Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Reshaped human anti-HM 1.24 antibody

United States Patent:  6,699,974

Issued:  March 2, 2004

Inventors:  Ono; Koichiro (Gotenba, JP); Ohtomo; Toshihiko (Gotenba, JP); Tsuchiya; Masayuki (Gotenba, JP); Yoshimura; Yasushi (Gotenba, JP); Koishihara; Yasuo (Gotenba, JP); Kosaka; Masaaki (Tokushima, JP)

Assignee:  Chugai Seiyaku Kabushiki Kaisha (Tokyo, JP)

Appl. No.:  269921

Filed:  April 1, 1999

PCT Filed:  October 3, 1997

PCT NO:  PCT/JP97/03553

371 Date:  April 1, 1999

102(e) Date:  April 1, 1999

PCT PUB.NO :  WO98/14580

PCT PUB. Date:  April 9, 1998

Abstract

A reshaped human anti-HM 1.24 antibody comprising: (A) an L chain comprising (1) the C region of a human L chain, and (2) the V region of an L chain comprising the FR of a human L chain and the CDR of the L chain of a mouse anti-HM 1.24 monoclonal antibody; and (B) an H chain comprising (1) the C region of a human H chain, and (2) the V region of an H chain comprising the FR of a human H chain and the CDR of the H chain of a mouse anti-HM 1.24 monoclonal antibody. Since most of this reshaped human antibody is derived from human antibody and the CDR has a low antigenicity, the reshaped human antibody of the present invention has a low antigenicity and, therefore, is expected to be-used for medical treatment.

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, the present invention provides chimeric antibodies and reshaped antibodies that specifically recognize a polypeptide having the amino 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 Oct. 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 DH5.alpha. (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 will be given hereinbelow.

Thus, the present invention also provides chimeric L chains comprising the constant region (C region) of a human light (L) chain and the variable (V) 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:

(1) 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

(2) an 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 the V region of the reshaped human L chain of anti-HM 1.24 antibody comprising:

(1) the framework region (FR) of the V region of a human L chain, and

(2) 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

(1) the FR of the V region of a human H chain, and

(2) the CDR of the V region of the H chain of an anti-HM 1.24 antibody.

The present invention further provides the reshaped human L chain of anti-HM 1.24 antibody comprising

(1) the C region of a human L chain, and

(2) 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

(1) the C region of a human H chains and

(2) 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:

(A) an L chain comprising

(1) the C region of a human L chain, and

(2) 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

(B) an H chain comprising

(1) the C region of a human H chain, and

(2) 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 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 anti-HM 1.24 antibody.

The present invention further provides

DNA encoding a chimeric L chain comprising

(1) the C region of a human L chain; and

(2) the V region of the L chain of an anti-HM 1.24 antibody, and

DNA encoding a chimeric H chain comprising

(1) the C region of a human H chain; and

(2) 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:

(1) the FR of the V region of a human L chain; and

(2) 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:

(1) the FR of the V region of a human H chain; and

(2) 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:

(1) the C region of a human L chain; and

(2) 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:

(1) the C region of a human H chain; and

(2) 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 a vector comprising any of the various DNAs mentioned above.

The present invention further provides a host cell 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 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: 129, 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: 129. As a pharmaceutical composition, there is specifically provided a therapeutic agent for myeloma.

MODE FOR CARRYING OUT THE INVENTION

1. Construction of a Chimeric Antibody

(1) Cloning of DNA Encoding the V Region of a 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 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 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: 40).

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, 85, 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 primer (SEQ ID NO: 77) and the constant region (C.gamma. 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 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 (J. Sambrook, et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989). 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) (J. 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 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 Services, 1983).

Many portions of the above four FRs take the .beta.-sheet structure with a result that three CDRs form loops. CDRs may sometimes form part of the .beta.-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.

(2) 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 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.

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, C.gamma.1, C.gamma.2, C.gamma.3, or C.gamma.4 of a human H chain, or C.lambda. or C.kappa. of an L chain.

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 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, C.gamma.1, C.gamma.2, C.gamma.3, or C.gamma.4.

Construction of an Expression Vector for a Chimeric H Chain Containing C.gamma.1 Genomic DNA

As an expression vector having genomic DNA for C.gamma.1 as the C region of an H chain, there can be used, for example HFE-PMh-g.gamma.1 (International Application Publication No. WO 92/19759) or DHFR-.DELTA.E-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 expression vector, and a chimeric H chain-expression vector comprising the C.gamma.1 DNA can be constructed.

Construction of an Expression Vector for the cDNA Chimeric H Chain

An expression vector having the cDNA of C.gamma.1 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-.DELTA.E-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 C.gamma.1 of the H chain of a human antibody (N. Takahashi, et al., Cell 29, 671-679, 1982) and the expression vector RV1-PM1a (International Application Publication No. WO 92/19759) encoding genomic DNA of the V region of the L chain of a humanized PM1 antibody and the C region of the .kappa. 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 C.gamma.1 of the H 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 C.gamma.1 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 C.gamma.1 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 with a suitable restriction enzyme, ligating to the above-mentioned cDNA containing the C region C.gamma.1 of the H chain, and inserting to an expression vector such as pCOS1 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 .kappa. chain or a .lambda. chain.

Construction of an Expression Vector for the .kappa. 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 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 .kappa. 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-PM1k-gk (see International Application Publication No. WO 92/19759) containing genomic DNA. An expression vector for the .kappa. 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 .kappa. chain C region of L chain by the PCR method, ligating the thus constructed V region of the mouse L chain to the .kappa. chain C region of L chain, and then inserting into an expression vector such as pCOS1 or pCHO1.

2. Construction of a Reshaped Human Antibody

(1) 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 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.

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 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 a reshaped human PM-1 antibody 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 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%.

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 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.

(2) 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-g.kappa..

The CDR grafting primers LIS (SEQ ID NO: 49), L2S (SEQ ID NO: 50), and L3S (SEQ ID NO: 51) have a sense DNA sequence. The CDR grafting primers LIA (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 L1S, L2S, and L3S, 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 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-g.kappa.. 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-g.kappa.. The amino acid sequence and the base sequence of the V region of the L chain contained in this plasmid HEF-RVLa-AHM-g.kappa. 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-g.kappa. 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-g.kappa. to obtain plasmid HEF-RVLb-AHM-g.kappa.. The amino acid sequence and the base sequence of the V region of the L chain contained in this plasmid HEF-RVLb-AHM-g.kappa. are shown in SEQ ID NO: 10.

(3) Construction of the V Region of the H Chain of a Reshaped Human Anti-HM 1.24 Antibody

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 5'-end 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 oligonucleotides are subjected to computer analysis for the secondary structure.

The sequences of the four oligonucleotides RVH1 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. 5).

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-g.gamma.1. After determination of 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-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHa-AHM-g.gamma.1 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 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-g.gamma.1 by the PCR method, version b is amplified to obtain plasmid HEF-RVHb-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHb-AHM-g.gamma.1 are shown in SEQ ID NO: 12.

Using as the mutagen primer CS (sequence 65) 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-g.gamma.1 by the PCR method, version c is amplified to obtain plasmid HEF-RVHc-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHc-AHM-g.gamma.1 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-g.gamma.1 by the PCR method, version d is amplified to obtain plasmid HEF-RVHd-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHd-AHM-g.gamma.1 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-g.gamma.1, version e is amplified to obtain plasmid HEF-RVHe-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHe-AHM-g.gamma.1 are shown in SEQ ID NO: 15.

3-2. Construction of the H Chain Hybrid V Region

By constructing a H chain hybrid V region, it is possible to investigate which FR of the V region of a humanized antibody contributes to the binding activity 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 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 FIGS. 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-g.gamma.1. The H chain hybrid construction primer HYS (SEQ ID NO: 73) is designed to have the sense 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-g.gamma.1 as a template, the external primer a, and the H chain hybrid primer HYA, and PCR using the plasmid HEF-RVHa-AHM-g.gamma.1 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.

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 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-g.gamma.1 as a template, the external primer a, and the H chain hybrid primer HYA, and PCR using the plasmid HEF-1.24H-g.gamma.1 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 (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-g.gamma.1 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. 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 region of the H chain of a reshaped human anti-HM 1.24 antibody was termed HEF-MH-RVH-AHM-g.gamma.1.

The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-MH-RVH-AHM-g.gamma.1 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-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-HM-RVH-AHM-g.gamma.1 are shown in SEQ ID NO: 76.

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, l, 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-g.gamma.1 by the PCR method, version f is amplified to obtain plasmid HEF-RVHf-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHf-AHM-g.gamma.1 are shown in SEQ ID NO: 16.

Using as the mutagen primer GS (sequence 80) 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-g.gamma.1, version g is amplified to obtain plasmid HEF-RVHg-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHg-AHM-g.gamma.1 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-g.gamma.1, version h is amplified to obtain the plasmid HEF-RVHh-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHh-AHM-g.gamma.1 are shown in SEQ ID NO: 18.

Using as the mutagen primer IS (sequence 82) 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-g.gamma.1, version i is amplified to obtain plasmid HEF-RVHi-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHi-AHM-g.gamma.1 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-g.gamma.1, version j is amplified to obtain plasmid HEF-RVHj-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHj-AHM-g.gamma.1 are shown in SEQ ID NO: 20.

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-g.gamma.1, version k is amplified to obtain plasmid HEF-RVHk-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHk-AHM-g.gamma.1 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-g.gamma.1, version 1 is amplified to obtain plasmid HEF-RVHl-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHl-AHM-g.gamma.1 are shown in SEQ ID NO: 22.

Using as the mutagen primer MS (SEQ ID NO: 90) 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 a template DNA the plasmid HEF-RVHh-AHM-g.gamma.1, version m is amplified to obtain plasmid HEF-RVHm-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHm-AHM-g.gamma.1 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-g.gamma.1, version n is amplified to obtain plasmid HEF-RVHh-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHn-AHM-g.gamma.1 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-g.gamma.1, version o is amplified to obtain plasmid HEF-RVHo-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHo-AHM-g.gamma.1 are shown in SEQ ID NO: 25.

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-g.gamma.1, version p is amplified by the PCR method to obtain plasmid HEF-RVHp-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHp-AHM-g.gamma.1 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-g.gamma.1, version q is amplified by the PCR method to obtain plasmid HEF-RVHq-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHq-AHM-g.gamma.1 are shown in SEQ ID NO: 27.

Using as the mutagen primer CS (SEQ ID NO: 65) and CA (SEQ ID NO: 66) and as a template DNA the plasmid HEF-RVHp-AHM-g.gamma.1, version r is amplified by the PCR method to obtain plasmid HEF-RVHr-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHr-AHM-g.gamma.1 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-g.gamma.1, version s is amplified to obtain plasmid HEF-RVHs-AHM-g.gamma.1. The amino acid sequence and the base sequence of the V region of the H chain contained in this plasmid HEF-RVHs-AHM-g.gamma.1 are shown in 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.

                                       TABLE 1
                The amino acid sequence of the V region of the L chain
                                             FRI                     CDR1
                                                    1         2          3
                                           12345678901234S67890123 45678901234
    AHM     (a region of SEQ ID NO: 1)     DIVMTQSHKFMSTSVGDRVSITC KASQDVNTAVA
              FR2               CDR2       FR3
                 4             5              6         7         8
            567890123456789    0123456     78901234567890123456789012345678
    AHM     WYQQKPGQSPKLLIY    SASNRYT     GVPDRITGSGSGTDFTFTISSVQAEDLALYYC
              CDR3          FR4
             9             10
            901234567     8901234567
    AHM     QQHYSTPFT     FGSGTKLEIK
                                             FRI                     CDR1
                                                    1         2          3
                                           12345678901234567890123 45678901234
    HuAGI   (SEQ ID NO: 130)               DIQMTQSPSSLSASVGDRVTITC
              FR2              CDR2          FR3
                 4            5               6         7         8
            567890123456789   0123456      78901234567890123456789012345678
    HuAGI   WYQQKPGKAPKLLIY                GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
              CDR3         FR4
             9            10
            901234567    8901234567
    HuAGI                FGQGTKVEIK
                                             FRI                     CDR1
                                                    1         2          3
                                           12345678901234567890123 45678901234
    REI     (SEQ ID NO: 131)               DIQMTQSPSSLSASVGDRVTITC
              FR2             CDR2           FR3
                 4           5                6         7         8
            567890123456789  0123456       78901234567890123456789012345678
    REI     MWYQQKPGKAPKLLIY               GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC
              CDR3         FR4
             9            10
            901234567    8901234567
    HuAGI                FGQGTKVEIK
                                             FR1                     CDR1
                                                    1         2          3
                                           12345678901234567890123
    45678901234
    RVLa    (a region of SEQ ID NO: 9      ------------------- -----------
               and SEQ ID NO: 106)
              FR2             CDR2           FR3
                 4           5                6         7         8
            567890123456789  0123456       78901234567890123456789012345678
    RVLa    ---------------  -------       --------------------------------
              CDR3        FR4
             9           10
            901234567   8901234567
    RVLa    ---------   ----------
                                             FRI                     CDR1
                                                    1         2          3
                                           12345678901234567890123 45678901234
    RVLb    (a region of SEQ ID NO:        ----------------------- -----------
    10)
              FR2             CDR2           FR3
                 4           5                6         7         8
            567890123456789  0123456       78901234567890123456789012345678
    RVLb    ---------------  -------       -------------Y------------------
              CDR3        FR4
             9           10
            901234567   8901234567
    RVLb    ---------   ----------
                                   TABLE 2
             The amino acid sequence of the V region of the H chain (1)
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    AHM     (a region of SEQ ID NO: 3)         QVQLQQSGAELARPGASVKLSCKASGYTFT
              CDR1           FR2
                               4
            12345          67890123456789
    AHM     PYWMQ          WVKQRPGQGLEWIG
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    HuSGI   (SEQ ID NO: 132)                   EVQLVQSGADVKKPGXSVXVSCKASGYTFS
              CDR1        FR2
                            4
            12345       67890123456789
    HuSGI               WVRQAPGXGLDWVG
                                                FRI
                                                        1         2         3
                                               123456789012345678901234567890
    HG3     (SEQ ID NO: 133)                   QVQLVQSGAEVKKPGASVKVSCKASGYTFN
               CDR1       FR2
                            4
            12345       67890123456789
    HG3                 WVRQAPGQGLEWMG
                                                FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHa    (a region of SEQ ID NO: 11)        -----------------------------T
              CDR1         FR2
                             4
            12345        67890123456789
    RVHa    -----        --------------
                                               FRI
                                                        1         2         3
            12345678901234567890123456789T
    RVHb    (a region of SEQ ID NO: 12)        ------------------------------
              CDR1          FR2
                              4
            12345         67890123456789
    RVHb    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHC    (a region of SEQ ID NO: 13)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHc    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHd    (a region of SEQ ID NO: 14)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHd    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHe    (a region of SEQ ID NO: 15)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHe    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHf    (a region of SEQ ID NO: 16)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHf    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHg    (a region of SEQ ID NO: 17)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHg    -----         ----R---------
                                               FRI
                                                        1        2          3
                                               123456789012345678901234567890
    RVHh    (a region of SEQ ID NO: 17)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHh    ----------------------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHi    (a region of SEQ ID NO: 19)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHi    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHi    (a region of SEQ ID NO: 20)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHi    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHk    (a region of SEQ ID NO: 21)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHk    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234S67890
    RVHl    (a region of SEQ ID NO: 22)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHl    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHm    (a region of SEQ ID NO: 23)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHm    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHn    (a region of SEQ ID NO: 24)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHn    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234S67890
    RVHo    (a region of SEQ ID NO: 25)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHo    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHp    (a region of SEQ ID NO: 26)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHp    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHq    (a region of SEQ ID NO: 27)        -----------------------------T
              CDR1          FR2
                              4
            12345         67890123456789
    RVHq    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHr    (a region of SEQ ID NO: 28         -----------------------------T
              and SEQ ID NO: 125)
              CDR1          FR2
                              4
            12345         67890123456789
    RVHr    -----         --------------
                                               FRI
                                                        1         2         3
                                               123456789012345678901234567890
    RVHs    (a region of SEQ ID NO: 102        -----------------------------T
              and SEQ ID NO: 128)
              CDR1          FR2
                              4
            12345         67890123456789
    RVHs    -----         --------------
                           TABLE 3
        The amino acid sequence of the V region of the H chain
                                          CDR2
                                        5          6
                                        012A3456789012345
    AHM       (a region of SEQ ID NO:3) SIFPGDGDTRYSQKFKG
                FR3
                  7         8            9
    67890123456789012ABC345678901234
    AHM       KATLTADKSSSTAYMQLSILAFEDSAVYYCAR
                                          CDR2
                                        5         6
                                        012A3456789012345
    HuSGI     (SEQ ID NO:134)
                FR3
                  7         8            9
              67890123456789012ABC345678901234
    HuSGI     RVTXTXDXSXNTAYMELSSLRSEDTAVYYCAR
                                          CDR2
                                        5         6
                                        012A3456789012345
    HG3       (SEQ ID NO:135)
                FR3
                  7
              67890123456789012ABC345678901234
    HG3       RVTMTRDTSTSTYYMELSSLRSEDTAVYYCAR
                                          CDR2
                                        5         6
                                        012A3456789012345
    RVHa      (a region of SEQ ID NO:11)-----------------
                FR3
                  7
    67890123456789012ABC345678901234
    RVHa      -----A--------------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHb      (a region of SEQ ID NO:12)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHb      K----A--------------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHC      (a region of SEQ ID NO:13)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHC      -----A-K------------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHd      (a region of SEQ ID NO:14)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHd      K----A-K------------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHe      (a region of SEQ ID NO:15)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHe      -A-L-A--------------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHf      (a region of SEQ ID NO:16)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHf      -A-L-A---S--A-------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHq      (a region of SEQ ID NO:17)-----------------
                FR3
                  7
              678901234S6789012ABC345678901234
    RVHg      -----A--------------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHh      (a region of SEQ ID NO:18)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHh      K----A---S--A-------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHi      (a region of SEQ ID NO:19)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHi      K----A---S--A-------AF----------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHj      (a region of SEQ ID NO:20)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHj      KA-L-A---S--A-------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHk      (a region of SEQ ID NO:21)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHk      K----A---S--A--Q----------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHl      (a region of SEQ ID NO:22)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHl      K----A---S--A--Q--I-------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHm      (a region of SEQ ID NO:23)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHm      K----A---S--A--Q--I-----S-------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHn      (a region of SEQ ID NO:24)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHn      K----A---S--A-------I-----------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHo      (a region of SEQ ID NO:25)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHo      K----A---S--A-----------S-------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHP      (a region of SEQ ID NO:26)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHp      -----A------A-------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHq      (a region of SEQ ID NO:27)-----------------
                FR3
                  7
              67890123456789012ABC345678901234
    RVHq      -----A---S---------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHr      (a region of SEQ ID NO:28 -----------------
                and SEQ ID NO: 125)
                FR3
                  7
              67890123456789012ABC345678901234
    RVHr      -----A-K----A-------------------
                                          CDR2
                                        5        6
                                        012A3456789012345
    RVHs      (a region of SEQ ID NO:102------------------
                and SEQ ID NO: 128)
                FR3
                  7
              67890123456789012ABC345678901234
    RVHs      ---I-A-K----A-------------------
                               TABLE 4
                The amino acid sequence of the V region of the
                                 H chain (3)
                                                CDR3            FR4
                                                  10                 11
                                               57890ABJK12     34567890123
    AHM     (a region of SEQ ID NO: 3)         GLRRGGYYFDY     WGQGTTLYVSS
                                                CDR3            FR4
                                                  10                 11
                                               57890ABJK12     34567890123
    HuSGI   (SEQ ID NO: 136)                                   WGQGTLVTVSS
                                                CDR3            FR4
                                                  10                 11
                                               57890ABJK12     34567890123
    JH6     (SEQ ID NO: 137)                                   WGQGTTVTVSS
                                                CDR3            FR4
                                                  10                 11
                                               57890ABJK12     34567890123
    RVHa    (a region of SEQ ID NO: 11)        -----------     -----------
                                                CDR3            FR4
                                                  10                 11
                                               57890ABJK12     34567890123
    RVHb    (a region of SEQ ID NO: 12)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHc    (a region of SEQ ID NO: 13)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHd    (a region of SEQ ID NO: 14)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHe    (a region of SEQ ID NO: 15)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               S7890ABJK12     34567890123
    RVHf    (a reqion of SEQ ID NO: 16)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHg    (a region of SEQ ID NO: 17)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHh    (a region of SEQ ID NO: 18)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHi    (a region of SEQ ID NO: 19)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHj    (a region of SEQ ID NO: 20)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34S67890123
    RVHk    (a region of SEQ ID NO: 21)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVH1    (a region of SEQ ID NO: 22)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHm    (a region of SEQ ID NO: 23)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHn    (a region of SEQ ID NO: 24)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHo    (a region of SEQ ID NO: 25)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHp    (a region of SEQ ID NO: 26)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHq    (a region of SEQ ID NO: 27)        -----------     -----------
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHr    (a region of SEQ ID NO: 28         -----------     -----------
                 and SEQ ID NO: 128)
                                                CDR3            FR4
                                                 10                  11
                                               57890ABJK12     34567890123
    RVHs    (a region of SEQ ID NO: 102        -----------     -----------
                 and SEQ ID NO: 128)


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 the enhancer/promoter system, or an expression vector comprising DNA encoding the V region of a humanized 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 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.

Subsequently, a host cell such as the mammalian cell is cotransformed using these vectors, and the transformed cells are cultured in vitro or in vivo to produce a chimeric antibody or a reshaped human antibody (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 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. WO 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 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 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-HC.gamma.1, HCMV-VL-HCk, etc. and which are derived from pSV2neo (International Application Publication No. WO 92-19759).

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 1.alpha. (HEF-1.alpha.), 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-1.alpha. 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

(1) Measurement of Antibody Concentration

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 .mu.l of goat anti-human IgG antibody at a concentration of 1 .mu.g/ml.

After blocking with 100 .mu.g/ml of a dilution buffer (for example 50 mM Tris-HCl, 1 mM MgCl2, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA), pH 8.1), 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 cels, or the purified chimeric antibody, hybrid antibody, or reshaped human antibody is added to each well. Then 100 .mu.l of alkaline phosphatase conjugated goat anti-human IgG antibody is added, 1 mg/ml of the substrate solution (Sigma104, p-nitrophenyl phosphate, manufactured by 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 IgG1.kappa. (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.

(2) 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 PBS(-), are fixed with 0.1% glutaraldehyde (manufactured by Nakalai tesque).

After blocking, 100 .mu.l 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, 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 .mu.l of 6N sulfuric acid, and then absorbance at 490 nm is measured using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad).

(3) Measurement of Binding Inhibition Activity

Binding inhibition activity by the biotinylated mouse anti-HM 1.24 antibody is measured by the Cell-ELISA using the human amniotic cell line WISH (ATCC CCL25). The Cell-ELISA plate may be prepared according to the above-mentioned (2). 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 PBS(-), are fixed with 0.1% glutaraldehyde (manufactured by Nakalai tesque).

After blocking, 50 .mu.l 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 .mu.l of 2 .mu.g/ml biotinylated mouse anti-HM 1.24 antibody is added, and then incubated at 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 .mu.l 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 51 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 (A-C)/(B-C)x100, 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 C.gamma.1 or human C.gamma.3 can 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 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 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 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, 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 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, lactose, pharmaceutically acceptable surfactants, and the like. Additives to be used may be selected from, but not limited to, the above or combinations thereof.

Claim 1 of 9 Claims

What is claimed is:

1. A light chain variable region of a humanized antibody, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:106, and wherein the humanized antibody having the light chain variable region binds to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 129 (HM1.24).




____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]