEQ ID No: 63) and BA (SEQ ID No: 64) are designed to enable arginine at position 66 to be mutated into lysine, plasmid HEF-RVHa-AHM-g gamma 1 is used as a template, and b type is amplified through a PCR method to obtain plasmid HEF-RVHb-AHM-g gamma 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHb-AHM-g γ 1 are shown in SEQ ID NO: 12.

mutagenesis primers CS (SEQ ID No: 65) and CA (SEQ ID No: 66) were designed to mutate threonine at position 73 to lysine, plasmid HEF-RVHa-AHM-g γ 1 was used as a template, and type C was amplified by PCR to obtain plasmid HEF-RVHc-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHc-AHM-g gamma 1 are shown in SEQ ID NO: 13.

mutagenesis primers DS (SEQ ID No: 67) and DA (SEQ ID No: 68) were designed to mutate arginine at position 66 to lysine, plasmid HEF-RVHa-AHM-g γ 1 was used as a template, and type d was amplified by PCR to obtain plasmid HEF-RVHd-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHd-AHM-g gamma 1 are shown in SEQ ID NO: 14.

mutagenesis primers ES (SEQ ID No: 69) and EA (SEQ ID No: 70) were designed so that valine at position 67 was mutated to alanine, methionine at position 69 was mutated to leucine, and plasmid HEF-RVHa-AHM-g γ 1 was used as a template to amplify type e by PCR to obtain plasmid HEF-RVHe-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHe-AHM-g γ 1 are shown in SEQ ID No: 15.

construction of H chain hybrid V region

Two H chain hybrid V regions were constructed. One is mouse-human hybrid anti-HM 1.24 antibody in which the amino acid sequences of FR1 and FR2 are derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 are derived from type a of the reconstructed H chain V region of human anti-HM 1.24 antibody. Another is a human mouse hybrid anti-HM 1.24 antibody in which the amino acid sequences of FR1 and FR2 are derived from the a-form of the H chain V region of the human anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 are derived from the mouse anti-HM 1.24 antibody. The amino acid sequences of the CDR regions were derived from mouse anti-HM 1.24 antibody.

Two H chain hybrid V regions were constructed by PCR. This process is schematically shown in fig. 6 and 7. To construct two H chain hybrid V regions, four primers were used. The outer primers a (SEQ ID No: 71) and h (SEQ ID No: 72) were designed to hybridize to the DNA sequence of the HEF expression vector HEF-VH-g γ 1. The H-strand hybrid construction primer HYS (SEQ ID No: 73) was designed to have a sense DNA sequence, and the H-strand hybrid primer HYA (SEQ ID No: 74) was designed to have an antisense DNA sequence so that the DNA sequences could be complementary to each other.

To construct an H chain hybrid V region in which the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from type a of the H chain V region of reconstituted human anti-HM 1.24 antibody, PCR with plasmid HEF-1.24H-g γ 1 as a template and outer primer a and H chain hybrid primer HYA and PCR with plasmid HEF-RVHa-AHM-g γ 1 as a template and H chain hybrid primer HYS (SEQ ID No: 73) and outer primer H (SEQ ID No: 72) were performed in the first stage of PCR, and each PCR product was purified. The two PCR products of the first stage can be assembled by their own complementarity (see International application publication No. WO 92-19759).

Then, the full-length DNA encoding the H chain hybrid V region was amplified in a second stage PCR by adding outer primers a (SEQ ID No: 71) and H (SEQ ID No: 72) in which the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from type a of the reconstructed human anti-HM 1.24 antibody H chain V region.

To construct an H chain hybrid V region in which the amino acid sequences of FR1 and FR2 were derived from type a of the H chain V region of reconstituted human anti-HM 1.24 antibody, type a of the H chain V region of human anti-HM 1.24 antibody of FR3 and FR4, and the amino acid sequences of FR3 and FR4 were derived from mouse anti-HM 1.24 antibody, PCR with plasmid HEF-HVHa-AHM-g.gamma.1 as a template and H chain hybrid primer HYA on outer primer a and PCR with plasmid HEF-1.24-g.gamma.1 as a template and H chain hybrid primer HYS and outer primer H were performed in the first stage of PCR, and each PCR product was purified. The two PCR products of the first stage can be assembled by their own complementarity (see International application publication No. WO 92-19759).

Next, the outer primers a and H were added, and the full length DNA encoding the H chain hybrid V region was amplified in the second stage PCR, in which the amino acid sequences of FR1 and FR2 were derived from type a reconstructing the H chain V region of human anti-HM 1.24 antibody, and the amino acid sequences of FR3 and FR4 were derived from mouse anti-HM 1.24 antibody.

The first-stage PCR, purification of PCR product, assembly, second-stage PCR and cloning into HEF expression vector HEF-VH-g.gamma.1 were carried out according to the method shown in "example 9. construction of L chain V region of human anti-HM 1.24 antibody".

After DNA sequencing, the plasmid containing the DNA fragment encoding the correct amino acid sequence of the V region of the H chain hybrid was designated HEF-MH-RVH-AHM-g.gamma.1, in which the amino acid sequences of FR1 and FR2 were derived from mouse anti-HM 1.24 antibody and the amino acid sequences of FR3 and FR4 were derived from type a of the reconstructed H chain V region of human anti-HM 1.24 antibody. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-MH-RVH-AHM-g.gamma.1 are shown in SEQ ID No. 75. The plasmid containing the DNA fragment encoding the correct amino acid sequence of the H chain hybrid V region was designated HEF-HM-RVH-AHM-g.gamma.1, in which the amino acid sequences of FR1 and FR2 were derived from type a of the reconstructed H chain V region of human anti-HM 1.24 antibody, and the amino acid sequences of FR3 and FR4 were derived from mouse anti-HM 1.24 antibody. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-HM-RVH-AHM-g.gamma.1 are shown in SEQ ID No: 76.

3-3 construction of reconstructed human anti-HM 1.24 antibody H chain V region f to s type

Each type f, g, H, i, j, k, l, m, n, o, p, q, r and s of the H chain V region of the human anti-HM 1.24 antibody reconstructed was constructed as follows.

Mutagenesis primers FS (SEQ ID No: 78) and FA (SEQ ID No: 79) are designed to mutate threonine 75 to serine, valine 78 to alanine, plasmid HEF-RVHe-AHM-g gamma 1 is used as template DNA, and type f is amplified by a PCR method to obtain plasmid HEF-RVHf-AHM-g gamma 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHf-AHM-g.gamma.1 are shown in SEQ ID No: 16.

mutagenic primers GS (SEQ ID No: 80) and GA (SEQ ID No: 81) were designed to mutate alanine at position 40 to arginine, plasmid HEF-RVHa-AHM-g γ 1 was used as template DNA, and type g was amplified to obtain plasmid HEF-RVHg-AHM-g γ 1. The amino acid sequence of the H chain V region contained in the plasmid HEF-RVHg-AHM-g γ 1 and the humanized reconstructed human antibody are shown in SEQ ID NO: 17.

mutagenesis primers FS (SEQ ID No: 78) and FA (SEQ ID No: 79) are designed, the plasmid HEF-RVHb-AHM-g gamma 1 is used as template DNA, and the h type is amplified to obtain the plasmid HEF-RVHh-AHM-g gamma 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHh-AHM-g gamma 1 are shown in SEQ ID No: 18.

mutagenesis primers IS (SEQ ID No: 82) and IA (SEQ ID No: 83) were designed to mutate arginine at position 83 to alanine, serine at position 84 to phenylalanine, plasmid HEF-RVHh-AHM-g γ 1 was used as template DNA, and type i was amplified to obtain plasmid HEF-RVHi-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHi-AHM-g gamma 1 are shown in SEQ ID No: 19.

mutagenesis primers JS (SEQ ID No: 84) and JA (SEQ ID No: 85) were designed to mutate arginine at position 66 to lysine, plasmid HEF-RVHf-AHM-g γ 1 was used as template DNA, and type j was amplified to obtain plasmid HEF-RVHj-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHj-AHM-g gamma 1 are shown in SEQ ID No: 20.

mutagenesis primers KS (SEQ ID No: 86) and KA (SEQ ID No: 98) were designed to mutate glutamic acid at position 81 to glutamine, plasmid HEF-RVHh-AHM-g γ 1 was used as template DNA, and type K was amplified to obtain plasmid HEF-RVHk-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHk-AHM-g gamma 1 are shown in SEQ ID NO: 21.

mutagenic primers LS (SEQ ID No: 88) and LA (SEQ ID No: 89) were designed to mutate glutamic acid at position 81 to glutamine, serine at position 82B to isoleucine, plasmid HEF-RVHh-AHM-g γ 1 as template DNA, and type 1 was amplified to obtain plasmid HEF-RVH1-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVH1-AHMH-g γ 1 are shown in SEQ ID No: 22.

mutagenic primers MS (SEQ ID No: 90) and MA (SEQ ID No: 91) were designed to mutate glutamic acid at position 81 to glutamine, serine at position 82b to isoleucine, threonine at position 87 to serine, plasmid HEF-RVHh-AHM-g γ 1 as template DNA, and m-type was amplified to obtain plasmid HEF-RVHm-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHm-AHM-g gamma 1 are shown in SEQ ID No: 23.

mutagenesis primers NS (SEQ ID No: 92) and NA (SEQ ID No: 93) were designed to mutate serine at position 82B to isoleucine, plasmid HEF-RVHh-AHM-g γ 1 was used as template DNA, and n-type was amplified to obtain plasmid HEF-RVHn-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHn-AHM-g gamma 1 are shown in SEQ ID NO: 24.

mutagenesis primers OS (SEQ ID No: 94) and OA (SEQ ID No: 95) were designed to mutate threonine 87 to serine, plasmid HEF-RVHh-AHM-g γ 1 was used as template DNA, and o-type was amplified to obtain plasmid HEF-RVHo-AHM-g γ 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHo-AHM-g γ 1 are shown in SEQ ID No: 25.

mutagenesis primers PS (SEQ ID No: 96) and PA (SEQ ID No: 97) were designed to mutate valine at position 78 to alanine. The plasmid HEF-RVHa-AHM-g gamma 1 is used as template DNA, and the p type is amplified by a PCR method to obtain the plasmid HEF-RVHp-AHM-g gamma 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHp-AHM-g gamma 1 are shown in SEQ ID NO: 26.

mutagenesis primers QS (SEQ ID No: 98) and QA (SEQ ID No: 99) are designed to mutate threonine at position 75 into serine, plasmid HEF-RVHa-AHM-g gamma 1 is used as template DNA, and the q type is amplified by a PCR method to obtain plasmid HEF-RVHq-AHM-g gamma 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHq-AHM-g gamma 1 are shown in SEQ ID NO: 27.

mutagenesis primers CS (SEQ ID No: 65) and CA (SEQ ID No: 66) are designed, plasmid HEF-RVHp-AHM-g gamma 1 is used as template DNA, and r type is amplified by a PCR method to obtain plasmid HEF-RVHr-AHM-g gamma 1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHr-AHM-g gamma 1 are shown in SEQ ID No: 28.

the S-form of the H chain V region of the reconstructed human anti-HM 1.24 antibody was constructed by mutagenesis using PCR. Mutagenesis primers SS (SEQ ID No: 100) and SA (SEQ ID No: 101) were designed to mutate methionine 69 to isoleucine.

After amplifying the above primers using the plasmid HEF-RVHr-AHM-g.gamma.1 as a template DNA, the final product was purified, digested with BamHI and HindIII, and the obtained DNA fragment was cloned into the HEF expression vector HEF-VH-g.gamma.1 to obtain the plasmid HEF-RVHs-AHM-g.gamma.1. The amino acid sequence and base sequence of the H chain V region contained in the plasmid HEF-RVHs-AHM-g gamma 1 are shown in SEQ ID No: 102.

the region encoding the HEF-RVLa-AHM-g kappa variable region and HEF-RVHr-AHM-g gamma 1 of each of the above plasmids were digested with restriction enzymes HindIII and BamHI to prepare restriction fragments. They were inserted into HindIII and BamHI sites of a plasmid vector pUC 19. Each plasmid was designated pUC19-RVLa-AHM-g κ and pUC19-RVHr-AHM-g γ 1.

Coli containing each plasmid pUC19-RVLa-AHM-gk and pUC19-RVHr-AHM-g gamma 1 were named E.coli DH5 alpha (pUC 19-RVLa-AHM-gk) and E.coli DH5 alpha (pUC19-RVHr-AHM-g gamma 1), respectively, and were stored under the terms of the Budapest treaty at the national institute of Life and human technology, agency of Industrial science and technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki Prefecture, Japan) by international convention on 8.29.1996, and were given accession numbers FERM BP-5645 and FERM BP-5643, respectively.

The region encoding the variable region of the above plasmid HEF-RVHs-AHM-g.gamma.1 was digested with the restriction enzymes HindIII and BamHI to prepare a restriction fragment. They were inserted into HindIII and BamHI sites of a plasmid vector pUC 19. The obtained plasmid was designated as pUC 19-RVHs-AHM-g.gamma.1.

Escherichia coli harboring the plasmid pUC 19-RVHs-AHM-g.gamma.1 was designated Escherichia coli DH5 alpha (pUC 19-RVHs-AHM-g.gamma.1) and was stored under the terms of the Budapest treaty on the national institute of Life sciences and technology, agency of Industrial science and technology, MITI (Higashi 1-Chrome 1-3, Tsukuba City, Ibalaki Prefecture, Japan) by the international convention on 9/29 of 1997 and was deposited under the accession number FERM BP-6127.

4. Construction of reconstructed human anti-HM 1.24 antibody, chimeric anti-HM 1.24 antibody, and H chain hybrid antibody

In order to evaluate each chain of the reconstituted human anti-HM 1.24 antibody, allowing the reconstituted human anti-HM 1.24 antibody and the chimeric anti-HM 1.24 antibody to be expressed as positive controls, in each b-type construction and after construction of the H chain V region of the reconstituted human anti-HM 1.24 antibody, in order to investigate which amino acid sequence in the FR should be substituted. Allowing expression of H chain hybrid antibodies. Furthermore, in order to evaluate the type a of L chain of the reconstructed human anti-HM 1.24 antibody, it was expressed in combination with a chimeric H chain.

4-1 expression of reconstituted human anti-HM 1.24 antibody (1)

Mu.g of each of the expression vector for the H chain of reconstituted human anti-HM 1.24 antibody (HEF-RVHa-AHM-g. gamma.1-HEF-RVHr-AHM-g. gamma.1) and the expression vector for the L chain of reconstituted human anti-HM 1.24 antibody (HEF-RVLa-AHM-gk or HEF-RVLb-AHM-gk) was co-transformed into COS-7 cells by electroporation using a gene pulser (produced by Biorad). Each DNA aliquot (10. mu.g) was added to 0.8ml of a solution containing 1X 10 DNA in PBS⁷Cells/ml of cells and pulsed at 1500V and 25UF capacitance.

After a recovery period of 10 minutes at room temperature, the electroporated cells were added to 30ml of DMEM medium (produced by GIBCO) containing 10% gamma-globulin-free fetal bovine serum. At 37 ℃ and 5% CO₂Under the condition of CO₂After culturing in an incubator BNA120D (manufactured by TABAI) for 72 hours, the culture supernatant was collected, centrifuged at 1000rpm for 5 minutes in a centrifuge 15PR-22 (manufactured by HITACHI) equipped with a centrifuge rotor O3 (manufactured by HITACHI) to remove cell debris, ultrafiltered in a microconcentrator (Centricon 100, manufactured by Amicon) using a centrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifuge rotor JA-20.1 (manufactured by BECKMAN), and used for cell ELISA.

Expression of the reconstructed human anti-HM 1.24 antibody (2)

Using a gene pulser (Biorad) 10. mu.g of each of the reconstructed human anti-HM 1.24 antibody H chain "S" -type expression vector (HEF-RVHs- -AHM-g. gamma.1) and the reconstructed human anti-HM 1.24 antibody L chain expression vector (HEF-RVLa-AHM-gk) was co-transformed into COS-7 cells by electroporation. Each DNA aliquot (10. mu.g) was added to 0.8ml of a solution containing 1X 10 DNA in PBS⁷Cells/ml of cells and pulsed at 1500V and 2.5UF capacitance.

After a recovery period of 10 minutes at room temperature, the electroporated cells were added to 30ml of DMEM medium containing 10 gamma-globulin-free fetal bovine serum (produced by GIBCO). At 37 ℃ and 5% CO₂Under conditions of CO₂After culturing in incubator BNA120D (manufactured by TABAI) for 72 hours, culture supernatant was collected and centrifuged in centrifuge O5PR-22 (manufactured by HITACHI) with centrifuge rotor O3 (manufactured by HITACHI)¹Cell debris was removed by centrifugation at 000rpm for 5 minutes, and the microconcentrator (Centricon 100, manufactured by Amicon) was concentrated by ultrafiltration using a centrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifuge rotor JA-20.1 (manufactured by BECKMAN), sterilized by filtration using a filter millex GVBmm (manufactured by Millipore), and then used for cell ELISA.

4-2 expression of chimeric anti-HM 1.24 antibody

The chimeric anti-HM 1.24 antibody for cell-ELISA was prepared according to the above-described reconstructed expression method of human anti-HM 1.24 antibody, using 10. mu.g of each of the expression vector HEF-1.24-g.gamma.1 for the H chain of chimeric anti-HM 1.24 antibody and the expression vector HF-1.24L-gk for the L chain of chimeric anti-HM 1.24 antibody.

4-3 expression of anti-HM 1.24 antibody comprising human type-a L chain and chimeric H chain

anti-HM 1.24 antibody containing human type-a L chain and chimeric H chain for cell ELISA was prepared according to the above-described expression method of the reconstructed human anti-HM 1.24 antibody using 10. mu.g of the expression vector HEF-1.24-g.gamma.1 of the chimeric anti-HM 1.24 antibody and the reconstructed expression vector HEF-RVLa-AHM-g.kappa.of type-a of the human anti-HM 1.24 antibody.

Expression of H chain hybrid antibodies

An H chain hybrid antibody for cell ELISA was prepared according to the above-described expression method of the reconstructed human anti-HM 1.24 antibody using 10. mu.g of an expression vector for H chain hybrid V region (HEF-MH-RVH-AHM-g γ 1 or HEF-HM-RVH-AHM-g γ 1) and an expression vector for L chain of the reconstructed human anti-HM 1.24 antibody HEF-RVLa-AHM-g κ.

4-5 determination of antibody concentration

The obtained antibody concentration was determined by ELISA. Add 100. mu.l of coating buffer (0.1M NaHCO)₃、0.02％ NaN₃pH9.6) to 1. mu.g/ml, each well of the 96-well ELISA plate (Maxi Sorp, manufactured by NUNC) was loaded with the goat anti-human IgG antibody (manufactured by BIOSOURCE). And incubated at room temperature for 1 hour. Dilution buffer (50mM Tris-HCl, 1mM MgCl) with 100. mu.l₂、0.15M NaCl、0.05％ Tween 20、0.02％ NaN₃After 1% Bovine Serum Albumin (BSA), pH8.1) blocking, 100. mu.l of each serial dilution of the ultrafiltrated concentrated COS-7 cell culture supernatant containing reconstituted human anti-HM 1.24 antibody, chimeric anti-HM 1.24 antibody or H chain hybrid antibody was added to each well and incubated at room temperature for 1 hour. After washing, 100. mu.l of an alkaline phosphatase-labeled goat anti-human IgG antibody (produced by DAKO) was added.

After incubation for 1 hour at room temperature and washing, 100. mu.l of lysis buffer (50mM NaHCO) was added₃、10mMMgcl₂pH9.8) was measured, and absorbance at 405nm was measured using a microplate reader 3550 type (manufactured by Bio Rad) after 1. mu.g/ml of the substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA). Human IgG1R (produced by binding Site) was used as a standard for concentration measurement.

5. Establishment of CHO cell line stably synthesizing reconstructed human anti-HM 1.24 antibody

5-1 construction of expression vector for reconstructed human anti-HM 1.24 antibody H chain

The plasmid HEF-RVHr-AHM-g.gamma.1 was digested with the restriction enzymes PvuI and BamHI, and the about 2.8kbp fragment containing DNA encoding the EF1 promoter and the reconstructed H chain V region of human anti-HM 1.24 antibody was purified by 1.5% low melting agarose gel. Next, the above DNA fragment was inserted into the expression vector DHFR-. DELTA.E-HEF-RVHr-AHM-g.gamma.1 of the H chain of human anti-HM 1.24 antibody reconstructed by construction of a 6kbp fragment prepared by digesting the expression vector DHFR-. DELTA.E-RVh-pmlf (International application publication No. WO92-19759) for human H chain expression with PvuI and BamHI, which contained the DHFR gene and the gene encoding the constant region of the human H chain.

5-2 introduction of Gene into CHO cells

To establish a stable synthesis system of the reconstructed human anti-HM 1.24 antibody, the genes of the above-described expression vectors DHFR-. DELTA.E-HEF-RVHr-AHM-g-. gamma.1 and HEF-RVLa-AHM-g-. kappa.which were linearized by PvuI digestion were simultaneously introduced into CHO cells DXB-11 by electroporation under conditions similar to those described above (transfected into the above-described COS-7 cells).

5-3 Gene amplification by MTX

Among CHO cells into which genes were introduced, only those into which both L chain and H chain expression vectors were introduced could survive in the nucleoside-free a-MEM medium (produced by GIBCO-BRL) to which 500. mu.g/ml of G418 (produced by GIBCO-BRL) and 10% fetal bovine serum were added. Next, 10nm MTX (produced by Sigma) was added to the above medium. Among the expanded clones, those capable of synthesizing a large amount of the reconstructed human anti-HM 1.24 antibody were selected. Clone 1, having an efficiency of about 3. mu.g/ml for the synthesis of reconstituted human anti-HM 1.24 antibody, was thus obtained and designated as reconstituted human anti-HM 1.24 antibody synthetic cell line.

5-4 construction of reconstructed human anti-HM 1.24 antibody

The reconstructed human anti-HM 1.24 antibody was constructed in the following method. The CHO cells capable of synthesizing and reconstructing human anti-HM 1.24 antibody were cultured at 37 ℃ and 5% CO₂Under the conditions of CO₂The incubator BNAS120D (manufactured by TABAI) was cultured for 10 days in the nucleoside-free α -MEM medium (manufactured by GIBCO-BRL) to which 500. mu.g/ml of G418 (manufactured by GIBCO-BRL) and 10% of gamma-globulin-free fetal calf serum were added. The culture solution was recovered at 8 th and 10 th days after the start of the culture, centrifuged at 2000rpm for 10 minutes using a centrifuge RL-500SP (manufactured by Tony Seiko) with a TS-9 rotor to remove cell debris, and centrifuged with a centrifuge having a straight bladeThe cells were sterilized by filtration through a 0.45-. mu.M filter membrane using a bottle top filter (manufactured by FALCON).

After an equal amount of PBS (-) was added to the CHO cell culture solution capable of synthesizing the reconstructed human anti-HM 1.24 antibody, the reconstructed human anti-HM 1.24 antibody was affinity-purified using PBS (-) as an adsorption/washing buffer, 0.1M sodium citrate buffer (pH3) as an elution buffer, and a high-speed antibody purification system ConSep LC100 (manufactured by MILLIPORE) and an ultra-D protein A column (Nippon Gaishi) according to the attached instructions. To the eluted fractions, 1M Tris-HCl (pH8.0) was immediately added to adjust to about pH7.2, followed by concentration and replacement of PBS (-) using a centrifugal ultrafiltration concentrator Centriprep-10 (manufactured by MILLIPORE), and filter sterilization was performed using a membrane filter MILLEX-GV (manufactured by MILLIPORE) having a pore size of 0.22. mu.M to obtain purified reconstructed human anti-HM 1.24 antibody. The antibody concentration was determined by absorbance at 280nm and calculated using 1.35OD corresponding to 1 mg/ml.

Example 11 determination of Activity of reconstituted human anti-HM 1.24 antibody

Evaluation of the following antigen-binding Activity and binding inhibition Activity of the reconstructed human anti-HM 1.24 antibody

1. Method for measuring antigen binding activity and binding inhibition activity

1-1. determination of antigen binding Activity

Antigen binding activity was determined by cell ELISA using WICH fine syncytial. Cell ELISA plates were prepared as described in examples 7.1-2 above.

After blocking, 100. mu.l of serial dilutions of reconstituted human anti-HM 1.24 antibody obtained from COS-7 cell culture supernatant concentrate or purified from CHO cell culture supernatant was added to each well. After incubation for 2 hours at room temperature and washing, peroxidase-labeled anti-human IgG antibody (produced by DAKO) was added. After incubation for 1 hour at room temperature and washing, 100. mu.l of substrate solution was added to each well. After the incubation, the reaction was terminated with 50. mu.l of 6N sulfuric acid, and the absorbance at 490nm was measured using a microplate reader 3550 type (manufactured by Bio-Rad).

1-2 measurement of binding inhibition Activity

The binding inhibition activity of the mouse anti-HM 1.24 antibody labeled with biotin was measured by cell ELISA using WISH cells. Cell ELISA plates were prepared as described in examples 7.1-2 above. After blocking, 50. mu.l of serial dilutions of reconstituted human anti-HM 1.24 antibody obtained from COS-7 cell culture supernatant concentrate or purified from CHO cell culture supernatant were added to each well, along with 50. mu.l of biotin-labeled mouse anti-HM 1.24 antibody. After incubation for 2 hours at room temperature and washing, peroxidase-labeled streptavidin (produced by DAKO) was added. After incubation for 1 hour at room temperature and washing, 100. mu.l of substrate solution was added to each well. After the incubation, the reaction was terminated with 50. mu.l of 6N sulfuric acid, and absorbance at 490nm was measured using a microplate reader 3550 type (manufactured by Bio-Rad).

2. Evaluation of reconstructed human anti-HM 1.24 antibody

The type a of the L chain of the reconstituted human anti-HM 1.24 antibody is evaluated as mentioned in the assay for antigen binding activity. As shown in fig. 8, when L chain version a and chimeric H chain were expressed in combination, it had a similar level of antigen binding activity. However, in view of further improvement in activity and compatibility with H chain, form b of L chain was constructed. When combined with H chain versions a, b, f and g, types a and b of L chain were put together to evaluate antigen binding activity and binding inhibition activity. As shown in FIGS. 9, 10, 11 and 12, the L chain type a has high activity in both activities in all the types a, b, f and h in the L chain than in the type b. Therefore, L chain a type of human anti-HM 1.24 antibody was reconstructed for the following experiment.

Forms a to e of H chain

The reconstructed human anti-HM 1.24 antibody H chain a to e forms were evaluated in combination with the L chain a form in the aforementioned measurement of antigen binding activity and binding inhibition activity. As shown in FIGS. 11, 13, 14, and 15, the results showed that all types were slightly weaker in both activities as compared with the chimeric anti-HM 1.24 antibody. Indicating that more amino acid substitutions are required.

H chain hybrid antibodies

The H chain hybrid antibodies were evaluated as mentioned with the antigen binding activity assay. As shown in FIG. 16, the results showed that the human murine hybrid anti-HM 1.24 antibody had an activity similar to that of the chimeric anti-HM 1.24 antibody against the antigen-binding activity. While the mouse human hybrid anti-HM 1.24 antibody had slightly weaker activity than the chimeric anti-HM 1.24 antibody. This indicates that in order to construct a reconstituted human anti-HM 1.24 antibody having an antigen-binding activity similar to that of the chimeric anti-HM 1.24 antibody, it is necessary to replace the amino acids included in the V region of the H chain, including those in FR3 to FR 4.

H chain of the f to r type

The reconstructed human anti-HM 1.24 antibody H chain form f was evaluated as mentioned with the determination of antigen binding activity. As shown in FIG. 17, the results showed that its binding activity was decreased as compared with the chimeric anti-HM 1.24 antibody, but its activity was increased as compared with the above types a to c, indicating that the newly substituted four amino acids at positions 67, 69, 75 and 78 in this type are responsible for the activity of the reconstructed human antibody.

The reconstructed human anti-HM 1.24 antibody H chain g form was evaluated as mentioned with the determination of antigen binding activity. As shown in FIGS. 18 and 19, the results showed that this type had similar activity to the above type a in most cases, revealing that the substituted amino acid at position 40 of this type was not responsible for the enhancement of the activity of the reconstructed human antibody as shown in the above H chain human mouse hybrid antibody.

The H to j forms of the H chain of the reconstituted human anti-HM 1.24 antibody were evaluated as mentioned in the assays for antigen binding activity and binding inhibition activity. As shown in FIGS. 20, 21, 22 and 23, the results showed that all types were weaker in both activities compared with the chimeric anti-HM 1.24 antibody and had similar activities to the above-mentioned form f. Indicating that amino acids 67 and 69 of the newly substituted four amino acids in the f-form are not responsible for the increased activity of the reconstructed human antibody.

The k to p forms of the H chain of the reconstituted human anti-HM 1.24 antibody were evaluated from the mentioned assays for antigen binding activity and binding inhibition activity. As shown in FIGS. 24, 25, 26 and 27, the results showed that all the types had weak activities in both activities as compared with the chimeric anti-HM 1.24 antibody and similar activities to those of the above version h, indicating that the amino acid at position 80 in these 6 types and the amino acid newly substituted thereafter were responsible for the improvement in the activity of the human antibody not to be reconstructed.

The q form of the H chain of reconstituted human anti-HM 1.24 antibody was evaluated as mentioned in the assays for antigen binding activity and binding inhibition activity. As shown in FIGS. 25 and 27, the results showed that this version was weaker in both activities than the above h-type or p-type and had similar activities to the above a-type, indicating that substitution of amino acid at position 78 was necessary for improvement of the activity of the reconstructed human antibody.

The r-form of the H chain of the reconstructed human anti-HM 1.24 antibody was evaluated by the method mentioned above. As shown in FIGS. 15 and 28, the results showed that the r-type and chimeric anti-HM 1.24 antibodies had similar levels of antigen binding activity and binding inhibition activity.

The above results show that in order to have an antigen binding activity at a level similar to that of the chimeric anti-HM 1.24 antibody of mouse anti-HM 1.24 antibody, the minimum substitutions required for the reconstituted human anti-HM 1.24 antibody are amino acids at positions 30, 71, and 78 and further amino acid at position 73.

The antigen binding activity and binding inhibition activity of the reconstructed human anti-HM 1.24 antibody H chain a to r types are summarized in Table 6.

TABLE 6

H chain type	Antigen binding Activity	Binding inhibition Activity
			abcdefghijklmnopqr	++++++++++++++++++++++++++++++	The + + + + + not determined +++++++++++++++++++++++++++++++++++++++++, is not determined

2.5H chain of S type

The reconstructed human anti-HM 1.24 antibody H chain S form was evaluated in combination with the aforementioned L chain a form as mentioned above in the measurement of antigen binding activity and binding inhibition activity. As shown in fig. 29 and 30, the results showed that S-type and r-type had similar levels of antigen binding activity and binding inhibition activity.

As mentioned above, the reconstructed human anti-HM 1.24 antibody of the present invention retains the ability to bind to the antibody even after one or more amino acid residues are replaced with other amino acids. The present invention therefore includes a reconstituted human anti-HM 1.24 antibody in which one or more amino acid residues may be substituted with other amino acids in the H chain or L chain variable region as long as it retains its original properties.

3. Evaluation of purified reconstituted human anti-HM 1.24 antibody

Purified reconstituted human anti-HM 1.24 antibody was evaluated with the above-described antigen binding activity and binding inhibition activity. As shown in FIGS. 31 and 32, the results showed that the reconstituted human anti-HM 1.24 antibody and the chimeric anti-HM 1.24 antibody had similar levels of antigen-binding activity and binding-inhibition activity. It was shown that the reconstituted human anti-HM 1.24 antibody and mouse anti-HM 1.24 antibody have the same antigen-binding activity.

Example 12 anti-tumor Effect of chimeric anti-HM 1.24 antibody on human myeloma mouse model

1. Preparation of administration antibodies

1-1 preparation of chimeric anti-HM 1.24 antibody

The purified chimeric anti-HM 1.24 antibody obtained in example 6 above was concentrated using a centrifugal ultrafiltration concentrator Centriprep 10 (manufactured by Amicon), and the buffer solution was replaced with PBS (-). The membrane filter MILLEX-GV (MILLIPORE) with a pore size of 0.22. mu.M was used for filter sterilization. The antibody was prepared to a concentration of 200. mu.g/ml using filter-sterilized PBS (-) and used in the following experiments. The concentration of the antibody was determined by absorbance at 280nm and calculated as 1.35OD equivalent to 1 mg/ml.

1-2 purification of control human IgG1

Human IgG1 used as a control for chimeric anti-HM 1.24 antibody was purified as follows. An equal amount of PBS (-) was added to the purified Hu IgG1 Kappa (produced by BINDING SITE), and the PBS (-) was used as an elution buffer to affinity-purify human IgG1 using the high-speed antibody purification system ConSep LC100 (produced by MILIPORE) and the Hyper D protein A column (produced by Nippon Gaishi) according to the attached instructions. 1M Tris-HCl (pH8.0) was immediately added to the eluted fractions to adjust pH7.4 or so, followed by concentration and replacement with PBS (-) buffer using a centrifugal ultrafiltration concentrator Centriprep 10 (manufactured by Amicon), and sterilized by filtration using a membrane filter MILLEX-GV1MILLIPORE having a pore size of 0.22. mu.M). Human IgG1 was adjusted to 200. mu.g/ml with filter-sterilized PBS (-) and used for the following experiments. The antibody concentration was determined by absorbance at 280nm and calculated using 1.35OD equivalent to 1 mg/ml.

2. Method for quantifying human serum IgG in mouse serum

Human IgG contained in mouse serum was quantified by the following ELISA. Mu.l of goat anti-human IgG diluted to 1. mu.g/ml with 0.1M bicarbonate buffer (pH9.6) was added to a 96-well plate (manufactured by NUNC), and incubated overnight at 4 ℃ to immobilize the antibody. After blocking, 100. mu.l of serially diluted mouse serum or human IgG as standard (produced by CAPPEL) was added and incubated at room temperature for 1 hour. After washing, 100. mu.l of alkaline phosphatase-labeled anti-human IgG (manufactured by CAPPEL) diluted 2000-fold was added and incubated, followed by measurement of absorbance at 405nm using a microplate reader model 3550 (manufactured by Bio-Rad).

3. Antitumor Effect of chimeric anti-HM 1.24 antibody on human myeloma cell-transplanted mice

3-1. establishment of human myeloma cell transplantation mouse

Human myeloma was established as followsCells were transplanted into mice. In 3X 10 of RPMI1640 medium supplemented with 10% fetal bovine serum (produced by GIBCOBRL)⁷KPMM2 cells were prepared at a concentration of cells/ml for in vivo passaging of SCID mice (fed by Nihon CLEA). Mu.l of the above KPMM2 cell suspension was injected via tail vein into SCID mice (male, 8 weeks, fed by Nihon (LEA)) that had been intraperitoneally injected with 100. mu.l of anti-asialo GM1 (manufactured by Wako Pure Chemical Industries Co., Ltd.) the previous day.

3-2 administration of antibodies

On day 12 after KPMM2 cell transplantation, sera were collected from the above human myeloma cell-transplanted mice, and human IgG in the sera were quantified using ELISA mentioned in 2 above. KPMM2 cells were confirmed to have entered the bone marrow by an increase in serum human IgG levels. On days 14, 21 and 28 after KPMM2 cell transplantation, 100 μ l of each antibody prepared in 1 above was intraperitoneally injected into these mice.

3-3 evaluation of antitumor Effect of chimeric anti-HM 1.24 antibody on human myeloma cell-transplanted mice

The antitumor effect of the gene anti-HM 1.24 antibody was evaluated by the survival time of the mouse. As shown in FIG. 33, mice receiving the chimeric anti-HM 1.24 antibody had an extended survival period compared to mice receiving control human IgG 1. Thus, it was confirmed that the chimeric anti-HM 1.24 antibody had an antitumor effect on human myeloma cell-transplanted mice.

Example 13 measurement of ADCC Activity of reconstituted human anti-HM 1.24 antibody

ADCC (antibody-dependent cellular cytotoxicity) was determined according to the method elucidated by current methods of immunology, edited by John e.coligan et al, John Wiley & sons.inc, 1993, chapter seven, in human immune studies.

1. Preparation of Effector cells

Monocytes are isolated from peripheral blood of healthy subjects by means of density centrifugation. Thus, an equal amount of PBS (-) was added to the peripheral blood of healthy subjects, which was at Ficoll-Paque PLUS (produced by Pharmacia), and centrifuged at 400g for 40 minutes. The monocyte layer was collected, washed four times with RPMI1640 medium (produced by GIBCO BRL) supplemented with 10% fetal bovine serum (produced by GIBCO BRL), and prepared to 5X 10 with the same medium⁶Cell density per ml.

LAK (lymphokine-activated killer cells) was induced from bone marrow cells of SCID mice (fed with Nihon CLEA). Bone marrow cells were isolated from mouse femurs and washed twice with RPMI1640 medium (manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum (manufactured by GIBCO BRL), and prepared to 2X 10 with the same medium⁵Cell density per ml. They were conjugated with 50ng/ml recombinant human IL-2 (R)&D SYSTEMS) and 10ng/ml recombinant mouse GM-CSF (R)&D SYSTEMS) together in CO₂The cells were cultured in an incubator (TABAI) for 7 days. The number of cells was adjusted to 2X 10 with the same medium⁶/ml。

2. Preparation of target cells

A radiolabeled human myeloma cell line KPMM2 (Japanese unexamined patent publication (Kokai) No. 7-236475) or ARH-77 from plasma cell leukemia [ American type culture Collection (CL-1621) ] was cultured at 37 ℃ for 60 minutes in RPMI1640 medium supplemented with 10% fetal bovine serum (manufactured by GIBCO BRL) and 0.1mCi of sodium 51Cr chromate (manufactured by ICN)]. After radiolabelling, cells were washed three times with the same medium and adjusted to 2X 10⁵/ml。

ADCC assay

To a 96-well U-shaped bottom plate (produced by Becton Dickinson), 50. mu.l of 2X 10 was added⁵Target cells/ml, 50. mu.l of reconstituted human anti-HM 1.24 antibody, mouse anti-HM 1.24 antibody, control human IgG1 (produced by THE BINDING SITE) or control mouse IgG2a (UPC10, produced by CAPPEL) and reacted at 4 ℃ for 15 minutes.

When the ratio (E: T) of effector cells (E) to target cells (T) is set to 0: 1, 3.2: 1, 8: 1, 20: 1 or 50: 1, 100. mu.l of effector cells are in CO₂The culture was carried out in an incubator for 4 hours.

Mu.l of the supernatant was removed and radioactivity released into the culture supernatant was measured by an r counter (ARC-300, manufactured by Aloka). For the determination of the maximum radioactivity, 1% NP-40 (manufactured by Nakalai) was used. Cytotoxicity was calculated by (A-C)/(B-C). times.100, where A is the radioactivity released in the presence of antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released by medium without antibody (cpm).

Fig. 34 shows the results obtained when cells prepared from peripheral blood of healthy subjects were used as effector cells and KPMM2 cells were used as target cells. FIG. 35 shows the results obtained when cells prepared from peripheral blood of healthy subjects were used as effector cells and ARH-77 was used as target cells. Cytotoxicity increased with increasing antibody concentration when reconstituted human anti-HM 1.24 antibody was added as compared to control human IgG1, indicating that reconstituted human anti-HM 1.24 antibody has ADCC activity.

In addition, when the reconstituted human anti-HM 1.24 antibody was added, cytotoxicity was significantly increased as compared with the mouse anti-HM 1.24 antibody, indicating that the reconstituted human anti-HM 1.24 antibody has higher ADCC activity than the mouse anti-HM 1.24 antibody. Furthermore, when KPMM2 was used as the target cell, addition of the reconstituted human anti-HM 1.24 antibody at a concentration of 0.1. mu.g/ml or more did not cause a change in cytotoxicity, indicating that the concentration of 0.1. mu.g/ml or more was sufficiently cytotoxic. When ARH-77 was used as the target cell, addition of the reconstituted human anti-HM 1.24 antibody at a concentration of 0.1. mu.g/ml or more did not cause a change in cytotoxicity, indicating that the concentration of 0.1. mu.g/ml or more was sufficiently cytotoxic.

FIG. 36 shows the results obtained when cells prepared from bone marrow of SCID mice were used as effector cells. Cytotoxicity increased with increasing antibody concentration when reconstituted human anti-HM 1.24 antibody was added as compared to control human IgG1, indicating that reconstituted human anti-HM 1.24 antibody has ADCC activity. Furthermore, addition of the reconstituted human anti-HM 1.24 antibody at a concentration of 0.1. mu.g/ml or more did not cause a change in cytotoxicity, indicating that the concentration of 0.1. mu.g/ml or more was sufficiently cytotoxic.

These results show that the reconstituted human anti-HM 1.24 antibody has ADCC activity when the effector cells used are derived from human or mouse.

Example 14 antitumor of reconstructed human anti-HM 1.24 antibody against human myeloma mouse model Effect

1. Preparation of administration antibodies

The reconstituted human anti-HM 1.24 antibody obtained by introducing the plasmid HEF-RVLa-AHM-g.gamma.1 and the plasmid HEF-RVHr-AHM-g.gamma.1 into CHO cells was diluted to concentrations of 40, 200 and 1000ug/ml with filter-sterilized PBS (-). The control human IgG1 obtained in example 12.1-2 was diluted to a concentration of 200ug/ml with filter-sterilized PBS (-) and used as the antibody for administration.

2. Antitumor Effect of the reconstructed human anti-HM 1.24 antibody on human myeloma cell-transplanted mice

2-1. establishment of human myeloma cell transplantation mouse

Human myeloma cell-transplanted mice were established according to example 12.3-1. The mice used were SCID mice (5 weeks) (fed with Nihon CLEA).

2-2 administration of antibodies

On day 9 after the KPMM2 cell transplantation, serum was collected from the human myeloma cell-transplanted mouse prepared in 2-1 above, and the human IgG in the serum was quantified by ELISA as mentioned in 12.2 above. The entry of KPMM2 cells into the bone marrow was confirmed by an increase in serum human IgG levels. On day 10 after KPMM2 cell transplantation, 100 μ l of each antibody prepared in 1 above was injected intraperitoneally into these mice.

2-3 evaluation of antitumor Effect of reconstructed human anti-HM 1.24 antibody on human myeloma cell transplantation mice

The antitumor effect of the reconstituted human anti-HM 1.24 antibody was evaluated by changes in the amount of human IgG in the mouse serum and the survival time of the mouse.

The change in the amount of human IgG in mouse serum was determined by measuring human IgG using ELISA mentioned in example 12.2 on serum collected on day 35 after KPMM2 cell transplantation.

As shown in fig. 37, the results showed that the amount of human IgG in plasma at day 35 after KPMM2 cell transplantation was increased by about 1000-fold in the control human IgG 1-administered group compared to day 9 (day before antibody administration), whereas the amount of human IgG in serum was almost equal to or lower than day 9 for any dose in the reconstituted human anti-HM 1.24 antibody-administered group, indicating that the reconstituted human anti-HM 1.24 antibody inhibited the growth of KPMM2 cells. On the other hand, as shown in FIG. 38, an increase in survival time was observed in the reconstituted human anti-HM 1.24 antibody-administered group, as compared with the control human IgG 1-administered group. These indicate that the reconstructed human anti-HM 1.24 antibody has an antitumor effect on human myeloma cell-transplanted mice.

Example 15 between the reconstituted human anti-HM 1.24 antibody and the survival drug melphalan Comparison of antitumor Effect on human myeloma mouse model

1. Preparation of administration drugs

1-1 preparation of administration antibodies

The reconstructed human anti-HM 1.24 antibody obtained by introducing the plasmid HEF-RVLa-AHM-gk and the plasmid HEF-RVHr-AHM-g.gamma.1 into CHO cells was diluted to a concentration of 40 and 200. mu.g/ml with filter-sterilized PBS (-) and the control human IgG1 obtained in example 12.1-2 was diluted to a concentration of 200. mu.g/ml with filter-sterilized PBS (-) and used as the administered antibody.

1-2 preparation of phenylalanine mustard

Melphalan (manufactured by SIGMA), which is a viable drug for myeloma, was formulated at a concentration of 0.1mg/ml with 0.2% hydroxymethylcellulose (CMC) (manufactured by Daicel Chemical Industries, Ltd.).

2. Antitumor Effect of reconstituted human anti-HM 1.24 antibody and melphalan on human myeloma cell-transplanted mice

2-1. establishment of human myeloma cell transplantation mouse

Human myeloma cell-transplanted mice were prepared according to example 14.2-1.

2-2 administration of drugs

On day 9 after the KPMM2 cell transplantation, serum was collected from the human myeloma cell-transplanted mouse prepared in 2-1 above, and the human IgG in the serum was quantified by ELISA as mentioned in 12.2 above. The entry of KPMM2 cells into the bone marrow was confirmed by an increase in serum human IgG levels. On day 10 after KPMM2 cell transplantation, 100. mu.l of each antibody prepared in 1-1 above was intraperitoneally injected into these mice. In addition, 200. mu.l of 0.2% CMC solution was orally administered once a day for 5 consecutive days from the 10 th day after transplantation. On the other hand, the phenylalanine nitrogen mustard administration group orally administered the phenylalanine nitrogen mustard solution prepared in 1-1 above in an amount of 100. mu.l per 10g body weight (i.e., 1mg phenylalanine nitrogen mustard/kg) for 5 consecutive days, once a day, for 5 consecutive days, from day 10 after the transplantation of KPMM2 cells.

2-3 evaluation of antitumor Effect of reconstructed anti-HM 1.24 antibody on human myeloma cell transplantation mice

The antitumor effect of the reconstituted anti-HM 1.24 antibody was evaluated by changes in the amount of human IgG in the mouse serum and the survival time of the mouse.

The change in the amount of human IgG in mouse serum was determined by measuring human IgG using ELISA described in example 12.2 on serum collected on day 35 after KPMM2 cell transplantation. As shown in fig. 39, the results showed that the amount of human IgG in serum at day 35 after KPMM2 cell transplantation was increased by about 1000-fold compared to day 9 (day before antibody administration) in the control human IgG 1-administered group, and it appeared that KPMM2 cells grew in these mice. Also in the melphalan-administered group, the amount of serum human IgG was much higher than before the drug administration, although not as high as in the control human IgG-administered group. This result indicates that alanine nitrogen mustard does not inhibit the growth of KPMM2 cells well. On the other hand, in the reconstituted human anti-HM 1.24 antibody administration group, the amount of human IgG in the serum was less than the 9 th day after transplantation for any dose, indicating that the reconstituted human anti-HM 1.24 antibody inhibits the growth of KPMM2 cells.

On the other hand, prolongation of survival time was observed in the reconstituted human anti-HM 1.24 antibody-administered group, as compared with the control human IgG 1-administered group or the melphalan-administered group, in survival as shown in FIG. 40. The results show that the reconstructed human anti-HM 1.24 antibody has anti-tumor effect on human myeloma cell transplantation mice, and the anti-tumor effect of the antibody is stronger than that of survival drug phenylalanine mustard.

The above results indicate that the mouse anti-HM 1.24 antibody is almost non-cytotoxic to human myeloma cells when human-derived effector cells are used, whereas the reconstructed human anti-HM 1.24 antibody and chimeric anti-HM 1.24 antibody have strong cytotoxicity. This fact indicates the importance of human antibodies and provides hope that the reconstituted human anti-HM 1.24 antibody will be useful in humans.

The reconstituted human anti-HM 1.24 antibody had a strong anti-tumor effect in SCID mice transplanted with human myeloma cells. Thus, in humans, the effector cells are derived from the human body. Lymphocytes are also usually present, and a stronger antitumor effect of the reconstituted human anti-HM 1.24 antibody is expected.

The reconstructed human anti-HM 1.24 antibody has a strong anti-tumor effect compared to a survival drug in a myeloma model, and thus the reconstructed human anti-HM 1.24 antibody is expected to be an epoch-making drug in the treatment of myeloma.

Reference example 1 construction of hybridoma synthesizing mouse anti-HM 1.24 monoclonal antibody

Hybridomas which synthesize mouse anti-HM 1.24 monoclonal antibody were prepared according to the method described in Goto, T.et al, blood (1994)84, 1992-1930.

Plasmacytic cell line KPC-32 (1X 10) negative for EB virus nucleic acid antigen (EBNA) from multiple myeloma patients⁷Cells) (Goto, T. et al, Japan clinical practiceJournal of hematology (11991), 32, 1400) BALB/c mice (Charles River breed) were intraperitoneally infused 2 times every 6 weeks.

To further increase the titer of antibody synthesis, 1.5X 10 animals were sacrificed 3 days before killing⁶KPC-32 cells were injected into the mouse spleen (Goto, t. et al, Tokushima j. exp. med (1990)37, 89). After killing the mice, the spleens were removed, according to Groth, de st.&Splenocytes obtained by Schreidegger (tumor research (1981)41, 3465) were cell fused with myeloma cell SP 210.

Hybridoma culture supernatants were screened for antibodies by ELISA (Posner, M.R. et al, J. Immunol. method (1982)48, 23) using KPC-32 cell-covered plates. Will be 5X 10⁴KPC-32 cells were suspended in 50ml of PBS and aliquoted into 96-well plates (U-shaped plates, produced by Corning, Iwaki). After blocking with PBS containing 1% Bovine Serum Albumin (BSA), hybridoma supernatants were added and incubated at 4 ℃ for 2 hours. Next, it was reacted with peroxidase-labeled goat anti-mouse IgG antibody (produced by Zymed) at 4 ℃ for 1 hour, washed 1 time, and reacted with an o-phenylenediamine substrate solution (produced by Sumitomo Bakelite) at room temperature for 30 minutes.

After the reaction was terminated with 2N sulfuric acid, the absorbance at 492nm was measured by an ELISA reader (manufactured by Bio-Rad). To obtain hybridomas that synthesize anti-human immunoglobulin antibodies, positive hybridoma culture supernatants were screened that had previously adsorbed to human serum and reacted with other subcellular components. Positive hybridomas were selected and their reactivity with various cell lines and human samples was studied by flow cytometry. The finally selected hybridoma clones were subjected to two asexual passages and injected into the peritoneal cavity of pristane-treated BALB/C mice, from which ascites fluid was then obtained.

The monoclonal antibody was purified from the mouse ascites by ammonium sulfate precipitation and protein A affinity chromatography kit (Ampure PA, produced by Amersham). The purified antibody was conjugated to Fluorescein Isothiocyanate (FITC) using Quick Tag FITC conjugation kit (produced by Boehringer Mannheim).

As a result, 30 hybridoma clones synthesized monoclonal antibodies reacted with KPC-32 and RPMI8226 cells. After clonal passage, the reactivity of these hybridoma supernatants with other cell lines and monocytes from peripheral blood was investigated.

Among them, three clones synthesized monoclonal antibodies specifically reactive with plasma cells. Out of these three clones, a hybridoma clone capable of synthesizing a monoclonal antibody useful for flow cytometric analysis and having completely dependent cytotoxicity to RPMI8226 cells was selected and designated HM 1.24. The subclass of the monoclonal antibody synthesized by this hybridoma was determined by ELISA using a rabbit anti-mouse antibody (produced by Zymed) of which subclass is specific. The anti-HM 1.24 antibody has the subclass IgG2 aK. Hybridomas synthesizing anti-HM 1.24 antibodies were stored under the terms of the Budapest treaty at 9/14.1995 by international convention at the national institute of bioscience and human technology, agency of Industrial science and technology, MITI (Higashi 1-Chome 1-3, Tsukuba City, Ibvalak iPrefecture, Japan), and were assigned the accession number FERM BP-5233.

Reference example 2 cloning of cDNA encoding HM1.24 antigen polypeptide

Construction of cDNA library

1) Preparation of Total RNA

A cDNA encoding the HM1.24 antigen, which is a polypeptide specifically recognized by the mouse anti-HM 1.24 monoclonal antibody, was isolated as follows. Total RNA was prepared from the human multiple myeloma cell line KPMM2 according to the method of Chirgwin et al (biochemistry, 18, 5294 (1979)). Thus 2.2X 10⁸KPPM2 cells were thoroughly homogenized in 20ml of 4M guanidinium thiocyanate (produced by Nakalai tesque).

The homogenate was placed on a 5.3M palladium chloride supernatant in a centrifuge tube and then centrifuged at 31,000rpm for 24 hours at 20 ℃ using a Beckman SW40 rotor to pellet the RNA. The RNA pellet was washed with 70% ethanol and dissolved in 300. mu.l of 10nm Tris-HCl (pH7.4) containing 1mM EDTA and 0.5% SDS. Pronase (manufactured by Boehringer) was added thereto to a concentration of 0.5mg/ml, followed by incubation at 37 ℃ for 30 minutes. The mixture was extracted with phenol and chloroform to precipitate RNA. The RNA pellet was then dissolved in 200. mu.l of 10mM Tris-HCl (pH7.4) containing 1mM EDTA.

2) Preparation of Poly (A) + RNA

Using about 500. mu.g of the above-prepared total RNA as a starting material, Poly (A) was purified using Fast Track 2.0mRNA isolation kit (manufactured by Invitrogen) according to the instructions attached to the kit⁺RNA。

3) construction of cDNA library

With 10. mu.g of above Poly (A)⁺RNA was used as a starting material, double-stranded cDNA was synthesized using cDNA synthesis kit Time Saver cDNA synthesis kit (produced by Pharmacia) according to the instructions attached to the kit, and EcoRI linker was ligated to the double-stranded cDNA using the directional cloning kit (produced by Pharmacia) according to the instructions attached to the kit. The EcoRI linker was treated with restriction enzyme NotI according to the instructions attached to the kit. Linker-ligated double-stranded cDNA of about 500bp or more was isolated and purified by 1.5% low melting point agarose gel (manufactured by SIGMA) to obtain about 40. mu.l of linker-ligated double-stranded cDNA.

The adaptor-ligated double-stranded cDNA prepared above was ligated to a pCOS1 vector (Japanese unexamined patent publication (Kokai) No. 8-255196) using T4 DNA ligase (manufactured by GIBCO BRL) to construct a cDNA library, wherein the pCOS1 vector was previously treated with restriction enzymes EcoRI and NotI and alkaline phosphatase (Takara Shuzo). The constructed cDNA library was transferred into E.coli DH 5. alpha. (produced by GIBCO BRL) and had a total size of about 2.5X 10⁶Independent cloning.

2. Cloning by directed expression

1) Transfection into COS-7 cells

By transducing the above transduced Escherichia coli 5X 10⁵Each clone was amplified by culturing it in 2-YT medium (molecular cloning: A laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press, (1989)) containing 50. mu.g/ml ampicillin, and the plasmid was recovered from E.coli by alkaline lysis (molecular cloning: a laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press, (1989)). ObtainThe resulting plasmid was transfected into COS-7 cells by electroporation using a gene pulser (manufactured by BioRad).

Thus, 10. mu.g of purified plasmid DNA was added at 1X 10⁷Cells/ml were suspended in 0.8ml COS-7 cells in PBS and pulsed for 1500V and 25UF capacitance. After a10 minute recovery period at room temperature, cells were electroporated at 37 ℃ and 5% CO₂Was cultured in DMEM (produced by GIBCO BRL) supplemented with 10% fetal bovine serum for 3 days under the conditions of (1).

2) Preparation of panning plates

Panning plates covered with mouse anti-HM 1.24 antibody were prepared using the method of B.seed et al (national academy of sciences USA, 84, 3365-. The mouse anti-HM 1.24 antibody was added to 50mM Tris-HCl (pH9.5) to a concentration of 10. mu.g/ml. 3ml of the antibody solution thus prepared was added to a combined culture plate having a diameter of 60mm, and incubated at room temperature for 2 hours. Washed 3 times with 0.5M NaCl solution and with 5% fetal bovine serum, 1mM EDTA and 0.02% NaN₃After blocking with PBS, these plates were used for the following clones.

3) cloning of cDNA library

COS-7 cells transfected as described above were detached with PBS containing 5mM EDTA and washed once with PBS containing 5% fetal bovine serum. These cells were suspended in a suspension containing 5% fetal bovine serum and 0.02% NaN₃To a concentration of 1X 10 in PBS⁶Cells/ml were then added to the panning plates prepared above and incubated for 2 hours at room temperature. Using a mixture of 5% fetal calf serum and 0.02% NaN₃After washing 3 times with PBS, plasmid DNA was recovered from the cells adhered to the panning plates using a solution containing 0.6% SDS and 10mM EDTA.

The recovered plasmid DNA was again transferred into E.coli DH5 α. After amplifying the plasmid DNA as described above, the plasmid DNA was extracted by an alkaline lysis method. The plasmid DNA recovered as described above was transfected into COS-7 cells by electroporation and the plasmid DNA was recovered from adherent cells. The same procedure was repeated once more and the recovered DNA was digested with restriction enzymes EcoRI and NotI. Thus confirming an insert having a size of 0.9 kbp. Part of the recovered plasmid DNA transduced E.coli was inoculated on 2-YT agar plates containing 50. mu.g/ml of ampicillin. After overnight incubation, plasmid DNA was recovered from the single clones. This plasmid was digested with the restriction enzymes EcoRI and NotI, giving clone p3.19 with a 0.9kbp insert.

The base sequence of this clone was determined by terminating the cycle sequencing kit (manufactured by Perkin Elmer) reaction with PRISM dye and sequencing with ABI 373A DNA sequencer (manufactured by Perkn Elmer) according to the instructions attached to the kit. The amino acid sequence and the base sequence thereof are shown in SEQ ID No: 103.

encoding the polypeptide with the sequence shown in SEQ ID No: 103 was inserted into the XbaI cleavage site of the pUC19 vector, and the resulting plasmid was designated pRS38-pUC 19. Escherichia coli containing plasmid pRS38-pUC19 was stored as Escherichia coli DH5 alpha (pRS38-pUC19) under the terms of the Budapest treaty at the national institute of bioscience and human technology, agency of Industrial science and technology, MITI (Higashi 1-Chome 1-3, Tsukuba City, Ibalaki Prefecture, Japan) by the international convention on 10.5.1993, and was given a accession number FERM BP-4434 (see Japanese unexamined patent publication (Kokai) No. 7-196694).

Industrial applications

Since the chimeric anti-HM 1.24 antibody contains the variable regions of the mouse anti-HM 1.24 antibody and the constant regions of the human antibody, the reconstructed human anti-HM 1.24 antibody contains the complementarity determining regions of the mouse anti-HM 1.24 antibody, the framework regions of the human antibody, and the constant regions of the human antibody, which has low antigenicity to the human body, and is promising as a pharmaceutical ingredient, particularly for the treatment of myeloma.

Deposited organism and reference to international depository

Name: national institute of Life sciences and human technology, agency of Industrial science and technology, MITI

Address: higashi 1-Chome 1-3, Tsukuba City, Ibalaki prefecture, Japan

1. Escherichia coli DH5 alpha (pRS38-pUC19)

The preservation number is as follows: FERM BP-4434

The preservation date is as follows: 1993 at 10 months and 5 days

2. Mouse-mouse hybridoma HM1.24

The preservation number is as follows: FERM BP-5233

The preservation date is as follows: 1995, 4 months and 27 days

3. Escherichia coli DH5 alpha (pUC19-RVHr-AHM-g gamma 1)

The preservation number is as follows: FERM BP-5643

The preservation date is as follows: 1996, 8, 29 months

4. Escherichia coli DH5 alpha (pUC19-1.24H-g gamma 1)

The preservation number is as follows: FERM BP-5644

The preservation date is as follows: 1996, 8, 29 months

5. Escherichia coli DH5 alpha (pUC19-RVLa-AHM-g kappa)

The preservation number is as follows: FREM BP-5645

The preservation date is as follows: 1996, 8, 29 months

6. Escherichia coli DH5 alpha (pUC19-1.24L-g kappa)

The preservation number is as follows: FERM BP-5646

The preservation date is as follows: 1996, 8, 29 months

7. Escherichia coli DH5 alpha (pUC19-RVHs-AHM-g gamma 1)

The preservation number is as follows: FERM BP-6127

The preservation date is as follows: 9/29/1997

Sequence listing

With respect to SEQ ID NO: 1 information

Length: 394

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GGC TTC AAG ATG GAG TCA CAT TTT CTG GTC TTT GTA TTC GTG TTT 48

Met Gly Phe Lys Met Glu Ser His Phe Leu Val Phe Val Phe Val Phe

-20 -15 -10

CTC TGG TTG TCT GGT GTT GAC GGA GAC ATT GTG ATG ACC CAG TCT CAC 96

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His

-5 -1 1 5

AAA TTC ATG TCC ACA TCA GTA GGA GAC AGG GTC AGC ATC ACC TGC AAG 144

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

10 15 20

GCC AGT CAG GAT GTG AAT ACT GCT GTA GCC TGG TAT CAA CAA AAA CCA 192

Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

25 30 35 40

GGA CAA TCG CCT AAA CTA CTG ATT TAC TCG GCA TCC AAC CGG TAC ACT 240

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr

45 50 55

GGA GTC CCT GAT CGC ATC ACT GGC AGT GGA TCT GGG ACG GAT TTC ACT 288

Gly Val Pro Asp Arg Ile Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

60 65 70

TTC ACC ATC AGC AGT GTG CAG GCG GAA GAC CTG GCA CTT TAT TAC TGT 336

Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

75 80 85

CAG CAA CAT TAT AGT ACT CCA TTC ACG TTC GGC TCG GGG ACA AAG TTG 384

Gln Gln His Tyr Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

90 95 100

GAA ATA AAA C 394

Glu Ile Lys

105

with respect to SEQ ID NO: 2 information

Length: 418

Type (2): nucleic acids

Topological structure: line shape

Molecular type: cDNA

Description of the sequence:

ATG GAA TGT AAC TGG ATA CTT CCT TTT ATT CTG TCA GTA ACT TCA GGT 48

Met Glu Cys Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly

-15 -10 -5

GCC TAC TCA CAG GTT CAA CTC CAG CAG TCT GGG GCT GAG CTG GCA AGA 96

Ala Tyr Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

-1 1 5 10

CCT GGG GCT TCA GTG AAG TTG TCC TGC AAG GCT TCT GGC TAC ACC TTT 144

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

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTA AAA CAG AGG CCT GGA CAG GGT CTG 192

Thr Pro Tyr Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

30 35 40 45

GAA TGG ATT GGG TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Ile Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAG GCC ACA TTG ACT GCA GAT AAA TCC TCC AGT 288

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

65 70 75

ACA GCC TAC ATG CAA CTC AGC ATC TTG GCA TTT GAG GAC TCT GCG GTC 336

Thr Ala Tyr Met Gln Leu Ser Ile Leu Ala Phe Glu Asp Ser Ala Val

80 85 90

TAT TAC TGT GCA AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 3 information

Length: 11

Type (2): amino acids

Topological structure: wire type

Molecular type: peptides

Description of the sequence:

Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala

5 10

with respect to SEQ ID NO: 4 information

Length: 7

Type (2): amino acids

Topological structure: wire type

Molecular type: peptides

Description of the sequence:

Ser Ala Ser Asn Arg Tyr Thr

5

with respect to SEQ ID NO: 5 information

Length: 9

Type (2): amino acids

Topological structure: wire type

Molecular type: peptides

Description of the sequence:

Gln Gln His Tyr Ser Thr Pro Phe Thr

5

regarding SE Q ID NO: 6 information

Length: 5

Type (2): amino acids

Topological structure: wire type

Molecular type: peptides

Description of the sequence:

Pro Tyr Trp Met Gln

5

with respect to SEQ ID NO: 7 information

Length: 16

Type (2): amino acids

Topological structure: wire type

Molecular type: peptides

Description of the sequence:

Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly

5 10 15

with respect to SEQ ID NO: 8 information

Length: 11

Type (2): amino acids

Topological structure: wire type

Molecular type: peptides

Description of the sequence:

Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

5 10

with respect to SEQ ID NO: 9 information

Length: 379

Type (2): nucleic acids

Topological structure: line shape

Molecular type: cDNA

Description of the sequence:

ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48

Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly

-15 -10 -5

GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96

Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

-1 1 5 10

AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCT AGT CAG GAT GTG 144

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val

15 20 25

AAT ACT GCT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG 192

Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

30 35 40 45

CTG CTG ATC TAC TCG GCA TCC AAC CGG TAC ACT GGT GTG CCA AGC AGA 240

Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg

50 55 60

TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC 288

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser

65 70 75

CTC CAG CCA GAG GAC ATC GCT ACC TAC TAC TGC CAG CAA CAT TAT AGT 336

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser

80 85 90

ACT CCA TTC ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA C 379

Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

95 100 105

with respect to SEQ ID NO: 10 information

Length: 379

Type (2): nucleic acids

Topological structure: line shape

Molecular type: cDNA

Description of the sequence:

ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48

Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly

-15 -10 -5

GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96

Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

-1 1 5 10

AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCT AGT CAG GAT GTG 144

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val

15 20 25

AAT ACT GCT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG 192

Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

30 35 40 45

CTG CTG ATC TAC TCG GCA TCC AAC CGG TAC ACT GGT GTG CCA AGC AGA 240

Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg

50 55 60

TTC AGC GGT AGC GGT AGT GGT ACC GAC TAC ACC TTC ACC ATC AGC AGC 288

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser

65 70 75

CTC CAG CCA GAG GAC ATC GCT ACC TAC TAC TGC CAG CAA CAT TAT AGT 336

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Sar

80 85 90

ACT CCA TTC ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA C 379

Thr Pro Phe Thr Phe GLy Gln Gly Thr Lys Val Glu Ile Lys

95 100 105

with respect to SEQ ID NO: 11 information

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 12 information

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 13 information

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 95

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACT ATG ACC GCA GAC AAG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 14 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC AAG TCC ACG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Lys Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 15 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GCC ACC CTG ACC GCA GAC ACG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 16 information

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GCC ACC CTG ACT GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAG TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAG TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 17 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG CGC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Arg Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 18 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 19 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG GCA TTT GAG GAC ACG GCC GTG 336

Thr Ala Tyr Met Glu Leu Ser Ser Leu Ala Phe Glu Asp Thr Ala Val

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 20 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GCC ACC CTG ACT GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 21 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG CAG CTG AGC AGC CTA AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 22 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG CAG CTG AGC ATC CTG AGA TCT GAG GAC ACG GCC GTG 336

Thr Ala Tyr Met Gln Leu Ser Ile Leu Arg Sar Glu Asp Thr Ala Val

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 23 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG CAG CTG AGC ATC CTG AGA TCT GAG GAC TCG GCC GTG 336

Thr Ala Tyr Met Gln Leu Ser Ile Leu Arg Ser Glu Asp Ser Ala Val

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 24 information

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC ATC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 25 of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC TCG GCC GTA 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 26 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 27 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATG ACC GCA GAC ACG TCC TCG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ser Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 28 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATG ACC GCA GAC AAG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 29 information of

Length: 40

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGAAGTTGC CTGTTAGGCT GTTGGTGCTG 40

with respect to SEQ ID NO: 30 information of

Length: 39

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGGAGWCAG ACACACTCCT GYTATGGGT 39

with respect to SEQ ID NO: 31 information of

Length: 40

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGAGTGTGC TCACTCAGGT CCTGGSGTTG 40

with respect to SEQ ID NO: 32 information of

Length: 43

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGAGGRCCC CTGCTCAGWT TYTTGGMWTC TTG 43

with respect to SEQ ID NO: 33 information of

Length: 40

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGGATTTWC AGGTGCAGAT TWTCAGCTTC 40

with respect to SEQ ID NO: 34 information of

Length: 37

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGAGGTKCY YTGYTSAGYT YCTGRGG 37

with respect to SEQ ID NO: 35 information of

Length: 41

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGGGCWTCA AGATGGAGTC ACAKWYYCWG G 41

with respect to SEQ ID NO: 36 of

Length: 41

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGTGGGGAY CTKTTTYCMM TTTTTCAATT G 41

with respect to SEQ ID NO: 37 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGGTRTCCW CASCTCAGTT CCTTG 35

with respect to SEQ ID NO: 38 information of

Length: 37

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGTATATAT GTTTGTTGTC TATTTCT 37

with respect to SEQ ID NO: 39 information of

Length: 38

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACTAGTCGAC ATGGAAGCCC CAGCTCAGCT TCTCTTCC 38

with respect to SEQ ID NO: 40 information

Length: 27

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GGATCCCGGG TGGATGGTGG GAAGATG 27

with respect to SEQ ID NO: 41 information

Length: 25

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

TAGAGTCACC GAGGAGCCAG TTGTA 25

with respect to SEQ ID NO: 42 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GGATCCCGGG AGTGGATAGA CCGATG 26

with respect to SEQ ID NO: 43 information

Length: 34

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GATAAGCTTC CACCATGGGC TTCAAGATGG AGTC 34

with respect to SEQ ID NO: 44 information of

Length: 34

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GATAAGCTTC CACCATGGAA TGTAACTGGA TACT 34

with respect to SEQ ID NO: 45 information

Length: 34

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GGCGGATCCA CTCACGTTTT ATTTCCAACT TTGT 34

with respect to SEQ ID NO: 46 of a mobile phone

Length: 34

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GGCGGATCCA CTCACCTGAG GAGACTGTGA GAGT 34

with respect to SEQ ID NO: 47 information

Length: 18

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

CAGACAGTGG TTCAAAGT 18

with respect to SEQ ID NO: 48 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GAATTCGGAT CCACTCACGT TTGATT 26

with respect to SEQ ID NO: 49 information

Length: 48

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGTCAGGATG TGAATACTGC TGTAGCCTGG TACCAGCAGA AGCCAGGA 48

with respect to SEQ ID NO: 50 information

Length: 39

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCATCCAACC GGTACACTGG TGTGCCAAGC AGATTCAGC 39

with respect to SEQ ID NO: 51 information

Length: 45

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

CAACATTATA GTACTCCATT CACGTTCGGC CAAGGGACCA AGGTG 45

with respect to SEQ ID NO: 52 information of

Length: 47

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCAGTATTCA CATCCTGACT GGCCTTACAG GTGATGGTCA CTCTGTC 47

with respect to SEQ ID NO: 53 information of

Length: 38

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACACCAGTGT ACCGGTTGGA TGCCGAGTAG ATCAGCAG 38

with respect to SEQ ID NO: 54 of the image data

Length: 41

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTGAATGGAG TACTATAATG TTGCTGGCAG TAGTAGGTAG C 41

with respect to SEQ ID NO: 55 information

Length: 31

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GGTACCGACT ACACCTTCAC CATCAGCAGC C 31

with respect to SEQ ID NO: 56 information

Length: 31

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GGTGAAGGTG TAGTCGGTAC CGCTACCGCT A 31

with respect to SEQ ID NO: 57 information

Length: 144

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ATGCCTTGCA GGAAACCTTC ACTGAGGCCC CAGGCTTCTT CACCTCAGCC CCAGACTGCA 60

CCAGCTGCAC CTGGGAGTGA GCACCTGGAG CTACAGCCAG CAAGAAGAAG ACCCTCCAGG 120

TCCAGTCCAT GGTGGAAGCT TATC 144

with respect to SEQ ID NO: 58 information

Length: 130

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

TCAGTGAAGG TTTCCTGCAA GGCATCTGGA TACACCTTCA CTCCCTACTG GATGCAGTGG 60

GTGCGACAGG CCCCTGGACA AGGGCTTGAG TGGATGGGAT CTATTTTTCC TGGAGATGGT 120

GATACTAGGT 130

with respect to SEQ ID NO: 59 information of

Length: 131

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AATACACGGC CGTGTCCTCA GATCTCAGGC TGCTCAGCTC CATGTAGACT GTGCTCGTGG 60

ACGTGTCTGC GGTCATGGTG ACTCTGCCCT TGAACTTCTG ACTGTACCTA GTATCACCAT 120

CTCCAGGAAA A 131

with respect to SEQ ID NO: 60 information

Length: 119

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GAGATCTGAG GACACGGCCG TGTATTACTG TGCGAGAGGA TTACGACGAG GGGGGTACTA 60

CTTTGACTAC TGGGGGCAAG GGACCACGGT CACCGTCTCC TCAGGTGAGT GGATCCGAC 119

with respect to SEQ ID NO: 61 information

Length: 25

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GATAAGCTTC CACCATGGAC TGGAC 25

with respect to SEQ ID NO: 62 information

Length: 25

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTCGGATCCA CTCACCTGAG GAGAC 25

with respect to SEQ ID NO: 63 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AAGTTCAAGG GCAAAGTCAC CATGAC 26

with respect to SEQ ID NO: 64 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTCATGGTGA CTTTGCCCTT GAACTT 26

with respect to SEQ ID NO: 65 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ATGACCGCAG ACAAGTCCAC GAGCAC 26

with respect to SEQ ID NO: 66 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTGCTCGTGG ACTTGTCTGC GGTCAT 26

with respect to SEQ ID NO: 67 information

Length: 46

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AAGTTCAAGG GCAAAGTCAC CATGACCGCA GACAAGTCCA CGAGCAC 46

with respect to SEQ ID NO: 68 information

Length: 47

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTGCTCGTGG ACTTGTCTGC GGTCATGGTG ACTTTGCCCT TGAACTT 47

with respect to SEQ ID NO: 69 of the information

Length: 38

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AAGTTCAAGG GCAGAGCCAC CCTGACCGCA GACACGTC 38

with respect to SEQ ID NO: 70 information of

Length: 38

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GACGTGTCTG CGGTCAGGGT GGCTCTGCCC TTGAACTT 38

with respect to SEQ ID NO: 71 information

Length: 18

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

CAGACAGTGG TTCAAAGT 18

with respect to SEQ ID NO: 72 information

Length: 17

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCCCCAAAGC CAAGGTC 17

with respect to SEQ ID NO: 73 information

Length: 23

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ATTTTTCCTG GAGATGGTGA TAC 23

with respect to SEQ ID NO: 74 information

Length: 23

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTATCACCAT CTCCAGGAAA TAT 23

with respect to SEQ ID NO: 75 information of

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAA TGT AAC TGG ATA CTT CCT TTT ATT CTG TCA GTA ACT TCA GGT 48

Met Glu Cys Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly

-15 -10 -5

GCC TAC TCA CAG GTT CAA CTC CAG CAG TCT GGG GCT GAG CTG GCA AGA 96

Ala Tyr Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

-1 1 5 10

CCT GGG GCT TCA GTG AAG TTG TCC TGC AAG GCT TCT GGC TAC ACC TTT 144

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

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTA AAA CAG AGG CCT GGA CAG GGT CTG 192

Thr Pro Tyr Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

30 35 40 45

GAA TGG ATT GGG TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Ile Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser

65 70 75

ACA GTC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 76 of a mobile communication terminal

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AAG GCC ACA TTG ACT GCA GAT AAA TCC TCC AGT 288

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

65 70 75

ACA GCC TAC ATG CAA CTC AGC ATC TTG GCA TTT GAG GAC TCT GCG GTC 336

Thr Ala Tyr Met Gln Leu Ser Ile Leu Ala Phe Glu Asp Ser Ala Val

80 85 90

TAT TAC TGT GCA AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GLy Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 77 information

Length: 38

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG 38

with respect to SEQ ID NO: 78 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCAGACACGT CCTCGAGCAC AGCCTACATG GAGCT 35

with respect to SEQ ID NO: 79 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGCTCCATGT AGGCTGTGCT CGAGGACGTG TCTGC 35

with respect to SEQ ID NO: 80 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

TGGGTGCGAC AGCGCCCTGG ACAAGG 26

with respect to SEQ ID NO: 81 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

CCTTGTCCAG GGCGCTGTCG CACCCA 26

with respect to SEQ ID NO: 82 information

Length: 41

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

TACATGGAGC TGAGCAGCCT GGCATTTGAG GACACGGCCG T 41

with respect to SEQ ID NO: 83 information of

Length: 41

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACGGCCGTGT CCTCAAATGC CAGGCTGCTC AGCTCCATGT A 41

with respect to SEQ ID NO: 84 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AAGTTCAAGG GCAAAGCCAC CCTGAC 26

with respect to SEQ ID NO: 85 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GTCAGGGTGG CTTTGCCCTT GAACTT 26

with respect to SEQ ID NO: 86 information of

Length: 23

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCCTACATGC AGCTGAGCAG CCT 23

with respect to SEQ ID NO: 87 of the

Length: 23

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGGCTGCTCA GCTGCATGTA GGC 23

with respect to SEQ ID NO: 88 information

Length: 38

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCCTACATGC AGCTGAGCAT CCTGAGATCT GAGGACAC 38

with respect to SEQ ID NO: 89 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GATCTCAGGA TGCTCAGCTG CATGTAGGCT GTGCT 35

with respect to SEQ ID NO: 90 information

Length: 50

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCCTACATGC AGCTGAGCAT CCTGAGATCT GAGGACTCGG CCGTGTATTA 50

with respect to SEQ ID NO: 91 information of

Length: 50

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACGGCCGAGT CCTCAGATCT CAGGATGCTC AGCTGCATGT AGGCTGTGCT 50

with respect to SEQ ID NO: 92 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GAGCTGAGCA TCCTGAGATC 20

with respect to SEQ ID NO: 93 information of

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GATCTCAGGA TGCTCAGCTC CATGTA 26

with respect to SEQ ID NO: 94 information

Length: 20

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGATCTGAGG ACTCGGCCGT 20

with respect to SEQ ID NO: 95 information

Length: 20

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

ACGGCCGAGT CCTCAGATCT 20

with respect to SEQ ID NO: 96 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCAGACACGT CCACGAGCAC AGCCTACATG GAGCT 35

with respect to SEQ ID NO: 97 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGCTCCATGT AGGCTGTGCT CGTGGACGTG TCTGC 35

with respect to SEQ ID NO: 98 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GCAGACACGT CCTCGAGCAC AGTCTACATG GAGCT 35

with respect to SEQ ID NO: 99 information

Length: 35

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGCTCCATGT AGACTGTGCT CGAGGACGTG TCTGC 35

with respect to SEQ ID NO: 100 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

AGAGTCACCA TCACCGCAGA CAAGTC 26

with respect to SEQ ID NO: 101 information

Length: 26

Type (2): nucleic acids

Topological structure: wire type

Molecular type: synthesis of DNA

Description of the sequence:

GACTTGTCTG CGGTGATGGT GACTCT 26

with respect to SEQ ID NO: 102 information

Length: 418

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly

-15 -10 -5

GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG 96

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

-1 1 5 10

CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

15 20 25

ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT 192

Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

30 35 40 45

GAG TGG ATG GGA TCT ATT TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT 240

Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser

50 55 60

CAG AAG TTC AAG GGC AGA GTC ACC ATC ACC GCA GAC AAG TCC ACG AGC 288

Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser

65 70 75

ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG 336

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

80 85 90

TAT TAC TGT GCG AGA GGA TTA CGA CGA GGG GGG TAC TAC TTT GAC TAC 384

Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

95 100 105

TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

110 115 120

with respect to SEQ ID NO: 103 information

Length: 1013

Type (2): nucleic acids

Topological structure: wire type

Molecular type: cDNA

Description of the sequence:

GAATTCGGCA CGAGGGATCT GG ATG GCA TCT ACT TCG TAT GAC TAT TGC 49

Met Ala Ser Thr Ser Tyr Asp Tyr Cys

1 5

AGA GTG CCC ATG GAA GAC GGG GAT AAG CGC TGT AAG CTT CTG CTG GGG 97

Arg Val Pro Met Glu Asp Gly Asp Lys Arg Cys Lys Leu Leu Leu Gly

10 15 20 25

ATA GGA ATT CTG GTG CTC CTG ATC ATC GTG ATT CTG GGG GTG CCC TTG 145

Ile Gly Ile Leu Val Leu Leu Ile Ile Val Ile Leu Gly Val Pro Leu

30 35 40

ATT ATC TTC ACC ATC AAG GCC AAC AGC GAG GCC TGC CGG GAC GGC CTT 193

Ile Ile Phe Thr Ile Lys Ala Asn Ser Glu Ala Cys Arg Asp Gly Leu

45 50 55

CGG GCA GTG ATG GAG TGT CGC AAT GTC ACC CAT CTC CTG CAA CAA GAG 241

Arg Ala Val Met Glu Cys Arg Asn Val Thr His Leu Leu Gln Gln Glu

60 65 70

CTG ACC GAG GCC CAG AAG GGC TTT CAG GAT GTG GAG GCC CAG GCC GCC 289

Leu Thr Glu Ala Gln Lys Gly Phe Gln Asp Val Glu Ala Gln Ala Ala

75 80 85

ACC TGC AAC CAC ACT GTG ATG GCC CTA ATG GCT TCC CTG GAT GCA GAG 337

Thr Cys Asn His Thr Val Met Ala Leu Met Ala Ser Leu Asp Ala Glu

90 95 100 105

AAG GCC CAA GGA CAA AAG AAA GTG GAG GAG CTT GAG GGA GAG ATC ACT 385

Lys Ala Gln Gly Gln Lys Lys Val Glu Glu Leu Glu Gly Glu Ile Thr

110 115 120

ACA TTA AAC CAT AAG CTT CAG GAC GCG TCT GCA GAG GTG GAG CGA CTG 433

Thr Leu Asn His Lys Leu Gln Asp Ala Ser Ala Glu Val Glu Arg Leu

125 130 135

AGA AGA GAA AAC CAG GTC TTA AGC GTG AGA ATC GCG GAC AAG AAG TAC 481

Arg Arg Glu Asn Gln Val Leu Ser Val Arg Ile Ala Asp Lys Lys Tyr

140 145 150

TAC CCC AGC TCC CAG GAC TCC AGC TCC GCT GCG GCG CCC CAG CTG CTG 529

Tyr Pro Ser Ser Gln Asp Ser Ser Ser Ala Ala Ala Pro Gln Leu Leu

155 160 165

ATT GTG CTG CTG GGC CTC AGC GCT CTG CTG CAG TGA GATCCCAGGA 575

Ile Val Leu Leu Gly Leu Ser Ala Leu Leu Gln ***

170 175 180

AGCTGGCACA TCTTGGAAGG TCCGTCCTGC TCGGCTTTTC GCTTGAACAT TCCCTTGATC 635

TCATCAGTTC TGAGCGGGTC ATGGGGCAAC ACGGTTAGCG GGGAGAGCAC GGGGTAGCCG 695

GAGAAGGGCC TCTGGAGCAG GTCTGGAGGG GCCATGGGGC AGTCCTGGGT CTGGGGACAC 755

AGTCGGGTTG ACCCAGGGCT GTCTCCCTCC AGAGCCTCCC TCCGGACAAT GAGTCCCCCC 815

TCTTGTCTCC CACCCTGAGA TTGGGCATGG GGTGCGGTGT GGGGGGCATG TGCTGCCTGT 875

TGTTATGGGT TTTTTTTGCG GGGGGGGTTG CTTTTTTCTG GGGTCTTTGA GCTCCAAAAA 935

AATAAACACT TCCTTTGAGG GAGAGCACAC CTTAAAAAAA AAAAAAAAAA AAAAAAAAAA 995

AAAATTCGGG CGGCCGCC 1013

Claims (6)

1. A reconstructed human antibody against HM1.24 antibody, consisting of

(A) L chain consisting of

(1) Human L chain C region, and

(2) an L chain V region consisting of the FR of human L chain and the L chain CDR of anti-HM 1.24 antibody; and

(B) h chain consisting of

(1) Human H chain C region, and

(2) an H chain V region consisting of the FR of human H chain and the CDR of H chain of anti-HM 1.24 antibody,

wherein the L chain CDR has an amino acid sequence represented by the following amino acid sequence,

CDR1：Lts Ala Ser Gln Asp Val Asn Thr Ala Val Ala(SEQ ID NO：3)

CDR2：Ser Ala Ser Asn Arg Tyr Thr(SEQ ID NO：4)

CDR3：Gln Gln His Tyr Ser Thr Pro Phe Thr(SEQ ID NO：5)

and the H chain CDR has an amino acid sequence represented by the following amino acid sequence,

CDR1：Pro Tyr Trp Met Gln(SEQ ID NO：6)

CDR2：Ser Ile Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly(SEQ ID NO：7)

CDR3：Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr(SEQ ID NO：8).

2. the reconstructed human antibody according to claim 1, wherein the L chain CDR consists of an amino acid sequence set forth in claim 1; the H chain CDR composed of the amino acid sequence shown in claim 1; the FR of the human L chain is derived from the FR of a human antibody of HSGI; the FR of the human H chain is derived from the FR of a human antibody of HSGI; the human L chain C region is a human C kappa region; and the human H chain C region is a human C gamma 1 region.

3. The reconstructed human antibody according to claim 1, wherein the FR of the L chain is derived from the FR of human antibody REI, the FR1-3 of the H chain is derived from human antibody HG3, and the FR4 of the H chain is derived from the FR4 of human antibody JH 6.

4. The reconstructed human antibody according to claim 1, wherein the L chain V region has an amino acid sequence represented by RVLa in Table 1.

5. The reconstructed human antibody according to claim 1, wherein the H chain V region has an amino acid sequence represented by any one of RVHf, RVHh, RVHi, RVHj, RVHk, RVH1, RVHm, RVHn, RVHo, RVHp, RVHr, or RVHs in Table 2 to Table 4.

6. A method for producing a reconstituted human antibody against HM1.24 antibody comprising the steps of: culturing a host cell co-transformed with an expression vector comprising DNA encoding the H chain of the antibody of any one of claims 1-5 and an expression vector comprising DNA encoding the L chain of the antibody of any one of claims 1-5; and recovering the desired antibody.