AU761036B2 - Reconstituted human anti-human 1.24 antibody - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

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0 Name of Applicant: Actual Inventors: .59.00 0 00e* 003* Chugai Seiyaku Kabushiki Kaisha Koichiro ONO, Toshihiko OHTOMO, Masayuki TSUCHIYA, Yasushi YOSHIMURA, Yasuo KOISHIHARA and Masaaki KOSAKA DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Address for Service: Invention Title: "Reconsitututed human anti-human 1.24 antibody" Details of Parent Application No: 19490/00 The following statement is a full description of this invention, including the best method of performing it known to us: -1A-

DESCRIPTION

RESHAPED HUMAN ANTI-HM 1.24 ANTIBODY Field of the Invention The present invention relates to reshaped human anti-HM 1.24 antibodies and chimeric anti-HM 1.24 antibodies, genes encoding them, methods for producing said antibodies, and the use of said antibodies. The reshaped human antibodies and the chimeric antibodies of the present invention are useful as a therapeutic agent, etc. for myeloma.

Background Art Human B cells go through a variety of processes that are classified based on the kind of surface antigens 15 being expressed, and finally mature into antibody-producing plasma cells. At the final stage of their differentiation, B cells, on one hand, acquire the ~ability of producing cytoplasmic immunoglobulins and, on the other, B cell-associated antigens such as cell 20 surface immunoglobulins, HLA-DR, CD20, Fc receptors, complement C3 receptors and the like disappear (Ling, N.R. et al., Leucocyte Typing III (1986) p320, Oxford, UK, Oxford).

So far, there have been reports on monoclonal antiboies such as anti-PCA-1 (Anderson, K.C. et al., J.

Immunol. (1983) 130, 1132), anti-PC-i (Anderson, K.C. et al., J. Immunol. (1983) 132, 3172), anti-MM4 (Tong, A.W.

et al., Blood (1987) 69, 238) and the like that recognize antigens on the cell membrane of the plasma cells.

However, anti-CD38 monoclonal antibody is still being used for detection of plasma cells and myeloma cells (Epstein, J. et al., N. Engl. J. Med. (1990) 322, 664, Terstappen, L.W.M.M. et al., Blood (1990) 76, 1739, Leo, R. et al., Ann. Hematol. (1992) 64, 132, Shimazaki, C. et al., Am J. Hematol. (1992) 39, 159, Hata, H. et al., Blood (1993) 81, 3357, Harada, H. et al., Blood (1993) 81, 2658, Billadeau, D. et al., J. Exp. Med. (1993) 178, 2 1023).

However, anti-CD38 monoclonal antibody is an antigen associated with activation of T cells rather than an antigen associated with differentiation of B cells, and is expressed on various cells in addition to B cells.

Furthermore, although CD38 is not expressed on some of the lymphoplasmacytoid, it is strongly expressed on the hemopoietic precursor cells. For these reasons, it is believed that anti-CD38 monoclonal antibody is not suitable for research on differentiation and maturation of human B cells or for treatment of diseases of plasma cells.

Goto, T. et al. have reported mouse anti-HM 1.24 monoclonal antibody that recognizes an antigen having a 15 molecular weight of 29 to 33 kDa which is specifically expressed on B cell lines (Blood (1994) 84, 1922-1930).

From the fact that the antigen recognized by anti-HM 1.24 .*monoclonal antibody is believed to be associated with the terminal differentiation of B cells (Goto, T. et al., 20 Jpn. J. Clin. Immun. (1992) 16, 688-691) and that the administration of anti-HM 1.24 monoclonal antibody to a plasmacytoma-transplanted mouse resulted in specific accumulation of the antibody at the tumor (Shuji Ozaki et al., The Program of General Assembly of the 19th Japan Myeloma Study Meeting, general presentation it has been suggested that anti-HM 1.24 monoclonal antibody, by labelling with a radioisotope, may be used for diagnosis of tumor localization, the missile therapy such as radioimmunotherapy, and the like.

Furthermore, the above-mentioned Blood describes that the anti-HM 1.24 monoclonal antibody has the complement-dependent cytotoxicity activity to the human myeloma cell line RPMI8226.

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 3 secrete immunoglobulins, or plasma cells, are monoclonally increased mainly in the bone marrow, and accordingly monoclonal immunoglobulins or the constituting components thereof, L chains or H chains, are detected in the serum (Masaaki Kosaka et al., Nippon Rinsho (1995) 53, 91-99).

Conventionally chemotherapeutic agents have been used for treatment of myeloma, but there have been found no effective therapeutic agents that can lead to remission of myeloma and elongation of the survival period of patients with myeloma. There is, therefore, a long-awaited need for the advent of drugs that have a therapeutic effect on myeloma.

Mouse monoclonal antiboies have high immunogenicity 15 (sometimes referred to as "antigenicity") in humans.

Accordingly, the medical therapeutic value of mouse monoclonal antibodies in humans is limited. For example, a mouse antibody administered into a human may be metabolized as a foreign substance so that the half life 20 of the mouse antibody in the human is relatively short and thereby it cannot fully exhibit its expected effects.

Furthermore, human anti-mouse antibodies that are raised against the administered mouse antibody may trigger immunological responses that are unfavorable and dangerous to the patients, such as serum disease, other allergic reactions, or the like. Therefore, mouse monoclonal antibody cannot be frequently administered into humans.

In order to resolve these problems, a method was developed for reducing the immunogenicity of non-human-derived antibodies such as mouse-derived monoclonal antibodies. As one such example, there is a method of producing a chimeric antibody in which the variable region (V region) of the antibody is derived from the original mouse and the constant region (C region) thereof is derived from an appropriate human antibody.

4 Since the chimeric antibody thus obtained contains the variable region of the original mouse antibody in the intact form, it is expected to bind to the antigen with a specificity identical to that of the original mouse antibody. Furthermore, in a chimeric antibody the ratio of the amino acid sequences derived from non-humans is substantially reduced, and so the antibody is expected to have a low immunogenicity compared to the original mouse antibody. A chimeric antibody may bind to the antigen -in an equal manner to the original mouse monoclonal antibody, and may include immunological responses against ~the mouse variable region though the immunogenicity is reduced (LoBuglio, A.F. et al., Proc. Natl. Acad. Sci.

USA, 86, 4220-4224, 1989).

15 The second method for reducing the immunogenicity of mouse antibody, though much more complicated, can reduce the potential immunogenicity of mouse antibody further greatly. In this method, only the complementarity determining region (CDR) of the variable region of a 20 mouse antibody is grafted to the variable region of a human antibody to prepare a "reshaped" human antibody variable region.

However, In order to make the structure of the CDR of a reshaped human antibody variable region as much close as possible to that of the original mouse antibody, if necessary, part of the amino acid sequence of the framework region (FR) that supports the CDR may be grafted from the variable region of the mouse antibody to the variable region of the human antibody. Subsequently, this V region of the humanized reshaped human antibody is linked to the constant region of a human antibody. The part that is derived from the non-human amino acid sequence in the finally reshaped humanized antibody is the CDR, and only part of the FR. A CDR is composed of hypervariable amino acid sequences which do not exhibit species-specific sequences. Therefore, the humanized antibody carrying the mouse CDR should not have an 5 immunogenicity stronger than the natural human antibody having the human antibody CDR.

For the humanized antibody, 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 Engng., 4, 773-783, 1991; Meada, H. et al., Human Antibodies and Hybridoma, 2, 124-134, 1991; 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, 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad.

Sci. USA, 89, 4285-4289, 1992; Co, M.S. et al., J.

Immunol., 148, 1149-1154, 1992; and Sato, K. et al, Cancer Res., 53, 851-856, 1993.

15 Queen et al. (International Application Publication No. WO 90-07861) describes a method for producing a humanized antibody of an anti-IL-2 receptor antibody Anti-Tac. However, it is difficult to completely humanize all antibodies even following the method as set 20 forth in WO 90-07861. Thus, WO 90-07861 does not describe a general method for humanizing of antibodies, but merely describes a method for humanizing of Anti-Tac antibody which is one of anti-IL-2 receptor antibodies.

Furthermore, even when the method of WO 90-07861 is completely followed, it is difficult to make a humanized antibody that has an activity completely identical to the original mouse antibody.

In general, the amino acid sequences of CDR/FR of individual antibodies are different. Accordingly, the determination of the amino acid residue to be replaced for the construction of a humanized antibody and the selection of the amino acid residue that replaces said amino acid residue vary with individual antibodies.

Therefore, the method for preparing humanized antibodies as set forth in WO 90-07861 cannot be applied to humanization of all antibodies.

Queen et al. Proc. Natl. Acad. Sci. USA, (1989) 86, 6 10029-10033 has a similar disclosure to that of WO 90-07861. This reference describes that only one third of the activity of the original mouse antibody was obtained for a humanized antibody produced according to the method as set forth in WO 90-07861. In other words, this shows that the method of WO 90-07861 itself cannot produce a complete humanized antibody that has an activity equal to that of the original mouse antibody Co et al., Cancer Research (1996) 56, 1118-1125 was published by the group of the above-mentioned Queen et al. This reference describes that a humanized antibody having an activity equal to that of the original mouse antibody could not be constructed even by the method for making humanized antibody as set forth in WO 90-07861.

S 15 Thus, the fact not only reveals that the method of WO 90-07861 itself cannot produce a complete humanized antibody having an activity equal to the original mouse antibody, but that the method for constructing humanized antibody as set forth in WO 90-07861 cannot be applied to 20 humanization of all antibodies.

Ohtomo et al., Molecular Immunology (1995) 32, 407-416 describes humanization of mouse ONS-M21 antibody.

This reference reveals that the amino acid residue which :.was suggested for humanization of the Anti-Tac antibody in WO 90-07861 has no relation with the activity and the method as set forth in WO 90-07861 cannot be applied.

Kettleborough et al, Protein Eng. (1991) 4, 773-783 discloses that several humanized antibodies were constructed from mouse antibody by substituting amino acid residues. However, the substitution of more amino acid residues than were suggested in the method of humanization of the Anti-Tac antibody as described in WO 90-07861 was required.

The foregoing references indicate that the method of producing humanized antibodies as set forth in WO 90-07861 is a technique applicable only to the Anti-Tac antibody described therein and that even the use of said 7 technology does not lead to the activity equal to that of the original mouse antibody.

The original mouse antibodies described in these references have different amino acid sequences from that of the Anti-Tac antibody described in WO 90-07861.

Accordingly, the method of constructing humanized antibody which was able to be applied to the Anti-Tac antibody could not be applied to 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, the method of *constructing humanized antibody for the Anti-Tac antibody cannot be applied. Furthermore, the successfully constructed humanized antibody of the present invention 15 has an amino acid sequence different from that of the humanized Anti-Tac antibody described in WO 90-07861.

This fact also indicates that the same method cannot be applied for humanization of antibodies having different CDR-FR sequences.

Thus, even if the original mouse antibody for humanization is known, the identity of the CDR-FR sequence of a humanized antibody having an activity is i confirmed only after trial and error experiments. WO 90-07861 makes no mention of the FR sequence which is combined in the humanized antibody constructed in the present invention and of the fact that an active humanized antibody could be obtained from the combination with FR, much less the sequence of the CDR.

As hereinabove mentioned, humanized antibodies are expected to be useful for therapeutic purposes, but humanized anti-HM 1.24 antibody is not known or not even suggested. Furthermore, there is no standardized method available that could be generally applied to any antibody for production of a humanized antibody, and a variety of contrivances are needed for constructing a humanized antibody that exhibits sufficient binding activity, binding inhibition activity, and neutralizing activity 8 (for example, Sato, K. et al., Cancer Res., 53, 851-856, 1993).

Disclosure of the Invention The present invention provides reshaped antibodies of anti-HM 1.24 antibody. The present invention further provides human/mouse chimeric antibodies that are useful in the process of constructing said reshaped antibodies.

The present invention further provides fragments of the reshaped antibodies. Furthermore, the present invention provides an expression system for production of chimeric antibodies, reshaped antibodies and the fragments thereof. The present invention further provides methods for producing chimeric antibodies of anti-HM 1.24 antibody and fragments thereof, as well as reshaped antibodies of anti-HM 1.24 antibody and fragments thereof.

More specifically, ttrepresent invention provides chimeric antibodies and reshaped antibodies that specifically recognize a polypeptide having the amino 20 acid sequence as set forth in SEQ ID NO: 103. cDNA that encodes said polypeptide has been inserted between the XbaI cleavage sites of pUC19 vector, and thereby been prepared as plasmid pRS38-pUC19. Escherichia coli that contains this plasmid pRS38-pUC19 has been internationally deposited on October 5,1993, with 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) as Escherichia coli DH5a (pRS38-pUC19) under the accession number FERM BP-4434 under the provisions of the Budapest Treaty (see Japanese Unexamined Patent Publication (Kokai) No. 7-196694).

As one embodiment of such chimeric antibodies or reshaped antibodies, there is mentioned a chimeric anti-HM 1.24 antibody or a reshaped human anti-HM 1.24 antibody. A detailed description of a chimeric anti-HM 1.24 antibody or a reshaped human anti-HM 1.24 antibody -9 will be given hereinbelow.

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

The present invention further provides chimeric antibodies comprising: an L chain comprising the C region of a human L chain and the V region of the L chain of an anti-HM 1.24 antibody; and an H chain comprising the C region of a human H 15 chain and the V region of the H chain of an anti-HM 1.24 antibody.

The present invention further provides the V region of the reshaped human L chain of anti-HM 1.24 antibody comprising: 20 the framework region (FR) of the V region of a human L chain, and the CDR of the V region of the L chain of an anti-HM 1.24 antibody; and the V region of the reshaped human H chain of anti-HM 1.24 antibody comprising the FR of the V region of a human H chain, and the CDR of the V region of the H chain of an anti-HM 1.24 antibody.

The present invention furtherprovides the reshaped human L chain of anti-HM 1.24 antibody comprising the C region of a human L chain, and the V region of an L chain comprising the FR of a human L chain and the CDR of the L chain of an anti-HM 1.24 antibody; and the reshaped human H chain of anti-HM 1.24 antibody comprising 10 the C region of a human H chain, and the V region of an H chain comprising the FR of a human H chain and the CDR of the H chain of an anti-HM 1.24 antibody.

The present invention further provides the reshaped human antibody of anti-HM 1.24 antibody comprising: an L chain comprising the C region of a human L chain, and the V region of an L chain comprising the FR of a human L chain and the CDR of the L chain of an anti-HM 1.24 antibody; and an H chain comprising the C region of a human H chain, and the V region of an H chain comprising the 15 FR of a human H chain and the CDR of the H chain of an anti-HM 1.24 antibody.

The present invention further provides DNA encoding the V region of the L chain of an anti-HM 1.24 antibody, and DNA encoding the V region of the H chain of an 20 anti-HM 1.24 antibody.

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

The present invention further provides DNA encoding the V region of the reshaped human L chain of anti-HM 1.24 antibody comprising: the FR of the V region of a human L chain; and the CDR of the V region of the L chain of an anti-HM 1.24 antibody; and DNA encoding the V region of the reshaped human H chain of anti-HM 1.24 antibody comprising: 11 the FR of the V region of a human H chain; and the CDR of the V region of the H chain of an anti-HM 1.24 antibody.

The present invention further provides DNA encoding the reshaped human L chain of an anti-HM 1.24 antibody comprising: the C region of a human L chain; and the V region of an L chain comprising the FR of a human L chain and the CDR of the L chain of an anti-HM 1.24 antibody; and DNA encoding the reshaped human H chain of an anti-HM 1.24 antibody comprising: the C region of a human H chain; and the V region of an H chain comprising the FR of 15 a human H chain and the CDR of the H chain of an anti-HM 1.24 antibody.

The present invention further provides a vector comprising any of the various DNAs mentioned above.

The present invention further provides a host cell 20 transformed with the above vector.

The present invention also provides methods for producing the chimeric antibody of an anti-HM 1.24 antibody comprising the steps of culturing a host cell which was cotransformed with an expression vector comprising DNA encoding said chimeric L chain and an expression vector comprising DNA encoding said H chain, and of-recovering the desired antibody.

The present invention further provides methods for producing the reshaped human antibody of an anti-HM 1.24 antibody comprising the steps of culturing a host cell which was cotransformed with an expression vector comprising DNA encoding said reshaped human L chain and an expression vector comprising DNA encoding said reshaped human H chain, and of recovering the desired antibody.

The present invention further provides pharmaceutical compositions, especially therapeutic 12 agents for myeloma, comprising said chimeric antibody or the reshaped human antibody.

The present invention further provides pharmaceutical compositions which contain as an active ingredient a chimeric antibody specifically recognizing a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 103, and pharmaceutical compositions which contain as an active ingredient a reshaped human antibody specifically recognizing a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 103. As a pharmaceutical composition, there is specifically provided a therapeutic agent for myeloma.

Brief Explanation of the Drawings Fig. 1 is a graph showing that, in the FCM analysis *15 using the human myeloma cell line KPMM2, the fluorescence intensity of a chimeric anti-HM 1.24 antibody is shifted in a similar manner to that of a mouse anti-HM 1.24 antibody as compared to the control antibody.

Fig. 2 is a graph showing that, in the Cell-ELISA 20 using the WISH cell, the chimeric anti-HM 1.24 antibody similarly to the mouse anti-HM 1.24 antibody inhibits the binding of the biotinylated mouse anti-HM 1.24 antibody to the WISH cells in a dose dependent manner.

*Fig. 3 is a graph showing that the control human IgGl or the mouse anti-HM 1.24 antibody has no cytotoxicity whereas the chimeric anti-HM 1.24 antibody exhibits increased cytotoxicity to the RPMI 8226 cell with the increased ratio of E/T.

Fig. 4 is a diagramatic representation of a method for constructing the L chain of a reshaped human anti-HM 1.24 antibody by CDR grafting in the PCR method.

Fig. 5 is a diagramatic representation of a method for assemblying the oligonucleotides of RVH1, RVH2, RVH3, and RVH4 by the PCR method in the preparation of the H chain of the reshaped human anti-HM 1.24 antibody.

Fig. 6 is a diagramatic representation of a method for constructing the V region of the H chain of a human 13 mouse hybrid anti-HM 1.24 antibody by the PCR method.

Fig. 7 is a diagramatic representation of a method for constructing the V region of the H chain of a mouse human hybrid anti-HM 1.24 antibody by the PCR method.

Fig. 8 is a graph showing that the version a of the L chain of a reshaped human anti-HM 1.24 antibody has an antigen biding activity equal to that of the chimeric anti-HM 1.24 antibody. -1 and -2 show that they are different lots.

Fig. 9 is a graph showing the antigen binding S. activity of a reshaped human anti-HM 1.24 antibody in which the version a of the L chain and the version a, b, f, or h of the H chain have been combined, and a chimeric anti-HM 1.24 antibody.

15 Fig. 10 is a graph showing the binding activity of a reshaped human anti-HM 1.24 antibody in which the version b of the L chain and the version a, b, f, or h of the H chain have been combined, and a chimeric anti-HM 1.24 antibody.

20 Fig. 11 is a graph showing the binding inhibition activity of a reshaped human anti-HM 1.24 antibody in which the version a of the L chain and H chain version a, b, f, or h have been combined, and a chimeric anti-HM 1.24 antibody.

Fig. 12 is a graph showing the binding inhibition activity of a reshaped human anti-HM 1.24 antibody in which the version b of the L chain and the version a, b, f, or h of the H chain have been combined, and a chimeric anti-HM 1.24 antibody.

Fig. 13 is a graph showing the antigen binding activity of the versions a, b, c, and d of the H chain of a reshaped human anti-HM 1.24 antibody, and a chimeric anti-HM 1.24 antibody.

Fig. 14 is a graph showing the antigen binding activity of the versions a and e of the H chain of a reshaped human anti-HM 1.24 antibody, and a chimeric anti-HM 1.24 antibody. -1 and -2 show that they are 14 different lots.

Fig. 15 is a graph showing the binding inhibition activity of the versions a, c, p, and r of the H chain of a reshaped human anti-HM 1.24 antibody, and a chimeric anti-HM 1.24 antibody.

Fig. 16 is a graph'showing the antigen binding activity of a human mouse hybrid anti-HM 1.24 antibody, a mouse human hybrid anti-HM 1.24 antibody, and a chimeric anti-HM 1.24 antibody.

Fig. 17 is a graph showing the antigen binding activity of the versions a, b, c, and f of the H chain of a reshaped human anti-HM 1.24 antibody, and a chimeric anti-HM 1.24 antibody.

Fig. 18 is a graph showing the antigen binding *15 activity of the versions a and g of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 19 is a graph showing the binding inhibition activity of the versions a and g of the H chain of a 20 reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 20 is a graph showing the antigen binding activity of the versions h and i of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 21 is a graph showing the antigen binding activity of the versions f, h, and j of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 22 is a graph showing the binding inhibition activity of the versions h and i of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 23 is a graph showing the binding inhibition activity of the versions f, h, and j of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

15 Fig. 24 is a graph showing the antigen binding activity of the versions h, k, 1, m, n, and O of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 25 is a graph showing the antigen binding activity of the versions a, h, p, and q of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 26 is a graph showing the inhibition activity of binding to the WISH cell of the versions h, k, 1, m, n, and o of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 27 is a graph showing the binding inhibition activity of the versions a, h, p, and q of the H chain of 15 a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 28 is a graph showing the antigen binding activity of the versions a, c, p, and r of the H chain of a reshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24 antibody.

Fig. 29 is a graph showing that the version s of a reshaped human anti-HM 1.24 antibody has an antigen binding activity equal to that of the version r of the reshaped human anti-HM 1.24 antibody.

Fig. 30 is a graph showing that the version s of a reshaped human anti-HM 1.24 antibody has a binding inhibition activity equal to that of the version r of the reshaped human anti-HM 1.24 antibody.

Fig. 31 is a graph showing that a purified reshaped human anti-HM 1.24 antibody has an antigen binding activity equal to that of a chimeric anti-HM 1.24 antibody.

Fig. 32 is a graph showing that a purified reshaped human anti-HM 1.24 antibody has a binding inhibition activity equal to that of a chimeric anti-HM 1.24 antibody.

Fig. 33 is a graph showing that the administration 16 of a chimeric anti-HM 1.24 antibody caused prolongation of the survival period as compared to the administration of the control human IgGl in a human myeloma cells-transplanted mouse.

Fig. 34 is a graph showing that when cells derived from the peripheral blood of healthy human are used as a effector cell the control human IgGl exhibits no cytotoxicity to the KPMM2 cells and a mouse anti-HM 1.24 antibody also has a weak cytotoxicity whereas a reshaped human anti-HM 1.24 antibody exhibits a strong cytotoxicity to the KPMM2 cells.

Fig. 35 is a graph showing that when cells derived from the peripheral blood of healthy human are used as a effector cell the control human IgGl exhibits no 15 cytotoxicity to the ARH-77 cells and a mouse anti-HM 1.24 antibody also has a weak cytotoxicity, whereas a reshaped human anti-HM 1.24 antibody exhibits a strong cytotoxicity to the ARH-77 cells.

Fig. 36 is a graph showing that when cells derived 20 from the bone marrow of SCID mice are used as a effector cell the control human IgGl exhibits no cytotoxicity to the KPMM2 cells, whereas a reshaped human anti-HM 1.24 antibody exhibits an increased cytotoxicity to the KPMM2 cells with the increase in the antibody concentration.

Fig. 37 is a graph showing that in a human myeloma cells-transplanted mouse the serum IgG human level is increased after the administration of the control human IgGl as compared to the level before the administration, whereas the administration of a reshaped human anti-HM 1.24 antibody inhibits the increase in the serum human IgG level.

Fig. 38 is a graph showing that in a human myeloma cells-transplanted mouse the administration of a reshaped human anti-HM 1.24 antibody causes prolongation of the survival period as compared to the administration of the control human IgGl.

Fig. 39 is a graph showing that in a human myeloma 17 cells-transplanted mouse the serum human IgG level is increased after the administration of melphalan and the control human IgG1 as compared to the level before the administration, whereas the administration of a reshaped human anti-HM 1.24 antibody inhibits the increase in the serum human IgG level.

Fig. 40 is a graph showing that in a human myeloma cells-transplanted mouse the administration of a reshaped human anti-HM 1.24 antibody causes prolongation of the survival period as compared to the administration of melphalan or the control human IgGl.

Mode for Carrying Out the Invention 1. Construction of a chimeric antibody Cloning of DNA encoding the V region of a 15 mouse anti-HM 1.24 monoclonal antibody Preparation of mRNA In order to clone DNA encoding the V region of a mouse anti-HM 1.24 monoclonal antibody, the total RNA is prepared from a recovered hybridoma using a 20 known method such as a guanidine-ultracentrifuge method (Chirgwin, J.M. et al., Biochemistry (1979), 18, 5294-5299), the AGPC method (Chomczynski, P. et al.

(1987), 162, 156-159), etc. and mRNA is prepared using the Oligo(dT)-cellulose spun column etc. attached with the mRNA Purification Kit (manufactured by Pharmacia), etc. Furthermore, by using the QuickPrep mRNA Purification Kit (manufactured by Pharmacia) mRNA can be prepared without the extraction step of the total RNA.

Preparation and Amplification of cDNA From the mRNA obtained in the above-mentioned Preparation of mRNA, each cDNA for the V regions of an L chain and an H chain is synthesized using a reverse transcriptase. The cDNA of the V region of the L chain is synthesized using the AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit. For the amplification of the synthesized cDNA, an appropriate primer that hybridizes with the leader sequence and the C 18 region of the antibody gene (for example, the MKV primer having the base sequences represented by the SEQ ID NO: 29 to 39, and the MKC primer having the base sequence represented by the SEQ ID NO: The synthesis and amplification of the cDNA of the V region of'an H chain can be carried out by PCR (polymerase chain reaction) by the 5'-RACE method (Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA, 8998-9002, 1988, Belyavsky, A. et al., Nucleic Acids Res.

17, 2919-2932, 1989) using the 5'-Ampli FINDER RACE kit (CLONTECH). To the 5'-end of the cDNA synthesized as above, the Ampli FINDER Anchor is ligated, and as a primer for amplification of the V region of the H chain, a primer that specifically hybridizes with the Anchor 15 primer (SEQ ID NO: 77) and the constant region (Cy region) of a mouse H chain (for example, the MHC2a primer having the base sequence represented by SEQ ID NO: 42) can be used.

Purification of DNA and the determination 20 of the base sequence thereof An agarose gel electrophoresis is conducted on the PCR product using a known method to excise the desired DNA fragment, and DNA is recovered and purified therefrom, which is then ligated to a vector

DNA.

DNA can be purified using a commercial kit (for example, GENECLEAN II; BIO101). A known vector DNA (for example, pUC19, Bluescript, etc.) can be used to retain DNA fragments.

The above DNA and the above DNA vector are ligated using a known ligation kit (manufactured by Takara Shuzo) to obtain a recombinant vector. The obtained recombinant vector is then introduced into Escherichia coli JM109, after which ampicillin resistant colonies are selected and a vector DNA is prepared based on a known method Sambrook, et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989).

19 After digesting the above vector DNA with restriction enzymes, the base sequence of the desired DNA is determined by a known method (for example, the dideoxy method) Sambrook, et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989). In accordance with the present invention, an automatic sequencing system (DNA Sequencer 373A; manufactured by ABI Co. Ltd.) can be used.

Complementarity Determining Region The V region of an H chain and the V region of an L chain form an antigen binding site, of which overall structures have similar properties. Thus, each of four framework regions (FR) has been ligated by three hypervariable regions, i.e. complementarity 15 determining regions (CDRs). The amino acid sequences of FRs have been relatively well conserved whereas variation is extremely high among the amino acid sequences of CDR regions (Kabat, E.A. et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human 20 Services, 1983).

Many portions of the above four FRs take the p-sheet structure with a result that three CDRs form loops. CDRs may sometimes form part of the p-sheet structure. The three CDRs are retained sterically in close proximity with one another and form an antigen binding site with three CDRs of the pairing region.

Based on these facts, the amino acid sequence of the variable region of a mouse anti-HM 1.24 antibody is fitted to the data base of the amino acid sequences of antibodies prepared by Kabat et al.

("Sequence of Proteins of Immunological Interest",

US

Dept. Health and Human Services, 1983) to investigate homology and thereby to find CDR regions.

Construction of expression vectors for a chimeric antibody Once a DNA fragment encoding the V regions of the mouse L chain and H chain of a mouse monoclonal 20 antibody is cloned, a chimeric anti-HM 1.24 antibody can be obtained by linking these mouse V regions to a DNA encoding the constant region of a human antibody and then by expressing them.

A basic method for constructing a chimeric antibody comprises linking the mouse leader sequence and the V region sequence present in the cloned cDNA to a sequence encoding the C region of a human antibody already present in an expression vector for mammalian cells. Alternatively it comprises linking the mouse leader sequence and the V region sequence present in the cloned cDNA to 'a sequence encoding the C region of a human antibody, which is then linked to an expression vector for mammalian cells.

15 The C region of a human antibody can be the C region of any H chain and the C region of any L chain. There can be mentioned, for example, Cyl, Cy2, Cy3, or Cy4 of a human H chain, or CX or CK of an L chain.

20 For production of a chimeric antibody two kinds of expression vectors are constructed: they are an expression vector comprising DNA encoding the V region of a mouse L chain and the C region of a human L chain under ~the control of an expression regulatory region such as the enhancer/promoter system, and an expression vector comprising DNA encoding the V region of a mouse H chain and the C region of a human H chain under the control of an expression regulatory region such as the enhancer/promoter system. Subsequently, using these expression vectors a host cell such as a mammalian cell is cotransformed, and the transformed cells are cultured in vitro or in vivo to produce a chimeric antibody (for example, WO 91-16928) Alternatively, DNA encoding the mouse leader sequence and the V region of an L chain and the C region of a human L chain and DNA encoding the mouse leader sequence and the V region of an H chain and the C 21 region of a human H chain present in the cloned cDNA are introduced into a single expression vector (see, International Application Publication No. WO 94-11523), and a host cell is transformed using said vector. The transformed host is then cultured in vitro or in vivo to produce the desired chimeric antibody.

1) Construction of a chimeric H chain An expression vector for the H chain of the chimeric antibody can be obtained by introducing cDNA encoding the V region of a mouse H chain into an appropriate expression vector containing genomic DNA or cDNA encoding the C region of the H chain of a human antibody. As the C region of an H chain there can be mentioned, for example, Cyl, Cy2, Cy3, or Cy4.

15 Construction of an expression vector for a chimeric H chain containing Cyl genomic

DNA

As an expression vector having genomic DNA for Cyl as the C region of an H chain, there can be used, 20 for example HFE-PMh-gyl (International Application Publication No. WO 92/19759) or DHFR-AE-RVh-PM1f (International Application Publication No. WO 92/19759).

In order to insert cDNA encoding the V region of a mouse H chain into these expression vectors, suitable base sequences may be introduced using the PCR method. These suitable base sequences can be introduced by the PCR method using a PCR primer designed to have a recognition sequence for a suitable restriction enzyme at the 5'-end and a Kozak consensus sequence immediately before the start codon, and a PCR primer designed to have at the 3'-end a recognition sequence for a suitable restriction enzyme and a splice donor site where a primary transcript of genomic DNA is properly spliced to become an mRNA.

The cDNA thus constructed encoding the V region of a mouse H chain is treated with suitable restriction enzymes, inserted into the above-mentioned 22 expression vector, and a chimeric H chain-expression vector comprising the Cyl DNA can be constructed.

Construction of an expression vector for the cDNA chimeric H chain An expression vector having the cDNA of Cyl as the C region of'an H chain may be constructed as follows. Thus, it can be constructed by preparing mRNA from a CHO cell in which the expression vector DHFR-AE-RVh-PM1f (International Application Publication- No. WO 92/19759) encoding genomic DNA of the V region of the H chain of a humanized PM1 antibody and the C region Cyl of the H chain of a human antibody Takahashi, et al., Cell 29, 671-679, 1982) and the expression vector RV1-PM1a (International Application Publication No. WO 15 92/19759) encoding genomic DNA of the V region of the L chain of a humanized PM1 antibody and the C region of the K chain of a human antibody L chain have been integrated; cloning cDNA comprising the V region of the H chain of the humanized PM1 antibody and the C region Cyl of the H 20 chain of the human antibody by the RT-PCR method, and; ligating to a suitable expression vector for animal cells using suitable restriction enzyme sites.

In order to directly ligate cDNA encoding the V region of a mouse H chain to cDNA containing the C region Cyl of the H chain of a human antibody, suitable base sequences can be introduced by the PCR method. For example, these suitable base sequences can be introduced by the PCR method using a PCR primer designed to have a recognition sequence for a suitable restriction enzyme at the 5'-end and a Kozak consensus sequence immediately before the start codon, and a PCR primer designed to have a recognition sequence for a suitable restriction enzyme used for direct ligation of the C region Cyl of an H chain at the 3'-end.

An expression vector containing a cDNA chimeric H chain can be constructed by treating the cDNA thus constructed encoding the V region of a mouse H chain 23 with a suitable restriction enzyme, ligating to the above-mentioned cDNA containing the C region Cyl of the H chain, and inserting to an expression vector such as pCOSI or pCHO1.

2) Construction of the L chain of a chimeric antibody An expression vector for the L chain of a chimeric antibody may be obtained by linking cDNA encoding the V region of a mouse L chain to genomic

DNA

or cDNA encoding the C region of the L chain of a human antibody, and then introducing it into a suitable expression vector. As the C region of an L chain there can be mentioned, for example a K chain or a A chain.

Construction of an expression vector for the K chain of a cDNA chimeric L chain In order to construct an expression vector containing cDNA encoding the V region of a mouse L chain, suitable base sequences can be introduced using the PCR method. For example, these suitable base sequences can i 20 be introduced by the PCR method using a PCR primer designed to have a recognition sequence for a suitable restriction enzyme and a Kozak consensus sequence at the 5'-end, and a PCR primer designed to have a recognition sequence for a suitable restriction enzyme at the 3'-end.

The K chain C region of a human L chain for linking to the V region of a mouse L chain can be constructed from, for example HEF-PMlk-gk (see International Application Publication No. WO 92/19759) containing genomic DNA. An expression vector for the K chain of the L chain of a cDNA chimeric antibody can be constructed by introducing recognition sequences of suitable restriction enzymes at the 5'-end or 3'-end of DNA encoding the K chain C region of L chain by the PCR method, ligating the thus constructed V region of the mouse L chain to the K chain C region of L chain, and then inserting into an expression vector such as pCOS1 or pCHO1.

24 2. Construction of a reshaped human antibody Designing of the V region of a reshaped human anti-HM 1.24 antibody In order to construct a reshaped human antibody in which the CDR of a mouse monoclonal antibody has been grafted to a human antibody, it is desirable that there is a high homology between the FR of the mouse monoclonal antibody and the FR of the human antibody.

Thus, the V regions of the L chain and the H chain of the mouse anti-HM 1.24 antibody are compared to the V regions of all known antibodies of which structures have been elucidated, using the Protein Data Bank.

The V region of the L chain of a mouse anti-HM 1.24 antibody is most similar to the consensus 15 sequence of the subgroup IV of the V region of the L chain of a human antibody (HSGIV) with a homology of 66.4%. On the other hand, it shows a homology of 56.9%, 55.8%, and 61.5% with HSGI, HSGII, and HSGIII, respectively.

20 The V region of L chain of a mouse anti-HM 1.24 antibody, when compared to the V region of the L chain of known human antibodies, shows a homology of 67.0% with the V region of the L chain of the human antibody REI, one of subgroup I of the V region of the L chain of the human antibody. Therefore, the FR of the REI was used as the starting material for construction of the V region of the L chain of the reshaped human anti-HM 1.24 antibody.

Version a of the V region of the L chain of the reshaped human anti-HM 1.24 antibody was designed.

In this version, the FR of the human antibody was made identical with the REI-based FR present in the reshaped human CA PATH-1H antibody (see Riechmann, L. et al., Nature 322, 21-25, (1988), the FR contained in version a of the V region of the L chain of a reshaped human PM-1 antibody described in International Application Publication No. WO 92-19759), and the mouse CDR was made 25 identical with the CDR in the V region of the L chain of the mouse anti-HM 1.24 antibody.

The V region of the H chain of a mouse anti-HM 1.24 antibody is most similar to the consensus sequence of the V region of the H chain of a human antibody (HSGI) with a homology of 54.7%. On the other hand, it shows a homology of 34.6% and 48.1% with HSGII and HSGIII, respectively. When the V region of the H chain of a mouse anti-HM 1.24 antibody is compared to the V region of the H chain of known human antibodies, FR1 to FR3 were most similar to the V region of the H chain of the human antibody HG3, one of subgroup I of the V region of a human H chain (Rechavi, G. et al., Proc. Natl. Acad.

Sci. USA, 80, 855-859), with a homology of 67.3%.

15 Therefore, the FR of the human antibody HG3 was used as the starting material for construction of the V region of the H chain of a reshaped human anti-HM 1.24 antibody.

However, since the amino acid sequence of 20 the FR4 of the human antibody HG3 has not been described, the amino acid sequence of the FR4 of the human antibody JH6 (Ravetch, J.V. et al., Cell, 27, 583-591) that shows the highest homology with the FR4 of a mouse anti-HM 1.24 antibody was used as FR4. The FR4 of JR6 has the same amino acid sequence as the FR4 of the H chain of a mouse anti-HM 1.24 antibody except only one amino acid.

In the first version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody, FR1 to FR3 were made identical with the FR1 to FR3 of the human antibody HG3, except that the amino acids at position 30 in the human FR1 and position 71 in the human FR3 were made identical with the amino acids of the mouse anti-HM 1.24 antibody, and the CDR was made identical with the CDR in the V region of the H chain of a mouse anti-HM 1.24 antibody.

26 Construction of the V region of the L chain of a reshaped human anti-HM 1.24 antibody The L chain of a reshaped human anti-HM 1.24 antibody is constructed by the CDR grafting in the PCR method. The method is schematically shown in Fig. 4.

Eight.PCR primers are used for construction of a reshaped human anti-HM 1.24 antibody (version a) having the FR derived from the human antibody REI. The external primers A (SEQ ID NO: 47) and H (SEQ ID NO: 48) are designed to hybridize with the DNA sequence of the HEF expression vector HEF-VL-gK.

The CDR grafting primers LIS (SEQ ID NO: 49), L2S (SEQ ID NO: 50), and L3S (SEQ ID NO: 51) have a 15 sense DNA sequence. The CDR grafting primers L1A (SEQ ID NO: 52), L2A (SEQ ID NO: 53), and L3A (SEQ ID NO: 54) have an antisense DNA sequence, each having a complementary DNA sequence (20 to 23 bp) to the DNA sequence at the 5'-end of the primers LIS, L2S, and L3S, 20 respectively.

In the first stage of PCR, the four reactions A-L1A, L1S-L2A, L2S-L3A, and L3S-H are conducted and each PCR product purities. The four PCR products from the first PCR are allowed to assemble with one another by their own complementarity (see WO 92-19759). Then, the external primers A and H are added to amplify the full-length DNA encoding the V region of the L chain of a reshaped human anti-HM 1.24 antibody (the second PCR). In the above-mentioned PCR, the plasmid HEF-RVL-M21a (see International Application Publication No. WO 95-14041) encoding the version a of the V region of the L chain of a reshaped human ONS-M21 antibody based on the human antibody REI-derived FR can be employed as a template.

In the first stage of PCR, template DNA and each of primers were used.

PCR products A-L1A (215 bp), L1S-L2A(98 27 bp), L2S-L3A (140 bp), and L3S-H (151 bp) are purified using 1.5% low melting point agarose gel and are assembled in the second PCR. In the second PCR, each product from the first PCR and each external primer (A and H) are used.

A 516 bp'DNA fragment resulting from the second PCR is purified using 1.5% low melting point agarose gel, digested with BamHI and HindIII, and the DNA fragments thus obtained are cloned into the HEF expression vector HEF-VL-gK. After determining the DNA sequence, the plasmid containing the DNA fragment having the correct amino acid sequence of the V region of the L chain of a reshaped human anti-HM 1.24 antibody was termed the plasmid HEF-RVLa-AHM-gK. The amino acid 15 sequence and the base sequence of the V region of the L chain contained in this plasmid HEF-RVLa-AHM-gK are shown in SEQ ID NO: 9.

The version b of the V region of the L chain of a reshaped human anti-HM 1.24 antibody can be constructed by mutagenesis using PCR. Mutagen primers FTY-1 (SEQ ID NO: 55) and FTY-2 (SEQ ID NO: 56) are so designed as to mutate phenylalanine at position 71 to tyrosine.

After the above primers are amplified using the plasmid HEF-RVLa-AHM-gK as a template, the final product is purified. By digesting with BamHI and HindIII, the DNA fragments obtained are cloned into the HEF expression vector HEF-VL-gK to obtain plasmid HEF-RVLb-AHM-gK. The amino acid sequence and the base sequence of the V region of the L chain contained in this plasmid HEF-RVLb-AHM-gK are shown in SEQ ID NO: Construction of the V region of the H chain of a reshaped human anti-HM 1.24 antibody 28 3-1. Construction of versions a to e of the V region of the H chain of a reshaped human anti-HM 1.24 antibody DNA encoding the V region of the H chain of a reshaped human anti-HM 1.24 antibody can be designed as follows. By linking the DNA sequence encoding the FRs 1 to 3 of the human antibody HG3 and the FR4 of the human antibody JH6 to the DNA sequence encoding the CDR of the V region of the H chain of a mouse anti-HM 1.24 antibody, a full length DNA encoding the V region of the H chain of a reshaped human anti-HM 1.24 antibody may be designed.

Then, the HindIII recognition site/KOZAK consensus sequence and the BamHI recognition site/splice donor sequence, respectively, are attached to the 15 and the 3'-end of this DNA sequence so as to allow insertion of the HEF expression vector.

The DNA sequence thus designed is divided into four oligonucleotides. Subsequently, oligonucleotides which potentially hinder the assembly of these 20 oligonucleotides are subjected to computer analysis for the secondary structure.

The sequences of the four oligonucleotides RVH1 0" to RVH4 are set forth in SEQ ID NO: 57 to 60. These oligonucleotides have a length of 119 to 144 bases and have a 25 to 26 bp overlapping region. Among the 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 for assembling these four oligonucleotides by the PCR method is shown in the figure (see Fig. PCR is carried out using the four oligonucleotides and RHP1 (SEQ ID NO: 60) and RHP2 (SEQ ID NO: 62) as the external primers.

The amplified 438 bp DNA fragment is purified, digested with HindIII and BamHI, and then cloned into the HEF expression vector HEF-VH-gyl. After determination of 29 the base sequence, the plasmid that contains the DNA fragment encoding the correct amino acid sequence of the V region of the H chain was termed HEF-RVHa-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHa-AHM-gyl are shown in SEQ ID NO:"11.

Each of versions b, c, d, and e of the V region of the H chain of a reshaped human anti-HM 1.24 antibody is constructed as follows. In constructing each of version b and after of the V region of the H chain of a reshaped human anti-HM 1.24 antibody, a three-dimensional structural model of the V region of a mouse anti-HM 1.24 antibody can be constructed in order to predict the position of the amino acid residue to be substituted in 15 the antibody molecule.

Using as the mutagen primer.BS (sequence 63) and BA (SEQ ID NO: 64) designed to mutate arginine at position 66 to lysine and as a template DNA the plasmid HEF-RVHa-AHM-gyl by the PCR method, version b is 20 amplified to obtain plasmid HEF-RVHb-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHb-AHM-gyl are shown in SEQ ID NO: 12.

Using as the mutagen primer CS (sequence and CA (SEQ ID NO: 66) designed to mutate threonine at position 73 to lysine and as a template DNA the plasmid HEF-RVHa-AHM-gyl by the PCR method, version c is amplified to obtain plasmid HEF-RVHc-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHc-AHM-gyl are shown in SEQ ID NO: 13.

Using as the mutagen primer DS (sequence 67) and DA (SEQ ID NO: 68) designed to mutate arginine at position 66 to lysine and threonine at position 73 to lysine and as a template DNA the plasmid HEF-RVHa-AHM-gyl by the PCR method, version d is amplified to obtain plasmid HEF-RVHd-AHM-gyl. The amino acid sequence and 30 the base sequence of the V region of the H chain contained in this plasmid HEF-RVHd-AHM-gyl are shown in SEQ ID NO: 14.

Using as the mutagen primer ES (sequence 69) and EA (SEQ ID NO: 70) designed to mutate valine at position 67 to alanine and methionine at position 69 to leucine and as a template DNA the plasmid HEF-RVHa-AHM-gyl, version e is amplified to obtain plasmid HEF-RVHe-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHe-AHM-gyl are shown in SEQ ID NO: 3-2. Construction of the H chain hybrid V region By constructing a H chain hybrid V region, it 15 is possible to investigate which FR of the V region of a humanized antibody contributes to the binding activity o' and the binding inhibition activity. Among the two that were constructed, the amino acid sequences of FR1 and FR2 are derived from a mouse anti-HM 1.24 antibody and those 20 of FR3 and FR4 are from version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody (mouse human hybrid anti-HM 1.24 antibody) in one, and the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from a mouse anti-HM 1.24 antibody (human mouse hybrid anti-HM 1.24 antibody) in the other. The amino acid sequences of the CDR regions are all derived from a mouse anti-HM 1.24 antibody.

Two H chain hybrid V regions are constructed by the PCR method. The method is schematically shown in Fig. 6 and 7. For the construction of two H chain hybrid V regions four primers can be used. The external primers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) are designed to hybridize with the DNA sequence of the HEF expression vector HEF-VH-gyl. The H chain hybrid construction primer HYS (SEQ ID NO: 73) is designed to have the sense 31 DNA sequence and the H chain hybrid primer HYA (SEQ ID NO: 74) to have the antisense DNA sequence so that the DNA sequences are complementary to each other.

For the construction of the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from a mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody, PCR using the plasmid HEF-1.24H-gyl as a template, the external primer a, and the H chain hybrid primer HYA, and PCR using the plasmid HEF-RVHa-AHM-gyl as a template, the H chain hybrid primer HYA, and the external primer h are carried out in the first stage of PCR and each PCR product is purified.

15 The two PCR products from the first PCR are allowed to assemble by their own complementarity (see International Application Publication No. WO 92-19759).

Then, by adding the external primers a and h, a full-length DNA encoding the H chain hybrid V region in 20 which the amino acid sequences of FR1 and FR2 are derived "from a mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody is amplified in the second PCR stage.

For the construction of the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from a mouse anti-HM 1.24 antibody, PCR using the plasmid HEF-RVHa-AHM-gyl as a template, the external primer a, and the H chain hybrid primer HYA, and PCR using the plasmid HEF-1.24H-gyl as a template, the H chain hybrid primer HYS, and the external primer h are carried out in the first stage of PCR and each PCR product is purified.

The two PCR purified products from the first PCR are allowed to assemble by their own complementarity 32 (see International Application Publication No. WO 92-19759). Then, by adding the external primers a and h, a full-length DNA encoding the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of a reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from a mouse anti-HM 1.24 antibody is amplified in the second PCR stage.

The methods of the first PCR, purification of PCR products, assembling, the second PCR, and cloning into the HEF expression vector HEF-VH-gyl are carried out according to the method shown in "Example 9. Construction of the V region of the L chain of a reshaped human anti-HM 1.24 antibody". After determination of the DNA 15 sequence, the plasmid that contains the DNA fragment encoding the correct amino acid sequence of the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from a mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V o 20 region of the H chain of a reshaped human anti-HM 1.24 antibody was termed HEF-MH-RVH-AHM-gyl.

The amino acid sequence and the base sequence ooo of the V region of the H chain contained in this plasmid HEF-MH-RVH-AHM-gyl are shown in SEQ ID NO: 75. Also, the plasmid that contains the DNA fragment encoding the correct amino acid sequence of the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from a version a reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from the V region of the H chain of a mouse anti-HM 1.24 antibody was termed HEF-HM-RVH-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gyl are shown in SEQ ID NO: 76.

33 3-3. Construction of versions f to s of the V region of the H chain of a reshaped human anti-HM 1.24 antibody Each of versions f, g, h, i, j, k, 1, m, n, o, p, q, r, and s of the V region of the H chain of a reshaped human anti-HM 1.24 antibody is constructed as follows. In constructing each of versions f and after of the V region of the H chain of a reshaped human anti-HM 1.24 antibody, a three-dimensional structural model of the V region of a mouse anti-HM 1.24 antibody can be constructed, as mentioned above, in order to predict the position of the amino acid residue to be substituted in the antibody molecule.

Using as the mutagen primer FS (sequence 78) and FA (SEQ ID NO: 79) designed to mutate threonine at position 75 to serine and valine at position 78 to alanine and as a template DNA the plasmid HEF-RVHe-AHM-gyl by the PCR method, version f is amplified to obtain plasmid HEF-RVHf-AHM-gyl. The amino 20 acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHf-AHM-gyl are shown in SEQ ID NO: 16.

Using as the mutagen primer GS (sequence and GA (SEQ ID NO: 81) designed to mutate alanine at position 40 to arginine and as a template DNA the plasmid HEF-RVHa-AHM-gyl, version g is amplified to obtain plasmid HEF-RVHg-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHg-AHM-gyl are shown in SEQ ID NO: 17.

Using as the mutagen primer FS and FA and as a template DNA the plasmid HEF-RVHb-AHM-gyl, version h is amplified to obtain the plasmid HEF-RVHh-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHh-AHM-gyl are shown in SEQ ID NO: 18.

Using as the mutagen primer IS (sequence 82) 34 and IA (SEQ ID NO: 83) designed to mutate arginine at position 83 to alanine and serine at position 84 to phenylalanine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version i is amplified to obtain plasmid HEF-RVHi-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHi-AHM-gyl are shown in SEQ ID NO: 19.

Using as the mutagen primer JS (SEQ ID NO: 84) and JA (SEQ ID NO: 85) designed to mutate arginine at position 66 to lysine and as a template DNA the plasmid HEF-RVHf-AHM-gyl, version j is amplified to obtain :plasmid HEF-RVHj-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain 15 contained in this plasmid HEF-RVHj-AHM-gyl are shown in SEQ ID NO: Using as the mutagen primer KS (SEQ ID NO: 86) and KA (SEQ ID NO: 87) designed to mutate glutamic acid at position 81 to glutamine and as a template DNA the 20 plasmid HEF-RVHh-AHM-gyl, version k is amplified to obtain plasmid HEF-RVHk-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHk-AHM-gyl are shown in SEQ ID NO: 21.

Using as the mutagen primer LS (SEQ ID NO: 88) and LA (SEQ ID NO: 89) designed to mutate glutamic acid at position 81 to glutamine and serine at position 82B to isoleucine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version 1 is amplified to obtain plasmid HEF-RVHI-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHl-AHM-gyl are shown in SEQ ID NO: 22.

Using as the mutagen primer MS (SEQ ID NO: and MA (SEQ ID NO: 91) designed to mutate glutamic acid at position 81 to glutamine, serine at position 82b to isoleucine, and threonine at position 87 to serine and as 35 a template DNA the plasmid HEF-RVHh-AHM-gyl, version m is amplified to obtain plasmid HEF-RVHm-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHm-AHM-gyl are shown in SEQ ID NO: 23.

Using as the mutagen primer NS (SEQ ID NO: 92) and NA (SEQ ID NO: 93) designed to mutate serine at position 82B to isoleucine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version n is amplified to obtain plasmid HEF-RVHn-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHn-AHM-gyl are shown in SEQ ID NO: 24.

Using as the mutagen primer OS (SEQ ID NO: 94) 15 and OA (SEQ ID NO: 95) designed to mutate threonine at position 87 to serine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version o is amplified to obtain plasmid HEF-RVHo-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHo-AHM-gyl are shown in "SEQ ID NO: Using as the mutagen primer PS (SEQ ID NO: 96) and PA (SEQ ID NO: 97) designed to mutate valine at position 78 to alanine and as a template DNA the plasmid HEF-RVHa-AHM-gyl, version p is amplified by the PCR method to obtain plasmid HEF-RVHp-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHp-AHM-gyl are shown in SEQ ID NO: 26.

Using as the mutagen primer QS (SEQ ID NO: 98) and QA (SEQ ID NO: 99) designed to mutate threonine at position 75 to serine and as a template DNA the plasmid HEF-RVHa-AHM-gyl, version q is amplified by the PCR method to obtain plasmid HEF-RVHq-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHq-AHM-gyl are shown in SEQ ID NO: 27.

36 Using as the mutagen primer CS (SEQ ID NO: and CA (SEQ ID NO: 66) and as a template DNA the plasmid HEF-RVHp-AHM-gyl, version r is amplified by the PCR method to obtain plasmid HEF-RVHr-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHr-AHM-gyl are shown in SEQ ID NO: 28.

Using as the mutagen primer SS (SEQ ID NO: 100) and SA (SEQ ID NO: 101) designed to mutate methionine at position 69 to isoleucine and as a template DNA the plasmid HEF-RVHr-AHM-gyl, version s is amplified to obtain plasmid HEF-RVHs-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHs-AHM-gyl are shown in 15 SEQ ID NO: 102.

The amino acid sequences of the V region of the L chain constructed are shown in Table 1, and the amino acid sequences of the V region of the H chain are shown in Tables 2 to 4.

oo** oo* 37 Table 1 The amino acid sequence of the V region of the L chain FRi CDRI FR2 1 2 3 4 12345678901234567890123 45678901234 567890123456789 AHM DIV&ITQSHKFMSTSVGDRVSITC KASQDVNTAVA

WYQQKPGQSPKLLIY

HuSG I DIQNITQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY

REI DIQNITQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY

R V L b- CDR2 FR3 *6 7 8 0123456 78901234567890123456789012345678 AHNI SASNRYT

GVPDRITGSGSGTDFTFTISSVQAEDLALYYC

HuSG I GVPSRFSGSGSGTDFTLT

ISSLQPEDFATYYC

RE!

GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC

R V L b CDR3 FR4 9 901234567 8901234567 A H NHM QQHYSTPFT

FGSGTKLEIK

HuSG I

FGQGTKVEIK

0RE! FGQGTKVEiK RVLa-- R'Lb 38 Table2 The amino acid sequence of the V region of the H chain (1) AH M H u SG I HG3 R IH a RVHb RVH c RVHd RVHe R V Hif R VI 0; RYH h R V H RVH i R'1H k RVH I RYH .n RVHn R VHo RV~pI R VH q R V H r R V Hs 2 3 123456789012345678901234567890 Q VQLQQS GAF LAR PG ASVK LS CKAS GYT FT E VQL VQS GAD VKK PGXS VXVS CKASG YT FS

QVQLVQSGAEVKKNPGASVKVSCKASGYTFN

T

T

T

T

T

'T

'T

'T

T

T

T

T

T

T

'T

'T

'T

'T

'T

CDRI FR2 4 12345 67890123456789 PYWMQ WVKQRPGQGLEWIG

WVRQAPGXGLDWVG

WVRQAPGQGLEWMG

39 Table 3 The amino acid sequence of the V region of the H chain (2)

S

S.

S S S. S

S.

S S

S

AHM

HUSG I HG3 RVH a RVHb RVHc RVHd RVHe R VH f RVHg RVHh

RVH

RVH

RVH k RYH 1 RVHm RVHn RVHo R~tVH p R IH q R V Hr ItVHS CDR2 6 012A34567890 12345 SI FPGDGDTRYSQKFKG FR3 7 8 9 6 7 89O12345'6789O12ABC3456-789O1234 KATLTADKSSSTAYMQLS

ILAFEDSAVYYCAR

RVTXTXDXSXNTAYMELSSLRSEDTAVYYCAR

R VT MTRD TSTSTY Y EL SS LR SB DTA V YYCAR A -K -A-L-A -AL A -S K KA-L-A K K A---SA I 40 Table 4 The amino acid sequence of the V region of the H chain (3) A HM HuSG I J H6 RVHa RVHb RVH c RVHd RVH e R VH f RVHg RVH h R V H R VH i RYH k RYH I RVHrn RVHn R VHo RVH p R VH q 1,V H r RVHs CDR3 57890ABJK12

GLRRGGYYFDY

FR4 34567890123

WGQGTTLTVSS

WGQGTLVTVSS

WGQGTTVTVSS

S

S

41- 3. Production of a chimeric antibody and a reshaped human antibody For the production of a chimeric antibody or a reshaped human antibody, two expression vectors for each are constructed, which comprises an expression vector comprising DNA encoding the V region of a mouse H chain and the C region of a human H chain under the control of an expression regulatory region such as the enhancer/promoter system and DNA encoding the V region'of a mouse L chain and the C region of a human L chain under the control of an expression regulatory region such as o the enhancer/promoter system, or an expression vector o So comprising DNA encoding the V region of a humanized H 555.

S.chain and the C region of a human H chain under the 15 control of an expression regulatory region such as the enhancer/promoter system and DNA encoding the V region of

*OS

a humanized L chain and the C region of a human L chain under the control of an expression regulatory region such as the enhancer/promoter system.

C 20 Subsequently, a host cell such as the mammalian S" cell is cotransformed using these vectors, and the transformed cells are cultured in vitro or in vivo to ooooo sce" produce a chimeric antibody or a reshaped human antibody *see(for example, International Application Publication No.

WO 91-16928). Furthermore, an antibody gene is introduced into mammals such as goat to produce a transgenic animal, from the milk of which a chimeric antibody or a reshaped human antibody can be obtained.

Also, the V region of an H chain and the C region of an H chain and the V region of an L chain and the C region of an L chain are ligated to a single vector to transform a suitable host cell and thereby to produce antibodies. Thus, for the expression of chimeric antibodies, DNA encoding the mouse leader sequence and the V region of the H chain and human H chain C region present in the cloned cDNA, and DNA encoding the mouse leader sequence and L chain V region and human L chain C 42 region are introduced into a single expression vector (see International Application Publication No. WO 94-11523).

For the expression of a reshaped human antibody, DNA encoding the V region of a humanized H chain and C region of a human H chain, and DNA encoding the V region of a humanized L chain and the C region of a human L chain are introduced into a single expression vector (see International Application Publication No. W0 94-11523). Using said vector a host cells are transformed, and the transformed host cells are cultured in vivo or in vitro to produce the desired chimeric antibody or the reshaped human antibody.

A transformant that was transformed, as 15 mentioned above, by a gene encoding the desired chimeric antibody or a reshaped human antibody is cultured, and the chimeric antibody or the reshaped human antibody produced can be isolated from the inside or the outside of the cells and purified to homogeneity.

The isolation and purification of the desired protein of the present invention, a chimeric antibody or a reshaped human antibody, may be carried out using an affinity column. As a column that employs protein A, for example, there is mentioned HyperD, POROS, Sepharose F.

F, etc. Alternatively, the conventional isolation and purification methods used for proteins can be used and the method is not limited in any way. For example, combinations of various chromatographic methods, ultrafiltration, salting-out, dialysis, and the like, as appropriate, would permit the isolation and purification of the chimeric antibody of the reshaped human antibody.

For the production of the chimeric anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody of the present invention, any expression method can be used including, for example, the eukaryotic cells such as animal cells, an established mammalian cell-line system, an insect cell system, a fungal cell system, and a yeast 43 cell system, and the procaryotic cells such as bacterial cells such as Escherichia coli cells, and the like.

Preferably, the chimeric antibody or the reshaped human antibody of the present invention may be expressed in the COS cells, the CHO cells, the Hela cells, the Vero cells, the myeloma cells or the BHK cells.

In these cases, common promoters that are useful for the expression of mammalian cells can be used.

For example, preferably the human cytomegalovirus immediate early (HCMV) promoter may be used. Examples of the expression vectors containing the HCMV promoter include those which are HCMV-VH-HCyl, HCMV-VL-HCK, etc.

Sand which are derived from pSV2neo (International Application Publication No. WO 92-19759).

S* 15 Furthermore, as a promoter for gene expression in the mammalian cells for use in the present invention, there can be used viral promoters such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV40), etc., and promoters derived from mammalian cells such as human polypeptide chain elongation factor la (HEF-la), etc.

For example, when the promoter of SV40 is used, expression can be easily carried out using the method of Mulligan et al. (Nature 277, 108(1979)), and when HEF-lc promoter is used the method of Mizushima, S. et al.

.(Nucleic Acids Research, 18, 5322, 1990) can be used.

As a source of replication, there can be used those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like, and for the amplification of the copy number of the gene in a host cell system, the expression vector can include, as a selective marker, aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (DHRF) gene, and the like.

4. The binding inhibition activity of a chimeric antibody or a reshaped human antibody Measurement of antibody concentration 44 The concentration of purified antibody may be measured by ELISA or the measurement of absorbance.

ELISA plates for measurement of antibody concentration may be prepared as follows. Each well of a 96-well ELISA plate (for example Maxisorp, manufactured by NUNC) is immobilized with 100 il of goat anti-human IgG antibody at a concentration of 1 gg/ml.

After blocking with 100 gg/ml of a dilution buffer (for example 50 mM Tris-HCl, 1 mM MgCl 2 0.15 M, NaCI, 0.05% Tween 20, 0.02% NaN 3 1% bovine serum albumin (BSA), pH serial dilutions of culture supernatant of cells in which the chimeric antibody, the hybrid antibody, or the reshaped human antibody was expressed, for example the culture supernatant of COS cells or CHO 15 cels, or the purified chimeric antibody, hybrid antibody, or reshaped human antibody is added to each well. Then 100 pl of alkaline phosphatase conjugated goat anti-human IgG antibody is added, 1 mg/ml of the substrate solution (Sigmal04, p-nitrophenyl phosphate, manufactured by 20 SIGMA) is added, and then the absorbance at 405 nm is measured using a microplate reader (Bio Rad). As the standard for the measurement of concentration, a human IgG1K (manufactured by The BInding Site) can be used.

The concentration of the purified antibody is obtained by measuring absorbance at 280 nm and calculating with 1 mg/ml as 1.35 OD.

Binding activity Binding activity can be measured by the Cell-ELISA using the human amniotic cell line WISH (ATCC CCL25). The Cell-ELISA plate may be prepared as follows.

WISH cells prepared at an appropriate concentration with PRMI 1640 medium supplemented with 10% fetal bovine serum are added to a 96-well plate, incubated overnight, and after washing twice with are fixed with 0.1% glutaraldehyde (manufactured by Nakalai tesque).

After blocking, 100 gl of serial dilutions of 45 the culture supernatant of cells in which the chimeric anti-HM 1.24 antibody, the hybrid anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody was expressed, for example the culture supernatant of COS cells or CHO cells, or the purified chimeric anti-HM 1.24 antibody, hybrid anti-RM 1.24 antibody or reshaped human anti-HM 1.24 antibody is added to each well, incubated at room temperature for two hours, and then peroxidase -labelled rabbit anti-human IgG antibody (manufactured'by DAKO) is added.

After icubating at room temperature for one hour, the substrate solution is added and then incubated.

Subsequently, the reaction is stopped by 50 l of 6N sulfuric acid, and then absorbance at 490 nm is measured 15 using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

Measurement of binding inhibition activity Binding inhibition activity by the biotinylated mouse anti-HM 1.24 antibody is measured by the Cell-ELISA 20 using the human amniotic cell line WISH (ATCC The Cell-ELISA plate may be prepared according to the above-mentioned WISH cells prepared at an S..appropriate concentration with PRMI 1640 medium supplemented with 10% fetal bovine serum are added to a .96-well plate, incubated overnight, and after washing twice with are fixed with 0.1% glutaraldehyde (manufactured by Nakalai tesque).

After blocking, 50 al of serial dilutions of the culture supernatant of cells in which the chimeric anti-HM 1.24 antibody, the hybrid anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody was expressed, for example the culture supernatant of COS cells or CHO cells, or the purified chimeric anti-HM 1.24 antibody, hybrid anti-HM 1.24 antibody or reshaped human anti-HM 1.24 antibody is added to each well, and simultaneously 50 il of 2 pg/ml biotinylated mouse anti-HM 1.24 antibody is added, and then incubated at 46 room temperature for two hours, and after washing, peroxidase-labelled streptavidin (manufactured by DAKO) is added.

After icubating at room temperature for one hour and after washing, the substrate solution is added and then incubated. Subsequently, the reaction is stopped by 50 pl of 6N sulfuric acid, and then absorbance at 490 nm is measured using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

Measurement of ADCC activity The ADCC activity of the chimeric antibody or the reshaped human antibody of the present invention can be measured as follows. First, mononuclear cells are separated from human peripheral blood or bone marrow by the density centrifugation method and prepared as the effector cell. Human myeloma cells are prepared as the target cell by labelling the RPMI 8226 cells (ATCC CCL 155) with Cr. Then, the chimeric antibody or the reshaped human antibody to be measured for ADCC activity is added to the labelled target cells and incubated, and then a suitable ratio of the effector cell is added to the target cell and incubated.

After incubation the supernatant is taken to be measured for radioactivity using a gamma counter. At this time, 1% NP-40 can be used for measurement of the maximum released radioactivity. Cytotoxicity can be calculated as x 100, wherein A is radioactivity (cpm) released in the presence of antibody, B is radioactivity (cpm) released by NP-40, and C is radioactivity (cpm) released by the culture liquid alone without antibody.

When ADCC activity or CDC activity is expected for the C region of antibody, human Cyl or human Cy3can be used as the C region of antibody. Furthermore, by adding, altering, or modifying part of the amino acid of the C region of antibody, a higher ADCC activity or CDC 47 activity can be induced.

For example, there are the IgM-like polymerization of IgG by amino acid substitution (Smith, R.I.F. Morrison, S.L, BIO/TECHNOLOGY (1994) 12, 683-688), the IgM-like polymerization of IgG by amino acid addition (Smith, R.I.F. et al., J. Immunol. (1995) 154, 2226-2236), expression by tandem linking of genes encoding an L chain (Shuford, W. et al., Science (1991) 252, 724-727), dimerization of IgG by amino acid substitution (Caron, P.C. et al., J. Exp. Med. (1992) 176, 1191-1195, Shopes, B.J. Immunology (1992) 148, 2918-2922, dimerization of IgG by chemical modification (Wolff, E.A. et al., Cancer Res. (1993) 53, 2560-2565), and the introduction of the effector function by amino 15 acid alteration at the hinge region of antibodies (Norderhaug, L. et al., Eur. J. Immunol (1991) 21, 2379-2384). They can be accomplished by the oligomer site directed mutagenesis using primers, addition of base sequences using restriction enzyme cleavage sites, and chemical modifiers that induces covalent bonding.

in vivo diagnostics for Myeloma The chimeric anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody of the present invention can be used as an in vivo diagnostics for myeloma by linking it to a labelled compound such as radioisotope and the like.

Furthermore, fragments of the chimeric anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody, such as Fab, F(ab')2, Fv, or single chain Fv (scFv) wherein the Fv or the Fv of an H chain and an L chain are linked by a suitable linker that has been bound to a label compound such as radioisotope etc. can be used as an in vivo diagnostics for myeloma.

Specifically these antibody fragments can be obtained by constructing the gene encoding these antibody fragments, introducing them into an expression vector, and then expressing in a suitable host cells, or 48 digesting the chimeric anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody with a suitable enzyme.

The above-mentioned in vivo diagnostics for myeloma can be systematically administered in a parenteral manner.

A pharmaceutical composition and a therapeutic agent for myeloma In order to confirm the therapeutic effects of the chimeric anti-HM 1.24 antibody or the humanized anti-HM 1.24 antibody of the present invention, said antibodies are administered to a myeloma cells-transplanted animal and the anti-tumor effects are evaluated.

As myeloma cells to be transplanted to animals, 15 human myeloma cells are preferred, and there can be mentioned, for example, KPMM2 (Japanese Unexamined Patent Publication (Kokai) No. 7-236475), RPMI8226 (ATCC CCL 155), ARH-77 (ATCC CRL 1621), and S6B45 (Suzuki, H. et al., Eur. J. Immunol. (1992) 22, 1989-1993). As the animals to which said cells are transplanted, animals in which immunological functions are decreased or lacking are preferred, and there can be mentioned nude mouse, SCID mouse, beige mouse, and nude rat.

Furthermore, the anti-tumor effects to be evaluated can be confirmed by variation in the amount of human immunoglobulins in the serum, measurement of tumor volume and/or weight, variation in the weight of human Bence Jones proteins in the urine, the survival period of animals, or the like.

Pharmaceutical compositions or therapeutic agents for myeloma that contain as an active ingredient the chimeric anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody of the present invention can be systematically or locally administered in a parenteral manner. For example, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, or subcutaneous injection can be selected and 49 the dosage regimen may be selected as appropriate depending on the age and the medical conditions of the patients.

Effective dosage is selected from the range of 0.01 mg to 1000 mg/kg body weight/dose. Alternatively, the dosage of 5 mg/body, preferably 50 to 100 mg/body, may be selected.

Pharmaceutical compositions or therapeutic agents for myeloma that contain as an active ingredient the chimeric anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibody of the present invention may contain pharmaceutically acceptable carriers or additives depending on the route of administration.

As examples of such carriers and additives, there may be mentioned water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, arabic gum, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, 25 lactose, pharmaceutically acceptable surfactants, and the like. Additives to be used may be selected from, but not limited to, the above or combinations thereof.

Examples Next, the present invention will be explained more specifically.

Example 1. Cloning of cDNA encoding the V region of a mouse anti-HM 1.24 antibody 1. Isolation of messenger RNA (mRNA) Using Fast Track mRNA Isolation Kit Version 3.2 (manufactured by Invitrogen) according to the instruction attached thereto, mRNA was isolated from 2 x 108 50 hybridoma cells (FERM BP-5233) that produce a mouse anti-HM 1.24 antibody.

2. Amplification of the gene encoding the variable region of antibody by the PCR method PCR was carried out using the amplification Thermal Cycler (manufactured by Perkin Elmer Cetus).

2-1. Amplification and fragmentation of the gene encoding the V region of a mouse L chain From the mRNA thus isolated, single stranded' cDNA was synthesized using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Life Science) and used for PCR. As primers used for PCR, MKV (Mouse Kappa Variable) primers (Jones, S.T. et al, Bio/Technology, 9, 88-89, (1991)) shown in SEQ ID NO: 29 15 to 39 that hybridize with the leader sequence of a mouse kappa type L chain was used.

100 tl of the PCR solution containing 10 mM Tris-HCl (pH 50 mM KC1, 0.1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl 2 5 units of DNA polymerase Ampli Tag (manufactured by Perkin Elmer Cetus), 0.25 mM of the MKV primers shown in SEQ ID NO: 29 to 39, 3 mM of the MKC primer shown in SEQ ID NO: 40, and 100 ng of single stranded cDNA was covered with 50 il of mineral I oil, and then heated at an initial temperature of 94°C 25 for 3 minutes, and then at 94 0 C for 1 minute, at 55 0 C for 1 minute and at 72 0 C for 1 minute in this order. After repeating this cycle for 30 times, the reaction mixture was incubated at 72 0 C for 10 minutes. The amplified DNA fragment was purified by the low melting point agarose (manufactured by Sigma), and digested with XmaI (manufactured by New England Biolabs) and SalI (manufactured by Takara Shuzo) at 37 0

C.

2-2. Amplification and fragmentation of cDNA encoding the V region of a mouse H chain The gene encoding the V region of a mouse H chain was amplified by the 5'-RACE method (Rapid 51 Amplification of cDNA ends; Frohman, M.A. et al., Proc.

Natl. Acad. Sci. USA, 85, 8998-9002, (1988), Edwards, et al., Nucleic Acids Res., 19, 5227-5232, (1991)). After cDNA was synthesized using primer P1 (SEQ ID NO: 41) that specifically hybridizes with the constant region of mouse IgG2a, cDNA encoding the V region of a mouse H chain was amplified by the 5'-AmpliFINDER

RACE

KIT (manufactured by CLONTECH) using the primer MHC2a (SEQ ID NO: 42) that specifically hybridizes with the constant region of mouse IgG2a and the anchor primer

(SEQ

ID NO: 77) attached to the kit. The amplified

DNA

fragment was purified with the low melting point agarose (manufactured by Sigma) and digested with EcoRI (manufactured by Takara Shuzo) and XmaI (manufactured by 15 New England Biolabs) at 37 0

C.

3. Linking and transformation The DNA fragment comprising the gene encoding the V region of the mouse kappa type L chain prepared as above was ligated to the pUC19 vector prepared by digesting with SalI and XmaI by. reacting in a reaction mixture containing 50 mM Tris-HCl (pH 10 mM MgCl 2 10 mM dithiothreitol, 1 mM ATP, 50 mg/ml of polyethylene glycol (8000) and one unit of T4 DNA ligase (manufactured by GIBCO-BRL) at 16 0 C for 2.5 hours. Similarly, the DNA fragment comprising the gene encoding the V region of the mouse H chain was reacted and ligated to pUC19 vector prepared by digesting with EcoRI and XmaI at 16 0 C for three hours.

Then 10 il of the above ligation mixture was added to 50 Al of the competent cells of Escherichia coli which was left on ice for 30 minutes, at 42 0 C for one minute, and again on ice for one minute.

Subsequently 400 Al of 2xYT medium (Molecular Cloning:

A

Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) was added thereto, incubated at 37 0 C for one hour, and then the E. coli was plated on the 52 2xYT agar medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) containing 50 ig/ml of ampicillin, and then incubated overnight at 37 0 C to obtain the E. coli transformant.

The transformant was cultured overnight at 37 0

C

in 10 ml of the 2xYT medium containing 50 gg/ml of ampicillin, and then from this culture plasmid DNA was prepared using the alkali method (Molecular Cloning: A' Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)).

The plasmid thus obtained containing the gene encoding the V region of the mouse kappa type L chain derived from the hybridoma that produces the anti-HM 1.24 15 antibody was termed pUCHMVL9. The plasmid obtained in the above-mentioned method containing the gene encoding the V region of the mouse H chain derived from the hybridoma that produces the anti-HM 1.24 antibody was termed pUCHMVHR16.

Example 2. Determination of the base sequence of DNA The base sequence of the cDNA coding region in the above-mentioned plasmid was determined using the automatic DNA sequencer (manufactured by Applied Biosystem Inc.) and Taq Dye Deoxy Terminator Cycle 25 Sequencing Kit (manufactured by Applied Biosystem Inc.) in the protocol indicated by the manufacturer.

The base sequence of the gene encoding the V region of the L chain of the mouse anti-HM 1.24 antibody contained in the plasmid pUCHMVL9 is shown in SEQ ID NO: 1. The base sequence of the gene encoding the V region of the H chain of the mouse anti-HM 1.24 antibody contained in the plasmid pUCHMVHR16 is shown in SEQ ID NO: 2.

ExamDle 3. Determination of CDR The overall structures of the V regions of an L chain and an H chain have similarity with each other, in which four framework portions are linked by three 53 hypervariable regions, i.e. complementarity determining regions (CDR). The amino acid sequence of the framework are relatively well conserved but variation in the amino acid sequence is extremely high (Kabat, et al., "Sequences of Proteins of Immunological Interest", 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 the amino acid sequences of antibodies in the database to investigate homology, and the CDR region was determined as shown in Table Table Plasmid Sequence No. CDR(1) CDR(2) CDR(3) pUCHMVL9 3-5 24-34 50-56 89-97 pUCHMVHR16 6-8 31-35 50-66 99-109 e Example 4. Confirmation of expression of the cloned cDNA (Construction of the chimeric anti-HM 1.24 antibody) 1. Construction of an expression vector In order to construct the expression vector 25 that expresses a chimeric anti-HM 1.24 antibody, cDNA clones pUCHMVL9 and pUCHMVHR16 encoding the V regions of the L.chain and the H chain of the mouse anti-HM 1.24 antibody, respectively, were modified by the PCR method, and then introduced into the HEF expression vector (International Application Publication No. WO 92-19759).

The backward primer ONS-L722S (SEQ ID NO: 43) for the V region of an L chain and the backward primer VHR16S (SEQ ID NO: 44) for the V region of an H chain were designed so that they hybridize to the DNA encoding the start of the leader sequence of the V region of each and they have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950, (1987)) and the 54 recognition site for HindIII restriction enzyme. The forward primer VL9A (SEQ ID NO: 45) for the V region of an L chain and the forward primer VHR16A (SEQ ID NO: 46) for the V region of an H chain were designed so that they hybridize to the DNA sequence encoding the end of the J region and they have a splice donor sequence and the recognition site for BamHI restriction enzyme.

100 pl of the PCR reaction mixture containing mM Tris-HCl (pH 50 mM KC1, 0.1 mM dNTPs MgCl 2 100 pmole each of each primer, 100 ng of template DNA (pUCHMVL9 or pUCHMVHR16), and 5 units of Ampli Taq enzyme was covered with 50 l of mineral oil, and then after the initial denaturation at 94 0 C, heated at 94°C for 1 minute, at 55 0 C for 1 minute and at 72 0 C for 1 15 minute for 30 cycles and finally incubated at 720C for minutes.

The PCR product was purified by the 1.5% low melting point agarose gel, and digested with HindIII and BamHI, and then cloned to HEF-VL-gK for the V region of the L chain and to HEF-VH-gyl for the V region of the H chain. After determination of the DNA sequence, the plasmids containing the DNA fragment that contains the correct DNA sequence were termed HEF-1.24L-gK and HEF-1.24H-gyl, respectively.

25 .The regions encoding the respective variable region from the above plasmids HEF-1.24L-gK and HEF-1.24H-gyl were digested with restriction enzymes HindIII and BamHI to make restriction fragments, which were inserted to the HindIII site and the BamHI sites of plasmid vector pUC19 and they were termed pUC19-1.24L-gK and pUC19-1.24H-gyl, respectively.

Escherichia coli containing respective plasmids pUC19-1.24L-gK and pUC19-1.24H-gyl were termed Escherichia coli DH5a (pUC19-1.24L-gK) and Escherichia coli DH5a (pUC19-1.24H-gyl), and were internationally deposited on August 29,1996, with the National Institute 55 a a a of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under the accession numbers FERM BP-5646 and FERM BP-5644, respectively, under the provisions of the Budapest Treaty.

2. Transfection into COS-7 cells In order to observe the transient expression of the chimeric anti-HM 1.24 antibody, the above expression vectors were tested in the COS-7 (ATCC CRL-1651) cells. HEF-1.24L-gK and HEF-1.24H-gyl were cotransformed into COS-7 cells by electroporation using the Gene Pulser instrument (manufactured by BioRad).

Each DNA (10 jg) was added to 0.8 ml aliquots of 1 x 107 cells/ml in PBS, and was subjected to pulses at 1500 V and a capacity of 25 UF.

After the recovery period of 10 minutes at room temperature, the electroporated cells were added to 30 ml of the DMEM culture liquid (manufactured by GIBCO) 20 containing 10% y-globulin free bovine fetal serum. After incubation of 72 hours in the CO 2 incubator BNA120D (manufactured by TABAI), the culture supernatant was collected, and the cell debris were removed by centrifugation, which were used for the following experiment.

3. FCM analysis The antigen binding activity of the chimeric anti-HM 1.24 antibody was investigated by FCM (flow cytometry) analysis using the KPMM2 cells. After 4.7 x 10 5 KPMM2 cells (Japanese Unexamined Patent Publication (Kokai) No. 7-236475) were washed with 50 gl of the culture of COS-7 cells that produces the above-mentioned chimeric anti-HM 1.24 antibody and 50 p1 of FACS buffer containing 2% bovine fetal serum and 0.1% sodium azide), or 5 tl of 500 jg/ml purified mouse anti-HM 1.24 antibody and 95 p1 of the FACS buffer 56 were added, and incubated on ice for one hour.

As a control, 50 1l of 2 pg/ml chimeric SK2 (International Application Publication No. WO 94-28159) and 50 pA of the FACS buffer, or 5 pl of 500 pg/ml purified mouse IgG2aK (UPC10) (manufactured by CAPPEL in stead of purified mouse anti-HM 1.24 antibody, and p1 of FACS buffer were added, and similarly incubated.

After washing with the FACS buffer, 100 pl of 25 pg/ml FITC conjugated goat anti-human antibody (GAH) (manufactured by CAPPEL) or 10 gg/ml FITC conjugated goat anti-mouse antibody (GAM) (manufactured by Becton Dickinson) were added, and incubated at a temperature of ice for 30 minutes. After washing with the FACS buffer, it was suspended in one ml of the FACS buffer, and 15 fluorescence intensity of each cell was measured by the FACScan (manufactured by Becton Dickinson).

As shown in Fig. 1, it was revealed that the chimeric anti-HM 1.24 antibody bound to the KPMM2 cell because the peak of fluorescence intensity shifted to the right in the chimeric anti-HM 1.24 antibody-added cells as 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 the mouse anti-HM 1.24 antibody.

Example 5. Establishment of the CHO cell line that stably produces a chimeric anti-HM 1.24 antibody 1. Construction of an expression vector for the chimeric H chain After digesting the above plasmid HEF-1.24H-gyl with the restriction enzymes PvuI and BamHI, an about 2.8 kbp fragment containing the EF1 promoter and the DNA encoding the V region of the H chain of the mouse anti-HM 1.24 antibody was purified using 1.5% low melting point agarose gel. Then, the above DNA fragment was inserted into an about 6 kbp fragment prepared by digesting the expression vector used for a human H chain expression 57 vector, DHFR-AE-Rvh-PM1f (see International Application Publication No. WO 92/19759), containing the DHFR gene and the gene encoding the constant region of a human H chain with PvuI and BamHI to construct an expression vector, DHFR-AE-HEF-1.24H-gyl, for the H chain of the chimeric anti-HM 1.24 antibody.

2. Gene introduction into CHO cells In order to establish a stable production system of the chimeric anti-HM 1.24 antibody, the genes of the above-mentioned expression vectors, HEF-1.24L-gK and DHFR-AE-HEF-1.24H-gyl, that were linearized by digestion with PvuI were simultaneously introduced into S. the CHO cell DXB11 (donated from the Medical Research Council Collaboration Center) by the electroporation 15 method under the condition similar to the above-mentioned one (the above-mentioned transfection into the COS-7 cells).

3. Gene amplification by MTX Among the gene-introduced CHO cells, only those CHO cells in which both of the L chain and the H chain expression vectors have been introduced can survive in the nucleoside-free a-MEM culture liquid (manufactured by GIBCO-BRL) to which 500 gg/ml G418 (manufactured by GIBCO-BRL) and 10% bovine fetal serum were added, and so they were selected. Subsequently, 10 nM MTX (manufactured by Sigma) was added to the above culture liquid. Among the clones that propagated, those that produce the chimeric anti-HM 1.24 antibody in large amounts were selected. As a result, clones #8 13 that exhibit a production efficiency of about 20 g/ml of the chimeric antibody were obtained and termed the chimeric anti-HM 1.24 antibody-producing cell lines.

Example 6. Construction of the chimeric anti-HM 1.24 antibody The chimeric anti-HM 1.24 antibody was constructed in the following method. The above chimeric anti-HM 1.24 antibody-producing CHO cells were subjected to continuous 58 culture for 30 days using as the medium Iscove's Modified Dulbecco's Medium (manufactured by GIBCO-BRL) containing y-globulin free newborn bovine serum (manufactured by GIBCO-BRL) by the high-density cell culture instrument Verax system 20 (manufactured by CELLEX BIOSCIENCE Inc.).

On day 13, 20, 23, 26, and 30 after starting the culture, the culture liquid was recovered using a pressurized filter unit SARTOBRAN (manufactured by Sartorius), and then the chimeric anti-HM 1.24 antibody was affinity-purified using a large-volume antibody collection system Afi-Prep System (manufactured by Nippon Gaishi) and Super Protein A column (bed volume: 100 ml, manufactured by Nippon Gaishi) using PBS as the absorption/wash buffer and 0.1 M sodium citrate buffer 15 (pH 3) as the elution buffer according to the attached instructions. The eluted fractions were adjusted to about pH 7.4 by immediately adding 1 M Tris-HCl (pH Antibody concentration was measured by absorbance at 280 Su nm and calculated with 1 Vg/ml as 1.35 OD.

Example 7. Determination of activity of the chimeric anti-HM 1.24 antibody Chimeric anti-HM 1.24 antibody was evaluated by the following binding inhibition activity.

1. Measurement of binding inhibition activity o* 25 1-1. Construction of a biotinylated anti-HM 1.24 antibody After the mouse anti-HM 1.24 antibody was diluted with 0.1 M bicarbonate buffer to 4 mg/ml, 4 Vl of mg/ml Biotin-N-hydroxy succinimide (manufactured by EY LABS Inc.) was added and reacted at room temperature for 3 hours. Thereafter, 1.5 ml of 0.2 M glycine solution was added thereto, incubated at room temperature for minutes to stop the reaction, and then the biotinylated IgG fractions were collected using the PD-10 column (manufactured by Pharmacia Biotech).

1-2. Measurement of binding inhibition activity The binding inhibition activity by the 59 biotinylated mouse anti-HM 1.24 antibody was measured by the Cell-ELISA using the human amniotic membrane cell line WISH cells (ATCC CCL 25). The Cell-ELISA plates were prepared as follows. To a 96-well plate was added 4 x 10 5 cells/ml prepared with PRMI 1640 medium supplemented with 10% fetal bovine serum, incubated overnight, and after washing twice with were immobilized with 0.1% glutaraldehyde (manufactured by Nakalai tesque).

After blocking, 50 gl of serial dilutions of the chimeric anti-HM 1.24 antibody or the mouse anti-HM 1.24 antibody obtained by affinity-purification was added to each well and simultaneously 50 gl of 2 g/ml biotinylated mouse anti-HM 1.24 antibody was added, incubated at room temperature for two hours, and then the peroxidase-labelled streptavidin (manufactured by DAKO) was added. After incubating-at room temperature for one hour and then washing, the substrate solution was added.

After stopping the reaction by adding 50 pl of 6N 20 sulfuric acid, absorbance at 490 nm was measured using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

The result, as shown in Fig. 2, revealed that the chimeric anti-HM 1.24 antibody has the identical binding inhibition activity with the mouse anti-HM 1.24 antibody to the biotinylated mouse anti-HM 1.24 antibody.

This indicates that the chimeric antibody had the same V region as the mouse anti-HM 1.24 antibody.

Example 8. Measurement of the ADCC activity of the chimeric anti-HM 1.24 antibody ADCC (Antibody-dependent Cellular Cytotoxicity) activity was measured according to the method as set forth in Current Protocols in Immunology, Chapter 7.

Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley Sons, Inc., 1993.

1. Preparation of effector cells 60 Monocytes were separated from the peripheral blood or bone marrow of healthy humans and patients with multiple myeloma by the density centrifugation method.

Thus, an equal amount of PBS(-) was added to the peripheral blood and the bone marrow of healthy humans and patients with multiple myeloma, which was layered on Ficoll (manufactured by Pharmacia)-Conrey (manufactured by Daiichi Pharmaceutical Co. Ltd.) (specific gravity, 1.077), and was centrifuged at 400 g for 30 minutes. The monocyte layer was collected, and washed twice with RPMI 1640 (manufactured by Sigma) supplemented with 10% fetal bovine serum (manufactured by Witaker), and prepared at a cell density of 5 x 10 /ml with the same culture liquid.

1"5 2. Preparation of target cells The human myeloma cell line RPMI 8226 (ATCC CCL 155) was radiolabelled by incubating in the RPMI 1640 (manufactured by Sigma) supplemented with 10% fetal bovine serum (manufactured by Witaker) together with 0.1 mCi of 51Cr-sodium chromate at 37 0 C for 60 minutes.

20 After radiolabelling, cells were washed three times with Hanks balanced salt solution (HBSS) and adjusted to a concentration of 2 x 10 /ml.

3. ADCC assay Into a 96-well U-bottomed plate (manufactured by Corning) were added 50 ul of 2 x 105 target cells/ml, 1 gg/ml of affinity-purified chimeric anti-HM 1.24 antibody and mouse anti-HM 1.24 antibody, or control human IgG (manufactured by Serotec), and reacted at 4 0

C

for 15 minutes.

Then, 100 ul of 5 x 106 effector cells/mi was added thereto, and cultured in the CO 2 incubator for 4 hours, when the ratio of the effector cells to the target cells was set at 0:1, 5:1, 20:1, or 50:1.

One hundred 1l of the supernatant was taken and the radioactivity released into the culture supernatant was measured by the gamma counter (ARC361, manufactured 61 by Aloka). For measurement of the maximum radioactivity, 1% NP-40 (manufactured by BRL) was used. Cytotoxicity was calculated by 100, wherein A is radioactivity (cpm) released in the presence of antibody, B is radioactivity (cpm) released by NP-40, and C is radioactivity (cpm) released by the culture liquid alone without antibody.

As shown in Fig. 3, when the chimeric anti-HM 1.24 antibody was added as compared to the control IgG1, cytotoxicity increased with the increase in the E:T ratio, which indicated that this chimeric anti-HM 1.24 antibody has ADCC activity. Furthermore, since there was no cytotoxicity observed even when the mouse anti-HM 1.24 antibody was added, it was shown that the Fc portion of 15 human antibody is required to obtain ADCC activity when the effector cell is a human-derived cell.

Example 9. Construction of the reshaped human anti-HM 1.24 antibody 1. Designing of the V region of the reshaped human 20 anti-HM 1.24 antibody- In order to construct the reshaped human antibody in which the CDR of mouse monoclonal antibody has been grafted to a human antibody, it is preferred that there is a high homology between the FR of the mouse antibody and the FR of the human antibody. Thus, the V regions of the L chain and the H chain of the mouse anti-HM 1.24 antibody were compared to the V regions of all known antibodies whose structure has been elucidated using the Protein Data Bank.

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

When the V region of the L chain of the mouse anti-HM 1.24 antibody is compared to the V region of the 62 L chain of known human antibodies, it has shown a homology of 67.0% with the V region REI of a human L chain, one of the subgroup I of the V region of a human L chain. Thus, the FR of REI was used as the starting material for construction of the V region of the L chain of the reshaped human.anti-HM 1.24 antibody.

Version a of the V region of the L chain of the reshaped human anti-HM 1.24 antibody was designed. In this version, human FR was made identical with the REI-based FR present in the reshaped human CAMPATH-1H antibody (see Riechmann, L. et al., Nature 322, 21-25, (1988), the FR contained in version a of the V region of the L chain of the reshaped human PM-1 described in "International Application Publication No. WO 92-19759), and the mouse CDR was made identical with the CDR in the V region of the L chain of the mouse anti-HM 1.24 antibody.

The H chain V region of the mouse anti-HM 1.24 antibody is most similar to the consensus sequence of HSGI of the V region of a human H chain with a homology of 54.7%. On the other hand, it shows a homology of 34.6% and 48.1% with HSGII and HSGIII, respectively.

When the V region of the H chain of the mouse anti-HM 1.24 antibody is compared to the V region of the H chain of known human antibodies, FR1 to FR3 were most similar to the V region of the H chain of the human antibody HG3, one of subgroup I of the V region of a human H chain (Rechavi, G. et al., Proc. Natl. Acad. Sci. USA, 855-859), with a homology of 67.3%.

Therefore, the FR of the human antibody HG3 was used as the starting material for construction of the V region of the H chain of the reshaped human anti-HM 1.24 antibody. However, since the amino acid sequence of the FR4 of human HG3 has not been described, the amino acid sequence of the FR4 of the human antibody JH6 (Ravetch, J.V. et al., Cell, 27, 583-591) that shows the highest homology with the FR4 of the H chain of the mouse anti-HM 63 1.24 antibody was used. The FR4 of JH6 has the same amino acid sequence as that of the FR4 of the H chain of the mouse anti-HM 1.24 antibody except one amino acid.

In the first version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody, FR1 to FR3 were made identical with the FR1 to FR3 of human HG3, and the CDR was made identical with the CDR of the V region of the H chain of the mouse anti-HM 1.24 antibody, except that the amino acids at position 30 in the human FR1 and position 71 in the human FR3 were made identical with the amino acids in the mouse anti-HM 1.24 antibody.

2. Construction of the V region of the L chain of the reshaped human anti-HM 1.24 antibody The L chain of the reshaped human anti-HM 1.24 15 antibody was constructed by the CDR grafting in the PCR method. The method is shown in Fig. 4. Eight PCR primers were used for construction of the reshaped human anti-HM 1.24 antibody (version a) having the FR derived from the human antibody REI. The external primers A (SEQ 20 ID NO: 47) and H (SEQ ID NO: 48) were designed to hybridize with the DNA sequence of the expression vector HEF-VL-gK.

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

In the first stage of PCR, the four reactions A-L1A, L1S-L2A, L2S-L3A, and L3S-H were conducted to purify each PCR product. The four PCR products from the first PCR were allowed to assemble with one another by their own complementarity (see International Application Publication No. WO 92-19759). Then, external primers A and H were added to amplify the full-length DNA encoding 64 the V region of the L chain of the reshaped human anti-HM 1.24 antibody (the second PCR). In the above-mentioned PCR, the plasmid HEF-RVL-M21a (see International Application Publication No. WO 95-14041) encoding the version a of the V region of the L chain of the reshaped human ONS-M21 antibody based on the human antibody REI-derived FR was employed as a template.

In the first stage of PCR, the PCR mixture containing 10 mM Tris-HC1 (pH 50 mM KC1, 0.1 mM dNTPs, 1.5 mM MgCl,, 100 ng of template DNA, 100 pmole of each primer, and 5 u of Ampli Taq was used. Each PCR tube was covered with 50 ul of mineral oil. Then after it was first denatured by heating at 94 0 C, it was subjected to a reaction cycle of 94 0 C for 1 minute, 55 0

C

for 1 minute and 72 0 C for 1 minute, and then was incubated at 72 0 C for 10 minutes.

PCR products A-L1A (215 bp), L1S-L2A(98 bp), L2S-L3A (140 bp), and L3S-H (151 bp) were purified using 1.5% low melting point agarose gel and were assembled in 20 the second PCR. In the second PCR, 98 il of PCR mixture containing 1 gg each of the first stage PCR products and 5 u of Ampli Taq was incubated for 2 cycles of 94 0 C for 2 .minutes, 55 0 C for 2 minutes, and 72 0 C for 2 minutes, and then 100 pmole each of the external primers (A and H) was added. The PCR tube was coated with 50 l of mineral oil and 30 cycles of PCR were conducted under the same condition as above.

A 516 bp DNA fragment resulting from the second PCR was purified using 1.5% low melting point agarose gel, digested with BamHI and HindIII, and the DNA fragments thus obtained were cloned into the HEF expression vector HEF-VL-gK. After determining the DNA sequence, the plasmid containing the DNA fragment having the correct amino acid sequence of the V region of the L chain of the reshaped human anti-HM 1.24 antibody was termed plasmid HEF-RVLa-AHM-gK. The amino acid sequence 65 and the base sequence of the V region of L chain contained in this plasmid HEF-RVLa-AHM-gK are shown in SEQ ID NO: 9.

The version b of the V region of the L chain of the reshaped human anti-HM 1.24 antibody was constructed by mutagenesis using PCR. Mutagen primers FTY-1 (SEQ ID NO: 55) and FTY-2 (SEQ ID NO: 56) were so designed as to mutate phenylalanine at position 71 to tyrosine.

After the above primers were amplified usingthe plasmid HEF-RVLa-AHM-gK as a template, the final product was purified and digested with BamHI and HindIII.

The DNA fragments obtained were cloned into the HEF

S.

expression vector HEF-VL-gK to obtain plasmid HEF-RVLb-AHM-gK. The amino acid sequence and the base 15 sequence of the V region of the L chain contained in this plasmid HEF-RVLb-AHM-gK are shown in SEQ ID NO: 3. Construction of the V region of the H chain of the reshaped human anti-HM 1.24 antibody 3-1. Construction of versions a to e of the V region 20 of the H chain of the-reshaped human anti-HM 1.24 antibody DNA encoding the V region of the H chain of the reshaped human anti-HM 1.24 antibody was designed as follows. By linking the DNA sequence encoding the FR1 to 3 of the human antibody HG3 and the FR4 of the human antibody JH6 to the DNA sequence encoding the CDR of the V region of the H chain of the mouse anti-HM 1.24 antibody, the full length DNA encoding the V region of the H chain of the reshaped human anti-HM 1.24 antibody was designed.

Then, to the 5'-end and the 3'-end of this DNA sequence the HindIII recognition site/KOZAK consensus sequence and BamHI recognition site/splice donor sequence, respectively, were attached so as to enable insertion of the HEF expression vector.

The DNA sequence thus designed was divided into four oligonucleotides. Subsequently, oligonucleotides 66 which potentially hinder assembly of these oligonucleotides were subjected to computer analysis for the secondary structure. The sequences of the four oligonucleotides RVH1 to RVH4 are shown in SEQ ID NO: 57 to 60. These oligonucleotides have a length of 119 to 144 bases and have the 25 to 26 bp overlapping region.

Among the oligonucleotides, RVH2 (SEQ ID NO: 58) and RVH4 (SEQ ID NO: 60) have the sense DNA sequence, and RVH1 (SEQ ID NO: 57) and RVH3 (SEQ ID NO: 59) have the antisense DNA sequence. The method for assembling these four oligonucleotides by the PCR method is shown in the figure (see Fig. The PCR mixture (98 gl) containing 100 ng each Sof the four oligonucleotides and 5 u of Ampli Taq was first denatured by heating at 94 0 C for 2 minutes, and was subjected to two cycles of incubation comprising 94 0 C for .2 minutes, 55 0 C for 2 minutes and 72 0 C for 2 minutes.

After 100 pmole each of RHP1 (SEQ ID NO: 61) and RHP2 (SEQ ID NO: 62) were added as the external primer, the PCR tube was coated with 50 il of mineral oil. Then it was first denatured by heating at 94 0 C for 1 minute, and then was subjected to 38 cycles of 94 0 C for 1 minute, 55 0 C for 1 minute and 72 0 C for 1 minute, and then was incubated at 72 0 C for 10 minutes.

The 438 bp DNA fragment was purified using low melting point agarose gel, digested with HindIII and BamHI, and then cloned into the HEF expression vector HEF-VH-gyl. After determination of the base sequence, the plasmid that contains the DNA fragment encoding the amino acid sequence of the correct V region of the H chain was termed HEF-RVHa-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHa-AHM-gyl are shown in SEQ ID NO: 11.

Each of versions b, c, d, and e of the V region of the H chain of the reshaped human anti-HM 1.24 antibody was constructed as follows.

67 Using as the mutagen primer BS (SEQ ID NO: 63) and BA (SEQ ID NO: 64) designed to mutate arginine at position 66 to lysine and as a template DNA the plasmid HEF-RVHa-AHM-gyl by the PCR method, version b was amplified to obtain plasmid HEF-RVHb-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHb-AHM-gyl are shown in SEQ ID NO: 12.

Using as the mutagen primer CS (SEQ ID NO: and CA (SEQ ID NO: 66) designed to mutate threonine at position 73 to lysine and as a template DNA the plasmid HEF-RVHa-AHM-gyl by the PCR method, version c was amplified to obtain plasmid HEF-RVHc-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHc-AHM-gyl are shown in SEQ ID NO: 13.

Using as the mutagen primer DS (SEQ ID NO: 67) and DA (SEQ ID NO: 68) designed to mutate arginine at position 66 to lysine and threonine at position 73 to 20 lysine and as a template DNA the plasmid HEF-RVHa-AHM-gyl by the PCR method, version d was amplified to obtain plasmid HEF-RVHd-AHM-gyl. The amino acid sequence and **the base sequence of the V region of the H chain contained in this plasmid HEF-RVHd-AHM-gyl are shown in SEQ ID NO: 14.

Using as the mutagen primer ES (SEQ ID NO: 69) and EA (SEQ ID NO: 70) designed to mutate valine at position 67 to alanine and methionine at position 69 to leucine and as a template DNA the plasmid HEF-RVHa-AHM-gyl, version e was amplified to obtain plasmid HEF-RVHe-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHe-AHM-gyl are shown in SEQ ID NO: 3-2. Construction of the H chain hybrid V region Two H chain hybrid V regions were constructed.

One is a mouse human hybrid anti-HM 1.24 antibody in 68 which the amino acid sequences of FR1 and FR2 are derived from the mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody, and the other is human mouse hybrid anti-HM 1.24 antibody in which the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from the mouse anti-HM 1.24 antibody. The amino acid sequences of the CDR regions are all derived from mouse anti-HM 1.24 antibody.

S: Two H chain hybrid V regions were constructed by the PCR method. The method is schematically shown in SFig. 6 and 7. For the construction of two H chain hybrid V regions, four primers were used. The external primers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) were designed to oo hybridize with the DNA sequence of the HEF expression vector HEF-VH-gyl. The H chain hybrid construction primer HYS (SEQ ID NO: 73) was designed to have the sense 20 DNA sequence and the H chain hybrid primer HYA (SEQ ID NO: 74) to have the antisense DNA sequence so that the DNA sequence are complementary to each other.

For the construction of the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from the mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody, PCR using the plasmid HEF-1.24H-gyl as a template, the external primer a, and the H chain hybrid primer HYA, and PCR using the plasmid HEF-RVHa-AHM-gyl as a template, the H chain hybrid primer HYS (SEQ ID NO: 73), and the external primer h (SEQ ID NO: 72) were carried out in the first stage of PCR and each PCR product was purified. The two PCR products from the first PCR were allowed to assemble by their own complementarity (see International Application Publication No. WO 92-19759).

69 Then, by adding the external primers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) a full-length DNA encoding the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from the mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody was amplified in the second PCR stage.

For the construction of the H chain hybrid V' region in which the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from the mouse anti-HM 1.24 antibody, PCR using the plasmid HEF-RVHa-AHM-gyl as a template, the external primer a, and the H chain hybrid primer HYA, and PCR using the plasmid HEF-1.24H-gyl as a template, the H chain hybrid primer HYS, and the external primer h were carried out in the first stage of PCR and each PCR product was purified. The two PCR purified products from S 20 the first PCR were allowed to assemble by their own complementarity (see International Application S..Publication No. WO 92-19759).

Then, by adding the external primers a and h, a full-length DNA encoding the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from the mouse anti-HM 1.24 antibody was amplified in the second PCR stage.

The methods of the first PCR, purification of PCR products, assembling, the second PCR, and cloning into the HEF expression vector HEF-VH-gyl were carried out according to the methods shown in "Example 9.

Construction of the V region of the L chain of the reshaped human anti-HM 1.24 antibody".

After determination of the DNA sequence, the plasmid that contains the DNA fragment encoding the 70 correct amino acid sequence of the H chain hybrid V region in which the amino acid sequences of FR1 and FR2 are derived from the mouse anti-HM 1.24 antibody and those of FR3 and FR4 are from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody was termed HEF-MH-RVH-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-MH-RVH-AHM-gyl are shown in SEQ ID NO: 75. Also, the plasmid that contains the DNA fragment encoding the correct amino acid sequence of the H chain hybrid V region in which the amino acid S:sequences of FR1 and FR2 are derived from version a of the V region of the H chain of the reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are from the mouse anti-HM 1.24 antibody was termed HEF-HM-RVH-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gyl are shown in SEQ ID NO: 76.

3-3. Construction of versions f to s of.the V region 20 of the H chain of the reshaped human anti-HM 1.24 antibody Each of versions f, g, h, i, j, k, 1, m, n, o, p, q, r, and s of the V region of the H chain of the reshaped human anti-HM 1.24 antibody were constructed as follows.

Using as the mutagen primer FS (SEQ ID NO: 78) and FA (SEQ ID NO: 79) designed to mutate threonine at position 75 to serine and valine at position 78 to alanine and as a template DNA the plasmid HEF-RVHe-AHM-gyl by the PCR method, version f was amplified to obtain plasmid HEF-RVHf-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHf-AHM-gyl are shown in SEQ ID NO: 16.

Using as the mutagen primer GS (SEQ ID NO: and GA (SEQ ID NO: 81) designed to mutate alanine at position 40 to arginine and as a template DNA the plasmid 71 HEF-RVHa-AHM-gyl, version g was amplified to obtain plasmid HEF-RVHg-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHg-AHM-gyl are shown in SEQ ID NO: 17.

Using as the mutagen primer FS (SEQ.ID NO: 78) and FA (SEQ ID NO: 79) and as a template DNA the plasmid HEF-RVHb-AHM-gyl, version h was amplified to obtain plasmid HEF-RVHh-AHM-gyl. The amino acid sequence andthe base sequence of the V region of the H chain contained in this plasmid HEF-RVHh-AHM-gyl are shown in SEQ ID NO: 18.

Using as the mutagen primer IS (SEQ ID NO: 82) and IA (SEQ ID NO: 83) designed to mutate arginine at 15 position 83 to alanine and serine at position 84 to phenylalanine as a template DNA the plasmid HEF-RVHh-AHM-gyl, version i was amplified to obtain plasmid HEF-RVHi-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain S 20 contained in this plasmid HEF-RVHi-AHM-gyl are shown in SEQ ID NO: 19.

Using as the mutagen primer JS (SEQ ID NO: 84) and JA (SEQ ID NO: 85) designed to mutate arginine at position 66 to lysine and as a template DNA the plasmid HEF-RVHf-AHM-gyl, version j was amplified to obtain plasmid HEF-RVHj-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHj-AHM-gyl are shown in SEQ ID NO: Using as the mutagen primer KS (SEQ ID NO: 86) and KA (SEQ ID NO: 87) designed to mutate glutamic acid at position 81 to glutamine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version k was amplified to obtain plasmid HEF-RVHk-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHk-AHM-gyl are shown in SEQ ID NO: 21.

72 Using as the mutagen primer LS (SEQ ID NO: 88) and LA (SEQ ID NO: 89) designed to mutate glutamic acid at position 81 to glutamine and serine at position 82B to isoleucine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version 1 was amplified to obtain plasmid HEF-RVH1-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVH1-AHM-gyl are shown in SEQ ID NO: 22.

Using as the mutagen primer MS (SEQ ID NO: and MA (SEQ ID NO: 91) designed to mutate glutamic acid at position 81 to glutamine, serine at position 82b to Sisoleucine, and threonine at position 87 to serine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version m 15 was amplified to obtain plasmid HEF-RVHm-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHm-AHM-gyl are shown in SEQ ID NO: 23.

Using as the mutagen primer NS (SEQ ID NO: 92) and NA (SEQ ID NO: 93) designed to mutate serine at position 82B to isoleucine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version n was amplified to obtain plasmid HEF-RVHn-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHn-AHM-gyl are shown in SEQ ID NO: 24.

Using as the mutagen primer OS (SEQ ID NO: 94) and OA (SEQ ID NO: 95) designed to mutate threonine at position 87 to serine and as a template DNA the plasmid HEF-RVHh-AHM-gyl, version o was amplified to obtain plasmid HEF-RVHo-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHo-AHM-gyl are shown in SEQ ID NO: Using as the mutagen primer PS (SEQ ID NO: 96) and PA (SEQ ID NO: 97) designed to mutate valine at position 78 to alanine and as a template DNA the plasmid 73 HEF-RVHa-AHM-gyl, version p was amplified by the PCR method to obtain plasmid HEF-RVHp-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHp-AHM-gyl are shown in SEQ ID NO: 26.

Using as the mutagen primer QS (SEQ.ID NO: 98) and QA (SEQ ID NO: 99) designed to mutate threonine at position 75 to serine and as a template DNA the plasmid HEF-RVHa-AHM-gyl, version q was amplified by the PCR method to obtain plasmid HEF-RVHq-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHq-AHM-gyl are shown in SEQ ID NO: 27.

Using as the mutagen primer CS (SEQ ID NO: 15 and CA (SEQ ID NO: 66) and as a template DNA the plasmid HEF-RVHp-AHM-gyl, version r was amplified by the PCR method to obtain plasmid HEF-RVHr-AHM-gyl. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHr-AHM-gyl are shown in SEQ ID NO: 28.

Version s of the V region of the H chain of the reshaped human anti-HM 1.24 antibody was constructed by mutagenesis using PCR. The mutagen primers SS (SEQ ID NO: 100) and SA (SEQ ID NO: 101) were designed to mutate methionine at position 69 to isoleucine.

After the above primer was amplified using plasmid HEF-RVHr-AHM-gyl as a template, the final product was purified, digested with BamHI and HindIII, and the DNA fragment obtained was cloned into the HEF expression vector HEF-VH-gyl to obtain plasmid HEF-RVHs-AHM-gyl.

The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHs-AHM-gyl are shown in SEQ ID NO: 102.

The regions encoding the variable region of each of the above-mentioned plasmids HEF-RVLa-AHM-gK and HEF-RVHr-AHM-gyl were digested to make restriction fragments with restriction enzymes HindIII and BamHI.

74 They were inserted into the HindIII and BamHI sites of plasmid vector pUC19. Each plasmid was termed pUC19-RVLa-AHM-gK and pUC19-RVHr-AHM-gyl.

The Escherichia coli that contains each of the plasmids pUC19-RVLa-AHM-gK and pUC19-RVHr-AHM-gyl was termed Escherichia coli DH5a (pUC19-RVLa-AHM-gK) and Escherichia coli DH5a (pUC19-RVHr-AHM-gyl), respectively, and has been internationally deposited on August 29,1996, with 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) under the accession number FERM BP-5645 and FERM BP-5643, respectively, under the provisions of the Budapest Treaty.

The regions encoding the variable region of the above-mentioned plasmid HEF-RVHs-AHM-gyl were digested to make a restriction fragment with restriction enzymes HindIII and BamHI. They were inserted into the HindIII and BamHI sites of plasmid vector pUC19. The plasmid 20 obtained was termed pUC19-RVHs-AHM-gyl.

SThe Escherichia coli that contains the plasmid pUC19-RVHs-AHM-gyl was termed Escherichia coli (pUC19-RVHs-AHM-gyl), and has been internationally pdeposited on September 29,1997, with the National 25 Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under the accession number FERM BP-6127 under the provisions of the Budapest Treaty.

4. Construction of the reshaped human anti-HM 1.24 antibody, the chimeric anti-HM 1.24 antibody, and the H chain hybrid antibody In order to evaluate each chain of the reshaped human anti-HM 1.24 antibody, the reshaped human anti-HM 1.24 antibody and the chimeric anti-HM 1.24 antibody as a positive control antibody were allowed to express. In constructing each of version b and after of the V region 75 of the H chain of the reshaped human anti-HM 1.24 antibody, the H chain hybrid antibody was allowed to express in order to investigate which amino acid sequence in the FR should be substituted. Furthermore, it was expressed in combination with the chimeric H chain in order to evaluate version a of L chain of the.reshaped human anti-HM 1.24 antibody.

4-1. Expression of the reshaped human anti-HM 1.24 antibody (1) Ten gg each of the expression vector (HEF-RVHa-AHM-gyl to HEF-RVHr-AHM-gyl) for the H chain of the reshaped human anti-HM 1.24 antibody and the expression vector (HEF-RVLa-AHM-gK or HEF-RVLb-AHM-gK) for the L chain of the reshaped human anti-HM 1.24 antibody were cotransformed into COS-7 cells by electroporation using the Gene Pulser instrument (manufactured by BioRad). Each DNA (10 jg) was added to 0.8 ml aliquots of 1 x 10 cells/ml in PBS, and was subjected to pulses at 1500 V and a capacity of 25 pF.

20 After the recovery period of 10 minutes at room temperature, the electroporated cells were added to 30 ml of DMEM culture medium (manufactured by GIBCO) containing y-globulin free fetal bovine serum. After incubation of 72 hours in the CO 2 incubator BNA120D (manufactured by TABAI) under the condition of 37 0 C and 5% CO 2 the culture supernatant was collected, the cell debris were removed by centrifugation at 1000 rpm for 5 minutes in a centrifuge 15PR-22 (manufactured by HITACHI) equipped with a centrifuge rotor 03 (manufactured by HITACHI), and the microconcentrator (Centricon 100, manufactured by Amicon) was ultrafiltrated using a centrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifuge rotor JA-20.1 (manufactured by BECKMAN), and was used for Cell-ELISA.

Expression of the reshaped human anti-HM 1.24 antibody (2) 76 Ten pg each of the expression vector (HEF-RVHs-AHM-gyl) for version of the H chain of the reshaped human anti-HM 1.24 antibody and the expression vector (HEF-RVLa-AHM-gK) for the L chain of the reshaped human anti-HM 1.24 antibody were cotransformed into COS-7 cells by electroporation using the Gene Pulser instrument (manufactured by BioRad). Each DNA (10 pg) was added to 0.8 ml aliquots of 1 x 107 cells/ml in PBS, and was subjected to pulses at 1500 V and a capacity of 25 pF.

After the recovery period of 10 minutes at room temperature, the electroporated cells were added to 30 ml of DMEM culture medium (manufactured by GIBCO) containing 10% y-globulin free fetal bovine serum. After incubation of 72 hours in the CO 2 incubator BNA120D (manufactured by 15 TABAI) under the condition of 37 0 C and 5% CO 2 the culture supernatant was collected, the cell debris were removed by centrifugation at 1000 rpm for 5 minutes in a centrifuge 05PR-22 (manufactured by HITACHI) equipped with a centrifuge rotor 03 (manufactured by HITACHI), and 20 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), and was filtration-sterilized using a filter, Millex GV13mm (manufactured by Millipore), which was used for Cell-ELISA.

4-2. Expression of the chimeric anti-HM 1.24 antibody Using Ten pg each of the expression vector HEF-1.24H-gyl for the H chain of the chimeric anti-HM 1.24 antibody and the expression vector HEF-1.24L-gK for the L chain of the chimeric anti-HM 1.24 antibody, the chimeric anti-HM 1.24 antibody to be used for Cell-ELISA was prepared according to the above-mentioned method for expression of the reshaped human anti-HM 1.24 antibody.

77 4-3. Expression of the anti-HM 1.24 antibody comprising version a of the humanized L chain and the chimeric H chain Using Ten gg each of the expression vector HEF-1.24H-gyl for the H chain of the chimeric anti-HM 1.24 antibody and the expression vector HEF-RVLa-AHM-gK for version a of the L chain of the reshaped human anti-HM 1.24 antibody, the anti-HM 1.24 antibody comprising version a of the humanized L chain and the chimeric H chain to be used for Cell-ELISA was prepared according to the above-mentioned method for expression of the reshaped human anti-HM 1.24 antibody.

4-4. Expression of the H chain hybrid antibody Using Ten gg each of the expression vector 15 (HEF-MH-RVH-AHM-gyl or HEF-HM-RVH-AHM-gyl) for the V region of the H chain hybrid and the expression vector HEF-RVLa-AHM-gK for the L chain of the reshaped human anti-HM 1.24 antibody, the H chain hybrid antibody to be used for Cell-ELISA was prepared according to the 20 above-mentioned method for expression of the reshaped human anti-HM 1.24 antibody.

Measurement of antibody concentration Concentration of the antibody obtained was measured by ELISA. Each well of a 96-well ELISA plate (Maxisorp, manufactured by NUNC) was immobilized by adding 100 pl of goat anti-human IgG antibody (manufactured by BIO SOURCE) prepared to a concentration of 1 pg/ml with the coating buffer (0.1 M NaHCO 3 0.02% NaN,, pH 9.6) and incubating at room temperature for one hour. After blocking with 100 pg of the dilution buffer mM Tris-HCl, 1 mM MgCl 2 0.15 M NaCl, 0.05% Tween 0.02% NaN 3 1% bovine serum albumin (BSA), pH 100 pi each of serial dilutions of the culture supernatant of cos-7 cells secrating the reshaped human anti-HM 1.24 antibody, the chimeric anti-HM 1.24 antibody, or the H chain hybrid antibody that were concentrated by 78 ultrafiltration were added to each well and incubated at room temperature for one hour. Then after washing, 100 gl of alkaline phosphatase-labelled goat anti-human IgG antibody (manufactured by DAKO) was added.

After incubating at room temperature for one hour and washing, 100 pl of 1 pg/ml substrate.solution (Sigma104, p-nitrophenyl phosphate, SIGMA) dissolved in the substrate buffer (50 mM NaHCO3, 10 mM MgCl 2 pH 9.8) was added, and then the absorbance at 405 run was measured using the MICROPLATE READER Model 3550 (manufactured by Bio Rad). As the standard for the measurement of concentration, human IgGlK (manufactured by The binding .,Site) was used.

5. Establishment of the CHO cell line that stably 15 produces the reshaped human anti-HM 1.24 antibody 5-1. Construction of the expression vector for the H **chain of the reshaped human anti-HM 1.24 antibody 20 By digesting plasmid HEF-RVHr-AHM-gyl with the restriction enzymes PvuI and BamHI, an about 2.8 kbp fragment containing the DNA encoding the EF1 promoter and the V region of the H chain of the reshaped human anti-HM 1.24 antibody was purified using 1.5% low melting point agarose gel. Then, the above DNA fragment was inserted into an about 6 kbp fragment that was prepared by digesting the expression vector used for a human H chain expression vector, DHFR-AE-RVh-PMlf (International Application Publication No. WO 92-19759), containing the DHFR gene and the gene encoding the constant region of a human H chain with PvuI and BamHI to construct an expression vector, DHFR-AE-HEF-RVHr-AHM-gyl, for the H chain of the reshaped anti-HM 1.24 antibody.

5-2. Gene introduction into CHO cells In order to establish a stable production system of the reshaped human anti-HM 1.24 antibody, the 79 genes of the above-mentioned expression vectors, DHFR-AE-HEF-RVHr-AHM-gyl and HEF-RVLa-AHM-gK, that were linearized by digestion with PvuI were simultaneously introduced into the CHO cell DXB-11 by the electroporation method under the condition similar to the above-mentioned one (transfection into the above-mentioned COS-7 cells).

5-3. Gene amplification by MTX Among the gene-introduced CHO cells, only those CHO cells in which both of L chain and H chain expression vectors have been introduced can survive in the nucleoside-free a-MEM culture medium (manufactured by :GIBCO-BRL) to which 500 gg/ml G418 (manufactured by GIBCO-BRL) and 10% fetal bovine serum were added, and so 15 they were selected. Subsequently, 10 nM MTX (manufactured by Sigma) was added to the above culture medium. Among the clones that propagated, those that produce the reshaped human anti-HM 1.24 antibody in large amounts were selected. As a result, clone 1 that exhibits a production efficiency of about 3 gg/ml of the reshaped human anti-HM 1.24 antibody was obtained and termed the reshaped human anti-HM 1.24 antibody-producing cell line.

5-4. Construction of the reshaped human anti-HM 1.24 antibody The reshaped anti-HM 1.24 antibody was constructed in the following method. The above CHO cells that produce the reshaped human anti-HM 1.24 antibody were cultured for 10 days using as the medium the nucleoside-free a-MEM culture medium (manufactured by GIBCO-BRL) to which 500 gg/ml G418 (manufactured by GIBCO-BRL) and 10% y-free fetal bovine serum were added using the CO 2 incubator BNAS120D (manufactured by TABAI) under the condition of 37 0 C and 5% CO z On day 8 and after starting the culture the culture liquid was recovered, the cell debris were removed by centrifuging 80 for 10 minutes at 2000 rpm using the centrifuge RL-500SP (manufactured by Tomy Seiko) equipped with the TS-9 rotor, and then filter-sterilized using a bottle top filter (manufactured by FALCON) having a membrane of 0.45 pm in diameter.

After an equal amount of PBS(-) was.added to the culture liquid of the CHO cells that produce the reshaped human anti-HM 1.24 antibody, then the reshaped human anti-HM 1.24 antibody was affinity-purified using the high-speed antibody purification system ConSep LC100 (manufactured by MILLIPORE) and Hyper D Protein A column S: (manufactured by Nippon Gaishi) using PBS(-) as the absorption/wash buffer and 0.1 M sodium citrate buffer (pH 3) as the elution buffer according to the attached 15 instructions. The eluted fractions were adjusted to about pH 7.4 by immediately adding 1 M Tris-HCi (pH and then using the centrifuging ultrafiltration concentrator Centriprep-10 (manufactured by MILLIPORE), concentration and substitution to PBS(-) was carried out and filter-sterilized using a membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 pm to obtain the purified reshaped human anti-HM 1.24 antibody. Antibody concentration was measured by absorbance at 280 run and calculated with 1 mg/ml as 1.35

OD.

Example 11. Determination of activity of the reshaped human anti-HM 1.24 antibody The reshaped human anti-HM 1.24 antibody was evaluated for the following antigen binding activity and binding inhibition activity.

1. The method of measurement of antigen binding activity and binding inhibition activity 1-1. Measurement of antigen binding activity Antigen binding activity was measured by the Cell-ELISA using WICH cells. Cell-ELISA plates were prepared as described in the above Example 7.1-2.

After blocking, 100 pi of serial dilutions of 81 the reshaped human anti-HM 1.24 antibody that was obtained from the concentrate of the culture supernatant of COS-7 cells or purified from the culture supernatant of CHO cells was added to each well. After it was incubated for 2 hours at room temperature and washed, peroxidase-labelled rabbit anti-human IgG antibody (manufactured by DAKO) was added. After it was incubated for 1 hour at room temperature and washed, 100 gl of substrate solution was added in each well. After incubation, the reaction was stopped by 50 ul of 6N sulfuric acid, and absorbance at 490 nm was measured using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

1-2. Measurement of binding inhibition activity 15 The binding inhibition activity by the biotin-labelled mouse anti-HM 1.24 antibody was measured by the Cell-ELISA using WISH cells. Cell ELISA plates were prepared as described in the above Example 7. 1-2.

After blocking, 50 gl of serial dilutions of the reshaped human anti-HM 1.24 antibody that was obtained from the concentrate of the culture supernatant of COS-7 cells or purified from the culture supernatant of CHO cells was :added to each well, and 50 gl of the biotin-labelled mouse anti-HM 1.24 antibody was added simultaneously.

After incubating at room temperature for two hours and washing, peroxidase-labelled streptavidin (manufactured by DAKO) was added. After incubating at room temperature for one hour and then washing, 100 ul of substrate solution was added in each well. After incubation, the reaction was stopped by 50 l of 6N sulfuric acid, and absorbance at 490 nm was measured using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

2. Evaluation of the reshaped human anti-HM 1.24 antibody 2-1. L chain Version a of the L chain of the reshaped human anti-HM 1.24 antibody was evaluated as mentioned for 82 measurement of antigen binding activity. As shown in Fig. 8, when version a of the L chain is expressed in combination with the chimeric H chain it has shown a similar level of antigen binding activity. However, in consideration of further increase in activity and of compatibility with the H chain, version b of the L chain was constructed. Versions a and b of the L chain were evaluated together for antigen binding activity and of binding inhibition activity when combined with versions a, b, f, or h of the H chain. As shown in Fig. 9, 11, and 12, version a of the L chain had a higher activity than version b in both activities in all versions a, b, f, and h of the H chain. Therefore, version a of the L chain of the reshaped human anti-HM 1.24 antibody was used for the following experiment.

2-2. H chain versions a to e Versions a to e of the H chain of the reshaped human anti-HM 1.24 antibody were evaluated in combination with the version a of the L chain as mentioned for measurement of antigen binding activity and binding inhibition activity. The result, as shown in Fig. 11, 13, 14, and 15, indicated that all versions were weaker in both activities as compared to the chimeric anti-HM 1.24 antibody, suggesting that further amino acid substitution is required.

2-3. The H chain hybrid antibody The H chain hybrid antibody was evaluated as mentioned for measurement of antigen binding activity.

The result, as shown in Fig. 16, indicated that the human-mouse hybrid anti-HM 1.24 antibody has shown a similar activity to that of the chimeric anti-HM 1.24 antibody for antigen binding activity, whereas the mouse human hybrid anti-HM 1.24 antibody had a weaker activity than the chimeric anti-HM 1.24 antibody. This indicated that in order to construct the reshaped human anti-HM 1.24 antibody having the antigen binding activity similar to that of the chimeric anti-HM 1.24 antibody, it is 83 necessary to convert amino acids included in FR3 or FR4 among those contained the V region of the H chain.

2-4. Versions f to r of the H chain Version f of the H chain of the reshaped human anti-HM 1.24 antibody was evaluated as mentioned for measurement of antigen binding activity. The.result, as shown in Fig. 17, indicated that its antigen binding activity is decreased as compared to the chimeric anti-HM 1.2.4 antibody, but is increased as compared to the above versions a to c, suggesting that any of the four amino acids at position 67, 69, 75, and 78 that were newly converted in this version is responsible for the activity of the reshaped human antibody.

Version g of the H chain of the reshaped human anti-HM 1.24 antibody was evaluated as mentioned for measurement of antigen binding activity. The result, as shown in Fig. 18 and 19, indicated that this version has *exhibited a similar level of activity to that of the above version a at most, revealing that, as shown for the above H chain human mouse hybrid antibody, the amino acid at position 40 that was converted in this version is not responsible for the increase in the activity of the reshaped human antibody.

.0 Versions h to j of the H chain of the reshaped human anti-HM 1.24 antibody were evaluated as mentioned for measurement of antigen binding activity and binding inhibition activity. The result, as shown in Fig. 21, 22, and 23, indicated that all versions were weaker for both activities as compared to the chimeric anti-HM 1.24 antibody and were similar to the above-mentioned version f, suggesting that the amino acids at position 67 and 69 among the four amino acids that were newly converted in version f are not responsible for the increase in the activity of the reshaped human antibody.

Versions k to p of the H chain of the reshaped human anti-HM 1.24 antibody were evaluated as mentioned for measurement of antigen binding activity and binding 84 inhibition activity. The result, as shown in Fig. 24, 26, and 27, indicated that all versions were weaker for both activities as compared to the chimeric anti-HM 1.24 antibody and were similar to the above-mentioned version h, suggesting that the amino acids at position and after that were newly converted in these six versions are not responsible for the increase in the activity of the reshaped human antibody.

Version q of the H chain of the reshaped human anti-HM 1.24 antibody was evaluated as mentioned for measurement of antigen binding activity and binding inhibition activity. The result, as shown in Fig. 25 and :27, indicated that this version was weaker for both activities as compared to the above version h or version p and was similar to that of the above-mentioned a, suggesting that substitution of the amino acid at position 78 is essential for the increase in the activity o *Cof the reshaped human antibody.

Version r of the H chain of the reshapedhuman anti-HM 1.24 antibody were evaluated by the method mentioned above. The result, as shown in Fig. 15 and 28, indicated that version r has a similar level of antigen binding activity and binding inhibition activity to that of the chimeric anti-HM 1.24 antibody.

The above results indicated that the minimum conversion required for the reshaped human anti-HM 1.24 antibody to have a similar level of antigen binding activity to that of the mouse anti-HM 1.24 antibody or the chimeric anti-HM 1.24 antibody is the amino acids at positions 30, 71, and 78, and furthermore 73.

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

85 H chain version Antigen a b Table 6 binding activity Binding inhibition activity 0 .e 040*6 6.00 0.60 not measured not measured see..

2-5. Version s of the H chain Version s of the H chain of the reshaped human anti-HM 1.24 antibody was evaluated in combination with the above-mentioned version a of the L chain as mentioned for measurement of antigen binding activity and binding inhibition activity. The result, as shown in Fig. 29 and indicated that version s has a similar level of antigen binding activity and binding inhibition activity to that of version r.

As mentioned above, the reshaped human anti-HM 1.24 antibody of the present invention retains the ability of binding to antigen even after one or more amino acid residues have been replaced with other amino acids. Accordingly, the present invention includes the reshaped human anti-HM 1.24 antibody in which one or more 86 amino acid residues have been replaced with other amino acids in the variable region of the H chain or the L chain as long as it retains the original properties.

3. Evaluation of the purified reshaped human anti-HM 1.24 antibody The purified reshaped human anti-HM 1.24 antibody was evaluated for the above-mentioned antigen binding activity and binding inhibition activity. The result, as shown in Fig. 31 and 32, indicated that the reshaped human anti-HM 1.24 antibody has a similar level of antigen binding activity and binding inhibition activity to that of the chimeric anti-HM 1.24 antibody.

see* This fact indicated that the reshaped human anti-HM 1.24 antibody has the same antigen binding activity as the 15 mouse anti-HM 1.24 antibody.

Examole 12. Anti-tumor effect of the chimeric anti-HM 1.24 antibody against the human myeloma 0oo* mouse model i. Preparation of antibody to be administered 20 1-1. Preparation of the chimeric anti-HM 1.24 antibody The purified chimeric anti-HM 1.24 antibody obtained in the above Example 6 was concentrated and the buffer solution was replaced by PBS(-) using the centrifuging ultrafiltration concentrator Centriprep (manufactured by Amicon). This was filter-sterilized using the membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 jim. This was prepared to a concentration of 200 pg/ml using the filter-sterilized which was used for the following experiments. The concentration of the antibody was measured by absorbance at 280 nm and calculated with 1 mg/ml as 1.35 OD.

1-2. Purification of the control human IgGl Human IgG1 to be used as a control for the chimeric anti-HM 1.24 antibody was purified as follows.

After an equal amount of PBS(-) was added to Hu IgG1 87 Kappa Purified (manufactured by BINDING SITE), it was affinity-purified using the high-speed antibody purification system ConSep LC100 (manufactured by MILLIPORE) and Hyper D Protein A column (manufactured by Nippon Gaishi) using PBS(-) as the absorption buffer and 0.1 M sodium citrate buffer (pH 3) as the elution buffer according to the attached instructions. The eluted fractions were adjusted to about pH 7.4 by immediately adding 1 M Tris-HCl (pH 8.0) and then using the 10 centrifuging ultrafiltration concentrator Centriprep :"(manufactured by Amicon) concentration and buffer substitution to PBS(-) was carried out, and filter-sterilized using the membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 pm.

This was adjusted to 200 pg/ml using the filter-sterilized PBS(-) and used for the following experiments. Antibody concentration was measured by absorbance at 280 nm and calculated with 1 mg/ml as 1.35

OD.

2. Method for quantitating of human serum IgG in the mouse serum Human IgG contained in the mouse serum was quantitated by the following ELISA. 100 pl of goat anti-human IgG diluted to 1 tg/ml with 0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate (manufactured by NUNC) and incubated at 4 0 C overnight to immobilize the antibody. After blocking, 100 pl of serially diluted mouse serum or human IgG as standard (manufactured by CAPPEL) was added and incubated at room temperature for one hour. After washing, 100 pl of 2000-fold diluted alkaline phosphatase-labelled anti-human IgG (manufactured by CAPPEL) was added and incubated at room temperature for one hour. After washing, the substrate solution was added and incubated, and then absorbance at 405 nm was measured using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

88 3. Anti-tumor effect of the chimeric anti-HM 1.24 antibody against the human myeloma cells-transplanted mouse 3-1. Construction the human myeloma cells-transplanted mouse The human myeloma cells-transplanted mouse was constructed as follows. KPMM2 cells passaged in vivo using SCID mice (breeded by Nihon CLEA) were prepared at a concentration of 3 x 107 cells/ml with RPMI 1640 medium 10 supplemented with 10% fetal bovine serum (manufactured by GIBCOBRL). Two hundred gl of the above KPMM2 cell suspension was injected via the tail vein to SCID mice S(male, 8-weeks old breeded by Nihon CLEA) to which 100 il of anti-asialo GM1 (manufactured by Wako Pure Chemical Industries Co., Ltd.) had been intraperitoneally given on the previous day.

3-2. Administration of antibody On day 12 after KPMM2 cell transplantation, serum was collected from the above human myeloma cells-transplanted mice, and human IgG in the serum was quantitated using the ELISA mentioned in the above 2.

Take of KPMM2 cells in the bone marrow was confirmed by the increase of human IgG level in the serum. On day 14, 21, and 28 after KPMM2 cell transplantation, 100 il each of the antibodies prepared in the above 1 was intraperitoneally given to these mice.

3-3. Evaluation of the anti-tumor effect of the chimeric anti-HM 1.24 antibody against the human myeloma cells-transplanted mouse The anti-tumor effect of the chimeric anti-HM 1.24 antibody was evaluated by the survival period of the mice. As shown in Fig. 33, the mice that were given the chimeric anti-HM 1.24 antibody showed a prolonged period of survival as compared to the mice that received control human IgGl. Thus, it was confirmed that the chimeric anti-HM 1.24 antibody has the anti-tumor effect against 89 the human myeloma cells-transplanted mouse.

Example 13. Measurement of ADCC activity of the reshaped human anti-HM 1.24 antibody ADCC (Antibody-dependent Cellular Cytotoxicity) activity was measured according to the method as set forth in Current Protocols in Immunology, Chapter 7, Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley Sons, Inc., 1993.

1. Preparation of effector cells Mononuclear cells were separated from the peripheral blood of healthy humans by the density *centrifugation method. Thus, an equal amount of PBS(-) was added to the peripheral blood of healthy humans, which was layered on Ficoll-Paque PLUS (manufactured by Pharmacia), and was centrifuged at 400 g for 40 minutes.

The mononuclear cells layer was collected, and washed four times with RPMI 1640 medium (manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum (manufactured by GIBCO BRL), and prepared at a cell 20 density of 5 x 10 /ml with the same culture medium.

LAK (Limphokine Activated Killer Cell) was induced from the bone marrow cells of SCID mice (breeded by Nihon CLEA). Thus, bone marrow cells were isolated from the femoral bone of the mice and washed twice with RPMI1640 medium (manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum (manufactured by GIBCO BRL), and prepared at a cell density of 2 x 10 /ml with the same culture medium. This was incubated together with ng/ml of recombinant human IL-2 (manufactured by R D SYSTEMS) and 10 ng/ml of recombinant mouse GM-CSF (manufactured by R D SYSTEMS) in the CO 2 incubator (manufactured by TABAI) for seven days. The cell number was adjusted to 2 x 10 6 /ml with the same culture medium.

2. Preparation of target cells The human myeloma cell line KPMM2 (Japanese Unexamined Patent Publication (Kokai) No. 7-236475) or 90 plasma cell leukemia-derived ARH-77 (ATCC CCL-1621) was radiolabelled by incubating in the RPMI 1640 medium (manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum (manufactured by GIBCO BRL) together with 0.1 mCi of 51Cr-sodium chromate (manufactured by ICN) at 37 0 C for 60 minutes. After radiolabelling, the cells were washed three times with the same culture medium and adjusted to 2 x 105/ml.

3. ADCC assay 10 Into a 96-well U-bottomed plate (manufactured by Becton Dickinson) were added 50 Al of 2 x 105 target cells/ml, 50 pl of the reshaped human anti-HM 1.24 antibody, the mouse anti-HM 1.24 antibody, control human IgGl (manufactured by THE BINDING SITE) or control mouse IgG2a (UPC10, manufactured by CAPPEL), and reacted at 4 0

C

for 15 minutes.

Then, 100 l of the effector cells was cultured in the CO 2 incubator for 4 hours, when the ratio of the effector cells to the target cells was set at 0:1, 3.2:1, 8:1, 20:1, or 50:1.

One hundred l1 of the supernatant was taken and the radioactivity released into the culture supernatant was measured by the gamma counter (ARC-300, manufactured by Aloka). For measurement of the maximum radioactivity, 1% NP-40 (manufactured by Nakalai) was used. Cytotoxicity was calculated by 100, wherein A is radioactivity (cpm) released in the presence of antibody, B is radioactivity (cpm) released by NP-40, and C is radioactivity (cpm) released by the culture medium alone without antibody.

Fig. 34 shows the result obtained when the cells prepared from the peripheral blood from the healthy human were used as the effector cell and KPMM2 cells were used as the target cell. Fig. 35 shows the result obtained when the cells prepared from the peripheral blood from the healthy human were used as the effector cell and 91 ARH-77 was used as the target cell. When the reshaped human anti-HM 1.24 antibody was added, cytotoxicity increased with the increase in antibody concentration as compared to the control human IgG1, indicating that the reshaped human anti-HM 1.24 antibody has ADCC activity.

Furthermore, when the reshaped human anti-HM 1.24 antibody was added, cytotoxicity evidently increased as compared to the mouse anti-HM 1.24 antibody, indicating that the reshaped human anti-HM 1.24 antibody has higher 0 ADCC activity than the mouse anti-HM 1.24 antibody.

Furthermore, when KPMM2 was used as the target cell, the addition of the reshaped human anti-HM 1.24 antibody at a concentration of 0.1 pg/ml or higher caused no change in :.cytotoxicity, indicating that the concentration of 0.1 pg/ml or higher has sufficient ADCC activity. When ARH-77 was used as the target cell, the addition of the reshaped human anti-HM 1.24 antibody at a concentration of 1 pg/ml or higher caused no change in cytotoxicity, "indicating that the concentration of 1 pg/ml or higher has sufficient ADCC activity.

Fig. 36 shows the result obtained when the cells prepared from the bone marrow of SCID mice were used as the effector cell. When the reshaped human anti-HM 1.24 antibody was added, cytotoxicity increased with the increase in antibody concentration as compared to the control human IgGl, indicating that the reshaped human anti-HM 1.24 antibody has ADCC activity. Furthermore, the addition of the reshaped human anti-HM 1.24 antibody at a concentration of 0.1 pg/ml or higher caused no change in cytotoxicity, indicating that the concentration of 0.1 pg/ml or higher has sufficient ADCC activity.

These results show that the reshaped human anti-HM 1.24 antibody has ADCC activity even when the effector cells used are derived from humans or mice.

92 Example 14. Anti-tumor effect of the reshaped anti-HM 1.24 antibody against the human myeloma mouse model 1. Preparation of antibody to be administered The reshaped anti-HM 1.24 antibody obtained by introduction of plasmid HEF-RVLa-AHM-gK and plasmid HEF-RVHr-AHM-gyl into CHO cells was prepared to a concentration of 40, 200, and 1000 jg/ml using the filter-sterilized and the control human IgGI 10 obtained in Example 12.1-2 was prepared to a concentration of 200 gg/ml using the filter-sterilized which were used as the antibodies to be administered.

2. Anti-tumor effect of the reshaped anti-HM 1.24 antibody against the human myeloma cells-transplanted mouse 2-1. Construction of the human myeloma cells-transplanted mouse The human myeloma cells-transplanted mice were 20 prepared according to Example 12.3-1. The mice used were SCID mice (five weeks old) (breeded by Nihon CLEA).

2-2. The administration of antibodies On day 9 after KPMM2 cell transplantation, serum was collected from the above human myeloma cells-transplanted mice prepared in the above 2-1, and human IgG in the serum was quantitated using the ELISA mentioned in the above 12.2. Take of KPMM2 cells on the bone marrow was confirmed by the increase of human IgG level in the serum. On day 10 after KPMM2 cell transplantation, 100 l each of the antibodies prepared in the above 1 was intravenously given to these mice.

2-3. Evaluation of the anti-tumor effect of the reshaped anti-HM 1.24 antibody against the human myeloma cells-transplanted mouse The anti-tumor effect of the reshaped anti-HM 1.24 antibody was evaluated by the change in the amount of human IgG in the mouse serum and in the survival 93 period of mice.

The change in the amount of human IgG in the mouse serum was quantitated for the serum collected on day 35 after the transplantation of KPMM2 cells by determining human IgG using the ELISA mentioned in Example 12.2. The result as shown in Fig. 37 revealed that in the control human IgGl-administration group the amount of human IgG in the serum on day 35 after the KPMM2 cell transplantation was increased by about 1000-fold as compared to that on day 9 (the day before antibody administration), whereas in the reshaped human anti-HM 1.24 antibody-administration group it was almost equal to or below that on day 9 for any dosage, indicating that the reshaped human anti-HM 1.24 antibody suppressed the growth of KPMM2 cells. On the other hand, for the survival period as shown in Fig. 38, prolongation was observed for the reshaped human anti-HM 1.24 antibody-administration group as compared to the control human IgGl-administration group. The foregoing shows 20 that the reshaped human anti-HM 1.24 antibody has the anti-tumor effect against the human myeloma cells-transplanted mouse.

ExamDle 15. Comparison of anti-tumor effect between the reshaoed human anti-HM 1.24 antibody and the existing drug melphalan against the human myeloma mouse model 1. Preparation of the drugs to be administered 1-1. Preparation of antibodies to be administered The reshaped human anti-HM 1.24 antibody obtained by the introduction of plasmid HEF-RVLa-AHM-gK and plasmid HEF-RVHr-AHM-gyl into CHO cells was prepared to a concentration of 40 and 200 ig/ml using the filter-sterilized and the control human IgGl obtained in Example 12.1-2 was prepared to a concentration of 200 tg/ml using the filter-sterilized which were used as the antibodies to be administered.

94 1-2. Preparation of melphalan Melphalan (manufactured by SIGMA) that is an existing drug for myeloma was prepared to a concentration of 0.1 mg/ml using 0.2% carboxymethyl cellulose (CMC) (manufactured by Daicel Chemical Industries, Ltd.).

2. The anti-tumor effect of the reshaped human anti-HM 1.24 antibody and melphalan against the human myeloma cells-transplanted mouse 2-1. Construction of human myeloma 10 cells-transplanted mouse The human myeloma cells-transplanted mice were prepared according to Example 14.2-1.

2-2. The administration of drug On day 9 after KPMM2 cells transplantation, serum was collected from the above human myeloma cells-transplanted mice prepared in the above 2-1, and human IgG in the serum was quantitated using the ELISA mentioned in the above 12.2. Take of KPMM2 cells on the bone marrow was confirmed by the increase of human IgG level in the serum. On day 10 after KPMM2 cell transplantation, 100 l each of the antibodies prepared in the above 1-1 were intravenously given to these mice.

Furthermore, 200 pl of 0.2% CMC solution was orally given once daily for five days from day 10 after transplantation. On the other hand, for the melphalan-administration group, the melphalan solution prepared in the above 1-2 was orally given at an amount of 100 pl per 10 g of body weight (1 mg/kg as melphalan) once daily for five days from day 10 after transplantation of KPMM2 cells.

2-3. Evaluation of the anti-tumor effect of the reshaped anti-HM 1.24 antibody against the human myeloma cells-transplanted mouse The anti-tumor effect of the reshaped anti-HM 1.24 antibody was evaluated by the change in the amount of human IgG in the mice serum and in the survival period 95 of mice.

The change in the amount of human IgG in the mice serum was quantitated for the serum collected on day after the transplantation of KPMM2 cells by determining human IgG using the ELISA mentioned in Example 12.2. The result as shown in Fig. 39 revealed that in the control human IgGl-administration group the amount of human IgG in the serum on day 35 after the KPMM2 cell transplantation was increased by about 10 1000-fold as compared to that on day 9 (the day before antibody administration), whereas it seemed that KPMM2 cells grew in these mice. In the melphalanadministration group as well, the amount of serum human IgG was more increased than that before the drug administration, though not so high as in the control human IgG-administration group. This result indicates that administration of melphalan did not suppress the growth of KPMM2 cells perfectly. On the other hand, in the reshaped human anti-HM 1.24 antibody-administration group, the amount of serum human IgG at day was less than at day 9 after transplantation for any dosage, indicating that the reshaped human anti-HM 1.24 antibody suppressed the growth of KPMM2 cells.

On the other hand, for the survival period also as shown in Fig. 40, prolongation was observed for the reshaped human anti-HM 1.24 antibody-administration group as compared to the control human IgGl-administration group or melphalan-administration group. From the foregoing, it was shown that the reshaped human anti-HM 1.24 antibody has the anti-tumor effect against the human myeloma cells-transplanted mice and that the anti-tumor effect of the present antibody is stronger than the existing drug melphalan.

The above results indicated that when the human-derived effector cells were used, the mouse anti-HM 1.24 antibody had little cytotoxicity to human myeloma cells, whereas the reshaped human anti-HM 1.24 antibody 96 and the chimeric anti-HM 1.24 antibody had strong cytotoxicity. This fact indicates the importance of humanizing antibody and provides hope on the usefulness of the reshaped human anti-HM 1.24 antibody in humans.

The reshaped human anti-HM 1.24 antibody have exhibited a very strong anti-tumor effect in the human myeloma cells-transplanted SCID mice. Since in humans the effector cells are derived from humans and lymphocytes are normally present, an even stronger 10 anti-tumor effect of the reshaped human anti-HM 1.24 antibody is expected.

In the myeloma model, the reshaped.human anti-HM 1.24 antibody have exhibited a strong anti-tumor effect as compared to the existing drug, and therefore, it is expected that the reshaped human anti-HM 1.24 antibody will make an epoch-making drug for treatment of myeloma.

Reference example 1. Construction of the hybridoma that produces the mouse anti-HM 1.24 monoclonal antibody The hybridoma that produces the mouse anti-HM 1.24 monoclonal antibody was prepared according to the method described in Goto, T. et al., Blood (1994) 84, 1992-1930.

The Epstein-Barr virus nuclear antigen (EBNA)-negative plasma cell line KPC-32 (1 x 107 cells) derived from the bone marrow of human patient with multiple myeloma (Goto, T. et al., Jpn. J. Clin. Hematol.

(11991) 32, 1400) was intraperitoneally given twice to BALB/c mice (breeded by Charles River) every six weeks.

In order to further elevate the titer of antibody production, 1.5 x 106 KPC-32 cells were injected into the spleen of the mise three days before sacrificing the animals (Goto, T. et al., Tokushima J. Exp. Med. (1990) 37, 89). After sacrificing the mice, the spleen were removed, and the spleen cells removed according to the method of Groth, de St. Schreidegger (Cancer Research 9 9 97 (1981) 41, 3465) were subjected to cell fusion with the myeloma cells Antibody in the supernatant of the hybridoma culture was screened by the ELISA (Posner, M.R. et al., J.

Immunol. Methods (1982) 48, 23) using the KPC-32 cell-coated plates. 5 x 104 KPC-32 cells were suspended in 50 ml of PBS and aliquoted into 96-well plates (U-bottomed, Corning, manufactured by Iwaki). After blocking with PBS containing 1% bovine serum albumin 10 (BSA), the supernatant of the hybridoma was added and incubated at 4 0 C for 2 hours. Subsequently, it reacted with peroxidase-labelled goat anti-mouse IgG antibody (manufactured by Zymed) at 4 0 C for 1 hour, washed once, and was reacted with o-phenylenediamine substrate solution (manufactured by Sumitomo Bakelite) at room temperature for 30 minutes.

After stopping the reaction with 2N sulfuric acid, absorbance at 492 nm was measured using the ELISA reader 0*oo&e (manufactured by Bio-Rad). In order to remove the hybridoma that produces antibody against human immunoglobulin, the positive hybridoma culture supernatant had previously been adsorbed to human serum, and the reactivity to other sub-cellular components were screened. Positive hybridomas were selected and their reactivity to various cell lines and human samples were investigated using flow cytometry. The finally selected hybridoma clones were cloned twice, which were injected into the abdominal cavity of the pristane-treated BALB/c mice and then the ascitic fluid was obtained therefrom.

Monoclonal antibody was purified from the mouse ascites by ammonium sulfate precipitation and Protein A affinity chromatography kit (Ampure PA, manufactured by Amersham). The purified antibody was conjugated to fluorescein isothiocyanate (FITC) using the Quick Tag FITC conjugation kit (manufactured by Boehringer Mannheim).

98

S

S

S

As a result, the monoclonal antibodies produced by hybridoma clones reacted with KPC-32 and RPMI 8226 cells. After cloning, the reactivity of the supernatant of these hybridomas with other cell lines and peripheral blood-derived mononuclear cells was investigated.

Among them, three clones produced monoclonal antibodies that specifically react with plasma cells.

Out of these three clones, the hybridoma clone that produce monocloned antibody that is most useful for flow cytometry analysis and that has complement-dependent cytotoxicity against RPUI 8226 cells was selected and termed HM1.24. The subclass of monoclonal antibody produced by this hybridoma was determined by the ELISA using subclass-specific rabbit anti-mouse antibody 15 (manufactured by Zymed). Anti-HM 1.24 antibody had a subclass of IgG2a K. The hybridoma that produces the anti-HM 1.24 antibody was internationally deposited on September 14, 1995, with the National Institute of Bioscience and Human-Technology, Agency of Industrial 20 Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under the accession number FERM BP-5233 under the provisions of the Budapest Treaty.

Reference example 2. Cloning of cDNA encoding the HM 1.24 antigen polypeptide 1. Construction of cDNA library 1) Preparation of total RNA The cDNA that encodes the HM 1.24 antigen which is a polypeptide specifically recognized by mouse anti-HM1.24 monoclonal antibody was isolated as follows.

From the human multiple myeloma cell line KPMM2, total RNA was prepared according to the method of Chirgwin et al. (Biochemistry, 18, 5294 (1979)). Thus, 2.2 x 10 a KPMM2 cells were completely homogenized in ml of 4 M guanidine thiocyanate (manufactured by Nakalai tesque).

99 The homogenate was layered on 5.3 M cesium chloride layer in the centrifuge tube, which was then centrifuged using Beckman SW40 rotor at 31,000 rpm at 0 C for 24 hours to precipitate RNA. The RNA precipitate was washed with 70% ethanol, and dissolved in 300 pl of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 0.5% SDS. After adding Pronase (manufactured by Boehringer) thereto to a concentration of 0.5 mg/ml, it was incubated at 37°C for 30 minutes. The mixture was extracted with phenol and chloroform to precipitate RNA.

Then, the RNA precipitate was dissolved in 200 l of mM Tris-HCl (pH 7.4) containing 1 mM EDTA.

2) Preparation of poly(A)+RNA Using about 500 gg of the total RNA 15 prepared as above as a raw material, poly(A)+RNA was purified using the Fast Track 2.0m RNA Isolation Kit (manufactured by Invitrogen) according to the instructions attached to the kit.

3) Construction of cDNA library 20 Using 10 pg of the above poly(A)+RNA as a raw material, double stranded cDNA was synthesized using the cDNA synthesizing kit TimeSaver cDNA Synthesis Kit (manufactured by Pharmacia) according to the instructions attached to the kit, and using the Directional Cloning Toolbox (manufactured by Pharmacia) EcoRI adapter was linked thereto according to the instructions attached to the kit. Kination and restriction enzyme NotI treatment of the EcoRI adapter were carried out according to the instructions attached to the kit. Furthermore, the adapter-attached double strand cDNA having a size of about 500 bp or higher was isolated and purified using low melting point agarose gel (manufactured by SIGMA) to obtain about 40 1l of adapter-attached double strand cDNA.

The adapter-attached double strand cDNA thus prepared was linked to pCOS1 vector (Japanese Unexamined Patent Publication (Kokai) 8-255196) that had 100 previously been treated with restriction enzymes EcoRI and NotI and alkaline phosphatase (manufactured by Takara Shuzo) using T4 to construct DNA ligase (manufactured by GIBCO BRL) to construct cDNA library. The constructed cDNA library was transduced into Escherichia coli strain (manufactured by GIBCO BRL) and the total size was estimated to be about 2.5 x 106 independent clones.

2. Cloning by direct expression 1) Transfection into COS-7 cells cDNA was amplified by culturing about 5 x 10 5 clones of the above transduced Escherichia coli in the 2-YT medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) containing 50 pg/ml of ampicillin, and plasmid 15 DNA was recovered from the Escherichia coli by the alkali method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)).

The plasmid DNA obtained was transfected into COS-7 cells by electroporation using the Gene Pulser instrument 20 (manufactured by BioRad).

S" Thus, 10 jg of the purified plasmid DNA was added to 0.8 ml of COS-7 cells that were suspended 7 S" into PBS at a concentration of 1 x 10 cells/ml, and was subjected to pulses at 1500 V and a capacity of 25 pF.

After 10 minutes of recovery period at room temperature, the electroporated cells were cultured in the DMEM (manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum under the condition of 37 0 C and 5% CO 2 for three days.

2) Preparation of the panning dish A panning dish coated with the mouse anti-HM 1.24 antibody was prepared by the method of B.

Seed et al. (Proc. Natl. Acad. Sci. USA, 84, 3365-3369 (1987)). Thus, the mouse anti-HM 1.24 antibody was added to 50 mM Tris-HCl, pH 9.5, to a concentration of pg/ml. Three ml of the antibody solution thus prepared 101 was added to a tissue culture plate with a diameter of mm and incubated at room temperature for 2 hours. After washing three times with 0.15 M NaCI solution and blocking with PBS containing 5% fetal bovine serum, 1 mM EDTA, and 0.02% NaN 3 these plates used for the following cloning.

3) Cloning of cDNA library The COS-7 cells transfected as described above were detached by PBS containing 5 mM EDTA, and then washed once with PBS containing 5% fetal bovine serum.

These cells were then suspended in PBS containing fetal bovine serum and 0.02% NaN 3 to a concentration of about 1 x 106 cells/ml, which was added to the panning dish prepared as above and incubated at room temperature 15 for 2 hours. After washing three times gently with PBS containing 5% fetal bovine serum and 0.02% NaN 3 plasmid DNA was recovered from the cells bound to the panning dish using a solution containing 0.6% SDS and 10 mM EDTA.

The recovered plasmid DNA was transduced 20 again to Escherichia coli DH5a. After amplifying plasmid SDNA as above, it was recovered by the alkali method. The recovered plasmid DNA was transfected into COS-7 cells by ~the electroporation method and plasmid DNA recovered from the bound cells as described above. The same procedure was repeated one more time, and the recovered plasmid DNA was digested with restriction enzymes EcoRI and NotI. As a result, concentration of the insert with a size of about 0.9 kbp was confirmed. Escherichia coli transduced with part of the recovered plasmid DNA was inoculated to the 2-YT agar plate containing 50 pg/ml of ampicillin.

After culturing overnight, plasmid DNA was recovered from single colony. It was digested with restriction enzymes EcoRI and NotI and clone p3.19 having an insert of 0.9 kbp was obtained.

The base sequence of this clone was determined by reacting using PRISM, Dye Terminater Cycle 102 Sequencing kit (manufactured by Perkin Elmer) according to the instructions attached to the kit and sequencing using ABI 373A DNA Sequencer (manufactured by Perkin Elmer). The amino acid sequence and the base sequence thereof are shown in SEQ ID NO: 103.

The cDNA encoding the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 103 was inserted into the XbaI cleavage site of pUC19 vector, and has been prepared as plasmid pRS38-pUC19. The Escherichia coli that contains this plasmid pRS38-pUC19 S. has been internationally deposited on October 5,1993, as Escherichia coli DH5a (pRS38-pUC19), with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 15 1-3, Tsukuba city, Ibalaki prefecture, Japan) under the accession number FERM BP-4434 under the provisions of the ~Budapest Treaty (see Japanese Unexamined Patent Publication (Kokai) No. 7-196694).

Industrial Applicability i 20 Since the chimeric anti-HM 1.24 antibody is composed of the variable region of the mouse anti-HM 1.24 antibody and the constant region of a human antibody, and the reshaped human anti-HM 1.24 antibody is composed of the complementarity determining region of the mouse anti-HM 1.24 antibody, the framework region of a human antibody, and the constant region of a human antibody, it has a low antigenicity against humans, and therefore, is expected to be used as a medical composition, especially for treatment of myeloma.

103 Reference to the organisms donated The international depository concerned Title: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI Address: Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan 1. Escherichia coli DH5a (pRS 38-pUC19) Accession No.: FERM BP-4434 Date of donation: October 5, 1993 2. Mouse-mouse hybridoma HM1.24 Accession No.: FERM BP-5233 Date of donation: April 27, 1995 3. Escherichia coli DH5a (pUC19-RVHr-AHM-gyl) 15 Accession No.: FERM BP-5643 Date of donation: August 29, 1996 4. Escherichia coli DH5a (pUC19-l.24H-gyl) Accession No.: FERM BP-5644 Date of donation: August 29, 1996 S* 20 5. Escherichia coli DH5a (pUC19-RVLa-AHM-gK) •Accession No.: FERM BP-5645 Date of donation: August 29, 1996 6. Escherichia coli DH5a (pUC19-1.24L-gK) Accession No.: FERM BP-5646 Date of donation: August 29, 1996 7. Escherichia coli DH5a (pUC19-RVHs-AHM-gyl) Accession No.: FERM BP-6127 Date of donation: September 29, 1997 EDITORIAL NOTE FOR 69655/00 THE FOLLOWING SEQUENCE LISTING IS PART OF THE DESCRIPTION THE CLAIMS FOLLOW ON PAGE 104 Sequence Listing SEQ ID NO: 1 LENGTH: 394 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GGC TTC AAG ATG GAG TCA CAT TTT CTG GTC TTT GTA TTC GTG TTT Met Gly CTC TGG Leu Trp AAA TTC Lys Phe GCC AGT Ala Ser 25 GGA CAA Gly Gin GGA GTC Gly Val Phe Lys Met -20 TTG TCT GGT Leu Ser Gly -5 ATG TCC ACA Met Ser Thr CAG GAT GTG Gin Asp Val TCG CCT AAA Ser Pro Lys CCT GAT CGC Pro Asp Arg Glu Ser His Phe Leu -15 GTT GAC GGA GAC ATT Val Asp Gly Asp Ile Val Phe Val Phe Val Phe TCA GTA Ser Val 15 -1

GGA

Gly 1

GAC

Asp GTG ATG ACC CAG TCT CAC Val Met Thr Gin Ser His GTC AGC ATC ACC TGC AAG Val Ser Ile Thr Cys Lys

AGG

Arg

TAT

Tyr AAT ACT GCT GTA GCC TGG Asn Thr Ala Val Ala Trp 30 35 CTA CTG ATT TAC TCG GCA Leu Leu Ile Tyr Ser Ala 50 ATC ACT GGC AGT GGA TCT Ile Thr Gly Ser Gly Ser CAA CAA AAA CCA Gin Gin Lys Pro TCC AAC CGG TAC ACT Ser Asn Arg Tyr Thr GGG ACG GAT TTC ACT Gly Thr Asp Phe Thr 144 192 240 288 336 384 TTC ACC ATC Phe Thr Ile CAG CAA CAT Gin Gin His

AGC

Ser

TAT

Tyr AGT GTG CAG GCG Ser Val Gin Ala 80 AGT ACT CCA TTC Ser Thr Pro Phe 65

GAA

Glu

ACG

Thr GAC CTG GCA CTT TAT TAC TGT Asp Leu Ala Leu Tyr Tyr Cys TTC GGC TCG GGG ACA AAG TTG Phe Gly Ser Gly Thr Lys Leu 95 100 GAA ATA AAA C Glu Ile Lys 105 SEQ ID NO: 2 LENGTH: 418 394 2 2.

TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAA TGT AAC TGG ATA GTT Met Glu cys Asn Trp Ile Leu COT TTT ATT GTG TGA OTA ACT TCA GGT Pro GGG TAG TCA cAG OTT Ala Tyr Ser Gin Val -1 1 CAA GTC GAG Gin Leu Gin 5 AAG TTG TCG Lys Leu Ser Phe Ile -10 GAG TOT Gin Ser TGG AAO Gys Lys Leu Ser Val Thr Ser Gly GGG GCT GAG CTG GGA AGA Gly Ala Giu Leu Ala Arg GGT TCT GGG TAG ACC TTT Ala Ser Gly Tyr Thr Phe .0.

so: 06 0 090 0 0 0* 0 0* 0 0 *00 GGT GGG GGT TCA GTG Pro Oly Ala Ser Val 15 ACT CCC TAG TGG ATG Thr Pro Tyr Trp Met 20 CG TGG Gin Trp GTA AAA GAG AGG Val Lys Gin Arg 40

CGT

Pro

GGA

Gly GAG GOT CG Gin Oly Leu 30 OAA TOO Oiu Trp 48 96 144 192 240 288 336 384 ATT 000 TGT Ile Oly Ser 50 TTT GGT OGA GAT Phe Pro Gly Asp 55 0CC AGA TTG ACT Ala Thr Leu Thr CAG AAG TTC Gin Lys Phe ACA 0CC TAG Thr Ala Tyr TAT TAC TOT Tyr Tyr Cys

AAO

Ly s

GC

Gly

AAO

Ly s GOT OAT ACT AGO TAG AOT Gly Asp Thr Arg Tyr Ser GCA OAT AAA TCG TCG AOT Ala Asp Lys Se'r Ser Ser TTT GAG OAG TCT OCO OTC Phe Oiu Asp Ser Ala Val 70 ATO CAA GTG AG ATG Met Gin Leu Ser Ile 85 OCA AGA OGA TTA GGA Ala Arg Oly Leu Arg 100 GG ACC ACT CTC ACA Oly Thr Thr Leu Thr TTO GA CGA 000 000 TAG TAG Arg Oly Oly Tyr Tyr 105 OTG TGG TGA 0 TTT GAG TAG Phe Asp Tyr TOO GG Trp Gly 110 SEQ ID

LENGTH:

CAA

Gin 428 Val Ser Ser 120 115 NO: 3 TYPE: amino acid TOPOLOGY: linear MOLECULAR TYPE: peptide SEQUENCE DESCRIPTION: Lys Ala Ser Gin Asp Val Asn Thr Ala Val Ala SEQ ID NO: 4 LENGTH: 7 TYPE: amino acid TOPOLOGY: linear MOLECULAR TYPE: peptide SEQUENCE DESCRIPTION: Ser Ala Ser Asn Arg Tyr Thr SEQ ID NO: LENGTH: 9 TYPE: amino acid TOPOLOGY: linear MOLECULAR TYPE: peptide SEQUENCE DESCRIPTION: Gin Gin His Tyr Ser Thr Pro Phe Thr SEQ ID NO: 6 LENGTH: TYPE: amino acid TOPOLOGY: linear **MOLECULAR TYPE: peptide SEQUENCE DESCRIPTION: Pro Tyr Trp Met Gin SEQ ID NO: 7 LENGTH: 17 TYPE: amino acid TOPOLOGY: linear MOLECULAR TYPE: peptide SEQUENCE DESCRIPTION: Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gin Lys Phe Lys Gly 10 SEQ ID NO: 8 LENGTH: 11 TYPE: amino acid 4 TOPOLOGY: linear MOLECULAR TYPE: peptide SEQUENCE DESCRIPTION: Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr SEQ ID NO: 9 LENGTH: 379 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT Met Gly Trp Ser Cys GTC CAC TCC GAC ATC Val His Ser Asp Ile Ile Ile Leu CAG ATG ACC Gin Met Thr 5 GTG ACC ATC Val Thr Ile Ser Leu -10 CAG AGC Gin Ser ACC TGT Thr Cys Val Ala Thr Ala Thr Gly AGC GTG Ser Val AAT ACT Asn Thr 1 GAC AGA Asp Arg CCA AGC AGC CTG AGC GCC Pro Ser Ser Leu Ser Ala AAG GCT AGT CAG GAT GTG Lys Ala Ser Gin Asp Val

CTG

Leu GCT GTA GCC TGG Ala Val Ala Trp 35

TAC

Tyr

TCC

Ser CAG CAG AAG CCA Gin Gin Lys Pro 40 AAC CGG TAC ACT Asn Arg Tyr Thr CTG ATC TAC TCG Leu Ile Tyr Ser

GCA

Ala TTC AGC GGT AGC Phe Ser Gly Ser CTC CAG CCA GAG Leu Gin Pro Glu ACT CCA TTC ACG Thr Pro Phe Thr

GGT

Gly 55 AGC GGT ACC GAC TTC ACC Ser Gly Thr Asp Phe Thr 70 GGA AAG GCT CCA AAG Gly Lys Ala Pro Lys GGT GTG CCA AGC AGA Gly Val Pro Ser Arg TTC ACC ATC AGC AGC Phe Thr Ile Ser Ser CAG CAA CAT TAT AGT Gin Gin His Tyr Ser GAA ATC AAA C Glu Ile Lys 105 48 96 144 192 240 288 336 379 GAC ATC GCT ACC TAC Asp Ile Ala Thr Tyr 85 TTC GGC CAA GGG ACC Phe Gly Gin Gly Thr 100 TAC TGC Tyr Cys AAG GTG Lys Val SEQ ID NO: LENGTH: 379 5 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT Met Gly Trp Ser Cys Ile Ile GTC CAC TCC GAC ATC CAG ATG Val His Ser Asp Ile Gin Met Leu Ser Leu Val -10 ACC CAG AGC CCA Thr Gin Ser Pro Ala Thr Ala Thr Gly r AGC GTG Ser Val AAT ACT Asn Thr -1

GGT

Gly AGA GTG ACC Arg Val Thr 20 5

ATC

Ile ACC TGT AAG Thr Cys Lys AGC AGC CTG AGC GCC Ser Ser Leu Ser Ala GCT AGT CAG GAT GTG Ala Ser Gin Asp Val

GGA

Gly GCT GTA GCC TGG Ala Val Ala Trp

TAC

Tyr CAG CAG AAG CCA Gin Gin Lys Pro 40 AAG GCT CCA AAG Lys Ala Pro Lys 48 96 144 192 240 288 336 379 ooooo *oo CTG CTG ATC TAC TCG Leu Leu Ile Tyr Ser TTC AGC GGT AGC GGT Phe Ser Gly Ser Gly 65 CTC CAG CCA GAG GAC Leu Gin Pro Glu Asp

GCA

Ala TCC AAC CGG TAC Ser Asn Arg Tyr 55 AGT GGT ACC GAC Ser Gly Thr Asp 70 ATC GCT ACC TAC Ile Ala Thr Tyr 85 GGC CAA GGG ACC Gly Gin Gly Thr

TAC

Tyr

TAC

Tyr ACT GGT GTG CCA AGC AGA Thr Gly Val Pro Ser Arg ACC TTC ACC ATC AGC AGC Thr Phe Thr Ile Ser Ser TGC CAG CAA CAT TAT AGT Cys Gin Gin His Tyr Ser ACT CCA TTC ACG Thr Pro Phe Thr SEQ ID NO: 11 LENGTH: 418

TTC

Phe AAG GTG GAA ATC AAA C Lys Val Glu Ile Lys 105 100 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -10 6 GCT CAC TCC CAG GTG CAG CTG GTG CAG Ala His Ser Gin Val Gin Leu Val Gin TCT GGG GCT GAG GTG AAG AAG Ser Gly Ala Glu Val Lys Lys 1 1 CCT GGG GCC TCA GTG Pro Gly Ala Ser Val ACT CCC TAC TGG ATG Thr Pro Tyr Trp Met

AAG

Lys GTT TCC TGC AAG GCA TCT GGA TAC ACC TTC 144 Val Ser Cys Lys Ala u

GAG

Glu

CAG

Gin TGG ATG GGA TCT Trp Met Gly Ser AAG TTC AAG GGC Lys Phe Lys Gly CAG TGG GTG CGA CAG GCC CCT Gin Trp Val Arg Gin Ala Pro 35 40 ATT TTT CCT GGA GAT GGT GAT Ile Phe Pro Gly Asp Gly Asp 55 AGA GTC ACC ATG ACC GCA GAC Arg Val Thr Met Thr.Ala Asp Gly Tyr Thr Phe GGA CAA GGG CTT Gly Gin Gly Leu ACT AGG TAC AGT Thr Arg Tyr Ser ACG TCC ACG AGC Thr Ser Thr Ser 192 240 288 336 384 ACA GTC TAC ATG Thr Val Tvr Met 70 GAG CTG AGC AGC CTG Glu Leu Ser Ser Leu AGA GGA TTA CGA CGA Arg Gly Leu Arg Arg AGA TCT GAG GAC ACG GCC GTG Arg Ser Glu Asp Thr Ala Val GGG GGG TAC TAC TTT GAC TAC Gly Gly Tyr Tyr Phe Asp Tyr 105

TAT

Tyr

TAC

Tyr

GCG

Ala TGG GGG CAA GGG ACC ACG Trp Gly Gin Gly Thr Thr 110 115 SEQ ID NO: 12 LENGTH: 418' TYPE: nucleic acid TOPOLOGY: linear 100

GTC

Val ACC GTC TCC TCA Thr Val Ser Ser 120 G 418 MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu TTC TTC TTG Phe Phe Leu -10 GTG CAG TCT Val Gin Ser CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys 1 1 7 CCT GGG GCC TCA GTG AAG GTT TCC Pro Gly ACT CCC Thr Pro Ala Ser Val Lys TAC TGG ATG CAG Tyr Trp Met Gin 35 ATG GGA TCT ATT Met Gly Ser Ile Val Ser 20 TGG GTG Trp Val TGC AAG GCA Cys Lys Ala CGA CAG GCC Arg Gin Ala 40

GAG

Glu

TGG

TrD TTT CCT GGA GAT GGT Phe Pro Gly Asp Gly 55 GTC ACC ATG ACC GCA Val Thr Met Thr Ala TCT GGA TAC ACC TTC Ser Gly Tyr Thr Phe CCT GGA CAA GGG CTT Pro Gly Gin Gly Leu GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser GAC ACG TCC ACG AGC Asp Thr Ser Thr Ser GAG GAC ACG GCC GTG Glu Asp Thr Ala Val TAC TAC TTT GAC TAC Tyr Tyr Phe Asp Tyr 144 192 240 288 336 384 CAG AAG TTC Gin Lys Phe ACA GTC TAC Thr Val Tyr TAT TAC TGT Tyr Tyr Cys

AAG

Lys

GGC

Gly

AAA

Lys 70 r ATG GAG CTG AGC AGC Met Glu Leu Ser Ser 85 GCG AGA GGA TTA CGA Ala Arg Gly Leu Arg 100 GGG ACC ACG GTC ACC Gly Thr Thr Val Thr

CTG

Leu AGA TCT Arg Ser TGG GGG Trp Gly 110 CGA GGG GGG Arg Gly Gly GTC TCC TCA Val Ser Ser 120 105

G

CAA

Gin 418 115 r r SEQ ID NO: 13 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu -1 1 CCT GGG GCC TCA GTG AAG GTT Pro Gly Ala Ser Val Lys Val 20 TTC TTC TTG Phe Phe Leu -10 GTG CAG TCT Val Gin Ser CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys 5 TCC TGC Ser Cys AAG GCA Lys Ala

TCT

Ser GGA TAC ACC TTC Gly Tyr Thr Phe 144 8 ACT CCC TAC TGG ATG CAG TGG GTG CGA CAG Arg Gin GCC CCT GGA CAA GGG CTT 192 Thr Pro GAG TGG Glu Trp Tyr Trp Met Gin Trp Val Ala Pro 40 GGT GAT Gly Asp Gly Gin Gly Leu ATG GGA TCT Met Gly Ser ATT TTT CCT GGA Ile Phe Pro Gly AGA GTC ACT ATG Arg Val Thr Met

GAT

Asp 55 ACT AGG Thr Arg TAC AGT Tyr Ser CAG AAG TTC AAG Gin Lys Phe Lys ACA GTC TAC ATG Thr Val Tvr Met

GGC

Gly r r r r r r 80 GAG CTG AGC AGC CTG Glu Leu Ser Ser Leu 85 ACC GCA GAC AAG TCC ACG AGC Thr Ala Asp Lys Ser Thr Ser AGA TCT GAG GAC ACG GCC GTG Arg Ser Glu Asp Thr Ala Val GGG GGG TAC TAC TTT GAC TAC Gly Gly Tyr Tyr Phe Asp Tyr 105 240 288 336 384 418 TAT TAC Tyr Tyr TGG GGG Trp Gly 110 TGT GCG AGA GGA TTA Cys Ala Arg Gly Leu 100 CAA GGG ACC ACG GTC Gin Gly Thr Thr Val 115 CGA CGA Arg Arg ACC GTC TCC TCA Thr Val Ser Ser 120

G

SEQ ID NO: 14 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG Ala His Ser Gin Val TTC TTC TTG Phe Phe Leu -10 GTG CAG TCT Val Gin Ser CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly CAG CTG Gin Leu GGG GCT Gly Ala CCT GGG Pro Gly ACT CCC Thr Pro GTG AAG GTT Val Lys Val 20 ATG CAG TGG Met Gin Trp 35

TCC

Ser

GTG

Val TGC AAG GCA TCT Cys Lys Ala Ser CGA CAG GCC CCT Are Gin Ala Pro GAG GTG AAG AAG Glu Val Lys Lys GGA TAC ACC TTC Gly Tyr Thr Phe GGA CAA GGG CTT Gly Gin Gly Leu 48 96 144 192 TAC TGG Tyr Trp 40 9 GAG TGG ATG GGA TCT ATT TTT CCT GGA Glu Trp Met Gly Ser Ile Phe GGC AAA GTC Gly Lys Val CAG AAG Gin Lys ACA GTC Thr Val TTC AAG Phe Lys TAC ATG Tyr Met Pro Gly ACC ATG Thr Met 70 GAT GGT GAT ACT AGG TAC AGT Asp Gly Asp Thr Arg Tyr Ser 55 ACC GCA GAC AAG TCC ACG AGC Thr Ala Asp Lys Ser Thr Ser GAG CTG AGC AGC CTG Glu Leu Ser Ser Leu 85 AGA GGA TTA CGA CGA Arg Gly Leu Arg Arg

AGA

Arg

AGA

Arg TCT GAG GAC ACG GCC GTG Ser Glu Asp Thr Ala Val GGG TAC TAC TTT GAC TAC Gly Tyr Tyr Phe Asp Tyr 240 288 336 384 TAT TAC TGT GCG Tyr Tyr Cys Ala 95 TGG GGG CAA GGG Trp Gly Gin Gly 110

GGG

Gly 100 ACC ACG GTC Thr Thr Val 115 105 ACC GTC TCC TCA Thr Val Ser Ser 120 G 418 SEQ ID NO: LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG Ala His Ser Gin Val TTC TTC TTG Phe Phe Leu -10 GTG CAG TCT Val Gin Ser CAG CTG Gin Leu CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys GCA TCT GGA TAC ACC TTC Ala Ser Gly Tyr Thr Phe

TCC

Ser CCT GGG Pro Gly ACT CCC Thr Pro GTG AAG GTT Val Lys Val 20 TGC AAG Cys Lys 48 96 144 192 240 TAC TGG ATG CAG Tyr Trp Met Gin ATG GGA TCT ATT Met Gly Ser lle

TGG

Trp

TTT

Phe GTG CGA CAG GCC CCT Val Arg Gin Ala Pro 40 CCT GGA GAT GGT GAT Pro Gly Asp Gly Asp GGA CAA GGG CTT Gly Gin Gly Leu ACT AGG TAC AGT Thr Arg Tyr Ser

TGG

Trp 10 CAG AAG TTC AAG Gin Lys Phe Lys ACA GTC TAC ATG Thr Val Tyr Met TAT TAC TGT GCG Tyr Tyr Cys Ala TGG GGG CAA GGG Trp Gly Gin Gly 110 SEQ ID NO: 16 LENGTH: 418 TYPE: nucleic GGC AGA GCC ACC Gly Arg Ala Thr CTG ACC Leu Thr 70 CTG AGA Leu Arg GCA GAC Ala Asp ACG TCC ACG AGC Thr Ser Thr Ser GAC ACG GCC GTG Asp Thr Ala Val GAG CTG AGC Glu Leu Ser AGA GGA TTA Arg Gly Leu 100 ACC ACG GTC Thr Thr Val 115

AGC

Ser 85

CGA

Arg TCT GAG Ser Glu GGG TAC Gly Tyr 288 336 384 CGA GGG Arg Gly

TAC

Tyr TTT GAC TAC Phe Asp Tyr 105 G ACC GTC TCC TCA Thr Val Ser Ser 120 418 acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESC ATG GAC TGG ACC Met Asp Trp Thr

RIPTION:

TGG

Trp AGG GTC TTC TTC TTG Arg Val Phe Phe Leu -10 CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GCT CAC TCC Ala His Ser -1 CCT GGG GCC Pro Glv Ala

CAG

Gin 1

TCA

Ser GTG CAG CTG GTG CAG Val Gin Leu Val Gin 5 GTG AAG GTT TCC TGC Val Lys Val Ser Cys 20 ATG CAG TGG GTG CGA Met Gin Trp Val Arg TCT GGG GCT GAG GTG AAG AAG Ser Gly Ala Glu Val Lys Lys AAG GCA TCT GGA TAC ACC TTC Lys Ala Ser Gly Tyr Thr Phe ACT CCC Thr Pro 48 96 144 192 240 288 TAC TGG Tyr Trp

GAG

Glu

CAG

Gin CAG GCC Gin Ala 40

CCT

Pro GGA CAA GGG CTT Gly Gln Gly Leu 35 TGG ATG GGA TCT Trp Met Gly Ser AAG TTC AAG GGC Lys Phe Lys Gly 35 ATT TTT CCT GGA GAT Ile Phe Pro Gly Asp 55 AGA GCC ACC CTG ACT Arg Ala Thr Leu Thr 70

GGT

Gly

GCA

Ala GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser GAC ACG TCC TCG AGC Asp Thr Ser Ser Ser 11 ACA CCC TAG ATG GAG GTG Thr Ala Tyr Met Giu Leu TAT TAG TGT GCG AGA GGA Tyr Tyr Cys Ala Arg Gly TGG GOG CAA GGG ACC AG Trp Cly Gin Gly Thr Thr 110 115 SEQ ID NO: 17 LENGTH: 418 TYPE: nucleic acid AGG AGG Ser Ser 85 TTA CGA Leu Arg CTG AGA TCT GAG GAG AG CCC GTG Leu Arg Ser Glu Asp Thr Ala Val CGA GCC GGG TAG TAG TTT GAG TAG Arg Gly Gly Tyr Tyr Phe Asp Tyr 105 GTG TGC TGA C 100u

GTG

Val1

ACC

Thr Val Ser Ser 120 TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAG TGG AGC TGG ACG C Met Asp Trp Thr Tr-p Arg V GCT GAG TOC GAG GTG GAG Ala His Ser Gin Val Gin

C,

TC TTG T TIC CTO ai Phe Phe Leu Leu -10 TG OTO GAG TGT CG eu Val Gin Ser. Gly GCT GTA CCT GGA GGT Ala Val Ala Pro Gly GCT GAG GTG AAO AAO Ala Giu Val Lys Lys 336 384 418 48 96 144 192 240 288 336 4- 5.55 CCT CCC Pro Gly ACT CCC Thr Pro 1

TCA

Ser OTO AAG GTT Val Lys Val 20 ATO GAG TCC Met Gin Trp TGG AAC Cys Lys

GCA

Ala ICT GGA TAG ACC TIC Ser Gly Tyr Thr Phe TAG TG Tyr Trp CTG CGA GAG CC Val Arg Gin Arg 40

GCT

Pro OGA CAA CCC CT Gly Gin Cly Leu

ATT

Ile TOG ATC COA TCT Trp Met Cly Ser TTT COT OCA CAT OGT Phe Pro Cly Asp Cly 55 CAT ACT ACG TAG ACT Asp Thr Arg Tyr Ser GAG AG TOG AGG AGO Asn Thr Ser Thr Ser GAG AAG TTG AAO Gin Lys Phe Lys ACA OTO TAG ATC Thr Val Tyr Met CCC AGA OTO AGO ATC Oly Arg Val Thr Met 70 GAG CG AGO AGO CTC Clu Leu Ser Ser Leu 85 AGO GA Thr Ala AG 000 GIG Thr Ala Val AGA TOT GAG GAG Arg Ser Clu Asp 12 TAT TAC TGT GCG AGA GGA TTA Tyr Tyr Cys Ala Arg Gly Leu 100 TGG GGG CAA GGG ACC ACG GTC Trp Gly Gin Gly Thr Thr Val 110 115 SEQ ID NO: 18 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val CGA CGA GGG GGG TAC TAC TTT GAC TAC Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 105 ACC GTC TCC TCA G Thr Val Ser Ser 120 i i r r r TTC TTC TTG CTG GCT GTA GCT CCA GGT Phe Phe Leu Leu Ala Val GCT CAC TCC CAG GTG Ala His Ser Gin Val -10 CAG CTG GTG CAG TCT Gin Leu Val Gin Ser Ala Pro Gly CCT GGG Pro Gly ACT CCC Thr Pro -1

GCC

Ala 1

TCA

Ser GTG AAG GTT Val Lys Val 20 5 TCC TGC AAG Ser Cys Lys GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys GCA TCT GGA TAC ACC TTC Ala Ser Gly Tyr Thr Phe 384 418 48 96 144 192 240 288 336 384 GTG CGA CAG GCC Val Arg Gin Ala

GAG

Glu TAC TGG ATG CAG Tyr Trp Met Gin 35

TGG

Trp TGG ATG GGA TCT Trp Met Gly Ser CAG AAG TTC AAG Gin Lys Phe Lys ACA GCC TAC ATG Thr Ala Tyr Met TAT TAC TGT GCG Tyr Tyr Cys Ala

GGC

Gly

GAG

Glu ATT TTT CCT GGA GAT Ile Phe Pro Gly Asp 55 AAA GTC ACC ATG ACC Lys Val Thr Met Thr 70 CTG AGC AGC CTG AGA Leu Ser Ser Leu Arz 40

GGT

Gly

GCA

Ala CCT GGA CAA GGG CTT Pro Gly Gin Gly Leu GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser GAC ACG TCC TCG AGC AsD Thr Ser Ser Ser TCT GAG GAC ACG GCC GTG Ser Glu Asp Thr Ala Val 85 AGA GGA TTA CGA Arg Gly Leu Arg 100 CGA GGG GGG TAC TAC TTT GAC TAC Arg Gly Gly Tyr Tyr Phe Asp Tyr 105 13 TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 110 115 120 SEQ ID NO: 19 LENGTH: 418 TYPE: nucleic acid 418

I-

'C 'C C

*CC.

TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val -15 GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu TTC TTC TTG CTG GCT GTA GCT CCA GGT Phe Phe Leu -10 GTG CAG TCT Val Gin Ser Leu Ala Val Ala Pro Gly CCT GGG Pro Gly ACT CCC Thr Pro 1

TCA

Ser GTG AAG GTT Val Lys Val 20 5

TCC

Ser GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys GCA TCT GGA TAC ACC TTC Ala Ser Gly Tyr Thr Phe TGC AAG Cys Lys Cs..

C

CC..

GAG

Glu

CAG

Gin TAC TGG ATG CAG Tyr Trp Met Gin 35

TGG

Trp

TTT

Phe GTG CGA CAG GCC Val Arg Gin Ala 40 CCT GGA GAT GGT Pro Gly Asp Gly CCT GGA Pro Gly CAA GGG CTT Gin Gly Leu TGG ATG GGA Trp Met Gly AAG TTC AAG Lys Phe Lys

TCT

Ser

ATT

Ile GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser 48 96 144 192 240 288 336 384 418 55 ACA GCC TAC ATG Thr Ala Tvr Met GGC AAA GTC ACC Gly Lys Val Thr GAG CTG AGC AGC Glu Leu Ser Ser AGA GGA TTA CGA Arg Gly Leu Arg

ATG

Met 70

ACC

Thr GCA GAC ACG TCC TCG AGC Ala Asp Thr Ser Ser Ser CTG GCA TTT GAG GAC ACG GCC GTG Leu Ala Phe Glu Asp Thr Ala Val CGA GGG GGG TAC TAC TTT GAC TAC Arg Gly Gly Tyr Tyr Phe Asp Tyr 105 TAT TAC Tyr Tyr TGG GGG Trp Gly 110

GCG

Ala 100 CAA GGG ACC ACG Gin Gly Thr Thr 115 GTC ACC GTC TCC TCA G Val Thr Val Ser Ser 120 SEQ ID NO: 14 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu TTC TTC TTG CTG GCT GTA GCT CCA GGT Phe Phe Leu -10 GTG CAG TCT Val Gin Ser

S

S

Leu Ala GGG GCT Gly Ala GCA TCT Ala Ser CCT GGG Pro Gly 15 ACT CCC Thr Pro -1

GCC

Ala 1 TCA GTG Ser Val AAG GTT Lys Val 20 5

TCC

Ser TGC AAG Cys Lys Val Ala Pro Gly GAG GTG AAG AAG Glu Val Lys Lys GGA TAC ACC TTC Gly Tyr Thr Phe GGA CAA GGG CTT Gly Gin Gly Leu ACT AGG TAC AGT Thr Arm Tvr Ser 30

GAG

Glu

CAG

Gin

ACA

Thr

TAT

Tyr TAC TGG ATG CAG Tyr Trp Met Gin 35 TGG GTG CGA CAG GCC Trp Val Arg Gin Ala 40 TTT CCT GGA GAT GGT Phe Pro Gly Asp Gly 55.

CCT

Pro

GAT

AsD 48 96 144 192 240 288 336 384 TGG ATG GGA Trp Met Gly AAG TTC AAG Lys Phe Lys

TCT

Ser

ATT

Ile GCC TAC Ala Tyr TAC TGT Tyr Cys GGC AAA GCC ACC CTG Gly Lys Ala Thr Leu GAG CTG AGC AGC CTG Glu Leu Ser Ser Leu 85 AGA GGA TTA CGA CGA Arg Gly Leu Arg Arg ACT GCA Thr Ala AGA TCT GAG GAC ACG GCC GTG Arg Ser Glu Asp Thr Ala Val GGG GGG TAC TAC TTT GAC TAC Gly Gly Tyr Tyr Phe Asp Tyr GAC ACG TCC TCG AGC Asp Thr Ser Ser Ser

GCG

Ala TGG GGG CAA GGG Trp Gly Gin Gly 110 SEQ ID NO: 21 LENGTH: 418 TYPE: nucleic 100 ACC ACG GTC Thr Thr Val 105 ACC GTC TCC TCA G Thr Val Ser Ser 120 418 acid TOPOLOGY: linear MOLECULAR TYPE: cDNA S 15 SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG Met Asp Trp Thr Trp GCT CAC TCC CAG GTG Ala His Ser Gin Val AGG GTC TTC TTC TTG Arg Val Phe Phe Leu -10 CAG CTG GTG CAG TCT Gin Leu Val Gin Ser 5 AAG GTT TCC TGC AAG Lys Val Ser Cys Lys CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys GCA TCT A TT GATAC ACC TTC Ala Ser Gly Tyr Thr Phe

S

5 CCT GGG Pro Gly 15 ACT CCC Thr Pro

GAG-TGG

Glu Trp -1

GCC

Ala TAC TGG ATG CAG Tyr Trp Met Gin 35 ATG GGA TCT ATT Met Gly Ser lle 20 TGG GTG Trp Val

GTG

Val CGA CAG GCC Arg Gin Ala 40

CCT

Pro GGA CAA GGG Gly Gin Gly

CTT

Leu TTT CCT GGA GAT Phe Pro Gly Asp 55

GGT

Gly GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser 96 144 192 240 288 336 384 418 CAG AAG TTC AAG Gin Lys Phe Lys ACA GCC TAC ATG Thr Ala Tyr Met TAT TAC TGT GCG Tyr Tyr Cys Ala GGC AAA GTC ACC ATG Gly Lys Val Thr Met 70 CAG CTG AGC AGC CTA Gin Leu Ser Ser Leu 85 AGA GGA TTA CGA CGA Arg Gly Leu Arg Arg ACC GCA GAC ACG TCC TCG AGC Thr Ala Asp Thr Ser Ser Ser AGA TCT GAG GAC ACG GCC GTG Arg Ser Glu Asp Thr Ala Val GGG GGG TAC TAC TTT GAC TAC Gly Gly Tyr Tyr Phe Asp Tyr

GGG

Gly 100 CAA GGG ACC ACG GTC Gin Gly Thr Thr Val 115 105 ACC GTC TCC TCA G Thr Val Ser Ser 120 SEQ ID NO: 22 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG-GCT GTA GCT CCA GGT Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -10 16 GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGG GCT Gin Ser Gly Ala GAG GTG Glu Val AAG AAG Lys Lys Ala His Ser -1 CCT GGG GCC Pro Gly Ala ACT CCC TAC Thr Pro Tyr Gin 1

TCA

Ser Val Gin Leu GTG AAG GTT Val Lys Val 20 Val 5

TCC

Ser TGC AAG GCA TCT GGA TAC ACC TTC Cys Lys Ala Ser Gly Tyr Thr Phe CGA CAG GCC CCT GGA CAA GGG CTT Arg Gin Ala Pro Gly Gin Gly Leu TGG ATG CAG Trp Met Gin 35 TGG GTG Trp Val a.

a a

GAG

Glu TGG ATG GGA TCT ATT Trp Met Gly Ser lle 40 TTT CCT GGA GAT GGT Phe Pro Gly Asp Gly 55 GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser CAG AAG Gin Lys ACA GCC Thr Ala TAT TAC Tyr Tyr 95 TGG GGG Trp Gly 110 TTC AAG Phe Lys GGC AAA Gly Lys GTC ACC ATG Val Thr Met TAC ATG CAG CTG AGC Tyr Met Gin Leu Ser TGT GCG AGA GGA TTA Cys Ala Arg Gly Leu 100 CAA GGG ACC ACG GTC Gin Gly Thr Thr Val 115 ACC GCA GAC ACG TCC TCG AGC Thr Ala Asp Thr Ser Ser Ser AGA TCT GAG GAC ACG GCC GTG Arg Ser Glu Asp Thr Ala Val GGG GGG TAC TAC TTT GAC TAC Gly.Gly Tyr Tyr Phe Asp Tyr 144 192 240 288 336 384 418 CGA CGA Arg Arg a a 105 ACC GTC TCC TCA G Thr Val Ser Ser 120 SEQ ID NO: 23 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu TTC TTC TTG Phe Phe Leu -10 GTG CAG TCT Val Gin Ser CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys 1 1 17 CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT Ala Ser GGA TAC ACC TTC Gly Tyr Thr Phe Pro Gly ACT CCC Thr Pro Ala Ser Val Lys TAC TGG ATG CAG Tyr Trp Met Gin 35 ATG GGA TCT ATT Met Gly Ser lle Val Ser Cys 20 TGG GTG CGA Trp Val Arg TTT CCT GGA Phe Pro Gly

GAG

Glu

CAG

Gin CAG GCC Gin Ala 40 GAT GGT Asp Gly

CCT

Pro GGA CAA GGG Gly Gin Gly Lys

CTT

Leu

TGG

TrD AAG TTC AAG Lys Phe Lys 55

ACC

Thr GAT ACT AGG TAC AGT Asp Thr Arg Tyr Ser GAC ACG TCC TCG AGC Asp Thr Ser Ser Ser 144 192 240 288 336 384 ACA GCC TAC Thr Ala-Tyr TAT TAC TGT Tyr Tyr Cys TGG GGG CAA Trp Gly Gin GGC AAA GTC ACC ATG Gly Lys Val Thr Met 70 CAG CTG AGC ATC CTG Gin Leu Ser Ile Leu 85 AGA GGA TTA CGA CGA Arg Gly Leu Arg Arg AGA TCT GAG GAC Arg Ser Glu Asp GGG GGG TAC TAC Gly Gly Tyr Tyr

TCG

Ser

GCC

Ala

GCA

Ala

GCG

Ala TTT GAC TAC Phe Asp Tyr 100 GGG ACC ACG GTC Gly Thr Thr Val 115 105 ACC GTC TCC TCA G Thr Val Ser.Ser 120 418 SEQ ID NO: 24 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu -1 1 CCT GGG GCC TCA GTG AAG GTT Pro Gly Ala Ser Val Lys Val 20 TTC TTC TTG Phe Phe Leu -10 GTG CAG TCT Val Gin Ser CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly GGG GCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys 48 96 144 5

TCC

Ser TGC AAG GCA TCT Cys Lys Ala Ser GGA TAC ACC TTC Gly Tyr Thr Phe ACT CCC TAG TGG Thr Pro Tyr Trp GAG TGG ATG GGA Giu Trp Met Gly ATG GAG TGG GTG CGA GAG GCC Met Gin Trp Val Arg Gin Ala 35 40 TGT ATT TTT CCT GGA GAT GGT Ser Ile Phe Pro Gly Asp Gly GCT GGA CAA GGG CTT Pro Gly Gin Gly Leu GAT ACT AGG TAG AGT Asp Thr Arg Tyr Ser GAG AAG TTC MAG Gin Lys Phe Lys ACA GCC TAG ATG Thr Ala Tyr Met

GGC

Gly

GAG

Giu MAA GTG ACC Lys Val Thr CTG AGC ATC Leu Ser Ile

ATG

Met 70 55

ACC

Thr GCA GAG AGG TCC TCG AGC Ala Asp Thr Ser Ser Ser 192 240 288 336 384 .00 S *000 000 0.00 80 TAT TAG Tyr Tyr TGG GGG Trp Gly 110

TGT

Cy s GCG AGA GGA Ala Arg Gly

TTA

Leu 100

GTC

Val1 85

CGA

Arg CTG AGA TCT GAG GAG ACG GCC GTG Leu Arg Ser Giu Asp Thr Ala Val CGA GGG GGG TAG TAG TTT GAG TAG Arg Gly Gly Tyr Tyr Phe Asp Tyr 105

G

GMA GGG ACC ACG Gin Gly Thr Thr 115 ACC GTC TGG TCA Thr Val Ser Ser 120 418 0 00000* 0 0000 0 0000 SEQ ID NO: LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAG TGG ACC TGG AGG GTG Met Asp Trp Thr Trp Arg Val GCT GAG TGG GAG GTG Ala His Ser Gin Val GAG CTG Gin Leu TTG TTG TTG Phe Phe Leu -10 GTG GAG TGT Val Gin Ser GTG OCT GTA OCT CGA GOT Leu Ala Val Ala Pro Gly GGG GGT GAG GTG MAG MAG Gly Ala Giu Val Lys Lys GGA TGT GGA TAG ACC TTG Ala Ser Gly Tyr Thr Phe 4+8 96 144 192 GGT GGG Pro Gly ACT CCC Thr Pro -i1 1 GCC TCA GTG MAG GTT Ala Ser Val Lys Val 20 TAG TGG ATG GAG TGG Tyr Trp Met Gin Trp 35 TGC MAG Gys Lys GTG CON GAG GCC Val Arg Gin Ala COT GCA CMA GGG GTT Pro Gly Gin Gly Leu 19 GAG TGG ATG GGA TCT Glu*Trp Met Gly Ser GAG MAG TTG MAG GO Gin Lys Phe Lys Gly ATT TTT CCT GGA GAT GGT GAT Ile Phe Pro Gly Asp Gly Asp 55 AAA GTC AGO ATG AGC GGA GAO Lys Val Thr Met Thr Ala Asp ACT AGG TAO AGT Thr Arg Tyr Set AOG TOO TOG AGO Thr Ser Ser Ser 240 288 336 384 0S S 0ee S *0 0@ S 0@S C

S.

S S @556

S.

S

0@ S e.g.

e.g.

0 @000 5056

S

50*5 ACA CG TAO Thr Ala Tyr TAT TAO TGT Tyr Tyr Gys 95 GAG GTG AGO AGO Glu Leu Ser Ser 85 AGA GGA TTA OGA Arg Glv Leu Arg

GOG

Ala Arg Ser Glu Asp Set Ala Val COG GGG TAO TAO TTT GAO TAO Gly Gly Tyr Tyr Phe Asp Tyr

OGA

Arg 1.00 TGG GGG GMA GOG AGO AG Trp Gly Gin Gly Thr Th 110 11 SEQ ID NO: 26 LENGTH: 418 TYPE: nucleic acid G GTO r Val 105 AGO GTO TOG TGA G Thr Val Ser Ser 120 418 TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE

DESCRIPTION:

0 0O** 0 5050 ATG GAO TOG AGO TGG AGG, Met Asp Trp Thr Trp Arg GOT GAO TOO GAG GTG GAG Ala His Ser Gin Val Gin GTO TTO TTO TTG OTO Val Phe Phe Leu Leu -1.0 OTG OTO GAG TOT GGG Leu Val Gin Ser Gly 5 GOT GTA GOT OGA GGT Ala Val Ala Pro Gly Ala -1 1 GOT 000 GOG TOA Pro Gly Ala Set ACT 000 TAG TG Thr Pro Tyr Trp GAG TOG ATG GGA Glu Trp Met Gly OTO MAG OTT Val Lys Val 20 ATO GAG TG Met Gin Trp TOO TOO MAG GA TOT Ser Gys Lys Ala Ser OTO OGA GAG 000 GOT.

Val Arg Gin Ala Pro Glu Val Lys Lys OGA TAO AGO TTO Gly Tyr Thr Phe GGA GMA 000 OTT Gly Gin Gly Leu ACT AGO TAO AGT 48 96 144 192 240 35 ATT TTT GOT GGA OAT Ile Phe Pro Gly Asp

TOT

Ser

OAT

Gly Asp Thr Arg Tyr Ser CAG AAG Gin Lys TTC AAG Phe Lys GGC AGA Gly Arg GAG CTG Glu Leu GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC Val Thr Met Thr Ala Asp Thr Ser Thr Ser 70 AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG Ser Ser Leu Arg Ser Glu Asp Thr Ala Val ACA GCC TAC ATG Thr Ala Tyr Met TAT TAC TGT GCG Tyr Tyr Cys Ala TGG GGG CAA GGG Trp Gly Gin Gly 110 SEQ ID NO: 27 LENGTH: 418 288 336 384 85 AGA GGA TTA Arg Gly Leu 100 ACC.ACG GTC Thr Thr Val 115 85 CGA CGA GGG GGG TAC Arg Arg Gly Gly Tyr 105 ACC GTC TCC TCA G Thr Val Ser Ser 120

TAC

Tyr TTT GAC TAC Phe Asp Tyr 418 .0*0 0 O 0.

.0 0 0.000 0.00.

0 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG Ala His Ser Gin Val TTC TTC TTG Phe Phe Leu.

-10 GTG CAG TCT Val Gin Ser CTG GCT GTA GCT CCA GGT Leu Ala Val Ala Pro Gly CAG CTG Gin Leu GGG GCT Gly Ala CCT GGG Pro Gly ACT CCC Thr Pro -1

GCC

Ala 5

TCC

Ser GTG AAG GTT Val Lys Val 20 TGC AAG GCA TCT Cys Lys Ala Ser GAG GTG AAG AAG Glu Val Lys Lys GGA TAC ACC TTC Gly Tyr Thr Phe GGA CAA GGG CTT Gly Gin Gly Leu ACT AGG TAC AGT Thr Arg Tyr Ser 48 96 144 192 240 288 TAC TGG ATG CAG Tyr Trp Met Gin 35

TGG

Trp GTG CGA CAG GCC CCT Val Arg Gin Ala Pro 40 GAG TGG ATG GGA TCT Glu Trp Met Gly Ser CAG AAG TTC AAG GGC Gin Lys Phe Lys Gly ATT TTT CCT GGA GAT Ile Phe Pro Gly Asp 55 AGA GTC ACC ATG ACC Arg Val Thr Met Thr 70 GGT GAT Gly Asp TCG AGC Ser Ser GCA GAC ACG TCC Ala Asp Thr Ser 21 ACA GTC TAG ATG GAG GTG Thr Val Tyr Met Glu Leu TAT TAG TGT GCG AGA GGA Tyr Tyr Gys Ala Arg Gly TGG GGG CAA GGG ACG ACG Trp Gly Gin Gly Thr Thr 110 115 SEQ ID NO: 28 LENGTH: 418 TYPE: nucleic acid AGG AGC CTG AGA TGT GAG GAG AGG GCC GTG Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 85 TTA GGA GGA GGG GGG TAG TAG TTT GAG TAG Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 100 105 GTG ACC GTG TCC TGA G Val Thr Val Ser Ser 120 336 384 418 TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAG TGG ACC TGG AGG G' Met Asp Trp Thr Trp Arg V TC TTG TTG TTG GTG GGT OTA GGT CCA GGT al Phe Phe Leu OCT CAC TCC CAG GTG Ala His Ser Gin Val -10 GAG GTG GTG GAG TCT Gin Leu Val Gin Ser Leu Ala Val Ala Pro Gly GGG OCT GAG GTG AAG AAG Gly Ala Glu Val Lys Lys CCT GGG Pro Gly ACT CCC Thr Pro 1

TCA

Ser GTG AAG GTT Val Lys Val 20 5 TCC TGG AAG GGA TCT Ser Gys Lys Ala Ser GTG CGA GAG GCG CCT Val Arg Gin Ala Pro

GAG

Clu TAG TGG ATG GAG Tyr Trp Met Gin 35

TGG

Trp TGG ATG OGA TCT Trp Met Oly Ser

ATT

Ile TTT CCT OGA OAT Phe Pro Oly Asp 55 40

GGT

Gly GGA TAG ACC TTC Gly Tyr Thr Phe GGA GAA GGG GTT Oly Gin Gly Leu ACT AOG TAG AGT Thr Arg Tyr Ser AAO TCC AGG AGG Lvs Ser Thr Ser 48 96 144 192 240 288 336 GAG AAG TTG AAO Gin Lys Phe Lys ACA GCC TAG ATO Thr Ala Tyr Met GGC AGA GTG ACC ATG Oly Arg Val Thr Met 70 GAG GTG AGG AGC CTG Glu Leu Ser Ser Leu 85 ACC GGA GAG Thr Ala AsD AGA TCT GAG GAG ACG GGC GTG Arg Ser Glu Asp Thr Ala Val TAT TAG TGT GCG AGA OGA TTA GGA GGA GGG GGG TAG TAG TTT GAG TAG 394 22 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg.Gly Gly Tyr Tyr Phe Asp Tyr 100 105 TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G 418 Trp Gly Gin Gly Thr Thr Val Thr Val Set Ser 110 115 120 SEQ ID NO: 29 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGAAGTTGC CTGTTAGGCT GTTGGTGCTG SEQ ID NO: LENGTH: 39 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGGAGWCAG ACACACTCCT GYTATGGGT 39 SEQ ID NO: 31 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGAGTGTGC TCACTCAGGT CCTGGSGTTG SEQ ID NO: 32 LENGTH: 43 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGAGGRCCC CTGCTCAGWT TYTTGGMWTC TTG 43 SEQ ID NO: 33 LENGTH: TYPE: nucleic acid TOPOLOGY: linear 23 MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGGATTTWC AGGTGCAGAT TWTCAGCTTC SEQ ID NO: 34 LENGTH: 37 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGAGGTKCY YTGYTSAGYT YCTGRGG 37 SEQ ID NO: .LENGTH: 41 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGGGCWTCA AGATGGAGTC ACAKWYYCWG G 41 SEQ ID NO: 36 LENGTH: 41 :TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGTGGGGAY CTKTTTYCMM TTTTTCAATT G 41 SEQ ID NO: 37 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGGTRTCCW CASCTCAGTT CCTTG SEQ ID NO: 38 LENGTH: 37 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGTATATAT GTTTGTTGTC TATTTCT 37 SEQ ID NO: 39 LENGTH: 38 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACTAGTCGAC ATGGAAGCCC CAGCTCAGCT TCTCTTCC 38 SEQ ID NO: LENGTH: 27 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GGATCCCGGG TGGATGGTGG GAAGATG 27 SEQ ID NO: 41 LENGTH: TYPE: nucleic acid TOPOLOGY: linear .:.**MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: TAGAGTCACC GAGGAGCCAG TTGTA SEQ ID NO: 42 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GGATCCCGGG AGTGGATAGA CCGATG 26 SEQ ID NO: 43 LENGTH: 34 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GATAAGCTTC CACCATGGGC TTCAAGATGG AGTC 34 SEQ ID NO: 44 25 LENGTH: 34 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GATAAGCTTC CACCATGGAA TGTAACTGGA TACT 34 SEQ ID NO: LENGTH: 34 TYPE: nucleic acid S. TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GGCGGATCCA CTCACGTTTT ATTTCCAACT TTGT 34 SEQ ID NO: 46 LENGTH: 34 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GGCGGATCCA CTCACCTGAG GAGACTGTGA GAGT 34 SEQ ID NO: 47 LENGTH: 18 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: CAGACAGTGG TTCAAAGT 18 SEQ ID NO: 48 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GAATTCGGAT CCACTCACGT TTGATT 26 SEQ ID NO: 49 LENGTH: 48 TYPE: nucleic acid 26 TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGTCAGGATG TGAATACTGC TGTAGCCTGG TACCAGCAGA AGCCAGGA 48 SEQ ID NO: LENGTH: 39 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCATCCAACC GGTACACTGG TGTGCCAAGC AGATTCAGC 39 SEQ ID NO: 51 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: CAACATTATA GTACTCCATT CACGTTCGGC CAAGGGACCA AGGTG SEQ ID NO: 52 LENGTH: 47 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCAGTATTCA CATCCTGACT GGCCTTACAG GTGATGGTCA CTCTGTC 47 SEQ ID NO: 53 LENGTH: 38 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACACCAGTGT ACCGGTTGGA TGCCGAGTAG ATCAGCAG 38 SEQ ID NO: 54 LENGTH: 41 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA 27 SEQUENCE DESCRIPTION: GTGAATGGAG TACTATAATG TTGCTGGCAG TAGTAGGTAG C 41 SEQ ID NO: LENGTH: 31 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GGTACCGACT ACACCTTCAC CATCAGCAGC C *31 SEQ ID NO: 56 :LENGTH: 31 .TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GGTCAAGGTG TAGTCGGTAC COCTACCGCT A 31 SEQ ID NO: 57 LENGTH: 144 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ATGCCTTCCA GGAAACCTTC ACTGAGCCCC CAGGCTTCTT CACCT.CAGCC CCAG.ACTGCA CCAGCTGCAC CTGGGAGTGA GCACCTGGAG CTACAGCCAG CAAGAACAAG ACCCTCCAGG 120 TCCAGTCCAT GGTGGAAGCT TATC 144 SEQ ID NO: 58 LENGTH: 130 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: TCAGTGAAGG TTTCCTGCAA CGCATCTCGA TACACCTTGA CTCCCTACTG GATGCAGTGG GTGCGACAGG CCCCTGGACA AGCGCTTGAG TGGATGGGAT CTATTTTTCC TCGAGATGGT 120 GATACTAGGT 130 SEQ ID NO: 59 LENGTH: 131 TYPE: nucleic acid 28 TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AATACACGGC CGTGTCCTCA GATCTCAGGC TGCTCAGCTC CATGTAGACT GTGCTCGTGG ACGTGTCTGC GGTCATGGTG ACTCTGCCCT TGAACTTCTG ACTGTACCTA GTATCACCAT 120 CTCCAGGAAA A 131 SEQ ID NO: LENGTH: 119 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GAGATCTGAG GACACGGCCG TGTATTACTG TGCGAGAGGA TTACGACGAG GGGGGTACTA CTTTGACTAC TGGGGGCAAG GGACCACGGT CACCGTCTCC TCAGGTGAGT GGATCCGAC 119 SEQ ID NO: 61 LENGTH: **TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GATAAGCTTC CACCATGGAC TGGAC SEQ ID NO: 62 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GTCGGATCCA CTCACCTGAG GAGAC SEQ ID NO: 63 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AAGTTCAAGG GCAAAGTCAC CATGAC 26 SEQ ID NO: 64 LENGTH: 26 29 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GTCATGGTGA CTTTGCCCTT GAACTT 26 SEQ ID NO: LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ATGACCGCAG ACAAGTCCAC GAGCAC 26 SEQ ID NO: 66 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GTGCTCGTGG ACTTGTCTGC GGTCAT 26 SEQ ID NO: 67 LENGTH: 46 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AAGTTCAAGG GCAAAGTCAC CATGACCGCA GACAAGTCCA CGAGCAC 46 SEQ ID NO: 68 LENGTH: 47 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GTGCTCGTGG ACTTGTCTGC GGTCATGGTG ACTTTGCCCT TGAACTT 47 SEQ ID NO: 69 LENGTH: 38 TYPE: nucleic acid TOPOLOGY: linear 30 MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AAGTTCAAGG GCAGAGCCAC CCTGACCGCA GACACGTC 38 SEQ ID NO: LENGTH: 38 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GACGTGTCTG CGGTCAGGGT GGCTCTGCCC TTGAACTT 38 SEQ ID NO: 71 LENGTH: 18 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: CAGACAGTGG TTCAAAGT 18 SEQ ID NO: 72 LENGTH: 17 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCCCCAAAGC CAAGGTC 17 SEQ ID NO: 73 LENGTH: 23 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ATTTTTCCTG GAGATGGTGA TAC 23 SEQ ID NO: 74 LENGTH: 23 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: 31 GTATCACCAT CTCCAGGAAA TAT 23 SEQ ID NO: LENGTH: 418 TYPE: nucleic acid

S.

5 55 5

S.

S

*S S,

S

n e

S

S

5.55

TOPOLOGY:

MOLECULAR

SEQUENCE

ATG GAA TGT Met Glu Cys GCC TAC TCA Ala Tyr Ser -1 CCT GGG GCT Pro Gly Ala linear TYPE: cDNA

DESCRIPTION:

AAC TGG ATA CTT CCT TTT ATT Asn Trp Ile Leu Pro Phe Ile -15 -10 CAG GTT CAA CTC CAG CAG TCT Gin Val Gin Leu Gin Gin Ser CTG TCA GTA ACT TCA GGT Leu Ser Val Thr Ser Gly GGG GCT GAG CTG GCA AGA Gly Ala Glu Leu Ala Arg GCT TCT GGC TAC ACC TTT Ala Ser Gly Tyr Thr Phe ACT CCC Thr Pro GTG AAG TTG Val Lys Leu 20 TCC TGC AAG Ser Cys Lys

GAA

Glu TAC TGG ATG CAG Tyr Trp Met Gin 35

TGG

Trp GTA AAA CAG AGG CCT GGA Val Lys Gin Arg Pro Gly 40 CAG GGT CTG Gin Gly Leu 48 96 144 192 240 288 336 384 TGG ATT GGG TCT Trp Ile Gly Ser

ATT

Ile

AGA

Arg TTT CCT GGA GAT GGT GAT ACT AGG TAC AGT Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 55 GTC ACC ATG ACC GCA GAC ACG TCC ACG AGC Val Thr Met Thr Ala Asp Thr Ser Thr Ser CAG AAG TTC AAG GGC Gin Lys Phe Lys Gly ACA GTC TAC ATG GAG Thr Val Tyr Met Glu 70 CTG AGC AGC CTG Leu Ser Ser Leu 85 GGA TTA CGA CGA Gly Leu Arg Arg TAT TAC TGT Tyr Tyr Cys AGA TCT GAG GAC ACG GCC GTG Arg Ser Glu Asp Thr Ala Val GGG GGG TAC TAC TTT GAC TAC Gly Gly Tyr Tyr Phe Asp Tyr 105 GCG AGA Ala Arg TGG GGG CAA GGG Trp Gly-Gln Gly 110 SEQ ID NO: 76 LENGTH: 418 100 ACC ACG GTC Thr Thr Val 115 ACC GTC TCC TCA Thr Val Ser Ser 120

G

418 TYPE: nucleic acid 32 TOPOLOGY: linear MOLECULAR TYPE: cDNA

SEQUENCEI

ATG GAG TGG Met Asp Trp OCT GAG TCC Ala His Ser -1

DESCRIPTION:

ACC TGG AGG GTG TTC TTC TTG OTG GCT GTA GCT OGA GGT Thr Trp GAG GTG Gin Val Arg Val Phe Phe Leu Leu -10 GAG OTO OTG GAG TOT GG Gin Leu Val Gin Ser Oly Ala Val Ala Pro Gly OCT GAG OTO AAG AAG Ala Olu Val Lys Lys 0O

SS

0 0S@ S

OS

S@ S

S

S@

SS

S

5S5* OCT GGG Pro Gly 15 ACT COG Thr Pro GAO TG Glu Trp GAG AAG Gin Lys 0CC Ala GTG AAO OTT TOG TG Val Lys Val Ser Gys 20 ATO GAG TOO OTO COA Met Gin Trp Val Arg AAG OCA TOT Lys Ala Ser CG 0CC COT Gin Ala Pro

OGA

Gly

OGA

Gly TAO TOO Tyr Trp TAG AGO TTC Tyr Thr Phe CAA 000 OTT Gin Oly Leu AGO TAO AOT Arg Tyr Ser 35 40

GT

Oly 48 96 144 192 240 288 336 384 ATO OGA Met Oly TTO AAO Phe Lys

TOT

Ser

ATT

Ile TTT OCT OGA OAT Phe Pro Oly Asp 55 OAT ACT AsD Thr 000 AAO 000 ACA TTO Oly Lys Ala Thr Leu 70 CAA OTO AGO ATO TTO Gin Leu Ser Ile Leu

ACT

Thr

GA

Ala GA OAT AAA TOO TOO AOT Ala Asp Lys Ser Ser Ser TTT GAG GAO TOT 000 OTO Phe Glu AsD Ser Ala Val 0*5556 etS.

5 0*#o ACA 000 TAO Thr Ala Tyr

ATO

Me t TAO TTT GAO TAO, Tyr Phe Asp Tyr TAT TAO Tyr Tyr TOG 000 Trp Oly 110 SEQ ID

LENGTH

TOT OCA AGA GGA TTA Cys Ala Arg Oly Leu 100 CAA 000 AGO ACT OTO Gin Gly Thr Thr Leu 115 OGA 000 000 Arg Oly Oly

TAO

Ty r 105 0 ACA OTO TOO TOA Thr Val Ser Ser 120 418 NO: 7 7 38 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: CTGOTTCGGO OOAOOTOTGA AGOTTOGAGA ATOGATAG 33 SEQ ID NO: 78 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCAGACACGT CCTCGAGCAC AGCCTACATG GAGCT SEQ ID NO: 79 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGCTCCATGT AGGCTGTGCT CGAGGACGTG TCTGC SEQ ID NO: LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: TGGGTGCGAC AGCGCCCTGG ACAAGG 26 SEQ ID NO: 81 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: CCTTGTCCAG GGCGCTGTCG CACCCA 26 SEQ ID NO: 82 LENGTH: 41 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: TACATGGAGC TGAGCAGCCT GGCATTTGAG GACACGGCCG T 41 SEQ ID NO: 83 LENGTH: 41 34 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACGGCCGTGT CCTCAAATGC CAGGCTGCTC AGCTCCATGT A 41 SEQ ID NO: 84 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AAGTTCAAGG GCAAAGCCAC CCTGAC 26 SEQ ID NO: LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GTCAGGGTGG CTTTGCCCTT GAACTT 26 SEQ ID NO: 86 LENGTH: 23 TYPE: nucleic acid TOPOLOGY: linear S* MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCCTACATGC AGCTGAGCAG CCT 23 SEQ ID NO: 87 LENGTH: 23 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGGCTGCTCA GCTGCATGTA GGC 23 SEQ ID NO: 88 LENGTH: 38 TYPE: nucleic acid TOPOLOGY: linear 35 MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCCTACATGC AGCTGAGCAT CCTGAGATCT GAGGACAC 38 SEQ ID NO: 89 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GATCTCAGGA TGCTCAGCTG CATGTAGGCT GTGCT SEQ ID NO: LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCCTACATGC AGCTGAGCAT CCTGAGATCT GAGGACTCGG CCGTGTATTA SEQ ID NO: 91 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACGGCCGAGT CCTCAGATCT CAGGATGCTC AGCTGCATGT AGGCTGTGCT SEQ ID NO: 92 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GAGCTGAGCA TCCTGAGATC SEQ ID NO: 93 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: S36 GATCTCAGGA TGCTCAGCTC CATGTA 26 SEQ ID NO: 94 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGATCTGAGG ACTCGGCCGT SEQ ID NO: LENGTH: TYPE: nucleic acid S. TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: ACGGCCGAGT CCTCAGATCT SEQ ID NO: 96 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCAGACACGT CCACGAGCAC AGCCTACATG GAGCT SEQ ID NO: 97 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGCTCCATGT AGGCTGTGCT CGTGGACGTG TCTGC SEQ ID NO: 98 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GCAGACACGT CCTCGAGCAC AGTCTACATG GAGCT SEQ ID NO: 99 37 LENGTH: TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGCTCCATGT AGACTGTGCT CGAGGACGTG TCTGC SEQ ID NO: 100 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: AGAGTCACCA TCACCGCAGA CAAGTC SEQ ID NO: 101 LENGTH: 26 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA SEQUENCE DESCRIPTION: GACTTGTCTG CGGTGATGGT GACTCT SEQ ID NO: 102 LENGTH: 418 TYPE: nucleic acid TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: ATG GAC TGG ACC TGG AGG GTC Met Asp Trp Thr Trp Arg Val GCT CAC TCC CAG GTG CAG CTG Ala His Ser Gin Val Gin Leu TTC TTC TTG CTG GCT GTA Phe Phe Leu Leu Ala Val -10 GTG CAG TCT GGG GCT GAG Val Gin Ser Gly Ala Glu GCT CCA GGT Ala Pro Gly GTG AAG AAG Val Lys Lys -1 1 5 CCT GGG GCC TCA GTG AAG GTT TCC TGC AAG GCA TCT Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 20 GGA TAC ACC TTC Gly Tyr Thr Phe 144 ACT CCC TAG TGG ATG GAG TGG GTG CGA GAG GGG CCT GGA CAA GGG CTT Ala Pro Gly Gin Gly Leu 192 Th r

GAG

G iu

GAG

Gin

ACA

Th r Pro Tyr Trp Met Gin Trp Val Arg Gin 35 TGG ATG GGA TGT ATT Trp Met Gly Ser Ile AAG TTG AAG GGG AGA Lys Phe Lys Gly Arg TTT GCT GGA GAT GGT Gly GAT ACT AGG Asp Thr Arg Phe Pro Gly GTG ACC ATG Val Thr Ile 70 AGG AGC CTG Ser Ser Leu Asp 55

ACC

Thr

AGA

Arg CCC TAG Ala Tyr 80 TAG TGT Tyr Gys

ATG

Met GGA GAC MAG TC Ala Asp Lys Ser TCT GAG GAG ACG Ser Giu Asp Thr TAG AGT Tyr Ser AGG AGG Thr Ser GGG GTC Aia Val.

GAG GTG Giu Leu 240 288 336 384 85

TAT

Ty r

TGG

Trp 110 95

GGG

Gly CG ACA GGA TTA Ala Arg Gly Leu 100 GGG AGC AGG GTG Cly Thr Thr Val GGA CGA GGG GGG TAG Arg Arg Gly Giy Tyr 105 ACC GTG TGG TGA G Thr Vai Ser Ser 120

TAG

Tyr Phe Asp Tyr TTT GAG TAG

CAA

Gin 418 SEQ ID NO: 103 LENGTH: 1013 TYPE: nucleic acid STRA1NDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: cDNA SEQUENCE DESCRIPTION: GAATTGGGA GCAGCATGT CC ATG CA TGT ACT TG Met Ala Ser Thr Ser TAT GAG TAT TG Tyr Asp Tyr Gys 1 AGA CTC CCC ATC CAA GAG CCC CAT AAC CGG TCT AAGCGTT CGCG CCC Arg Val Pro Met Clu Asp Cly Asp Lys Arg Gys Lys Leu Leu Leu Cly 15 20 ATA CCA ATT CTC CTGCGTG CTC ATG ATCGCTC ATT CG CCC CTC CCC TTC Ile Cly Ile Leu Val Leu Leu Ile Ile Val Ile Leu Cly Val Pro Leu 35 ATT ATC TTG ACC ATC AAC CCC MGC AC GAG CCC TCC CGC GACGGCC CTT Ile Ile Phe Thr Ile Lys Ala Asn Ser.Giu Ala Cys Ara Asp Cly Leu 50 49 97 145 193 CGG GCA GTG ATG GAG TGT CGC AAT GTC ACC CAT CTC CTG CAA CAA GAG Arg Ala Vai Met Giu Cys Arg Asn Val Thr His Leu Leu Gin Gin Giu CTG ACC Leu Thr ACC TGC Thr Cys

GAG

Glu GCC GAG Ala Gin AAG GGC Lys Gly 80 TTT GAG GAT' GTG GAG GC Phe Gin Asp Val Giu Ala CCC OTA ATG GOT TOO OTG Ala Leu Met Ala Ser Leu GAG GCO Gin Ala GAT GCA Asp Ala

GC

Ala

GAG

Giu 105

AAG

Ly s MAC GAG ACT GTG Asn His Thr Val 95 CAA GGA CAA AAG Gin Gly Gin Lys 100

OTT

Leu

ATG

Met

GC

Ala AAA GTG Lys Val a a a e..

GAG GAG Giu Giu 115 GAG GGA GAG ATO ACT Glu Gly Glu Ile Thr 120 110 289 337 385 433 481 529 ACA TTA AAC CAT Thr Leu Asn His 125 AGA ACA GMA AC Arg Arg Giu Asn 140 TAO CCC AGO TOO Tyr Pro Ser Ser

MAG

Ly s OTT GAG GAO GOG TOT Leu Gin Asp Ala Ser 130 GCA GAG GTG GAG CGA CTG Ala Giu Val Glu Arg Leu 135 GAG GTO TTA AGO Gin Val Leu Ser 145 CAG GAG TOO AGO Gin Asp Ser Ser 160 OTO AGA ATO CG GAO AAG MAG TAO Val Arg Ile Ala Asp Lys Lys Tyr 150 TOO GOT 000 CG CCC GAG OTG OTO Ser Ala.Ala Ala Pro Gin Leu Leu 155 ATT GTG Ile Val 170 AGO TGOOA

TOATOAGT

GAGMOG

AGTCGCCT

TOTTGTOT

TOT TATGC OTO OTO GOC OTO Leu Leu Gly Leu 175 CA TOTTGGAACG TC TO TOAGOGGOTO AT CO TOTOGAGOAG 01 TO ACOCACOGOT 01 C0 CACOOTOACA TT OT TTTTTTTGC CC AGO GOT Ser Ala OTO CTG CAG Leu Leu Gin 180 165 TCA GATCOAGGA 575 G TOOT GO

GGGGCMAC

'OTGGAGGG

OTOOCTCO

'GGGCATGG

GGGGGTTG

TCGGOTTTTO

ACGGTTAGOG

GCCATOGGOC

AGAGCTOCO

CO TOO 0TOT

CTTTTTTCTG

GOTTGAAOAT

GGGAGAGOAC

AGTOOTGGGT

TOCGAOMT

CGGGOCATO

GGGTCTTTGA

TCOTTOATO

GGOTAGOOG

OTGOGACAC

GAO TOCC T GOTGC CTO T

GCTCCAAAMA

635 695 755 815 875 935

AATAAACAOT

AAAA T T C G TOO TTTGAGG

CGGCOGC

GAGAGOACAC CTTAAAAAAA AAAAAAAMAA AAAAAAAAAA 995 1013

Claims (9)

1. An antibody which binds to a polypeptide having the amino acid sequence shown in SEQ ID NO:103, and has an ADCC activity.

2. The antibody according to Claim 1 wherein the antibody is a monoclonal antibody.

3. The antibody according to Claim 2, wherein the monoclonal antibody is a chimeric antibody comprising the c region of a human antibody.

4. The antibody according to Claim 2, wherein the monoclonal antibody is a humanized antibody.

5. The antibody according to Claim 4, wherein the humanized antibody is a humanized anti-HM 1.24 antibody.

6. A pharmaceutical composition comprising an antibody according to any one of Claims 1 to

7. The pharmaceutical composition according to Claim 6, useful for treatment of myeloma.

8. The pharmaceutical composition according to Claim 7, wherein the myeloma is multiple myeloma.

9. An antibody according to any one of Claims 1 to or a pharmaceutical composition according to any one of Claims 6 to 8 substantially as hereinbefore defined with reference to the Figures and/or Examples. P:\Opa\Vpa\2338023.ch.ugai.div~cims.306.doc-01/1 1100 -105- DATED this first day of November 2000. Chugai Seiyaku Kabushiki Kaisha by DAVIES COLLISION CAVE Patent Attorneys for the Applicant