Humanized antibodies to ganglioside GM.sub.2

Chimeric human antibody expression vectors are constructed by inserting the antibody heavy chain variable region-encoding cDNA and antibody light chain variable region-encoding cDNA isolated from hybridomas producing a mouse or rat monoclonal antibody reacting with the ganglioside GM.sub.2 respectively into an expression vector for use in animal cells which contains the human antibody heavy chain constant region- or human antibody light chain constant region-encoding cDNA. The expression vectors are introduced into animal cells and the transformant thus obtained is cultured for the production of a chimeric human antibody reacting with the ganglioside GM.sub.2. In contrast to mouse monoclonal antibodies, the chimeric human antibodies of the invention will not cause anti-mouse immunoglobulin antibody production in the patient's body but shows a prolonged blood half-life, with a reduced frequency of adverse effects, so that it can be expected to be superior to mouse monoclonal antibodies in the efficacy in the treatment of human cancer, for instance.

FIELD OF THE INVENTION 
The present invention relates to humanized antibodies reacting with the 
ganglioside GM.sub.2. The humanized antibodies do not cause production of 
anti-mouse immunoglobulins in the patient's body as compared with mouse 
monoclonal antibodies, hence the incidence of adverse effects possibly 
caused by them is much lower, their blood half-lives are longer and, 
further, their anti-tumor effector effect is greater. Therefore, the 
humanized antibodies are expected to produce improved therapeutic effects 
as compared with mouse monoclonal antibodies. 
BACKGROUND OF THE INVENTION 
When administered to humans, mouse antibodies are generally recognized as 
foreign matters, inducing production of anti-mouse immunoglobulin 
antibodies in the human body. It is known that the former antibodies react 
with the latter antibodies to produce adverse effects J. Clin. Oncol., 2, 
881 (1984); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80, 932 (1988); 
Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)! and that the mouse 
antibodies undergo rapid clearance J. Nucl. Med., 26, 1011 (1985); Blood, 
65, 1349 (1985); J. Natl. Cancer Inst., 80, 937 (1988)!, thus showing only 
a reduced efficacy J. Immunol., 135, 1530 (1985); Cancer Res., 46, 6489 
(1986)!. Attempts have been made to solve these problems by deriving, from 
mouse monoclonal antibodies, chimeric human antibodies or CDR 
(complementarity determining region)-transplanted antibodies (reshaped 
antibodies) using gene engineering technique. In a human chimeric 
antibody, the variable regions thereof are of mouse origin and the 
constant regions thereof are of human origin Proc. Natl. Acad. Sci. 
U.S.A., 81, 6851 (1984)! and it is reported that when administered to 
humans, said antibody causes litte human anti-mouse immunoglobulin 
antibody production, its blood half-life being 6-fold longer Proc. Natl. 
Acad. Sci. U.S.A., 86, 4220 (1989)!. The CDR-transplanted antibodies are 
antibodies resulting from replacement of the CDRs in a human antibody 
alone with the CDRs from an animal other than the human Nature, 321, 522 
(1986)! and, in an experiment with monkeys, such antibodies showed reduced 
immunogenicity and 4- to 5-fold higher serum half-lives as compared with 
mouse antibodies J. Immunol., 147, 1352 (1991)!. 
As regards the cytocidal activity of antibodies, it is reported that the Fc 
region of a human antibody is more potent in activating human complement 
and human effector cells than the Fc region of a mouse antibody. Thus, for 
instance, a chimeric antibody derived from a mouse monoclonal antibody to 
the ganglioside GD.sub.2 and containing a human antibody Fc region 
enhances the human effector cell-mediated antitumor effect J. Immunol., 
144, 1382 (1990)!. Similar results are reported for CDR-transplanted 
antibodies Nature, 332, 323 (1988)!. Such results indicate that, for 
clinical use, humanized monoclonal antibodies are preferred to mouse 
monoclonal antibodies. 
The antibody classes include IgA, IgM, IgG, IgD and IgE and, in mice, the 
class IgG includes four subclasses, namely IgG.sub.1, IgG.sub.2 a, 
IgG.sub.2 b and IgG.sub.3 (in humans, IgG.sub.1, IgG.sub.2, IgG.sub.3 and 
IgG.sub.4). When antigens are administered to animals, the antibodies 
produced mostly belong to the classes IgM or IgG. IgG molecules have a 
molecular weight of about 160,000 daltons and a dimeric structure and are 
relatively easy to handle. IgM molecules are large with a molecular weight 
of about 900,000 daltons and occur in the form of a complicated pentameric 
structure coupled with the joining (J) chain, hence they have the 
following drawbacks: they are difficult to purify; they tend to 
agglutinate, hence are difficult to store; they are readily inactivated by 
partial decomposition in the presence of a protease, hence it is difficult 
to prepare Fab fragments; and they lose their binding activity in many 
instances upon chemical modification, for example chemical binding of an 
anticancer agent or a toxin J. W. Goding: Monoclonal Antibodies: 
Principles and Practice, Academic Press, 1986!. As to which are superior 
in therapeutic effect against cancer, IgG class monoclonal antibodies or 
IgM class monoclonal antibodies, reference may be made to a detailed study 
made by Bernstein et al. using an IgG class monoclonal antibody and an IgM 
class monoclonal antibody to the lymphocyte Thy-1 antigen Monoclonal 
Antibodies, edited by R. H. Kennet, T. J. Mckearn and K. B. Bechtol, 
Plenum Press, 1980, p. 275!. According to the reference, an IgG class 
monoclonal antibody and an IgM class monoclonal antibody comparable in 
terms of reactivity to Thy-1 antigen-positive lymphocytes, were compared 
in terms of antitumor effect. While the IgM monoclonal antibody was 
superior in in vitro complement-dependent antitumor effect, the IgG class 
monoclonal antibody showed a significant antitumor effect in in vivo 
antitumor effect in cancer-bearing mice, with no antitumor effect being 
observed with the IgM class monoclonal antibody. It was further revealed 
that, as compared with the IgG class monoclonal antibody, the IgM class 
monoclonal antibody showed a very short half-life in the blood after 
administration, in an isotope-labeled form, to mice. These results 
indicate that the monoclonal antibodies to be used clinically in humans 
should preferably be of the IgG class. 
Gangliosides, a class of glycolipids, are constituents of animal cell 
membranes. These molecules are composed of a carbohydrate chain, which 
constitutes a hydrophilic side chain, and sphingosine and a fatty acid, 
which constitute hydrophobic side chains. It is known that the ganglioside 
species expressed and the amount thereof differ between cell species, 
organ species, and animal species, among others. Furthermore, it has been 
reported that the ganglioside expressed changed quantitatively and 
qualitatively during the process of cancer development Cancer Res., 45, 
2405 (1985)!. For example, expression of the gangliosides GD.sub.2, 
GD.sub.3 and GM.sub.2 has been reported in neuroblastoma, lung small cell 
carcinoma, and melanoma, which are highly malignant neural ectodermal 
tumors J. Exp. Med., 155, 1133 (1982); J. Biol. Chem., 257, 12752 (1982); 
Cancer Res., 47, 225 (1987); ibid., 47, 1098 (1987); ibid., 45, 2642 
(1985); Proc. Natl. Acad. Sci. U.S.A., 80, 5392 (1983)!. 
GM.sub.2, one of the gangliosides that are sialic acid residue containing 
glycolipids, occurs only in trace amounts in normal cells but is found in 
increased amounts in cancer cells in lung small cell carcinoma, melanoma, 
neuroblastoma, etc. Monoclonal antibodies to GM.sub.2 are considered to be 
useful in the treatment of such cancers Lancet, 4, 786 (1989)!. However, 
those monoclonal antibodies to GM.sub.2 that have so far been reported are 
of the human IgM class or of the rat IgM, mouse IgM or mouse IgG class 
Cancer Res., 46, 4116 (1986); Proc. Natl. Acad. Sci. U.S.A., 79, 7629 
(1982); Cancer Res., 48, 6154 (1988); J. Biol. Chem., 264, 12122 (1989)!. 
Anti-ganglioside GM.sub.2 monoclonal antibodies, if produced in the form of 
humanized antibodies, for example chimeric human antibodies or 
CDR-transplanted antibodies, which are not expected to induce anti-mouse 
immunoglobulin antibody production in the patient's body, produce reduced 
adverse effects and show a prolonged blood half-life and an enhanced 
antitumor effector effect. These antibodies are thus expected to be 
superior in therapeutic effect to the corresponding mouse monoclonal 
antibodies. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide humanized antibodies to 
the ganglioside GM.sub.2 (hereinafter, "humanized anti-GM.sub.2 
antibodies") which are useful in the treatment of cancers of neural 
ectodermal origin, among others. 
The present inventors prepared the antibody heavy chain (hereinafter, "H 
chain") variable region (hereinafter "V.sub.H ") cDNA and light chain 
(hereinafter, "L chain") variable region (hereinafter, "V.sub.L ") cDNAs 
from mRNAs isolated from the hybridomas KM750 and KM796, described in 
EP-A-0 508 472. These hybridomas produce IgG.sub.3 class mouse monoclonal 
antibodies to the ganglioside GM.sub.2. V.sub.H and V.sub.L cDNAs were 
also prepared from mRNAs isolated from the hybridoma KM603, which produces 
an IgM class rat monoclonal antibody to the ganglioside GM.sub.2. Chimeric 
human antibody expression vectors were constructed by inserting the cDNA 
into an expression vector containing human antibody H chain constant 
region (hereinafter, "C.sub.H ") or human antibody L chain constant region 
(hereinafter, "C.sub.L ") encoding sequences. Such vectors were then 
introduced into animal cells to effect the production of anti-ganglioside 
GM.sub.2 chimeric human antibodies. Among the chimeric antibodies 
produced, the anti-ganglioside GM.sub.2 chimeric human antibody, KM966, 
was found to react with the ganglioside GM.sub.2 and show cytocidal 
activity. The H chain variable region of KM966 contains an amino acid 
sequence segment as defined by SEQ ID NO:91 and includes the 1st to 120th 
amino acids of that sequence and the L chain variable region of KM966 
contains an amino acid sequence segment as defined by SEQ ID NO:92 and 
includes the 1st to 107th amino acids of said sequence. The present 
invention is based, at least in part, on these findings. 
The present invention thus relates to a humanized antibody reacting with 
the ganglioside GM.sub.2.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to humanized antibodies specific for the 
ganglioside GM.sub.2. The antibodies can be of any of the immunoglobulin 
(Ig) classes, it is preferable, however, that the antibodies be of the IgG 
type. The term "humanized antibody", as used herein, includes within its 
meaning, chimeric human antibody and CDR-grafted antibody. Chimeric human 
antibodies of the invention include the V.sub.H and V.sub.L of an antibody 
of an animal other than a human and the C.sub.H and C.sub.L of a human 
antibody. The CDR-transplanted antibodies of the invention result from the 
replacement of CDRs of the V.sub.H and V.sub.L of a human antibody with 
those of the V.sub.H and V.sub.L, respectively, of an antibody of an 
animal other than a human. 
An example of a chimeric human antibody of the invention is an antibody the 
V.sub.H of which contains an amino acid sequence segment as defined by SEQ 
ID NO:91, including the 1st to 120th amino acids of that sequence, and the 
V.sub.L of which contains an amino acid sequence segment as defined by SEQ 
ID NO:92, including the 1st to 107th amino acids of that sequence. 
An example of a CDR-transplanted antibody of the invention is an antibody 
the V.sub.H CDRs of which have the amino acid sequences defined by SEQ ID 
NO:94, SEQ ID NO:95 and SEQ ID NO:96 and the V.sub.L CDRs of which have 
the amino acid sequences defined by SEQ ID NO:97, SEQ ID NO:98 and SEQ ID 
NO:99. 
The chimeric human antibodies of the invention can be produced in the 
following manner: 
(1) Preparation of cDNAs coding for the V.sub.H and V.sub.L of an antibody 
of nonhuman animal 
cDNAs coding for the V.sub.H and V.sub.L of an antibody of a nonhuman 
animal, for example a mouse anti-GM.sub.2 monoclonal antibody, can be 
prepared as follows. 
mRNAs can be extracted from hybridomas producing the mouse anti-GM.sub.2 
monoclonal antibody, for example hybridomas producing the mouse 
anti-GM.sub.2 monoclonal antibody KM796, and cDNAs reverse transcribed 
therefrom. Using the cDNAs, a library can be constructed using phage or 
plasmid vectors. The recombinant phage or recombinant plasmid containing 
the cDNA coding for the V.sub.H, and the recombinant phage or recombinant 
plasmid containing the cDNA coding for the V.sub.L can be isolated from 
the library using a constant region portion or a variable region portion 
of an antibody of a nonhuman animal, for example a mouse antibody, as a 
probe. The base sequences of the V.sub.H -encoding cDNA and V.sub.L 
-encoding cDNA in the recombinant phage or recombinant plasmid can then be 
determined. Examples of the nonhuman animals include mice, rats, hamsters 
and monkeys. 
(2) Construction of a vector for chimeric human antibody expression 
Expression of chimeric human antibody H chain and L chains can be effected 
using expression vectors suitable for use in animal cells, inserted into 
which are the cDNAs coding for the human C.sub.H and C.sub.L. Any 
expression vector suitable for use in animal cells can be used, provided 
that it allows integration and expression of the human antibody constant 
region-encoding cDNAs. Examples include pAGE107 Cytotechnology, 3, 133 
(1990)!, pAGE103 J. Biochem., 101, 1307 (1987)!, pHSG274 Gene, 27, 223 
(1984)!, pKCR Proc. Natl. Acad. Sci. U.S.A., 78, 1527 (1981)! and 
pSG1.beta.d2-4 Cytotechnology, 4, 173 (1990)!, among others. Examples of 
promoters and enhancers suitable for use in such expression vectors 
include the SV40 early promoter and enhancer J. Biochem., 101, 1307 
(1987)!, the Moloney mouse leukemia virus LTR (long terminal repeat) 
promoter and enhancer Biochem. Biophys. Res. Commun., 149, 960 (1987)! 
and the immunoglobulin H chain promoter Cell, 41, 479 (1985)! and 
enhancer Cell, 33, 717 (1983)!. The promoters and enhancers are located 
in the expression vector in operable linkage with the coding sequences. 
(3) Construction of a chimeric human antibody expression vector 
The vector for chimeric human antibody H chain and L chain expression, as 
obtained in (2), is provided with a cloning site upstream of the human 
constant region, for insertion of a cDNA coding for the variable region of 
an antibody of a nonhuman animal. Insertion, at this cloning site, of the 
cDNA coding for the variable region of a nonhuman animal antibody, using a 
synthetic DNA comprising a 5' terminal base sequence of the human antibody 
constant region and a 3' terminal base sequence of the variable region of 
the nonhuman animal antibody and having restriction enzyme sites on both 
ends, gives a chimeric human antibody expression vector with the cDNA 
coding for the human antibody constant region and the cDNA coding for the 
variable region of the nonhuman animal antibody joinedly inserted therein 
via the synthetic DNA for producing appropriate restriction enzyme sites. 
The synthetic DNA can be synthesized using a DNA synthesizer based on the 
5' terminal base sequence of the human antibody constant region and the 
base sequence of said 3' terminal base sequence of the nonhuman animal 
antibody variable region. 
(4) Construction of a chimeric human antibody H chain expression vector 
A vector for chimeric human antibody H chain expression is constructed, for 
example, by excising that portion of the human antibody C.sub.H -encoding 
cDNA which covers from the ApaI site near the 5' terminus to the 3' 
terminus and inserting that portion into an expression vector suitable for 
use in animal cells. This vector for chimeric human antibody H chain 
expression is provided with a cloning site for insertion of a cDNA coding 
for a nonhuman animal V.sub.H. cDNA coding for the nonhuman animal 
V.sub.H, excised using an appropriate restriction enzyme, is inserted into 
the vector at the cloning site using a synthetic DNA comprising that 
portion of the human antibody C.sub.H gene which covers from the 5' 
terminus to the ApaI site and the base sequence of a 3' terminal portion 
of the nonhuman animal antibody V.sub.H gene and having restriction enzyme 
sites on both ends, to give a chimeric human antibody H chain expression 
vector which allows no change in the amino acid sequence of V.sub.H upon 
expression thereof and has appropriate restriction enzyme sites. 
(5) Construction of a chimeric human antibody L chain expression vector 
A vector for chimeric human antibody L chain expression is constructed, for 
example by introducing an EcoRV site into the human antibody C.sub.L 
-encoding cDNA in the vicinity of the 5' terminus by mutagenesis, excising 
that portion which covers from the EcoRV site to the 3' terminus and 
inserting that portion into a plasmid, such as the plasmid pIg1SE1d4. This 
vector for chimeric human antibody L chain expression is provided with a 
cloning site for insertion of the cDNA coding for nonhuman animal V.sub.L. 
The nonhuman animal antibody V.sub.L -encoding cDNA, excised with an 
appropriate restriction enzyme, is inserted into the vector at the cloning 
site using a synthetic DNA comprising that portion of the human antibody 
C.sub.L gene which covers from the 5' terminus to the EcoRV site and the 
base sequence of a 3' terminal portion of the nonhuman animal antibody 
V.sub.L gene and having restriction enzyme sites on both ends, to give a 
chimeric human antibody L chain expression vector which allows no change 
in the amino acid sequence of V.sub.L upon expression thereof. 
(6) Introduction of the chimeric human antibody expression vectors into 
host cells 
Introduction of the chimeric human antibody H chain expression vector and 
chimeric human antibody L chain expression vector into host cells gives a 
transformant producing the chimeric human antibody. In introducing the 
vectors into host cells, a splicing signal may be introduced into the 
chimeric human antibody H chain and L chain expression vectors for mRNA 
stabilization Cell, 17, 737 (1979)!. 
The chimeric human antibody H chain and L chain vectors can be introduced 
into host cells, for example, simultaneously by electroporation 
JP-A-2-257891 (the term "JP-A" used herein means an unexamined published 
Japanese patent application.); Cytotechnology, 3, 133 (1990)!. In 
addition, an expression vector containing genes coding for both the 
chimeric human antibody H chain and L chain tandem expression vector! can 
be introduced into host cells BIO/TECHNOLOGY, 9, 64 (1991)!. The use of a 
tandem expression vector is preferred since a higher level of chimeric 
human antibody expression can be attained thereby, with approximately 
equal H chain and L chain expression levels. 
An example of a method of producing the CDR-transplanted antibodies of the 
invention is described as follows. 
First, a CDR-transplanted antibody expression vector can be constructed by 
the method of Winter et al. Nature, 332, 323 (1988)! as follows. 
Three synthetic DNAs are constructed designed so as to comprise the cDNAs 
coding for three CDR peptides of the V.sub.H of a nonhuman animal 
antibody, for example, peptides having the amino acid sequences defined by 
SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, with DNAs coding for amino 
acid sequences comprising of several amino acids from both ends of the 
corresponding CDRs of the V.sub.H of a human antibody being located at the 
respective both ends of the cDNAs, DNA synthesis is carried out with a 
plasmid containing the human antibody V.sub.H gene as a template. An 
example of the human antibody V.sub.H gene-containing plasmid is the M13 
plasmid containing a human antibody NEW gene-derived sequence J. Biol. 
Chem., 253, 585 (1978); Nature, 332, 323 (1988)!. 
The DNA obtained is inserted into the vector for chimeric human antibody H 
chain expression in the same manner as in the construction of the chimeric 
human antibody expression vector mentioned above to give a 
CDR-transplanted antibody H chain expression vector. 
Similarly, using, as primers, three synthetic DNAs designed to comprise the 
cDNAs coding for three CDR peptides of the V.sub.L of a nonhuman animal 
antibody, for example, the peptides having the amino acid sequences 
defined by SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, with DNAs coding 
for amino acid sequences comprising several amino acids from both ends of 
the corresponding CDRs of the human antibody V.sub.L being located at the 
respective both ends of said cDNAs, DNA synthesis is carried out with a 
human antibody V.sub.L gene-containing plasmid as a template. An example 
of the human antibody V.sub.L gene-containing plasmid is the M13 plasmid 
containing a human myeloma protein (Bence-Jones protein) REI gene-derived 
sequence Eur. J. Biochem., 45, 513 (1974); Nature, 332, 323 (1988)!. 
By inserting the DNA obtained into a vector for chimeric human L chain 
expression in the same manner as described in respect of the construction 
of the chimeric human antibody expression vector, a CDR-transplanted 
antibody L chain expression vector can be constructed. 
It is also possible to construct the CDR-transplanted antibody H chain and 
L chain expression vectors by synthesizing DNAs coding for the peptides 
having amino acid sequences resulting from replacement of the three CDRs 
each of the H chain and L chain of a human antibody with the corresponding 
CDRs of the H chain and L chain of a nonhuman animal antibody and then 
inserting the DNAs into a vector for chimeric human antibody H chain or L 
chain expression in the same manner as described in respect of the 
construction of the chimeric human antibody expression vector mentioned 
above. 
The CDR-transplanted antibody expression vector can be introduced into host 
cells in the same manner as the chimeric human antibody expression vector 
to give a transformant producing the CDR-transplanted antibody. 
The host cells suited for the introduction thereinto of the chimeric human 
antibody or CDR-transplanted antibody expression vector may be any host 
cells provided that the chimeric human antibody or CDR-transplanted 
antibody can be expressed therein. Examples include mouse SP2/0-Ag14 cells 
(ATCC CRL1581; hereinafter, "SP2/0 cells"), mouse P3X63-Ag8.653 cells 
(ATCC CRL1580), CHO cells deficient in the dihydrofolate reductase gene 
(hereinafter, "dhfr") Urlaub et al.: Proc. Natl. Acad. Sci. U.S.A., 77, 
4216 (1980)! and rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL1662; 
hereinafter, "YB2/0 cells"), with YB2/0 cells being preferred. 
The transformants producing the chimeric human antibody or CDR-transplanted 
antibody are selected by the method disclosed in JP-A-2-257891 using 
PRMI1640 medium containing G418 and fetal calf serum. A particular example 
of the chimeric human antibody-producing transformant is the transformant 
KM966 producing a chimeric human antibody that reacts with the ganglioside 
GM.sub.2. Examples of human CDR-transplanted antibody-producing 
transformants include the transformants KM8966 and KM8967 each producing a 
human CDR-transplanted antibody that reacts with the ganglioside GM.sub.2. 
KM966 has been deposited with the Fermentation Research Institute, Agency 
of Industrial Science and Technology, of 1-3, Higashi 1-chome, 
Tsukuba-shi, Ibaraki 305 JAPAN, as of Jul. 15, 1992 under the deposit 
number FERM BP-3931. KM8966 and KM8967 have also deposited with the 
above-described institute as of May 23, 1995 under the deposit numbers 
FERM BP-5105 and FERM BP-5106, respectively. 
When the transformant obtained is cultivated in a medium, the chimeric 
human antibody or CDR-transplanted antibody can be produced and 
accumulated in the culture fluid. The activity of the chimeric human 
antibody or CDR-transplanted antibody in the medium can be determined by 
an enzyme-linked immunosorbent assay (ELISA; E. Harlow et al. (ed.): 
Antibodies--A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). The 
antibody productivity of the transformant can be increased by utilizing a 
dhfr amplification system as disclosed in JP-A-2-257891. 
The chimeric human antibody and CDR-transplanted antibody can be purified 
from the culture supernatants obtained as mentioned above using a protein 
A column (E. Harlow et al. (ed.): Antibodies--A Laboratory Manual, Cold 
Spring Harbor Laboratory, 1988). As noted above, the chimeric human 
antibody KM966, which reacts with the ganglioside GM.sub.2, is a specific 
example of the thus-obtained chimeric human antibodies and 
CDR-transplanted antibodies. 
The reactivity of the chimeric human antibody or CDR-transplanted antibody 
of the invention can be checked by ELISA. The molecuar weight of the 
purified antibody H chain or L chain or whole antibody molecule can be 
determined by polyacrylamide gel electrophoresis (SDS-PAGE) or Western 
blotting (E. Harlow et al. (ed.): Antibodies--A Laboratory Manual, Cold 
Spring Harbor Laboratory, 1988). 
The binding activity of the chimeric human antibody or CDR-transplanted 
antibody that reacts with the ganglioside GM.sub.2 of cultured cancer 
cells can be measured, for example, by the fluorescent antibody technique 
or by ELISA. The complement dependent cytotoxic activity (CDC activity) 
and antibody dependent cell mediated cytotoxic activity (ADCC activity) of 
the chimeric human antibody or CDR-transplanted antibody are measured by 
the methods of Ohta et al. Cancer Immunol. Immunother., 36, 260 (1993)!. 
The humanized antibodies of the invention specifically bind to human cancer 
cells and exhibit CDC activity and ADCC activity against human cancer 
cells and therefore are useful in the treatment of human cancers, among 
others. 
The humanized antibodies according to the present invention can be used 
alone as an anticancer agent. They may be formulated into an anticancer 
composition together with at least one pharmaceutically acceptable 
carrier. For instance, the humanized antibodies are dissolved in 
physiological saline, an aqueous solution of glucose, lactose or mannitol 
and the like. The powder of the humanized antibodies for injection can be 
prepared by lyophilizing the humanized antibodies in accordance with the 
conventional method and mixing the lyophilized products with sodium 
chloride. The anticancer composition may further contain additives 
conventionally used well known in the art of medical preparation, for 
example, pharmaceutically acceptable salts. 
The humanized antibodies according to the present invention can be 
administered in the form of the above-described anticancer composition to 
mammals including human in a dose of 0.2 to 20 mg/kg/day. The dose may 
vary depending on the age, condition, etc. of patients. The administration 
of the anticancer composition can be effected by intravenous injection 
once a day (single administration or consecutive administration) or 
intermittently one to three times a week or once every two to three weeks. 
The anticancer composition is expected to be useful for treating cancer 
such as melanoma, neuroblastoma and glioma. 
The following Examples and Reference Examples are further illustrative of 
the present invention, but are not to be construed to limit the scope of 
the present invention. 
EXAMPLE 1 
Production of Chimeric Human Anti-GM.sub.2 Antibodies 
1. Isolation of mRNAs From Hybridoma Cells Producing the Mouse 
Anti-GM.sub.2 Monoclonal Antibody KM-796 or KM-750 and From Hybridoma 
Cells Producing the Rat Anti-GM.sub.2 Monoclonal Antibody KM-603 
Using mRNA extraction kit Fast Track (product number K1593-02) manufactured 
by Invitorogen and following the description of the manual attached to the 
kit, mRNAs were isolated from 1.times.10.sup.8 cells each of the mouse 
anti-GM.sub.2 monoclonal antibody KM-796-producing hybridoma cell line 
(FERM BP-3340), the mouse anti-GM.sub.2 monoclonal antibody 
KM-750-producing hybridoma cell line (FERM BP-3339) and the rat 
anti-GM.sub.2 monoclonal antibody KM-603-producing hybridoma cell line 
(FERM BP-2636). 
2. Construction of Monoclonal Antibody KM-796 and KM-750 H Chain and L 
Chain cDNA Libraries 
Using cDNA Synthesis Kit (product number 27-9260-01) manufactured by 
Pharmacia and following the manual attached to the kit, cDNA having the 
EcoRI adapter on both ends was synthesized from 5 .mu.g each of the 
KM-796- and KM-750-derived mRNAs obtained as described in Paragraph 1 
above. About 6 .mu.g of each cDNA product obtained was dissolved in 10 
.mu.l of sterilized water and fractionated by agarose gel electrophoresis, 
and a cDNA fragment (about 1.8 kb) corresponding to the IgG antibody H 
chain and a cDNA fragment (about 1.0 kb) corresponding to the L chain were 
recovered (about 0.1 .mu.g each). Then, 0.1 .mu.g of each cDNA fragment of 
about 1.8 kb and 0.1 .mu.g of each cDNA fragment of about 1.0 kb were 
respectively dissolved in 11.5 .mu.l of T4 ligase buffer, together with 1 
.mu.g of the Lambda ZAPII vector (cleaved with EcoRI and then treated with 
calf intestine alkaline phosphatase; product of Stratagene). After 
addition of 175 units of T4 DNA ligase, each solution was incubated at 
12.degree. C. for 24 hours and then at room temperature for 2 hours. A 
4-.mu.l portion of each reaction mixture was subjected to packaging into 
the lambda phage in the conventional manner Maniatis et al. (ed.): 
Molecular Cloning, 2.95 Cold pring Harbor Laboratory, 1989! using Giga Pak 
Gold (Stratagene), followed by transfection, in the conventional manner 
Maniatis et al. (ed.): Molecular Cloning, 2.95-107, Cold Spring Harbor 
Laboratory, 1989! of the Escherichia coli strain XL1-Blue Biotechniques, 
5, 376 (1987)! attached to Giga Pak Gold, to give about 4.times.10.sup.3 
phage clones each as a KM-796 or KM-750 H chain or L chain cDNA library. 
Then the phage clones of each library were fixed on a nitrocellulose 
filter in the conventional manner Maniatis et al. (ed.): Molecular 
Cloning, 2.112, Cold Spring Harbor Laboratory, 1989!. 
3. Construction of KM-603 H Chain and L Chain cDNA Libraries 
Using 5 ug of the KM-603 MRNA obtained as mentioned above in Paragraph 1 
and cDNA Synthesis Kit (product number 27-9260-01) manufactured by 
Pharmacia, cDNA having the EcoRI adapter on both ends was synthesized. 
About 6 .mu.g of the cDNA produced was dissolved in 10 .mu.l of sterilized 
water and fractionated by agarose gel electrophoresis. A cDNA fragment 
(about 2.2 kb) corresponding to the IgG antibody H chain and a cDNA 
fragment (about 1.0 kb) corresponding to the L chain were recovered (about 
0.1 .mu.g each). Then 0.1 .mu.g of the cDNA fragment of about 2.2 kb and 
0.1 .mu.g of the cDNA fragment of about 1.0 kb were respectively dissolved 
in 11.5 .mu.l of T4 ligase buffer, together with 1 .mu.g of the Lambda 
ZAPII vector (cleaved with EcoRI and then treated with calf intestine 
alkaline phosphatase; product of Stratagene) and, after addition of 175 
units of T4 DNA ligase, the resultant solution was incubated at 12.degree. 
C. for 24 hours and then at room temperature for 2 hours. A 4-.mu.l 
portion of each reaction mixture was subjected to packaging into the 
lambda phage in the conventional manner Maniatis et al. (ed.): Molecular 
Cloning, 2.95, Cold Spring Harbor Laboratory, 1989! using Giag Pak Gold 
(Stratagene), followed by transfection, in the conventional manner 
Maniatis et al. (ed.): Molecular Cloning, 2.95-107, Cold Spring Harbor 
Laboratory, 1989!, of the Escherichia coli strain XL-Blue attached to Giga 
Pak Gold, whereby about 1.times.10.sup.4 phage clones were obtained each 
as a KM-603 H chain or L chain cDNA library. Then, the phage clones of 
each library were fixed on a nitrocellulose filter in the conventional 
manner Maniatis et al. (ed.): Molecular Cloning, 2.112, Cold Spring 
Harbor Laboratory, 1989!. 
4. Cloning of the KM-796 and KM-750 H Chain and L Chain cDNAs 
From among the KM-796 and KM-750 H chain cDNA libraries and L chain cDNA 
libraries constructed as described above in Paragraph 2, phage clones 
firmly bound at 65.degree. C. to a probe prepared by labeling a mouse 
immunoglobulin constant region cDNA for the H chain, the BamHI-XhoI 
fragment of the mouse C.gamma.3 cDNA (Wels et al: EMBO J., 3, 2041-2046, 
1984); for the L chain, the HpaI-XhoI fragment of the mouse C.kappa. cDNA 
(Hieter et at.: Cell, 22, 197-207, 1980)! with .sup.32 P were recovered in 
the conventional manner Maniatis et al.: Molecular Cloning, 2.108, Cold 
Spring Harbor Laboratory, 1989!. Then, using a ZAP-cDNA Synthesis Kit 
(cDNA synthesis kit; product number sc200400) manufactured by Stratagene, 
phage clones were converted into pBluescript plasmids, and a KM-796 H 
chain cDNA-containing recombinant plasmid (pKM796H1) and a KM-796 L chain 
cDNA-containing recombinant plasmid (pKM796L1) (FIG. 1) as well as a 
KM-750 H chain cDNA-containing recombinant plasmid (pKM750H1) and a KM-750 
L chain cDNA-containing recombinant plasmid (pKM750L1) (FIG. 2) were 
obtained. Cleavage of pKM796H1, pKM750H1, pKM796L1 and pKM750L1 with EcoRI 
revealed that a cDNA fragment of about 1.8 kb had been inserted into 
pKM796H1 and pKM750H1 and a cDNA fragment of about 0.9 kb into pKM796L1 
and pKM750L1. 
5. Cloning of KM-603 H Chain and L Chain cDNAs 
Phage clones firmly bound at 65.degree. C. to a probe prepared by labeling 
a mouse immunoglobulin constant region chromosomal gene mouse C.mu. 
gene-containing SmaI-KpnI fragment of about 11.5 kb (Kataoka et al.: Proc. 
Natl. Acad. Sci. U.S.A., 77, 919-923, 1980) and mouse C.kappa. 
gene-containing HindIII-BamHI fragment of about 3 kb (Sakano et al.: 
Nature, 280, 288, 1979)! with .sup.32 P were isolated from the KM-603 H 
chain cDNA library and L chain cDNA library constructed as mentioned above 
in Paragraph 3 in the conventional manner Maniatis et al. (ed.): 
Molecular Cloning, 2.108, Cold Spring Harbor Laboratory, 1989!. Then, 
using ZAP-cDNA Synthesis kit (product number sc200400) manufactured by 
Stratagene, the phage clones were converted to pBluescript plasmids and a 
KM-603 H chain cDNA-containing recombinant plasmid, pKM603H1, and a KM-603 
L chain cDNA-containing recombinant plasmid, pKM603L1, were obtained (FIG. 
3). Cleavage of pKM603H1 and pKM603L1 revealed that pKM603H1 contained a 
cDNA fragment of about 2.0 kb as inserted therein and pKM603L1 a cDNA 
fragment of about 0.9 kb as inserted therein. 
6. Base Sequences of the Variable Regions in the H Chain cDNA and L Chain 
cDNA 
The base sequences of the variable regions in the H chain cDNA and L chain 
cDNA obtained as mentioned above in Paragraphs 4 and 5 were determined by 
the dideoxy method Maniatis et al. (ed.): Molecular Cloning, 13.42, Cold 
Spring Harbor Laboratory, 1989! using Sequenase Version 2.0 DNA Sequencing 
Kit manufactured by United States Biochemical Corporation. All the cDNA 
had a methionine codon, presumably the initiation codon ATG, at the 5' 
terminus and were leader sequence-containing full-length cDNAs. Based on 
the base sequences of the respective cDNAs, the amino acid sequences of 
the H chain and L chain of KM-796, KM-750 and KM-603 were deduced. The 
amino acid sequence of the KM-796 H chain is shown in SEQ ID NO:91, that 
of the L chain of KM-796 and KM-750 in SEQ ID NO:92, that of the KM-750 H 
chain in SEQ ID NO:93, that of the KM-603 H chain in SEQ ID NO:4 and that 
of the KM-603 L chain in SEQ ID NO:5. 
7. Construction of KM-796- and KM-750-Derived Chimeric Human Antibody H 
Chain and L Chain Expression Vectors 
(1) Construction of a vector, pAGE147, carrying the Moloney mouse leukemia 
virus terminal repeat promoter/enhancer 
The plasmid pPMOL1 (2 .mu.g), described in JP-A-1-63394, was dissolved in 
30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM 
magnesium chloride and 6 mM 2-mercaptoethanol, 20 units of SmaI was added, 
and digestion was carried out at 30.degree. C. for 3 hours. Then, sodium 
chloride was added to a concentration of 50 mM, 20 units of ClaI was 
added, and digestion was conducted at 37.degree. C. for 2 hours. The 
reaction mixture was subjected to agarose gel electrophoresis, and a DNA 
fragment (about 0.6 kb) containing the Moloney mouse leukemia virus 
terminal repeat promoter/enhancer was recovered. 
Then, the following two synthetic DNAs were synthesized using an automatic 
DNA synthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.). 
##STR1## 
The thus-obtained synthetic DNAs (25 picomoles each) were dissolved in 10 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mM 
magnesium chloride, 5 mM DTT (dithiothreitol), 0.1 mM EDTA and 0.5 mM 
adenosine triphosphate (hereinafter, "ATP"), 5 units of T4 DNA kinase was 
added, and 5'-phosphorylation was carried out at 37.degree. C. for 30 
minutes. 
The plasmid pPMOL1-derived ClaI-SmaI fragment (0.6 kb, 0.05 .mu.g) and two 
5'-phosphorylated synthetic DNAs (1 picomole each), obtained as described 
above, and a HindIII linker (5'-pCAAGCTTG-3'; Takara Shuzo) (1 picomole) 
were dissolved in 30 .mu.l of 66 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6.6 mM magnesium chloride, 10 mM DTT and 0.1 mM ATP, 200 units 
of T4 DNA ligase (Takara Shuzo; hereinafter the same shall apply) were 
added, and ligation was carried out at 12.degree. C. for 16 hours. The 
resultant DNA fragment was recovered by ethanol precipitation and 
dissolved in 20 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride, 100 mM sodium chloride and 6 mM 
2-mercaptoethanol, 10 units of HindIII and 10 units of XhoI were added, 
and digestion was carried out at 37.degree. C. for 2 hours. The reaction 
was terminated by phenol-chloroform extraction, and the DNA fragment was 
recovered by ethanol precipitation. 
Separately, 1 .mu.g of the plasmid pAGE107 Cytotechnology, 3, 133 (1990)! 
was dissolved in 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride, 100 mM sodium chloride and 6 mM 
2-mercaptoethanol, 10 units of HindIII and 10 units of XhoI were added, 
and digestion was carried out at 37.degree. C. for 2 hours. The reaction 
mixture was subjected to agarose gel electrophoresis, and a DNA fragment 
(about 6.0 kb) containing the G418 resistance gene and ampicillin 
(hereinafter, "Ap") resistance gene was recovered. 
The plasmid pAGE107-derived HindIII-XhoI fragment (6.0 kb, 0.3 .mu.g) and 
plasmid pPMOL1-derived HindIII-XhoI fragment (0.63 kb, 0.01 .mu.g) 
obtained as mentioned above were dissolved in 20 .mu.l of 66 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6.6 mM magnesium chloride, 
10 mM DTT and 0.1 mM ATP, 200 units of T4 DNA ligase were added, and 
ligation was carried out at 12.degree. C. for 16 hours. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101, and 
the plasmid pAGE147 shown in FIG. 4 was obtained. 
(2) Construction of a vector, pAGE148, carrying the .beta.-globin 3' 
splicing signal (SP) 
For introducing the .beta.-globin 3' splicing signal into the chimeric 
human antibody expression vector at a site downstream from the antibody 
constant region gene, a vector (pAGE148), was constructed as follows, 
which contained the .beta.-globin 3' splicing signal and the same genes as 
those in the chimeric human antibody expression vector (except for the 
human antibody constant region gene). 
Two .mu.g of pSE1UK1SEd1-3, described in JP-A-2-257851, were added to 30 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride, 50 mM sodium chloride and 1 mM DTT. After addition of 
10 units of HindIII, digestion was carried out at 37.degree. C. for 4 
hours. The reaction mixture was subjected to phenol-chloroform extraction 
and then to ethanol precipitation. The precipitate was dissolved in 20 
.mu.l of DNA polymerase I buffer, 5 units of Escheichia coli-derived DNA 
polymerase I Klenow fragment were added, and the 5' cohesive ends produced 
by HindIII digestion were rendered blunt by incubation at 22.degree. C. 
for 30 minutes. The reaction mixture was subjected to phenol-chloroform 
extraction and then to ethanol precipitation, 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT and 10 units of KpnI were added, and digestion was effected at 
37.degree. C. for 4 hours. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, 30 .mu.l 
of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 100 mM sodium chloride and 1 mM DTT and 10 units of XhoI were 
added, and digestion was carried out at 37.degree. C. for 4 hours. The 
reaction mixture was fractionated by agarose gel electrophoresis and two 
DNA fragments, about 6.67 kb and about 1.98 kb in size, were recovered 
(about 0.2 .mu.g each). 
Then, 2 .mu.g of pAGE147 obtained in (1) was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of KpnI was added, and digestion was effected at 
37.degree. C. for 4 hours. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, 30 .mu.l 
of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 100 mM sodium chloride and 1 mM DTT and 10 units of XhoI were 
added, and digestion was carried out at 37.degree. C. for 4 hours. The 
reaction mixture was fractionated by agarose gel electrophoresis and about 
0.2 .mu.g of a DNA fragment of about 0.66 kb was recovered. 
Then, 0.1 .mu.g of the XhoI-HindIII fragment (about 6.67 kb) of 
pSE1UK1SEd1-3, as obtained above, 0.1 .mu.g of the KpnI-HindIII fragment 
(about 1.98 kb), obtained above, and 0.1 .mu.g of the XhoI-KpnI fragment 
(about 0.66 kb) of pAGE147, as obtained above, were dissolved in a total 
of 20 .mu.l of T4 ligase buffer. Three hundred fifty units of T4 ligase 
were added to the solution, and ligation was carried out at 4.degree. C. 
for 24 hours. The thus-obtained recombinant plasmid DNA was used to 
transform Escherichia coli HB101, and the plasmid pAGE148 shown in FIG. 5 
was obtained. 
(3) Construction of KM-796- and KM-750-derived chimeric human antibody H 
chain expression vectors 
First, the cDNA coding for the antibody variable region in the plasmid 
pKM796H1 or pKM750H1 was excised by cleavage at the 5'-terminal EcoRI site 
and the MaeIII site near the 3' end of said cDNA and joined, together with 
a synthetic DNA having the base sequence shown in SEQ ID NO:12, to the 
chimeric human antibody H chain expression vector pChi641HAM1, as follows 
(FIG. 6). 
Three .mu.g of pKM796H1 or pKM750H1, obtained in Paragraph 4, were added to 
30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride, 100 mM sodium chloride and 1 mM DTT. Further, 10 units 
of EcoRI and 10 units of MaeIII were added, and digestion was effected at 
37.degree.0 C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.3 .mu.g of a DNA fragment of about 
0.43 kb was recovered. Then, 3 .mu.g of pChi641HAM1, obtained in Reference 
Example 2, was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of EcoRI 
and 10 units of ApaI were also added, and digestion was carried out at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 1.0 .mu.g of a DNA fragment of about 
9.0 kb was recovered. Then, 0.1 .mu.g of the EcoRI-MaeIII fragment (about 
0.43 kb) of pKM796H1 or pKM750H1, as obtained above, 0.1 .mu.g of the 
EcoRI-ApaI fragment (about 9.0 kb) of pChi641HAM1, as obtained above, and 
0.3 .mu.g of a synthetic DNA having the base sequence shown in SEQ ID 
NO:12 were dissolved in a total of 20 .mu.l of T4 ligase buffer, 350 units 
of T4 ligase was further added to the solution, and ligation was carried 
out at 4.degree. C. for 24 hours. The thus-obtained recombinant plasmid 
DNA was used to transform Escherichia coli HB101. In this way, the 
plasmids pChi796HM1 and pChi750HM1, shown in FIG. 6, were obtained. 
Then, the .beta.-globin 3' splicing signal was introduced into the plasmids 
pChi796HM1 and pChi750HM1 by the method described below to construct 
KM796- and KM-750-derived chimeric human antibody H chain expression 
vectors (FIG. 7). 
Three .mu.g of pChi796HM1 or pChi750HM1 were added to 30 .mu.l of 33 mM 
Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mM 
potassium acetate, 0.5 mM DTT and 0.01% bovine serum albumin (hereinafter, 
"BSA"). Ten units of XhoI and 10 units of KpnI were also added, and 
digestion was carried out at 37.degree. C. for 4 hours. The reaction 
mixture was fractionated by agarose gel electrophoresis and about 0.3 
.mu.g of a DNA fragment of about 3.4 kb was recovered. Then, 3 .mu.g of 
pAGE148 obtained in (2) was added to 30 .mu.l of 33 mM Tris-acetate buffer 
(pH 7.9) containing 10 mM magnesium acetate, 66 mM potassium acetate, 0.5 
mM DTT and 0.01% BSA; 10 units of XhoI and 10 units of KpnI were further 
added, and digestion was effected at 37.degree. C. for 4 hours. The 
reaction mixture was fractionated by agarose gel electrophoresis and about 
0.3 .mu.g of a DNA fragment of about 8.7 kb was recovered. Then, 0.1 .mu.g 
of the XhoI-KpnI fragment of pChi796HM1 or pKM750HM1 and 0.1 .mu.g of the 
XhoI-KpnI fragment of pAGE148 were dissolved in a total of 20 .mu.l of T4 
ligase buffer, 350 units of T4 ligase was further added to the solution, 
and ligation was carried out at 4.degree. C. for 24 hours. The 
thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli HB101. The plasmids pChi796HMS1 and pChi750HMS1 shown in FIG. 7 were 
thus obtained. 
(4) Construction of KM-796- and KM-750-derived chimeric human antibody L 
chain expression vectors 
First, the cDNA coding for the antibody variable region in the plasmid 
pKM796L1 or PKM750L1 was excised by cleavage at the 5'-terminal EcoRI site 
and the Af1III site near the 3' end of said cDNA and joined, together with 
a synthetic DNA having the base sequence shown in SEQ ID NO:13, to the 
chimeric human antibody L chain expression vector pChiIgLA1, as follows 
(FIG. 8). 
Three .mu.g of pKM796L1 or pKM750L1 were added to 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT. Further, 10 units of EcoRI and 10 
units of Af1III were added, and digestion was effected at 37.degree. C. 
for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a DNA fragment of about 0.39 kb was 
recovered. Then, 3 .mu.g of pChiIgLA1 obtained in Reference Example 1 was 
added to 30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 
10 mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of 
EcoRI and 10 units of EcoRV were further added, and digestion was carried 
out at 37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 1 .mu.g of a DNA fragment of about 
8.6 kb was recovered. 
Then, 0.1 .mu.g of the EcoRI-Af1III fragment of pKM796L1 or pKM750L1, as 
obtained above, 0.1 .mu.g of the EcoRI-EcoRV fragment of pChiIgLA1, as 
obtained above, and 0.3 .mu.g of a synthetic DNA, having the base sequence 
shown in SEQ ID NO:13, were dissolved in a total of 20 .mu.l of T4 ligase 
buffer; 350 units of T4 ligase was further added to the solution, and 
ligation was carried out at 4.degree. C. for 24 hours. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101. In 
this way, the plasmids pChi796LI1 and pChi750LI1 shown in FIG. 8 were 
obtained. 
Then, the Moloney mouse leukemia virus terminal repeat promoter/enhancer 
was introduced into the plasmids pChi796LI1 and pChi750LI1 in the 
following manner (FIG. 9). 
Three .mu.g of pChi796LI1 and pChi750LI1 were added to 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT. Further, 10 units of EcoRI and 10 
units of XhoI were added, and digestion was effected at 37.degree. C. for 
4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a DNA fragment of about 8.2 kb was 
recovered Then, 3 .mu.g of the chimric human antibody H chain expression 
vector pChi641HAM1 obtained in Reference Example 2 was added to 30 .mu.l 
of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10 
units of XhoI were further added, and digestion was carried out at 
37.degree.0 C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.3 .mu.g of a DNA fragment of about 
0.6 kb was recovered. 
Then, 0.1 .mu.g of the EcoRI-XhoI fragment of pChi796LI1 or pKM750LI1 as 
obtained above and 0.1 .mu.g of the EcoRI-XhoI fragment of pChi641HAM1 as 
obtained above were dissolved in a total of 20 .mu.l of T4 ligase buffer, 
350 units of T4 ligase was further added to the solution, and ligation was 
carried out at 4.degree. C. for 24 hours. The thus-obtained recombinant 
plasmid DNA was used to transform Escherichia coli HB101. In this way, the 
plasmids pChi796LM1 and pChi750LM1 shown in FIG. 9 were obtained. 
Then, the .beta.-globin 3' splicing signal was introduced into the plasmids 
pChi796LM1 and pChi750LM1 in the manner mentioned below to construct 
KM-796- and KM-750-derived chimeric human antibody L chain expression 
vectors (FIG. 10). 
Three .mu.g of pChi796LM1 or pChi750LM1 were added to 30 .mu.l of 33 mM 
Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mM 
potassium acetate, 0.5 mM DTT and 0.01% BSA. Further, 10 units of XhoI and 
10 units of KpnI were added, and digestion was carried out at 37.degree. 
C. for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a DNA fragment of about 2.0 kb was 
recovered. Then, 3 .mu.g of pAGE148 obtained in (2) was added to 30 .mu.l 
of 33 mM Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 
66 mM potassium acetate, 0.5 mM DTT and 0.0% BSA; 10 units of XhoI and 10 
units of KpnI were further added, and digestion was carried out at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.3 .mu.g of a DNA fragment of about 
8.7 kb was recovered. Then 0.1 .mu.g of the XhoI-KpnI fragment of 
pChi796LM1 or pKM750LM1 as obtained above and 0.1 .mu.g of the XhoI-KpnI 
fragment of pAGE148 were dissolved in a total of 20 .mu.l of T4 ligase 
buffer, 350 units of T4 ligase was further added, and ligation was carried 
out at 4.degree. C. for 24 hours. The thus-obtained recombinant plasmid 
DNA was used to transform Escherichia coli HB101. In this way, the 
plasmids pChi796LMS1 and pChi750LMS1 shown in FIG. 10 were obtained. 
8. Construction of KM-796- and KM-750-Derived Chimeric Human Antibody H 
Chain and L Chain Tandem Expression Vectors 
Tandem expression vectors containing the chimeric human antibody H 
chain-encoding cDNA and L chain-encoding cDNA on one and the same vector 
were constructed (FIG. 11 and FIG. 12). 
Three .mu.g of pChi796HMS1 or pChi750HMS1, obtained in Paragraph 7, were 
added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 
10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT. Further, 10 
units of MluI and 10 units of SalI were added, and digestion was carried 
out at 37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis. In each case, about 0.3 .mu.g of a DNA 
fragment of about 5.9 kb was recovered. Then, 2 .mu.g of pAGE107 described 
in EP-A-0 405 285 was dissolved in 30 .mu.l of 10 mM Tris-hydrochloride 
buffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride 
and 1 mM DTT; 10 units of MluI and 10 units of SalI were further added, 
and digestion was carried out at 37.degree. C. for 4 hours. The reaction 
mixture was fractionated by agarose gel electrophoresis and about 0.2 
.mu.g of a DNA fragment of about 3.55 kb was recovered. Then, 0.1 .mu.g of 
the MluI-SalI fragment of pChi796HMS1 or pChi750HMS1 and 0.1 .mu.g of the 
MluI-SalI fragment of pAGE107 were dissolved in a total of 20 .mu.l of T4 
ligase buffer, 350 units of T4 ligase was added, and ligation was carried 
out at 4.degree. C. for 24 hours. The thus-obtained recombinant plasmid 
DNA was used to transform Escherichia coli HB101 to give the plasmid 
pChi796H107 or pChi750H107 shown in FIG. 11. 
Then, 3 .mu.g of pChi796H107 or pChi750H107 was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 50 
mM sodium chloride and 1 mM DTT, 10 units of ClaI was further added, and 
digestion was carried out at 37.degree. C. for 4 hours. The reaction 
mixture was subjected to phenol-chloroform extraction and then to ethanol 
precipitation. The precipitate was dissolved in 20 .mu.l of DNA polymerase 
I buffer, 5 units of Escherichia coli-derived DNA polymerase I Klenow 
fragment was added, and the mixture was incubated at 22.degree. C. for 30 
minutes for rendering the cohesive ends formed upon ClaI digestion 
blunt-ended. The reaction mixture was further subjected to 
phenol-chloroform extraction and then to ethanol precipitation. To the 
precipitate were added 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM 
DTT, and 10 units of MluI. Digestion was carried out at 37.degree. C. for 
4 hours and the reaction mixture was fractionated by agarose gel 
electrophoresis. In each case, about 0.3 .mu.g of a DNA fragment of about 
7.5 kb was recovered. Then, 3 .mu.g of pChi796LMS1 or pChi750LMS1 was 
added to 30 .mu.l of 20 mM Tris-hydrochloride buffer (pH 8.5) containing 
10 mM magnesium chloride, 100 mM potassium chloride and 1 mM DTT, 10 units 
of XhoI was further added, and digestion was carried out at 37.degree. C. 
for 4 hours. The reaction mixture was subjected to phenol-chloroform 
extraction and then to ethanol precipitation. The precipitate was 
dissolved in 20 .mu.l of DNA polymerase I buffer, 5 units of Escherichia 
coli-derived DNA polymerase I Klenow fragment was added, and the mixture 
was incubated at 22.degree. C. for 30 minutes for rendering the cohesive 
ends formed upon XhoI digestion blunt-ended. The reaction mixture was 
further subjected to phenol-chloroform extraction and then to ethanol 
precipitation. To the precipitate was added 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 50 
mM sodium chloride and 1 mM DTT as well as 10 units of MluI. Digestion was 
carried out at 37.degree. C. for 4 hours and the reaction mixture was 
fractionated by agarose gel electrophoresis. In each case, about 0.3 .mu.g 
of a DNA fragment of about 9.3 kb was recovered. Then, 0.1 .mu.g of the 
MluI-ClaI fragment of pChi796H107 or pChi750H107, as obtained above, and 
0.1 .mu.g of the MluI-XhoI fragment of pChi796LMS1 or pChi750LMS1, as 
obtained above, were dissolved in a total of 20 .mu.l of T4 ligase buffer; 
350 units of T4 ligase was further added, and ligation was carried out at 
4.degree. C. for 24 hours. The thus-obtained recombinant plasmid DNA was 
used to transform Escherichia coli HB101, and the plasmid pChi796HL1 or 
pChi750HL1 shown in FIG. 12 was obtained. 
Construction of a KM-603-Derived Chimeric Human Antibody H Chain Expression 
Vector 
First, the antibody variable region-encoding cDNA of the plasmid pKM603H1 
was excised by cleavage at the 5'terminal EcoRI site and the StyI site 
near the 3' end of said cDNA and joined to the chimeric human antibody H 
chain expression vector pChi641HAM1 together with a synthetic DNA having 
the base sequence shown in SEQ ID NO:14 in the following manner (FIG. 13). 
Three .mu.g of pKM603H1 obtained in Paragraph 5 were added to 30 .mu.l of 
50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 100 mM sodium chloride and 1 mM DTT, followed by further 
addition of 10 units of EcoRI and 10 units of StyI. Digestion was carried 
out at 37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.3 .mu.g of a 0.4-kb DNA fragment 
was recovered. Then, 3 .mu.g of pChi641HAM1, obtained in Reference Example 
2, was added to 30 .mu.l of 10 mM Tris-hydrochloride (pH 7.5) containing 
10 mM magnesium chloride and 1 mM DTT, 10 units of EcoRI and 10 units of 
ApaI were further added, and digestion was effected at 37.degree. C. for 4 
hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 1.0 .mu.g of a DNA fragment of about 9.0 kb was 
recovered. Then, 0.1 .mu.g of the EcoRI-StyI fragment (about 0.4 kb) of 
pKM603H1, as obtained above, and 0.1 .mu.g of the EcoRI-ApaI fragment 
(about 9.0 kb) of pChi641HAM1, as obtained above, were dissolved, together 
with 0.3 .mu.g of a synthetic DNA having the base sequence shown in SEQ ID 
NO:14, in a total of 20 .mu.l of T4 ligase buffer; 350 units of T4 ligase 
was added to the solution, and ligation was effected at 4.degree. C. for 
24 hours. The thus-obtained recombinant plasmid DNA was used to transform 
Escherichia coli HB101 and the plasmid pChi603HM1 shown in FIG. 13 was 
obtained. 
Then, a KM-603-derived chimeric human antibody H chain expression vector 
was constructed by introducing the .beta.-globin 3' splicing signal into 
the plasmid pChi603HM1 in the following manner (FIG. 14). 
Three .mu.g of pChi603HM1 obtained above were added to 30 .mu.l of 33 mM 
Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mM 
potassium acetate, 0.5 mM DTT and 0.01% BSA. Further, 10 units of XhoI and 
10 units of KpnI were added, and digestion was carried out at 37.degree. 
C. for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a DNA fragment of about 3.3 kb was 
recovered. 
Then, 3 .mu.g of pAGE148 obtained in Paragraph 7 (2) was added to 30 .mu.l 
of 33 mM Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 
66 mM sodium acetate, 0.5 mM DTT and 0.01% BSA; 10 units of XhoI and 10 
units of KpnI were further added, and digestion was carried out at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.3 .mu.g of a DNA fragment of about 
8.7 kb was recovered. Then, 0.1 .mu.g of the XhoI-KpnI fragment of 
pChi603HM1, as obtained above, and 0.1 .mu.g of the XhoI-KpnI fragment of 
pAGE148, as obtained above, were dissolved in a total of 20 .mu.l of T4 
ligase buffer; 350 units of T4 ligase was added to the solution, and 
ligation was carried out at 4.degree. C. for 24 hours. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101 and 
the plasmid pChi603HMS1 shown in FIG. 14 was obtained. 
10. Construction of a KM-603-Derived Chimeric Human Antibody L Chain 
Expression Vector 
First, the antibody variable region cDNA in the plasmid pKM603L1 was 
excised by cleavage at the 5' terminal EcoRI site and the Af1III site near 
the 3' end and joined to the chimeric human antibody L chain expression 
vector pChiIgLA1 together with a synthetic DNA having the base sequence 
defined by SEQ ID NO:15 (FIG. 15). 
Thus, 3 .mu.g of pKM603L1 obtained in Paragraph 5 was added to 30 .mu.l of 
50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10 
units of Af1III were further added, and digestion was carried out at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.3 .mu.g of a DNA fragment of about 
0.4 kb was recovered. Then, 3 .mu.g of pChiIgLA1 obtained in Reference 
Example 1 was added to 30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mM 
DTT, 10 units of EcoRI and 10 units of EcoRV were further added, and 
digestion was carried out at 37.degree. C. for 4 hours. The reaction 
mixture was fractionated by agarose gel electrophoresis and about 1 .mu.g 
of a DNA fragment of about 8.6 kb was recovered. Then, 0.1 .mu.g of the 
EcoRI-Af1III fragment of pKM603L1, as obtained above, 0.1 .mu.g of the 
EcoRI-EcoRV fragment of pChiIgLA1, as obtained above, and 0.3 .mu.g of a 
synthetic DNA, having the base sequence defined by SEQ ID NO:15, were 
dissolved in a total of 20 .mu.l of T4 ligase buffer; 350 units of T4 
ligase was added to the solution, and ligation was carried out at 
4.degree. C. for 24 hours. The thus-obtained recombinant plasmid DNA was 
used to transform Escherichia coli HB101 and the plasmid pChi603LI1 shown 
in FIG. 15 was obtained. 
Then, the Moloney mouse leukemia virus terminal repeat promoter/enhancer 
was introduced into the plasmid pChi603LI1 in the following manner (FIG. 
16). 
Thus, 3 .mu.g of pChi603LI1 obtained above was added to 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10 units of 
XhoI were further added, and digestion was carried out at 37.degree. C. 
for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a DNA fragment of about 8.3 kb was 
recovered. Then, 3 .mu.g of pChi641HAM1 obtained in Reference Example 2 
was added to 30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 
10 units of EcoRI and 10 units of XhoI were further added, and digestion 
was effected at 37.degree. C. for 4 hours. The reaction mixture was 
fractionated by agarose gel electrophoresis and about 0.3 .mu.g of a DNA 
fragment of about 0.6 kb was recovered. Then, 0.1 .mu.g of the EcoRI-XhoI 
fragment of pChi603LI1, as obtained above, and 0.1 .mu.g of the EcoRI-XhoI 
fragment of pChi641HAM1, as obtained above, were dissolved in a total of 
20 .mu.l of T4 ligase buffer; 350 units of T4 ligase was added to the 
solution, and ligation was effected at 4.degree. C. for 24 hours. The 
thus-obtained recombinant plasmid DNA was used to transform Eschrichia 
coli HB101 to give the plasmid pChi603LM1 shown in FIG. 16. 
A KM-603-derived chimeric human antibody L chain expression vector was then 
constructed by introducing the .beta.-globin 3' splicing signal into the 
plasmid pChi603LM1, as follows (FIG. 17). 
Thus, 3 .mu.g of pChi603LM1 obtained above was added to 30 .mu.l of 33 mM 
Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mM 
potassium acetate, 0.5 mM DTT and 0.01% BSA, 10 units of XhoI and 10 units 
of KpnI were further added, and digestion was effected at 37.degree. C. 
for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a DNA fragment of about 2.0 kb was 
recovered. Then, 3 .mu.g of pAGE148 obtained in Paragraph 7 (2) was added 
to 30 .mu.l of 33 mM Tris-acetate buffer (pH 7.9) containing 10 mM 
magnesium acetate, 66 mM potassium acetate, 0.5 mM DTT and 0.01% BSA, 10 
units of XhoI and 10 units of KpnI were further added, and digestion was 
effected at 37.degree. C. for 4 hours. The reaction mixture was 
fractionated by agarose gel electrophoresis and about 0.3 .mu.g of a DNA 
fragment of about 8.7 kb was recovered. Then, 0.1 .mu.g of the XhoI-KDnI 
fragment of pChi603LM1, as obtained above, and 0.1 .mu.g of the XhoI-KpnI 
fragment of pAGE148, as obtained above, were dissolved in a total of 20 
.mu.l of T4 ligase buffer; 350 units of T4 ligase was added to the 
solution, and ligation was carried out at 4.degree. C. for 24 hours. The 
thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli HB101 to give the plasmid pChi603LMS1 shown in FIG. 17. 
11. Expression of the KM-796- and KM-750-Derived Chimeric Human Anti-GM2 
Antibody in YB2/0 Cells 
The plasmids were introduced into YB2/0 cells by the electroporation method 
of Miyaji et al. Cytotechnbology, 3, 133-140 (1990)!. 
After introduction of 4 .mu.g of pChi750HL1 or pChi796HL1 obtained in 
Paragraph 8 into 4.times.10.sup.6 YB2/0 (ATCC CRL1581) cells, the cells 
were suspended in 40 ml of RPMI-1640-FCS(10) RPMI1640 medium (Nissui 
Pharmaceutical) containing 10% of FCS, 1/40 volume of 7.5% NaHCO.sub.3, 3% 
of 200 mM L-glutamine solution (Gibco) and 0.5% of penicillin-streptomycin 
solution (Gibco; containing 5,000 units/ml penicillin and 5,000 .mu.g/ml 
streptomycin)!, and the suspension was distributed in 200-.mu.l portions 
into wells of 96-well microtiter plates. After 24 hours of incubation at 
37.degree. C. in a CO.sub.2 incubator, G418 (Gibco) was added to a 
concentration of 0.5 mg/ml and then incubation was continued for 1 to 2 
weeks. Transformant colonies appeared, the culture fluid was recovered 
from each well in which the cells had grown to confluence and an 
enzyme-linked immunosorbent assay (ELISA) was conducted for anti-GM.sub.2 
chimeric human antibody activity measurement. 
Enzyme-Linked Immunosorbent Assay (ELISA) 
In a solution of 5 ng of phosphatidylcholine (Sigma) and 2.5 ng of 
cholesterol (Sigma) in 2 ml of ethanol was dissolved 2 ng of GM.sub.2 
(N-acetyl-GM.sub.2 ; Boehringer Mannheim) or some other ganglioside. The 
solution or dilutions thereof were respectively distributed in 20-.mu.l 
portions into wells of 96-well microtiter plates (Greiner) and, after air 
drying, blocking was effected with PBS containing 1% BSA. Each culture 
supernatant for each transformant, each purified mouse monoclonal antibody 
solution and each purified chimeric human antibody solution were 
distributed in 50- to 100-.mu.l portions into the wells and the reaction 
was allowed to proceed at room temperature for 1 to 2 hours. The wells 
were then washed with PBS, and 50 to 100 .mu.l of peroxidase-labeled 
antibody were added thereto followed by reaction at room temperature for 1 
to 2 hours. The wells were washed with PBS and an ABTS substrate solution 
prepared by dissolving 550 mg of 
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in 
0.1M citrate buffer (pH 4.2) and adding, just prior to use, hydrogen 
peroxide to a concentration of 1 .mu.l/ml! was added in 50- to 100-.mu.l 
portions to each well for color development, followed by OD.sub.415 
measurement. 
The clone showing the highest activity in ELISA among the clones obtained 
gave a chimeric human anti-GM.sub.2 antibody content of about 1.0 .mu.g/ml 
of culture fluid. 
Cells of the clone showing the above-mentioned chimeric human anti-GM.sub.2 
antibody activity were suspended in RPMI1640-FCS(10) medium containing 0.5 
mg/ml G418 and 50 nM methotrexate (hereinafter, "MTX") to a concentration 
of 1 to 2.times.10.sup.5 cells/ml, and the suspension was distributed in 
2-ml portions into wells of 24-well plates. Incubation was performed at 
37.degree. C. in a CO.sub.2 incubator for 1 to 2 weeks to induce 50 nM 
MTX-resistant clones. At the time of confluence, the chimeric human 
anti-GM.sub.2 antibody activity in each culture fluid was determined by 
ELISA. The 50 nM MTX-resistance clone showing the highest activity among 
the clones obtained showed a chimeric human anti-GM.sub.2 antibody content 
of about 5.0 .mu.g/ml. 
Cells of the above 50 nM MTX-resistant clone were suspended in 
RPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to a 
concentration of 1 to 2.times.10.sup.5 cells/ml, and the suspension was 
distributed in 2-ml portions into wells of 24-well plates. Incubation was 
carried out at 37.degree. C. in a CO.sub.2 incubator for 1 to 2 weeks to 
induce 200 nM MTX-resistant clones. At the time of confluence, each 
culture fluid was assayed for chimeric human anti-GM.sub.2 antibody 
activity by ELISA. The 200 nM MTX-resistant clone showing the highest 
activity among the clones obtained had a chimeric human anti-GM.sub.2 
antibody content of about 10 .mu.g/ml. The 200 nM MTX-resistant clones 
obtained from pChi750HL1 and pChi796HL1 were named transformants "KM966" 
(KM-796-derived chimeric human antibody KM966-producing strain) and 
"KM967" (KM-750-derived chimeric human antibody KM967-producing strain), 
respectively. 
The following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed 
that the above transformants KM966 and KM967 express the respective 
chimeric human anti-GM.sub.2 antibodies. 
The transformants KM966 and KM967 were each suspended in GIT medium (Nippon 
Pharmaceutical) containing 0.5 mg/ml G418 and 200 nM MTX to a 
concentration of 1 to 2.times.10.sup.5 cells/ml. Each suspension was 
distributed in 100-ml portions into 175 cm.sup.2 flasks (Greiner). 
Cultivation was performed at 37.degree. C. in a CO.sub.2 incubator for 5 
to 7 days. At the time of confluence, the culture fluid was recovered. 
Treatment of about 1 liter of the culture fluid with Affi-Gel Protein A 
MAPS-II kit (Bio-Rad) gave about 5 mg of a purified chimeric human 
anti-GM.sub.2 antibody for each transformant. About 2 .mu.g of the 
purified chimeric human anti-GM.sub.2 antibody KM966 or KM967 was 
electrophoresed by the conventional method Laemmli: Nature, 227, 680 
(1970)! for molecular weight checking. The results are shown in FIG. 18. 
As shown in FIG. 18, both KM966 and KM967 gave an antibody H chain 
molecular weight of about 50 kilodaltons and an antibody L chain molecular 
weight of about 25 kilodaltons under reducing conditions, indicating the 
correctness in molecular weight of the H chain and L chain expressed. For 
each of KM966 and KM967, the molecular weight of the chimeric human 
antibody under nonreducing conditions was about 150 kilodaltons, 
indicating that the antibody expressed was composed of two H chains and 
two L chains and was correct in size. 
12. Expression of KM-603-Derived Chimeric Human Anti-GM.sub.2 Antibodies in 
SP2/0 Cells 
A 2-.mu.g portion of the plasmid pChi603HMS1 or pChi603LMS1 obtained in 
Paragraph 9 was introduced into 4.times.10.sup.6 cells of YB2/0 (ATCC 
CRL1581) by electroporation in the same manner in Paragraph 11. The cells 
were suspended in 40 ml of RPMI1640-FCS(10) and the suspension was 
distributed in 200-.mu.l portions into wells of 96-well microtiter plates. 
After 24 hours of incubation in a CO.sub.2 incubator at 37.degree. C., 
G418 (Gibco) was added to a concentration of 0.5 mg/ml and incubation was 
continued for 1 to 2 weeks. Transformant colonies appeared. The culture 
fluid was recovered from confluent wells and the chimeric human 
anti-GM.sub.2 antibody activity was measured by ELISA as described above. 
The clone showing the highest chimeric human anti-GM.sub.2 antibody 
activity among the clones obtained gave a chimeric human anti-GM.sub.2 
antibody content of about 0.1 .mu.g/ml of culture fluid. 
Cells of the clone showing the above-mentioned chimeric human anti-GM.sub.2 
antibody activity were suspended in RPMI1640-FCS(10) medium containing 0.5 
mg/ml G418 and 50 nM MTX to a concentration of 1 to 2.times.10.sup.5 
cells/ml and the suspension was distributed in 2-ml portions into wells of 
24-well plates. Clones resistant to 50 nM MTX were induced by incubating 
in a CO.sub.2 incubator at 37.degree. C. for 2 to 3 weeks. When confluence 
was attained, the culture fluids were subjected to ELISA for chimeric 
human anti-GM.sub.2 antibody activity measurement. Among the 50 nM 
MTX-resistant clones obtained, the clone showing the highest activity gave 
a chimeric human anti-GM.sub.2 antibody content of about 0.3 .mu.g/ml of 
culture fluid. 
Cells of the above 50 nM MTX-resistant clone were suspended in 
RPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to a 
concentration of 1 to 2.times.10.sup.5 cells/ml and the suspensions as 
distributed in 2-ml portions into well of 24-well plates. Clones resistant 
to 200 nM MTX were induced by following incubation in a CO.sub.2 incubator 
at 37.degree. C. for 2 to 3 weeks. When confluence was attained, the 
chimeric human anti-GM.sub.2 antibody activity in the culture fluid was 
measured by ELISA. Among the 200 nM MTX-resistant clones obtained, the 
clone showing the highest activity gave a chimeric human anti-GM.sub.2 
antibody content of about 0.5 .mu.g/ml of culture fluid. 
Cells of the above 200 nM MTX-resistant clone were suspended in 
RPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 500 nM MTX to a 
concentration of 1 to 2.times.10.sup.5 cells/ml and the suspension was 
distributed in 2-ml portions into well of 24-well plates. Clones resistant 
to 500 nM MTX were induced following incubation in a CO.sub.2 incubator at 
37.degree. C. for 1 to 2 weeks. When confluence was attained, the chimeric 
human anti-GM.sub.2 antibody activity in the culture fluid was determined 
by ELISA. Among the 500 nM MTX-resistant clones obtained, the one showing 
the highest activity gave a chimeric human anti-GM.sub.2 antibody content 
of about 1.0 .mu.g/ml of culture fluid. This 500 nM MTX-resistant clone 
was named "transformant KM968". 
The following SDS-PAGE confirmed the expression of a chimeric human 
anti-GM.sub.2 antibody in the above transformant KM968. 
Cells of the transformant KM968 were suspended in GIT medium (Nippon 
Pharmaceutical) containing 0.5 mg/ml G418 and 500 nM MTX to a 
concentration of 1 to 2.times.10.sup.5 cells/ml and the suspension was 
distributed in 100-ml portions into 175 cm.sup.2 flasks (Greiner). 
Cultivation was conducted in a CO.sub.2 incubator at 37.degree. C. for 5 
to 7 days and, when confluence was attained, the culture fluid was 
recovered. Treatment of about 3.0 liters of the culture fluid with 
Affi-Gel Protein A MAPS-II kit (Bio-Rad) gave about 1 mg of a purified 
chimeric human anti-GM.sub.2 antibody. About 2 .mu.g of this purified 
chimeric human anti-GM.sub.2 antibody KM968 was electrophoresed by the 
conventional method Laemmli: Nature, 227, 680 (1970)! for molecular 
weight checking. The results are shown in FIG. 19. Under reducing 
conditions, the molecular weight of the antibody H chain was about 50 
kilodaltons and the molecular weight of the antibody L chain was about 25 
kilodaltons, thus confirming the expression of the H chain and L chain 
having the correct molecular weight. Under nonreducing conditions, the 
molecular weight of the chimeric human antibody was about 150 kilodaltons, 
confirming that the antibody expressed was composed of two H chains and 
two L chains and was correct in size. 
13. Reaction Specificity of the Chimeric Human Anti-GM.sub.2 Antibodies 
The reactivities of the chimeric anti-GM.sub.2 antibodies with ganglioside 
GM.sub.1, N-acetyl-GM.sub.2 (Boehringer Mannheim), N-glycolyl-GM.sub.2, 
N-acetyl-GM.sub.3, N-glycolyl-GM.sub.3, GD.sub.1a, GD.sub.1b (Iatron), 
GD.sub.2, GD.sub.3 (Iatron) and GQ.sub.1b (Iatron) were examined by the 
technique of ELISA. The results are shown below in Table 1. GM.sub.1 and 
GD.sub.1a were purified from the bovine brain, N-glycolyl-GM.sub.2 and 
N-glycolyl-GM.sub.3 from the mouse liver, N-acetyl-GM.sub.3 from canine 
erythrocytes, and GD.sub.2 from the cultured cell line IMR32 (ATCC 
CCL127), by a known method J. Biol. Chem., 263, 10915 (1988)!. 
As shown in Table 1, it was confirmed that the chimeric human anti-GM.sub.2 
antibodies KM966 and KM967 specifically react with GM.sub.2. The 
reactivity of KM966 was greater than that of KM967, however. On the 
contrary, KM968 (KM-603-derived chimeric human antibody) did not show any 
reactivity for GM.sub.2. 
TABLE 1 
______________________________________ 
Binding activity of antibody (OD.sub.415) 
Ganglioside KM966 (5 .mu.g/ml) 
KM967 (5 .mu.g/ml) 
______________________________________ 
GM.sub.1 0.105 0.000 
N-Acetyl-GM.sub.2 
0.870 0.423 
N-Glycolyl-GM.sub.2 
0.774 0.065 
N-Acetyl-GM.sub.3 
0.002 0.000 
N-Glycolyl-GM.sub.3 
0.122 0.001 
GD.sub.1a 0.004 0.000 
GD.sub.1b 0.002 0.000 
GD.sub.2 0.095 0.001 
GD.sub.3 0.004 0.000 
GQ.sub.1b 0.005 0.000 
______________________________________ 
14. Reactivities of the Chimeric Human Anti-GM.sub.2 Antibodies KM966 and 
KM967 With Cancer Cells (Fluorescent Antibody Technique) 
Suspended in PBS were 1.times.10.sup.6 cells of cultured human lung small 
cell carcinoma cell line QC90 Cancer Res., 49, 2683 (1989)!, NCI-H69 
(ATCC HTB119), NCI-H128 (ATCC HTB120), SBC-1 (JCRB 0816), SBC-2 (JCRB 
0817), SBC-3 (JCRB 0818), SBC-5 (JCRB 0819), RERF-LC-MA (JCRB 0812), 
Lu-134-A-H (JCRB 0235), Lu-139 (RCB 469), Lu-130 (RCB 465), Lu-135 (RCB 
468), Lu-134-B (RCB 467), Lu-140 (RCB 470), PC-6 Naito et al.: Gann to 
Kagaku Ryoho (Cancer and Chemotherapy), 5 (suppl.), 89 (1978)!, cultured 
human lung squamous carcinoma cell line PC-1 Naito et al.: Gann to Kagaku 
Ryoho, 5 (suppl.), 89 (1978)!, PC-10 Naito et al.: Gann to Kagaku Ryoho, 
5 (suppl.), 89 (1978)!, Colol6 Moor et al.: Cancer Res., 35, 2684 
(1975)!, Calu-1 (ATCC HTB54), SK-LC-4 Proc. Natl. Acad. Sci. U.S.A., 85, 
4441 (1988)!, cultured human lung adenocarcinoma cell line PC-7 Hayata et 
al.: Hito Gansaibo no Baiyo (Human Cancer Cell Culture), 131 (1975)!, PC-9 
Kinjo et al.: Brit. J. Cancer, 39, 15 (1979)!, PC-12 (ATCC CRL1721), 
RERF-LC-MS (JCRB 0081), HLC-1 (RCB 083), cultured human lung large cell 
carcinoma cell line PC-13 Oboshi et al.: Tanpakushitsu, Kakusan, Koso 
(Protein, Nucleic Acid, Enzyme), 23, 697 (1978)!, Lu65 (JCRB 0079), CALU-6 
(ATCC HTB56), SK-LC-6 Proc. Natl. Acad. Sci. U.S.A., 85, 4441 (1988)!, 
cultured human neuroblastoma cell line YT-nu Ishikawa et al.: Acta Path. 
Jap., 27, 697 (1977)!, NAGAI Ishikawa et al.: Acta Path. Jap., 29, 289 
(1979)!, NB-1 Ishikawa et al.: Acta Path. Jap., 27, 697 (1977)!, IMR32 
(ATCC CCL127), GOTO (JCRB 0612), NB-9 (RCB 477), SK-N-MC (ATCC HTB10), 
cultured human brain tumor (glioma) cell line SK-MG-4 EMBO J., 6, 2939 
(1987)!, A172 (ATCC CRL1620), T98G (ATCC CRL1690), U-118MG (ATCC HTB15), 
cultured human leukemia cell line HSB-2 (ATCC CCL120.1), ATN-1, U-937 
(ATCC CRL1593), HPB-ALL Oboshi et al.: Tanpakushitsu, Kakusan, Koso, 23, 
697 (1978)!, CCRF-SB (ATCC CCL120), KOPN-K Hanei et al.: Haigan (Lung 
Cancer), 25, 524 (1985)!, TYH Haranaka et al.: Int. J. Cancer, 36, 313 
(1985)!, MOLT-3 (ATCC CRL1552), CCRF-CEM (ATCC CCL119), TALL-1 (JCRB 
0086), NALL-1 Oboshi et al.: Tanpakushitsu, Kakusan, Koso, 23, 697 
(1978), CCRF-SB (JCRB 0032), THP-1 (ATCC TIB202), HEL92-1-7 (ATCC TIB180), 
cultured human maligant melanoma cell line C24.cndot.32 (EP-A-0 493686), 
KHm-3/P J. Natl. Cancer Inst., 59, 775 (1977)! or G361 (ATCC CRL1424). 
The suspension was placed in a microtube (Tref) and centrifuged (3,000 
rpm, 2 minutes) to wash the cells, 50 .mu.l of KM966 or KM967 (50 
.mu.g/ml) was added, the mixture was stirred, and the reaction was allowed 
to proceed at 4.degree. C. for 1 hour. Then, the cells were washed three 
times by centrifugation with PBS, 20 .mu.l of fluorescein 
isocyanate-labeled protein A (30-fold dilution; Boehringer Mannheim 
Yamanouchi) was added and, after stirring, the reaction was allowed to 
proceed at 4.degree. C. for 1 hour. Then, the cells were washed three 
times by centrifugation with PBS, then suspended in PBS and subjected to 
analysis using flow cytometer EPICS Elite (Coulter). In a control run, the 
same procedure as described above was followed without adding the chimeric 
human antibody. The results thus obtained are shown in Table 2. The 
chimeric human antibody KM966 reacted with 9 (NCI-H-128, SBC-1, SBC-3, 
SBC-5, Lu-139, Lu-130, Lu-135, Lu-134-B and Lu-140) of the 14 lung small 
cell carcinoma cell lines, 2 (PC-10 and Calu-1) of the 5 lung squamous 
carcinoma cell lines, 2 (PC-9 and RERF-LC-MS) of the 5 lung adenocarcinoma 
cell lines, 2 (PC-13 and SK-LC-6) of the 4 lung large cell carcinoma cell 
lines, 7 (YT-nu, NAGAI, NB-1, IMR32, GOTO, NB-9 and SK-N-MC) of the 7 
neuroblastoma cell lines and 4 (SK-MG-4, A172, T98G and U-118MG) of the 4 
brain tumor (glioma) cell lines. On the other hand, the chimeric human 
antibody KM967 did not react with any of the cultured cell lines. The 
above results indicate that the chimeric human antibody KM966 is useful in 
the diagnosis and treatment of brain tumors, peripheral nervous system 
tumors and lung cancer, among others. 
TABLE 2 
______________________________________ 
KM966 (%) KM967 (%) 
Cell line (50 .mu.g/ml) (50 .mu.g/ml) 
______________________________________ 
Lung small cell carcinoma 
9/14 (64) 0/14 (0) 
Lung squamous cell 
2/5 (40) 0/5 (0) 
carcinoma 
Lung adenocarcinoma 
2/5 (40) 0/5 (0) 
Lung large cell carcinoma 
2/4 (50) 0/4 (0) 
Neuroblastoma 7/7 (100) 0/7 (0) 
Brain tumor (glioma) 
4/4 (100) 0/4 (0) 
Leukemia 0/14 (0) 0/14 (0) 
Malignant melanoma 
0/3 (0) 0/3 (0) 
______________________________________ 
15. In Vitro Antitumor Activity of the Chimeric Human Anti-GM.sub.2 
Antibody KM966: Complement Dependent Cytotoxicity (CDC) 
(1) Preparation of target cells 
The target cells SBC-3, Lu-135, PC-10, RERF-LC-MS, PC-13, NAGAI, GOTO or 
A172, cultured in RPMI1640 medium supplemented with 10% FCS, were adjusted 
to a cell concentration of 5.times.10.sup.6 cells/ml, Na.sub.2.sup.51 
CrO.sub.4 was added to a concentration of 100 .mu.Ci/5.times.10.sup.6 
cells, then the reaction was allowed to proceed at 37.degree. C. for 1 
hours, and the cells were washed three times with the medium. The cells 
were then allowed to stand in the medium at 4.degree. C. for 30 minutes 
for spontaneous dissociation and then, after centrifugation, the medium 
was added to adjust the cell concentration to 1.times.10.sup.6 cells/ml. 
(2) Preparation of the complement 
Sera from three healthy subjects were combined and used as a complement 
source. 
(3) CDC activity measurement 
The chimeric human anti-GM.sub.2 antibody KM966 or mouse anti-GM.sub.2 
antibody KM696 (FERM BP-3337) was added to wells of 96-well U-bottom 
plates within the final concentration range of 0.5 to 50 .mu.g/ml and then 
5.times.10.sup.4 cells/well of the target cells prepared in (1) were 
added. The reaction was allowed to proceed at room temperature for 30 
minutes. After centrifugation, the supernatants were discarded, 150 .mu.l 
of the human serum obtained in (2) was added to each well (final 
concentration 15% v/v), and the reaction was allowed to proceed at 
37.degree. C. for 1 hour. After centrifugation, the amount of .sup.51 Cr 
in each supernatant was determined using a gamma counter. The amount of 
spontaneously dissociated .sup.51 Cr was determined by adding to the 
target cells the medium alone in lieu of the antibody and complement 
solutions and measuring the amount of .sup.51 Cr in the supernatant in the 
same manner as mentioned above. The total amount of dissociated .sup.51 Cr 
was determined by adding to the target cells 5 N sodium hydroxide in lieu 
of the antibody and complement solutions and measuring the amount of 51Cr 
in the supernatant in the same manner as mentioned above. The CDC activity 
was calculated as follows: 
##EQU1## 
The results thus obtained are shown in FIGS. 20 to 23. It was shown that 
the chimeric human antibody KM966 show CDC activity against all the cells 
tested. 
16. In Vitro Antitumor Activity of the Chimeric Human Anti-GM.sub.2 
Antibody KM966: Antibody Dependent Cell Mediated Cytotoxicity (ADCC) 
(1) Preparation of target cells 
The target cells SBC-3, Lu-135, PC-10, RERF-LC-MS, PC-13, NAGAI, GOTO or 
A172, cultured in RPMI1640 medium supplemented with 10% FCS, were adjusted 
to a cell concentration of 1.times.10.sup.6 cells/ml, Na.sub.2.sup.51 
CrO.sub.4 was added to a concentration of 50 .mu.Ci/1.times.10.sup.6 
cells, then the reaction was allowed to proceed at 37.degree. C. for 1 
hour, and the cells were washed three times with the medium. The cells 
were then allowed to stand in the medium at 4.degree. C. for 30 minutes 
for spontaneous dissociation and then, after centrifugation, the medium 
was added to adjust the cell concentration to 2.times.10.sup.5 cells/ml. 
(2) Preparation of effector cells 
Human venous blood (25 ml) was collected, 0.5 ml of heparin sodium (Takeda 
Chemical Industries; 1,000 units/ml) was added, and the mixture was gently 
stirred. This mixture was centrifuged (1,500 to 1,800 g, 30 minutes) using 
Polymorphprep (Nycomed Pharma AS), the lymphocyte layer was separated and 
washed three times by centrifugation with RPMI1640 medium (15,00 to 1,800 
g, 15 minutes), and the cells were suspended in RPMI1640 medium 
supplemented with 10% FCS (5.times.10.sup.6 cells/ml) for use as effector 
cells. 
(3) ADCC activity measurement 
The chimeric human anti-GM.sub.2 antibody KM966 or mouse anti-GM.sub.2 
antibody KM696 were added to wells of 96-well U-bottom plates within the 
final concentration range of 0.05 to 5 .mu.g/ml and then 50 .mu.l 
(1.times.10.sup.4 cells/well) of the target cell suspension prepared in 
(1) and 100 .mu.l (5.times.10.sup.5 cells/well) of the effector cell 
suspension prepared in (2) were added to each well (the ratio between the 
effector cells and target cells being 50:1). The reaction was allowed to 
proceed at 37.degree. C. for 4 hours and, after centrifugation, the amount 
of .sup.51 Cr in each supernatant was measured using a gamma counter. The 
amount of spontaneously dissociated .sup.51 Cr was determined by adding to 
the target cells the medium alone in lieu of the antibody and effector 
cells and measuring the amount of .sup.51 Cr in the supernatant in the 
same manner as mentioned above. The total amount of dissociated .sup.51 Cr 
was determined by adding to the target cells 5N sodium hydroxide in lieu 
of the antibody and effector cells and measuring the amount of .sup.51 Cr 
in the supernatant in the same manner as mentioned above. The ADCC 
activity was calculated as follows: 
##EQU2## 
The results thus obtained are shown in FIGS. 24 to 27. The chimeric 
antibody KM966 showed ADCC activity against all the cells whereas the 
mouse anti-GM.sub.2 antibody KM696 showed no or low ADCC activity. The 
above results indicate that the chimeric human antibody KM966 is more 
effective in the treatment of human cancer than the mouse antibody KM-696. 
REFERENCE EXAMPLE 1 
Construction of the Vector pChiIgLA1 for Chimeric Human Antibody L Chain 
Expression 
1. Isolation of the KM50 Cell-Derived Immunoglobulin H Chain Promoter and 
Enhancer Genes 
(1) Preparation of chromosomal DNAs from KM50 cells, P3U1 cells and rat 
kidney 
Chromosal DNAs were prepared by the conventional method Maniatis et al. 
(ed.): Molecular Cloning, 1989, p. 9.16!, as follows. 
KM50 cells (1.2.times.10.sup.8 cells), P3U1 cells (ATCC CRL1597) 
(2.times.10.sup.8 cells) and a rat kidney sample (frozen at -80.degree. C. 
and then smashed to a sufficient extent using a wooden hammer) (1.6 g) 
were suspended in 2 ml of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 150 mM sodium chloride and 10 mM ethylenediaminetetraacetic 
acid disodium salt (hereinafter, "EDTA"), 0.8 mg of proteinase K (Sigma) 
and 10 mg of sodium lauryl sulfate (hereinafter, "SDS"), were added to 
each suspension, and the suspension was incubated at 37.degree. C. for 10 
hours. Then, each mixture was extracted once with an equal volume of 
phenol, twice with an equal volume of chloroform and then once with an 
equal volume of ether, and dialyzed for 10 hours against 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 1 mM EDTA. The DNA solution 
was recovered from the dialysis tube and ribonuclease A (Sigma) was added 
to the solution to a final concentration of 20 .mu.g/ml. Each resultant 
solution was incubated at 37.degree. C. for 6 hours for sufficient 
decomposition of RNA, 15 mg of SDS and 1 mg of proteinase K were then 
added and the mixture was incubated at 37.degree. C. for 10 hours. The 
mixture was then extracted twice with an equal volume of phenol, twice 
with an equal volume of chloroform and twice with an equal volume of ether 
and then dialyzed for 10 hours against 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 1 mM EDTA. The DNA solution was recovered from the 
dialysis tube for use as a chromosomal DNA sample. DNA concentration 
measurement in terms of the absorbance at 260 nm revealed that the yield 
of chromosomal DNA from 1.2.times.10.sup.8 KM50 cells was 1.6 mg, that 
from 2.times.10.sup.8 P3U1 cells 1.5 mg, and that from 1.6 g of rat liver 
1.9 mg. 
(2) Identification of the active-form immunoglobulin H chain gene in KM50 
cells by Southern blotting 
The KM50 cell, p3U1 cell and rat kidney chromosomal DNAs obtained in (1) (3 
.mu.g each) were dissolved in 25 .mu.l of 10 mM Tris-hydrochloride buffer 
(pH 7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 15 
units of XbaI (Takara Shuzo; hereinafter the restriction enzymes used were 
products of Takara Shuzo) was added and incubation was carried out at 
37.degree. C. for 2 hours for effecting cleavage at the XbaI sites. Each 
reaction mixture was subjected to agarose gel electrophoresis, then DNA 
transfer onto a nitrocellulose filter was effected by the method of 
Southern et al. J. Mol. Biol., 98, 503 (1975)! and hybridization was 
carried out by the conventional method Kameyama et al.: FEBS Letters, 
244, 301-306 (1989)! using the mouse JH probe described in the last-cited 
reference. The KM50 cell DNA alone gave a band at a site corresponding to 
about 9.3 kb. Therefore, the immunoglobulin XbaI fragment DNA was 
considered to code for the active-form immunoglobulin H chain gene in KM50 
cells. (3) Construction of a KM50 cell genomic DNA library A 60-.mu.g 
portion of the KM50 cell-derived chromosomal DNA obtained in (1) was 
dissolved in 250 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride and 100 mM sodium chloride, 150 units 
of XbaI was added, and incubation was conducted at 37.degree. C. for 2 
hours for causing cleavage at the XbaI sites. The reaction mixture was 
fractionated by agarose gel electrophoresis and a KM50 cellderived 9.3 kb 
DNA fraction sample (about 2 .mu.g) was recovered using, for example, the 
DEAE paper method Maniatis et al. (ed.): Molecular Cloning, 1989, p. 
6.24!. Separately, 3 .mu.g of Lambda ZAP (Stratagene), for use as the 
vector, was dissolved in 200 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 50 
units of XbaI was added, and the mixture was incubated at 37.degree. C. 
for 2 hours to effect cleavage at the XbaI sites. The reaction mixture was 
subjected to phenol-chloroform extraction and then to ethanol 
precipitation, whereby about 3 .mu.g of DNA was recovered. This DNA was 
dissolved in 100 .mu.l of 100 mM Tris-hydrochloride buffer (pH 7.5), 1 
unit of alkaline phosphatase (Takara Shuzo) was added, dephosphorylation 
was effected at the restriction enzyme cleavage ends of the vector DNA. 
The reaction mixture was subjected to phenol-chloroform extraction and 
then to ethanol precipitation, whereby 2 .mu.g of DNA was recovered. This 
DNA was dissolved in 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 1 mM EDTA for use as a vector sample. Two tenths .mu.g of the 
vector DNA sample and 0.2 .mu.g of the KM50 cell-derived 9.3 kb DNA sample 
were dissolved in 5 .mu.l of T4 ligase buffer, 175 units of T4 ligase 
(Takara Shuzo) was added, and the mixture was incubated at 4.degree. C. 
for 3 days. A 2-.mu.l portion of this mixture was packaged into the lambda 
phage by the conventional method Maniatis et al. (ed.): Molecular 
Cloning, 1989, p. 2.95! using Giga Pak Gold (Stratagene), and the 
packaging mixture was used to transfect Escherichia coli BB4 to give 
200,000 phage clones. Among them, 100,000 clones were fixed on a 
nitrocellulose filter by the conventional method Maniatis et al. (ed.): 
Molecular Cloning, 1989, p. 2.112!. 
(4) Selection of a recombinant DNA containing the gene for the H chain 
variable region of an immunoglobulin occurring as an active form in KM50 
cells (anti-human serum albumin) 
From among the phage library composed of 100,000 clones, as constructed in 
(3), two clones firmly associable at 65.degree. C. with the .sup.32 
P-labeled mouse JH probe labeled by the method of Kameyama et al. FEBS 
Letters, 44, 301-306 (1989)!! were isolated. The phage DNA was recovered 
from them by the conventional method Maniatis et al. (ed.): Molecular 
Cloning, 1989, p. 2.118-2.169!, whereupon the 9.3 kb XbaI fragment of the 
KM50 cell-derived chromosomal DNA was found to have been inserted therein. 
(5) Base sequence of the gene for the H chain variable region of the 
immunoglobulin occurring as an active form in KM50 cells (anti-human serum 
albumin) 
For the two clones obtained in (4), restriction enzyme cleavage maps were 
prepared by conducting digestion using various restriction enzymes, 
whereby it was revealed that the same DNA fragment (9.3 kb) had been 
inserted therein (FIG. 28). Therefore, those portions of this 9.3 kb DNA 
fragment which were supposed to be coding for the rat immunoglobulin H 
chain promoter region and variable region were sequenced by the method of 
Sanger Sanger et al.: Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977); 
AMERSHAM M13 cloning and sequencing handbook!. In SEQ ID NO:16, the 
portion containing the octamer sequence such as ATGCAAAT and the TATA box 
sequence such as TTGAAAA is considered to be the immunoglobulin promoter 
region. 
2. Construction of Heterologous Protein Expression Vectors Using the 
Promoter and Enhancer For the H Chain Variable Region Gene for an 
Immunoglobulin Occurring as an Active Form in KM50 cells (anti-human serum 
albumin) 
(1) Construction of pKMB11 
A 1-.mu.g portion of the 9.3 kb immunoglobulin H chain variable region gene 
fragment obtained in Paragraph 1 (5) was dissolved in 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 10 units each of BglII and HindIII were added, and 
the mixture was incubated at 37.degree. C. for 2 hours for causing 
cleavage at the BglII and HindIII sites. The reaction mixture was 
subjected to agarose gel electrophoresis and 0.01 .mu.g of a DNA fragment 
containing the 0.8 kb immunoglobulin promoter was recovered. Then, 1 .mu.g 
of the plasmid pBR322-BglII Kuwana et al.: FEBS Letters, 219, 360 (1987)! 
was dissolved in 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 units of 
BglII and 10 units of HindIII were added, and the mixture was incubated at 
37.degree. C. for 2 hours to effect cleavage at the BglII and HindIII 
sites. The reaction mixture was subjected to agarose gel electrophoresis 
and a DNA fragment of about 4.2 kb in size was recovered. The 
thus-obtained pBR322-BglII-derived DNA fragment (about 4.2 kb, 0.1 .mu.g) 
and immunoglobulin promoter-containing DNA fragment (0.01 yg) were 
dissolved in 20 .mu.l of T4 ligase buffer, 175 units of T4 DNA ligase 
(Takara Shuzo) was added, and the mixture was incubated at 4.degree. C. 
for 1 day. The reaction mixture was used to transform Escherichia coli 
HB101 J. Mol. Biol., 41, 459 (1969)! by the method of Scott et al. 
Masaru Shigesada: Saibo Kokagu (Cell Engineering), 2, 616 (1983)! to give 
an Ap-resistant colony. The recombinant plasmid DNA was recovered from 
this colony. Plasmid pKMB11, shown in FIG. 29, was thus obtained. 
(2) Construction of pKMD6 
For providing an appropriate restriction enzyme site downstream from the 
immunoglobulin promoter, the plasmid pKMB11 constructed in (1) was 
digested at the NcoI site using the nuclease BAL31. Thus, 10 .mu.g of the 
plasmid pKMB11 was dissolved in 100 .mu.l of 10 mM Tris-hydrochloride 
buffer (pH 7.5) containing 6 mM magnesium chloride and 50 mM potassium 
chloride, 30 units of NcoI was added, and the mixture was incubated at 
37.degree. C. for 2 hours to effect cleavage at the NcoI site. The 
reaction mixture was subjected to phenol-chloroform extraction and then to 
ethanol precipitation, the whole amount of the DNA fragment was dissolved 
in 100 .mu.l of BAL31 buffer (20 mM Tris-hydrochloride buffer (pH 8.0) 
containing 600 mM sodium chloride, 12 mM calcium chloride, 12 mM magnesium 
chloride and 1 mM EDTA!, 0.25 unit of BAL31 Bethesda Research 
Laboratories (BRL)!! was added, and digestion was carried out at 
37.degree. C. for 5 seconds. The reaction was terminated by extraction 
with phenol and subjected to chloroform extraction and then to ethanol 
precipitation, and 1 .mu.g of DNA was recovered. A 0.1-.mu.g portion of 
this DNA and 0.01 .mu.g of a synthetic DNA linker (SalI) were dissolved in 
20 .mu.l of T4 ligase buffer, 175 units of T4 DNA ligase was added, and 
the mixture was incubated at 4.degree. C. for 1 day. The reaction mixture 
was used to transform Escherichia coli HB101 by the method of Scott et al. 
An Ap-resistant colony was obtained and the recombinant plasmid DNA was 
recovered from this colony to give the plasmid pKMD6 shown in FIG. 30. For 
this plasmid, the portion of BAL31 digestion was sequenced by the method 
of Sanger, whereupon deletion was found to the third base (303rd base in 
SEQ ID NO:16) toward the upstream of the initiation codon ATG for 
immunoglobulin. 
(3) Construction of pEPKMA1, pEPKMB1 and pAGE501 
The original immunoglobulin promoter and enhancer are positionally 
separated. Therefore, it was necessary to construct a vector containing 
the promoter and enhancer connected to each other for use of said vector 
as a heterologous protein expression vector. Accordingly, the following 
procedure was followed. 
Thus, 1 .mu.g of the 9.3 kb immunoglobulin H chain variable region gene 
obtained in Paragraph 1 (5) was dissolved in 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 10 units of EcoRV and 10 units of XbaI were added, 
and the mixture was incubated at 37.degree. C. for 2 hours for causing 
cleavage at the EcoRV and XbaI sites. The reaction mixture was subjected 
to agarose gel electrophoresis and 0.1 .mu.g of a DNA fragment (about 1 
kb) containing the immunoglobulin enhancer region was recovered. 
Separately, 1 .mu.g of the plasmid pKMD6 obtained in (2) was dissolved in 
100 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM 
magnesium chloride and 100 mM sodium chloride, 10 units of BglII was 
added, and the mixture was incubated at 37.degree. C. for 2 hours to 
effect cleavage at the BglII site. After phenol-chloroform extraction, the 
DNA was precipitated with ethanol and dissolved in a total of 40 .mu.l of 
DNA polymerase I buffer, 6 units of Escherichia coli-derived DNA 
polymerase I Klenow fragment was added, and the reaction was allowed to 
proceed at 16.degree. C. for 90 minutes for rendering the 5' protruding 
ends formed upon BglII digestion blunt-ended. The reaction was terminated 
by extraction with phenol, the mixture was extracted with chloroform and 
then subjected to ethanol precipitation, the DNA obtained was dissolved in 
30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM 
magnesium chloride and 50 mM sodium chloride, 10 units of HindIII was 
added, and the mixture was incubated at 37.degree. C. for 2 hours to 
effect cleavage at the HindIII site. The reaction mixture was subjected to 
agarose gel electrophoresis and 0.1 .mu.g of a DNA fragment (about 0.8 kb) 
containing the immunoglobulin promoter region was recovered. Then, 0.2 
.mu.g of the plasmid pUC18 Messing: Methods in enzymology 101, 20 (1983)! 
was dissolved in 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 units of 
HindIII and 10 units of XbaI were added, and the mixture was incubated at 
37.degree. C. for 2 hours for causing cleavage at the HindIII and XbaI 
sites. The reaction mixture was subjected agarose gel electrophoresis and 
0.1 .mu.g of a DNA fragment of about 2.7 kb in size was recovered. The 
thus-obtained pPKMD6-derived 0.8 kb DNA fragment (0.1 .mu.g), 
immunoglobulin enhancer region-containing DNA fragment (0.02 .mu.g) and 
pUC18 (0.1 .mu.g) were dissolved in 20 .mu.l of T4 ligase buffer, 175 
units of T4 DNA ligase was added, and the mixture was incubated at 
4.degree. C. for 1 day. The reaction mixture was used to transform 
Escherichia coli HB101 to give an Ap-resistant colony. The recombinant 
plasmid DNA was recovered from this colony to give pEPKMA1 shown in FIG. 
31. 
Then, 1 .mu.g of the plasmid pEPKMA1 was dissolved in 100 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 10 units of XbaI was added, and the mixture was 
incubated at 37.degree. C. for 2 hours for causing cleavage at the XbaI 
site. After phenol-chloroform extraction, the resultant DNA fragment was 
precipitated with ethanol and dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 6 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was allowed to proceed at 
16.degree. C. for 90 minutes for rendering the cohesive ends formed upon 
XbaI digestion blunt-ended. The reaction was terminated by extraction with 
phenol and, after chloroform extraction, the DNA fragment was recovered by 
ethanol precipitation. This DNA fragment and a synthetic DNA linker XhoI 
(Takara Shuzo) (0.01 .mu.g) were dissolved in 20 .mu.l of T4 ligase 
buffer, 175 units of T4 DNA ligase was added, and the mixture was 
incubated at 4.degree. C. for 1 day. The reaction mixture was used to 
transform Escherichia coli HB101 to give an Ap-resistant colony. The 
recombinant plasmid DNA was recovered from this colony to give pEPKMB1 
shown in FIG. 32. 
Then, the SV40 early gene promoter and enhancer regions (hereinafter 
abbreviated as P.sub.SE) of the heterologous gene expression vector 
pAGE107 for use in animals Miyaji et al.: Cytotechnology, 3, 133-140 
(1990)! were replaced with the KM50-derived immunoglobulin H chain 
promoter and enhancer (hereinafter abbreviated as P.sub.IH) of pEPKMB1 in 
the following manner. 
One .mu.g of the plasmid pAGE107 was dissolved in 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
150 mM sodium chloride, 10 units of SalI and 10 units of XhoI were added, 
and the mixture was incubated at 37.degree. C. for 2 hours to effect 
cleavage at the SalI and XhoI sites. The reaction mixture was subjected to 
agarose gel electrophoresis and 0.5 .mu.g of a DNA fragment (about 5.95 
kb) containing the G418 resistance gene, among others, was recovered. 
Then, 1 .mu.g of the plasmid pEPKMB1 was dissolved in 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
150 mM sodium chloride, 10 units of SalI and 10 units of XhoI were added, 
and the mixture was incubated at 37.degree. C. for 2 hours to effect 
cleavage at the SalI and XhoI sites. The reaction mixture was subjected to 
agarose gel electrophoresis and 0.1 .mu.g of a DNA fragment (about 1.7 kb) 
containing the immunoglobulin promoter and enhancer regions was recovered. 
The thus-obtained pAGE107-derived 5.95 kb DNA fragment (0.1 .mu.g) and 
immunoglobulin promoter and enhancer region-containing DNA fragment (0.02 
.mu.g) were dissolved in 20 .mu.l of T4 ligase buffer, 175 units of T4 DNA 
ligase was added, and the mixture was incubated at 4.degree. C. for 1 day. 
The reaction mixture was used to transform Escherichia coli HB101. An 
Ap-resistant colony was isolated and the recombinant plasmid DNA was 
recovered therefrom to give pAGE501 shown in FIG. 33. 
(4) Construction of pAGE109 
A plasmid, pAGE109, derived from pAGE106 by deletion of one of the two 
EcoRI sites in pAGE106 was constructed as follows. 
Thus, 2 .mu.g of the heterologous gene expression vector pAGE106 for use in 
animal cells as described in EP-A-0 405 285 was added to 100 .mu.l of 10 
mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride 
and 50 mM sodium chloride; 10 units each of EcoRI and SacI were further 
added, and digestion was conducted at 37.degree. C. for 4 hours. The 
reaction mixture was fractionated by agarose gel electrophoresis and about 
1.5 .mu.g of a DNA fragment (4.3 kb) resulting from cleavage of pAGE106 
with EcoRI and SacI and containing the SV40 early gene promoter and G418 
resistance gene was recovered. Then, this DNA fragment was dissolved in a 
total of 40 .mu.l of DNA polymerase I buffer, 5 units of Escherichia 
coli-derived DNA polymerase I large fragment was added, and the reaction 
was conducted at 16.degree. C. for 2 hours for rendering the 3' protruding 
ends formed upon SalI digestion and the 5' protruding ends formed upon 
EcoRI digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was dissolved in 20 .mu.l of T4 ligase buffer; 350 units of T4 
DNA ligase was further added to the mixed solution, and ligation was 
carried out at 4.degree. C. for 4 hours. The thus-obtained recombinant 
plasmid DNA was used to transform Escherichia coli HB101 to give the 
plasmid pAGE109 shown in FIG. 34. 
(5) Construction of pAGE502 
For replacing the SV40 promoter and enhancer of pAGE107 with the 
immunoglobulin H chain promoter and enhancer, a plasmid named pAGE502 was 
constructed as follows. 
Two .mu.g of pAGE107 described in EP-A-0 405 285 was added to 100 .mu.l of 
10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium 
chloride and 50 mM sodium chloride, 10 units of HindIII was further added, 
and digestion was effected at 37.degree. C. for 4 hours. The reaction 
mixture was subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was conducted at 16.degree. C. 
for 2 hours for rendering the 5' protruding ends formed upon HindIII 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 
units of XhoI was further added, and digestion was effected at 37.degree. 
C. for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 1.5 .mu.g of a DNA fragment (about 5.95 kb), 
resulting from cleavage of pAGE107 with XhoI and HindIII and containing 
the G418 resistance gene and Ap resistance, was recovered. 
Two .mu.g of pAGES01 obtained in (3) was added to 100 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
175 mM sodium chloride, 10 units of SalI was further added, and digestion 
was carried out at 37.degree. C. for 4 hours. The reaction mixture was 
subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was conducted at 16.degree. C. 
for 2 hours for rendering the 5' protruding ends formed upon SalI 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 
units of XhoI was further added, and digestion was effected at 37.degree. 
C. for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.2 .mu.g of a DNA fragment (1.8 kb) resulting 
from cleavage of pAGE501 with XhoI and SalI and containing the KM50 cell 
immunoglobulin H chain promoter and enhancer was recovered. 
Then, 0.1 .mu.g of the HindIII-XhoI fragment (about 5.95 kb) of pAGE107 as 
obtained above and 0.1 .mu.g of the SalI-XhoI fragment (about 1.8 kb) of 
pAGE501 were dissolved in a total of 20 .mu.l of T4 ligase buffer; 350 
units of T4 DNA ligase was added to the solution, and the mixture was 
incubated at 4.degree. C. for 1 day. The thus-obtained recombinant plasmid 
DNA was used to transform Escherichia coli HB101 to give the plasmid 
PAGE502 shown in FIG. 35. 
(6) Construction of pAGE503 
A plasmid named pAGE503 derived from pAGE502 by deletion of one of the two 
EcoRI sites was constructed as follows. 
Two .mu.g of pAGE109 obtained in (4) was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
50 mM sodium chloride; 10 units of HindIII and 10 units of ClaI were 
further added, and digestion was carried out at 37.degree. C. for 4 hours. 
The reaction mixture was fractionated by agarose gel electrophoresis and 
about 0.2 .mu.g of a DNA fragment (about 1 kb) resulting from cleavage of 
pAGE109 with ClaI and HindIII and containing the poly-A signal gene for 
the beta globulin and SV40 early genes was recovered. 
Then, 2 .mu.g of pAGE502 obtained in (5) was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
50 mM sodium chloride, 10 units of HindIII and 10 units of ClaI were 
further added, and digestion was conducted at 37.degree. C. for 4 hours. 
The reaction mixture was fractionated by agarose gel electrophoresis and 
about 1 .mu.g of a DNA fragment (about 6.1 kb) resulting from cleavage of 
pAGE502 with HindIII and ClaI and containing the KM50 cell immunoglobulin 
H chain promoter and enhancer genes, the Ap resistance gene and the G418 
resistance gene was recovered by the DEAE paper method. Then, 0.1 .mu.g of 
the HindIII-ClaI fragment (about 1 kb) of pAGE109 as obtained above and 
0.1 .mu.g of the HindIII-ClaI fragment (about 6.1 kb) of pAGE502 as 
obtained above were dissolved in a total of 20 .mu.l of T4 ligase buffer, 
350 units of T4 DNA ligase was added to the solution, and the mixture was 
incubated at 4.degree. C. for 1 day. The thus-obtained recombinant plasmid 
DNA was used to transform Escherichia coli HB101 and the plasmid pAGES03 
shown in FIG. 36 was obtained. 
(7) Construction of pSE1d1 
A plasmid named pSEldl was constructed by introducing the dhfr gene into 
pAGE107, as follows. 
Two .mu.g of pAGE107 described in EP-A-0 405 825 was added to 100 .mu.l of 
100 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium 
chloride and 50 mM sodium chloride, 10 units of EcoRI was further added, 
and digestion was effected at 37.degree. C. for 4 hours. The reaction 
mixture was subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was conducted at 16.degree. C. 
for 2 hours for rendering the 5' protruding ends formed upon EcoRI 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride; 10 
units of HindIII was further added, and digestion was effected at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 1.5 .mu.g of a DNA fragment (about 
5.6 kb) resulting from cleavage of pAGE107 with EcoRI and HindIII and 
containing the G418 resistance gene and Ap resistance gene was recovered. 
Two .mu.g of pSV2-dhfr Subramani et al.: Mol. Cell. Biol., 1, 854 (1981)! 
was added to 100 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 units of 
BglII was further added, and digestion was carried out at 37.degree. C. 
for 4 hours. The reaction mixture was subjected to phenol-chloroform 
extraction and then to ethanol precipitation, the precipitate was 
dissolved in a total of 40 .mu.l of DNA polymerase I buffer, 5 units of 
Escherichia coli-derived DNA polymerase I Klenow fragment was added, and 
the reaction was conducted at 16.degree. C. for 2 hours for rendering the 
5' protruding ends formed upon BglII digestion blunt-ended. The reaction 
mixture was subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 10 units of HindIII was further added, and 
digestion was effected at 37.degree. C. for 4 hours. The reaction mixture 
was fractionated by agarose gel electrophoresis and about 0.2 .mu.g of a 
pSV2-dhfr DNA fragment (0.76 kb) resulting from cleavage with BglII and 
HindIII and containing the dehydrofolate reductase (dhfr) gene was 
recovered. 
Then, 0.1 .mu.g of the HindIII-EcoRI fragment (about 5.6 kb) of pAGE107, as 
obtained above, and 0.1 .mu.g of the BglII-HindIII fragment (about 0.76 
kb) of pSV2-dhfr, as obtained above, were dissolved in a total of 20 .mu.l 
of T4 ligase buffer; 350 units of T4 DNA ligase was added to the solution, 
and the mixture was incubated at 4.degree. C. for 1 day. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101 and 
the plasmid pSE1d1 shown in FIG. 37 was obtained. 
(8) Construction of pSE1d2 
A plasmid named pSE1d2 was constructed by deleting the HindIII cleavage 
site from pSE1d1, as follows. 
Thus, 2 .mu.g of pSE1d1 obtained in (7) was added to 100 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
50 mM sodium chloride, 10 units of HindIII was further added, and 
digestion was effected at 37.degree. C. for 4 hours. The reaction mixture 
was subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was conducted at 16.degree. C. 
for 2 hours for rendering the 5' protruding ends formed upon HindIII 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was dissolved in 20 .mu.l of T4 ligase buffer, 350 units of T4 
DNA ligase was added to the solution, and the mixture was incubated at 
4.degree. C. for 1 day. The thus-obtained recombinant plasmid DNA was used 
to transform Escherichia coli HB101 and the plasmid pSE1d2 shown in FIG. 
38 was obtained. 
(9) Construction of pIg1SE1d2 
A plasmid named pIg1SE1d2 was constructed by introducing the dhfr gene into 
pAGE503, as follows. 
Two .mu.g of pAGE503 obtained in (6) was added to 100 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
50 mM sodium chloride, 10 units of ClaI was further added, and digestion 
was effected at 37.degree. C. for 4 hours. The reaction mixture was 
subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was conducted at 16.degree. C. 
for 2 hours for rendering the 5' protruding ends formed upon ClaI 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride; 10 
units of MluI was further added, and digestion was effected at 37.degree. 
C. for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 1 .mu.g of a DNA fragment (about 5.4 kb) 
resulting from cleavage of pAGE503 with ClaI and MluI and containing the 
KM50 immunoglobulin H chain promoter and enhancer was recovered. 
Then, 2 .mu.g of pSE1d2 obtained in (8) was added to 100 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 10 units of XhoI was further added, and digestion 
was carried out at 37.degree. C. for 4 hours. The reaction mixture was 
subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was conducted at 16.degree. C. 
for 2 hours for rendering the 5' protruding ends formed upon XhoI 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 
units of MluI was further added, and digestion was effected at 37.degree. 
C. for 4 hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 1 .mu.g of a DNA fragment (about 3.8 kb) 
resulting from cleavage of pSE1d2 with XhoI and MluI and containing the 
dhfr gene was recovered. 
Then, 1 .mu.g of the ClaI-MluI fragment (about 5.4 kb) of pAGE503 as 
obtained above and 1 .mu.g of the XhoI-MluI fragment (about 3.8 kb) of 
pSE1d2 as obtained above were dissolved in a total of 20 .mu.l of T4 
ligase buffer, 350 units of T4 DNA ligase was added to the solution, and 
the mixture was incubated at 4.degree. C. for 1 day. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101 and 
the plasmid pIg1SE1d2 shown in FIG. 39 was obtained. 
(10) Construction of pIg1SE1d3 
A plasmid named pIg1SE1d3 was constructed by deleting the ApaI cleavage 
site from pIg1SE1d2, as follows. 
Two .mu.g of pIg1SE1d2 obtained in (9) was added to 100 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride, 10 
units of ApaI was further added, and digestion was carried out at 
37.degree. C. for 4 hours. The reaction mixture was subjected to 
phenol-chloroform extraction and then to ethanol precipitation, the 
precipitate was dissolved in a total of 40 .mu.l of DNA polymerase I 
buffer, 5 units of Escherichia coli-derived DNA polymerase I Klenow 
fragment was added, and the reaction was carried out at 16.degree. C. for 
2 hours for rendering the 3' protruding ends formed upon ApaI digestion 
blunt-ended. The reaction mixture was subjected to phenol-chloroform 
extraction and then to ethanol precipitation, the precipitate was 
dissolved in 20 .mu.l of T4 ligase buffer, 350 units of T4 ligase was 
added to the solution, and ligation was effected at 4.degree. C. for 24 
hours. The thus-obtained recombinant plasmid DNA was used to transform 
Escherichia coli HB101 and the plasmid pIg1SE1d3 shown in FIG. 40 was 
obtained. 
(11) Construction of pIg1SE1d4 
For providing pIg1SE1d3 with a cloning site between the HindIII cleavage 
site and EcoRI cleavage site, a plasmid named pIg1SE1d4 was constructed 
containing the synthetic DNA defined by SEQ ID NO:17 as an insert, as 
follows. 
Two .mu.g of pIg1SE1d3 obtained in (10) was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
50 mM sodium chloride, 10 units each of HindIII and EcoRI were further 
added, and digestion was carried out at 37.degree. C. for 4 hours. The 
reaction mixture was fractionated by agarose gel electrophoresis and about 
1 .mu.g of a DNA fragment (about 9.2 kb) resulting from cleavage of 
pIg1SE1d3 with HindIII and EcoRI and containing the KM50 cell 
immunoglobulin H chain promoter, enhancer, Ap resistance gene, G418 
resistance gene and dhfr gene was recovered. 
Then, 0.1 .mu.g of the HindIII-EcoRI fragment (about 9.2 kb) of pIg1SE1d3 
as obtained above and 10 ng of the synthetic DNA (SEQ ID NO:17) were a 
total of 20 .mu.l of T4 ligase buffer, 350 units of T4 DNA ligase was 
added to the solution, and the mixture was incubated at 4.degree. C. for 1 
day. The thus-obtained recombinant plasmid DNA was used to transform 
Escherichia coli HB101 and the plasmid pIg1SE1d4 shown in FIG. 41 was 
obtained. 
3. Preparation of the Moloney Mouse Leukemia Virus Long Terminal Repeat 
(hereinafter abbreviated as "MoLTR") 
It is known that MoLTR has promoter and enhancer activity Kuwana et al.: 
Biochem. Biophys. Res. Commun., 149, 960 (1987)!. Therefore, for using 
MoLTR as a promoter and enhancer in vectors for chimeric human antibody 
expression, a plasmid, pPMOL3, containing MoLTR was constructed as 
follows. 
Three .mu.g of pPMOL1 described in JP-A-1-63394 was added to 30 .mu.l of 10 
mM Tris-hydrochloride buffer (pH 7.5) containing 7 mM magnesium chloride 
and 6 mM 2-mercaptoethanol, 10 units of ClaI was further added, and 
digestion was carried out at 37.degree. C. for 4 hours. The reaction 
mixture was subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment was added, and the reaction was carried out at 16.degree. 
C. for 2 hours for rendering the 5' protruding ends formed upon ClaI 
digestion blunt-ended. The reaction was terminated by extraction with 
phenol, the reaction mixture was subjected to chloroform extraction and 
then to ethanol precipitation, and 2 .mu.g of a DNA fragment was 
recovered. This DNA fragment and 0.01 .mu.g of a synthetic DNA linker XhoI 
(Takara Shuzo) were dissolved in 20 .mu.l of T4 ligase buffer, 175 units 
of T4 DNA ligase was added, and the mixture was incubated at 4.degree. C. 
for 1 day. The reaction mixture was used to transform Escherichia coli 
HB101 and the plasmid pPMOL2 shown in FIG. 42 was obtained. Then, 3 .mu.g 
of pPMOL2 was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 7 mM magnesium chloride, 10 mM sodium chloride and 6 mM 
2-mercaptoethanol, 10 units of SmaI was further added, and digestion was 
conducted at 37.degree. C. for 4 hours. The reaction mixture was subjected 
to phenol-chloroform extraction and then to ethanol precipitation, and 2 
.mu.g of a DNA fragment was recovered. This DNA fragment and 0.01 .mu.g of 
a synthetic DNA linker (EcoRI; Takara Shuzo) were dissolved in 20 .mu.l of 
T4 ligase buffer, 175 units of T4 DNA ligase was added, and the mixture 
was incubated at 4.degree. C. for 1 day. The reaction mixture was used to 
transform Escherichia coli HB101 and the plasmid pPMOL3 shown in FIG. 43 
was obtained. 
4. Cloning of the Human Immunoglobulin IgGl H Chain Constant Region 
(C.gamma.1) cDNA and L Chain Constant Region (C.kappa.) cDNA 
(1) Isolation of mRNA from the chimeric antibody producer cell line SP2-PC 
Chimera-1 
Using mRNA extraction kit Fast Track (product number K1593-02) manufactured 
by Invitrogen, MRNA (6.2 .mu.g) was isolated from 1.times.10.sup.8 cells 
of the chimeric antibody producer cell line SP2-PC Chimera-1 described in 
FEBS Letters, 244, 301-306 (1989) and capable of producing a chimeric 
antibody having anti-phosphorylcholine activity. 
(2) Construction of an SP2-PC Chimera-1 cDNA library and cloning of the 
human immunoglobulin H chain constant region (C.gamma.1) cDNA and L chain 
constant region (C.kappa.) cDNA 
Starting with 2 .mu.g of the MRNA obtained in (1) and using cDNA Synthesis 
Kit (product number 27-9260-01) manufactured by Pharmacia, EcoRI adapter 
joining was performed, followed by phosphorylation. The cDNA solution 
obtained was subjected to phenol-chloroform extraction and then to ethanol 
precipitation, and 4 .mu.g of cDNA was recovered. This cDNA was dissolved 
in 20 .mu.l of sterilized water and then fractionated by agarose gel 
electrophoresis, and about 0.3 .mu.g each of two DNA fragments, about 1.8 
kb and about 1.0 kb in size, were recovered. 
Then, 5 .mu.g of the vector pUC18 was added to 100 .mu.l of 100 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 50 units of EcoRI was further added, and digestion 
was carried out at 37.degree. C. for 4 hours for cleaving the pUC18 DNA at 
the EcoRI site. The reaction mixture was subjected to phenol-chloroform 
extraction and then to ethanol precipitation, and about 3 .mu.g of a DNA 
fragment resulting from cleavage of pUC18 at the EcoRI site thereof was 
recovered. 
Then, 0.1 .mu.g of the EcoRI fragment (about 2.7 kb) of pUC18 as obtained 
above and 0.1 .mu.g each of the 1.8 kb and 1.0 kb cDNA fragments prepared 
from SP2-PC Chimera-1 cells were dissolved in a total of 20 .mu.l of T4 
ligase buffer; 350 units of T4 DNA ligase was added to the solution, and 
ligation was effected at 4.degree. C. for 24 hours. 
The thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli LE392. About 3,000 colonies obtained were fixed onto a nitrocellulose 
filter. From among the strains firmly bound at 65.degree. C. to probes 
prepared by labeling the human immunoglobulin constant region chromosomal 
genes (IgG1 H chain constant region C.gamma.1 and L chain constant region 
C.gamma.) Kameyama et al.: FEBS Letters, 244, 301 (1989)! with .sup.32 P, 
a plasmid (pPCVHhCGI1) associable with C.gamma.1 and another (pPCVLhCK1) 
associable with C.kappa. were isolated. 
(3) Introduction of an EcoRV site into the human Ig.kappa. chain constant 
region gene 
An EcoRV site was introduced into the human Ig.kappa. chain constant region 
at a site near the 5' end thereof by site-directed mutagenesis using a kit 
(catalog number Q6210) manufactured by Promega. The plasmid pPCVLhCK1 (2 
.mu.g) was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride and 50 mM sodium chloride, 10 units of 
EcoRI and 10 units of KpnI were further added, and digestion was conducted 
at 37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 0.2 .mu.g of a DNA fragment (about 
0.8 kb) resulting from cleavage of pPCVLhCK1 with KpnI and EcoRI and 
containing the human immunoglobulin L chain constant region gene was 
recovered. 
Then, 2 .mu.g of pSELECT1 (a kit manufactured by Promega; catalog number 
Q6210) was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 6 mM magnesium chloride and 50 mM sodium chloride, 10 units 
each of EcoRI and KpnI were further added, and digestion was carried out 
at 37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 1 .mu.g of a DNA fragment (about 5.7 
kb) resulting from cleavage of pSELECT1 with EcoRI and KpnI was recovered. 
Then, 0.1 .mu.g of the EcoRI-KpnI fragment (about 0.8 kb) of pPCVLhCK1 as 
obtained above and 0.1 .mu.g of the EcoRI-KpnI fragment (about 5.7 kb) of 
pSELECT1 as obtained above were dissolved in a total of 20 .mu.l of T4 
ligase buffer; 350 units of T4 DNA ligase was added to the solution, and 
ligation was effected at 4.degree. C. for 24 hours. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli JM109 and 
the plasmid pchCKA7 shown in FIG. 44 was obtained. 
Then, using pchCKA7 and using the synthetic DNA defined by SEQ ID NO:18 as 
a mutagenic primer, the sequence covering the 12th base to 14 base from 
the N terminus of the human immunoglobulin L chain constant region, namely 
ACC, was converted to GAT and thus an EcoRV site was introduced into that 
site, to give a plasmid named pchCKB1 (FIG. 45). 
Then, the EcoRV site of pchCKB1 was converted to a HindIII cleavage site in 
the following manner. 
Thus, 2 .mu.g of the plasmid pchCKB1 was added to 10 .mu.l of 100 mM 
Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride and 
100 mM sodium chloride, 10 units of EcoRI was further added, and digestion 
was effected at 37.degree. C. for 4 hours. The reaction mixture was 
subjected to phenol-chloroform extraction and then to ethanol 
precipitation, the precipitate was dissolved in a total of 40 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived polymerase I 
Klenow fragment was added, and the reaction was carried out at 37.degree. 
C. for 30 minutes for rendering the 5' protruding ends formed upon EcoRI 
digestion blunt-ended. The reaction mixture was subjected to 
phenol-chloroform extraction and then ethanol precipitation, the 
precipitate was dissolved, together with 0.1 .mu.g of a HindIII linker 
(Takara Shuzo), in 20 .mu.l of T4 ligase buffer; 350 units of T4 ligase 
was added to the solution, and ligation was effected at 4.degree. C. for 
24 hours. The thus-obtained recombinant plasmid DNA was used to transform 
Escherichia coli HB101 and the plasmid pchCKC1 shown in FIG. 46 was 
obtained. 
5. Construction of Vectors for Chimeric Human Antibody H Chain Expression 
(1) Construction of a vector to be used in constructing chimeric human 
antibody H chain expression vectors (vector for chimeric human antibody H 
chain expression) 
The plasmid pIg1SE1d4 obtained in Paragraph 2 (11) (2 .mu.g) was added to 
30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM 
magnesium chloride and 100 mM sodium chloride, 10 units each of EcoRV and 
ApaI were further added, and digestion was effected at 37.degree. C. for 4 
hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 1.5 .mu.g of a DNA fragment (about 9.2 kb) 
resulting from cleavage of pIg1SE1d4 with EcoRV and ApaI was recovered. 
Then, 2 .mu.g of pPCVHhCGI1 obtained in Paragraph 4 (2) was added to 30 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM 
magnesium chloride, 10 units of ApaI and 10 units of SmaI were further 
added, and digestion was conducted at 37.degree. C. for 1 hour. The 
reaction mixture was fractionated by agarose gel electrophoresis and about 
0.2 .mu.g of a DNA fragment (about 1 kb) resulting from cleavage of 
pPCVHhCGI1 with ApaI and SmaI and containing the human immunoglobulin H 
chain constant region gene was recovered. 
Then, 0.1 .mu.g of the EcoRV-ApaI fragment (about 9.2 kb) of pIg1SE1d4 as 
obtained above and 0.1 .mu.g of the ApaI-SmaI fragment (about 1 kb) of 
pPCVHhCGI1 as obtained above were dissolved in a total of 20 .mu.l of T4 
ligase buffer; 350 units of T4 DNA ligase was added to the solution, and 
ligation was conducted at 4.degree. C. for 24 hours. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101 and 
the vector pCHiIgHB2 for chimeric human antibody H chain expression as 
shown in FIG. 47 was obtained. 
(2) Construction of a vector to be used in constructing chimeric human 
antibody L chain expression vectors (vector for chimeric human antibody L 
chain expression) 
The plasmid pIg1SE1d4 obtained in Paragraph 2 (11) (2 .mu.g) was added to 
30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM 
magnesium chloride and 100 mM sodium chloride, 10 units of EcoRV and 10 
units of HindIII were further added, and digestion was effected at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 1.5 .mu.g of a DNA fragment (about 
9.2 kb) resulting from cleavage of pIg1SE1d4 with EcoRV and HindIII was 
recovered. 
Then, 2 .mu.g of pckCKC1 obtained in Paragraph 4 (3) was added to 30 .mu.l 
of 10 mM Tris-hydrochloride (pH 7.5) containing 6 mM magnesium chloride 
and 100 mM sodium chloride, 10 units of EcoRV and 10 units of HindIII were 
further added, and digestion was carried out at 37.degree. C. for 1 hour. 
The reaction mixture was fractionated by agarose gel electrophoresis and 
about 0.2 .mu.g of a DNA fragment (about 0.6 kb) resulting from cleavage 
of pPCVLhCK1 with EcoRV and HindIII and containing the human 
immunoglobulin L chain constant region gene was recovered. 
Then, 0.1 .mu.g of the EcoRV-HindIII fragment (about 9.2 kb) of pIg1SE1d4 
as obtained above and 0.1 .mu.g of the EcoRV-HindIII fragment (about 0.6 
kb) of pchCKC1 as obtained above were dissolved in a total of 20 .mu.l of 
T4 ligase buffer, 350 units of T4 DNA ligase was added to the solution, 
and ligation was carried out at 4.degree. C. for 24 hours. The 
thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli HB101 and the vector pChiIgLA1 for chimeric human antibody L chain 
expression as shown in FIG. 48 was obtained. 
REFERENCE EXAMPLE 2 
Construction of a Chimeric Human Antibody H Chain Expression Vector, 
pChi641HA1 
1. Isolation of MRNA from mouse anti-GD.sub.3 monoclonal antibody 
KM-641-producing hybridoma cells 
Using mRNA extraction kit Fast Track (product number K1593-02) manufactured 
by Invitrogen, 34 .mu.g of mRNA was isolated from 1.times.10.sup.8 mouse 
anti-GD.sub.3 monoclonal antibody KM-641-producing hybridoma cells 
obtainable as in Reference Example 1. 
2. Construction of a KM-641 H chain cDNA library and a KM-641 L chain cDNA 
library 
Using 3 .mu.g of the mRNA obtained in Paragraph 1 and using cDNA synthesis 
kit ZAP-cDNA Synthesis Kit (product number sc200400) manufactured by 
Stratagene, cDNA having an EcoRI adapter at the 5' terminus and cDNA 
having an XhoI adapter at the 3' terminus were synthesized. About 6 .mu.g 
of each cDNA was dissolved in 10 .mu.l of sterilized water and 
fractionated by agarose gel electrophoresis. In this way, about 0.1 .mu.g 
of a cDNA fragment having a size of about 1.8 kb and corresponding to the 
H chain and a cDNA fragment having a size of about 1.0 kb and 
corresponding to the L chain were recovered. Then, 0.1 .mu.g of the cDNA 
fragment of about 1.8 kb in size, 0.1 .mu.g of the cDNA fragment of about 
1.0 kb in size and 1 .mu.g of Uni-ZAP XR (Stratagene; derived from the 
Lambda ZAPII vector by cleavage with EcoRI and XhoI, followed by treatment 
with calf intestine alkaline phosphatase), to be used as the vector, were 
dissolved in T4 ligase buffer; 175 units of T4 DNA ligase was added, and 
the mixture was incubated at 12.degree. C. for 10 hours and further at 
room temperature for 2 hours. A 4-.mu.l portion of the reaction mixture 
was packaged into the lambda phage by the conventional method Maniatis et 
al. (ed.): Molecular Cloning, 1989, p. 2.95! using Giga Pak Gold 
(Stratagene), followed by transfection of Escherichia coli PLK-F with the 
packaging mixture by the conventional method Maniatis et al. (ed.): 
Molecular Cloning, 1989, p. 2.95-107!. As an H chain cDNA library and as 
an L chain cDNA library, about 10,000 phage clones were respectively 
obtained. The phages were then fixed onto nitrocellulose filters by the 
conventional method Maniatis et al. (ed.): Molecular Cloning, 1989, p. 
2.112!. 
3. Cloning of the monoclonal antibody KM-641 H chain and L chain cDNAs 
Using probes prepared by labeling a mouse C.gamma.1 gene (mouse 
immunoglobulin constant region chromosomal gene)-containing EcoRI fragment 
(about 6.8 kb) Roeder et al.: Proc. Natl. Acad. Sci. U.S.A., 78, 474 
(1981)! and a mouse C.kappa. gene-containing HindIII-BamHI fragment (about 
3 kb) Sakano et al.: Nature, 280, 288 (1979)! with .sup.32 P, one phage 
clone strongly associable with the former probe at 65.degree. C. and one 
phage clone strongly associable with the latter probe at 65.degree. C. 
were isolated from the H chain cDNA library and L chain cDNA library 
constructed in Paragraph 2 in accordance with the conventional method 
Maniatis et al. (ed.): Molecular Cloning, 1989, p. 2.108!. Then, by 
converting the phage clones to pBluescript plasmids using cDNA synthesis 
kit ZAP-cDNA Synthesis Kit (product number sc200400) manufactured by 
Stratagene, a KM-641 H chain cDNA-containing recombinant plasmid, 
pKM641HA3, and a KM-641 L chain cDNA-containing recombinant plasmid, 
pKM641LA2, were obtained. Cleavage of pKM641HA3 and pKM641LA2 with EcoRI 
and XhoI revealed that a cDNA fragment of about 1.6 kb and a cDNA fragment 
of about 0.9 kb had been inserted therein, respectively (FIG. 49). 
4. Base sequences of the immunoglobulin variable regions in the KM-641 H 
chain cDNA (pKM641HA3) and KM-641 L chain cDNA (pKM641LA2) 
The base sequences of the immunoglobulin regions in pKM641HA3 and pKM641LA2 
obtained in Paragraph 3 were determined by the dideoxy method Maniatis et 
al. (ed.): Molecular Cloning, 1989, p. 13.42! using Sequenase Version 2.0 
DNA Sequencing Kit (United States Biochemical Corporation). The results 
obtained are shown in SEQ ID NO:19 and SEQ ID NO:20. In pKM641LA2, a 
methionine codon, presumably the initiation codon ATG, was found in the 
vicinity of the 5' terminus and the cDNA was a leader sequence-containing 
full-length one. In pKM641HA3, no methionine initiation codon was found 
and the leader sequence was partly lacking. 
5. Construction of a KM-641-derived chimeric human antibody H chain 
expression vector 
A chimeric human antibody H chain expression vector was constructed by 
joining the H chain variable region gene obtained by cleaving the plasmid 
pKM641HA3 at the AluI site near the 5' terminus of the variable region 
gene and at the StyI site near the 3' terminus of the variable region gene 
to the vector for chimeric human antibody H chain expression as obtained 
in Reference Example 1 using the synthetic DNAs defined by SEQ ID NO:21 
and SEQ ID NO:22 (FIG. 50). 
First, the DNA defined by SEQ ID NO:22 composed of the base sequence from 
the 3' terminus of the immunoglobulin H chain variable region in pKM641HA3 
to the StyI cleavage site near said 3'terminus and the base sequence from 
the 5' terminus of the immunoglobulin H chain constant region in pAGE28 to 
the ApaI cleavage site near said 5' terminus and having a StyI cleavage 
site and an ApaI cleavage site on the respective termini (cf. FIG. 50) was 
synthesized using a DNA synthesizer. This synthetic DNA was then 
introduced into the plasmid pKM641HA3 in the following manner. 
Three .mu.g of pKM641HA3 was added to 30 .mu.l of 50 mM Tris-hydrochloride 
buffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride 
and 1 mM DTT, 10 units of EcoRI and 10 units of StyI were further added, 
and digestion was effected at 37.degree. C. for 4 hours. The reaction 
mixture was fractionated by agarose gel electrophoresis and about 0.3 
.mu.g of a 0.41 kb DNA fragment was recovered. Then, 3 .mu.g of pAGE28 
Mizukami et al.: J. Biochem., 101, 1307-1310 (1987)! was added to 30 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7 mM 
magnesium chloride and 6 mM 2-mercaptoethanol, 10 units of EcoRI and 10 
units of ApaI were further added, and digestion was carried out at 
37.degree. C. for 4 hours. The reaction mixture was fractionated by 
agarose gel electrophoresis and about 2 .mu.g of a 2.45 kb DNA fragment 
was recovered. Then, 0.1 .mu.g of the EcoRI-StyI fragment (about 0.41 kb) 
of pKM641HA3, as obtained above, 0.1 .mu.g of the EcoRI-ApaI fragment 
(about 2.45 kb) of pAGE28, as obtained above, and 0.3 .mu.g of the 
synthetic DNA, defined by SEQ ID NO:22, were dissolved in a total of 20 
.mu.l of T4 ligase buffer; 350 units of T4 ligase was added to the 
solution, and ligation was conducted at 4.degree. C. for 24 hours. The 
thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli HB101 and the plasmid pKM641HE1 shown in FIG. 51 was obtained. 
Since pKM641HE1 had no leader sequence, the following measure was taken to 
supplement the deficit using the synthetic DNA defined by SEQ ID NO:21. 
pKM641HE1 (3 .mu.g) was added to 30 .mu.l of 10 mM Tris-hydrochloride 
buffer (pH 7.5) containing 7 mM magnesium chloride and 6 mM 
2-mercaptoethanol, 10 units of EcoRI and 10 units of ApaI were further 
added, and digestion was effected at 37.degree. C. for 4 hours. The 
reaction mixture was fractionated by agarose gel electrophoresis and about 
0.4 .mu.g of a DNA fragment of about 0.42 kb in size was recovered. The 
EcoRI-ApaI fragment (about 0.42 kb; 0.4 .mu.g) of pKM641HE1 was added to 
30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7 mM 
magnesium chloride, 50 mM sodium chloride and 6 mM 2-mercaptoethanol, 10 
units of AluI was further added, and digestion was effected at 37.degree. 
C. for 4 hours. The reaction mixture was subjected to phenol-chloroform 
extraction and then to ethanol precipitation, and about 0.3 .mu.g of a DNA 
fragment of about 0.4 kb in size was recovered. 
Then, 0.1 .mu.g of the AluI-ApaI fragment (about 0.4 kb) of pKM641HE1 as 
obtained above, 0.1 .mu.g of the EcoRI-ApaI fragment (about 2.45 kb) of 
pAGE28 as obtained above and 0.3 .mu.g of the synthetic DNA defined by SEQ 
ID NO:21 were dissolved in a total of 20 .mu.l of T4 ligase buffer; 350 
units of T4 ligase was added to the solution, and ligation was carried out 
at 4.degree. C. for 24 hours. The thus-obtained recombinant plasmid DNA 
was used to transform Escherichia coli HB101 and the plasmid pKM641HF1 
shown in FIG. 52 was obtained. 
Then, the immunoglobulin H chain variable region of pKM641HF1 was 
introduced into the vector pChiIgHB2 for chimeric human antibody H chain 
expression, as follows. pKM641HF1 (3 .mu.g) was added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 7 mM magnesium chloride and 
6 mM 2-mercaptoethanol, 10 units of EcoRI and 10 units of ApaI were 
further added, and digestion was effected at 37.degree. C. for 4 hours. 
The reaction mixture was fractionated by agarose gel electrophoresis and 
about 0.5 .mu.g of a 0.44 kb DNA fragment was recovered. Then, 3 .mu.g of 
pChiIgHB2 was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 7 mM magnesium chloride and 6 mM 2-mercaptoethanol, 10 
units of EcoRI and 10 units of ApaI were further added, and digestion was 
conducted at 37.degree. C. for 4 hours. The reaction mixture was subjected 
to phenol-chloroform extraction and about 3 .mu.g of DNA was recovered. 
Then, 0.1 .mu.g of the EcoRI-ApaI fragment (about 0.44 kb) of pKM641HF1 as 
obtained above and 0.1 .mu.g of the EcoRI-ApaI fragment (about 10.1 kb) of 
pChiIgHB2 as obtained above were dissolved in a total of 20 .mu.l of T4 
ligase buffer; 350 units of T4 ligase was added to the solution, and 
ligation was carried out at 4.degree. C. for 24 hours. The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101 and 
the plasmid pChi641HA1 shown in FIG. 53 was obtained. 
Then, the KM50-derived immunoglobulin H chain promoter and enhancer region 
of pChi641HA1 was replaced with MoLTR, as follows. 
pChi641HA1 (3 .mu.g) was added to 30 .mu.l of 50 mM Tris-hydrochloride 
buffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride 
and 1 mM DTT, 10 units of EcoRI and 10 units of XhoI were further added, 
and digestion was effected at 37.degree. C. for 4 hours. The reaction 
mixture was fractionated by agarose gel electrophoresis and about 0.2 
.mu.g of a DNA fragment of about 8.8 kb in size was recovered. pPMOL3 (3 
.mu.g) obtained in Example 1, Paragraph 2 was added to 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 50 
mM sodium chloride and 1 mM DTT; 10 units of EcoRI and 10 units of XhoI 
were further added, and digestion was carried out at 37.degree. C. for 4 
hours. The reaction mixture was fractionated by agarose gel 
electrophoresis and about 0.3 .mu.g of a MoLTR-containing DNA fragment 
(0.63 kg) was recovered. Then, 0.1 .mu.g of the EcoRI-XhoI fragment of 
pChi641HA1 and 0.1 .mu.g of the EcoRI-XhoI fragment of pPMOL3 were 
dissolved in 20 .mu.l of T4 ligase buffer, 175 units of T4 DNA ligase was 
added, and the mixture was incubated at 4.degree. C. for 1 day. The 
reaction mixture was used to transform Escherichia coli HB101 and the 
KM-641-derived chimeric human H chain expression vector pChi641HAM1 shown 
in FIG. 54 was obtained. 
EXAMPLE 2 
Production of human CDR-transplanted anti-GM.sub.2 antibodies (1) 
1. Construction of DNAs Each Coding For Human CDR-Transplanted 
Anti-GM.sub.2 Antibody H Chain Variable Region and Human CDR-transplanted 
Anti-GM.sub.2 Antibody L Chain Variable Region 
(1) Construction of DNA coding for human CDR-transplanted anti-GM.sub.2 
antibody H chain variable region 
A DNA coding for a human CDR-transplanted anti-GM.sub.2 antibody H chain 
variable region, hKM796H, which contains amino acid sequences of SEQ ID 
NO:94, SEQ ID NO:95 and SEQ ID NO:96, was constructed in the following 
manner. 
NEWM BIO/TECHNOLOGY, 9, 266 (1991)! was used as human antibody H chain 
variable region-encoding DNA to which each CDR was to be transplanted. 
DNAs set forth in SEQ ID NO:23 through NO:29 corresponding to NEWM in 
which each CDR was replaced with amino acid sequences of SEQ ID NO:94, SEQ 
ID NO:95 and SEQ ID NO:96 were synthesized using an automatic DNA 
synthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.). The 
thus-obtained synthetic DNAs (50 picomoles each) were dissoloved in 20 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mM 
magnesium chloride, 5 mM DTT, 0.1 mM EDTA and 0.5 mM ATP, 5 units of T4 
polynucleotide kinase was added, and 5'-phosphorylation was carried out at 
37.degree. C. for 30 minutes. Ten picomoles each of the resulting 
phosphorylated synthetic DNAs, which had restriction enzyme sites on both 
ends, were ligated in the order of SEQ ID NO. (SEQ ID NO:23 throuth NO:29) 
using a DNA ligation kit (Takara Shuzo) in accordance with the 
manufacturer's instruction attached to the kit to obtain a DNA, hKM796H, 
shown in FIG. 55. The amino acid sequence corresponding to hKM796H is 
shown in SEQ ID NO:100. 
(2) Construction of DNA coding for human CDR-transplanted anti-GM.sub.2 
antibody L chain variable region 
A DNA coding for a human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region, hKM796L, which contains amino acid sequences of SEQ ID 
NO:97, SEQ ID NO:98 and SEQ ID NO:99, was constructed in the following 
manner. 
REI BIO/TECHNOLOGY, 9, 266 (1991)! was used as human antibody L chain 
variable region-encoding DNA to which each CDR was to be transplanted. 
DNAs set forth in SEQ ID NO:30 through NO:35 corrresponding to REI in 
which each CDR was replaced with amino acid sequences of SEQ ID NO:97, SEQ 
ID NO:98 and SEQ ID NO:99 were synthesized using an automatic DNA 
synthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.). The 
thus-obtained synthetic DNAs (50 picomoles each) were dissoloved in 20 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mM 
magnesium chloride, 5 mM DTT, 0.1 mM EDTA and 0.5 mM ATP, 5 units of T4 
polynucleotide kinase was added, and 5'-phosphorylation was carried out at 
37.degree. C. for 30 minutes. Ten picomoles each of the resulting 
phosphorylated synthetic DNAs, which had restriction enzyme sites on both 
ends, were ligated in the order of SEQ ID NO. (SEQ ID NO:30 through NO:35) 
using a DNA ligation kit (Takara Shuzo) in accordance with the 
manufacturer's instruction attached to the kit to obtain a DNA, hKM796L, 
shown in FIG. 56. The amino acid sequence corresponding to hKM796L is 
shown in SEQ ID NO:101. 
2. Construction of Human CDR-Transplanted Antibody H Chain Expression 
Vector and Human CDR-Transplanted Antibody L Chain Expression Vector 
(1) Construction of human CDR-transplanted antibody H chain expression 
vector 
A NotI-ApaI fragment of the DNA coding for human CDR-transplanted antibody 
H chain variable region, obtained in Paragraph 1(1) of Example 2, was 
ligated to the plasmid pChi796HM1, obtained in Paragraph 7(3) of Example 
1, in the following manner (FIG. 57). 
Three .mu.g of pChi796HM1, obtained in Paragraph 7(3) of Example 1, were 
dissolved in 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of ApaI were 
added thereto and the mixture was allowed to react at 37.degree. C. for 1 
hour. The resulting mixture was subjected to ethanol precipitation and the 
thus-obtained precipitate was dissolved in 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT. Ten units of NotI were added thereto 
to allow the mixture to react at 37.degree. C. for 1 hour. The reaction 
mixture was fractionated by agarose gel electrophoresis to recover about 2 
.mu.g of a DNA fragment of about 9.0 kb. Then, about 0.1 .mu.g of the 
thus-obtained ApaI-NotI fragment of pChi796HM1 was ligated to 0.5 pmoles 
of the NotI-ApaI fragment of the DNA coding for human CDR-transplanted 
antibody H chain variable region, obtained in Paragraph 1(1) of Example 2, 
using a DNA ligation kit (Takara Shuzo). The resulting recombinant plasmid 
DNA was used to transform Escherichia coli HB101 and the plasmid 
phKM796HM1 shown in FIG. 57 was obtained. 
Then, a human CDR-transplanted antibody H chain expression vector was 
constructed by introducing .beta.-globin 3' splicing signal into the 
plasmid phKM796HM1 in the following manner (FIG. 58). 
Three .mu.g of phKM796HM1 were added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 1 unit of KpnI was added thereto. The mixture was allowed to 
react at 37.degree. C. for 10 minutes to effect partial digestion. The 
resulting mixture was subjected to ethanol precipitation and the 
thus-obtained precipitate was dissolved in 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT. After adding 1 unit of XhoI, the 
mixture was allowed to react at 37.degree. C. for 10 minutes to effect 
partial digestion. The reaction mixture was fractionated by agarose gel 
electrophoresis to recover about 0.2 .mu.g of a DNA fragment of about 2.1 
kb. Separately, 3 .mu.g of pAGE148, obtained in Paragraph 7(2) of Example 
1, were added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT. Ten units of KpnI were 
added thereto to allow the mixture to react at 37.degree. C. for 1 hour. 
The reaction mixture was subjected to ethanol precipitation and the 
thus-obtained precipitate was dissloved in 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT. After adding 10 units of XhoI, the 
mixture was allowed to react at 37.degree. C. for 1 hour and then 
fractionated by agarose gel electrophoresis to recover about 1 .mu.g of a 
DNA fragment of about 8.7 kb. One tenth .mu.g of the thus-obtained 
XhoI-KpnI fragment of phKM796HM1 was ligated to 0.1 .mu.g of the XhoI-KpnI 
fragment of pAGE148 using a DNA ligation kit (Takara Shuzo). The 
thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli HB101 to obtain the plasmid phKM796HMS1 shown in FIG. 58. 
(2) Construction of human CDR-transplanted antibody L chain expression 
vector 
An EcoRI fragment having blunt ends of the DNA coding for human 
CDR-transplanted antibody L chain variable region, obtained in Paragraph 1 
(2) of Example 2, was ligated to the chimeric human antibody L chain 
expression vector pChiIgLA1 in the following manner (FIG. 59). 
Three .mu.g of pChiIgLA1, obtained in Reference Example 1, were added to 30 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI 
and 10 units of EcoRV were added thereto and the mixture was allowed to 
react at 37.degree. C. for 1 hour. The reaction mixture was fractionated 
by agarose gel electrophoresis to recover about 1 .mu.g of a DNA fragment 
of about 8.6 kb. Then, about 0.1 .mu.g of the thus-obtained EcoRI-EcoRV 
fragment of pChiIgLA1 was ligated to 0.5 pmoles of the EcoRI fragment 
having blunt ends derived from the DNA coding for human CDR-transplanted 
antibody L chain variable region, obtained in Paragraph 1(2) of Example 2, 
using a DNA ligation kit (Takara Shuzo). The resulting recombinant plasmid 
DNA was used to transform Escherichia coli HB101 and the plasmid 
phKM796LI1 shown in FIG. 59 was obtained. 
Then, P.sub.MO was introduced into the plasmid phKM796LI1 in the following 
manner (FIG. 60). 
Three .mu.g of phKM796LI1 were added to 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10 units of 
XhoI were added thereto, and the mixture was allowed to react at 
37.degree. C. for 1 hour. The reaction mixture was fractionated by agarose 
gel electrophoresis to recover about 1 .mu.g of a DNA fragment of about 
8.2 kb. Separately, 3 .mu.g of the chimeric human antibody H chain 
expression vector pChi641HAM1, obtained in Reference Example 2, were added 
to 30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride, 100 mM sodium chloride and 1 mM DTT. Ten units of 
EcoRI and 10 units of XhoI were added thereto to allow the mixture to 
react at 37.degree. C. for 1 hour. The reaction mixture was fractionated 
by agarose gel electrophoresis to recover about 0.3 .mu.g of a DNA 
fragment of about 0.6 kb. One tenth .mu.g of the thus-obtained EcoRI-XhoI 
fragment of pChi641HAM1 was ligated to 0.1 .mu.g of the EcoRI-XhoI 
fragment of phKM796LI1 using a DNA ligation kit (Takara Shuzo). The 
thus-obtained recombinant plasmid DNA was used to transform Escherichia 
coli HB101 to obtain the plasmid phKM796LM1 shown in FIG. 60. 
Then, a human CDR-transplanted antibody L chain expression vector was 
constructed by introducing .beta.-globin 3' splicing signal into the 
plasmid phKM796LM1 in the following manner (FIG. 61). 
Three .mu.g of phKM796LM1 were added to 30 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of KpnI were added thereto, and the mixture was allowed 
to react at 37.degree. C. for 1 hour. The resulting mixture was subjected 
to ethanol precipitation and the thus-obtained precipitate was dissolved 
in 30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride, 100 mM sodium chloride and 1 mM DTT. After adding 10 
units of XhoI, the mixture was allowed to react at 37.degree. C. for 1 
hour. The reaction mixture was fractionated by agarose gel electrophoresis 
to recover about 0.3 .mu.g of a DNA fragment of about 1.6 kb. Separately, 
3 .mu.g of pAGE148, obtained in Paragraph 7(2) of Example 1, were added to 
30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mm 
magnesium chloride and 1 mM DTT. Ten units of KpnI were added thereto to 
allow the mixture to react at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to ethanol precipitation and the thus-obtained 
precipitate was dissloved in 30 .mu.l of 50 mM Tris-hydrochloride buffer 
(pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 
mM DTT. After adding 10 units of XhoI, the mixture was allowed to react at 
37.degree. C. for 1 hour and then fractionated by agarose gel 
electrophoresis to recover about 1 .mu.g of a DNA fragment of about 8.7 
kb. One tenth .mu.g of the thus-obtained XhoI-KpnI fragment of phKM796LM1 
was ligated to 0.1 .mu.g of the XhoI-KpnI fragment of pAGE148 using a DNA 
ligation kit (Takara Shuzo). The thus-obtained recombinant plasmid DNA was 
used to transform Escherichia coli HB101 to obtain the plasmid phKM796LMS1 
shown in FIG. 61. 
3. Construction of Human CDR-Transplanted Antibody H Chain and L Chain 
Tandem Expression Vector 
A tandem expression vector containing both of cDNA coding for human 
CDR-transplanted antibody H chain and cDNA coding for human 
CDR-transplanted antibody L chain was constructed in the following manner 
(FIG. 62 and FIG. 63). Three .mu.g of phKM796HMS1, obtained in Paragraph 
2(1) of Example 2, were dissolved in 30 .mu.l of 50 mM Tris-hydrochloride 
buffer (pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium 
chloride and 1 mM DTT, 1 unit of SalI was added thereto and the mixture 
was allowed to react at 37.degree. C. for 10 minutes to effect partial 
digestion. The resulting mixture was subjected to ethanol precipitation 
and the thus-obtained precipitate was dissolved in 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT. Ten units of MluI was added thereto 
to allow the mixture to react at 37.degree. C. for 1 hour. The reaction 
mixture was fractionated by agarose gel electrophoresis to recover about 
0.2 .mu.g of a DNA fragment of about 5.9 kb. Then, about 2 .mu.g of 
pAGE107 as described in EP-A-0 405 285 was added to 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride, 
100 mM sodium chloride and 1 mM DTT, 10 units of MluI and 10 units of SalI 
were added thereto, and the mixture was allowed to react at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis to recover about 0.2 .mu.g of a DNA fragment of about 3.35 
kb. Then, 0.1 .mu.g of the thus-obtained MluI-SalI fragment of phKM796HMS1 
was ligated to 0.1 .mu.g of the MluI-SalI fragment of pAGE107 using a DNA 
ligation kit (Takara Shuzo). The thus-obtained recombinant plasmid DNA was 
used to transform Escherichia coli HB101 to obtain the plasmid phKM796H107 
shown in FIG. 62. 
Then, 3 .mu.g of phKM796H107 were added to 30 .mu.l of 10 mM 
Tris-hydrochloride (pH 7.5) containing 10 mM magnesium chloride, 100 mM 
sodium chloride and 1 mM DTT, 10 units of ClaI was added thereto and the 
mixture was allowed to react at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to phenol-chloroform extraction and ethanol 
precipitation. The resulting precipitate was dissolved in 20 .mu.l of DNA 
polymerase I buffer, 5 units of Escherichia coli-derived DNA polymerase I 
Klenow fragment were added, and the 5' cohesive ends produced by ClaI 
digestion were rendered blunt by incubation at 22.degree. C. for 30 
minutes. The reaction mixture was fractionated by agarose gel 
electrophoresis to recover about 0.2 .mu.g of a DNA fragment of about 3.35 
kb. The reaction mixture was also subjected to phenol-chloroform 
extraction and then to ethanol precipitation. The resulting precipitate 
was dissolved in 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 
10 units of MluI were added thereto and the mixture was allowed to react 
at 37.degree. C. for 1 hour. The reaction mixture was fractionated by 
agarose gel electrophoresis to recover about 0.3 .mu.g of a DNA fragment 
of about 7.5 kb. Separately, 3 .mu.g of phKM796LMS1 were added to 30 .mu.l 
of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of XhoI were added 
and the mixture was allowed to react at 37.degree. C. for 1 hour. The 
reaction mixture was subjected to phenol-chloroform extraction and then to 
ethanol precipitation. The resulting precipitate was dissloved in 20 .mu.l 
of DNA polymerase I buffer, 5 units of Escherichia coli-derived DNA 
polymerase I Klenow fragment were added, and the 5' cohesive ends produced 
by XhoI digestion were rendered blunt by incubation at 22.degree. C. for 
30 minutes. The reaction mixture was subjected to phenol-chloroform 
extraction followed by ethanol precipitation. The resulting precipitate 
was added to 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT, 
10 units of MluI were added thereto, and the mixture was allowed to react 
at 37.degree. C. for 1 hour. The reaction mixture was fractionated by 
agarose gel electrophoresis to recover about 0.3 .mu.g of a DNA fragment 
of about 9.3 kb. Then, 0.1 .mu.g of the thus-obtained MluI-ClaI fragment 
of phKM796H107 was ligated to 0.1 .mu.g of the MluI-XhoI fragment of 
phKM796LMS1 using a DNA ligation kit (Takara Shuzo). The thus-obtained 
recombinant plasmid DNA was used to transform Escherichia coli HB101 to 
obtain the plasmid phKM796HL1 shown in FIG. 63. 
4. Expression of Human CDR-Transplanted Anti-GM.sub.2 Antibody in YB2/0 
Cells 
The plasmids were introduced into YB2/0 cells by the electroporation method 
of Miyaji et al. Cytotechnology, 3, 133 (1990)!. 
After introduction of 4 .mu.g of phKM796HL1 obtained in Paragraph 3 of 
Example 2 into 4.times.10.sup.6 YB2/0 (ATCC CRL1581) cells, the cells were 
suspended in 40 ml of RPMI1640-FCS(10) RPMI1640 medium (Nissui 
Pharmaceutical) containing 10% of FCS, 1/4 volume of 7.5% NaHCO.sub.3, 3% 
of 200 mM L-glutamine solution (Gibco) and 0.5% of penicillin-streptomycin 
solution (Gibco; containing 5,000 units/ml penicillin and 5,000 .mu.g/ml 
streptomycin)!, and the suspension was distributed in 200-.mu.l portions 
into wells of 96-well microtiter plates. After 24 hours of incubation at 
37.degree. C. in a CO.sub.2 incubator, G418 (Gibco) was added to a 
concentration of 0.5 mg/ml and then incubation was continued for 1 to 2 
weeks. Transformant colonies appeared, the culture fluid was recovered 
from each well in which the cells had grown to confluence and an 
enzyme-linked immunosorbent assay (ELISA) described in Paragraph 11 of 
Example 1 was conducted for anti-GM.sub.2 human CDR-transplanted antibody 
activity measurement. 
The clone showing the highest activity in ELISA among the clones obtained 
gave a human CDR-transplanted anti-GM.sub.2 antibody content of about 0.1 
.mu.g/ml of culture fluid. 
Cells of the clone showing the above-mentioned human CDR-transplanted 
anti-GM.sub.2 antibody activity were suspended in RPMI1640-FCS(10) medium 
containing 0.5 mg/ml G418 and 50 nM MTX to a concentration of 1 to 
2.times.10.sup.5 cells/ml, and the suspension was distributed in 2-ml 
portions into wells of 24-well plates. Incubation was performed at 
37.degree. C. in a CO.sub.2 incubator for 1 to 2 weeks to induce 50 nM 
MTX-resistant clones. At the time of confluence, the human 
CDR-transplanted anti-GM.sub.2 antibody activity in each culture fluid was 
determined by ELISA. The 50 nM MTX-resistant clone showing the highest 
activity among the clones obtained showed a human CDR-transplanted 
anti-GM.sub.2 antibody content of about 1.0 .mu.g/ml. 
Cells of the above 50 nM MTX-resistant clone were suspended in 
RPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to a 
concentration of 1 to 2.times.10.sup.5 cells/ml, and the suspension was 
distributed in 2-ml portions into wells of 24-well plates. Incubation was 
carried out at 37.degree. C. in a CO.sub.2 incubator for 1 to 2 weeks to 
induce 200 nM MTX-resistant clones. At the time of confluence, each 
culture fluid was assayed for human CDR-transplanted anti-GM.sub.2 
antibody activity by ELISA. The 200 nM MTX-resistant clone showing the 
highest activity among the clones obtained had a human CDR-transplanted 
anti-GM.sub.2 antibody content of about 5.0 .mu.g/ml. 
As described in detail hereinabove, the present invention provides 
humanized antibodies reacting with the ganglioside GM.sub.2 . 
EXAMPLE 3 
Production of Human CDR-Transplanted Anti-GM.sub.2 Antibodies (2) 
1. Construction of Tandem Cassette Type Humanized Antibody Expression 
Vector, pKANTEX93 
A tandem cassette type humanized antibody expression vector, pKANTEX93, for 
the expression of a human CDR-transplanted antibody in mammalian cells was 
constructed based on the plasmid pSE1UK1SEd1-3 described in JP-A-2-257891 
by inserting a DNA fragment coding for a human CDR-transplanted 
anti-GM.sub.2 antibody H chain variable region and a DNA fragment coding 
for a human CDR-transplanted anti-GM.sub.2 antibody L chain variable 
region into said plasmid upstream of the human antibody .gamma. 1H chain 
constant region cDNA and human antibody .kappa. L chain constant region 
cDNA, respectively, in the following manner. 
(1) Modification of ApaI and EcoRI restriction enzyme sites occurring in 
rabbit .beta.-globin gene splicing and poly A signals 
For making it possible to construct a human CDR-transplanted antibody 
expression vector by inserting human CDR-transplanted antibody variable 
regions cassette-wise in the form of NotI-ApaI (H chain) and EcoRI-SplI (L 
chain) restriction fragments into a vector for human CDR-transplanted 
antibody expression, the ApaI and EcoRI restriction sites occurring in the 
rabbit .beta.-globin gene splicing and poly A signals of the plasmid 
pSE1UK1SEd1-3 were modified in the following manner. 
Three .mu.g of the plasmid pBluescript SK(-) (Stratagene) was added to 10 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI 
(Takara Shuzo) was further added, and the digestion reaction was allowed 
to proceed at 37.degree. C. for 1 hour. The reaction mixture was subjected 
to ethanol precipitation, and the 3' cohesive ends resulting from ApaI 
digestion were rendered blunt using DNA Blunting Kit (Takara Shuzo), 
followed by ligation using DNA Ligation Kit (Takara Shuzo). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101. Thus was obtained a plasmid, pBSA, shown in FIG. 
64. Furthermore, 3 .mu.g of the plasmid pBSA thus obtained was added to 10 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of the 
restriction enzyme EcoRI (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to ethanol precipitation, and the 5' cohesive ends 
resulting from EcoRI digestion were rendered blunt using DNA Blunting Kit 
(Takara Shuzo), followed by ligation using DNA Ligation Kit (Takara 
Shuzo). The thuso-btained recombinant plasmid DNA solution was used to 
transform Escherichia coli HB101. Thus was obtained the plasmid pBSAE 
shown in FIG. 65. 
Then, 3 .mu.g of the thus-obtained plasmid pBSAE was added to 10 .mu.l of 
10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride, 50 mM sodium chloride and 1 mM DTT, 10 units of the restriction 
enzyme HindIII (Takara Shuzo) was further added, and the reaction was 
allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
subjected to ethanol precipitation, the precipitate was dissolved in 20 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, and the solution was divided into two 
10-.mu.l portions. To one portion, 10 units of the restriction enzyme 
SacII (Toyobo) was further added and, to the other, 10 units of the 
restriction enzyme KpnI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. Both the reaction 
mixtures were fractionated by agarose gel electrophoresis, whereby about 
0.3 .mu.g each of a HindIII-SacII fragment (about 2.96 kb) and a 
KpnI-HindIII fragment (about 2.96 kb) were recovered. 
Then, 3 .mu.g of the plasmid pSE1UK1SEd1-3 was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of the restriction enzyme SacII (Toyobo) and 10 units 
of the restriction enzyme KonI (Takara Shuzo) were further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to ethanol precipitation, the precipitate was 
dissolved in 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT, 
10 units of the restriction enzyme HindIII (Takara Shuzo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, whereby about 0.2 .mu.g each of a HindIII-SacII fragment 
(about 2.42 kb) and a KpnI-HindIII fragment (about 1.98 kb) were 
recovered. 
Then, 0.1 .mu.g of the thus-obtained HindIII-SacII fragment of 
pSE1UK1SEd1-3 and 0.1 .mu.g of the above HindIII-SacII fragment of pBSAE 
were dissolved in a total of 20 .mu.l of sterilized water and ligated to 
each other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101 and, as a result, a plasmid, pBSH-S, shown in FIG. 
66 was obtained. Further, 0.1 .mu.g of the above-mentioned KpnI-HindIII 
fragment of pSE1UK1SEd1-3 and 0.1 .mu.g of the above-mentioned 
KpnI-HindIII fragment of pBSAE were dissolved in a total of 20 .mu.l of 
sterilized water and ligated to each other using Ready-To-Go T4 DNA Ligase 
(Pharmacia Biotech). The thus-obtained recombinant plasmid DNA solution 
was used to transform Escherichia coli HB101, and the plasmid pBSK-H shown 
in FIG. 67 was obtained. 
Then, 3 .mu.g each of the thus-obtained plasmids pBSH-S and pBSK-H were 
respectively added to 10-.mu.l portions of 10 mM Tris-hydrochloride buffer 
(pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added to each mixture, 
and the reaction was allowed to proceed at 37.degree. C. for 1 hour. Both 
the reaction mixtures were subjected to ethanol precipitation. With each 
precipitate, the 3' cohesive ends resulting from ApaI digestion were 
rendered blunt using DNA Blunting Kit (Takara Shuzo), followed by ligation 
using DNA Ligation Kit (Takara Shuzo). The thus-obtained recombinant DNA 
solution were used to transform Escherichia coli HB101, and the plasmids 
pBSH-SA and pBSK-HA shown in FIG. 68 were obtained. 
Then, 5 .mu.g each of the thus-obtained plasmids pBSH-SA and pBSK-HA were 
respectively added to 10-.mu.l portions of 50 mM Tris-hydrochloride buffer 
(pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 
mM DTT, 1 unit of the restriction enzyme EcoRI (Takara Shuzo) was further 
added to each mixture, and the reaction was allowed to proceed at 
37.degree. C. for 10 minutes for partial digestion. Both the reaction 
mixtures were subjected to ethanol precipitation. With each precipitate, 
the 5' cohesive ends resulting from EcoRI digestion were rendered blunt 
using DNA Blunting Kit (Takara Shuzo), followed by fractionation by 
agarose gel electrophoresis, whereby about 0.5 .mu.g each of a fragment 
about 5.38 kb in length and a fragment about 4.94 kb in length were 
recovered. The thus-recovered fragments (0.1 .mu.g each) were each 
dissolved in a total of 20 .mu.l of sterilized water and subjected to 
ligation treatment using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). 
The thus-obtained recombinant DNA solutions were respectively used to 
transform Escherichia coli HB101, and the plasmids pBSH-SAE and pBSK-HAE 
shown in FIG. 69 were obtained. 
Then, 3 .mu.g each of the thus-obtained plasmids pBSH-SAE and pBSK-HAE were 
respectively added to 10-.mu.l portions of 50 mM Tris-hydrochloride buffer 
(pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 
mM DTT, 10 units of the restriction enzyme EcoRI (Takara Shuzo) was 
further added to each mixture, and the reaction was allowed to proceed at 
37.degree. C. for 1 hour. Both the reaction mixtures were subjected to 
ethanol precipitation. With each precipitate, the 5' cohesive ends 
resulting from EcoRI digestion were rendered blunt using DNA Blunting Kit 
(Takara Shuzo), followed by ligation using DNA Ligation Kit (Takara 
Shuzo). The thus-obtained recombinant plasmid DNA solutions were each used 
to transform Escherichia coli HB101, and two plasmids, pBSH-SAEE and 
pBSK-HAEE, shown in FIG. 70 were obtained. Ten .mu.g each of the 
thus-obtained plasmids were subjected to sequencing reaction according to 
the instructions attached to AutoRead Sequencing Kit (Pharmacia Biotech), 
followed by base sequence determination by electrophoresis on A.L.F. DNA 
Sequencer (Pharmacia Biotech), whereby it was confirmed that both the ApaI 
and EcoRI sites had disappeared as a result of the above modification. 
(2) SalI restriction site introduction downstream from rabbit .beta.-globin 
gene splicing and poly A signals and SV40 early gene poly A signal 
For making it possible to exchange the antibody H chain and L chain 
expression promoters of the human CDR-transplanted antibody expression 
vector each for an arbitrary promoter, a SalI restriction site was 
introduced into the plasmid pSE1UK1SEd1-3 downstream from the rabbit 
.beta.-globin gene splicing and poly A signals and from the SV40 early 
gene poly A signal in the following manner. 
Three .mu.g of the plasmid pBSK-HAEE obtained in Paragraph 1 (1) of Example 
3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme NaeI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was dissolved in 
20 .mu.l of 50 mM Tris-hydrochloride buffer (pH 9.0) containing 1 mM 
magnesium chloride, 1 unit of alkaline phosphatase (E. coli C75, Takara 
Shuzo) was further added, and the reaction was allowed to proceed at 
37.degree. C. for 1 hour for dephosphorylation at the 5' termini. The 
reaction mixture was further subjected to phenol-chloroform extraction and 
then to ethanol precipitation, and the precipitate was dissolved in 20 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 8.0) containing 1 mM disodium 
ethylenediaminetetraacetate (hereinafter briefly referred to as TE 
buffer). One .mu.l of said reaction solution and 0.1 .mu.g of a 
phosphorylated SaIlI linker (Takara Shuzo) were added to sterilized water 
to make a total volume of 20 .mu.l, followed by ligation treatment using 
Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and a plasmid, pBSK-HAEESal, shown in FIG. 71 was obtained. Ten 
.mu.g of the plasmid thus obtained was subjected to sequencing reaction 
according to the instructions attached to AutoRead Sequencing Kit 
(Pharmacia Biotech), followed by electrophoresis on A.L.F. DNA Sequencer 
(Pharmacia Biotech) for base sequence determination, whereby it was 
confirmed that one SalI restriction site had been introduced downstream 
from the rabbit .beta.-globin gene splicing and poly A signals and from 
the SV40 early gene poly A signal. 
(3) Modification of ApaI restriction site occurring in poly A signal of 
Herpes simplex virus thymidine kinase (hereinafter referred to as HSVtk) 
gene 
The ApaI restriction site occurring in the HSVtk gene poly A signal 
downstream from the Tn5 kanamycin phosphotransferase gene of the plasmid 
pSE1UK1SEd1-3 was modified in the following manner. 
Three .mu.g of the plasmid pBSA obtained in Paragraph 1 (1) of Example 3 
was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme SacII (Toyobo) was further added, and the reaction was 
allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
subjected to ethanol precipitation, the precipitate was added to 10 .mu.l 
of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium 
chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme XhoI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 1 .mu.g of 
a SacII-XhoI fragment (about 2.96 kb) was recovered. 
Then, 5 .mu.g of the plasmid pSE1UK1SEd1-3 was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of the restriction enzyme SacII (Toyobo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was subjected to ethanol precipitation, the 
precipitate was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 
7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM 
DTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) was further 
added, and the reaction was fractionated by agarose gel electrophoresis, 
whereby about 1 .mu.g of a SacII-XhoI fragment (about 4.25 kb) was 
recovered. 
Then, 0.1 .mu.g of the above SacII-XhoI fragment of pBSA and the above 
SacII-XhoI fragment of pSE1UK1SEd1-3 were added to a total of 20 gl of 
sterilized water, followed by ligation using Ready-To-Go T4 DNA Ligase 
(Pharmacia Biotech). The thus-obtained recombinant plasmid DNA solution 
was used to transform Escherichia coli HB101, and the plasmid pBSX-S shown 
in FIG. 72 was obtained. 
Then, 3 .mu.g of the thus-obtained plasmid pBSX-S was added to 10 .mu.l of 
10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI (Takara 
Shuzo) was further added, and the reaction was allowed to proceed at 
37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the 3' cohesive ends resulting from ApaI digestion were 
rendered blunt using DNA Blunting Kit (Takara Shuzo) and then ligation was 
carried out using DNA Ligation Kit (Takara Shuzo). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and a plasmid, pBSX-SA, shown in FIG. 73 was obtained. Ten .mu.g of 
the thus-obtained plasmid was subjected to sequencing reaction according 
to the instructions attached to AutoRead Sequencing Kit (Pharmacia 
Biotech), followed by electrophoresis on A.L.F. DNA Sequencer (Pharmacia 
Biotech) for base sequence determination, whereby it was confirmed that 
the ApaI restriction site in the HSVtk gene poly A signal had disappeared. 
(4) Construction of human CDR-transplanted antibody L chain expression unit 
A plasmid, pMohC.kappa., containing a human antibody .kappa. L chain 
constant region cDNA downstream from the promoter/enhancer of the Moloney 
mouse leukemia virus long terminal repeat and having a human 
CDR-transplanted antibody L chain expression unit allowing cassette-wise 
insertion thereinto of a human CDR-transplanted antibody L chain variable 
region was constructed in the following manner. 
Three .mu.g of the plasmid pBluescript SK(-) (Stratagene) was added to 10 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme SacI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ClaI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, and the cohesive ends resulting from SacI and ClaI 
digestion were rendered blunt using DNA Blunting Kit (Takara Shuzo), 
followed by fractionation by agarose gel electrophoresis, whereby about 1 
.mu.g of a DNA fragment about 2.96 kb in length was recovered. A 0.1-.mu.g 
portion of the DNA fragment recovered was added to a total of 20 .mu.l of 
sterilized water and subjected to ligation reaction using Ready-To-Go T4 
DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNA 
solution was used to transform Escherichia coli HB101, and the plasmid 
pBSSC shown in FIG. 74 was obtained. 
Then, 3 .mu.g of the thus-obtained plasmid pBSSC was added to 10 .mu.l of 
10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride and 1 mM DTT, 10 units of the restriction enzyme KpnI (Takara 
Shuzo) was further added, and the reaction was allowed to proceed at 
37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was dissolved in 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme 
XhoI (Takara Shuzo) was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 1 hour. The reaction mixture was fractionated 
by agarose gel electrophoresis, whereby about 1 .mu.g of a KpnI-XhoI 
fragment (about 2.96 kb) was recovered. 
Then, 5 .mu.g of the plasmid pAGE147 described in JP-A-6-205694 was added 
to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme KpnI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was dissolved in 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme 
XhoI (Takara Shuzo) was further added, and the reaction was fractionated 
by agarose gel electrophoresis, whereby about 0.3 .mu.g of a KpnI-XhoI 
fragment (about 0.66 kb) containing the Moloney mouse leukemia virus long 
terminal repeat promoter/enhancer was recovered. 
Then, 0.1 .mu.g of the KpnI-XhoI fragment of pBSSC and 0.1 .mu.g of the 
KpnI-XhoI fragment of pAGE147 each obtained as mentioned above were 
dissolved in a total of 20 .mu.l of sterilized water and subjected to 
ligation using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pBSMo shown in FIG. 75 was 
obtained. 
Then, 3 .mu.g of the above plasmid pBSMo was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of the restriction enzyme KpnI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was subjected to ethanol precipitation, 
the precipitate was dissolved in 10 .mu.l of 10 mM Tris-hydrochloride 
buffer (pH 7.5) containing 50 mM sodium chloride, 10 mM magnesium chloride 
and 1 mM DTT, 10 units of the restriction enzyme HindIII (Takara Shuzo) 
was further added, and the reaction was allowed to proceed at 37.degree. 
C. for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, whereby about 1 .mu.g of a KpnI-HindIII fragment (about 
3.62 kb) was recovered. 
Then, synthetic DNAs respectively having the base sequences shown in SEQ ID 
No:38 and SEQ ID No:39 were synthesized using an automatic DNA synthesizer 
(Applied Biosystems model 380A) To 15 .mu.l of sterilized water were added 
0.3 .mu.g each of the thus-obtained synthetic DNAs, and the mixture was 
heated at 65.degree. C. for 5 minutes. The reaction mixture was allowed to 
stand at room temperature for 30 minutes and then 2 .mu.l of 10-fold 
concentrated buffer 500 mM Tris-hydrochloride (pH 7.6), 100 mM magnesium 
chloride, 50 mM DTT! and 2 .mu.l of 10 mM ATP were added, 10 units of T4 
polynucleotide kinase was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 30 minutes for phosphorylation of the 5' 
termini. To a total of 20 .mu.l of sterilized water were added 0.1 .mu.g 
of the above KpnI-HindIII fragment (3.66 kb) derived from the plasmid 
pBSMo and 0.05 .mu.g of the phsophorylated synthetic DNA pair, and 
ligation was effected using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). 
The thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pBSMoS shown in FIG. 76 was 
obtained. Ten .mu.g of the plasmid thus obtained was subjected to 
sequencing reaction according to the instructions attached to AutoRead 
Sequencing Kit (Pharmacia Biotech), followed by electrophoresis on A.L.F. 
DNA Sequencer (Pharmacia Biotech) for base sequence determination, whereby 
it was confirmed that the synthetic DNA pair had been introduced as 
desired. 
Then, 3 .mu.g of the plasmid pChiIgLA1 described in JP-A-5-304989 was 
dissolved in 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 
10 units each of the restriction enzymes EcoRI (Takara Shuzo) and EcoRV 
(Takara Shuzo) were further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was fractionated by 
agarose gel electrophoresis, whereby about 1 .mu.g of an EcoRI-EcoRV 
fragment (about 9.70 kb) was recovered. 
Then, synthetic DNAs respectively having the base sequences shown in SEQ ID 
NO:40 and SEQ ID NO:41 were synthesized using an automatic DNA synthesizer 
(Applied Biosystems model 380A). To 15 .mu.l of sterilized water were 
added 0.3 .mu.g each of the thus-obtained synthetic DNAs, and the mixture 
was heated at 65.degree. C. for 5 minutes. The reaction mixture was 
allowed to stand at room temperature for 30 minutes. Then, 2 .mu.l of 
10-fold concentrated buffer 500 mM Tris-hydrochloride (pH 7.6), 100 mM 
magnesium chloride, 50 mM DTT! and 2 .mu.l of 10 mM ATP were added, 10 
units of T4 polynucleotide kinase was further added, and the reaction was 
allowed to proceed at 37.degree. C. for 30 minutes for phosphorylation of 
the 5' termini. To a total of 20 .mu.l of sterilized water were added 0.1 
.mu.g of the above EcoRI-EcoRV fragment (9.70 kb) derived from the plasmid 
pChiIgLA1 and 0.05 .mu.g of the phsophorylated synthetic DNA, and ligation 
was effected using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pChiIgLA1S shown in FIG. 77 was 
obtained. 
Then, 3 .mu.g of the plasmid pBSMoS obtained in the above manner was 
dissolved in 10 .mu.l of 20 mM Tris-hydrochloride buffer (pH 8.5) 
containing 100 mM potassium chloride, 10 mM magnesium chloride and 1 mM 
DTT, 10 units of the restriction enzyme HpaI (Takara Shuzo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was subjected to ethanol precipitation, the 
precipitate was dissolved in 10 .mu.l of 50 mM Tris-hydrochloride buffer 
(pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 
mM DTT, 10 units of the restriction enzyme EcoRI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, whereby about 1 .mu.g of an HpaI-EcoRI fragment (about 
3.66 kb) was recovered. 
Then, 10 .mu.g of the plasmid pChiIgLA1S obtained as mentioned above was 
dissolved in 10 .mu.l of 20 mM Tris-acetate buffer (pH 7.9) containing 50 
mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT and 100 .mu.g/ml 
BSA, 10 units of the restriction enzyme NlaIV (New England BioLabs) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was subjected to ethanol precipitation, 
the precipitate was dissolved in 10 .mu.l of 50 mM Tris-hydrochloride 
buffer (pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium 
chloride and 1 mM DTT, 10 units of the restriction enzyme EcoRI (Takara 
Shuzo) was further added, and the reaction was allowed to proceed at 
37.degree. C. for 1 hour. The reaction mixture was fractionated by agarose 
gel electrophoresis, whereby about 0.3 .mu.g of an NlaIV-EcoRI fragment 
(about 0.41 kb) was recovered. 
Then, 0.1 .mu.g of the above HpaI-EcoRI fragment of pBSMoS and 0.1 .mu.g of 
the above NlaIV-EcoRI fragment of pChiIgLA1S were added to a total of 20 
.mu.l of sterilized water, and ligation was effected using Ready-To-Go T4 
DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNA 
solution was used to transform Escherichia coli HB101, and the plasmid 
pMohC.kappa. shown in FIG. 78 was obtained. 
(5) Construction of human CDR-transplanted antibody H chain expression unit 
A plasmid, pMohC.gamma.1, containing a human antibody .gamma.1 H chain 
constant region cDNA downstream from the pro-moter/enhancer of the Moloney 
mouse leukemia virus long terminal repeat and having a human 
CDR-transplanted antibody H chain expression unit allowing cassette-wise 
insertion thereinto of a human CDR-transplanted antibody H chain variable 
region was constructed in the following manner. 
Three .mu.g of the plasmid pBSMo obtained in Paragraph 1 (4) of Example 3 
was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 
10 units of the restriction enzyme XhoI (Takara Shuzo) was further added, 
and the reaction was allowed to proceed at 37.degree. C. for 1 hour. The 
reaction mixture was subjected to ethanol precipitation, the precipitate 
was dissolved in 10 .mu.l of 30 mM sodium acetate buffer (pH 5.0) 
containing 100 mM sodium chloride, 1 mM zinc acetate and 10% glycerol, 10 
units of Mung bean nuclease (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 10 minutes. The 
reaction mixture was subjected to phenol-chloroform extraction and then to 
ethanol precipitation, the cohesive ends of the precipitate were rendered 
blunt using DNA Blunting Kit (Takara Shuzo) and ligation was effected 
using DNA Ligation Kit (Takara Shuzo). The thus-obtained recombinant 
plasmid DNA solution was used to transform Escherichia coli HB101, and the 
plasmid pBSMoSal shown in FIG. 79 was obtained. A 10-.mu.g portion of the 
plasmid obtained was subjected to sequencing reaction according to the 
instructions attached to AutoRead Sequencing Kit (Pharmacia Biotech), 
followed by electrophoresis on A.L.F. DNA Sequencer (Pharmacia Biotech) 
for base sequence determination, whereby it was confirmed that the XhoI 
restriction site upstream of the Moloney mouse leukemia virus long 
terminal repeat promoter/enhancer had disappeared. 
Then, 3 .mu.g of the plasmid pBSMoSal obtained as mentioned above was added 
to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme KpnI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was dissolved in 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme 
HindIII (Takara Shuzo) was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 1 hour. The reaction mixture was fractionated 
by agarose gel electrophoresis, whereby about 1 .mu.g of a KpnI-HindIII 
fragment (about 3.66 kb) was recovered. 
Then, synthetic DNAs respectively having the base sequences shown in SEQ ID 
NO:42 and SEQ ID NO:43 were synthesized using an automatic DNA synthesizer 
(Applied Biosystems model 380A). To 15 .mu.l of sterilized water were 
added 0.3 .mu.g each of the thus-obtained synthetic DNAs, and the mixture 
was heated at 65.degree. C. for 5 minutes. The reaction mixture was 
allowed to stand at room temperature for 30 minutes. Then, 2 .mu.l of 
10-fold concentrated buffer 500 mM Tris-hydrochloride (pH 7.6), 100 mM 
magnesium chloride, 50 mM DTT! and 2 .mu.l of 10 mM ATP were added, 10 
units of T4 polynucleotide kinase was further added, and the reaction was 
allowed to proceed at 37.degree. C. for 30 minutes for phosphorylation of 
the 5' termini. To a total of 20 .mu.l of sterilized water were added 0.1 
.mu.g of the above KpnI-HindIII fragment (3.66 kb) derived from the 
plasmid pBSMoSal and 0.05 .mu.g of the phosphorylated synthetic DNA, and 
ligation was effected using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). 
The thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pBSMoSa1S shown in FIG. 80 was 
obtained. A 10-.mu.g portion of the thus-obtained plasmid was subjected to 
sequencing reaction according to the instructions attached to AutoRead 
Sequencing Kit (Pharmacia Biotech), followed by electrophoresis on A.L.F. 
DNA Sequencer (Pharmacia Biotech), for base sequence determination whereby 
it was confirmed that the synthetic DNA had been introduced as desired. 
Then, 10 Ag of the plasmid pChiIgHB2 described in JP-A-5-304989 was 
dissolved in 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 
10 units of the restriction enzyme Eco52I (Toyobo) was further added, and 
the reaction was allowed to proceed at 37.degree. C. for 1 hour. The 
reaction mixture was subjected to ethanol precipitation, the precipitate 
was dissolved in 10 .mu.l of 30 mM sodium acetate buffer (pH 5.0) 
containing 100 mM sodium chloride, 1 mM zinc acetate and 10% glycerol, 10 
units of Mung bean nuclease (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 10 minutes. The 
reaction mixture was subjected to phenolchloroform extraction and then to 
ethanol precipitation, and the cohesive ends were rendered blunt using DNA 
Blunting Kit (Takara Shuzo). After ethanol precipitation, the precipitate 
was dissolved in 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 0.7 .mu.g 
of ApaI-blunt end fragment (about 0.99 kb) was recovered. 
Then, 3 .mu.g of the plasmid pBluescript SK(-) (Stratagene) was added to 10 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was dissolved in 10 .mu.l of 33 mM 
Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mM 
potassium acetate, 0.5 mM DTT and 100 .mu.g/ml BSA, 10 units of the 
restriction enzyme SmaI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 1 .mu.g of 
an ApaI-SmaI fragment (about 3.0 kb) was recovered. 
Then, 0.1 .mu.g of the ApaI-blunt end fragment of pChiIgHB2 and 0.1 .mu.g 
of the ApaI-SmaI fragment of pbluescript SK(-), each obtained as mentioned 
above, were added to a total of 20 .mu.l of sterilized water and ligated 
to each other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pBShC.gamma.1 shown in FIG. 81 was 
obtained. 
Then, 5 .mu.g of the above plasmid pBShC.gamma.1 was dissolved in 10 .mu.l 
of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium 
chloride and 1 mM DTT, 10 units of the restriction enzyme ApAI (Takara 
Shuzo) was further added, and the reaction was allowed to proceed at 
37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was dissolved in 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme SpeI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was fractionated by 
agarose gel electrophoresis, whereby about 1 .mu.g of an ApaI-SpeI 
fragment (about 1.0 kb) was recovered. 
Then, 3 .mu.g of the plasmid pBSMoSalS obtained as mentioned above was 
dissolved in 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was dissolved in 
10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme SpeI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 1 .mu.g of 
an ApaI-SpeI fragment (about 3.66 kb) was recovered. 
Then, 0.1 .mu.g of the ApaI-SpeI fragment of pBShC.gamma.1 and 0.1 .mu.g of 
the ApaI-SpeI fragment of pBSMoSa1S, each obtained as mentioned above, 
were added to a total of 20 .mu.l of sterilized water and ligated to each 
other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pMohC.gamma.1 shown in FIG. 82 was 
obtained. 
(6) Construction of tandem cassette type humanized antibody expression 
vector pKANTEX93 
A tandem cassette type humanized antibody expression vector, pKANTEX93, was 
constructed using the various plasmids obtained in Paragraphs (1) through 
(5) of Example 3 in the following manner. 
Three .mu.g of the plasmid PBSH-SAEE obtained in Paragraph 1 (1) of Example 
3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 50 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 
10 units of the restriction enzyme HindIII (Takara Shuzo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was subjected to ethanol precipitation, the 
precipitate was dissolved in 10 .mu.l of 50 mM Tris-hydrochloride buffer 
(pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 
mM DTT, 10 units of the restriction enzyme SalI (Takara Shuzo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, whereby about 1 .mu.g of a HindIII-SalI fragment (about 
5.42 kb) was recovered. 
Then, 5 .mu.g of the plasmid PBSK-HAEE obtained in Paragraph 1 (1) of 
Example 3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (ph 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme KpnI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was dissolved in 
10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme HindIII (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was fractionated by agarose gel electrophoresis, whereby about 0.8 
.mu.g of a KpnI-HindIII fragment (about 1.98 kb) containing the rabbit 
.beta.-globin gene splicing and poly A signals, the SV40 early gene poly A 
signal and the SV40 early gene promoter was recovered. 
Then, 5 .mu.g of the plasmid pMohC.gamma.1 obtained in Paragraph 1 (5) of 
Example 3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme KpnI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was dissolved in 
10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme SalI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 0.8 .mu.g 
of a human CDR-transplanted antibody H chain expression unit-containing 
KpnI-SalI fragment (about 1.66 kb) was recovered. 
Then, 0.1 .mu.g of the HindIII-SalI fragment of PBSH-SAEE, 0.1 .mu.g of the 
KpnI-HindIII fragment of pBSK-HAEE and 0.1 .mu.g of the KpnI-SalI fragment 
of pMohC.gamma.1, each obtained as mentioned above, were added to a total 
of 20 .mu.l of sterilized water and ligated together using Ready-To-Go T4 
DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNA 
solution was used to transform Escherichia coli HB101, and the plasmid 
pMo.gamma.1SP shown in FIG. 83 was obtained. 
The, 3 .mu.g of the above plasmid pMo.gamma.1SP was added to 10 .mu.l of 50 
mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 
10 mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme 
SalI (Takara Shuzo) and 10 units of the restriction enzyme XhoI were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, whereby about 1 .mu.g of a SalI-XhoI fragment (about 9.06 
kb) was recovered. 
Then, 5 .mu.g of the plasmid pBSK-HAEESal obtained in Paragraph 1 (2) of 
Example 3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme KpnI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was. dissolved in 
10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme SalI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 0.7 .mu.g 
of a KpnI-SalI fragment (about 1.37 kb) containing the rabbit 
.beta.-globin gene splicing and poly A signals and the SV40 early gene 
poly A signal was recovered. 
Then, 5 .mu.g of the plasmid pMohC.kappa. obtained in Paragraph 1 (4) of 
Example 3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1mM DTT, 10 units of the 
restriction enzyme KpnI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was dissolved in 
10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme XhoI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 0.7 .mu.g 
of a human CDR-transplanted antibody L chain expression unit-containing 
KpnI-XhoI fragment (about 1.06 kb) was recovered. 
Then, 0.1 .mu.g of the SalI-XhoI fragment of pMo.gamma.1SP, 0.1 .mu.g of 
the KpnI-SalI fragment of pBSK-HAEESal and 0.1 .mu.g of the KpnI-XhoI 
fragment of pMohC.kappa., each obtained as mentioned above, were added to 
a total of 20 .mu.l of sterilized water and ligated together using 
Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and the plasmid pMo.kappa..gamma.1SP shown in FIG. 84 was obtained. 
Then, 3 .mu.g of the above plasmid pMo.kappa..gamma.1 SP was added to 10 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium 
chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme XhoI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was added to 10 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 1 units of the restriction enzyme SacII 
(Toyobo) was further added, and the reaction was allowed to proceed at 
37.degree. C. for 10 minutes for partial digestion. The reaction mixture 
was fractionated by agarose gel electrophoresis, and about 0.2 .mu.g of a 
SacII-XhoI fragment (about 8.49 kb) was recovered. 
Then, 3 .mu.g of the plasmid pBSX-SA obtained in Paragraph 1 (4) of Example 
3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme SacII (Toyobo) was further added, and the reaction was 
allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
subjected to ethanol precipitation, the precipitate was dissolved in 10 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium 
chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme XhoI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, and about 1 .mu.g of a 
SacII-XhoI fragment (about 4.25 kb) was recovered. 
Then, 0.1 .mu.g of the SacII-XhoI fragment of pMo.kappa..gamma.1SP and 0.1 
.mu.g of the SacII-XhoI fragment of pBSX-SA, each obtained as mentioned 
above, were added to a total of 20 .mu.l of sterilized water and ligated 
to each other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pKANTEX93 shown in FIG. 85 was 
obtained. 
2. Expression of Human Anti-GM.sub.2 Chimera Antibody Using Humanized 
Antibody Expression Vector pKANTEX93 
Human anti-GM.sub.2 chimera antibody expression was effected using the 
humanized antibody expression vector pKANTEX93 mentioned above in 
Paragraph 1 of Example 3 in the following manner. 
(1) Construction of plasmid pBSH3 containing mouse anti-GM.sub.2 antibody 
KM796 H chain variable region cDNA 
Three .mu.g of the plasmid pBluescript SK(-) (Stratagene) was added to 10 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units each of the restriction enzymes 
SacII (Toyobo) and KpnI (Takara Shuzo) were further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to ethanol precipitation, and the precipitate was 
subjected to blunting treatment for rendering blunt the 3' cohesive ends 
resulting from the restriction enzyme digestion using DNA Blunting Kit 
(Takara Shuzo) and then to fractionation by agarose gel electrophoresis, 
and about 1 .mu.g of a DNA fragment about 2.95 kb in size was recovered. 
Then, synthetic DNAs respectively having the base sequences shown in SEQ ID 
NO:44 and SEQ ID NO:45 were synthesized using an automatic DNA synthesizer 
(Applied Biosystems model 380A). To 15 .mu.l of sterilized water were 
added 0.3 .mu.g each of the synthetic DNAs obtained, and the mixture was 
heated at 65.degree. C. for 5 minutes. The reaction mixture was allowed to 
stand at room temperature for 30 minutes and then 2 .mu.l of 10-fold 
concentrated buffer 500 mM Tris-hydrochloride (pH 7.6), 100 mM magnesium 
chloride, 50 mM DTT! and 2 .mu.l of 10 mM ATP were added, 10 units of T4 
polynucleotide kinase was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 30 minutes for phosphorylating the 5' 
termini. To a total of 20 .mu.l of sterilized water were added 0.1 .mu.g 
of the DNA fragment (2.95 kb) derived from the plasmid pbluescript SK(-) 
and 0.05 .mu.g of the phosphorylated synthetic DNA, each obtained as 
mentioned above, followed by ligation to each other using Ready-To-Go T4 
DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNA 
solution was used to transform Escherichia coli HB101, and the plasmid 
pBSNA shown in FIG. 86 was obtained. Ten .mu.g of the plasmid obtained was 
subjected to sequencing reaction treatment according to the instructions 
attached to AutoRead Sequencing Kit (Pharmacia Biotech), followed by 
electrophoresis on A.L.F. DNA Sequencer (Pharmacia Biotech) for base 
sequence determination, whereby it was confirmed that the synthetic DNA 
had been introduced as desired. 
Then, 3 .mu.g of the plasmid pBSNA obtained as mentioned above was added to 
10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, the precipitate was added to 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, 1 mM DTT, 100 .mu.g/ml BSA and 0.01% Triton X-100, 
10 units of the restriction enzyme NotI (Takara Shuzo) was further added, 
and the reaction was allowed to proceed at 37.degree. C. for 1 hour. The 
reaction mixture was fractionated by agarose gel electrophoresis, and 
about 1 .mu.g of a DNA fragment about 2.95 kb in size was recovered. 
Then, 10 .mu.g of the plasmid pChi796HM1 obtained in Paragraph 7 (3) of 
Example 1 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was added to 10 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium 
chloride, 10 mM magnesium chloride, 1 mM DTT, 100 .mu.g/ml BSA and 0.0% 
Triton X-100, 10 units of the restriction enzyme NotI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 0.3 .mu.g of a DNA fragment about 0.45 kb in 
size was recovered. 
Then, 0.1 .mu.g of the Apa-NotI fragment of PBSNA and 0.1 .mu.g of the 
Apa-NotI fragment of pChi796HM1, each obtained as mentioned above, were 
added to a total of 20 .mu.l of sterilized water and ligated to each other 
using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and the plasmid pBSH3 shown in FIG. 87 was obtained. 
(2) Construction of plasmid pBSL3 containing mouse anti-GM.sub.2 antibody 
KM796 L chain variable region cDNA 
Three .mu.g of the plasmid pBluescript SK(-) (Stratagene) was added to 10 
.mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme KpnI 
(Takara Shuzo) was further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was subjected to ethanol 
precipitation, and the precipitate was subjected to blunting treatment for 
rendering blunt the 3' cohesive ends resulting from KpnI digestion using 
DNA Blunting Kit (Takara Shuzo) and then to ethanol precipitation, the 
precipitate was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme SacI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was fractionated by agarose gel electrophoresis, whereby about 1 .mu.g of 
a DNA fragment about 2.95 kb in size was recovered. 
Then, synthetic DNAs respectively having the base sequences shown in SEQ ID 
NO:46 and SEQ ID NO:47 were synthesized using an automatic DNA synthesizer 
(Applied Biosystems model 380A). To 15 .mu.l of sterilized water were 
added 0.3 .mu.g each of the synthetic DNAs obtained, and the mixture was 
heated at 65.degree. C. for 5 minutes. The reaction mixture was allowed to 
stand at room temperature for 30 minutes. Then, 2 .mu.l of 10-fold 
concentrated buffer 500 mM Tris-hydrochloride (pH 7.5), 100 mM magnesium 
chloride, 50 mM DTT! and 2 .mu.l of 10 mM ATP were added, 10 units of T4 
polynucleotide kinase was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 30 minutes for phosphorylating the 5' termini 
The, 0.1 .mu.g of the DNA fragment (2.95 kb) derived from the plasmid 
pbluescript SK(-) and 0.05 .mu.g of the phosphorylated synthetic DNA, each 
obtained as mentioned above, were added to a total of 20 .mu.l of 
sterilized water and ligated to each other using Ready-To-Go T4 DNA Ligase 
(Pharmacia Biotech). The thus-obtained recombinant plasmid DNA solution 
was used to transform Escherichia coli HB101, and the plasmid pBSES shown 
in FIG. 88 was obtained. Ten .mu.g of the plasmid obtained was subjected 
to sequencing reaction treatment according to the instructions attached to 
AutoRead Sequencing Kit (Pharmacia Biotech), followed by electrophoresis 
on A.L.F. DNA Sequencer (Pharmacia Biotech) for base sequence 
determination, whereby it was confirmed that the synthetic DNA had been 
introduced as desired. 
Then, 3 .mu.g of the plasmid pBSES obtained as mentioned above was added to 
10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 
10 units each of the restriction enzymes EcoRI (Takara Shuzo) and SplI 
(Takara Shuzo) were further added, and the reaction was allowed to proceed 
at 37.degree. C. for 1 hour. The reaction mixture was fractionated by 
agarose gel electrophoresis, and about 1 .mu.g of a DNA fragment about 
2.95 kb in size was recovered. 
The, 5 .mu.g of the plasmid pKM796L1 obtained in Paragraph 4 of Example 1 
was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and AflIII (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 0.3 .mu.g of an EcoRI-AflIII fragment about 
0.39 kb in size was recovered. 
The, synthetic DNAs respectively having the base sequences shown in SEQ ID 
NO:48 and SEQ ID NO:49 were synthesized using an automatic DNA synthesizer 
(Applied Biosystems model 380A). To 15 .mu.l of sterilized water were 
added 0.3 .mu.g each of the synthetic DNAs obtained, and the mixture was 
heated at 65.degree. C. for 5 minutes. The reaction mixture was allowed to 
stand at room temperature for 30 minutes. Then, 2 .mu.l of 10-fold 
concentrated buffer 500 mM Tris-hydrochloride (pH 7.6), 100 mM magnesium 
chloride, 50 mM DTT! and 2 .mu.l of 10 mM ATP were added, 10 units of T4 
polynucleotide kinase was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 30 minutes for phosphorylating the 5' 
termini. 
Then, 0.1 .mu.g of the pBSES-derived EcoRI-SplI fragment (2.95 kb), 0.1 
.mu.g of the pKM796L1-derived EcoRI-AflIII fragment and 0.05 .mu.g of the 
phosphorylated synthetic DNA, each obtained as mentioned above, were added 
to a total of 20 .mu.l of sterilized water and ligated together using 
Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and the plasmid pBSL3 shown in FIG. 89 was obtained. Ten .mu.g of 
the plasmid obtained was subjected to sequencing reaction treatment 
according to the instructions attached to AutoRead Sequencing Kit 
(Pharmacia Biotech), followed by electrophoresis on A.L.F. DNA Sequencer 
(Pharmacia Biotech) for base sequence determination, whereby it was 
confirmed that the synthetic DNA had been introduced as desired. 
(3) Construction of human anti-GM.sub.2 chimera antibody expression vector 
pKANTEX796 
An human anti-GM.sub.2 chimera antibody expression vector, pKANTEX796, was 
constructed using the plasmid pKANTEX93 obtained in Paragraph 1 of Example 
3 and the plasmids pBSH3 and pBSL3 respectively obtained in Paragraph 2 
(1) and (2) of Example 3, in the following manner. 
Three .mu.g of the plasmid pBSH3 was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was subjected to ethanol precipitation, 
the precipitate was dissolved in 10 .mu.l of 50 mM Tris-hydrochloride 
buffer (pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium 
chloride, 1 mM DTT, 100 .mu.g/ml BSA and 0.01% Triton X-100, 10 units of 
the restriction enzyme NotI (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was fractionated by agarose gel electrophoresis, and about 0.3 
.mu.g of an ApaI-NotI fragment about 0.46 kb in size was recovered. 
Then, 3 .mu.g of the plasmid pKANTEX93 was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was subjected to ethanol precipitation, 
the precipitate was dissolved in 10 .mu.l of 50 mM Tris-hydrochloride 
buffer (pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium 
chloride, 1 mM DTT, 100 .mu.g/ml BSA and 0.01% Triton X-100, 10 units of 
the restriction enzyme NotI (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was fractionated by agarose gel electrophoresis, whereby about 1 
.mu.g of an ApaI-NotI fragment about 12.75 kb in size was recovered. 
Then, 0.1 .mu.g of the pBSH3-derived ApaI-NotI fragment and 0.1 .mu.g of 
the pKANTEX93-derived ApaI-NotI fragment, each obtained as mentioned 
above, were added to a total of 20 .mu.l of sterilized water and ligated 
to each other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pKANTEX796H shown in FIG. 90 was 
obtained. 
Then, 3 .mu.g of the plasmid pBSL3 was added to 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 0.3 .mu.g of an EcoRI-SplI fragment about 0.4 
kb in size was recovered. 
Then, 3 .mu.g of the plasmid pKANTEX796H was added to 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 1 .mu.g of an EcoRI-SplI fragment about 13.20 
kb in size was recovered. 
Then, 0.1 .mu.g of the pBSL3-derived EcoRI-SplI fragment and 0.1 .mu.g of 
the pKANTEX796H-derived EcoRI-SplI fragment, each obtained as mentioned 
above, were added to a total of 20 .mu.l of sterilized water and ligated 
to each other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pKANTEX796 shown in FIG. 91 was 
obtained. 
(4) Expression of human anti-GM.sub.2 chimera antibody by pKANTEX796 
According to the procedure described in Paragraph 11 of Example 1, 
pKANTEX796 was introduced into YB2/0 (ATCC CRL 1581) cells and, as a 
result of selection by means of G418 (0.5 mg/ml) and MTX (200 nM), a cell 
line capable of producing about 1 to 2 .mu.g/ml of human anti-GM.sub.2 
chimera antibody was obtained. It was confirmed that efficient and stable 
humanized antibody expression is possible using expression vector 
pKANTEX93. 
3. Transient Humanized Antibody Expression in COS-7 (ATCC CRL 1651) Cells 
For enabling more rapid activity evaluation of various versions of human 
CDR-transplanted anti-GM.sub.2 antibody, transient expression of human 
anti-GM.sub.2 chimera antibody expression was caused in COS-7 cells by the 
Lipofectamine method using pKANTEX796 and a variant thereof in the 
following manner. 
(1) Construction of variant of pKANTEX796 
Since transient antibody expression in animal cells is dependent on the 
copy number of an expression vector introduced, it was supposed that an 
expression vector smaller in size would show a higher expression 
efficiency. Therefore, a smaller humanized antibody expression vector, 
pT796, was constructed by deleting a region supposedly having no effect on 
humanized antibody expression from pKANTEX796 in the following manner. 
Thus, 3 .mu.g of the plasmid pKANTEX796 was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10 mM 
magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme 
HindIII (Takara Shuzo) was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 1 hour. The reaction mixture was subjected to 
ethanol precipitation, the precipitate was dissolved in 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzyme 
MluI (Takara Shuzo) was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 1 hour. The reaction mixture was subjected to 
ethanol precipitation, and the 5' cohesive ends resulting from the 
restriction enzyme digestion were rendered blunt using DNA Blunting Kit 
(Takara Shuzo). The reaction mixture was fractionated by agarose gel 
electrophoresis and about 1 .mu.g of a DNA fragment about 9.60 kb in size 
was recovered. A 0.1-.mu.g portion of the thus-recovered DNA fragment was 
added to a total of 20 .mu.l of sterilized water and subjected to ligation 
treatment using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pT796 shown in FIG. 92 was 
obtained. 
(2) Transient expression of human anti-GM.sub.2 chimera antibody using 
pKANTEX796 and pT796 
A 1.times.10.sup.5 cells/ml suspension of COS-7 cells was distributed in 
2-ml portions into wells of a 6-well plate (Falcon) and cultured overnight 
at 37.degree. C. Two .mu.g of pKANTEX796 or pT796 was added to 100 .mu.l 
of OPTI-MEM medium (Gibco), a solution prepared by adding 10 .mu.l of 
LIPOFECTAMINE reagent (Gibco) to 100 .mu.l of OPTI-MEM medium (Gibco) was 
further added, and the reaction was allowed to proceed at room temperature 
for 40 minutes to cause DNA-liposome complex formation. The COS-7 cells 
cultured overnight were washed twice with 2 ml of OPTI-MEM medium (Gibco), 
the complex-containing solution was added, and the cells were cultured at 
37.degree. C. for 7 hours. Then, the solution was removed, 2 ml of DMEM 
medium (Gibco) containing 10% FCS was added to each well, and the cells 
were cultured at 37.degree. C. After 24 hours, 48 hours, 72 hours, 96 
hours and 120 hours of cultivation, the culture supernatant was recovered 
and, after concentration procedure as necessary, evaluated for human 
anti-GM.sub.2 chimera antibody activity in the culture supernatant by the 
ELISA method described in Paragraph 11 of Example 1. The results are shown 
in FIG. 93. As shown in FIG. 93, higher levels of transient human 
anti-GM.sub.2 chimera antibody expression was observed with pT796 as 
compared with pKANTEX796. For pT796, the level of expression was highest 
at 72 to 96 hours, the concentration being about 30 ng/ml (in terms of 
GM.sub.2 binding activity). The above results indicate that construction 
of a pKANTEX93-derived vector having a reduced size and introduction 
thereof into COS-7 cells make it possible to make activity evaluation of 
expression vector-derived humanized antibodies in a transient expression 
system. Furthermore, for close activity comparison of various versions of 
human CDR-transplanted anti-GM.sub.2 antibody as mentioned hereinafter, 
the ELISA method described below under (3) was used to determine antibody 
concentrations in transient expression culture supernatants. 
(3) Determination by ELISA of humanized antibody concentrations in 
transient expression culture supernatants 
A solution prepared by 400-fold dilution of goat anti-human IgG (.gamma. 
chain) antibody (Igaku Seibutugaku Kenkyusho) with PBS was distributed in 
50-.mu.l portions into wells of a 96-well microtiter plate and allowed to 
stand overnight at 4.degree. C. for binding to the wells. After removing 
the antibody solution, blocking was effected with 100 .mu.l of PBS 
containing 1% BSA at 37.degree. C. for 1 hour. Fifty .mu.l of a transient 
expression culture supernatant or purified human anti-GM.sub.2 chimera 
antibody was added thereto and allowed to react at room temperature for 1 
hour. Thereafter, the solution was removed, the wells were washed with 
PBS, and 50 .mu.l of a solution prepared by 500-fold dilution of 
peroxidase-labeled mouse anti-human .kappa. L chain antibody (Zymet) with 
PBS was added and allowed to react at room temperature for 1 hour. After 
washing with PBS, 50 .mu.l of an ABTS substrate solution prepared by 
dissolving 550 mg of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) 
diammonium salt in 0.1M citrate buffer (pH 4.2) and adding, just before 
use, 1 .mu.l/ml of hydrogen peroxide! was added for causing color 
development, and the OD.sub.415 was measured. 
4. Production of human CDR-transplanted anti-GM.sub.2 antibody using 
humanized antibody expression vector pKANTEX93 
A human CDR-transplanted anti-GM.sub.2 antibody higher in GM.sub.2 binding 
activity than the human CDR-transplanted anti-GM.sub.2 antibody described 
in Example 2 was produced in the following manner. 
(1) Modification of human CDR-transplanted anti-GM.sub.2 antibody H chain 
variable region described in Paragraph 1 (1) of Example 2 
DNAs coding for some versions of the human CDR-transplanted anti-GM.sub.2 
antibody H chain variable region described in Example 2 as derived by 
replacing several amino acids in regions other than the CDR (framework; 
hereinafter referred to as FR) with original mouse antibody amino acids 
were constructed in the following manner. Based on a computer model for 
the variable region of mouse KM796, those amino acid residues that were 
expected to contribute to restoration of antigen-binding activity as a 
result of mutation were selected as the amino acid residues to be mutated. 
First, DNAs respectively having the base sequences of SEQ ID NO:50 and SEQ 
ID NO:51 were synthesized using an automatic DNA synthesize (Applied 
Biosystems model 380A). 
Then, a version (version 2) of human CDR-transplanted antibody H chain 
variable region shown in SEQ ID NO:52 and having mutation in positions 78 
(threonine in lieu of glutamine), 79 (alanine in lieu of phenylalanine) 
and 80 (tyrosine in lieu of serine) was constructed in the same manner as 
in Paragraph 1 (1) of Example 2 using a synthetic DNA of SEQ ID NO:50 in 
lieu of the synthetic DNA of SEQ ID NO:27. 
Then, another version (version 4) of human CDR-transplanted antibody H 
chain variable region shown in SEQ ID NO:53 and having mutations in 
positions 27 (tyrosine in lieu of phenylalanine), 30 (threonine in lieu of 
serine), 40 (serine in lieu of proline) and 41 (histidine in lieu of 
proline) was constructed in the same manner as in Paragraph 1 (1) of 
Example 2 using a synthetic DNA of SEQ ID NO:51 in lieu of the synthetic 
DNA of SEQ ID NO:25. 
(2) Construction of human CDR-transplanted anti-GM.sub.2 antibody H chain 
variable region using known common human antibody H chain variable region 
According to Kabat et al. (Kabat E. A. et al., "Sequences of Proteins of 
Immunological Interest", US Dept. of Health and Human Services, 1991), 
known human antibody H chain variable regions are classifiable into 
subgroups I to III (HSG I to III) based on the homology of their FR 
regions, and common sequences have been identified for respective 
subgroups. Therefore, a human CDR-transplanted anti-GM.sub.2 antibody H 
chain variable region was constructed based on those common sequences. 
First, for selecting common sequences to serve as the base, the homology 
was examined between the FR of the mouse KM796 H chain variable region and 
the common sequence FR of the human antibody H chain variable region of 
each subgroup (Table 3). 
TABLE 3 
______________________________________ 
Homology (%) between mouse KM796 H chain variable region 
FR and human antibody H chain variable region common 
sequence FR 
HSG I HSG II HSG III 
______________________________________ 
72.1 52.9 58.6 
______________________________________ 
As a result, it was confirmed that subgroup I shows the greatest 
similarity. Thus, based on the common sequences of subgroup I, a human 
CDR-transplanted anti-GM.sub.2 antibody H chain variable region was 
constructed by the PCR method in the following manner. 
Synthetic DNAs respectively having the base sequences of SEQ ID NO:54 
through SEQ ID NO:59 were synthesized using an automatic DNA synthesizer 
(Applied Systems model 380A). The DNAs synthesized were added, each to a 
final concentration of 0.1 .mu.M, to 50 .mu.l of 10 mM Tris-hydrochloride 
buffer (pH 8.3) containing 50 mM potassium chloride, 1.5 mM magnesium 
chloride, 0.00% gelatin, 200 .mu.M dNTP, 0.5 .mu.M M13 primer RV (Takara 
Shuzo), 0.5 .mu.M M13 primer M4 (Takara Shuzo) and 2 units of TaKaRa Taq 
DNA polymerase, the mixture was covered with 50 .mu.l of mineral oil, a 
DNA thermal cycler (Perkin Elmer model PJ480) was loaded with the mixture, 
and 30 PCR cycles (2 minutes at 94.degree. C., 2 minutes at 55.degree. C. 
and 2 minutes at 72.degree. C. per cycle) were conducted. The reaction 
mixture was purified using QIAquick PCR Purification Kit (Qiagen) and then 
made into a solution in 30 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was added to 10 
.mu.l of 50 mM Tris-hydrochloride (pH 7.5) containing 100 mM sodium 
chloride, 10 mM magnesium chloride, 1 mM DTT, 100 .mu.g/ml BSA and 0.0% 
Triton X-100, 10 units of the restriction enzyme NotI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 0.2 .mu.g of an ApaI-NotI fragment about 0.44 
kb in size was recovered. 
Then, 3 .mu.g of the plasmid pBSH3 obtained in Paragraph 2 (1) of Example 3 
was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture 
was subjected to ethanol precipitation, the precipitate was added to 10 
.mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium 
chloride, 10 mM magnesium chloride, 1 mM DTT, 100 .mu.g/ml BSA and 0.01% 
Triton X-100, 10 units of the restriction enzyme NotI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agrose gel 
electrophoresis, and about 1 .mu.g of an ApaI-NotI fragment about 2.95 kb 
in size was recovered. 
Then, 0.1 .mu.g of the ApaI-NotI fragment of the human CDR-transplanted 
anti-GM.sub.2 antibody H chain variable region and 0.1 .mu.g of the 
ApaI-NotI fragment of pBSH3, each obtained as mentioned above, were added 
to a total of 20 .mu.l of sterilized water and ligated to each other using 
Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101. Plasmid DNAs were prepared from 10 transformant clones and their 
base sequences were determined. As a result, a plasmid, pBSH10, shown in 
FIG. 94 and having the desired base sequence was obtained. The amino acid 
sequence and base sequence of the human CDR-transplanted anti-GM.sub.2 
antibody H chain variable region contained in pBSH10 are shown in SEQ ID 
NO:60. In the amino acid sequence of the thus-constructed human 
CDR-transplanted anti-GM.sub.2 antibody H chain variable region, the amino 
acids in positions 67, 72, 84 and 98 in the FR as selected based on a 
computer model for the variable region are those amino acids found in the 
mouse KM796 H chain variable region. This is for the purpose of retaining 
the antigenbinding capacity of mouse KM796. 
(3) Modification of human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region described in Paragraph 1 (2) of Example 2 
First, a DNA having the base sequence of SEQ ID NO:61 was synthesized using 
an automatic DNA synthesizer (Applied Biosystems model 380A), and a human 
CDR-transplanted anti-GM.sub.2 antibody L chain variable region cDNA with 
a 3' terminus capable of pairing with the restriction enzyme SplI was 
constructed by following the same reaction procedure as in Paragraph 1 (2) 
of Example 2 using the synthetic DNA in lieu of the synthetic DNA of SEQ 
ID NO:35. 
Then, 3 .mu.g of the plasmid pBSL3 obtained in Paragraph 2 (2) of Example 3 
was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 
100 .mu.g/ml BSA, 10 units each of the restriction enzymes EcoRI (Takara 
Shuzo) and SplI (Takara Shuzo) were further added, and the reaction was 
allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
fractionated by agarose gel electrophoresis, and about 1 .mu.g of an 
EcoRI-SplI fragment about 2.95 kb in size was recovered. 
Then, 0.1 .mu.g of the EcoRI-SplI fragment of the human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region obtained as mentioned above 
and 0.1 .mu.g of the above EcoRI-SplI fragment of pBSL3 were added to a 
total of 20 .mu.l of sterilized water and ligated to each other using 
Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and the plasmid pBSL16 shown in FIG. 95 was obtained. 
Then, DNAs coding for certain versions of the human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region contained in the above 
plasmid pBSL16 were constructed by replacing a certain number of amino 
acids in the FR with original mouse antibody amino acids by mutagenesis by 
means of PCR in the following manner (FIG. 96). Based on a computer model 
for the variable region of mouse KM796, those amino acid residues that 
were expected to contribute to restoration of antigen-binding activity as 
a result of mutation were selected as the amino acid residues to be 
mutated. 
Antisense and sense DNA primers for introducing mutations were synthesized 
using an automatic DNA synthesizer (Applied Biosystems model 380A). A 
first PCR reaction was conducted in the same manner as in Paragraph 4 (2) 
of Example 3 using a final concentration each of 0.5 .mu.M of M13 primer 
RV (Takara Shuzo) and the antisense DNA primer and of M13 primer M4 
(Takara Shuzo) and the sense DNA primer, with 1 ng of pBSL16 as the 
template. Each reaction mixture was purified using QIAquick PCR 
Purification Kit (Qiagen) with elution with 20 .mu.l of 10 mM 
Tris-hydrochloride (pH 8.0). Using 5 .mu.l of each eluate, a second PCR 
reaction was conducted in the same manner as in Paragraph 4 (2) of Example 
3. The reaction mixture was purified using QIAaquick PCR Purification Kit 
(Qiagen) and then made into a solution in 30 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 0.2 .mu.g of an EcoRI-SplI fragment (about 0.39 
kb) of each mutant version of the human CDR-transplanted anti-GM.sub.2 
antibody L chain variable region was recovered. 
Then, 0.1 .mu.g of the above EcoRI-SplI fragment of each mutant version of 
the human CDR-transplanted anti-GM.sub.2 antibody L chain variable region 
and 0.1 .mu.g of the EcoRI-SplI fragment of pBSL3 were added to a total of 
20 .mu.l of sterilized water and ligated to each other using Ready-To-Go 
T4 DNA ligase (Pharmacia Biotech). The thus-obtained recombinant plasmid 
DNA solution was used to transform Escherichia coli HB101, and a plasmid 
DNA was prepared from a transformant clone, and the base sequence of said 
plasmid was determined. In this way, plasmids respectively containing a 
base sequence having a desired mutation or mutations were obtained. 
Thus, a plasmid, pBSLV1, containing version 1, shown in SEQ ID NO:64, of 
the human CDR-transplanted anti-GM.sub.2 antibody L chain variable region 
was obtained following the above procedure using the synthetic DNA of SEQ 
ID NO:62 as the mutant antisense primer and the synthetic DNA of SEQ ID 
NO:63 as the mutant sense primer. In the amino acid sequence of the 
version 1 human CDR-transplanted anti-GM.sub.2 antibody L chain variable 
region, the amino acid in position 15 in the FR is that amino acid found 
in the mouse KM796 L chain variable region. This is for the purpose of 
retaining the antigen-binding capacity of mouse KM796. 
A plasmid, pBSLV2, containing version 2, shown in SEQ ID NO:67, of the 
human CDR-transplanted anti-GM.sub.2 antibody L chain variable region was 
obtained following the above procedure using the synthetic DNA of SEQ ID 
NO:65 as the mutant antisense primer and the synthetic DNA of SEQ ID NO:66 
as the mutant sense primer. In the amino acid sequence of the version 2 
human CDR-transplanted anti-GM.sub.2 antibody L chain variable region, the 
amino acid in positions 46 in the FR is that amino acid found in the mouse 
KM796 L chain variable region. This is for the purpose of retaining the 
antigen-binding capacity of mouse KM796. 
A plasmid, pBSLV3, containing version 3, shown in SEQ ID NO:70, of the 
human CDR-transplanted anti-GM.sub.2 antibody L chain variable region was 
obtained following the above procedure using the synthetic DNA of SEQ ID 
NO:68 as the mutant antisense primer and the synthetic DNA of SEQ ID NO:69 
as the mutant sense primer. In the amino acid sequence of the version 3 
human CDR-transplanted anti-GM.sub.2 antibody L chain variable region, the 
amino acids in position 79 and 82 in the FR are those amino acids found in 
the mouse KM796 L chain variable region. This is for the purpose of 
retaining the antigen-binding capacity of mouse KM796. 
Then, a plasmid, pBSLV1+2, containing a human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region having both the version 1 
and version 2 mutations was constructed in the following manner. 
Three .mu.g of the plasmid pBSLV1 obtained as mentioned above was added to 
10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units each of 
the restriction enzymes EcoRI (Takara Shuzo) and HindIII (Takara Shuzo) 
were further added, and the reaction was allowed to proceed at 37.degree. 
C. for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 0.2 .mu.g of an EcoRI-HindIII fragment about 
0.20 kb in size was recovered. 
Then, 3 .mu.g of the plasmid pBSLV2 obtained as mentioned above was added 
to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units each of 
the restriction enzymes EcoRI (Takara Shuzo) and HindIII (Takara Shuzo) 
were further added, and the reaction was allowed to proceed at 37.degree. 
C. for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 1 .mu.g of an EcoRI-HindIII fragment about 3.2 
kb in size was recovered. 
Then, 0.1 .mu.g of the EcoRI-HindIII fragment of pBSLV1 and 0.1 .mu.g of 
the EcoRI-HindIII fragment of pBSLV2, each obtained as mentioned above, 
were added to a total of 20 .mu.l of sterilized water and ligated to each 
other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The 
thus-obtained recombinant plasmid DNA solution was used to transform 
Escherichia coli HB101, and the plasmid pBSLV1+2 shown in FIG. 97 was 
obtained. 
Then, the PCR reaction procedure mentioned above was followed using 1 ng of 
the plasmid pBSLV1+2 obtained as mentioned above as the template, a 
synthetic DNA having the base sequence of SEQ ID NO:71 as the mutant 
antisense primer and a synthetic DNA having the base sequence of SEQ ID 
NO:72 as the mutant sense primer, whereby a plasmid, pBSLV4, containing a 
version 4 human CDR-transplanted anti-GM.sub.2 antibody L chain variable 
region set forth in SEQ ID NO:73 was obtained. In the amino acid sequence 
of the version 4 human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region, the amino acids in positions 15, 46, 69, 70 and 71 in the 
FR are those amino acids that are found in the mouse KM796 L chain 
variable region. This is for the purpose of retaining the antigen-binding 
capacity of mouse KM796. 
Then, the PCR reaction procedure mentioned above was followed using 1 ng of 
the plasmid pBSLV1+2 obtained as mentioned above as the template, a 
synthetic DNA having the base sequence of SEQ ID NO:74 as the mutant 
antisense primer and a synthetic DNA having the base sequence of SEQ ID 
NO:75 as the mutant sense primer, whereby a plasmid, pBSLV8, containing a 
version 8 human CDR-transplanted anti-GM.sub.2 antibody L chain variable 
region set forth in SEQ ID NO:76 was obtained. In the amino acid sequence 
of the version 8 human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region, the amino acids in positions 15, 46, 69, 70, 71, 76, 77 
and 78 in the FR are those amino acids that are found in the mouse KM796 L 
chain variable region. This is for the purpose of retaining the 
antigen-binding capacity of mouse KM796. 
Then, the PCR reaction procedure mentioned above was followed using 1 ng of 
the plasmid pBSLV4 obtained as mentioned above as the template, a 
synthetic DNA having the base sequence of SEQ ID NO:77 as the mutant 
antisense primer and a synthetic DNA having the base sequence of SEQ ID 
NO:78 as the mutant sense primer, whereby a plasmid, pBSLm-2, containing a 
version Lm-2 human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region set forth in SEQ ID NO:79 was obtained. In the amino acid 
sequence of the version Lm-2 human CDR-transplanted anti-GM.sub.2 antibody 
L chain variable region, the amino acids in positions 15, 35, 46, 69, 70 
and 71 in the FR are those amino acids that are found in the mouse KM796 L 
chain variable region. This is for the purpose of retaining the 
antigen-binding capacity of mouse KM796. 
Then, the PCR reaction procedure mentioned above was followed using 1 ng of 
the plasmid pBSLV4 obtained as mentioned above as the template, a 
synthetic DNA having the base sequence of SEQ ID NO:80 as the mutant 
antisense primer and a synthetic DNA having the base sequence of SEQ ID 
NO:81 as the mutant sense primer, whereby a plasmid, pBSLm-8, containing a 
version Lm-8 human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region set forth in SEQ ID NO:82 was obtained. In the amino acid 
sequence of the version Lm-8 human CDR-transplanted anti-GM.sub.2 antibody 
L chain variable region, the amino acids in positions 15, 46, 69, 70, 71, 
72 and 77 in the FR are those amino acids that are found in the mouse 
KM796 L chain variable region. This is for the purpose of retaining the 
antigen-binding capacity of mouse KM796. 
Then, a plasmid, pBSLm-28, containing a human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region having both the version 
Lm-2 and version Lm-8 mutations was constructed in the following manner. 
Three .mu.g of the plasmid pBSLm-2 obtained as mentioned above was added to 
10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme EcoRI (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to ethanol precipitation, the precipitate was added 
to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mM 
sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 
10 units of the restriction enzyme XbaI (Takara Shuzo) was further added, 
and the reaction as allowed to proceed at 37.degree. C. for 1 hour. The 
reaction mixture was fractionated by agarose gel electrophoresis, and 
about 0.2 .mu.g of an EcoRI-XbaI fragment about 0.24 kb in size was 
recovered. 
Then, 3 .mu.g of the plasmid pBSLm-8 obtained as mentioned above was added 
to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme EcoRI (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was subjected to ethanol precipitation, the precipitate was added 
to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mM 
sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 
10 units of the restriction enzyme XbaI (Takara Shuzo) was further added, 
and the reaction was allowed to proceed at 37.degree. C. for 1 hour. The 
reaction mixture was fractionated by agarose gel electrophoresis, and 
about 1 .mu.g of an EcoRI-XbaI fragment about 3.16 kb in size was 
recovered. 
Then, 0.1 .mu.g of the EcoRI-XbaI fragment of pBSLm-2 and 0.1 .mu.g of the 
EcoRI-XbaI fragment of pBSLm-8, each obtained as mentioned above, were 
added to a total of 20 .mu.l of sterilized water and ligated to each other 
using Ready-To-go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and the plasmid pBSLm-28 shown in FIG. 98 was obtained. The version 
Lm-28 human CDR-transplanted anti-GM.sub.2 antibody L chain variable 
region contained in the plasmid pBSLm-28 is shown in SEQ ID NO:83. In the 
amino acid sequence of the version Lm-28 human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region thus constructed, the amino 
acids in positions 15, 35, 46, 69, 70, 71, 72 and 77 are those amino acids 
that are found in the mouse KM796 L chain variable region. This is for the 
intended purpose of retaining the antigen-binding capacity of mouse KM796. 
(4) Construction of human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region using known common human antibody L chain variable region 
According to Kabat et al. (Kabat E. A. et al., "Sequences of Proteins of 
Immunological Interest", US Dept. of Health and Human Services, 1991), 
known human antibody L chain variable regions are classifiable into 
subgroups I to IV based on the homology of their FR regions, and common 
sequences have been identified for respective subgroups. Therefore, a 
human CDR-transplanted anti-GM.sub.2 antibody L chain variable region was 
constructed based on those common sequences. First, for selecting common 
sequences to serve as the base, the homology was examined between the FR 
of the mouse KM796 L chain variable region and the common sequence FR of 
the human antibody L chain variable region of each subgroup (Table 4). 
TABLE 4 
______________________________________ 
Homology (%) between mouse KM796 L chain variable region FR 
and human antibody L chain variable region common sequence FR 
HSG I HSG II HSG III HSG IV 
______________________________________ 
70.0 65.0 68.8 67.5 
______________________________________ 
As a result, it was confirmed that subgroup I shows the greatest 
similarity. Thus based on the common sequence of subgroup I, a human 
CDR-transplanted anti-GM.sub.2 antibody L chain variable region was 
constructed by the PCR method in the following manner. 
Synthetic DNAs respectively having the base sequences of SEQ ID NO:84 
through SEQ ID NO:89 were synthesized using an automatic DNA synthesizer 
(Applied Systems model 380A). The DNAs synthesized were added, each to a 
final concentration of 0.1 .mu.M, to 50 .mu.l of 10 mM Tris-hydrochloride 
buffer (pH 8.3) containing 50 mM potassium chloride, 1.5 mM magnesium 
chloride, 0.001% gelatin, 200 .mu.M dNTP, 0.5 .mu.M M13 primer RV (Takara 
Shuzo), 0.5 .mu.M M13 primer M4 (Takara Shuzo) and 2 units of TaKaRa Taq 
DNA polymerase. The mixture was covered with 50 .mu.l of mineral oil, a 
DNA thermal cycler (Perkin Elmer model PJ480) was loaded with the mixture, 
and 30 PCR cycles (2 minutes at 94.degree. C., 2 minutes at 55.degree. C. 
and 2 minutes at 72.degree. C. per cycle) were conducted. The reaction 
mixture was purified using QIAquick PCR Purification Kit (Qiagen) and then 
made into a solution in 30 .mu.l of 50 mM Tris-hydrochloride buffer (pH 
7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT 
and 100 .mu.g/ml BSA, 10 units each of the restriction enzymes EcoRI 
(Takara Shuzo) and SplI (Takara Shuzo) were further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. The reaction 
mixture was fractionated by agarose gel electro-phoresis, and about 0.2 
.mu.g of an EcoRI-SplI fragment about 0.39 kb in size was recovered. 
Then, 0.1 .mu.g of the above EcoRI-SplI fragment of the human 
CDR-transplanted anti-GM.sub.2 antibody L chain variable region and 0.1 
.mu.g of the EcoRI-SplI fragment of pBSL3 were added to a total of 20 
.mu.l of sterilized water and ligated to each other using Ready-To-Go T4 
DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNA 
solution was used to transform Escherichia coli HB101. Plasmid DNAs were 
prepared from 10 transformant clones and their base sequences were 
determined. As a result, a plasmid, PBSHSGL, shown in FIG. 99 and having 
the desired base sequence was obtained. The amino acid sequence and base 
sequence of the human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable region contained in pBSHSGL are shown in SEQ ID NO:90. In the 
amino acid sequence of the thus-constructed human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region, the amino acids in 
positions 4, 11, 15, 35, 42, 46, 69, 70, 71, 77 and 103 in the FR as 
selected based on a computer model for the variable region are those amino 
acids found in the mouse KM796 L chain variable region. This is for the 
intended purpose of retaining the antigen-binding capacity of mouse MK796. 
(5) Activity evaluation of mutant versions of human CDR-transplanted 
anti-GM.sub.2 antibody in terms of transient expression 
Various mutant version human CDR-transplanted anti-GM.sub.2 antibodies 
composed of the human CDR-transplanted anti-GM.sub.2 antibody H chain and 
L chain variable regions constructed in Paragraphs (1) through (4) of 
Example 3 and having varying mutations were evaluated for activity in 
terms of transient expression in the following manner. 
First, for evaluating the human CDR-transplanted anti-GM.sub.2 antibody H 
chain variable regions having varying mutations, expression vectors, 
pT796HCDRHV2, pT796HCDRHV4 and pT796HCDRH10, were constructed by replacing 
the mouse H chain variable region of the human anti-GM.sub.2 chimera 
antibody transient expression vector pT796 obtained in Paragraph 3 (1) of 
Example 3 with the human CDR-transplanted anti-GM.sub.2 antibody H chain 
variable regions having varying mutations, in the following manner. For 
comparison, an expression vector, pT796HCDR was constructed by replacing 
the mouse H chain variable region of pT796 with the human CDR-transplanted 
anti-GM.sub.2 antibody H chain variable region obtained in Paragraph 1 (1) 
of Example 1. 
Three .mu.g of the plasmid pT796 was added to 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, lmM DTT and 100 .mu.g/ml BSA, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 1 .mu.g of an EcoRI-SplI fragment about 9.20 kb 
in size was recovered. 
Then, 3 .mu.g of the plasmid pBSL16 obtained in Paragraph 4 (3) of Example 
3 was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 
100 .mu.g/ml BSA, 10 units each of the restriction enzymes EcoRI (Takara 
Shuzo) and SplI (Takara Shuzo) were further added, and the reaction was 
allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
fractionated by agarose gel electrophoresis, and about 0.3 .mu.g of an 
EcoRI-SplI fragment about0.39 kb in size was recovered. 
Then, 0.1 .mu.g of the EcoRI-SplI fragment of pT796 and 0.1 .mu.g of the 
EcoRI-SplI fragment of pBSL16, each obtained as mentioned above, were 
added to a total of 20 .mu.l of sterilized water and ligated to each other 
using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtained 
recombinant plasmid DNA solution was used to transform Escherichia coli 
HB101, and the plasmid pT796LCDR shown in FIG. 100 was obtained. 
Then, 3 .mu.g of the above plasmid pT796LCDR was added to 10 .mu.l of 10 mM 
Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride and 
1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) was 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was subjected to ethanol precipitation, 
the precipitate was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer 
(pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM 
DTT, 100 .mu.g/ml BSA and 0.01% Triton X-100, 10 units of the restriction 
enzyme NotI (Takara Shuzo) was further added, and the reaction was allowed 
to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
fractionated by agarose gel electrophoresis, and about 1 .mu.g of an 
ApaI-NotI fragment about 9.11 kb in size was recovered. 
Then, 0.1 .mu.g of the human CDR-transplanted anti-GM.sub.2 antibody H 
chain variable region obtained in Paragraph 1 (1) of Example 2 or the 
mutant version 2 or 4 human CDR-transplanted anti-GM.sub.2 antibody H 
chain variable region obtained in Paragraph 4 (1) of Example 3 and 0.1 
.mu.g of the ApaI-NotI fragment of pT796LCDR were added to a total of 20 
.mu.l of sterilized water and ligated to each other using Ready-To-Go T4 
DNA Ligase (Pharmacia Biotech). Each recombinant plasmid DNA solution thus 
obtained was used to transform Escherichia coli HB101. The plasmids 
pT796HLCDR, pT796HLCDRHV2 and pT796HLCDRHV4 shown in FIG. 101 were 
obtained. Then, 3 .mu.g of the plasmid pBSH10 obtained in Paragraph 4 (2) 
of Example 3 was added to 10 .mu.l of 10 mM Tris-hydrochloride buffer (pH 
7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme ApaI (Takara Shuzo) was further added, and the 
restriction was allowed to proceed at 37.degree. C. for 1 hour. The 
reaction mixture was subjected to ethanol precipitation, the precipitate 
was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100 
.mu.g/ml BSA and 0.01% Triton X-100, 10 units of the restriction enzyme 
NotI (Takara Shuzo) was further added, and the reaction was allowed to 
proceed at 37.degree. C. for 1 hour. The reaction mixture was fractionated 
by agarose gel electrophoresis, and about 0.3 .mu.g of an ApaI-NotI 
fragment about 0.44 kb in size was recovered. 
Then, 0.1 .mu.g of the ApaI-NotI fragment of pBSM10 and 0.1 .mu.g of the 
ApaI-NotI fragment of pT796LCDR were added to a total of 20 .mu.l of 
sterilized water and ligated to each other using Ready-To-Go T4 DNA Ligase 
(Pharmacia Biotech). The thus-obtained recombinant plasmid DNA solution 
was used to transform Escherichia coli HB101, and the plasmid 
pT796HLCDRH10 shown in FIG. 102 was obtained. 
Then, 3 .mu.g each of the plasmids pT796HLCDR, pT796HLCDRHV2, pT796HLCDRHV4 
and pT796HLCDRH10 were respectively added to 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. Each reaction mixture was fractionated by agarose gel 
electrophoresis, and about 1 .mu.g of an EcoRI-SplI fragment about 9.15 kb 
in size was recovered. 
Then, 5 .mu.g of the plasmid pBSL3 obtained in Paragraph 2 (2) of Example 3 
was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) 
containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 
100 .mu.g/ml BSA, 10 units each of the restriction enzymes EcoRI (Takara 
Shuzo) and SplI (Takara Shuzo) were further added, and the reaction was 
allowed to proceed at 37.degree. C. for 1 hour. The reaction mixture was 
fractionated by agarose gel electrophoresis, and about 0.4 .mu.g of an 
EcoRI-SplI fragment about 0.39 kb in size was recovered. 
Then, 0.1 .mu.g of the EcoRI-SplI fragment of each of pT796HLCDR, 
pT796HLCDRHV2, pT796HLCDRHV4 and pT796HLCDRH10 and 0.1 .mu.g of the 
EcoRI-SplI fragment of pBSL3 were added to a total of 20 .mu.l of 
sterilized water and ligated to each other using Ready-To-Go DNA Ligase 
(Pharmacia Biotech). Each recombinant plasmid DNA solution thus obtained 
was used to transform Escherichia coli HB101. In this way, the plasmids 
pT796HCDR, pT796HCDRHV2, pT796HCDRHV4 and pT796HCDRH10 shown in FIG. 103 
were obtained. 
Then, 2 .mu.g each of the plasmids pT796HCDR, pT796HCDRHV2, pT796HCDRHV4 
and pT796HCDRH10 thus obtained were used for transient human 
CDR-transplanted anti-GM.sub.2 antibody expression and for culture 
supernatant human CDR-transplanted anti-GM.sub.2 antibody activity 
evaluation by the procedures described in Paragraph 3 (2) and (3) of 
Example 3, respectively. After introduction of each plasmid, the culture 
supernatant was recovered at 72 hours, and the GM.sub.2 -binding activity 
and antibody concentration in the culture supernatant were determined by 
ELISA and the relative activity was calculated with the activity of the 
positive control chimera antibody taken as 100%. The results are shown in 
FIG. 104. 
The results revealed that the amino acid mutations alone in mutant versions 
2 and 4 have little influence on the restoration of the antigen-binding 
activity of the human CDR-transplanted anti-GM.sub.2 antibody but that the 
use of the pBSH10-derived human CDR-transplanted antibody H chain variable 
region constructed based on the known human antibody H chain variable 
region common sequence, contributes to the restoration of the 
antigen-binding activity. 
In view of the above results, the human CDR-transplanted anti-GM.sub.2 
antibody H chain variable region constructed based on the known human 
antibody H chain variable region common sequence as shown in SEQ ID NO:60 
was selected as a novel human CDR-transplanted anti-GM.sub.2 antibody H 
chain variable region. 
Then, for evaluating the human CDR-transplanted anti-GM.sub.2 antibody L 
chain variable regions having varying variations, expression vectors, 
pT796HLCDRLV1, pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4, pT796HLCDRLV8, 
pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 and pT796HLCDRHSGL, were 
constructed in the following manner by replacing the mouse L chain 
variable region of the vector pT796HCDRH10 for transient human 
CDR-transplanted anti-GM.sub.2 antibody expression obtained as mentioned 
above with the human CDR-transplanted anti-GM.sub.2 antibody L chain 
variable regions having varying mutations. 
Thus, 3 .mu.g of the plasmid pT796HCDRH10 was added to 10 .mu.l of 50 mM 
Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 
mM magnesium chloride, 1 mM DTT and 100 .mu.g/ml BSA, 10 units each of the 
restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) were 
further added, and the reaction was allowed to proceed at 37.degree. C. 
for 1 hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 1 .mu.g of an EcoRI-SplI fragment about 9.15 kb 
in size was recovered. 
Then, 3 .mu.g of the plasmid pBSLV1, pBSLV2, pBSLV3, pBSLV4, pBSLV8, 
pBSLm-2, pBSLm-8, pBSLm-28 or PBSHSGL obtained in Paragraph (3) or (4) of 
Example 3 was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 
7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT 
and 100 .mu.g/ml BSA, 10 units each of the restriction enzymes EcoRI 
(Takara Shuzo) and SplI (Takara Shuzo) were further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. Each reaction 
mixture was fractionated by agarose gel electrophoresis, and about 0.3 
.mu.g of an EcoRI-SplI fragment about 0.39 kb in size was recovered. 
Then, 0.1 .mu.g of the EcoRI-SplI fragment of the pT796HCDRH10 and 0.1 
.mu.g of the EcoRI-SplI fragment of each mutant version human 
CDR-transplanted anti-GM.sub.2 antibody L chain variable region were added 
to a total of 20 .mu.l of sterilized water and ligated to each other using 
Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Each recombinant plasmid 
DNA solution thus obtained was used to transform Escherichia coli HB101. 
In this way, the plasmids pT796HLCDRLV1, pT796HLCDRLV2, pT796HLCDRLV3, 
pT796HLCDRLV4, pT796HLCDRLV8, pT796HLCDRLm-2, pT796HLCDRLm-8, 
pT796HLCDRLm-28 and pT796HLCDRHSGL were obtained as shown in FIG. 105. 
Then, 2 .mu.g each of the thus-obtained plasmids pT796HLCDRLV1, 
pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4, pT796HLCDRLV8, 
pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 and pT796HLCDRHSGL and of 
the plasmid pT796HLCDR described in Example 2 and capable of expressing 
human CDR-transplanted anti-GM.sub.2 antibody were used for transient 
human CDR-transplanted anti-GM.sub.2 antibody expression and for culture 
supernatant human CDR-transplanted anti-GM.sub.2 antibody activity 
evaluation by the procedures described in Paragraph 3 (2) and (3) of 
Example 3, respectively. After introduction of each plasmid, the culture 
supernatant was recovered at 72 hours, and the GM.sub.2 -binding activity 
and antibody concentration in the culture supernatant were determined by 
ELISA and the relative activity was calculated with the activity of the 
positive control chimera antibody taken as 100%. The results are shown in 
FIG. 106. 
The results revealed that the amino acid mutations alone in mutant versions 
1, 2, 3, 4 and 8 have little influence on the restoration of the 
antigen-binding activity of the human CDR-transplanted anti-GM.sub.2 
antibody but that the amino acid mutations in mutant versions Lm-2 and 
Lm-8 contributes to the restoration of the antigen-binding activity. 
Furthermore, version Lm-28 having both the amino acid mutations of Lm-2 
and Lm-8 showed a high level of antigen-biding activity almost comparable 
to that of the chimera antibody, revealing that those amino acids mutated 
in producing Lm-28 were very important from the antigen-binding activity 
viewpoint. 
In view of the above results, the version Lm-28 human CDR-transplanted 
anti-GM.sub.2 antibody L chain variable region shown in SEQ ID NO:83 was 
selected as a first novel human CDR-transplanted anti-GM.sub.2 antibody L 
chain variable region. 
It was further revealed that the antigen-binding activity can be restored 
when the pBSHSGL-derived human CDR-transplanted anti-GM.sub.2 antibody L 
chain variable region, namely the human CDR-transplanted anti-GM.sub.2 
antibody L chain variable region constructed based on the known human 
antibody L chain variable region common sequence, is used. 
In view of the above result, the human CDR-transplanted anti-GM.sub.2 
antibody L chain variable region constructed based on the known human 
antibody L chain variable region common sequence as set forth in SEQ ID 
NO:90 was selected as a second novel human CDR-transplanted anti-GM.sub.2 
antibody L chain variable region. 
It is to be noted that in those human CDR-transplanted anti-GM.sub.2 
antibody L chain variable regions that showed high binding activity 
against GM.sub.2, certain amino acid residues which cannot be specified by 
deduction from known human CDR-transplanted antibody production examples 
have been replaced with amino acids found in the mouse L chain variable 
region. Thus, obviously, it was very important, in human CDR-transplanted 
anti-GM.sub.2 antibody production, to identify these amino acid residues. 
Furthermore, the fact that the human CDR-transplanted anti-GM.sub.2 
antibodies having those human CDR-transplanted anti-GM.sub.2 antibody H 
chain and L chain variable regions based on the known human antibody 
variable region common sequences showed high antigen binding activity is 
proof of the usefulness of the present process in human CDR-transplanted 
antibody production. 
(6) Acquisition of cell lines for stable production of human 
CDR-transplanted anti-GM.sub.2 antibodies 
Based on the results of Paragraph 4 (5) of Example 3, two cell lines, 
KM8966 and KM8967, capable of stably expressing KM8966, which has the 
amino acid sequence set forth in SEQ ID NO:60 as the H chain variable 
region and the amino acid sequence set forth in SEQ ID NO:83 as the L 
chain variable region, and KM8967, which has the amino acid sequence set 
forth in SEQ ID NO:60 as the H chain variable region and the amino acid 
sequence set forth in SEQ ID NO:90 as the L chain variable region, 
respectively as human CDR-transplanted anti-GM.sub.2 antibodies having 
higher antigen-binding activity than the human CDR-transplanted 
anti-GM.sub.2 antibody described in Example 2 were obtained in the 
following manner. 
Three .mu.g each of the plasmids pT796HLCDRLm-28 and pT796HLCDRHSGL 
obtained in Paragraph 4 (5) of Example 3 were respectively added to 10 
.mu.l of 20 mM Tris-hydrochloride buffer (pH 8.5) containing 100 mM 
potassium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme BamHI (Takara Shuzo) was further added, and the 
reaction was allowed to proceed at 37.degree. C. for 1 hour. Each reaction 
mixture was subjected to ethanol precipitation, the precipitate was added 
to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM 
sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of the 
restriction enzyme XhoI (Takara Shuzo) was further added, and the reaction 
was allowed to proceed at 37.degree. C. for 1 hour. Each reaction mixture 
was fractionated by agarose gel electrophoresis, and about 1 .mu.g of a 
BamHI-XhoI fragment about 4.93 kb in size was recovered. 
Then, 3 .mu.g of the plasmid pKANTEX93 obtained in Paragraph 1 of Example 3 
was added to 10 .mu.l of 20 mM Tris-hydrochloride buffer (pH 8.5) 
containing 100 mM potassium chloride, 10 mM magnesium chloride and 1 mM 
DTT, 10 units of the restriction enzyme BamHI (Takara Shuzo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was subjected to ethanol precipitation, the 
precipitate was added to 10 .mu.l of 50 mM Tris-hydrochloride buffer (pH 
7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM 
DTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) was further 
added, and the reaction was allowed to proceed at 37.degree. C. for 1 
hour. The reaction mixture was fractionated by agarose gel 
electrophoresis, and about 1 .mu.g of a BamHI-XhoI fragment about 8.68 kb 
in size was recovered. 
Then, 0.1 .mu.g of the BamHI-XhoI fragment of pT796HLCDRLm-28 or 
pT796HLCDRHSGL and 0.1 .mu.g of the BamHI-XhoI fragment of pKANTEX93, each 
obtained as mentioned above, were added to a total of 20 .mu.l of 
sterilized water and ligated to each other using Ready-To-Go T4 DNA Ligase 
(Pharmacia Biotech). Each recombinant plasmid DNA solution thus obtained 
was used to transform Escherichia coli HB101. In this way, the plasmids 
pKANTEX796HLCDRLm-28 and pKANTEX796HLCDRHSGL shown in FIG. 107 were 
obtained. 
Then, 4 .mu.g each of the above plasmids pKANTEX796HLCDRLm-28 and 
pKANTEX796HLCDRHSGL were respectively used to transform YB2/0 (ATCC CRL 
1581) cells according to the procedure described in Paragraph 11 of 
Example 1 and, after final selection using G418 (0.5 mg/ml) and MTX (200 
nM), a transformant cell line, KM8966, capable of producing about 40 
.mu.g/ml of KM8966, i.e. the pKANTEX796HLCDRLm-28-derived human 
CDR-transplanted anti-GM.sub.2 antibody, and a transformant cell line, 
KM8967, capable of producing about 30 .mu.g/ml of KM8966, i.e. the 
pKANTEX796HLCDRHSGL-derived human CDR-transplanted anti-GM.sub.2 antibody, 
were obtained. 
(7) Purification of human CDR-transplanted anti-GM.sub.2 antibodies KM8966 
and KM8967 
The transformant cell lines KM8966 and 8967 obtained in Paragraph 4 (6) of 
Example 3 were respectively suspended in GIT medium (Nippon 
Pharmaceutical) containing 0.5 mg/ml G418 and 200 nM MTX and, according to 
the procedure of Paragraph 11 of Example 1, 18 mg of purified human 
CDR-transplanted anti-GM.sub.2 antibody KM8966 and 12 mg of purified 
KM8967 were obtained each from about 0.5 liter of culture fluid. Three 
.mu.g each of the purified human CDR-transplanted anti-GM.sub.2 antibodies 
obtained and the human anti-GM.sub.2 chimera antibody KM966 were subjected 
to electrophoresis by the known method Laemli, Nature, 227, 680 (1979)! 
for molecular weight determination. The results are shown in FIG. 108. As 
shown, under reducing conditions, both antibody H chains showed a 
molecular weight of about 50 kilodaltons and both antibody L chains showed 
a molecular weight of about 25 kilodaltons. Expression of H and L chains 
of correct molecular weights was thus confirmed. Under non-reducing 
conditions, both human CDR-transplanted anti-GM.sub.2 antibodies showed a 
molecular weight of about 150 kilodaltons and it was thus confirmed that 
antibodies each composed of two H chains and two L chains and having a 
correct size had been expressed. Furthermore, the H and L chains of each 
human CDR-transplanted anti-GM.sub.2 antibody were analyzed for N-terminal 
amino acid sequence by automatic Edman degradation using a protein 
sequencer (Applied Biosystems model 470A), whereby an amino acid sequence 
deducible from the base sequence of the variable region DNA constructed 
was revealed. 
5. In vitro reactivity of human CDR-transplanted anti-GM.sub.2 antibodies 
KM8966 and KM8967 against GM.sub.2 
The human anti-GM.sub.2 chimera antibody KM966 and the purified human 
CDR-transplanted anti-GM.sub.2 antibodies KM8966 and KM8967 were tested 
for reactivity against GM.sub.2 by ELISA as described in Paragraph 11 of 
Example 1. The results are shown in FIG. 109. GM.sub.2 (N-acetyl-GM.sub.2) 
used was purified from cultured cell line HPB-ALL Oboshi et al., 
Tanpakushitsu, Kakusan & Koso (Protein, Nucleic acid & Enzyme), 23, 697 
(1978) ! in accordance with the known method J. Biol. Chem., 263, 10915 
(1988)!. As shown, it was found that the purified human CDR-transplanted 
anti-GM.sub.2 antibody KM8966 exerted the binding activity comparable to 
that of the human anti-GM.sub.2 chimera antibody KM966. On the other hand, 
the binding activity of purified human CDR-transplanted anti-GM.sub.2 
antibody KM8967 was about 1/4 to 1/5 of that of the human anti-GM.sub.2 
chimera antibody KM966. 
6. Reaction specificity of human CDR-transplanted anti-GM.sub.2 antibodies 
KM8966 and KM8967 
The human anti-GM.sub.2 chimera antibody KM966 and the human 
CDR-transplanted anti-GM.sub.2 antibodies KM8966 and KM8967 were tested 
for reactivity against the gangliosides GM.sub.1, N-acetyl-GM.sub.2, 
N-glycolyl-GM.sub.2, N-acetyl-GM.sub.3, N-glycolyl-GM.sub.3, GD.sub.1a, 
GD.sub.1b (Iatron), GD.sub.2, GD.sub.3 (Iatron) and GQ.sub.1b (Iatron) by 
ELISA as described in Paragraph 11 of Example 1. The results are shown in 
FIG. 110. GM.sub.1 and GD.sub.1a were purified from bovine brain, 
N-acetyl-GM.sub.2 from cultured cell line HPB-ALL Oboshi et al., 
Tanpakushitsu, Kakusan & Koso (Protein, Nucleic acid & Enzyme), 23, 697 
(1978)!, N-glycolyl-GM.sub.2 and N-glycolyl-GM.sub.3 from mouse liver, 
N-acetyl-GM.sub.3 canine erythrocytes, and GD.sub.2 from cultured cell 
line IMR32 (ATCC CCL127), respectively by the per se known method J. 
Biol. Chem., 263, 10915 (1988)!. Each antibody was used in a concentration 
of 10 .mu.g/ml. 
As shown in FIG. 110, it was confirmed that the human CDR-transplanted 
anti-GM.sub.2 antibodies KM8966 and KM8967 react specifically with 
GM.sub.2 (N-acetyl-GM.sub.2 and N-glycolyl-GM.sub.2) like the human 
anti-GM.sub.2 chimera antibody KM966. 
7. Reactivity of human CDR-transplanted anti-GM.sub.2 antibodies KM8966 and 
KM8967 against cancer cells 
The human lung small cell carcinoma culture cell line SBC-3 (JCRB 0818) 
(1.times.10.sup.6 cells) was suspended in PBS, the suspension was placed 
in a microtube (TREF) and centrifuged (1200 rpm, 2 minutes). To the 
thus-washed cells was added 50 .mu.l (50 .mu.g/ml) of the human 
anti-GM.sub.2 chimera antibody KM966 or the purified human 
CDR-transplanted anti-GM.sub.2 antibody KM8966 or KM8967, followed by 
stirring and 1 hour of standing at 4.degree. C. After the above reaction 
step, the cells were washed three times with PBS, each time followed by 
centrifugation. Then, 20 .mu.l of fluorescein isocyanate-labeled protein A 
(30-fold dilution, Boehringer Mannheim) was added and, after stirring, the 
reaction was allowed to proceed at 4.degree. C. for 1 hour. Thereafter, 
the cells were washed three times with PBS, each time followed by 
centrifugation, then further suspended in PBS and subjected to analysis 
using a flow cytometer, EPICS Elite (Coulter). In a control run, the above 
procedure was followed without addition of the human CDR-transplanted 
anti-GM.sub.2 antibody and analyzed. The results are shown in FIG. 111. It 
was found that the purified human CDR-transplanted anti-GM.sub.2 
antibodies KM8966 and KM8967 strongly reacted with the human lung small 
cell carcinoma culture cell line SBC-3 like the human anti-GM.sub.2 
chimera antibody KM966. 
The results shown indicate that, like the human chimera antibodies, the 
human CDR-transplanted anti-GM.sub.2 antibodies are useful in the 
diagnosis and treatment of human cancer, among others. 
While the invention has been described in detail and with reference to 
specific examples thereof, it will be apparent to one skilled in the art 
that various changes and modifications can be made therein without 
departing from the spirit and scope thereof. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 103 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 449 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 50..66 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..109 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- CTCCACAGTC CCTGAAGACA CTGACTCTAA CC ATG GGA TGG AGC - # TGG ATC TTT 
53 
#Met Gly Trp Ser Trp Ile Phe 
15 
- CTC TTC CTC CTG TCA GGA ACT GCA GGT GTC CT - #C TCT GAG GTC CAG CTG 
101 
Leu Phe Leu Leu Ser Gly Thr Ala Gly Val Le - #u Ser Glu Val Gln Leu 
# 1 
- CAG CAG TCT GGA CCT GAG CTG GTG AAG CCT GG - #G GCT TCA GTG AAG ATA 
149 
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile 
# 20 
- TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT GA - #C TAC AAC ATG GAC TGG 
197 
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p Tyr Asn Met Asp Trp 
# 35 
- GTG AAG CAG AGC CAT GGA AAG AGC CTT GAG TG - #G ATT GGA TAT ATT TAT 
245 
Val Lys Gln Ser His Gly Lys Ser Leu Glu Tr - #p Ile Gly Tyr Ile Tyr 
# 50 
- CCT AAC AAT GGT GGT ACT GGC TAC AAC CAG AA - #G TTC AAG AGC AAG GCC 
293 
Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Ly - #s Phe Lys Ser Lys Ala 
# 65 
- ACA TTG ACT GTA GAC AAG TCC TCC AGC ACA GC - #C TAC ATG GAG CTC CAC 
341 
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu His 
# 80 
- AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TA - #C TGT GCA ACC TAC GGT 
389 
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ala Thr Tyr Gly 
#100 
- CAT TAC TAC GGC TAC ATG TTT GCT TAC TGG GG - #C CAA GGG ACT CTG GTC 
437 
His Tyr Tyr Gly Tyr Met Phe Ala Tyr Trp Gl - #y Gln Gly Thr Leu Val 
# 115 
# 449 
Thr Val Ser Ala 
120 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 393 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE TO TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
#TTC CTG CTA ATC AGT 48AG ATT TTC AGC 
Met His Phe Gln Val Gln Il - #e Phe Ser Phe Leu Leu Ile Ser 
10 
- GCC TCA GTC ATA ATG TCC AGA GGA CAA ATT GT - #T CTC ACC CAG TCT CCA 
96 
Ala Ser Val Ile Met Ser Arg Gly Gln Ile Va - #l Leu Thr Gln Ser Pro 
# 5 1 
- GCA ATC ATG TCT GCA TCT CCA GGG GAG AAG GT - #C ACC ATA ACC TGC AGT 
144 
Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Va - #l Thr Ile Thr Cys Ser 
# 20 
- GCC AGC TCA AGT GTA AGT TAC ATG CAC TGG TT - #C CAG CAG AAG CCA GGC 
192 
Ala Ser Ser Ser Val Ser Tyr Met His Trp Ph - #e Gln Gln Lys Pro Gly 
# 40 
- ACT TCT CCC AAA CTC TGG ATT TAT AGC ACA TC - #C AAC CTG GCT TCT GGA 
240 
Thr Ser Pro Lys Leu Trp Ile Tyr Ser Thr Se - #r Asn Leu Ala Ser Gly 
# 55 
- GTC CCT GCT CGC TTC AGT GGC AGT GGA TCT GG - #G ACC TCT TAC TCT CTC 
288 
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gl - #y Thr Ser Tyr Ser Leu 
# 70 
- ACA ATC AGC CGA ATG GAG GCT GAA GAT GCT GC - #C ACT TAT TAC TGC CAG 
336 
Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Al - #a Thr Tyr Tyr Cys Gln 
# 85 
- CAA AGG AGT AGT TAC CCG TAC ACG TTC GGA GG - #G GGG ACC AAG CTG GAA 
384 
Gln Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gl - #y Gly Thr Lys Leu Glu 
# 100 
# 393 
Ile Lys Arg 
105 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 443 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 55..66 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..107 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISEHD 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- CTCCACAGTC CCTGAAGACA CTGACTCTAA CC ATG GGA TGG AGC - # TGG ATC TTT 
53 
#Met Gly Trp Ser Trp Ile Phe 
15 
- CTC TTC CTC CTG TCA GGA ACT GCA GGT GTC CT - #C TCT GAG GTC CAG CTG 
101 
Leu Phe Leu Leu Ser Gly Thr Ala Gly Val Le - #u Ser Glu Val Gln Leu 
# 1 
- CAG CAG TCT GGA CCT GAG CTG GTG AAG CCT GG - #G GCT TCA GTG AAG ATA 
149 
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile 
# 20 
- TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT GA - #C TAC AAC ATG GAC TGG 
197 
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p Tyr Asn Met Asp Trp 
# 35 
- GTG AAG CAG AGC CAT GGA AAG AGC CTT GAG TG - #G ATT GGA TAT ATT TAT 
245 
Val Lys Gln Ser His Gly Lys Ser Leu Glu Tr - #p Ile Gly Tyr Ile Tyr 
# 50 
- CCT AAC AAT GGT GGT ACT GGC TAC AAC CAG AA - #G TTC AAG AGC AAG GCC 
293 
Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Ly - #s Phe Lys Ser Lys Ala 
# 65 
- ACA TTG ACT GTA GAC AAG TCC TCC AGC ACA GC - #C TAC ATG GAG CTC CAC 
341 
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu His 
# 80 
- AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TA - #C TGT GCA AGA GCG GGG 
389 
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ala Arg Ala Gly 
#100 
- AGG TAT TAC TAC GCC TGG GAC TGG GGC CAA GG - #G ACT CTG GTC ACT GTC 
437 
Arg Tyr Tyr Tyr Ala Trp Asp Trp Gly Gln Gl - #y Thr Leu Val Thr Val 
# 115 
# 443 
Ser Ala 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 405 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to mRNA 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: 10..66 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 157..171 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 214..261 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 358..369 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- CATCACAGC ATG GCT GTC CTG GTG CTG TTG CTC TGC - # CTG GTG ACA TTT 
48 
#Leu Leu Leu Cys Leu Val Thr Phe 
10 
- CCA AGC TGT GTC CTG TCC CAA GTG CAG CTG AA - #G GAG TCA GGA CCT GGT 
96 
Pro Ser Cys Val Leu Ser Gln Val Gln Leu Ly - #s Glu Ser Gly Pro Gly 
# 10 
- CTG GTG CAG CCC TCA CAG ACC CTG TCC CTC AC - #C TGC ACT GTC TCT GGG 
144 
Leu Val Gln Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly 
# 25 
- TTC TCA TTA ACC AGC TAT ACT GTA AGC TGG GT - #T CGC CAG CCT CCA GGA 
192 
Phe Ser Leu Thr Ser Tyr Thr Val Ser Trp Va - #l Arg Gln Pro Pro Gly 
# 40 
- AAG GGT CTG GAG TGG ATT GCA GCA ATA TCA AG - #T GGT GGA AGC ACA TAT 
240 
Lys Gly Leu Glu Trp Ile Ala Ala Ile Ser Se - #r Gly Gly Ser Thr Tyr 
# 55 
- TAT AAT TCA GCT CTC AAA TCA CGA CTG AGC AT - #C AGC AGG GAC ACC TCC 
288 
Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser Il - #e Ser Arg Asp Thr Ser 
# 70 
- AAG AGC CAA GTT TTC TTA AAA ATG AAC AGT CT - #G CAA ACT GAA GAC ACA 
336 
Lys Ser Gln Val Phe Leu Lys Met Asn Ser Le - #u Gln Thr Glu Asp Thr 
# 90 
- GCC ATG TAC TTC TGT GCC CCT TCT GAG GGG GC - #C TGG GGC CAA GGA GTC 
384 
Ala Met Tyr Phe Cys Ala Pro Ser Glu Gly Al - #a Trp Gly Gln Gly Val 
# 105 
# 405 TCA GAG 
Met Val Thr Val Ser Ser Glu 
110 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 402 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to mRNA 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: 19..78 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 148..180 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABILSHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 226..246 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 343..369 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
#TTA GGG CTG CTG 51 ATG GCT CCA GTC CAG CTC 
#Leu Leuet Ala Pro Val Gln Leu Leu Gly 
10 
- CTG ATT TGG CTC CCA GCC ATG AGA TGT GAC AT - #C CAG ATG ACC CAG TCT 
99 
Leu Ile Trp Leu Pro Ala Met Arg Cys Asp Il - #e Gln Met Thr Gln Ser 
#1 5 
- CCT TCA TTC CTG TCT GCA TCT GTG GGA GAC AG - #A GTC ACT ATC AAC TGC 
147 
Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Ar - #g Val Thr Ile Asn Cys 
# 20 
- AAA GCA AGT CAG AAT ATT AAC AAG TAC TTA AA - #C TGG TAT CAG CAA AAG 
195 
Lys Ala Ser Gln Asn Ile Asn Lys Tyr Leu As - #n Trp Tyr Gln Gln Lys 
# 35 
- CTT GGA GAA GCT CCC AAA CGC CTG ATA TAT AA - #T ACA AAC AAT TTG CAA 
243 
Leu Gly Glu Ala Pro Lys Arg Leu Ile Tyr As - #n Thr Asn Asn Leu Gln 
# 55 
- ACG GGC ATT CCA TCA AGG TTC AGT GGC AGT GG - #A TCT GGT ACA GAT TAC 
291 
Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gl - #y Ser Gly Thr Asp Tyr 
# 70 
- ACA CTC ACC ATC AGC AGC CTG CAG CCT GAA GA - #T TTT GCC ACA TAT TTC 
339 
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu As - #p Phe Ala Thr Tyr Phe 
# 85 
- TGC TTG CAG CAT AAT AGT TTT CCG AAC ACG TT - #T GGA GCT GGG ACC AAG 
387 
Cys Leu Gln His Asn Ser Phe Pro Asn Thr Ph - #e Gly Ala Gly Thr Lys 
# 100 
# 402 GG 
Leu Glu Leu Lys Arg 
105 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 15 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 15 AC 
Asp Tyr Asn Met Asp 
1 5 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 51 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- TAT ATT TAT CCT AAC AAT GGT GGT ACT GGC TA - #C AAC CAG AAG TTC AAG 
48 
Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Ty - #r Asn Gln Lys Phe Lys 
# 15 
# 51 
Ser 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 33 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
# 33T TAC TAC GGC TAC ATG TTT GCT TA - #C 
Tyr Gly His Tyr Tyr Gly Tyr Met Phe Ala Ty - #r 
# 10 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 30 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA(genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 30 GT GTA AGT TAC ATG CAC 
Ser Ala Ser Ser Ser Val Ser Tyr Met His 
# 10 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
#21 TG GCT TCT 
Ser Thr Ser Asn Leu Ala Ser 
1 5 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 27 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 27 GT TAC CCG TAC ACG 
Gln Gln Arg Ser Ser Tyr Pro Tyr Thr 
1 5 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 31 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
# 31 CA GCC TCC ACC AAG GGC C 
Val Thr Val Ser Ala Ala Ser Thr Lys Gly 
# 10 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 51 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
#AAA CGA ACT GTG GCT 46 CTG GAA ATA 
Thr Phe Gly Gly Gly Thr Lys Leu Glu I - #le Lys Arg Thr Val Ala 
# 15 
# 51 
Ala 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 43 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- CAA GGA GTC ATG GTC ACA GTC TCG AGC GCC TC - #C ACC AAG GGC 
# 42 
Gln Gly Val Met Val Thr Val Ser Ser Ala Se - #r Thr Lys Gly 
# 10 
# 43 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 51 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
#AAA CGA ACT GTG GCT 46 CTT GAG CTC 
Thr Phe Gly Ala Gly Thr Lys Leu Glu L - #eu Lys Arg Thr Val Ala 
# 15 
# 51 
Ala 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 812 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (vi) ORIGINAL SOURCE: 
(B) STRAIN: HYBRIDOMA K - #M50 
- (ix) FEATURE: 
(A) NAME/KEY: TATA.sub.-- - #signal 
(B) LOCATION: 261..267 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- AAAGTCAGAC AACTTTGTAG AGTAGGTTCT ATCAATCCTA CTGCAATCCA AC - #ATCACTGA 
60 
- GGACAAATGT TTATACTGAG GAACCTGGTC TTGTGTGATA CGTACTTTCT GT - #GGGAAGCA 
120 
- GATACGCACT CTCATGTGGC TCCTGAATTT CCCATCACAG AATGATACAT CT - #TGAGTCCT 
180 
- AAAATTTAAG TACACCATCA GTGTCAGCAC CTGGTGAGGA AATGCAAATC TC - #TCCTGGAT 
240 
- CCACCCAACC TTGGGTTGAA AAGCCAAAGC TGGGCCTGGG TACTCACTGG TG - #TGCAGCC 
299 
- ATG GAC AGG CTT ACT TCC TCA TTC CTA CTG CT - #G ATG GTC CCT GCA 
34 - #4 
Met Asp Arg Leu Thr Ser Ser Phe Leu Leu Le - #u Met Val Pro Ala 
- TGTGAGTACC AAAGCTTCCT AAGTGATGAA CTGTTCTATC CTCACCTGTT CA - #AACCTGAC 
404 
- CTCCTCCCCT TTGATTTCTC CACAG AT GTC CTG TCT CAG G - #TT ACT CTG AAA 
455 
#Val Thr Leu Lysl Leu Ser Gln 
# 5 1 
- GAA TCT GGC CCT GGG ATA TTG CAG CCC TCC CA - #G ACC CTC AGT CTG ACT 
503 
Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gl - #n Thr Leu Ser Leu Thr 
# 20 
- TGC TCT TTC TCT GGG TTT TCA CTG AGC ACT TA - #T GGT ATG TGT GTG GGC 
551 
Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ty - #r Gly Met Cys Val Gly 
# 35 
- TGG ATT CGT CAG TCT TCA GGG AAG GGT CTG GA - #G TGG CTG GCA AAC GTT 
599 
Trp Ile Arg Gln Ser Ser Gly Lys Gly Leu Gl - #u Trp Leu Ala Asn Val 
# 50 
- TGG TGG AGT GAT GCT AAG TAC TAC AAT CCA TC - #T CTG AAA AAC CGG CTC 
647 
Trp Trp Ser Asp Ala Lys Tyr Tyr Asn Pro Se - #r Leu Lys Asn Arg Leu 
# 65 
- ACA ATC TCC AAG GAC ACC TCC AAC AAC CAA GC - #A TTC CTC AAG ATC ACC 
695 
Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Al - #a Phe Leu Lys Ile Thr 
# 85 
- AAT ATG GAC ACT GCA GAT ACT GCC ATA TAC TA - #C TGT GCT GGG AGA GGG 
743 
Asn Met Asp Thr Ala Asp Thr Ala Ile Tyr Ty - #r Cys Ala Gly Arg Gly 
# 100 
- GCT ACG GAG GGT ATA GTG AGC TTT GAT TAC TG - #G GGC CAC GGA GTC ATG 
791 
Ala Thr Glu Gly Ile Val Ser Phe Asp Tyr Tr - #p Gly His Gly Val Met 
# 115 
# 812 GGTAAG 
Val Thr Val Ser Ser 
120 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 46 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
# 46ATA TCAAGCTTGT CGACTCTAGA GGTACC 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 29 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
# 29 GCAG CCACAGTTC 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 408 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to mRNA 
- (vi) ORIGINAL SOURCE: 
(B) STRAIN: HYBRIDOMA K - #M-641 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: 25..84 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- AATTCGGCAC GAGTCAGCCT GGAC ATG ATG TCC TCT GCT C - #AG TTC CTT GGT 
51 
#Gln Phe Leu Glyt Ser Ser Ala 
15 
- CTC CTG TTG CTC TGT TTT CAA GGT ACC AGA TG - #T GAT ATC CAG ATG ACA 
99 
Leu Leu Leu Leu Cys Phe Gln Gly Thr Arg Cy - #s Asp Ile Gln Met Thr 
# 5 1 
- CAG ACT GCA TCC TCC CTG CCT GCC TCT CTG GG - #A GAC AGA GTC ACC ATC 
147 
Gln Thr Ala Ser Ser Leu Pro Ala Ser Leu Gl - #y Asp Arg Val Thr Ile 
# 20 
- AGT TGC AGT GCA AGT CAG GAC ATT AGT AAT TA - #T TTA AAC TGG TAT CAA 
195 
Ser Cys Ser Ala Ser Gln Asp Ile Ser Asn Ty - #r Leu Asn Trp Tyr Gln 
# 35 
- CAG AAA CCA GAT GGA ACT GTT AAA CTC CTG AT - #C TTT TAC TCA TCA AAT 
243 
Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Il - #e Phe Tyr Ser Ser Asn 
# 50 
- TTA CAC TCG GGA GTC CCA TCA AGG TTC AGT GG - #C GGT GGG TCC GGG ACA 
291 
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gl - #y Gly Gly Ser Gly Thr 
# 65 
- GAT TAT TCT CTC ACC ATC AGC AAC CTG GAG CC - #T GAA GAT ATT GCC ACT 
339 
Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pr - #o Glu Asp Ile Ala Thr 
# 85 
- TAC TTT TGT CAT CAG TAT AGT AAG CTT CCG TG - #G ACG TCC GGT GGA GGC 
387 
Tyr Phe Cys His Gln Tyr Ser Lys Leu Pro Tr - #p Thr Ser Gly Gly Gly 
# 100 
# 408 AAA CGG 
Thr Lys Leu Glu Ile Lys Arg 
105 
- (2) INFORMATION FOR SEQ ID NO:20: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 403 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to mRNA 
- (vi) ORIGINAL SOURCE: 
(B) STRAIN: HYBRIDOMA K - #M-641 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: 14..43 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- AATTCGGCAC GAG CTT GTC CTT GTT TTC AAA GGT GT - #T CAG TGT GAA GTG 
49 
#Leu Val Phe Lys Gly Val Gln Cys Glu Val 
# 1 
- ACG CTG GTG GAG TCT GGG GGA GAC TTT GTG AA - #A CCT GGA GGG TCC CTG 
97 
Thr Leu Val Glu Ser Gly Gly Asp Phe Val Ly - #s Pro Gly Gly Ser Leu 
# 15 
- AAA GTC TCC TGT GCA GCC TCT GGA TTC GCT TT - #C AGT CAT TAT GCC ATG 
145 
Lys Val Ser Cys Ala Ala Ser Gly Phe Ala Ph - #e Ser His Tyr Ala Met 
# 30 
- TCT TGG GTT CGC CAG ACT CCG GCG AAG AGG CT - #G GAA TGG GTC GCA TAT 
193 
Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Le - #u Glu Trp Val Ala Tyr 
# 50 
- ATT AGT AGT GGT GGT AGT GGC ACC TAC TAT TC - #A GAC AGT GTA AAG GGC 
241 
Ile Ser Ser Gly Gly Ser Gly Thr Tyr Tyr Se - #r Asp Ser Val Lys Gly 
# 65 
- CGA TTC ACC ATT TCC AGA GAC AAT GCC AAG AA - #C ACC CTG TAC CTG CAA 
289 
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys As - #n Thr Leu Tyr Leu Gln 
# 80 
- ATG CGC AGT CTG AGG TCT GAG GAC TCG GCC AT - #G TAT TTC TGT ACA AGA 
337 
Met Arg Ser Leu Arg Ser Glu Asp Ser Ala Me - #t Tyr Phe Cys Thr Arg 
# 95 
- GTT AAA CTG GGA ACC TAC TAC TTT GAC TCC TG - #G GGC CAA GGC ACC ACT 
385 
Val Lys Leu Gly Thr Tyr Tyr Phe Asp Ser Tr - #p Gly Gln Gly Thr Thr 
# 110 
# 403 CA GCT 
Leu Thr Val Ser Ser Ala 
115 1 - #20 
- (2) INFORMATION FOR SEQ ID NO:21: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 35 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
# 35 TTT GGG CTC AGC TGG CTT TTT 
Met Glu Phe Gly Leu S - #er Trp Leu Phe 
# 5 1 
- (2) INFORMATION FOR SEQ ID NO:22: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 43 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- CAA GGT ACC ACG TTA ACT GTC TCC TCA GCC TC - #C ACC AAG GGC 
# 42 
Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Se - #r Thr Lys Gly 
# 10 
# 43 
- (2) INFORMATION FOR SEQ ID NO:23: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 80 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- GGCCGCACCA TGGGATGGAG CTGGATCTTT CTCTTCCTCC TGTCAGGAAC TG - #CTGGTGTC 
60 
# 80 TGCA 
- (2) INFORMATION FOR SEQ ID NO:24: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 59 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- GGAGAGCGGT CCAGGTCTTG TGAGGCCTAG CCAGACCCTG AGCCTGACCT GC - #ACCGTGT 
59 
- (2) INFORMATION FOR SEQ ID NO:25: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 60 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- CCGGATTCAC CTTCAGCGAC TACAACATGG ACTGGGTGAG ACAGCCACCT GG - #ACGAGGTC 
60 
- (2) INFORMATION FOR SEQ ID NO:26: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 81 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- TCGAGTGGAT TGGATATATT TATCCTAACA ATGGTGGTAC TGGCTACAAC CA - #GAAGTTCA 
60 
#81 GCTG G 
- (2) INFORMATION FOR SEQ ID NO:27: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 61 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- TCGACACCAG CAAGAACCAG TTCAGCCTGA GACTCAGCAG CGTGACAGCC GC - #CGACACCG 
60 
# 61 
- (2) INFORMATION FOR SEQ ID NO:28: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 66 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- GGTCTATTAT TGTGCGCGCT ACGGTCATTA CTACGGCTAC ATGTTTGCTT AC - #TGGGGTCA 
60 
# 66 
- (2) INFORMATION FOR SEQ ID NO:29: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 35 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
# 35 TCAG CCTCCACCAA GGGCC 
- (2) INFORMATION FOR SEQ ID NO:30: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 77 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- AATTCACCAT GCATTTTCAA GTGCAGATTT TCAGCTTCCT GCTAATCAGT GC - #CTCAGTCA 
60 
# 77 T 
- (2) INFORMATION FOR SEQ ID NO:31: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 62 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- ATCCAGCTGA CCCAGAGCCC AAGCAGCCTG AGCGCTAGCG TGGGTGACAG AG - #TGACCATG 
60 
# 62 
- (2) INFORMATION FOR SEQ ID NO:32: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 65 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- GTGCAGTGCC AGCTCAAGTG TAAGTTACAT GCACTGGTAT CAGCAGAAGC CA - #GGTAAGGC 
60 
# 65 
- (2) INFORMATION FOR SEQ ID NO:33: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 45 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
#45 CACA TCCAACCTGG CTTCTGGTGT GCCAT 
- (2) INFORMATION FOR SEQ ID NO:34: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 76 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
- CTAGATTCAG CGGTAGCGGT AGCGGTACAG ACTTCACCTT CACCATCAGC AG - #CCTCCAGC 
60 
# 76 
- (2) INFORMATION FOR SEQ ID NO:35: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 84 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
#synthetic DNAECULE TYPE: other nucleic acid, 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
- GTACTACTGC CAGCAAAGGA GTAGTTACCC GTACACGTTC GGCGGGGGGA CC - #AAGGTGGA 
60 
# 84GCTG CACC 
- (2) INFORMATION FOR SEQ ID NO:36: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 442 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
- GGCCGCACC ATG GGA TGG AGC TGG ATC TTT CTC TTC - # CTC CTG TCA GGA 
48 
#Ile Phe Leu Phe Leu Leu Ser Gly 
10 
- ACT GCT GGT GTC CTC TCT CAG GTC CAA CTG CA - #G GAG AGC GGT CCA GGT 
96 
Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly 
# 10 
- CTT GTG AGG CCT AGC CAG ACC CTG AGC CTG AC - #C TGC ACC GTG TCC GGA 
144 
Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly 
# 25 
- TTC ACC TTC AGC GAC TAC AAC ATG GAC TGG GT - #G AGA CAG CCA CCT GGA 
192 
Phe Thr Phe Ser Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Pro Pro Gly 
# 40 
- CGA GGT CTC GAG TGG ATT GGA TAT ATT TAT CC - #T AAC AAT GGT GGT ACT 
240 
Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr 
# 55 
- GGC TAC AAC CAG AAG TTC AAG AGC AGA GTG AC - #A ATG CTG GTC GAC ACC 
288 
Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr 
# 70 
- AGC AAG AAC CAG TTC AGC CTG AGA CTC AGC AG - #C GTG ACA GCC GCC GAC 
336 
Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp 
# 90 
- ACC GCG GTC TAT TAT TGT GCG CGC TAC GGT CA - #T TAC TAC GGC TAC ATG 
384 
Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met 
# 105 
- TTT GCT TAC TGG GGT CAA GGT ACC ACC GTC AC - #A GTC TCC TCA GCC TCC 
432 
Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser 
# 120 
# 442 
Thr Lys Gly 
125 
- (2) INFORMATION FOR SEQ ID NO:37: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 409 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
#TTC CTG CTA ATC AGT 50TG CAG ATT TTC AGC 
Met His Phe Gln Val G - #ln Ile Phe Ser Phe Leu Leu Ile Ser 
10 
- GCC TCA GTC ATA ATG TCC AGA GGA GAT ATC CA - #G CTG ACC CAG AGC CCA 
98 
Ala Ser Val Ile Met Ser Arg Gly Asp Ile Gl - #n Leu Thr Gln Ser Pro 
# 5 1 
- AGC AGC CTG AGC GCT AGC GTG GGT GAC AGA GT - #G ACC ATC ACG TGC AGT 
146 
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Va - #l Thr Ile Thr Cys Ser 
# 20 
- GCC AGC TCA AGT GTA AGT TAC ATG CAC TGG TA - #T CAG CAG AAG CCA GGT 
194 
Ala Ser Ser Ser Val Ser Tyr Met His Trp Ty - #r Gln Gln Lys Pro Gly 
# 40 
- AAG GCT CCA AAG CTT CTG ATC TAC AGC ACA TC - #C AAC CTG GCT TCT GGT 
242 
Lys Ala Pro Lys Leu Leu Ile Tyr Ser Thr Se - #r Asn Leu Ala Ser Gly 
# 55 
- GTG CCA TCT AGA TTC AGC GGT AGC GGT AGC GG - #T ACA GAC TTC ACC TTC 
290 
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gl - #y Thr Asp Phe Thr Phe 
# 70 
- ACC ATC AGC AGC CTC CAG CCA GAG GAC ATC GC - #T ACG TAC TAC TGC CAG 
338 
Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Al - #a Thr Tyr Tyr Cys Gln 
# 85 
- CAA AGG AGT AGT TAC CCG TAC ACG TTC GGC GG - #G GGG ACC AAG GTG GAA 
386 
Gln Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gl - #y Gly Thr Lys Val Glu 
# 100 
# 409TG GCT GCA CC 
Ile Lys Arg Thr Val Ala Ala 
105 1 - #10 
- (2) INFORMATION FOR SEQ ID NO:38: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 32 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
# 32 AGGA GCAGGTGAAT TC 
- (2) INFORMATION FOR SEQ ID NO:39: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
# 40 TCCT CAGTTAACAC TGAGTGGTAC 
- (2) INFORMATION FOR SEQ ID NO:40: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
#21 GCAC C 
- (2) INFORMATION FOR SEQ ID NO:41: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
# 17 G 
- (2) INFORMATION FOR SEQ ID NO:42: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 26 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
# 26 CCGC GGCCGC 
- (2) INFORMATION FOR SEQ ID NO:43: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
# 34 CCAC TAGTCGCGAG GTAC 
- (2) INFORMATION FOR SEQ ID NO:44: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
# 20 CCCG 
- (2) INFORMATION FOR SEQ ID NO:45: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
# 20 CCAC 
- (2) INFORMATION FOR SEQ ID NO:46: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 36 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
# 36 GGTT CGATAAATCG ATACCG 
- (2) INFORMATION FOR SEQ ID NO:47: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
# 40 AACC GTACGAAGAA TTCATGAGCT 
- (2) INFORMATION FOR SEQ ID NO:48: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 35 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
# 35 ACCA AGCTGGAAAT AAAAC 
- (2) INFORMATION FOR SEQ ID NO:49: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 35 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
# 35 GCTT GGTCCCCCCT CCGAA 
- (2) INFORMATION FOR SEQ ID NO:50: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 61 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
- TCGACACCAG CAAGAACACA GCCTACCTGA GACTCAGCAG CGTGACAGCC GC - #CGACACCG 
60 
# 61 
- (2) INFORMATION FOR SEQ ID NO:51: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 59 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
- CCGGATACAC ATTCACTGAC TACAACATGG ACTGGGTGAG ACAGAGCCAT GA - #CGAGGTC 
59 
- (2) INFORMATION FOR SEQ ID NO:52: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 442 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 50..66 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..109 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
- GGCCGCACC ATG GGA TGG AGC TGG ATC TTT CTC TTC - # CTC CTG TCA GGA 
48 
#Ile Phe Leu Phe Leu Leu Ser Gly 
10 
- ACT GCT GGT GTC CTC TCT CAG GTC CAA CTG CA - #G GAG AGC GGT CCA GGT 
96 
Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly 
# 10 
- CTT GTG AGG CCT AGC CAG ACC CTG AGC CTG AC - #C TGC ACC GTG TCC GGA 
144 
Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly 
# 25 
- TTC ACC TTC AGC GAC TAC AAC ATG GAC TGG GT - #G AGA CAG CCA CCT GGA 
192 
Phe Thr Phe Ser Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Pro Pro Gly 
# 40 
- CGA GGT CTC GAG TGG ATT GGA TAT ATT TAT CC - #T AAC AAT GGT GGT ACT 
240 
Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr 
# 55 
- GGC TAC AAC CAG AAG TTC AAG AGC AGA GTG AC - #A ATG CTG GTC GAC ACC 
288 
Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr 
# 70 
- AGC AAG AAC ACA GCC TAC CTG AGA CTC AGC AG - #C GTG ACA GCC GCC GAC 
336 
Ser Lys Asn Thr Ala Tyr Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp 
# 90 
- ACC GCG GTC TAT TAT TGT GCA ACC TAC GGT CA - #T TAC TAC GGC TAC ATG 
384 
Thr Ala Val Tyr Tyr Cys Ala Thr Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met 
# 105 
- TTT GCT TAC TGG GGT CAA GGT ACC ACC GTC AC - #A GTC TCC TCA GCC TCC 
432 
Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser 
# 120 
# 442 
Thr Lys Gly 
125 
- (2) INFORMATION FOR SEQ ID NO:53: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 442 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 50..66 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..109 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
- GGCCGCACC ATG GGA TGG AGC TGG ATC TTT CTC TTC - # CTC CTG TCA GGA 
48 
#Ile Phe Leu Phe Leu Leu Ser Gly 
10 
- ACT GCT GGT GTC CTC TCT CAG GTC CAA CTG CA - #G GAG AGC GGT CCA GGT 
96 
Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly 
# 10 
- CTT GTG AGG CCT AGC CAG ACC CTG AGC CTG AC - #C TGC ACC GTG TCC GGA 
144 
Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly 
# 25 
- TAC ACA TTC ACT GAC TAC AAC ATG GAC TGG GT - #G AGA CAG AGC CAT GGA 
192 
Tyr Thr Phe Thr Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Ser His Gly 
# 40 
- CGA GGT CTC GAG TGG ATT GGA TAT ATT TAT CC - #T AAC AAT GGT GGT ACT 
240 
Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr 
# 55 
- GGC TAC AAC CAG AAG TTC AAG AGC AGA GTG AC - #A ATG CTG GTC GAC ACC 
288 
Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr 
# 70 
- AGC AAG AAC CAG TTC AGC CTG AGA CTC AGC AG - #C GTG ACA GCC GCC GAC 
336 
Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp 
# 90 
- ACC GCG GTC TAT TAT TGT GCA ACC TAC GGT CA - #T TAC TAC GGC TAC ATG 
384 
Thr Ala Val Tyr Tyr Cys Ala Thr Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met 
# 105 
- TTT GCT TAC TGG GGT CAA GGT ACC ACC GTC AC - #A GTC TCC TCA GCC TCC 
432 
Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser 
# 120 
# 442 
Thr Lys Gly 
125 
- (2) INFORMATION FOR SEQ ID NO:54: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 100 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
- CAGGAAACAG CTATGACGCG GCCGCCACCA TGGGATGGAG CTGGATCTTT CT - #CTTCCTCC 
60 
# 100 TGTC CTCTCTGAGG TGCAGCTGGT 
- (2) INFORMATION FOR SEQ ID NO:55: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 100 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
- AGTCAGTGAA GGTGTATCCG GAAGCCTTGC AGGAGACCTT CACTGAGGCC CC - #AGGCTTCT 
60 
# 100 CTGC ACCAGCTGCA CCTCAGAGAG 
- (2) INFORMATION FOR SEQ ID NO:56: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 100 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
- CGGATACACC TTCACTGACT ACAACATGGA CTGGGTGCGA CAGGCCCCTG GA - #CAAGGGCT 
60 
# 100 ATTT ATCCTAACAA TGGTGGTACT 
- (2) INFORMATION FOR SEQ ID NO:57: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 94 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
- AGCTCCATGT AGGCTGTGCT CGTGGATGTG TCTACGGTAA TGGTGACCTT GC - #TCTTGAAC 
60 
# 94 TACC ACCATTGTTA GGAT 
- (2) INFORMATION FOR SEQ ID NO:58: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 96 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
- AGCACAGCCT ACATGGAGCT GCACAGCCTG AGATCTGAGG ACACGGCCGT GT - #ATTACTGT 
60 
# 96 ACTA CGGCTACATG TTTGCT 
- (2) INFORMATION FOR SEQ ID NO:59: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 90 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
- GTTTTCCCAG TCACGACGGG CCCTTGGTGG AGGCTGAGGA GACGGTGACC AG - #GGTTCCCT 
60 
# 90 CATG TAGCCGTAGT 
- (2) INFORMATION FOR SEQ ID NO:60: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 432 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#an established consensu 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 50..66 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..109 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
- ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT 
48 
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly 
5 
- GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG 
96 
Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys 
# 10 
- CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC 
144 
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe 
# 25 
- ACT GAC TAC AAC ATG GAC TGG GTG CGA CAG GC - #C CCT GGA CAA GGG CTC 
192 
Thr Asp Tyr Asn Met Asp Trp Val Arg Gln Al - #a Pro Gly Gln Gly Leu 
# 45 
- GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC 
240 
Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn 
# 60 
- CAG AAG TTC AAG AGC AAG GTC ACC ATT ACC GT - #A GAC ACA TCC ACG AGC 
288 
Gln Lys Phe Lys Ser Lys Val Thr Ile Thr Va - #l Asp Thr Ser Thr Ser 
# 75 
- ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG 
336 
Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val 
# 90 
- TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC 
384 
Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr 
# 105 
- TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC 
432 
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly 
110 1 - #15 1 - #20 1 - 
#25 
- (2) INFORMATION FOR SEQ ID NO:61: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 68 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
- GTACTACTGC CAGCAAAGGA GTAGTTACCC GTACACGTTC GGCGGGGGGA CC - #AAGGTGGA 
60 
# 68 
- (2) INFORMATION FOR SEQ ID NO:62: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
# 25 TAGC GCTCA 
- (2) INFORMATION FOR SEQ ID NO:63: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
# 25 TGAC AGAGT 
- (2) INFORMATION FOR SEQ ID NO:64: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT CTG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA GA - #C TTC ACC TTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Phe Thr Ile 
# 70 
- AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:65: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
# 25 GCTT TGGAG 
- (2) INFORMATION FOR SEQ ID NO:66: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
# 25 CTAC AGCAC 
- (2) INFORMATION FOR SEQ ID NO:67: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC GTG GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA GA - #C TTC ACC TTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Phe Thr Ile 
# 70 
- AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:68: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
# 25 AGCC TGGAG 
- (2) INFORMATION FOR SEQ ID NO:69: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
# 25 CTGC TACGT 
- (2) INFORMATION FOR SEQ ID NO:70: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC GTG GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT CTG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA GA - #C TTC ACC TTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Phe Thr Ile 
# 70 
- AGC AGC CTC CAG GCT GAA GAT GCT GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Ser Leu Gln Ala Glu Asp Ala Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:71: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
# 25 ATGT ACCGC 
- (2) INFORMATION FOR SEQ ID NO:72: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
# 25 TTTC ACCAT 
- (2) INFORMATION FOR SEQ ID NO:73: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT TTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Phe Thr Ile 
# 70 
- AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:74: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
# 40 TGAT GGTGAAAGAG TAAGATGTAC 
- (2) INFORMATION FOR SEQ ID NO:75: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
# 40 CACC ATCAGCCGAA TGGAGCCAGA 
- (2) INFORMATION FOR SEQ ID NO:76: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT TTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Phe Thr Ile 
# 70 
- AGC CGA ATG GAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Arg Met Glu Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:77: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
# 20 GCAT 
- (2) INFORMATION FOR SEQ ID NO:78: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
# 20 AGAA 
- (2) INFORMATION FOR SEQ ID NO:79: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TTC CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT TTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Phe Thr Ile 
# 70 
- AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:80: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
# 25 TGAG AGAGT 
- (2) INFORMATION FOR SEQ ID NO:81: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
# 25 CCGA CTCCA 
- (2) INFORMATION FOR SEQ ID NO:82: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile 
# 70 
- AGC CGA CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Arg Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:83: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC 
144 
Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TTC CAG CA - #G AAG CCA GGT AAG GCT 
192 
Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ala 
# 40 
- CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile 
# 70 
- AGC CGA CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG 
336 
Ser Arg Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:84: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 94 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
- CAGGAAACAG CTATGACGAA TTCCACCATG CATTTTCAAG TGCAGATTTT CA - #GCTTCCTG 
60 
# 94 TCAT AATGTCCAGA GGAG 
- (2) INFORMATION FOR SEQ ID NO:85: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 88 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
- ACAAGTGATG GTGACTCTGT CTCCTGGAGA TGCAGACATG GAGGATGGAG AC - #TGGGTCAG 
60 
# 88 GACA TTATGACT 
- (2) INFORMATION FOR SEQ ID NO:86: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 92 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 
- ACAGAGTCAC CATCACTTGT AGTGCAAGTT CAAGTGTAAG TTACATGCAC TG - #GTTTCAGC 
60 
# 92 ACCT AAGCTCTGGA TC 
- (2) INFORMATION FOR SEQ ID NO:87: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 87 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 
- AAGATGTACC GCTACCGCTA CCGCTGAATC TAGATGGCAC ACCAGAAGCT AA - #ATTTGAAG 
60 
# 87 CTTA GGTGATT 
- (2) INFORMATION FOR SEQ ID NO:88: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 89 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 
- TAGCGGTAGC GGTACATCTT ACTCTCTCAC CATCAGCAGC ATGCAGCCTG AA - #GATTTTGC 
60 
# 89 CAAA GGAGTAGTT 
- (2) INFORMATION FOR SEQ ID NO:89: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 84 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: 
- GTTTTCCCAG TCACGACCGT ACGTTTGATT TCCAGCTTGG TCCCCTGGCC GA - #ACGTGTAC 
60 
# 84GCTG ACAG 
- (2) INFORMATION FOR SEQ ID NO:90: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 390 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapi - #ens and mouse 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR1"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR2"ER INFORMATION: 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # by similarity with known 
sequence 
#established consensun 
#/product= "CDR3"ER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: 
- ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA 
48 
Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser 
10 
- GTC ATA ATG TCC AGA GGA GAC ATC CAG CTG AC - #C CAG TCT CCA TCC TCC 
96 
Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser 
# 10 
- ATG TCT GCA TCT CCA GGA GAC AGA GTC ACC AT - #C ACT TGT AGT GCA AGT 
144 
Met Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser 
# 25 
- TCA AGT GTA AGT TAC ATG CAC TGG TTT CAG CA - #G AAA CCA GGG AAA TCA 
192 
Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ser 
# 40 
- CCT AAG CTC TGG ATC TAC TCA ACT TCA AAT TT - #A GCT TCT GGT GTG CCA 
240 
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro 
# 55 
- TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC 
288 
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile 
# 70 
- AGC AGC ATG CAG CCT GAA GAT TTT GCA ACT TA - #T TAC TGT CAG CAA AGG 
336 
Ser Ser Met Gln Pro Glu Asp Phe Ala Thr Ty - #r Tyr Cys Gln Gln Arg 
# 90 
- AGT AGT TAC CCG TAC ACG TTC GGC CAG GGG AC - #C AAG CTG GAA ATC AAA 
384 
Ser Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Th - #r Lys Leu Glu Ile Lys 
# 105 
# 390 
Arg Thr 
- (2) INFORMATION FOR SEQ ID NO:91: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 139 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 50..66 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..109 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: 
#Met Gly Trp Ser Trp Ile Phe 
15 
- Leu Phe Leu Leu Ser Gly Thr Ala Gly Val Le - #u Ser Glu Val Gln Leu 
# 1 
- Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile 
# 20 
- Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p Tyr Asn Met Asp Trp 
# 35 
- Val Lys Gln Ser His Gly Lys Ser Leu Glu Tr - #p Ile Gly Tyr Ile Tyr 
# 50 
- Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Ly - #s Phe Lys Ser Lys Ala 
# 65 
- Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu His 
# 80 
- Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ala Thr Tyr Gly 
#100 
- His Tyr Tyr Gly Tyr Met Phe Ala Tyr Trp Gl - #y Gln Gly Thr Leu Val 
# 115 
- Thr Val Ser Ala 
120 
- (2) INFORMATION FOR SEQ ID NO:92: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 129 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
22..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE TO TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 24..33 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 49..55 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 88..96 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: 
- Met His Phe Gln Val Gln Il - #e Phe Ser Phe Leu Leu Ile Ser 
10 
- Ala Ser Val Ile Met Ser Arg Gly Gln Ile Va - #l Leu Thr Gln Ser Pro 
# 5 1 
- Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Va - #l Thr Ile Thr Cys Ser 
# 20 
- Ala Ser Ser Ser Val Ser Tyr Met His Trp Ph - #e Gln Gln Lys Pro Gly 
# 40 
- Thr Ser Pro Lys Leu Trp Ile Tyr Ser Thr Se - #r Asn Leu Ala Ser Gly 
# 55 
- Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gl - #y Thr Ser Tyr Ser Leu 
# 70 
- Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Al - #a Thr Tyr Tyr Cys Gln 
# 85 
- Gln Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gl - #y Gly Thr Lys Leu Glu 
# 100 
- Ile Lys Arg 
105 
- (2) INFORMATION FOR SEQ ID NO:93: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 137 amino 
(B) TYPE: amino acids 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
19..-1 (B) LOCATION: 
(C) IDENTIFICATION METHOD: 
BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN 
#CONSENSUS ESTABLISHED 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 31..35 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 1" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 55..66 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 2" 
- (ix) FEATURE: 
(A) NAME/KEY: domain 
(B) LOCATION: 99..107 
(C) IDENTIFICATION METHOD: - # BY SIMILARITY 
WITH KNOW - #N SEQUENCE OR TO AN ESTABLISEHD 
CONSENSUS 
#/product= "HYPERVARIABLE REGION 3" 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: 
#Met Gly Trp Ser Trp Ile Phe 
15 
- Leu Phe Leu Leu Ser Gly Thr Ala Gly Val Le - #u Ser Glu Val Gln Leu 
# 1 
- Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile 
# 20 
- Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p Tyr Asn Met Asp Trp 
# 35 
- Val Lys Gln Ser His Gly Lys Ser Leu Glu Tr - #p Ile Gly Tyr Ile Tyr 
# 50 
- Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Ly - #s Phe Lys Ser Lys Ala 
# 65 
- Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu His 
# 80 
- Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ala Arg Ala Gly 
#100 
- Arg Tyr Tyr Tyr Ala Trp Asp Trp Gly Gln Gl - #y Thr Leu Val Thr Val 
# 115 
- Ser Ala 
- (2) INFORMATION FOR SEQ ID NO:94: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 5 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 
- Asp Tyr Asn Met Asp 
1 5 
- (2) INFORMATION FOR SEQ ID NO:95: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 17 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95: 
- Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Ty - #r Asn Gln Lys Phe Lys 
# 15 
- Ser 
- (2) INFORMATION FOR SEQ ID NO:96: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 11 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: 
- Tyr Gly His Tyr Tyr Gly Tyr Met Phe Ala Ty - #r 
# 10 
- (2) INFORMATION FOR SEQ ID NO:97: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 10 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: 
- Ser Ala Ser Ser Ser Val Ser Tyr Met His 
# 10 
- (2) INFORMATION FOR SEQ ID NO:98: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 7 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: 
- Ser Thr Ser Asn Leu Ala Ser 
1 5 
- (2) INFORMATION FOR SEQ ID NO:99: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 9 amino 
(B) TYPE: amino acids 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: 
- Gln Gln Arg Ser Ser Tyr Pro Tyr Thr 
1 5 
- (2) INFORMATION FOR SEQ ID NO:100: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 144 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: 
#Ile Phe Leu Phe Leu Leu Ser Gly 
10 
- Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly 
# 10 
- Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly 
# 25 
- Phe Thr Phe Ser Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Pro Pro Gly 
# 40 
- Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr 
# 55 
- Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr 
# 70 
- Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp 
# 90 
- Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met 
# 105 
- Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser 
# 120 
- Thr Lys Gly 
125 
- (2) INFORMATION FOR SEQ ID NO:101: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 133 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: 
- Met His Phe Gln Val G - #ln Ile Phe Ser Phe Leu Leu Ile Ser 
10 
- Ala Ser Val Ile Met Ser Arg Gly Asp Ile Gl - #n Leu Thr Gln Ser Pro 
# 5 1 
- Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Va - #l Thr Ile Thr Cys Ser 
# 20 
- Ala Ser Ser Ser Val Ser Tyr Met His Trp Ty - #r Gln Gln Lys Pro Gly 
# 40 
- Lys Ala Pro Lys Leu Leu Ile Tyr Ser Thr Se - #r Asn Leu Ala Ser Gly 
# 55 
- Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gl - #y Thr Asp Phe Thr Phe 
# 70 
- Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Al - #a Thr Tyr Tyr Cys Gln 
# 85 
- Gln Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gl - #y Gly Thr Lys Val Glu 
# 100 
- Ile Lys Arg Thr Val Ala Ala 
105 1 - #10 
- (2) INFORMATION FOR SEQ ID NO:102: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 27 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: 
# 27 CACC ATCTGTC 
- (2) INFORMATION FOR SEQ ID NO:103: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 27 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: 
# 27 CACC ATCTGTC 
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