DESCRIPTION
Pharmaceutical Composition for Treatment and Prevention of Cancer
TECHNICAL FIELD
[0001]
The present invention relates to a novel pharmaceutical use of an antibody
against CD 179b or a fragment thereof, as an agent for therapy and/or prophylaxis of
cancer.
BACKGROUND ART
[0002]
Cancers are the commonest cause for death among all of the causes for death,
and the therapies currently carried out therefor are mainly surgical treatment in
combination with radiotherapy and chemotherapy. In spite of the developments of
new surgical methods and discovery of new anti-cancer agents in recent years,
treatment results of cancers are not improved very much at present except for some
cancers. In recent years, by virtue of development in molecular biology and cancer
immunology, cancer antigens recognized by antibodies and cytotoxic T cells which
are specifically reactive with cancers, as well as the genes encoding the cancer
antigens, were identified, and expectations for therapeutic methods specifically
targeting cancer antigens have been raised (Non-patent Literature 1).
[0003]
In a therapeutic method for cancer, to reduce side effects, it is desired that the
peptide, polypeptide or protein recognized as the antigen exist hardly in normal cells
and exist specifically in cancer cells. In 1991, Boon et al. in Ludwig Institute in
Belgium isolated a human melanoma antigen MAGE 1 recognized by CD8-positive
T cells by a cDNA-expression cloning method using an autologous cancer cell line
and cancer-reactive T cells (Non-patent Literature 2). Thereafter, the SEREX
(serological identifications of antigens by recombinant expression cloning) method,
wherein tumor antigens recognized by antibodies produced in the living body of a
cancer patient in response to the cancer of the patient himself are identified by
application of a gene expression cloning method, was reported (Non-patent Literature
3; Patent Literature 1), and several cancer antigens which are hardly expressed in
normal cells while being specifically expressed in cancer cells have been isolated by
this method (Non-patent Literatures 4 to 9). Further, using a part thereof as targets,
clinical tests for cell therapies using immunocytes specifically reactive with the
cancer antigens, and cancer-specific immunotherapies such as those using vaccines
containing the cancer antigens have been carried out.
[0004]
On the other hand, in recent years, various antibody drugs for therapy of
cancer have become conspicuous in the world, which drugs target antigen proteins on
cancer cells. Since certain levels of pharmacological effects can be obtained with
such antibody drugs as cancer-specific therapeutic agents, they are drawing attention,
but most of the antigen proteins to be targeted are those also expressed in normal
cells, so that, as a result of administration of the antibody, not only cancer cells, but
also normal cells expressing the antigen are damaged, resulting in occurrence of side
effects, which has been problematic. Thus, it is expected that identification of
cancer antigens specifically expressed on the surfaces of cancer cells and
employment of antibodies targeting these as drugs will allow therapy with antibody
drugs with less side effects.
[0005]
CD179b is known to be a part of the surrogate light chain of immunoglobulin
and expressed on the membrane surfaces of precursor cells of B cells (pre-B cells and
pro-B cells). It disappears upon differentiation of B cells and is not expressed in
mature B cells. However, CD 179b is known to be expressed in leukemia (pre-B
cell leukemia) cells produced by cancerization of pre-B cells (Non-patent Literatures
10 and 11). Further, CD 179b is known to be expressed also in lymphoma (pre-B
cell lymphoma) cells produced by cancerization of pre-B cells, and able to be used as
a diagnostic marker for pre-B cell lymphoma (Non-patent Literature 12). However,
its specific expression has not been reported for leukemia cells other than pre-B cell
leukemia cells, lymphomas other than pre-B cell lymphoma, breast cancer cells and
the like. Further, there has been no report suggesting that antibodies against
CD 179b are useful for therapy and/or prophylaxis of cancer.
PRIOR ART LITERATURES
Patent Literature
[0006]
Patent Literature 1: US 5698396 B
Non-patent Literatures
[0007]
Non-patent Literature 1: Tsuyoshi Akiyoshi, "Cancer and Chemotherapy",
1997, Vol. 24, pp. 551-519
Non-patent Literature 2: Bruggen P. et al., Science, 254:1643-1647 (1991)
Non-patent Literature 3: Proc. Natl. Acad. Sci. USA, 92:11810-11813 (1995)
Non-patent Literature 4: Int.J.Cancer,72:965-971 (1997)
Non-patent Literature 5: Cancer Res., 58:1034-1041 (1998)
Non-patent Literature 6: Int. J. Cancer, 29:652-658 (1998)
Non-patent Literature 7: Int. J. Oncol., 14:703-708 (1999)
Non-patent Literature 8: Cancer Res., 56:4766-4772 (1996)
Non-patent Literature 9: Hum. Mol. Genet 6:33-39 (1997)
Non-patent Literature 10: Adv. Immunol., 63:1-41 (1996)
Non-patent Literature 11: Blood, 92:4317-4324 (1998)
Non-patent Literature 12: Modern Pathology, 17:423-429 (2004)
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0008]
The present invention aims to identify cancer antigen proteins specifically
expressed on the surfaces of cancer cells and provide uses of antibodies targeting
them as agents for therapy and/or prophylaxis of cancer.
MEANS FOR SOLVING THE PROBLEMS
[0009]
The present inventors intensively studied to obtain, by the SEREX method
using serum from a patient dog from which a canine breast cancer tissue-derived
cDNA library was prepared, cDNA encoding a protein which binds to antibodies
existing in the serum derived from the cancer-bearing living body, and, based on a
human gene homologous to the obtained gene, human CD179b having the amino
acid sequence shown in SEQ ID NO:3 was prepared. Further, the present inventors
discovered that CD 179b is hardly expressed in normal tissues, but specifically
expressed in breast cancer, leukemia and lymphoma cells. Further, the present
inventors discovered that antibodies against such CD 179b damage cancer cells
expressing CD 179b, thereby completing the present invention.
[0010]
Thus, the present invention has the following characteristics.
[0011]
The present invention provides a pharmaceutical composition for therapy
and/or prophylaxis of cancer comprising as an effective component an antibody or a
fragment thereof, the antibody having immunoreactivity to a CD 179b protein having
the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having a
sequence identity of not less than 60% with the amino acid sequence, or to a
fragment thereof comprising not less than 7 continuous amino acids.
[0012]
In its mode, the above cancer is a cancer expressing the CD 179b gene.
[0013]
In another mode, the above cancer is breast cancer, leukemia or lymphoma.
[0014]
In another mode, the antibody is a monoclonal antibody or a polyclonal
antibody.
[0015]
In another mode, the antibody is a human antibody, humanized antibody,
chimeric antibody, single-chain antibody or bispecific antibody.
[0016]
In another mode, the above antibody is an antibody comprising a heavy chain
variable region having the amino acid sequences shown in SEQ ID NOs:103, 104 and
102 and a light chain variable region having the amino acid sequences shown in SEQ
ID NOs:106,107 and 108, the antibody having immunoreactivity to a CD 179b
protein.
[0017]
In another mode, the above antibody is an antibody comprising a heavy chain
variable region having the amino acid sequence shown in SEQ ID NO: 105 and a light
chain variable region having the amino acid sequence shown in SEQ ID NO: 109, the
antibody having immunoreactivity to a CD 179b protein.
[0018]
The present invention further provides the following antibodies.
[0019]
(i) An antibody comprising a heavy chain variable region having the amino
acid sequences shown in SEQ ID NOs:103,104 and 102 and a light chain variable
region having the amino acid sequences shown in SEQ ID NOs:106, 107 and 108, the
antibody having immunoreactivity to a CD 179b protein.
[0020]
(ii) An antibody comprising a heavy chain variable region having the amino
acid sequence shown in SEQ ID NO: 105 and a light chain variable region having the
amino acid sequence shown in SEQ ID NO: 109, the antibody having
immunoreactivity to a CD 179b protein.
[0021]
(iii) The antibodies of the above (i) and (ii), having cytotoxic activity.
[0022]
(iv) The antibodies of the above (i) and (ii), each of which is a humanized
antibody, chimeric antibody, single-chain antibody or bispecific antibody.
[0023]
The present invention further provides the following polypeptides or DNAs.
[0024]
(v) A DNA encoding a polypeptide having the amino acid sequence shown in
SEQ ID NO: 105, or a DNA encoding the polypeptide.
[0025]
(vi) A DNA encoding a polypeptide having the amino acid sequence shown in
SEQ ID NO: 109, or a DNA encoding the polypeptide.
[0026]
(vii) A DNA having the base sequence shown in SEQ ID NO: 110.
[0027]
(viii) A DNA having the base sequence shown in SEQ ID NO: 111.
[0028]
(ix) A heavy-chain complementarity-determining region (CDR) polypeptide
selected from the group consisting of the amino acid sequences shown in SEQ ID
NOs:103, 104 and 102, or a DNA encoding the polypeptide.
[0029]
(x) A light-chain complementarity-determining region (CDR) polypeptide
selected from the group consisting of the amino acid sequences shown in SEQ ID
NOs:106,107 and 108, or a DNA encoding the polypeptide.
EFFECT OF THE INVENTION
[0030]
The antibody against CD 179b, which is used in the present invention
damages cancer cells. Therefore, the antibody against CD 179b is useful for therapy
and/or prophylaxis of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031]
Fig. 1 is a diagram showing the expression patterns of the gene encoding the
CD 179b protein in normal tissues and tumor cell lines. Reference numeral 1
represents the expression pattern of the gene encoding the CD 179b protein; and
reference numeral 2 represents the expression pattern of the GAPDH gene.
Fig. 2 is a diagram showing an anti-tumor effect of an antibody against
CD 179b (anti-CD 179b monoclonal antibody #8) in nude mice to which a human
cancer cell line Namalwa expressing CD 179b was transplanted. Reference numeral
3 represents the size of the tumor in miceto which the anti-CD 179b monoclonal
antibody #8 was administered; and reference numeral 4 represents the size of the
tumor in mice to which PBS(-) was administered.
BEST MODE FOR CARRYING OUT THE INVENTION
[0032]
The amino acid sequence shown in SEQ ID NO:3 in SEQUENCE LISTING
disclosed in the present invention is the amino acid sequence of CD 179b isolated, by
the SEREX method using serum from a patient dog from which a canine mammary
gland cancer tissue-derived cDNA library was prepared, as a human homologous
factor (homologue) of a polypeptide which binds to antibodies specifically existing in
the serum derived from the cancer-bearing dog (see Example 1). The antibody
against CD 179b used in the present invention may be any type of antibody as long as
the antibody can exert an anti-tumor activity, and examples thereof include
monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific
antibodies, human antibodies, humanized antibodies, chimeric antibodies, single
chain antibodies (scFv), and antibody fragments such as Fab and F(ab')2. These
antibodies and fragments thereof can be prepared by methods known to those skilled
in the art. In the present invention, the antibody can preferably specifically bind to a
CD179b protein, and, in cases where the subject is a human, the antibody is
preferably a human antibody or a humanized antibody in order to avoid or suppress
rejection reaction.
[0033]
Here, the term "specifically bind to a CD 179b protein" means that the
antibody specifically binds to a CD 179b protein and does not substantially bind to
other proteins.
[0034]
In the present invention, the antibody against CD 179b employed may be
commercially available. Examples of known antibodies against human CD 179b
include clones such as GA170, H-60, HP6054, A-19, C-16, SLC1, SLC2, SLC3,
SLC4 and HSL11, which are available.
[0035]
The anti-tumor activity of the antibody which may be used in the present
invention can be assayed in vitro by investigating whether or not the antibody shows
cytotoxicity against tumor cells expressing the polypeptide via immunocytes or
complement, as mentioned later.
[0036]
Further, the subject of the present invention to be subjected to the therapy
and/or prophylaxis of cancer is a mammal such as a human, pet animal, domestic
animal or sport animal, and the subject is preferably human.
[0037]
The terms "cancer" and "tumor" used in the present specification mean a
malignant neoplasm, and are used interchangeably.
[0038]
Preparation of antigens, preparation of antibodies, and pharmaceutical
compositions, related to the present invention will now be described.
[0039]
Preparation of antigens for preparation of antibodies>
The animal species from which the protein or a fragment thereof used as a
sensitizing antigen to obtain an antibody against CD 179b used in the present
invention is derived is not restricted, and examples thereof include human, dog,
bovine, mouse and rat. However, the animal species is preferably selected in
consideration of the compatibility with the parent cells used for cell fusion, and, in
general, a protein derived from a mammal, especially human, is preferred. For
example, in cases where the CD179b is human CD179b, a human CD179b protein or
a partial peptide thereof, cells expressing human CD179b, or the like may be used.
[0040]
The base sequences and the amino acid sequences of human CD179b and homologues
thereof can be obtained by, for example, accessing GenBank (NCBI, USA) and using an
algorithm such as BLAST or FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci. U. S. Av
90:5873-5877 1993; Altschul et al., Nucleic Acids Res., 25:3389-3402,1997). CD179b is
also called as \5, IGLL1, Vpreb2, LOC608248 or the like, but "CD179b" is used as a
representative in the present specification. For example, human CD179b is registered
under the numbers such as NM_152855 and NM_020070: murine Vpreb2 is registered
under the numbers such as NM_016983; and canine LOC608248 is registered under the
numbers such as XM_845215.
NM_020070; murine Vpreb2 is registered under the numbers such as NMO16983;
and canine LOC608248 is registered under the numbers such as XM845215.
[0041]
In the present invention, in cases where the base sequence or the amino acid
sequence of human CD 179b is used as a standard, a nucleic acid or a protein is
targeted which has a sequence showing a sequence identity of 50% to 100%,
preferably 60% to 100%, more preferably 80% to 100%, still more preferably 90% to
100%, most preferably 95% to 100%, for example, 97% to 100%, 98% to 100%,
99% to 10% or 99.5% to 100% to the sequence shown in SEQ ID NO: 1 or 3. Here,
the term "% sequence identity" means the percentage (%) of identical amino acids (or
bases) with respect to the total number of amino acids (or bases) when two sequences
are aligned with each other such that the maximum similarity is achieved
therebetween with or without introduction of a gap(s).
[0042]
The length of the fragment of the CD179b protein is not less than the length
of amino acids of the epitope (antigenic determinant), which is the shortest unit
recognized by the antibody, and less than the total length of the protein. The length
of the epitope is normally within the range of 7 to 12 continuous amino acids.
[0043]
The above-described human CD 179b protein and polypeptides containing its
partial peptides can be synthesized by a chemical synthesis method such as the Fmoc
method (fluorenyl-methyloxycarbonyl method) or the tBoc method (t-
butyloxycarbonyl method). Further, they can be synthesized by conventional
methods using various types of commercially available peptide synthesizers.
Further, the polypeptide of interest can be obtained using known genetic engineering
techniques, by preparing a polynucleotide encoding the above polypeptide and
incorporating the polynucleotide into an expression vector, which is then introduced
into a host cell, followed by allowing the polypeptide to be produced in the host cell.
[0044]
The polynucleotide encoding the above polypeptide can be easily prepared by
a known genetic engineering technique or a conventional method using a
commercially available nucleic acid synthesizer. For example, DNA having the
base sequence shown in SEQ ID NO:1 can be prepared by carrying out PCR using
human chromosomal DNA or a human cDNA library as a template, and a pair of
primers designed such that the base sequence shown in SEQ ID NO: 1 can be
amplified therewith. The reaction conditions for the PCR can be set appropriately,
and examples thereof include, but are not limited to, repeating the reaction process of
94°C for 30 seconds (denaturation), 55°C for 30 seconds to 1 minute (annealing) and
72°C for 2 minutes (extension) for, for example, 30 cycles, followed by the reaction
at 72°C for 7 minutes. Further, the desired DNA can be isolated by preparing an
appropriate probe(s) or primer(s) based on the information of the base sequence and
the amino acid sequence shown in SEQ ID NOs:l and 3, respectively, in
SEQUENCE LISTING in the present specification, and using the probe(s) or
primer(s) for screening of a cDNA library of human or the like.
[0045]
The cDNA library is preferably prepared from cells, an organ or a tissue
expressing the protein of SEQ ID NO:3. Examples of such cells and a tissue
include bone marrow, leukemia cells, breast cancer cells and lymphoma cells. The
above-described operations such as preparation of the probe(s) or primer(s),
construction of a cDNA library, screening of the cDNA library and cloning of the
gene of interest are known to those skilled in the art, and can be carried out according
to the methods described in, for example, Sambrook et al., Molecular Cloning,
Second Edition, Current Protocols in Molecular Biology (1989). From the thus
obtained DNA, a DNA encoding a human CD 179b protein or a partial peptide
thereof can be obtained.
[0046]
The above-described host cells may be any cells as long as they can express
the above polypeptide, and examples of prokaryotic cells include, but are not limited
to, E. coli, and examples of eukaryotic cells include, but are not limited to,
mammalian cultured cells such as the monkey kidney cells COS 1, Chinese hamster
ovary cells CHO, human fetal kidney cell line HEK 293 and mouse embryonic skin
cell line NIH3T3; yeast cells such as budding yeasts and fission yeasts; silkworm
cells; and Xenopus egg cells.
[0047]
In cases where prokaryotic cells are used as the host cells, the expression
vector employed in the prokaryotic cells has a replication origin, promoter, ribosome
binding site, multicloning site, terminator, drug resistant gene, nutrient
complementary gene and/or the like. Examples of the expression vector for E. coli
include the pUC system, pBluescriptll, pET expression system and pGEX expression
system. By incorporating a DNA encoding the above polypeptide into such an
expression vector and transforming prokaryotic host cells with the vector, followed
by culturing the resulting transformants, the polypeptide encoded by the DNA can be
expressed in the prokaryotic host cells. In this process, the polypeptide can also be
expressed as a fusion protein with another protein (e.g., green fluorescent protein
(GFP) or glutathione S-transferase (GST)).
[0048]
In cases where eukaryotic cells are used as the host cells, an expression vector
for eukaryotic cells having a promoter, splicing site, poly(A) addition site and/or the
like is used as the expression vector. Examples of such an expression vector
include pKAl, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector,
pRS, pcDNA3, pMSG and pYES2. In the same manner as described above, by
incorporating a DNA encoding the above polypeptide into such an expression vector
and transforming eukaryotic host cells with the vector, followed by culturing the
resulting transformants, the polypeptide encoded by the DNA can be expressed in the
eukaryotic host cells. In cases where pIND/V5-His, pFLAG-CMV-2, pEGFP-N 1,
pEGFP-Cl or the like is used as the expression vector, the above polypeptide can be
expressed as a fusion protein to which a tag such as His tag (e.g., (His)6 to (His)io),
FLAG tag, myc tag, HA tag or GFP was added.
[0049]
For the introduction of the expression vector into the host cells, well-known
methods such as electroporation, the calcium phosphate method, the liposome
method, the DEAE dextran method and microinjection can be used.
[0050]
Isolation and purification of the polypeptide of interest from the host cells can
be carried out by a combination of known separation operations. Examples of the
known separation operations include, but are not limited to, treatment with a
denaturant such as urea, or a surfactant; ultrasonication treatment; enzyme digestion;
salting-out or solvent fractional precipitation; dialysis; centrifugation; ultrafiltration;
gel filtration; SDS-PAGE; isoelectric focusing; ion-exchange chromatography;
hydrophobic chromatography; affinity chromatography; and reversed-phase
chromatography.
[0051]

An antibody is usually a heteropolymeric glycoprotein having at least two
heavy chains and two light chains. Except for IgM, it is a heterotetrameric
glycoprotein of about 150 kDa constituted by two identical light (L) chains and two
identical heavy (H) chains. Typically, each light chain is linked to a heavy chain via
a single disulfide covalent bond, but the number of disulfide bonds between the
heavy chains varies among various immunoglobulin isotypes. Each of the heavy
chains and the light chains also has intrachain disulfide bonds. Each heavy chain
has a variable domain (VH region) in its one end, and the variable domain is
followed by several constant regions. Each light chain has a variable domain (VL
region), and has one constant region at the opposite end thereof. The constant
region of each light chain is aligned with the first constant region of a heavy chain,
and each light chain variable domain is aligned with a heavy chain variable domain.
Each variable domain of an antibody has particular regions showing particular
variabilities, called the complementarity-determining regions (CDRs), which give a
binding specificity to the antibody. Parts in each variable region, which parts are
relatively conserved are called the framework regions (FRs). Each of the complete
variable domains of the heavy chains and the light chains has four FRs linked via
three CDRs. In each heavy chain, the three CDRs are called CDRH1, CDRH2 and
CDRH3 in the order from the N-terminus, and, in each light chain, they are called
CDRL1, CDRL2 and CDRL3 in a similar manner. For the binding specificity of an
antibody against an antigen, CDRH3 is most important. Further, the CDRs in each
strand are held together by the FR regions such that the CDRs are close to one
another, thereby contributing to formation of an antigen-binding site together with
the CDRs from another strand. Although the constant region does not directly
contribute to binding of the antibody to an antigen, it shows various effector
functions such as involvement in antibody-dependent cell-mediated cytotoxicity
(ADCC), phagocytosis via binding to the Fey receptor, the half life/clearance rate via
the Neonatal Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC) via
the Clq component of the complement cascade.
[0052]
Preparation of the Antibody>
The anti-CD179b antibody in the present invention means an antibody having an
immunological reactivity with the total length of the CD179b protein or a fragment
thereof. Here the term "immunological reactivity" means a property by which the
antibody and a CD179b antigen are bound to each other, and the function to damage (to
cause death, suppression or regression of )tumors is exerted by such binding. That is
type of the antibody used in the present invention is not restricted as long as the
antibody can be bound to a CD179b protein to damage tumors such as breast cancer,
leukemia, and lymphoma.
[0053]
Examples of the antibody include monoclonal antibodies, polyclonal
antibodies, synthetic antibodies, multispecific antibodies, human antibodies,
humanized antibodies, chimeric antibodies, single chain antibodies, and antibody
fragments (e.g., Fab and (Aab')2). Further, the antibody belongs to an arbitrary
class of an immunoglobulin molecule, such as IgG, IgE, IgM, IgA, IgD or IgY, or to
an arbitrary subclass such as IgGl, IgG2, IgG3, IgG4, IgAl or IgA2.
[0054]
The antibody may be further modified by glycosylation, acetylation,
formylation, amidation, phosphorylation, pegylation (PEG) and/or the like.
[0055]
Preparation examples of various antibodies are described below.
[0056]
In cases where the antibody is a monoclonal antibody, for example, a
leukemia cell line Namalwa expressing CD 179b is administered to a mouse to
immunize the mouse, and spleen is extracted from the mouse. Cells are separated
and fused with mouse myeloma cells, and, from the obtained fused cells
(hybridomas), a clone producing an antibody having a cancer cell growth suppressing
action is selected. By isolating the monoclonal antibody-producing hybridoma
having a cancer cell growth suppressing action, and culturing the hybridoma,
followed by purifying the antibody from the culture supernatant by a commonly-used
affinity purification method, the antibody can be prepared.
[0057]
A hybridoma which produces a monoclonal antibody can also be prepared, for
example, as follows.
[0058]
First, according to a known method, an animal is immunized with a
sensitizing antigen. In general, the method is carried out by intraperitoneal or
subcutaneous injection of the sensitizing antigen to a mammal. More particularly,
the sensitizing antigen is diluted to an appropriate volume with PBS (Phosphate-
Buffered Saline) or physiological saline and suspended, followed by mixing, as
desired, an appropriate amount of a normal adjuvant such as Freund's complete
adjuvant with the suspension. This is followed by emulsification, and then
administration of the emulsion to a mammal every 4 to 21 days for several times.
Further, it is also possible to use an appropriate carrier when the immunization with
the sensitizing antigen is carried out.
[0059]
After such immunization of a mammal and confirmation of increase in the
serum level of the desired antibody, immunocytes are collected from the mammal
and subjected to cell fusion. Examples of preferred immunocytes especially include
spleen cells.
[0060]
As the other parent cells to be fused with the immunocytes, mammalian
myeloma cells are used. Examples of the myeloma cells preferably employed
include various known cell lines such as P3U1 (P3-X63Ag8Ul), P3 (P3x63Ag8.653)
(J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.l (Current Topics in Microbiology
and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol.
(1976) 6, 511-519), MPC-11 (Margulies. D.H. et al., Cell (1976) 8, 405-415), SP2/0
(Shulman, M. et al., Nature (1978) 276,269-270), FO (deSt. Groth, S. F. et al., J.
Immunol. Methods (1980) 35,1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978)
148,313-323) and R210 (Galfre, G. et al., Nature (1979) 277, 131-133).
[0061]
The cell fusion between the immunocytes and the myeloma cells can be
carried out basically according to a known method, for example, a method by Kohler
and Milstein (Kohler. G. and Milstein, C, Methods Enzymol. (1981) 73, 3-46).
[0062]
More particularly, the cell fusion is carried out, for example, in the presence
of a cell fusion-promoting agent, in a normal nutrient medium. Examples of the
fusion-promoting agent include polyethylene glycol (PEG) and Sendai virus (HVJ),
and, in order to enhance the fusion efficiency, an auxiliary agent such as
dimethylsulfoxide may also be added as desired.
[0063]
The ratio between the immunocytes and the myeloma cells to be used may be
arbitrarily set. For example, it is preferred to use 1 to 10 times more immunocytes
than the myeloma cells. Examples of the medium which can be used for the cell
fusion include the RPMI1640 medium which is preferred for the growth of the
myeloma cell line; MEM medium; and other media normally used for this kind of
cell culture. Further, a serum replacement such as fetal calf serum (FCS) can also
be used in combination.
[0064]
During the cell fusion, prescribed amounts of the immunocytes and the
myeloma cells are mixed together well in the medium, and a PEG solution (with an
average molecular weight of about 1000 to 6000, for example) preheated to about
37°C is added to a concentration of normally 30 to 60% (w/v), followed by mixing
the resulting mixture to form the hybridoma of interest. Subsequently, by repeating
the operation of successive addition of an appropriate medium and removal of the
supernatant by centrifugation, cell fusion agents and the like which are not preferred
for the growth of the hybridoma are removed.
[0065]
The thus obtained hybridoma is selected by being cultured in a normal
selection medium such as the HAT medium (a medium containing hypoxanthine,
aminopterin and thymidine). The culture in the above HAT medium is continued
for a length of time sufficient for the cells other than the hybridoma of interest
(unfused cells) to die (normally, for several days to several weeks). Thereafter, a
normal limiting dilution method is carried out for screening and cloning of a single
hybridoma producing the antibody of interest.
[0066]
In addition to the method in which the above hybridoma is obtained by
immunizing a non-human animal with the antibody, there is also a method in which
human lymphocytes, such as human lymphocytes infected with EB virus, are
sensitized in vitro with a protein, protein-expressing cells or a lysate thereof, and the
sensitized lymphocytes are fused with human-derived myeloma cells having a
permanent division potential, for example, U266 (registration number TIB 196), to
obtain a hybridoma producing a human antibody having a desired activity (cell
growth suppression activity, for example).
[0067]
The thus prepared hybridoma producing a monoclonal antibody can be
subcultured in a normal medium, and can be stored in liquid nitrogen for a long
period.
[0068]
That is, the hybridoma can be prepared by a process wherein the desired
antigen or cells expressing the desired antigen is/are used as a sensitizing antigen to
carry out immunization according a conventional immunization method, thereby
obtaining immunocytes, which are then fused with known parent cells by a
conventional cell fusion method, followed by screening of monoclonal antibody-
producing cells (hybridomas) by a conventional screening method.
[0069]
Another example of the antibody which can be used in the present invention
is a polyclonal antibody. The polyclonal antibody can be obtained, for example, as
follows.
[0070]
A naturally occurring CD 179b protein, or a recombinant CD 179b protein
expressed as a fusion protein with GST in a microorganism such as E. coli, or a
partial peptide thereof is used for immunization of a small animal such as a mouse,
human antibody-producing mouse or rabbit, and serum is obtained from the small
animal. A polyclonal antibody is prepared by purifying the serum by, for example,
ammonium sulfate precipitation, protein A and protein G columns, DEAE ion-
exchange chromatography, or an affinity column coupled with a CD 179b protein or a
synthetic peptide.
[0071]
Here, known examples of the human antibody-producing mouse include the
KM mouse (Kirin Pharma/Medarex) and XenoMouse (Amgen). When such a
mouse is immunized with a CD 179b protein or a fragment thereof, a complete human
polyclonal antibody can be obtained from blood. Further, by removing spleen cells
from the immunized mouse and subjecting the cells to the fusion method with
myeloma cells, a human-type monoclonal antibody can be prepared.
[0072]
The antigen can be prepared according to a method using animal cells
(Japanese Translated PCT Patent Application Laid-open No. 2007-530068), a
method using a baculovirus (e.g., WO98/46777), or the like. In cases where the
immunogenicity of the antigen is low, the immunization may be carried out after
binding the antigen to a macromolecule having immunogenicity, such as albumin.
[0073]
Further, a gene recombinant antibody can also be used, which antibody was
prepared by cloning the antibody gene from the hybridoma and incorporating it into
an appropriate vector, which was then introduced transfected to a host, followed by
allowing the host to produce the antibody by the genetic recombination technique
(for example, see Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC
MONOCLONAL ANTIBODIES, Published in the United Kingdom by
MACMILLAN PUBLISHERS LTD, 1990).
[0074]
More particularly, cDNA of the variable region (V region) of the antibody is
synthesized from mRNA of the hybridoma using a reverse transcriptase. After
obtaining the DNA encoding the V region of the antibody of interest, the DNA is
linked to DNA encoding the antibody constant region (C region) of interest, and the
resultant is incorporated into an expression vector. Alternatively, the DNA
encoding the V region of the antibody may be incorporated into an expression vector
having the DNA of the antibody C region. The incorporation is carried out such
that the expression is allowed under the controls by expression control regions such
as an enhancer and/or a promoter. Subsequently, host cells can be transformed with
this expression vector to allow expression of the antibody.
[0075]
The anti-CD 179b antibody of the present invention is preferably a
monoclonal antibody. However, it may also be a polyclonal antibody, genetically
modified antibody (such as a chimeric antibody or humanized antibody) or the like.
[0076]
Examples of the monoclonal antibody include human monoclonal antibodies
and non-human animal monoclonal antibodies (e.g., mouse monoclonal antibodies,
rat monoclonal antibodies and chicken monoclonal antibodies). The monoclonal
antibody can be prepared by culturing a hybridoma obtained by fusion of spleen cells
from a non-human mammal (e.g., mouse or human antibody-producing mouse)
immunized with a CD 179b protein, with myeloma cells. In Examples below, a
mouse monoclonal antibody #8 was prepared, with which an anti-tumor effect was
confirmed. The antibody #8 has a heavy chain variable (VH) region having the
amino acid sequence shown in SEQ ID NO: 105 and a light chain variable region
(VL) having the amino acid sequence shown in SEQ ID NO: 109. Here, the VH
region has the amino acid sequences shown in SEQ ID NO: 103 (CDR1), SEQ ID
NO: 104 (CDR2) and SEQ ID NO: 102 (CDR3); and the VL region has the amino acid
sequences shown in SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2) and SEQ ID
NO:108(CDR3).
[0077]
A chimeric antibody is an antibody prepared by combining sequences derived
from different animals. Examples thereof include an antibody having variable
regions of the heavy chain and the light chain of a mouse antibody and the constant
regions of the heavy chain and the light chain of a human antibody. Preparation of
the chimeric antibody can be carried out using a known method. For example, it
can be obtained by linking a DNA encoding an antibody V region to a DNA
encoding a human antibody C region, followed by incorporating the resultant to an
expression vector and introducing transfecting the vector to a host, thereby allowing
production of a chimeric antibody.
[0078]
Examples of the polyclonal antibody include antibodies obtained by
immunizing a human antibody-producing animal (mouse, for example) with a
CD179b protein
EQ079]
A humanized antibody is a modified antibody also called as a reshaped human
antibody. A humanized antibody can be constructed by transplantation of the CDRs
of an antibody derived from an immunized animal to the complementarity-
determining regions of a human antibody. A common genetic recombination
technique therefor is known.
£0080]
More particularly, a DNA sequence designed such that the CDRs of a mouse
antibody are linked to the framework regions (FRs) of a human antibody is
synthesized by the PCR method from several oligonucleotides prepared such that the
oligonucleotides have overlapped regions in their ends. The obtained DNA is
linked to a DNA encoding the human antibody constant region, and the resultant is
incorporated into an expression vector, followed by introducing the vector to a host,
to obtain a humanized antibody (see European Patent Application Publication No. EP
239400 and International Patent Application Publication No. WO96/02576). The
FRs of the human antibody linked via the CDRs are selected such that the
complementarity-determining regions form a good antigen-binding site. As
required, amino acids in the framework regions in the variable regions of the
antibody may be substituted such that the complementarity-determining regions of
the reshaped human antibody form an appropriate antigen-binding site (Sato, K. et al.,
Cancer Res. (1993) 53, 851-856). Further, the framework regions may be
substituted with framework regions derived from various human antibodies (see
International Patent Application Publication No. WO99/51743).
After preparation of a chimeric antibody or a humanized antibody, amino
acids in the variable regions (FRs, for example) and/or the constant regions may be
substituted with other amino acids.
The number of the amino acids to be substituted is, for example, less than 15,
less than 10, not more than 8, not more than 7, not more than 6, not more than 5, not
more than 4, not more than 3 or not more than 2, preferably 1 to 5, more preferably 1
or 2, and the substituted antibody should be functionally equivalent to the
unsubstituted antibody. The substitutions are preferably conservative amino acid
substitutions, which are substitutions among amino acids having similar properties of
charges, side chains, polarities, aromaticities and/or the like. The amino acids
having similar properties can be classified, for example, into basic amino acids
(arginine, lysine and histidine), acidic amino acids (aspartic acid and glutamic acid),
uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine,
cysteine and tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine,
proline, phenylalanine, tryptophan and methionine), branched chain amino acids
(threonine, valine and isoleucine) and aromatic amino acids (phenylalanine, tyrosine,
tryptophan and histidine).
fee«4r
Examples of the modified antibody include antibodies bound to various
molecules such as polyethylene glycol (PEG). In the modified antibody of the
present invention, the substance to which the antibody is bound is not restricted.
Such a modified antibody can be obtained by chemical modification of the obtained
antibody. These methods are already established in the art.
[0085]
Here, the term "functionally equivalent" means, for example, that the subject
antibody has a similar biological or biochemical activity, more particularly, a
function to damage tumors, and does not essentially cause the rejection reaction
when it is applied to human. Examples of such an activity may include a cell
growth suppressing activity and a binding activity.
[0086]
As a method well-known to those skilled in the art for preparation of a
polypeptide functionally equivalent to a certain polypeptide, introduction of a
mutation(s) to a polypeptide is known. For example, those skilled in the art can use
site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275;
Zoller, MJ, and Smith, M.(1983) Methods Enzymol. 100, 468-500; Kramer, W. et al.
(1984) Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz HJ (1987) Methods.
Enzymol. 154, 350-367; Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492;
Kunkel (1988) Methods Enzymol. 85,2763-2766) or the like to introduce, as
appropriate, a mutation(s) to the antibody of the present invention, to prepare an
antibody functionally equivalent to this antibody.
[0087]
The antibody which recognizes the epitope of the CD 179b protein to be
recognized by the above-described anti-CD 179b antibody can be obtained by a
method known to those skilled in the art. Examples of the method by which it can
be obtained include a method wherein the epitope of the CD 179b protein recognized
by the anti-CD179b antibody is determined by a normal method (e.g., epitope
mapping) and an antibody is prepared using as an immunogen a polypeptide having
an amino acid sequence included in the epitope; and a method wherein the epitope of
the antibody is determined by a normal method, followed by selecting an antibody
having the same epitope as that of the anti-CD 179b antibody. Here, the term
"epitope" means a polypeptide fragment having antigenicity or immunogenicity in a
mammal, preferably human, and its minimum unit has about 7 to 12 amino acids.
[0088]
The affinity constant Ka (Kon/Koff) of the antibody of the present invention is
preferably at least 107 M'1, at least 108 M"1, at least 5><108 M'1, at least 109 M"1, at
least 5xlO9 M"1, at least 1010 M'1, at least 5xl010 M"1, at least 1011 M"1, at least
5xl0n NT1, at least 1012 M'\ at least 1013 M"1.
[0089]
The antibody of the present invention can be conjugated with an antitumor
agent. The binding between the antibody and the antitumor agent can be carried out
via a spacer having a group (e.g., succinimidyl group, formyl group, 2-pyridyldithio
group, maleimidyl group, alkoxycarbonyl group or hydroxy group) reactive with an
amino group, carboxyl group, hydroxy group, thiol group and/or the like.
[0090]
Examples of the antitumor agent include the following antitumor agents
known in literatures and the like, that is, paclitaxel, doxorubicin, daunorubicin,
cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan,
piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine,
triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide,
trimethylolomelamine, bullatacin, bullatacinone, camptothecin, bryostatin, callystatin,
cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin,
sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxyhydrochloride,
melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard,
carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine,
calicheamicin, dynemicin, clodronate, esperamicin, aclacinomycin, actinomycin,
authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin,
carzinophilin, chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C,
mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-
mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine;
androgens such as calusterone, dromostanolone propionate, epitiostanol,
mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid,
aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine,
bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine,
elliptinium acetate, epothilone, etoglucid, lentinan, lonidamine, maytansine,
ansamitocine, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,
phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,
procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid,
triaziquone, roridine A, anguidine, urethane, vindesine, dacarbazine, mannomustine,
mitobronitol, mitolactol, pipobroman, gacytosine, doxetaxel, chlorambucil,
gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin,
vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone,
teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan,
topoisomerase inhibitors, difluoromethylornithine (DMFO), retinoic acid and
capecitabine, and pharmaceutically acceptable salts and derivatives thereof.
[0091]
Alternatively, the antibody of the present invention can be linked to a known
radioisotope described in a literature or the like, such as At21', I131,1125, Y90, Re186,
Re188, Sm153, Bi212, P32 or Lu. The radioisotope is preferably effective for therapy
and/or diagnosis of a tumor.
[0092]
The antibody of the present invention is an antibody having an immunological
reactivity with CD 179b, or an antibody which specifically recognizes CD 179b. The
antibody should be an antibody having a structure by which the rejection reaction can
be mostly or completely avoided in the subject animal to which the antibody was
administered. Examples of such an antibody include, for example, in cases where
the subject animal is human, human antibodies, humanized antibodies, chimeric
antibodies (e.g., human-mouse chimeric antibodies), single chain antibodies and
bispecific antibodies. Each of these antibodies is a recombinant antibody wherein:
each variable region in the heavy chain and the light chain is derived from a human
antibody; each variable region in the heavy chain and the light chain is constituted by
the complementarity-determining regions (CDR1, CDR2 and CDR3) of an antibody
derived from a non-human animal and the framework regions derived from a human
antibody; or each variable region in the heavy chain and the light chain is derived
from a non-human animal; which recombinant antibody has human antibody-derived
constant regions in the heavy chain and the light chain. The first two antibodies are
preferred.
[0093]
These recombinant antibodies can be prepared as follows. A DNA encoding
a monoclonal antibody (for example, human monoclonal antibody, mouse
monoclonal antibody, rat monoclonal antibody or chicken monoclonal antibody)
against human CD 179b is cloned from antibody-producing cells such as hybridomas,
and, using this as a template, DNAs encoding the light chain variable region and the
heavy chain variable region of the antibody is prepared by, for example, the RT-PCR
method, followed by determining the sequence of the variable region or the
sequences of CDR1, CDR2 and CDR3 in each of the light chain and the heavy chain
according to the Kabat EU numbering system (Kabat et al., Sequences of Proteins of
Immunological Interest, 5Th Ed. Public Health Service, National Institute of Health,
Bethesda, Md. (1991)). Further, DNAs encoding the respective variable regions or
DNAs encoding the respective CDRs are prepared using the genetic recombination
technique (Sambrook et al., Molecular Cloning, Second Edition, Current Protocols in
Molecular Biology (1989)) or a DNA synthesizer. Here, the above-described
human monoclonal antibody-producing hybridoma can be prepared by immunizing a
human antibody-producing animal (e.g., mouse) with human CD 179b, followed by
fusing spleen cells excised from the immunized animal with myeloma cells. In
addition, as required, DNAs encoding the variable region and the constant region in
the light chain or the heavy chain derived from a human antibody are prepared using
the genetic recombination technique or a DNA synthesizer.
[0094]
In the case of a humanized antibody, a DNA encoding the humanized
antibody can be prepared by a process wherein the CDR sequences in a DNA
encoding the variable region of the light chain or the heavy chain derived from a
human antibody are substituted with the corresponding CDR sequences of an
antibody derived from a non-human animal (e.g., mouse, rat or chicken) to prepare a
DNA, and the thus obtained DNA is linked to a DNA encoding the constant region in
the light chain or the heavy chain, respectively, derived from a human antibody.
[0095]
In the case of a chimeric antibody, a DNA encoding the chimeric antibody can
be prepared by a process wherein a DNA encoding the variable region in the light
chain or the heavy chain derived from a non-human animal (e.g., mouse, rat or
chicken) is linked to a DNA encoding the constant region of the light chain or the
heavy chain, respectively, derived from a human antibody
[0096]
In the case of a single-chain antibody, which is an antibody having a heavy
chain variable region and a light chain variable region linearly linked to each other
via a linker, a DNA encoding the single-chain antibody can be prepared by a process
wherein a DNA encoding the heavy chain variable region, a DNA encoding the linker
and a DNA encoding the light chain variable region are linked together. Here, each
of the heavy chain variable region and the light chain variable region is either derived
from a human antibody or derived from a human antibody in which only the CDRs
were replaced by the CDRs of an antibody derived from a non-human animal (e.g.,
mouse, rat or chicken). Further, the linker has 12 to 19 amino acids, and examples
thereof include (G4S)3 having 15 amino acids (Kim, GB. et al., Protein Engineering
Design and Selection 2007,20(9):425-432).
[0097]
In the case of a bispecific antibody (diabody), which is an antibody capable of
binding specifically to two different epitopes, a DNA encoding the bispecific
antibody can be prepared, for example, by a process wherein a DNA encoding a
heavy chain variable region A, a DNA encoding a light chain variable region B, a
DNA encoding a heavy chain variable region B and a DNA encoding a light chain
variable region A are linked together in this order (however, the DNA encoding a
light chain variable region B and the DNA encoding a heavy chain variable region B
are linked to each other via a DNA encoding a linker as described above). Here,
each of the heavy chain variable region and the light chain variable region is either
derived from a human antibody or derived from a human antibody in which only the
CDRs were replaced by the CDRs of an antibody derived from a non-human animal
(e.g., mouse, rat or chicken).
[0098]
A recombinant antibody can be prepared by incorporating the thus prepared
recombinant DNA(s) into one or more appropriate vector(s) and introducing the
resulting vector(s) into host cells (e.g., mammalian cells, yeast cells and insect cells),
followed by allowing (co-)expression of the recombinant DNA(s) (P.J. Delves.,
ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY; P.
Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY
PRESS; J.W. Goding., Monoclonal Antibodies: principles and practice., 1993
ACADEMIC PRESS).
[0099]
The antibody of the present invention prepared by the above method is an
antibody comprising, for example, a heavy chain variable region having the amino
acid sequences shown in SEQ ID NOs:103,104 and 102 and a light chain variable
region having the amino acid sequences shown in SEQ ID NOs:106, 107 and 108.
Here, the amino acid sequences shown in SEQ ID NOs:103, 104 and 102 are those
for CDR1, CDR2 and CDR3, respectively, of a mouse antibody heavy chain variable
region, and the amino acid sequences shown in SEQ ID NOs:106, 107 and 108 are
those for CDR1, CDR2 and CDR3, respectively, of a mouse antibody light chain
variable region. Therefore, the humanized antibody, chimeric antibody, single-
chain antibody or bispecific antibody of the present invention is the following
antibody, for example.
[0100]
(i) An antibody comprising: a heavy chain variable region having the amino
acid sequences shown in SEQ ID NOs:103, 104 and 102 and the amino acid
sequences of the framework regions derived from a human antibody; and a light
chain variable region having the amino acid sequences shown in SEQ ID NOs:106,
107 and 108 and the amino acid sequences of the framework regions derived from a
human antibody.
[0101]
(ii) An antibody comprising: a heavy chain variable region having the amino
acid sequences shown in SEQ ID NOs:103,104 and 102 and the amino acid
sequences of the framework regions derived from a human antibody; a heavy chain
constant region having an amino acid sequence derived from a human antibody; a
light chain variable region having the amino acid sequences shown in SEQ ID
NOs:106,107 and 108 and the amino acid sequences of the framework regions
derived from a human antibody; and a light chain constant region having an amino
acid sequence derived from a human antibody.
[0102]
(iii) An antibody comprising: a heavy chain variable region having the amino
acid sequence shown in SEQ ID NO: 105; and a light chain variable region having the
amino acid sequence shown in SEQ ID NO: 109.
[0103]
(iv) An antibody comprising: a heavy chain variable region having the amino
acid sequence shown in SEQ ID NO: 105; a heavy chain constant region having an
amino acid sequence derived from a human antibody; a light chain variable region
having the amino acid sequence shown in SEQ ID NO: 109; and a light chain constant
region having an amino acid sequence derived from a human antibody.
[0104]
Sequences of the constant regions and the variable regions of human antibody
heavy chains and light chains can be obtained from, for example, NCBI (USA:
GenBank, UniGene and the like). Examples of the sequences which may be
referred to include the accession number J00228 for the human IgGl heavy chain
constant region, the accession number J00230 for the human IgG2 heavy chain
constant region, the accession number X03604 for the human IgG3 heavy chain
constant region, the accession number K01316 for the human IgG4 heavy chain
constant region, the accession numbers V00557, X64135, X64133 and the like for
the human light chain k constant region, and the accession numbers X64132, X64134
and the like for the human light chain % constant region.
[0105]
The above antibody preferably has cytotoxic activity and therefore can exert
an anti-tumor effect.
[0106]
Further, the specific sequences of the variable regions of the heavy chain and
the light chain and the CDRs in the above antibodies are presented for the illustration
purpose only, and it is apparent that they are not restricted to the specific sequences.
A hybridoma which can produce another human antibody or non-human animal
antibody (e.g., mouse antibody) against human CD179b is prepared, and the
monoclonal antibody produced by the hybridoma is recovered, followed by judging
whether or not it is an antibody of interest using as indices its immunological affinity
and cytotoxicity to human CD 179b. By this, a monoclonal antibody-producing
hybridoma of interest is identified, and DNAs encoding the variable regions of the
heavy chain and the light chain of the antibody of interest are prepared from the
hybridoma as described above, followed by determining the sequences of the DNAs
and then using the DNAs for preparation of the another antibody.
[0107]
Further, the above antibody of the present invention may have substitution,
deletion and/or addition of 1 or several (preferably, 1 or 2) amino acid(s) especially
in a framework region sequence(s) and/or constant region sequence(s) in each of the
antibodies (i) to (iv) above, as long as the antibody has a specificity allowing specific
recognition of CD179b. Here, the term "several" means 2 to 5, preferably 2 or 3.
[0108]
The present invention further provides a DNA encoding the above antibody of
the present invention, a DNA encoding the heavy chain or the light chain of the
above antibody or a DNA encoding the variable region of the heavy chain or the light
chain of the above antibody. Examples of such a DNA include: DNAs encoding
heavy chain variable regions having the base sequences encoding the amino acid
sequences shown in SEQ ID NOs:103,104 and 102; DNAs encoding light chain
variable regions having the base sequences encoding the amino acid sequences
shown in SEQ ID NOs:106,107 and 108; and the like.
[0109]
Since the complementarity-determining regions (CDRs) encoded by DNAs
having these sequences are regions which determine the specificity of the antibody,
the sequences encoding the other regions in the antibody (that is, the constant regions
and the framework regions) may be those derived from another antibody. Here,
although the another antibody includes antibodies derived from non-human
organisms, it is preferably derived from human in view of reduction of side effects.
That is, in the above-described DNA, the regions encoding the respective framework
regions and the constant regions of the heavy chain and the light chain preferably
have base sequences encoding corresponding amino acid sequences derived from a
human antibody.
[0110]
Other examples of the DNA encoding the antibody of the present invention
include DNAs encoding the heavy chain variable region having a base sequence
encoding the amino acid sequence shown in SEQ ID NO: 105 and DNAs wherein the
region encoding the light chain variable region has a base sequence encoding the
amino acid sequence shown in SEQ ID NO: 109. Here, examples of the base
sequence encoding the amino acid sequence shown in SEQ ID NO: 105 include the
base sequence shown in SEQ ID NO:110. Further, examples of the base sequence
encoding the amino acid sequence shown in SEQ ID NO: 109 include the base
sequence shown in SEQ ID NO: 111. Among these DNAs, preferred are those
comprising the region encoding the constant region of each of the heavy chain and
the light chain, having a base sequence encoding a corresponding amino acid
sequence derived from a human antibody.
[0111]
The DNA of the present invention can be obtained by, for example, the above
method or the following method. First, from a hybridoma related to the antibody of
the present invention, total RNA is prepared using a commercially available RNA
extraction kit, and cDNA is synthesized by a reverse transcriptase using random
primers or the like. Subsequently, by the PCR method using as primers
oligonucleotides having sequences conserved in the variable region of each of a
known mouse antibody heavy chain gene and light chain gene, cDNAs encoding the
antibody are amplified . The sequence encoding each constant region can be
obtained by amplifying a known sequence by the PCR method. The base sequences
of the DNAs can be determined by a conventional method by, for example,
incorporating the sequences into plasmids or phages for sequence determination.
[0112]
The anti-tumor effect of the anti-CD 179b antibody used in the present
invention against CD179b-expressing cancer cells is considered to be caused by the
following mechanism.
[0113]
The antibody-dependent cell-mediated cytotoxicity (ADCC) by effector cells
against CD179b-expressing cells; and
the complement-dependent cytotoxicity (CDC) against CD179b-expressing
cells.
[0114]
Thus, evaluation of the activity of the anti-CD 179b antibody used in the
present invention can be carried out, as particularly shown in Examples below, by
measuring the above-described ADCC activity or CDC activity against cancer cells
expressing CD 179b in vitro.
[0115]
Since the anti-CD 179b antibody used in the present invention binds to a
CD 179b protein on cancer cells and exhibits an anti-tumor action due to the above
activities, the antibody is considered to be effective for therapy and/or prophylaxis of
cancer. That is, the present invention provides a pharmaceutical composition for
therapy and/or prophylaxis of cancer comprising as an effective component an anti-
CD 179b antibody. In cases where the anti-CD 179b antibody is used for the purpose
of administration to a human body (antibody therapy), the antibody is preferably
prepared as a human antibody or a humanized antibody in order to reduce its
immunogenicity.
[0116]
A higher binding affinity of the anti-CD 179b antibody to the CD 179b protein
on the cancer cell surface causes a stronger anti-tumor activity by the anti-CD 179b
antibody. Thus, if an anti-CD 179b antibody having a higher binding affinity to the
CD 179b protein can be obtained, a higher anti-tumor effect can be expected, and
therefore the antibody can be applied as a pharmaceutical composition for the
purpose of therapy and/or prophylaxis of cancer. In terms of the higher binding
affinity, the affinity constant Ka (Kon/Koff) is preferably at least 107 M'1, at least 108
M"1, at least 5*108 M"1, at least 109 M"\ at least 5*109 NT1, at least 1010 M"1, at least
5xl010 M"1, at least 1011 M"1, at least 5xl0n M"1, at least 1012 M"1, at least 1013 M"1,
as previously mentioned.
[0117]

The target of the pharmaceutical composition of the present invention for
therapy and/or prophylaxis of cancer is not restricted as long as it is a cancer (cell)
expressing the CD 179b gene, and preferably a cancer (cell) selected from the group
consisting of leukemia, lymphoma and breast cancer, including also mammary gland
cancer, combined mammary gland cancer, mammary gland malignant mixed tumor,
intraductal papillary adenocarcinoma, mastocytoma, chronic lymphocytic leukemia,
gastrointestinal lymphoma, digestive organ lymphoma and small/medium cell
lymphoma.
[0118]
Further, the antibody or a fragment thereof used in the present invention can
be used for therapy and/or prophylaxis of the above-described cancers.
[0119]
When the antibody used in the present invention is used as a pharmaceutical
composition, it can be formulated by a method known to those skilled in the art.
For example, it can be parenterally used in the form of an injection solution
containing a sterile solution or suspension prepared with another pharmaceutically
acceptable liquid. For example, the composition may be used in combination with a
pharmaceutically acceptable carrier(s) and/or medium/media, such as sterile water,
physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer,
flavoring agent, excipient, vehicle, antiseptic and/or binder, which is/are mixed into
the form of a unit dose required for carrying out formulation which is generally
accepted. The amount of the effective component in the formulation is determined
such that an appropriate volume is obtained within the prescribed range.
[0120]
The sterile composition for injection can be prescribed using a vehicle such as
distilled water for injection, according to a conventional formulation method.
[0121]
Examples of the aqueous solution include isotonic solutions containing
physiological saline, glucose and/or an adjunct(s) such as D-sorbitol, D-mannose, D-
mannitol and/or sodium chloride, which may be used in combination with an
appropriate solubilizer(s) such as an alcohol, in particular, ethanol; polyalcohol such
as propylene glycol; polyethylene glycol; nonionic surfactant such as polysorbate 80
(TM); and/or HCO-60.
[0122]
Examples of the oily liquid include sesame oils and soybean oils, which may
be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer.
Further, a buffering agent such as phosphate buffer or sodium acetate buffer;
soothing agent such as procaine hydrochloride; and/or stabilizer such as benzyl
alcohol, phenol or antioxidant may also be blended. The prepared injection solution
is usually filled into an appropriate ampoule.
[0123]
The administration is carried out orally or parenterally, preferably parenterally,
and particular examples thereof include the injection solution type, nasal
administration type, pulmonary administration type and percutaneous administration
type. Examples of the injection solution type include intravenous injection,
intramuscular injection, intraperitoneal administration and subcutaneous injection, by
which the injection solution can be administered systemically or topically.
[0124]
Further, the method of administration can be appropriately selected depending
on the age, symptom, sex and the like of the patient. The dose of the
pharmaceutical composition containing the antibody or a fragment thereof can be
selected within the range of, for example, 0.0001 mg to 1000 mg per 1 kg of the body
weight per one time. Alternatively, the dose can be selected within the range of
0.001 to 100000 mg/body per patient, but the dose is not restricted to these values.
The dose and the method of administration vary depending on the body weight, age,
symptom and the like of the patient, and those skilled in the art can appropriately
select them.
[0125]

The present invention further provides the following polypeptides and DNAs
related to the above antibody.
[0126]
(i) A polypeptide having the amino acid sequence shown in SEQ ID NO: 105,
and a DNA encoding the polypeptide.
[0127]
(ii) A polypeptide having the amino acid sequence shown in SEQ ID NO: 109,
and a DNA encoding the polypeptide.
[0128]
(iii) A DNA having the base sequence shown in SEQ ID NO:110.
[0129]
(iv) A DNA having the base sequence shown in SEQ ID NO: 111.
[0130]
(v) A heavy chain CDR polypeptide selected from the group consisting of the
amino acid sequences shown in SEQ ID NOs:103,104 and 102, and a DNA encoding
the polypeptide.
[0131]
(vi) A light chain CDR polypeptide selected from the group consisting of the
amino acid sequences shown in SEQ ID NOs:106,107 and 108, and a DNA encoding
the polypeptide.
[0132]
These polypeptides and DNAs may be prepared using the genetic
recombination technique as described above.
EXAMPLES
[0133]
The present invention will now be described concretely by way of Examples,
but the scope of the present invention is not restricted by these particular examples.
[0134]
Example 1: Identification of a Novel Cancer Antigen by the SEREX Method
(1) Preparation of a cDNA Library
From a canine mammary gland cancer tissue removed by surgery, total RNA
was extracted by the Acid guanidium-Phenol-Chloroform method, and poly(A)+
RNA was purified using the Oligotex-dT30 mRNA purification Kit (manufactured by
Takara Shuzo Co., Ltd.) according to the protocol described in the attached
instructions.
[0135]
Using the thus obtained mRNA (5 |xg), a canine mammary gland cancer-
derived cDNA phage library was synthesized. For preparation of the cDN A phage
library, cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit and ZAP-cDNA GigapacklH
Gold Cloning Kit (manufactured by STRATAGENE) were used according to the
protocols described in the attached instructions. The size of the prepared cDNA
phage library was 2.99x105 pfu/ml.
[0136]
(2) Screening of the cDNA Library by Serum
Using the canine mammary gland cancer-derived cDNA phage library
prepared as described above, immunoscreening was carried out. More particularly,
host E. coli (XL 1-Blue MRF') was infected with the library such that 2340 clones
were included in a 090x15 mm NZY agarose plate, followed by culture at 42°C for 3
to 4 hours to allow formation of plaques. The plate was covered with a
nitrocellulose membrane (Hybond C Extra; manufactured by GE Healthcare Bio-
Science) impregnated with IPTG (isopropyl-P-D-thiogalactoside) at 37°C for 4 hours,
to allow induction and expression of proteins, thereby transferring the proteins to the
membrane. Thereafter, the membrane was recovered and soaked in TBS (10 mM
Tris-HCl, 150 mM NaCl pH 7.5) supplemented with 0.5% non-fat dry milk, followed
by being shaken at 4°C overnight to suppress nonspecific reactions. This filter was
allowed to react with 500-fold diluted patient dog serum at room temperature for 2 to
3 hours.
[0137]
As the above-described patient dog serum, a total of 3 serum samples were
used which were collected from each of the dog from which the above mammary
gland cancer was removed and another mammary gland cancer patient dog. These
sera were stored at -80°C and pretreated immediately before use. The pretreatment
of the sera was carried out by the following method. That is, host E. coli (XL1-
BLue MRF) was infected with X ZAP Express phage into which no exogenous gene
was inserted, and cultured on a NZY plate at 37°C overnight. Subsequently, 0.2 M
NaHCO3 buffer (pH 8.3) containing 0.5 M NaCl was added to the plate, and the plate
was left to stand at 4°C for 15 hours, followed by recovering the supernatant as an E.
co/zVphage extract. Thereafter, the recovered E. co/i/phage extract was passed
through a NHS-column (manufactured by GE Healthcare Bio-Science) to immobilize
the proteins derived from the E. co///phage. The serum from the patient dog was
passed through this protein-immobilized column and allowed to react with the
proteins, thereby removing antibodies that adsorb to E. coli and the phage from the
serum. The serum fraction passed through the column without being adsorbed was
500-fold diluted with TBS supplemented with 0.5% non-fat dry milk, and the
resulting dilution was used as a material for the immunoscreening.
[0138]
The membrane to which the thus treated serum and the above-described
fusion proteins were blotted was washed with TBS-T (0.05% Tween 20/TBS) 4 times,
and a goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated; manufactured by
BETHYL Laboratories, Inc.) which was 5000-fold diluted with TBS supplemented
with 0.5% non-fat dry milk was allowed, as a secondary antibody, to react at room
temperature for 1 hour. Detection was carried out by an enzymatic coloring reaction
using the NBT/BCIP reaction solution (manufactured by Roche), and colonies whose
positions were identical to those of positive sites of the coloring reaction were
collected from the <15mm NZY agarose plate, and dissolved into 500 ul of SM
buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl, 0.01% gelatin, pH7.5).
The second and third screenings were carried out by repeating the same method as
described above until the colonies positive in the coloring reaction became single
colonies, thereby isolating 45 positive clones after screening of 92820 phage clones
reactive with IgG in the serum.
[0139]
Homology Search of the Isolated Antigen Genes
To subject the 45 positive clones isolated by the above method to sequence
analysis, an operation to convert the phage vector to a plasmid vector was carried out.
More particularly, 200 ul of a solution prepared such that the host E. coli (XL 1-Blue
MRF1) was contained to an absorbance OD600 of 1.0,250 ul of the purified phage
solution and 1 ul of ExAssist helper phage (manufactured by STRATAGENE) were
mixed together, and the resulting mixture was allowed to react at 37°C for 15
minutes, followed by adding 3 ml of LB broth thereto and culturing the resultant at
37°C for 2.5 to 3 hours. This was immediately followed by 20 minutes of
incubation in a water bath at 70°C and centrifugation at 1000xg for 15 minutes, after
which the supernatant was collected as a phagemid solution. Subsequently, 200 ul
of a solution prepared such that the phagemid host E. coli (SOLR) was contained to
an absorbance OD600 of 1.0 and 10 ul of the purified phagemid solution were mixed
together, and the resulting mixture was allowed to react at 37°C for 15 minutes,
followed by plating a 50 ul aliquot of the resultant on LB agar medium supplemented
with ampicillin (50 ug/ml final concentration) and culturing at 37°C overnight.
Single colonies of the transformed SOLR were picked up and cultured in LB medium
supplemented with ampicillin (50 |xg/ml final concentration) at 37°C, followed by
purifying plasmid DNAs having inserts of interest using QIAGEN plasmid Miniprep
Kit (manufactured by QIAGEN).
[0140]
Each purified plasmid was subjected to analysis of the full-length sequence of
the insert by the primer walking method using the T3 primer shown in SEQ ID
NO:94 and the T7 primer shown in SEQ ID NO:95. By this sequence analysis, the
gene sequences shown in the even number IDs of SEQ ID NOs:4 to 92 were obtained.
Using the base sequences and the amino acid sequences (odd number IDs of SEQ ID
NOs: 5 to 93) of these genes, homology search against known genes were carried out
using a homology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/),
and, as a result, it was revealed that all the obtained 45 genes were those encoding
CD 179b. The homologies among the 45 genes were 94 to 99% in terms of the base
sequences and 96 to 99% in terms of the amino acid sequences. The homologies
between these genes and the gene encoding a human homologous factor were 62 to
82% in terms of the base sequences and 69 to 80% in terms of the amino acid
sequences, in the region translated to a protein. The base sequence of the human
homologous factor is shown in SEQ ID NO:1, and the amino acid sequences of the
human homologous factor are shown in SEQ ID NOs:2 and 3.
[0141]
(4) Analysis of Gene Expression in Each Tissue
Expressions of the genes obtained by the above method in dog and human
normal tissues and various cell lines were investigated by the RT-PCR (Reverse
Transcription-PCR) method. The reverse transcription reaction was carried out as
follows. That is, from 50 to 100 mg of each tissue or 5-10><106 cells of each cell
line, total RNA was extracted using the TRIZOL reagent (manufactured by
INVITROGEN) according to the protocol described in the attached instructions.
Using this total RNA, cDNA was synthesized by the Superscript First-Strand
Synthesis System for RT-PCR (manufactured by INVITROGEN) according to the
protocol described in the attached instructions. As the cDNAs of human normal
tissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA
(manufactured by INVITROGEN), QUICK-Clone cDNA (manufactured by
CLONETECH) and Large-Insert cDNA Library (manufactured by CLONETECH)
were used. The PCR reaction was carried out as follows, using primers specific to
the obtained dog genes (shown in SEQ ID NOs:96 and 97) and their human
homologous gene (shown in SEQ ID NOs:98 and 99). That is, reagents and an
attached buffer were mixed such that concentrations/amounts of 0.25 ul of a sample
prepared by the reverse transcription reaction, 2 uM each of the above primers, 0.2
mM each of dNTPs, and 0.65 U ExTaq polymerase (manufactured by Takara Shuzo
Co., Ltd.) were attained in a total volume of 25 ul, and the reaction was carried out
by repeating 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30
seconds using a Thermal Cycler (manufactured by Bio-Rad Laboratories, Inc.). The
above-described primers specific to genes having the base sequences shown in SEQ
ID NOs: 96 and 97 were for amplification of the positions 32 to 341 in the base
sequence shown in SEQ ID NO:4, and for amplification of the region common to all
the dog CD 179b genes shown in the even number IDs of SEQ ID NOs: 4 to 92.
Further, the primers specific to genes having the base sequences shown in SEQ ID
NOs:98 and 99 were for amplification of the positions 216 to 738 in the base
sequence shown in SEQ ID NO:1. As a control for comparison, primers specific to
GAPDH (shown in SEQ ID NOs: 100 and 101) were used at the same time. As a
result, as shown in Fig. 1, the obtained dog genes did not show expression in normal
dog tissues at all, but showed strong expression in canine breast cancer tissues. In
terms of expression of the human homologous gene, bone marrow was the only
human normal tissue wherein its expression was confirmed, but, in human cancer
cells, its expression was detected in leukemia cell lines and breast cancer cell lines,
so that specific expression of CD 179b in the leukemia cell lines and the breast cancer
cell lines was confirmed.
[0142]
In Fig. 1, reference numeral 1 in the ordinate represents the expression pattern
of the gene identified as above, and reference numeral 2 represents the expression
pattern of the GAPDH gene as the control for comparison.
[0143]
(5) Analysis of Expression of the Antigen Protein on Cancer Cells
Subsequently, each cancer cell line wherein expression of the CD 179b gene
was confirmed was investigated for whether or not the CD 179b protein is expressed
on the cell surface. In a 1.5 ml microcentrifuge tube, 106 cells of each human
cancer cell line for which expression of the gene was observed were placed, which
tube was then centrifuged. To this tube, 5 ul of mouse anti-human CD 179b
antibody (clone name: GA170; manufactured by Santa Cruz Biotechnology) was
added, and the resultant was suspended in 95 ul of PBS supplemented with 0.1%
fetal calf serum, followed by leaving the resulting suspension to stand on ice for 1
hour. After washing the cells with PBS, the cells were suspended in 5 ul of FITC-
labeled rabbit anti-mouse IgG2a monoclonal antibody (manufactured by BD
Pharmingen) and 95 ul of PBS supplemented with 0.1% fetal bovine serum, and left
to stand on ice for 1 hour. After washing the cells with PBS, fluorescence intensity
was measured by FACSCalibur manufactured by Beckton Dickinson. On the other
hand, the same operation as described above was carried out to prepare the cells as a
control, using mouse IgG2a Isotype control (manufactured by MBL) instead of the
mouse anti-human CD 179b antibody. As a result, the cells to which the anti-human
CD 179b antibody was added showed a fluorescence intensity not less than 10%
higher than that of the control, and therefore it was confirmed that the CD 179b
protein is expressed on the cell membrane surface of the above human cancer cell
line.
[0144]
Example 2: Anti-tumor Effect, against Cancer Cells, of the Antibody against CD 179b
(1) The ADCC Activity
Thereafter, whether or not the antibody against CD 179b can damage tumor
cells expressing CD 179b was studied. The evaluation was carried out using a
commercially available mouse antibody against human CD 179b (clone name:
GA170). Into a 50 ml centrifuge tube, 106 cells belonging to each of the 3 types of
human leukemia cells, Namalwa, BDCM and RPMI1788 (all of these were
purchased from ATCC), whose expression of CD 179b was confirmed in Example
1(5), were collected, and 100 uCi of chromium 51 was added to the tube, followed by
incubation at 37°C for 2 hours. Thereafter, the cells were washed 3 times with
RPMI medium supplemented with 10% fetal calf serum, and placed in a 96-well V-
bottom plate in an amount of 103 cells/well. To each well, 1 ug of GA170 was
added, and 2x 105 lymphocytes separated from mouse spleen were further added
thereto, followed by culture under the conditions of 37°C, 5% CO2 for 4 hours.
Thereafter, the amount of chromium 51 in the culture supernatant released from
damaged tumor cells was measured, and the ADCC activity by GA170 against each
type of cancer cells was calculated. As a result, ADCC activities of 32.6%, 32.3%
and 28.3% were confirmed for Namalwa, BDCM and RPMI1788, respectively. On
the other hand, when an isotype control (clone name: 6H3) of GA170 was used for
the same operation, the above activity was not detected. Thus, it was revealed that,
by the ADCC activity induced using an antibody against CD 179b, tumor cells
expressing CD 179b can be damaged.
[0145]
The cytotoxic activity was obtained as a result of a process wherein the
antibody against CD 179b used in the present invention, mouse lymphocytes, and 103
cells of each leukemia cell line were mixed together, followed by culturing the cells
for 4 hours, measuring the amount of chromium 51 released into the medium after
the culture, and calculating the cytotoxic activity against the leukemia cell line
according to the following calculation equation*.
[0146]
*Equation: Cytotoxic activity (%) = the amount of chromium 51 released
from Namalwa, BDCM or RPMI1788 upon addition of the antibody against CD179b
and mouse lymphocytes / the amount of chromium 51 released from the target cells
upon addition of IN hydrochloric acid * 100.
[0147]
(2) The CDC Activity
Blood collected from a rabbit was placed in an Eppendorf tube, and left to
stand at room temperature for 60 minutes, followed by centrifugation at 3000 rpm for
5 minutes to prepare serum for measurement of the CDC activity. Into a 50 ml
centrifuge tube, 106 cells belonging to each of the 3 types of human leukemia cells,
Namalwa, BDCM and RPMI1788 were collected, and 100 uCi of chromium 51 was
added to the tube, followed by incubation at 37°C for 2 hours and washing the cells 3
times with RPMI medium supplemented with 10% fetal calf serum. Thereafter, the
cells were suspended in RPMI medium containing the rabbit serum prepared as
above in an amount of 50%, and placed in a 96-well V-bottom plate in an amount of
103 cells/well. To each well, 1 ug of GA170 was added, followed by culture under
the conditions of 37°C, 5% CO2 for 4 hours. Thereafter, the amount of chromium
51 in the culture supernatant released from damaged tumor cells was measured, and
the CDC activity by GA170 against each cancer cells was calculated. As a result,
CDC activities of 30.5%, 21.2% and 30.5% were confirmed for Namalwa, BDCM
and RPMI1788, respectively. On the other hand, when an isotype control (clone
name: 6H3) of GA170 was used for the same operation, the above activity was not
detected. Thus, it was revealed that, by the CDC activity induced using an antibody
against CD 179b, tumor cells expressing CD 179b can be damaged.
[0148]
The cytotoxic activity was obtained, as in the above (1), as a result of
calculation of the cytotoxic activity against each leukemia cell line according to the
following calculation equation*.
[0149]
?Equation: Cytotoxic activity (%) = the amount of chromium 51 released
from Namalwa, BDCM or RPMI1788 upon addition of the antibody against CD 179b,
and rabbit serum / the amount of chromium 51 released from the target cells upon
addition of IN hydrochloric acid x 100.
[0150]
Example 3: Preparation of a monoclonal antibody
(1) Preparation of an Antigen Protein
The human CD179b protein was prepared by the method of lipofection to animal cells.
The human CD179b gene (SEQ ID NO:22) was introduced to a vector encoding the
human IgGlFc
region, the SRalgGlFc vector, via restriction sites Xhol and BamHl. The SRalgG 1 Fc vector is a
vector prepared by introduction of the gene for the human IgGlFc region into the pcDL-SRa296
vector (manufactured by DNAX). Subsequently, 24 ug of the plasmid was mixed with 60 ul of
Lipofectamine 2000 (manufactured by Invitrogen), and OPTI-MEM (manufactured by Invitrogen)
was added to the resulting mixture to attain a total volume of 3 ml, followed by leaving the mixture
to stand at room temperature for not less than 20 minutes. To CHO-K1 cells preliminarily prepared
to 2*106 cells/12 ml OPTI=MEM, 3 ml of the above-mentioned mixed solution of the plasmid was
added, followed by 8 hours of culture under the conditions of 37°C and 5% CO2. The medium was
replaced with 10 ml of CHO-S-SFM medium (manufactured by Invitrogen), and culture was then
carried out for 4 to 5 days. Purification of the antigen protein produced in the obtained culture
supernatant was carried out using ProteinA sepharose HP (manufactured by GE Healthcare).
ProteinA sepharose HP was sufficiently equilibrated with 20 mM phosphate buffer (pH 7.4)/0.15 M
NaCl (equilibration buffer/washing buffer), and a solution prepared by mixing the culture
supernatant with the equilibration buffer at a ratio of 1:1 was introduced thereto. Subsequently, the
column was washed sufficiently with the washing buffer, and elution was carried out with 0.2 M
Glycine buffer (pH 2.5). The eluted solution was neutralized by addition of 1 M Tris (pH 9), and
the buffer was exchanged by ultrafiltration using 20 mM phosphate buffer (pH 7.4)/0.15 M NaCl, to
prepare the human CD 179b protein.
[0151]
(2) Obtaining Hybridomas
With an equal amount of the MPL+TDM adjuvant (manufactured by Sigma),
100 jig of the antigen protein (human CD179b protein) prepared in (1) was mixed, to
prepare an antigen solution for each individual of mouse. The antigen solution was
intraperitoneally administered to a Balb/c mouse (Japan SLC, Inc.) of 6 weeks old,
and 3 more times of administrations were then carried out at intervals of 1 week,
thereby completing immunization. Spleen removed 3 days after the last
immunization was placed between 2 sterile slide glasses and ground, followed i>y
repeating 3 times of operations wherein the cells were washed with PBS(-)
(manufactured by Nissui Pharmaceutical Co., Ltd.) and centrifuged at 1500 rpm for
10 minutes to remove the supernatant, thereby obtaining spleen cells. The obtained
spleen cells and mouse myeloma cells SP2/0 (purchased from ATCC) were mixed
together at a ratio of 10:1, and a PEG solution warmed to 37°C prepared by mixing
200 ul of RPMI1640 medium supplemented with 10% fetal calf serum and 800 ul of
PEG1500 (manufactured by Boehringer) together was added to the resulting mixture,
followed by leaving the mixture to stand for 5 minutes, thereby carrying out cell
fusion. The supernatant was removed by 5 minutes of centrifugation at 1700 rpm,
and the cells were suspended in 150 ml of RPMI1640 medium (HAT selection
medium) supplemented with 15% fetal calf serum, to which 2% equivalent of HAT
solution manufactured by Gibco was added. On each well of 15 96-well plates
(manufactured by Nunc), 100 ul of the cell suspension was seeded. The cells were
cultured for 7 days under the environment of 37°C, 5% CO2, to obtain hybridomas
produced by fusion of the spleen cells and the myeloma cells.
[0152]
(3) Selection of the Hybridomas
Using as indices the binding affinities, against the human CD 179b protein, of
the antibodies produced by the prepared hybridomas, hybridomas were selected. In
each well of a 96-well plate, 100 ul of 1 ug/ml solution of the human CD179b
protein prepared in the above (1) was placed, and the solution was left to stand at 4°C
for 18 hours. Each well was washed with PBS-T 3 times, and 400 ul of 0.5% BSA
(Bovine Serum Albumin) solution (manufactured by Sigma) was added to each well,
followed by leaving the plate to stand at room temperature for 3 hours. The solution
was removed, and the wells were washed 3 times with 400 ul/well of PBS-T,
followed by adding 100 ul/well of the culture supernatant of each of the hybridomas
obtained in the above (2) and leaving the plate at room temperature for 2 hours.
After washing the wells 3 times with PBS-T, 100 ul of an HRP-labeled anti-mouse
IgG (H+L) antibody (manufactured by Invitrogen) 5000-fold diluted with PBS was
added to each well, and the plate was left to stand at room temperature for 1 hour.
The wells were washed 3 times with PBS-T, and 100 ul of TMB substrate solution
(manufactured by Thermo) was added to each well, followed by leaving the plate to
stand for 15 to 30 minutes to carry out coloring reaction. After allowing coloration,
100 ul of 1 N sulfuric acid was added to each well to stop the reaction, and the
absorbance at 450 nm to 595 nm was measured using an absorption spectrometer.
As a result, hybridomas producing the antibodies showing the highest absorbance
was selected.
[0153]
The selected hybridomas were placed in a 96-well plate such that each well
contains 0.5 cell, and cultured. One week later, hybridomas forming single colonies
in the wells were observed. The cells in these wells were further cultured to obtain
60 cloned hybridoma cell lines.
[0154]
Subsequently, a hybridoma cell line was selected using as indices the binding
affinities, against leukemia cells, of the antibodies produced by the above 60
hybridoma cell lines. In each well of a 96-well plate, 100 ul of 1 mg/ml poly-L-
lysine (manufactured by Sigma)-PBS solution was placed, and the plate was left to
stand at room temperature for 30 minutes. After removing the poly-L-lysine-PBS
solution, an operation of filling sterile distilled water in each well and discarding it
was repeated 3 times, followed by air-drying of the plate in a clean bench.
Namalwa, a human leukemia cell line for which expression of CD 179b was
confirmed was suspended in PBS(-) such that a cell density of 106 cells/ml was
attained, and 100 ul of the resulting suspension was added to each well of the above
plate, followed by leaving the plate to stand at room temperature for 15 minutes.
After centrifugation at 1700 rpm for 5 minutes, the supernatant was removed, and
100 ul of 0.05% glutaraldehyde (manufactured by Sigma)-PBS solution was added to
each well, followed by leaving the plate to stand at room temperature for 10 minutes.
Each well was washed with PBS-T 3 times, and 300 ul of 0.5% BSA solution was
added to each well, followed by leaving the plate to stand at 4°C for 18 hours. After
washing the wells 3 times with PBS-T, 100 ul of the culture supernatant of each of
the 60 hybridoma cell lines obtained as above was added to the well, and the plate
was left to stand at room temperature for 2 hours. The supernatant was removed
and the wells were washed 3 times with PBS-T, followed by adding 100 ul of an
HRP-labeled anti-mouse IgG (H+L) antibody 5000-fold diluted with PBS to each
well and leaving the plate to stand at room temperature for 1 hour. The wells were
washed 3 times with PBS-T, and 100 ul of TMB substrate solution (manufactured by
Thermo) was added to each well, followed by leaving the plate to stand for 30
minutes to carry out coloring reaction. After allowing coloration, 100 ul of 1 N
sulfuric acid was added to each well to stop the reaction, and the absorbance at 450
nm to 595 nm was measured using an absorption spectrometer. As a result, the
hybridoma cell line #8, which produces the antibody showing the highest absorbance,
was selected.
[0155]
The isotype of the anti-CD 179b monoclonal antibody #8 produced by the
hybridoma cell strain #8 selected as described above was determined by the ELISA
method. The culture supernatant of the hybridoma cell strain #8 was evaluated with
the sub-isotyping kit (COSMO BIO Co., Ltd.) according to the protocols described in
the attached instructions, and, as a result, the anti-CD 179b monoclonal antibody was
revealed to be IgG3.
[0156]
Example 4: The Anti-tumor Effect of the Anti-CD179b Monoclonal Antibody #8
(1) Preparation of the Anti-CD 179b Monoclonal Antibody #8
The hybridoma cell strain #8 was cultured in Hybridoma SFM (manufactured
by Invitrogen). The culture fluid was centrifuged at 1500 rpm for 10 minutes, and
passed through a filter system 0.22 um. For purification of the antibody, a Hitrap
Protein A Sepharose FF (manufactured by GE Healthcare) column was used. The
column was washed with PBS for equilibration. Subsequently, the culture
supernatant was introduced to the column, followed by washing the column with
PBS. Elution was carried out with 0.1M Glycine-HCl (pH2.5) to obtain a purified
antibody.
[0157]
The Anti-tumor Effect in Vitro (on Cells)
The ADCC Activity
Whether or not the anti-CD 179b monoclonal antibody #8 can damage tumor
cells expressing human CD 179b was studied. Human leukemia cells Namalwa, for
which expression of human CD179b was confirmed, were collected into a 50 ml
centrifuge tube in an amount of 106 cells, and 10 uCi of chromium 51 was added to
the tube, followed by incubation at 37°C for 2 hours. Thereafter, the cells were
washed 3 times with RPMI medium supplemented with 10% fetal calf serum, and
placed in a 96-well V-bottom plate in an amount of 103 cells/well. To each well, 2
ug of the anti-CD 179b monoclonal antibody #8 was added, and 2><105 lymphocytes
separated from mouse spleen were further added thereto, followed by culture under
the conditions of 37°C, 5% CO2 for 4 hours. Thereafter, the amount of chromium
51 in the culture supernatant released from damaged tumor cells was measured, and
the ADCC activity by the anti-CD 179b monoclonal antibody #8 against the Namalwa
cells was calculated. As a result, an ADCC activity of 60.6% was confirmed for
Namalwa in each well. On the other hand, when an isotype control (clone name:
ME07) was used in a similar operation, the above activity was not detected. Thus,
it was revealed that the anti-CD179 monoclonal antibody #8 can damage tumor cells
expressing CD 179b by its ADCC activity.
[0158]
The CDC Activity
Blood collected from a rabbit was placed in an Eppendorf tube, and left to
stand at room temperature for 60 minutes, followed by centrifugation at 3000 rpm for
5 minutes to prepare serum for measurement of the CDC activity. Into a 50 ml
centrifuge tube, 106 cells of Namalwa, which are human leukemia cells, were
collected, and 100 uCi of chromium 51 was added to the tube, followed by
incubation at 37°C for 2 hours and washing the cells 3 times with RPMI medium
supplemented with 10% fetal calf serum. Thereafter, the cells were suspended in
RPMI medium containing the rabbit serum prepared as described above in an amount
of 50%, and placed in a 96-well V-bottom plate in an amount of 103 cells/well. To
each well, 2 ug of the anti-CD 179b monoclonal antibody #8 was added, followed by
culture under the conditions of 37°C, 5% CO2 for 4 hours. Thereafter, the amount
of chromium 51 in the culture supernatant released from damaged tumor cells was
measured, and the CDC activity by the anti-CD179b monoclonal antibody #8 against
the Namalwa cells was calculated. As a result, a CDC activity of 30.5% was
confirmed for Namalwa. On the other hand, when an isotype control (clone name:
ME07) was used in a similar operation, the above activity was not detected. Thus,
it was revealed that the anti-CD 179b monoclonal antibody #8 can damage tumor
cells expressing CD 179b by its CDC activity.
[0159]
(3) The Anti-tumor Effect in the Living Body of a Mouse
The anti-tumor activity of the anti-CD 179b monoclonal antibody #8 in the
living body of a tumor-bearing mouse was evaluated. The antibody used was
prepared by purifying the culture supernatant of the hybridoma cell strain #8 by a
column in the same manner as described above.
[0160]
Using a tumor-bearing mouse to which a cancer cell line derived from human
which expresses CD 179b was transplanted, the anti-tumor activity of the anti-
CD 179b monoclonal antibody #8 was evaluated. The Namalwa cells were
subcutaneously transplanted to the back of each of 20 nude mice (BALB/c Slc-nu/nu,
derived from Japan SLC, Inc.) in an amount of 106 cells, and the tumor was allowed
to grow to a size of about 7 mm in diameter. Among these mice, each of 10 tumor-
bearing mice was subjected to administration of 107 lymphocytes separated from
peripheral blood of BALB/c mice (BALB/c Cr Sic, derived from Japan SLC, Inc.)
and 300 ug of the anti-CD179b monoclonal antibody #8 from a caudal vein.
Thereafter, the same amounts of the mouse lymphocytes and the antibody were
administered to each tumor-bearing mouse from a caudal vein a total of 3 times in 2
days, and the size of the tumor was measured every day, thereby evaluating the anti-
tumor effect. On the other hand, to each of the remaining 10 tumor-bearing mice,
PBS(-) was administered instead of the above antibody, to provide a control group.
As a result of this study, in the group wherein the anti-CD 179b antibody was
administered, the tumor volume reduced to 65% on Day 8 with respect to the tumor
volume at the beginning of the administration of the antibody, which was defined as
100%. On Day 11, Day 17 and Day 20, the tumor regressed to 52%, 45% and 35%,
respectively (see Fig. 2). On the other hand, in the control group, on Day 8, Day 11,
Day 17 and Day 20, the tumor grew to about 180%, 220%, 350% and 420% (see Fig.
2). From these results, it was shown that the obtained anti-CD 179b monoclonal
antibody #8 exerts a strong anti-tumor effect in the living body, against cancer cells
expressing CD 179b. In terms of the size of the tumor, the volume was calculated
using the calculation equation: longer diameterxshorter diameterxshorter
diameterx0.5.
[0161]
Example 5: Cloning of the Gene for the Variable Region of the Anti-CD 179b
Monoclonal Antibody #8
From the hybridoma cell line #8, mRNA was extracted, and the genes for the
heavy chain variable (VH) region and the light chain variable (VL) region of the anti-
CD 179b monoclonal antibody #8 were obtained by the RT-PCR method using
primers specific to a mouse leader sequence and the antibody constant region of IgG3.
For determination of their sequences, these genes were cloned into the pCR2.1 vector
(manufactured by Invitrogen).
[0162]
(1) RT-PCR
From 106 cells of the hybridoma cell strain #8, mRNA was prepared using the
mRNA micro purification kit (manufactured by GE Healthcare), and the obtained
mRNA was reverse-transcribed to synthesize cDNA using the Superscript II 1 st
strand synthesis kit (manufactured by Invitrogen). These operations were carried
out according to the protocols described in the attached instructions of the respective
kits.
[0163]
Using the obtained cDNA, the antibody genes were amplified by the PCR
method. To obtain the gene for the VH region, a primer specific to the mouse
leader sequence (SEQ ID NO:112) and a primer specific to the mouse IgG3 constant
region (SEQ ID NO: 113) were used. Further, to obtain the gene for the VL region,
a primer specific to the mouse leader sequence (SEQ ID NO: 114) and a primer
specific to the mouse k chain constant region (SEQ ID NO: 115) were used. These
primers were designed referring to Jones ST and Bending MM Bio/technology 9, 88-
89 (1991). For the PCR, Ex Taq (manufactured by TAKARA BIO INC.) was used.
To 5 ul of IOxEX Taq Buffer, 4 ul of dNTP Mixture (2.5 mM), 2 ul each of the
primers (1.0 uM) and 0.25 ul of Ex Taq (5 units/ ul), a cDNA sample was added, and
sterile water was added to the resulting mixture to a total volume of 50 ul. The
reaction was carried out under the conditions of 2 minutes of treatment at 94°C
followed by 30 cycles of the combination of denaturation at 94°C for 1 minute,
annealing at 58°C for 30 seconds and the extension reaction at 72°C for 1 minute.
[0164]
(2) Cloning
Using each of the PCR products obtained as described above, electrophoresis
was carried out with agarose gel, and the DNA band corresponding to each of the VH
region and the VL region was excised. Each DNA fragment was processed using
the QIAquick Gel purification kit (manufactured by QIAGEN) according to the
protocol described in the attached instructions. Each purified DNA was cloned into
the pCR2.1 vector using the TA cloning kit (manufactured by Invitrogen). The
vector to which the DNA was linked was used for transformation of DH5a competent
cells (manufactured by TOYOBO) according to a conventional method. Ten clones
each of the transfonnants were cultured in a medium (100 ug/ml ampicillin) at 37°C
overnight, and each plasmid DNA was purified using the Qiaspin Miniprep kit
(manufactured by QIAGEN).
[0165]
(3) Determination of the Sequences
The analysis of the gene sequences of the VH region and the VL region was
carried out by analyzing the plasmid DNAs in (2) using the Ml 3 forward primer
(SEQ ID NO: 116) and the Ml3 reverse primer (SEQ ID NO:117), by a fluorescent
sequencer (DNA sequencer 3130XL manufactured by ABI), using the BigDye
Terminator Ver. 3.1 Cycle Sequencing kit according to the protocol in the attached
instructions. As a result, the respective gene sequences were determined (identical
among the 10 clones for each gene). The amino acid sequence of the VH region of
the anti-CD179b monoclonal antibody #8 is shown in SEQ ID NO:105, and the
amino acid sequence of the VL region of the antibody is shown in SEQ ID NO: 109.
INDUSTRIAL APPLICABILITY
[0166]
The antibody of the present invention is useful for therapy and/or prophylaxis
of cancer.
[0167]
SEQUENCE LISTING FREE TEXT
SEQ ID NOs:94, 96 to 99: primers
SEQ ID NO:95:T7 primer
SEQ ID NOs:100 and 101: GAPDH primers
SEQ ID NOs: 116 and 117: primers
We claim:
1. aA pharmaceutical composition for therapy and/or prophylaxis of cancer comprising as an
effective component an antibody or a fragment thereof, said antibody having immunoreactivity to a
CD179b protein having the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence
having a sequence identity of not less than 60% with the amino acid sequence, or to a fragment
thereof comprising not less than 7 continuous amino acids.
2. aThe pharmaceutical composition according to claim 1, wherein said cancer is breast cancer,
leukemia or lymphoma.
3. aThe pharmaceutical composition according to claim 1 or 2, wherein said antibody is a
monoclonal antibody or a polyclonal antibody.
4. aThe pharmaceutical composition according to any one of claims 1 to 3, wherein said
antibody is a human antibody, humanized antibody, chimeric antibody, single-chain antibody or
bispecific antibody.
5. aAn antibody comprising a heavy chain variable region having the amino acid sequences
shown in SEQ ID NOs:103,104 and 102 and a light chain variable region having the amino acid
sequences shown in SEQ ID NOs:106,107 and 108, said antibody having immunoreactivity to a
CD 179b protein.
6. aAn antibody comprising a heavy chain variable region having the amino acid sequence
shown in SEQ ID NO:105 and a light chain variable region having the amino acid sequence shown
in SEQ ID NO:109, said antibody having immunoreactivity to a CD179b protein.
7. aThe antibody according to claim 5 or 6, which is a humanized antibody, chimeric antibody,
single-chain antibody or bispecific antibody.
8. aThe pharmaceutical composition according to any one of claims 1 to 4, wherein said
antibody is the antibody according to any one of claims 5 to 7.
9. aA method of therapy and/or prophylaxis of cancer using the antibody or a fragment thereof
said antibody having immunoreactivity to a CD 179b protein
having the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having a
sequence identity of not less than 60% with the amino acid sequence, or to a fragment thereof
comprising not less than 7 continuous amino acids.
10. aA polypeptide having the amino acid sequence shown in SEQ ID NO: 105.
11. aA polypeptide having the amino acid sequence shown in SEQ ID NO: 109.
12. aA heavy-chain CDR polypeptide selected from the group consisting of the amino acid
sequences shown in SEQ ID NOs:103,104 and 102.
13. aA light-chain CDR polypeptide selected from the group consisting of the amino acid
sequences shown in SEQ ID NOs:106,107 and 108.
14. aA DNA encoding the polvpeDtide accordins to any one of claims 10ti 15.

An antibody or a fragment thereof having immunoreactivity to a polypeptide
comprising not less than 7 continuous amino acids in the CD 179b protein, which was
identified as a cancer antigen protein specifically expressed on the surfaces of cancer
cells, can be used as a pharmaceutical composition for therapy and/or prophylaxis of
cancer.