DESCRIPTION
Pharmaceutical Composition for Treating and/or Preventing Cancer
Technical Field
[0001]
The present invention relates to a novel pharmaceutical use of an
antibody against CAPRIN-1 or a fragment thereof, as an agent, for treating and/or
preventing a cancer.
Background Art
[0002]
Cancer is the leading cause of death. Currently conducted therapy
comprises mainly surgical therapy in combination with radiation therapy and
chemotherapy. In spite of the development of new operative procedures and the
discovery of new anticancer agents in recent years, cancer treatment results have
not been much improved recently, excluding that for some types of cancer.
Recent advances in molecular biology or cancer immunology lead to identification
of antibodies specifically reacting with cancer, cancer antigens to be recognized
by cytotoxic T cells, genes encoding cancer antigens, and the like. Demands on
specific cancer therapies targeting cancer antigens are increasing (Non-patent
Literature 1).
[0003]
In cancer therapy, it is desirable that peptides, polypeptides, or proteins
recognized as antigens be almost absent in normal cells, but they be present
specifically in cancer cells, in order to alleviate side effects. In 1991, Boon et al.,
(Ludwig Institute for Cancer Research, Belgium) isolated a human melanoma
antigen MAGE1 recognized by CD8-positive T cells by the cDNA expression
cloning method using autologous cancer cell lines and cancer-reactive T cells
(Non-patent Literature 2). Thereafter, the SEREX (serological identification of
antigens by recombinant expression cloning) method that comprises identifying
tumor antigens recognized by antibodies that are produced in vivo in response to
autologous cancer of a cancer patient by gene expression cloning techniques was
reported (Non-patent Literature 3 and Patent Literature 1). With the use of this
method, some cancer antigens, which are almost never expressed in normal cells
but are specifically expressed in cancer cells, were isolated (Non-patent
Literatures 4-9). Furthermore, clinical trials were conducted with cell therapies
targeting some cancer antigens using immunocytes specifically reactive with
cancer antigens, or cancer-specific immunotherapies using vaccines or the like
containing cancer antigens.
[0004]
Meanwhile, in recent years, various antibody medicines which target
antigenic proteins on cancer cells for cancer treatment have appeared throughout
the world. Antibody medicines exhibit some pharmacological effects as cancer
specific therapeutic agents and are thus attracting attention. However, most
antigen proteins to be targeted are also expressed in normal cells, so that not only
cancer cells, but also normal cells expressing antigens are also damaged as a result
of antibody administration. The resulting side effects cause for concern.
Therefore, it is expected that identification of cancer antigens that are specifically
expressed on the surface of a cancer cell and use of antibodies targeting the cancer
antigens as pharmaceuticals will realize treatment with antibody medicines with
lower side effects.
[0005]
Cytoplasmic- and proliferation-associated protein 1 (CAPRIN-1) is
expressed when normal cells at the resting phase are activated or undergo cell
division, and it is an intracellular protein known to form intracellular stress
granules with RNA within cells, so as to be involved in mRNA transport and
translational regulation. Meanwhile, many other names that represent CAPRIN-1
exist, such as GPI-anchored membrane protein 1 or membrane component surface
marker 1 protein (MUSI), as if such proteins had been known to be cell
membrane proteins. These names originated from a report that the gene sequence
of CAPRIN-1 is a membrane protein having a GPI-binding region and expressed in
colorectal cancer cells (Non-patent Literature 10). However, the gene sequence
of CAPRIN-1 provided in this report was later revealed to be wrong. The
following has recently been reported; i.e., deletion of a single nucleotide in the
gene sequence of CAPRIN-1 registered at GenBank or the like causes a frame shift,
so that 80 amino acids are lost from the C-terminus, resulting in generation of an
artifact (74 amino acids) which corresponds to the GPI-binding portion in the
previous report, and additionally, another error is also present 5' of the gene
sequence, so that 53 amino acids were lost from the N-terminus (Non-patent
Literature 11). It has been also recently reported that the protein encoded by the
gene sequence of CAPRIN-1 registered at GenBank or the like is not a cell
membrane protein (Non-patent Literature 11).
[0006]
In addition, on the basis of the report of Non-patent Literature 10 that
CAPRIN-1 is a cell membrane protein, Patent Literatures 2 and 3 describe that
CAPRIN-1 (as a cell membrane protein) under the name of MUSI can be used as
a target of an antibody medicine in cancer therapy, although working examples do
not describe treatment using an antibody against the protein. However, as
reported in Non-patent Literature 11, it has been commonly believed from the time
of the filing of Patent Literature 2 to date that CAPRIN-1 is not expressed on the
surface of a cell. The contents of Patent Literatures 2 and 3 based only on
incorrect information that CAPRIN-1 is a cell membrane protein should not
clearly be understood as common general knowledge for persons skilled in the art.
Prior Art Literature
Patent Literature
[0007]
Patent Literature 1: U.S. Patent No. 5698396
Patent Literature 2: US2008/0075722
Patent Literature 3: WO2005/100998
Non-patent Literature
[0008]
Non-patent Literature 1: Tsuyoshi Akiyoshi, "Gan To Kagaku-Ryoho (Cancer
and Chemotherapy)," 1997, Vol. 24, p551-519 (Cancer and Chemotherapy
Publishers, Inc., Japan)
Non-patent Literature 2 Bruggen P. et al., Science, 254: 1643-1647 (1991)
Non-patent Literature 3: Proc. Natl. Acad. Sci. U.S.A, 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. Genet6: 33-39, 1997
Non-patent Literature 10: J. Biol. Chem., 270: 20717-20723, 1995
Non-patent Literature 11: J. Immunol., 172: 2389-2400, 2004
Summary of the Invention
Problem to be Solved by the Invention
[0009]
Objects of the present invention are to identify a cancer antigen protein
specifically expressed on the surface of a cancer cell and to provide the use of an
antibody targeting the cancer antigen protein as an agent for treating and/or
preventing a cancer.
Means for Solving the Problem
[0010]
As a result of intensive studies, the present inventors have now obtained a
cDNA encoding a protein that binds to an antibody existing in sera from dogs with
breast cancer by the SEREX method using both cDNA libraries prepared from dog
testis tissues and sera of dogs with breast cancer. The present inventors have
now further prepared CAPRIN-1 proteins having the even-numbered amino acid
sequences of SEQ ID NOS: 2 to 30 and antibodies against such CAPRIN-1
proteins based on the obtained dog gene and the corresponding human, cattle,
horse, mouse, and chicken homologous genes. Thus, the present inventors have
now found that CAPRIN-1 is specifically expressed in breast cancer, brain tumor,
leukemia, lymphoma, lung cancer, uterine cervix cancer, bladder cancer,
esophageal cancer, colorectal cancer, gastric cancer, renal cancer, ovarian cancer,
prostate cancer, and fibrosarcoma, and that a portion of the CAPRIN-1 protein is
specifically expressed on the surface of each cancer cell. The present inventors
have thus now found that an antibody or antibodies against the portion of
CAPRIN-1 expressed on the surface of each cancer cell is/are cytotoxic to the
CAPRIN-1-expressing cancer cells. On the basis of these findings, the present
invention as described below was completed.
[0011]
The present invention has the following characteristics.
[0012]
The present invention provides a pharmaceutical composition for treating and/or
preventing a cancer, comprising an antibody or a fragment thereof as an active
ingredient having immunological reactivity with a partial polypeptide of
CAPRIN-1, wherein CAPRIN-1 is represented by any of the even-numbered
sequences of SEQ ID NOS: 2 to 30, and wherein the partial polypeptide comprises
the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence
having 80% or more sequence identity with the amino acid sequence of SEQ ID
NO: 37.
[0013]
In an embodiment, the above caner is breast cancer, brain tumor,
leukemia, lymphoma, lung cancer, uterine cervix cancer, bladder cancer,
esophageal cancer, colorectal cancer, gastric cancer, renal cancer, ovarian cancer,
prostate cancer, or fibrosarcoma.
[0014]
In another embodiment, the antibody is a monoclonal antibody or a
polyclonal antibody.
[0015]
In another embodiment, the antibody is a human antibody, humanized
antibody, chimeric antibody, single chain antibody, or bispecific antibody.
[0016]
This description includes all or part of the contents as disclosed in the
descriptions and/or drawings of Japanese Patent Application Nos. 2010-023454
and 2010-183162, from which the present application claims the priority.
Effects of the Invention
Effects of the Invention
[0017]
The antibody against CAPRIN-1 used in the present invention is
cytotoxic to cancer cells. As such, the antibody against CAPRIN-1 is useful for
treating or preventing cancers.
Brief Description of the Drawings
[0018]
Fig. 1 shows the expression patterns of genes encoding CAPRIN-1
proteins in normal tissues and tumor cell lines. Reference No. 1 indicates the
expression patterns of genes encoding CAPRIN-1 proteins, and Reference No. 2
indicates the expression patterns of GAPDH genes.
Fig. 2 shows the cytotoxicity to the MDA-MB-157 breast cancer cell line
expressing CAPRIN-1 by anti-CAPRIN-1 polyclonal antibodies that are reactive
with the surfaces of the cancer cells. Reference No. 3 indicates the activity
exhibited when the anti-CAPRIN-1 polyclonal antibody #1 was added.
Reference No. 4 indicates the activity exhibited when a control antibody from a
rabbit not immunized with an antigen was added. Reference No. 5 indicates the
activity exhibited when PBS was added instead of the antibodies.
Mode for Carrying Out the Invention
[0019]
The anti-tumor activity of an antibody against a polypeptide represented
by any of the even-numbered sequences of SEQ ID NOS: 2 to 30 used in the
present invention can be evaluated by examining in vivo suppression of tumor
growth in animals with cancer, or, examining whether or not the antibody exhibits
cytotoxicity via imrnunocytes or complements to tumor cells expressing the
polypeptide in vitro, as described later.
[0020]
In the context, the nucleotide sequences of polynucleotides encoding
proteins comprising the even-numbered amino acid sequences (i.e., SEQ ID NOS:
2, 4, 6, ... , 28, 30) of SEQ ID NOS: 2 to 30 are represented by the odd-numbered
sequences (i.e., SEQ ID NOS: 1, 3, 5, ... , 27, 29) of SEQ ID NOS: 1 to 29.
[0021]
The amino acid sequences that are represented by SEQ ID NOS: 6, 8, 10,
12, and 14 in the Sequence Listing disclosed herein are the amino acid sequences
of CAPRIN-1 isolated as polypeptides, which bind to antibodies specifically
existing in serum from a dog with cancer, through the SEREX method using a
cDNA library from dog testis tissue and the serum of a dog with breast cancer.
The amino acid sequences represented by SEQ ID NOS: 2 and 4 are the amino acid
sequences of CAPRIN-1 isolated as human homologues. The amino acid
sequence represented by SEQ ID NO: 16 is the amino acid sequence of CAPRIN-1
isolated as a cattle horaologue. The amino acid sequence represented by SEQ ID
NO: 18 is the amino acid sequence of CAPRIN-1 isolated as a horse homologue.
The amino acid sequences represented by SEQ ID NOS: 20 to 28 are the amino
acid sequences of CAPRIN-1 isolated as mouse homologues. The amino acid
sequence represented by SEQ ID NO: 30 is the amino acid sequence of CAPRIN-1
isolated as a chicken homologue (see Example 1 described later). CAPRIN-1 is
known to be expressed when normal cells in the resting phase are activated or give
rise to cell division.
[0022]
It was known that CAPRIN-1 was not expressed on cell surfaces.
However, as a result of the examination by the present inventors, it has now
revealed that a portion of the CAPRIN-1 protein is expressed on the surfaces of
various cancer cells. It has thus been now revealed that an antibody recognizing
a partial polypeptide of the CAPRIN-1 protein, which comprises the amino acid
sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or
more, preferably 85% or more, more preferably 90% or more, further preferably
95% or more sequence identity with the amino acid sequence of SEQ ID NO: 37,
exhibits anti-tumor activity. Examples of the antibody of the present invention
include all antibodies which bind to a fragment of the above CAPRIN-1 protein
and exhibit anti-tumor activity.
[0023]
The above-described anti-CAPRIN-1 antibody used in the present
invention may be any type of antibody as long as it can exhibit anti-tumor activity.
Examples of such antibodies include monoclonal antibodies, polyclonal antibodies,
recombinant antibodies, such as synthetic antibodies, multispecific antibodies,
humanized antibodies, chimeric antibodies, and single chain antibodies (scFv),
human antibodies, and fragments thereof, such as Fab, F(ab')2, and Fv. These
antibodies and fragments thereof can be prepared by methods known by persons
skilled in the art. In the present invention, antibodies having immunological
reactivity with CAPRIN-1 proteins or partial (poly)peptides thereof (that is,
binding to CAPRIN-1 proteins via antigen-antibody reaction) and preferably
antibodies capable of specifically binding to CAPRIN-1 proteins are desired.
Preferably, they are monoclonal antibodies. Polyclonal antibodies may also be
used as long as homogenous antibodies can be stably produced. Also, when a
subject is a human, human antibodies or humanized antibodies are desired in order
to avoid or suppress rejection. The term "specifically binding to a CAPRIN-1
protein" as used herein means that the antibody specifically binds to a CAPRIN-1
protein, but does not substantially bind to proteins other than the CAPRIN-1
protein."
[0024]
The anti-tumor activity of an antibody that can be used in the present
invention can be evaluated as described below by examining in vivo the
suppression of the tumor growth in animals with cancer, or, by examining whether
or not it exhibits in vitro an activity of cytotoxicity, which is mediated by
immunocytes or complements, to tumor cells expressing the polypeptide.
[0025]
Furthermore, examples of the subject for cancer treatment and/or
prevention in the present invention include mammals, such as humans, pet animals,
domestic animals, and animals for competition. A preferable subject is a human.
[0026]
Preparation of antigens and antibodies and pharmaceutical compositions
relating to the present invention are described below.
[0027]

Proteins or fragments thereof to be used as sensitizing antigens for
obtaining anti-CAPRIN-1 antibodies used in the present invention may be derived
from any animal species without particular limitation, such as humans, dogs, cattle,
horses, mice, rats, and chickens. However, proteins or fragments thereof are
preferably selected in consideration of compatibility with parent cells used for cell
fusion. In general, mammal-derived proteins are preferred and, in particular,
human-derived protein is preferred. For example, when CAPRIN-1 is human
CAPRIN-1, the human CAPRIN-1 protein, a partial peptide thereof, or cells
expressing human CAPRIN-1 can be used.
[0028]
The nucleotide sequences and the amino acid sequences of human
CAPRIN-1 and homologues thereof can be obtained by accessing GenBank (NCBI,
U.S.A.) and using an algorithm such as BLAST or FASTA (Karlin and Altschul,
Proc. Natl. Acad. Sci. U.S.A., 90: 5873-5877, 1993; Altschul et al., Nucleic Acids
Res. 25: 3389-3402, 1997).
[0029]
In the present invention, on the basis of the nucleotide sequence (SEQ
ID NO: 1 or 3) or the amino acid sequence (SEQ ID NO: 2 or 4) of human
CAPRIN-1, a target nucleic acid or a target protein comprises a sequence having
70% to 100%, preferably 80% to 100%, more preferably 90% to 100%, even more
preferably 95% to 100% (e.g., 97% to 100%, 98% to 100%, 99% to 100%, or
99.5% to 100%>) sequence identity with the nucleotide sequence or the amino acid
sequence of the ORF or the mature portion of human CAPRIN-1. As use herein,
the term "% sequence identity" refers to a percentage (%) of identical amino acids
(or nucleotides) relative to the total number of amino acids (or nucleotides), when
two sequences are aligned to achieve the highest similarity with or without
introduction of gaps.
[0030]
The length of a fragment of CAPRIN-1 protein ranges from the amino
acid length of an epitope (antigenic determinant), which is the minimum unit
recognized by an antibody, to a length less than the full length of the protein.
The term "epitope" refers to a polypeptide fragment having antigenicity or
immunogenicity in mammals, preferably in humans, and the minimum unit of the
epitope consists of about 7 to 12 amino acids, for example 8 to 11 amino acids.
Therefore, the antibody of the present invention is characterized by recognizing a
fragment consisting of about 7 to 12 amino acids (e.g., 8 to 11 amino acids) in the
amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence
having 80% or more, preferably 85% or more, more preferably 90% or more,
further preferably 95% or more sequence identity with the amino acid sequence of
SEQ ID NO: 37.
[0031]
The polypeptides comprising the above-mentioned human CAPRIN-1
protein or partial peptides of the protein, can be synthesized by a chemical
synthesis method, such as the Fmoc method (fluorenylmethyloxycarbonyl method)
or the tBoc method (t-butyloxycarbonyl method) (Edited by The Japanese
Biochemical Society, Seikagaku Jikken Koza (Biochemical Experimental Lecture
Series) 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis,
TOKYO KAGAKU DOZIN (Japan), 1981). Alternatively, the above-mentioned
polypeptides may also be synthesized by conventional methods using various
commercially available peptide synthesizers. Furthermore, with the use of
known genetic engineering techniques (e.g., Sambrook et al., Molecular Cloning,
2nd Edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor
Laboratory Press, Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition,
A compendium of Methods from Current Protocols in Molecular Biology (1995),
John Wiley & Sons), a polynucleotide encoding the above polypeptide is prepared
and then incorporated into an expression vector, which is subsequently introduced
into a host cell in order to produce a polypeptide of interest in the host cell, and
then recover it.
[0032]
The polynucleotides encoding the above polypeptides can be easily
prepared by known genetic engineering techniques or conventional techniques
using a commercially available nucleic acid synthesizer. For example, DNA
comprising the nucleotide sequence of SEQ ID NO: 1 can be prepared by PCR
using a human chromosomal DNA or cDNA library, as a template, and a pair of
primers designed to be able to amplify the nucleotide sequence represented by
SEQ ID NO: 1. PCR conditions can be appropriately determined. For example,
PCR conditions comprise conducting 30 cycles of the reaction cycle of:
denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds to 1 minute;
and extension at 72°C for 2 minutes, using a thermostable DNA polymerase (e.g.,
Taq polymerase or Pfu polymerase) and PCR buffer containing Mg2+, followed by
reacting at 72°C for 7 minutes. However, the PCR conditions are not limited to
the above example. PCR techniques, conditions, and the like are described in
Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A compendium
of Methods from Current Protocols in Molecular Biology (1995), John Wiley &
Sons (particularly Chapter 15).
[0033]
Also, on the basis of the nucleotide sequence and amino acid sequence
information represented by SEQ ID NOS: 1 to 30 in the Sequence Listing
described herein, appropriate probes or primers are prepared, and then a cDNA
library of a human or the like is screened using them, so that desired DNA can be
isolated. A cDNA library is preferably constructed from cells, organs or tissues,
which express proteins having even-numbered sequences of SEQ ID NOS: 2 to 30.
Examples of such cells or tissues include cells or tissues derived from testis, and
cancers or tumors, such as leukemia, breast cancer, lymphoma, brain tumor, lung
cancer, colorectal cancer, and the like. Procedures such as the preparation of
probes or primers, construction of a cDNA library, screening of a cDNA library,
and cloning of target genes are known by a person skilled in the art and can be
carried out by the methods described in Sambrook et al., Molecular Cloning, 2n
Edition, Current Protocols in Molecular Biology (1989), Ausbel et al., (above),
and the like. DNA encoding a human CAPRIN-1 protein or a partial peptide
thereof can be obtained from the thus obtained DNA.
[0034]
The host cells may be any cells, as long as they can express the
above-mentioned polypeptide. Examples of prokaryotic cells include, but are not
limited to, Escherichia coli and the like. Examples of eukaryotic cells include,
but are not limited to, mammalian cells, such as monkey kidney cells (COS1) and
Chinese hamster ovary cells (CHO), human fetal kidney cell line (HEK293), fetal
mouse skin cell line (NIH3T3), yeast cells such as budding yeast and fission yeast,
silkworm cells, and Xenopus oocyte.
[0035]
When prokaryotic cells are used as host cells, an expression vector used
herein contains an origin replicable within prokaryotic cells, a promoter, a
ribosome-binding site, a multiple cloning site, a terminator, a drug resistance gene,
an auxotrophic complementary gene, and the like. Examples of Escherichia coli
expression vector include a pUC-based vector, pBluescript II, a pET expression
system, and a pGEX expression system. DNA encoding the above polypeptide is
incorporated into such an expression vector, prokaryotic host cells are transformed
with the vector, the thus obtained transformed cells are cultured, and thus the
polypeptide encoded by the DNA can be expressed in prokaryotic host cells. At
this time, the polypeptide can also be expressed as a fusion protein with another
protein.
[0036]
When eukaryotic cells are used as host cells, an expression vector used
herein is an expression vector for eukaryotic cells, which contains a promoter, a
splicing region, a poly(A) addition site, and the like. Examples of such an
expression vector include pKAl, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV,
pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2. In a manner similar to the
above, DNA encoding the above polypeptide is incorporated into such an
expression vector, eukaryotic host cells are transformed with the vector, the thus
obtained transformed cells are cultured, and thus the polypeptide encoded by the
DNA can be expressed in eukaryotic host cells. When pIND/V5-His,
pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, or the like is used as an expression vector,
the above polypeptide can be expressed as a fusion protein to which a tag from
among various tags such as a His tag (e.g., (His)6-(His)10), a FLAG tag, a myc tag,
an HA tag, and GFP has been added.
[0037]
For introduction of an expression vector into host cells, a known method
can be employed, such as electroporation, a calcium phosphate method, a liposome
method, a DEAE dextran method, microinjection, viral infection, lipofection, and
binding to a cell membrane-permeable peptide.
[0038]
The polypeptide of interest can be isolated and purified from host cells
by a combination of known separation procedures. Examples of such procedures
include, but are not limited to, treatment with a denaturing agent such as urea or a
surfactant, ultrasonication, enzymatic digestion, salting-out or solvent
fractionation and precipitation, dialysis, centrifugation, ultrafiltration, gel
filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography,
hydrophobic chromatography, affinity chromatography, and reverse phase
chromatography.
[0039]

An antibody is a heteromultimeric glycoprotein that generally contains
at least two heavy chains and two light chains. Antibodies other than IgM is an
antibody is an about 150-kDa heterotetramer glycoprotein composed of two
identical light (L) chains and two identical heavy (H) chains. Typically, each
light chain is connected to a heavy chain via one disulfide covalent bond, however,
the number of disulfide bonds between heavy chains of various immunoglobulin
isotypes is varied. Each heavy chain or each light chain also has an intrachain
disulfide bond. Each heavy chain has a variable domain (VH region) on one end
followed by several constant regions. Each light chain has a variable domain
(VL region) and has one constant region on an end opposite to the other end. The
constant region of a light chain is aligned with the first constant region of a heavy
chain, and a light chain variable domain is aligned with a heavy chain variable
domain. A specific region of an antibody variable domain exhibits specific
variability that is referred to as a complementarity determining region (CDR), so
that it imparts binding specificity to the antibody. A portion of a variable region,
which is relatively conserved, is referred to as a framework region (FR).
Complete heavy chain and light chain variable domains separately contains four
FRs ligated via three CDRs. The three CDRs in a heavy chain are referred to as
CDRH1, CDRH2, and CDRH3 in this order from the N-terminus. Similarly, in
the case of a light chain, CDRLs are referred to as CDRL1, CDRL2, and CDRL3.
CDRH3 is most important for the binding specificity of an antibody to an antigen.
Also, the CDRs of each chain are retained together in a state of being adjacent to
each other due to the FR regions, contributing to the formation of the antigen
binding site of the antibody together with CDRs from the other chain. A constant
region does not directly contribute to the binding of an antibody to an antigen, but
exhibits various effector functions, such as involvement in antibody-dependent
cell-mediated cytotoxicity (ADCC), phagocytosis via binding to an Fey receptor,
the rate of half-life/clearance via a neonate Fc receptor (FcRn), and
complement-dependent cytotoxicity (CDC) via a Clq constituent of the
complement cascade.
[0040]

The term "anti-CAPRIN-1 antibody" as used herein refers to an antibody
having immunological reactivity with a full-length CAPRIN-1 protein or a
fragment thereof.
[0041]
As used herein, the term "immunological reactivity" refers to the property
of in vivo binding of an antibody to a CAPRIN-1 antigen. Through such an in
vivo binding, the function of damaging tumor (e.g., death, suppression, or
degeneration) is exhibited. Specifically, an antibody used in the present
invention may be any type of antibody, as long as it binds to a CAPRIN-1 protein
so as to be able to damage tumor, such as leukemia, lymphoma, breast cancer,
brain tumor, lung cancer, esophageal cancer, gastric cancer, renal cancer,
colorectal cancer, ovarian cancer, prostate cancer, or fibrosarcoma.
[0042]
Examples of an antibody include a monoclonal antibody, a polyclonal
antibody, a synthetic antibody, a multispecific antibody, a human antibody, a
humanized antibody, a chimeric antibody, a single chain antibody, and an antibody
fragment (e.g., Fab and F(ab')2). Also, an antibody may be an immunoglobulin
molecule of any class such as IgG, IgE, IgM, IgA, IgD, or IgY, or any subclass
such as IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.
[0043]
The antibody may further be modified by, in addition to glycosylation,
acetylation, formylation, amidation, phosphorylation, pegylation (PEG), or the
like.
[0044]
Various antibody preparation examples are as described below.
[0045]
When the antibody is a monoclonal antibody, for example, the breast
cancer cell line SK-BR-3 expressing CAPRIN-1 is administered to a mouse for
immunization, the spleen is removed from the mouse, cells are separated, and then
the cells and mouse myeloma cells are fused. From among the thus obtained
fusion cells (hybridomas), a clone producing an antibody having the effect of
suppressing cancer cell proliferation is selected. A hybridoma producing a
monoclonal antibody that has the effect of suppressing cancer cell proliferation is
isolated, the hybridoma is cultured, and then an antibody is purified from the
culture supernatant by general affinity purification, so that the antibody can be
prepared.
[0046]
The hybridoma producing a monoclonal antibody can also be prepared as
described below, for example. First, an animal is immunized with a sensitizing
antigen according to a known method. A general method is carried out by
injecting a sensitizing antigen to a mammal intraperitoneally or subcutaneously.
Specifically, a sensitizing antigen is diluted with PBS (Phosphate-Buffered Saline),
saline, or the like to an appropriate amount, followed by suspension. The
resultant is then mixed with an appropriate amount of a general adjuvant as
necessary, such as Freund's complete adjuvant. After emulsification, the solution
was administered to a mammal several times every 4 to 21 days. Furthermore, an
appropriate carrier can also be used upon immunization with a sensitizing antigen.
[0047]
A mammal is immunized as described above. After confirmation of a
rise in a desired serum antibody level, immunized cells are collected from the
mammal and then subjected to cell fusion. Preferable immunized cells are
particularly splenocytes.
[0048]
Mammalian myeloma cells are used as the other parent cells to be fused
with the immunized cells. As the myeloma cells, various known cell lines are
preferably used, such as P3U1 (P3-X63Ag8Ul), P3 (P3x63Ag8. 653) (J. Immunol.
(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).
[0049]
Fusion of the immunized cell and the myeloma cell can be carried out
according to basically a known method such as Kohler and Milstein's technique
(Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46), for example.
[0050]
More specifically, the above cell fusion is carried out, for example, in
the presence of a cell fusion accelerator in a usual nutrient culture medium. As
this fusion accelerator, polyethyleneglycol (PEG), Sendai virus (HVJ), or the like
is used. If desired, an auxiliary agent such as dimethylsulfoxide may be added
and used in order to enhance fusion efficiency.
[0051]
The ratio of the immunized cells to the myeloma cells to be used herein
can be arbitrarily set. For example, the number of immunized cells that are
preferably used is one to ten times the number of myeloma cells. As a culture
medium to be used for the above-mentioned cell fusion, an RPMI1640 culture
medium suitable for proliferation of the above-mentioned myeloma cell line, an
MEM culture medium, and other culture media usually used for culturing this kind
of cell can be used. Further, liquid that is supplemental to serum such as fetal
bovine serum (FCS) can be used together therewith.
[0052]
Cell fusion can be performed by thoroughly mixing the predetermined
amounts of the above immunized cells and the myeloma cells in the above culture
medium, and a PEG solution (for example, having an average molecular weight
ranging from about 1000 to 6000) prewarmed at about 37°C is added usually at a
concentration of 30%-60% (w/v) and mixed, thereby forming a culture containing
hybridomas of interest. Next, a suitable culture medium is successively added to
the thus-obtained culture, which is then centrifuged to remove the supernatant, and
this procedure is repeated to remove the cell fusion agent or the like which is not
preferable for the growth of hybridomas.
[0053]
The thus obtained hybridomas are cultured for selection in a usual
selection culture medium (e.g., a HAT culture medium containing hypoxanthine,
aminopterin and thymidine). Culturing in this HAT culture medium is continued
for a sufficient period of time (usually several days to several weeks) so that the
cells (non-fused cells) other than the target hybridomas die. Subsequently,
screening and single cloning of the hybridoma which produces an antibody of
interest are performed using the general limiting dilution method.
[0054]
The above hybridomas are obtained by an immunizing non-human
animal with an antigen. In addition to this method, hybridomas that produce a
human antibody having desired activity (e.g., activity of suppressing cell
proliferation) can also be obtained by in vitro sensitizing human lymphocytes,
such as human lymphocytes that have been infected with the EB virus, with a
protein, a protein-expressing cell, or a lysate thereof, followed by fusing of the
thus sensitized lymphocytes with human-derived myeloma cells having an ability
to permanently divide, such as U266 (registration no. TIB 196).
[0055]
The thus prepared hybridoma that produces a monoclonal antibody of
interest can be passaged in a general culture medium and can be stored in liquid
nitrogen over a long period of time.
[0056]
Specifically, a hybridoma can be prepared by immunizing by a general
immunization method using, as a sensitizing antigen, a desired antigen or a cell
that expresses the desired antigen, fusing the thus obtained immunized cell with a
known parent cell by a general cell fusion method, and then screening for a
monoclonal antibody-producing cell (i.e., a hybridoma) by a general screening
method.
[0057]
Another example of an antibody that can be used in the present invention
is a polyclonal antibody. A polyclonal antibody can be obtained as described
below, for example.
[0058]
A small animal, such as a mouse, a human antibody-producing mouse, or
a rabbit, is immunized with a natural CAPRIN-1 protein, a recombinant
CAPRIN-1 protein expressed in a microorganism such as Escherichia coli in the
form of a fusion protein with GST or the like, or a partial peptide thereof, and then
serum is obtained. The serum is purified by ammonium sulfate precipitation,
protein A column, protein G column, DEAE ion exchange chromatography,
affinity column to which a CAPRIN-1 protein or a synthetic peptide has been
coupled, or the like, so that a polyclonal antibody can be prepared.
[0059]
As a human antibody-producing mouse, a KM mouse (Kirin
Pharma/Medarex) and a Xeno mouse (Amgen) are known (e.g., International
Patent Publications WO02/43478 and WO02/092812), for example. When such a
mouse is immunized with a CAPRIN-1 protein or a fragment thereof, a complete
human polyclonal antibody can be obtained from blood. Also, splenocytes are
collected from the immunized mouse and then a human-type monoclonal antibody
can be prepared by a method for fusion with myeloma cells.
[0060]
An antigen can be prepared according to a method using animal cells (JP
Patent Publication (Kohyo) No. 2007-530068) or baculovirus (e.g., International
Patent Publication W098/46777), for example. When an antigen has low
immunogenicity, the antigen may be bound to a macromolecule having
immunogenicity, such as albumin, and then immunization is carried out.
[0061]
Furthermore, an antibody gene is cloned from said hybridoma and then
incorporated into an appropriate vector. The vector is then introduced into a host,
and then the genetically recombined antibody produced using gene recombination
techniques can be used (e.g., see Carl, A. K. Borrebaeck, James, W. Larrick,
THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United
Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, the cDNA
of a variable region (V region) of an antibody is synthesized from the mRNA of
the hybridoma using reverse transcriptase. When DNA encoding the V region of
an antibody of interest can be obtained, this DNA is ligated to DNA encoding the
constant region (C region) of a desired antibody, and then the resultant fusion
product is incorporated into an expression vector. Alternatively, DNA encoding
the V region of an antibody may be incorporated into an expression vector
containing the DNA for the C region of an antibody. At this time, the DNA can
be incorporated into an expression vector so that it is expressed under the control
of expression control regions, such as enhancer and promoter. Next, host cells
are transformed with the expression vector, so that the antibody can be expressed.
[0062]
The anti-CAPRIN-1 antibody of the present invention is preferably a
monoclonal antibody. However, the anti-CAPRIN-1 antibody may also be a
polyclonal antibody or a genetically-modified antibody (e.g., a chimeric antibody
or a humanized antibody), for example.
[0063]
Examples of a monoclonal antibody include human monoclonal
antibodies, non-human animal monoclonal antibodies (e.g., a mouse monoclonal
antibody, a rat monoclonal antibody, a rabbit monoclonal antibody, and a chicken
monoclonal antibody), and chimeric monoclonal antibodies. A monoclonal
antibody can be prepared by culturing a hybridoma obtained by cell fusion of a
splenocyte from a non-human mammal (e.g., a mouse, a human
antibody-producing mouse, a chicken, or a rabbit) immunized with a CAPRIN-1
protein, with a myeloma cell. A chimeric antibody is prepared by combining
sequences from different animals, such as an antibody comprising heavy chain and
light chain variable regions of a mouse antibody and heavy chain and light chain
constant regions of a human antibody. A chimeric antibody can be prepared
using a known method. For example, a chimeric antibody can be obtained by
ligating DNA encoding an antibody V region to DNA encoding a human antibody
C region, incorporating the resultant fusion product into an expression vector, and
then introducing the vector into a host for production of the chimeric antibody.
[0064]
In Examples described later, monoclonal antibodies having
immunological reactivity with a partial polypeptide of CAPRIN-1 were prepared,
wherein CAPRIN-1 is represented by any of the even-numbered sequences of SEQ
ID NOS: 2 to 30, and wherein the partial polypeptide comprises the amino acid
sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or
more sequence identity with the amino acid sequence of SEQ ID NO: 37. The
anti-tumor effects of the monoclonal antibodies were confirmed. These
monoclonal antibodies comprise a heavy chain variable (VH) region comprising
the amino acid sequence of SEQ ID NO: 43 or 63 and a light chain variable (VL)
region comprising the amino acid sequence of SEQ ID NO: 47, 51, or 67, wherein:
the VH region comprises CDR1 represented by the amino acid sequence of SEQ ID
NO: 40 or 60, CDR2 represented by the amino acid sequence of SEQ ID NO: 41 or
61, and CDR3 represented by the amino acid sequence of SEQ ID NO: 42 or 62;
and the VL region comprises CDR1 represented by the amino acid sequence of
SEQ ID NO: 44, 48, or 64, CDR2 represented by the amino acid sequence of SEQ
ID NO: 45, 49, or 65, and CDR3 represented by the amino acid sequence of SEQ
ID NO: 46, 50, or 66.
[0065]
Examples of a polyclonal antibody include an antibody obtained by
immunizing a human antibody-producing animal (e.g., a mouse) with a CAPRIN-1
protein.
[0066]
A humanized antibody is a modified antibody that is also referred to as a
reshaped human antibody. A humanized antibody can be constructed by
transplanting CDRs of an antibody from an immunized animal into the
complementarity determining regions of a human antibody. General gene
recombination techniques therefor are also known.
[0067]
Specifically, DNA sequences designed to have each of the CDRs of a
mouse or chicken antibody ligated to each of the framework regions (FRs) of a
human antibody are synthesized by the PCR method from several oligonucleotides,
which are prepared so as to have overlap portions at their terminal portions, for
example. A humanized antibody can be obtained by ligating the thus obtained
DNA to DNA encoding the constant region of a human antibody, incorporating the
resultant fusion product into an expression vector, introducing the vector into a
host, and thus causing the host to produce the gene product (see European Patent
Publication No. 239400 and International Patent Publication WO96/02576). As
the FRs of a human antibody, which is ligated via CDRs, FRs that allow the
formation of an antigen-binding site with good complementarity determining
regions are selected. If necessary, for the formation of an antigen-binding site
having the appropriate complementarity determining regions of a reshaped human
antibody, the amino acids of the framework regions of an antibody variable region
may be substituted (Sato, K. et al., Cancer Research, 1993, 53: 851-856). Also,
the amino acids of FRs may be substituted with those of framework regions from
various human antibodies (see International Patent Publication W099/51743).
[0068]
As the framework regions (FRs) of a human antibody, which are ligated
via CDRs, FRs that allows the formation of an antigen-binding site with good
complementarity determining regions are selected. If necessary, for the
formation of an antigen-binding site having the appropriate complementarity
determining regions of a reshaped human antibody, the amino acids of the
framework regions of an antibody variable region may be substituted (Sato K. et
al., Cancer Research 1993, 53: 851-856).
[0069]
After preparation of a chimeric antibody or a humanized antibody, amino
acids in a variable region (e.g., FR) or a constant region may be substituted with
other amino acids.
[0070]
Amino acid substitution is a substitution of, for example, less than 15,
less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less
amino acids and is preferably a substitution of 1 to 5 amino acids, and more
preferably 1 or 2 amino acids. A substituted antibody should be functionally
equivalent to an unsubstituted antibody. Substitution is desirably a substitution
of a conservative amino acid(s) between amino acids having analogous properties
such as electric charge, side chain, polarity, and aromaticity. Amino acids having
analogous properties can be classified 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), for example.
[0071]
Examples of a modified antibody product include antibodies bound to
various molecules such as polyethylene glycol (PEG). Substances to be bound in
the modified antibody product of the present invention are not limited. Such a
modified antibody product can be obtained by subjecting the thus obtained
antibody to chemical modification. Methods therefor have already been
established in the art.
[0072]
As used herein, the term "functionally equivalent" refers to that a
subject antibody has biological or biochemical activity similar to that of the
antibody of the present invention, and specifically refers to that a subject antibody
has the function of impairing tumor without essentially causing rejection upon its
application to a human, for example. An example of such activity includes an
activity to suppress cell proliferation or a binding activity.
[0073]
As a method well known by persons skilled in the art for preparation of a
polypeptide functionally equivalent to a polypeptide, a method for introducing a
mutation into a polypeptide is known. For example, persons skilled in the art can
prepare an antibody functionally equivalent to the antibody of the present
invention by appropriately introducing a mutation into the antibody using
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.
U.S.A. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766), for
example.
[0074]
An antibody that recognizes an epitope of a CAPRIN-1 protein
recognized by the above anti-CAPRIN-1 antibody can be obtained by a method
known by persons skilled in the art. For example, such an antibody can be
obtained by a method that involves determining an epitope of a CAPRIN-1 protein
recognized by an anti-CAPRIN-1 antibody, by a general method (e.g., epitope
mapping) and then preparing an antibody using a polypeptide having an amino
acid sequence contained in the epitope as an immunogen, or a method that
involves determining an epitope of such an antibody prepared by a general method,
and then selecting an antibody having the epitope identical with that of an
anti-CAPRIN-1 antibody. As used herein, the term "epitope" refers to, in a
mammal and preferably a human, a polypeptide fragment having antigenicity or
immunogenicity. The minimum size unit thereof consists of about 7 to 12 amino
acids, and preferably 8 to 11 amino acids.
[0075]
The affinity constant Ka (kon/k0ff) of the antibody of the present
invention is preferably at least 107 M"1, at least 108 M'\ at least 5 x 108 M"1, at
least 109 M"1, at least 5 x 109 M"1, at least 1010 M"1, at least 5 x 1010 M"\ at least
1011 M"1, at least 5 x 1011 M"1, at least 1012 M"1, or at least 1013 M"!.
[0076]
The antibody of the present invention can be conjugated with an
antitumor agent. Conjugation of the antibody with an antitumor agent can be
carried out via a spacer having a group reactive to an amino group, a carboxyl
group, a hydroxy group, a thiol group or the like (e.g., a succinimidyl succinate
group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxy
carbonyl group, and a hydroxy group).
[0077]
Examples of the antitumor agent include the following known antitumor
agents as in prior art literatures and the like, such as paclitaxel, doxorubicin,
daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan,
improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa,
altretamine, triethylenemelamine, triethylenephosphoramide,
triethilenethiophosphoraniide, trimethylolomelamine, bullatacin, bullatacinone,
camptothecin, bryostatin, callystatin, cryptophycinl, cryptophycin8, dolastatin,
duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin,
chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, 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, mitomycinC, 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, floxuridine,
androgens (e.g., calusterone, dromostanolone propionate, epitiostanol,
mepitiostane, and testolactone), aminoglutethimide, mitotane, trilostane, frolinic
acid, aceglatone, aldophosphamideglycoside, aminolaevulinic acid, eniluracil,
amsacrine, bestrabucil, bisantrene, edatraxate, defo famine, demecolcine,
diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, lenthinan,
lonidamine, maytansine, ansamitocine, mitoguazone, mitoxantrone, mopidanmol,
nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid,
2-ethyl hydrazide, procarbazine, razoxane, rhizoxin, schizophyllan,
spirogermanium, tenuazonic acid, triaziquone, roridine A, anguidine, urethane,
vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,
gacytosine, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine,
cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide,
mitoxanthrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate,
daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor,
difluoromethylolnitine (DMFO), retinoic acid, capecitabine, and pharmaceutically
acceptable salts or derivatives thereof.
[0078]
Through administration of the antibody of the present invention in
combination with an antitumor agent, even higher therapeutic effects can be
obtained. This technique is applicable to both before and after surgery of a
cancer patient with the expression of CAPRIN-1. Particularly after surgery, more
effective prevention of cancer recurrences or prolonged survival period can be
obtained against cancer with the expression of CAPRIN-1, which has been
conventionally treated with an antitumor agent alone.
[0079]
Examples of the antitumor agent to be administered in combination with
the antibody of the present invention include the following known antitumor
agents as in prior art literatures or the like, such as paclitaxel, doxorubicin,
daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan,
improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa,
altretamine, triethylenemelamine, triethylenephosphoramide,
triethilenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone,
camptothecin, bryostatin, callystatin, cryptophycinl, cryptophycin8, dolastatin,
duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin,
chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, 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-norIeucine, adriamycin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycinC, 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, floxuridine,
calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone,
aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone,
aldophosphamideglycoside, aminolaevulinic acid, eniluracil, amsacrine,
bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone,
elfornithine, elliptinium acetate, epothilone, etoglucid, lenthinan, lonidamine,
maytansine, ansamitocine, mitoguazone, mitoxantrone, mopidanmol, nitraerine,
pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethyl
hydrazide, procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium,
tenuazonic acid, triaziquone, roridine A, anguidine, urethane, vindesine,
dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine,
docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin,
oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide, mitoxanthrone,
vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin,
aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor,
difluoromethylolnitine (DMFO), retinoic acid, capecitabine, and pharmaceutically
acceptable (known) salts or (known) derivatives thereof. Of the above examples,
particularly cyclophosphamide, paclitaxel, docetaxel, and vinorelbine are
preferably used.
[0080]
Alternatively, a known radio isotope as in prior art literatures or the like,
such as 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 175Lu, or 176Lu can be
bound to the antibody of the present invention. A desired radio isotope is
effective for treatment or diagnosis of tumor.
[0081]
The antibody of the present invention is an antibody having
immunological reactivity with CAPRIN-1, an antibody specifically recognizing
CAPRIN-1, or an antibody specifically binding to CAPRIN-1, which exhibits
cytotoxic activity against cancer or the effect of suppressing tumor growth. The
antibody should have a structure such that rejection is almost or completely
avoided in a subject animal to which the antibody is administered. Examples of
such an antibody include, when a subject animal is human, human antibody,
humanized antibody, chimeric antibody (e.g., human-mouse chimeric antibody),
single chain antibody, and bispecific antibody. These antibodies are:
recombinant antibodies having heavy chain and light chain variable regions from a
human antibody; recombinant antibodies having heavy chain and light chain
variable regions composed of complementarity determining regions (CDRs)
(CDR1, CDR2, and CDR3) from a non-human animal antibody and framework
regions from a human antibody; or recombinant antibodies having heavy chain and
light chain variable regions from a non-human animal antibody; said recombinnat
antibodies also having heavy chain and light chain constant regions from a human
antibody. Preferable antibodies are the former two antibodies.
[0082]
These recombinant antibodies can be prepared as follows by cloning
DNA encoding an anti-human CAPRIN-1 monoclonal antibody (e.g., a human
monoclonal antibody, a mouse monoclonal antibody, a rat monoclonal antibody, a
rabbit monoclonal antibody, or a chicken monoclonal antibody) from an
antibody-producing cell such as a hybridoma, preparing DNA encoding a light
chain variable region and a heavy chain variable region of the antibody by an
RT-PCR method using it as a template, and then determining the sequence of each
variable region of light chain and heavy chain or each sequence of CDR1, CDR2,
and CDR3 based on a 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)).
[0083]
Furthermore, DNA encoding each of these variable regions or DNA
encoding each CDR is prepared using gene recombination techniques (Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory
Press (1989)) or a DNA synthesizer. Here, the above human monoclonal
antibody-producing hybridoma can be prepared by immunizing a human
antibody-producing animal (e.g., a mouse) with human CAPRIN-1 and then fusing
splenocytes excised from the immunized animal to myeloma cells. Alternatively,
DNAs encoding a light chain or heavy chain variable region and a constant region
from a human antibody are prepared as necessary using gene recombination
techniques or a DNA synthesizer.
[0084]
In the case of humanized antibody, DNA is prepared by substituting a
CDR coding sequence in DNA encoding a variable region of light chain or heavy
chain derived from a human antibody, with a CDR coding sequence corresponding
thereto of an antibody derived from a non-human animal (e.g., a mouse, a rat, or a
chicken) and then ligating the DNA thus obtained to DNA encoding a constant
region of light chain or heavy chain derived from a human antibody. Thus, DNA
encoding humanized antibody can be prepared.
[0085]
In the case of chimeric antibody, DNA encoding a chimeric antibody can
be prepared by ligating DNA encoding a light chain or heavy chain variable region
of an antibody from a non-human animal (e.g., a mouse, a rat, and a chicken) to
DNA encoding a light chain or heavy chain constant region from a human
antibody.
[0086]
In the case of single chain antibody, this antibody is an antibody
prepared by linearly ligating a heavy chain variable region to a light chain variable
region via a linker. Thus, DNA encoding a single chain antibody can be prepared
by binding DNA encoding a heavy chain variable region, DNA encoding a linker,
and DNA encoding a light chain variable region. Herein, a heavy chain variable
region and a light chain variable region are both from a human antibody, or, only
CDRs are substituted with CDRs of an antibody from a non-human animal (e.g., a
mouse, a rat, and a chicken) although the other regions are from a human antibody.
Also, a linker comprises 12 to 19 amino acids, such as (G4S)3 of 15 amino acids (G.
-B. Kim et al., Protein Engineering Design and Selection 2007, 20 (9): 425-432).
[0087]
In the case of bispecific antibody (diabody), this antibody is capable of
specifically binding to two different epitopes. For example, DNA encoding a
bispecific antibody can be prepared by linking DNA encoding a heavy chain
variable region A, DNA encoding a light chain variable region B, DNA encoding a
heavy chain variable region B, and DNA encoding a light chain variable region A
in this order (here, DNA encoding a light chain variable region B is bound to DNA
encoding a heavy chain variable region B via DNA encoding the above linker).
Here, a heavy chain variable region and a light chain variable region are both from
a human antibody, or, a human antibody in which only CDRs have been substituted
with CDRs of an antibody from a non-human animal (e.g., a mouse, a rat, or a
chicken).
[0088]
The above-prepared recombinant DNA is incorporated into one or a
plurality of appropriate vectors, they are introduced into host cells (e.g.,
mammalian cells, yeast cells, or insect cells), and then (co)expression is caused, so
that a recombinant antibody can be prepared (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).
[0089]
Examples of the antibody of the present invention prepared by the above
method include the following antibody (a), (b), or (c) obtained in Examples below:
[0090]
(a) an antibody (e.g., the antibody composed of the heavy chain variable region of
SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 47) comprising
a heavy chain variable region comprising SEQ ID NOS: 40, 41, and 42 and a light
chain variable region comprising SEQ ID NOS: 44, 45, and 46; and
[0091]
(b) an antibody (e.g., the antibody composed of the heavy chain variable region of
SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51) comprising
a heavy chain variable region comprising SEQ ID NOS: 40, 41, and 42 and a light
chain variable region comprising SEQ ID NOS: 48, 49, and 50.
[0092]
(c) an antibody (e.g., the antibody composed of the heavy chain variable region of
SEQ ID NO: 63 and the light chain variable region of SEQ ID NO: 67) comprising
a heavy chain variable region comprising SEQ ID NOS: 60, 61, and 62 and a light
chain variable region comprising SEQ ID NOS: 64, 65, and 66.
[0093]
The amino acid sequences represented by SEQ ID NOS: 40, 41, and 42,
and SEQ ID NOS: 60, 61, and 63 are CDR1, CDR2, and CDR3, respectively, of
mouse antibody heavy chain variable regions. Also, the amino acid sequences
represented by SEQ ID NOS: 44, 45, and 46, SEQ ID NOS: 48, 49, and 50, and
SEQ ID NOS: 64, 65, and 66 are CDR1, CDR2, and CDR3, respectively, of mouse
antibody light chain variable regions.
[0094]
Also, the humanized antibody, the chimeric antibody, the single chain
antibody, or the bispecific antibody of the present invention is the following
antibody (exemplified as "antibody (a)"), for example:
[0095]
(i) an antibody wherein the heavy chain variable region comprises the amino acid
sequences of SEQ ID NOS: 40, 41, and 42 and the amino acid sequences of
framework regions from a human antibody, and, a light chain variable region
comprises the amino acid sequences of SEQ ID NOS: 44, 45, and 46 and the amino
acid sequences of framework regions from a human antibody (preferably, the
antibody wherein the heavy chain variable region comprises the amino acid
sequence of SEQ ID NO: 43, and, the light chain variable region comprises the
amino acid sequence of SEQ ID NO: 47); and
[0096]
(ii) an antibody wherein a heavy chain variable region comprises the amino acid
sequences of SEQ ID NOS: 40, 41, and 42 and the amino acid sequences of
framework regions from a human antibody, and, a heavy chain constant region
comprises an amino acid sequence from a human antibody, and, a light chain
variable region comprises the amino acid sequences of SEQ ID NOS: 44, 45, and
46 and the amino acid sequences of framework regions from a human antibody,
and a light chain constant region comprises an amino acid sequence from a human
antibody (preferably, the antibody wherein a heavy chain variable region
comprises the amino acid sequence of SEQ ID NO: 43, and, a heavy chain constant
region comprises an amino acid sequence from a human antibody, as well as, a
light chain variable region comprises the amino acid sequence of SEQ ID NO: 47,
and, a light chain constant region comprises an amino acid sequence from a human
antibody).
[0097]
In addition, the sequences of human antibody heavy chain and light
chain constant regions and variable regions can be obtained from NCBI (e.g.,
U.S.A.: GenBank, UniGene), for example. For example, the sequence of
Accession No. J00228 can be referred to for a human IgGl heavy chain constant
region, the sequence of Accession No. J00230 can be referred to for a human IgG2
heavy chain constant region, the sequence of Accession No. X03604 can be
referred to for a human IgG3 heavy chain constant region, the sequence of
Accession No. KOI316 can be referred to for a human IgG4 heavy chain constant
region, the sequences of Accession Nos. V00557, X64135, X64133, and the like
can be referred to for human light chain k constant regions, and the sequences of
Accession Nos. X64132, X64134, and the like can be referred to for human light
chain X constant regions.
[0098]
The above antibodies preferably have cytotoxic activity and thus can
exhibit anti-tumor effects.
[0099]
Also, the specific sequences of heavy chain and light chain variable
regions or CDRs in the above antibodies are given simply for illustrative purposes,
and thus are clearly not limited to such specific sequences. A hybridoma capable
of producing another human antibody or non-human animal antibody (e.g., a
mouse antibody) against human CAPRIN-1 is prepared, a monoclonal antibody
that is produced by the hybridoma is collected, and then whether or not it is a
target antibody is determined by immunological binding property with human
CAPRIN-1 and cytotoxic activity as indicators. After identification of a
hybridoma producing the target monoclonal antibody in this manner, DNA
encoding heavy chain and light chain variable regions of the target antibody is
prepared from the hybridoma as described above, sequencing is carried out, and
then the DNA is used for preparation of another antibody.
[0100]
Furthermore, regarding the above antibody, the sequence of the above
antibodies (a) to (c), particularly the sequence of the framework region and/or the
sequence of the constant region of each of the antibodies may have a substitution,
a deletion, or an addition of one or several amino acids, as long as it has
specificity for specific recognition of CAPRIN-1. Here the term "several" refers
to preferably 2 to 5, and more preferably 2 or 3.
[0101]
The present invention further provides DNA encoding the above antibody
of the present invention, or, DNA encoding the above antibody heavy chain or
light chain, or, DNA encoding the above antibody heavy chain or light chain
variable region. Examples of such DNA include, in the case of antibody (a),
DNA encoding a heavy chain variable region comprising the nucleotide sequences
encoding the amino acid sequences of SEQ ID NOS: 40, 41, and 42 and DNA
encoding a light chain variable region comprising the nucleotide sequences
encoding the amino acid sequences of SEQ ID NOS: 44, 45, and 46.
[0102]
Complementarity determining regions (CDRs) encoded by the sequences
of DNA are regions for determining the specificity of an antibody. Thus,
sequences encoding regions in an antibody other than CDRs (specifically, a
constant region and a framework region) may be from other antibodies. Here,
examples of such "other antibodies" include antibodies from non-human
organisms, and are preferably from a human in view of reduction of side effects.
Thus, in the case of the above DNA, regions encoding each framework region and
each contact region of heavy chains and light chains preferably comprise
nucleotide sequences encoding corresponding amino acid sequences from a human
antibody.
[0103]
Further alternative examples of DNA encoding the antibody of the
present invention include, in the case of antibody (a), DNA encoding a heavy
chain variable region comprising the nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 43 and DNA encoding a light chain variable region
comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID
NO: 47. Here, an example of the nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 43 is the nucleotide sequence of SEQ ID NO: 52. Also,
an example of the nucleotide sequence encoding the amino acid sequence of SEQ
ID NO: 47 is the nucleotide sequence of SEQ ID NO: 53. In these DNAs, regions
encoding each constant region of heavy chains and light chains preferably
comprise nucleotide sequences encoding the corresponding amino acid sequences
from a human antibody.
[0104]
The DNAs of these antibodies can be obtained by the above methods or
the following method, for example. First, total RNA is prepared from a
hybridoma relating to the antibody of the present invention using a commercially
available RNA extraction kit, and then cDNA is synthesized with reverse
transcriptase using random primers, and the like. Subsequently, cDNA encoding
an antibody is amplified by a PCR method using as primers the oligonucleotides of
sequences conserved in each variable region of known mouse antibody heavy
chain and light chain genes. The sequence encoding a constant region can be
obtained by amplifying a known sequence by a PCR method. The nucleotide
sequence of DNA can be determined by a conventional method such as insertion of
it into a plasmid or a phage for sequencing.
[0105]
An anti-CAPRIN-1 antibody to be used in the present invention is
considered to exhibit the anti-tumor effects against CAPRIN-1-expressing cancer
cells through the following mechanism:
[0106]
effector-cell antibody-dependent cytotoxicity (ADCC) of
CAPRIN-1-expressing cells, and the complement-dependent cytotoxicity (CDC) of
CAPRIN-1-expressing cells.
[0107]
Therefore, the activity of an anti-CAPRIN-1 antibody to be used in the
present invention can be evaluated by, as specifically described in Examples below,
measuring ex vivo the above ADCC activity or CDC activity against
CAPRIN-1-expressing cancer cells.
[0108]
An anti-CAPRIN-1 antibody to be used in the present invention binds to
a CAPRIN-1 protein on a cancer cell and exhibits anti-tumor effects due to the
above activity, and thus it is useful for treating or preventing cancer. Specifically,
the present invention provides a pharmaceutical composition for treating and/or
preventing cancer, which comprises an anti-CAPRIN-1 antibody as an active
ingredient. When the anti-CAPRIN-1 antibody is used for administration thereof
to a human body (antibody therapy), it is preferably human antibody or humanized
antibody in order to decrease immunogenicity.
[0109]
In addition, the higher the binding affinity between an anti-CAPRIN-1
antibody and a CAPRIN-1 protein on the cancer cell surfaces, the stronger the
anti-tumor activity of the anti-CAPRIN-1 antibody that can be obtained. Therefore,
when an anti-CAPRIN-1 antibody having high binding affinity with a CAPRIN-1
protein can be acquired, stronger anti-tumor effects can be expected and such
antibody's application as a pharmaceutical composition for the purpose of cancer
treatment and/or prevention becomes possible. Such high binding affinity is
desirably as follows. As described above, binding constant (affinity constant) Ka
(kon/koff) is preferably at least 107 M"1, at least 108 M"1, at least 5 x 108 M"1, at least
109 M"1, at least 5 x 109 M"1, at least 1010 M"1, at least 5 x 1010 M"1, at least 1011
M*1, at least 5 x 1011 M"\ at least 1012 M"1, or, at least 1013 M"1.
[0110]

The capacity of an antibody to bind to CAPRIN-1 can be specified by
binding assay using ELISA, a Western blot method, immuno-fluorescence and
flow cytometric analysis, or the like as described in Examples.
[0111]

An antibody that recognizes CAPRIN-1 can be tested for reactivity to
CAPRIN-1 by a method for immunohistochemistry known by persons skilled in
the art using paraformaldehyde- or acetone-fixed frozen sections or
paraformaldehyde-fixed paraffin-embedded tissue sections, which is prepared
from tissue samples obtained from a patient during surgery, or tissue samples
obtained from an animal having heterotransplant tissue inoculated with a cell line
expressing CAPRIN-1, naturally or after transfection.
[0112]
An antibody reactive to CAPRIN-1 can be stained by various methods
for immunohistochemical staining. For example, a horseradish
peroxidase-conjugated goat anti-mouse antibody or goat anti-chicken antibody is
caused to undergo reaction, a target antibody can be visualized.
[0113]
Pharmaceutical composition>
The present invention further provides a pharmaceutical composition for
treating and/or preventing cancer, which is characterized by containing the above
antibody or a fragment thereof as an active ingredient that has immunological
reactivity with partial polypeptides of CAPRIN-1 represented by even-numbered
SEQ ID NOS: 2 to 30, wherein the polypeptide has the amino acid sequence
represented by SEQ ID NO: 37, or an amino acid sequence having 80% or more
sequence identity with the amino acid sequence of SEQ ID NO: 37.
[0114]
A target of the pharmaceutical composition for treating and/or
preventing cancer of the present invention is not particularly limited, as long as it
is cancer (cell) expressing a CAPRIN-1 gene.
[0115]
The term "tumor" and "cancer" as used herein refers to malignant
neoplasm and is used interchangeably.
[0116]
Cancer to be subjected to the present invention is cancer expressing
genes encoding CAPRIN-1 proteins having amino acid sequences of
even-numbered SEQ ID NOS: 2 to 30. Examples of such cancer include
preferably breast cancer, brain tumor, leukemia, lung cancer, lymphoma,
mastocytoma, renal cancer, uterine cervix cancer, bladder cancer, esophageal
cancer, gastric cancer, colorectal cancer, ovarian cancer, prostate cancer, and
fibrosarcoma.
[0117]
Examples of such specific cancer include, but are not limited to, breast
adenocarcinoma, composite type breast adenocarcinoma, mammary gland
malignant mixed tumor, intraductal papillary adenocarcinoma, lung
adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell
carcinoma, glioma that is neural epithelial tissue tumor, ependymoma,
neurocytoma, fetal neuroectodermal tumor, schwannoma, neurofibroma,
meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal
lymphoma, digestive lymphoma, small-cell to medium-cell lymphoma, cancer of
cecum, ascending colon cancer, descending colon cancer, transverse colon cancer,
sigmoid colon cancer, rectal cancer, epithelial ovarian cancer, germ cell tumor, and
interstitial cell tumor.
[0118]
Moreover, preferable subjects are mammals including primates, pet
animals, domestic animals, animals for race, and the like and are particularly
preferably humans, dogs, and cats.
[0119]
When an antibody to be used in the present invention is used as a
pharmaceutical composition, it can be formulated by a method known by persons
skilled in the art. For example, the antibody can be used parenterally in the form
of an injection preparation such as an aseptic solution or a suspension prepared
with water or a pharmacologically acceptable solution other than water. For
example, it can be formulated by mixing in a unit dosage form required by
generally accepted pharmaceutical practice in appropriate combination with a
pharmacologically acceptable carrier or medium, specifically, sterile water or
saline, vegetable oil, an emulsifier, a suspension, a surfactant, a stabilizer, a
flavoring compound, an excipient, a vehicle, an antiseptic, a binder, and the like.
The amounts of active ingredients in these preparations are determined so that an
appropriate dose within the indicated range can be obtained.
[0120]
An aseptic composition for injection can be prescribed according to
general pharmaceutical practice using a vehicle such as distilled water for
injection.
[0121]
Examples of an aqueous solution for injection include saline, an isotonic
solution containing dextrose or other adjuvants, such as D-sorbitol, D-mannose,
D-mannitoI, and sodium chloride. These examples may be used in combination
with an appropriate solubilizing agent such as alcohol, specifically ethanol and
polyalcohol (e.g., propylene glycol and polyethylene glycol), and nonionic
surfactant (e.g., polysorbate 80 (TM) and HCO-60).
[0122]
Examples of the oil include sesame oil and soybean oil, which can be
used in combination with a solubilizing agent such as benzyl benzoate or benzyl
alcohol. Also, a buffering agent such as phosphate buffer or sodium acetate
buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl
alcohol or phenol, and an antioxidant may be combined therewith. An
appropriate amplus is generally filled with the thus prepared injection solution.
[0123]
Administration is peroral or perenteral administration and is preferably
perenteral administration. Specific examples of the route of administration
include injection, transnasal administration, pulmonary administration, and
transdermal administration. Examples of injection include intravenous injection,
intramuscular injection, intraperitoneal injection, and subcutaneous injection, so
that systemic or local administration is possible.
[0124]
Also, administration methods can be appropriately selected depending
on a patient's age, body weight, gender, symptoms, and the like. The dosage per
administration of a pharmaceutical composition containing an antibody or a
polynucleotide encoding the antibody can be selected from the range between
0.0001 mg and 1000 mg per kg of body weight, for example. Alternatively, for
example, dosage can be selected from the range between 0.001 mg/body and
100000 mg/body per patient. However, the dosage range is not always limited to
these numerical values. The dosage and administration method are varied
depending on a patient's body weight, age, gender, symptoms, and the like, but can
be appropriately selected by persons skilled in the art.
[0125]
The above pharmaceutical composition containing the antibody or a
fragment thereof of the present invention is administered to a subject, so that
cancer, preferably, breast cancer, brain tumor, leukemia, lung cancer, lymphoma,
mastocytoma, renal cancer, uterine cervix cancer, bladder cancer, esophageal
cancer, gastric cancer, and colorectal cancer can be treated and/or prevented.
[0126]
The present invention further encompasses a method for treating and/or
preventing cancer, comprising administering to a subject the pharmaceutical
composition of the present invention in combination with the above exemplified
antitumor agent or pharmaceutical composition containing such antitumor agent.
The antibody or a fragment thereof of the present invention and an antitumor agent
may be administered simultaneously or separately to a subject. They can be
separately administered regardless of the order of administration. The
administration intervals, dosage, the route of administration, and the frequency of
administration can be appropriately selected by a specialist. Examples of the
other pharmaceutical formulation to be administered simultaneously include
pharmaceutical compositions obtained by mixing the antibody or a fragment
thereof of the present invention with an antitumor agent in a pharmacologically
acceptable carrier (or a medium) followed by formulation. Furthermore, to either
the above pharmaceutical composition containing an antitumor agent or
formulation, explanations concerning prescription, formulation, the route of
administration, dose, cancer, and the like for administration of a pharmaceutical
composition containing the antibody of the present invention and formulation are
applicable.
[0127]
Therefore, the present invention also provides a pharmaceutical
combination for treating and/or preventing cancer, comprising the pharmaceutical
composition of the present invention, and the above exemplified pharmaceutical
composition containing an antitumor agent. Also, the present invention provides
a pharmaceutical composition for treating and/or preventing cancer, comprising
the antibody or a fragment thereof of the present invention and an antitumor agent
together with a pharmacologically acceptable carrier.
[0128]
Polypeptide and DNA>
The present invention further provides the following polypeptides and
DNAs relating to the above antibody (a), (b), or (c).
[0129]
(i) A polypeptide comprising the amino acid sequences of SEQ ID NOS: 43 and 63,
and DNA encoding the polypeptide, wherein the DNA comprises the nucleotide
sequences of SEQ ID NOS: 52 and 68.
[0130]
(ii) A polypeptide comprising the amino acid sequences of SEQ ID NOS: 47, 51,
and 67, and DNA encoding the polypeptide, wherein the DNA comprises the
nucleotide sequences of SEQ ID NOS: 70, 53, and 69.
[0131]
(iii) A heavy chain CDR polypeptide selected from the group consisting of the
amino acid sequences represented by SEQ ID NOS: 40, 41, and 42 and SEQ ID
NOS: 60, 61, and 62, and DNA encoding the polypeptide.
[0132]
(iv) A light chain CDR polypeptide selected from the group consisting of the
amino acid sequences represented by SEQ ID NOS: 44, 45, and 46, SEQ ID NOS:
48, 49, and 50, and SEQ ID NOS: 64, 65, and 66, and DNA encoding the
polypeptide.
[0133]
These polypeptides and DNAs can be prepared using gene recombination
techniques as described above.
[0134]

The above-explained present invention is as summarized as follows.
[0135]
(1) A pharmaceutical composition for treating and/or preventing a cancer,
comprising an antibody or a fragment thereof as an active ingredient that has
immunological reactivity with a partial polypeptide of CAPRIN-1, wherein
CAPRIN-1 is represented by any of the even-numbered sequences of SEQ ID
NOS: 2 to 30, and wherein the partial polypeptide comprises the amino acid
sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or
more sequence identity with the amino acid sequence of SEQ ID NO: 37.
[0136]
(2) The pharmaceutical composition according to (1) above, wherein the cancer is
breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mastocytoma, renal
cancer, uterine cervix cancer, bladder cancer, esophageal cancer, gastric cancer, or
colorectal cancer.
[0137]
(3) The pharmaceutical composition according to (1) or (2) above, wherein the
antibody is a monoclonal antibody or a polyclonal antibody.
[0138]
(4) The pharmaceutical composition according to any one of (1) to (3) above,
wherein the antibody is a human antibody, humanized antibody, chimeric antibody,
single chain antibody, or a bispecific antibody.
[0139]
(5) An antibody having immunological reactivity with a polypeptide that comprise
the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence
having 80% or more sequence identity with the amino acid sequence of SEQ ID
NO: 37.
[0140]
(6) The antibody according to (5) above, which has a cytotoxic activity against a
cancer cell expressing a CAPRIN-1 protein.
[0141]
(7) An antibody, which comprises a heavy chain variable region comprising SEQ
ID NOS: 40, 41, and 42 and a light chain variable region comprising SEQ ID NOS:
44, 45, and 46, and has immunological reactivity with a CAPRIN-1 protein.
[0142]
(8) An antibody, which comprises a heavy chain variable region comprising SEQ
ID NOS: 40, 41, and 42 and a light chain variable region comprising SEQ ID NOS:
48, 49, and 50, and has immunological reactivity with a CAPRIN-1 protein.
[0143]
(9) An antibody, which comprises a heavy chain variable region comprising SEQ
ID NOS: 60, 61, and 62 and a light chain variable region comprising SEQ ID NOS:
64, 65, and 66, and has immunological reactivity with a CAPRIN-1 protein.
[0144]
(10) The antibody according to any one of (5) to (9) above, which is a human
antibody, humanized antibody, chimeric antibody, single chain antibody, or
bispecific antibody.
[0145]
(11) A pharmaceutical composition for treating and/or preventing a cancer,
comprising the antibody or a fragment thereof of any one of (5) to (10) above as an
active ingredient.
[0146]
(12) The pharmaceutical composition according to (11) above, wherein the cancer
is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mastocytoma,
renal cancer, uterine cervix cancer, bladder cancer, esophageal cancer, gastric
cancer, or colorectal cancer.
[0147]
(13) A pharmaceutical combination for treating and/or preventing a cancer,
comprising the pharmaceutical composition of any one of (1) to (4) above or the
pharmaceutical composition of (11) or (12) above, and a pharmaceutical
composition containing an antitumor agent.
[0148]
(14) A method for treating and/or preventing a cancer, comprising administering to
a subject the antibody or a fragment thereof of any one of (5) to (10) above or the
pharmaceutical composition of (11) or (12) above.
[0149]
(15) A method for treating and/or preventing a cancer, comprising using
pharmaceutical compositions of the pharmaceutical combination of (13) above in
combination in a subject.
Examples
[0150]
The present invention is described more specifically based on Examples,
but the scope of the present invention is not limited by these specific examples.
[0151]
Example 1 Identification of novel cancer antigen protein by SEREX method
(1) Preparation of cDNA library
Total RNA was extracted from a testis tissue of a healthy dog by an acid
guanidium-phenol-chloroform method. PolyA RNA was purified according to
protocols attached to an Oligotex-dT30 mRNA purification Kit (Takara Shuzo Co.,
Ltd.) using the kit.
[0152]
A dog testis cDNA phage library was synthesized using the thus obtained
mRNA (5 ng). For preparation of the cDNA phage library, a cDNA synthesis kit,
a ZAP-cDNA synthesis kit, and a ZAP-cDNA gigapack III gold cloning kit
(STRATAGENE) were used and the library was prepared according to protocols
attached to the kit. The size of the thus prepared cDNA phage library was 7.73 x
105pfu/ml.
[0153]
(2) Screening of cDNA library using serum
Immunoscreening was carried out using the above-prepared dog testis
cDNA phage library. Specifically, host Escherichia coli (XLl-Blue MRF') was
infected with the phage so that 2210 clones were present on a 90 x 15 mm NZY
agarose plate. Cells were cultured at 42°C for 3 to 4 hours, so as to cause plaque
formation. The plate was covered with a nitrocellulose membrane (Hybond C
Extra: GE HealthCare Bio-Sciences) impregnated with IPTG
(isopropyl-p-D-thiogalactoside) at 37°C for 4 hours. Proteins were induced,
expressed, and then transferred to the membrane. Subsequently, the membrane
was recovered, immersed, and shaken in TBS (10 mM Tris-HCl, 150 mM NaCl pH
7.5) containing 0.5% powdered skim milk at 4°C overnight, so that nonspecific
reaction was suppressed. The filter was caused to react with 500-fold diluted
sera of dogs with cancer at room temperature for 2 to 3 hours.
[0154]
As the above sera from dogs with cancer, sera collected from dogs with
breast cancer were used. The sera were stored at -80°C and then subjected to
pretreatment immediately before use. Pretreatment for sera was performed by the
following method. Specifically, host Escherichia coli (XLl-Blure MRF') was
infected with X ZAP Express phage into which no foreign gene had been inserted,
and then cultured on NZY plate medium at 37°C overnight. Subsequently, a 0.2
M NaHC03 buffer (pH 8.3) containing 0.5 M NaCi was added to the plate and then
the plate was left to stand at 4°C for 15 hours. The supernatants were collected
as Escherichia co/z'/phage extracts. Next, the collected Escherichia coli/phage
extract was passed through a NHS-column (GE HealthCare Bio-Sciences), so as to
immobilize the Escherichia co/z-phage-derived protein. The serum of a dog with
cancer was passed through the column to which the protein had been immobilized
for reaction, thereby removing Escherichia coli and antibodies adsorbed to the
phage from the serum. Each serum fraction that had passed through the column
was diluted 500-fold with TBS containing 0.5% powdered skim milk, and the
resultant was used as an immunoscreening material.
[0155]
A membrane, to which the thus treated serum and the fusion protein had
been blotted, was washed 4 times with TBS-T (0.05% Tween20/TBS). The
membrane was reacted with goat anti-dog IgG (Goat anti Dog IgG-h+I HRP
conjugated: BETHYL Laboratories) diluted 5000-fold as a secondary antibody
with TBS containing 0.5% powdered skim milk at room temperature for 1 hour.
Detection was carried out by enzyme color reaction using an NBT/BCIP reaction
solution (Roche). Colonies corresponding to the color reaction positive site were
collected from the (|)90 x 15 mm NZY agarose plate, and then dissolved in 500 ul
of SM buffer (100 mM NaCl, 10 mM MgClS04, 50 mM Tris-HCl, 0.01% gelatin,
pH 7.5). Until unification of color reaction positive colonies, secondary
screening and tertiary screening were repeated by a method similar to the above.
Thus, 30940 phage clones that had reacted with serum IgG were screened so that 5
positive clones were isolated.
[0156]
(3) Homology search for isolated antigen gene
A procedure for conversion of phage vectors to plasmid vectors was
performed for the 5 positive clones isolated by the above method for the purpose
of subjecting the clones to nucleotide sequence analysis. Specifically, 200 ul of
a solution of host Escherichia coli (XLl-Blue MRF') prepared to give an
absorbance ODgoo of 1.0, 250 ul of a purified phage solution, and 1 ul of ExAssist
helper phage (STRATAGENE) were mixed and allowed to react at 37°C for 15
minutes. After that, 3 ml of LB medium was added, cells were cultured at 37°C
for 2.5 to 3 hours, and then the resultant was immediately put in water bath at
70°C for incubation for 20 minutes. Centrifugation was carried out at 4°C, 1000
x g for 15 minutes, and then the supernatant was collected as a phagemid solution.
Subsequently, 200 jal of a solution prepared from phagemid host Escherichia coli
SOLR to give an absorbance OD6oo of 1.0 and 10 u.1 of the purified phage solution
were mixed, followed by 15 minutes of reaction at 37°C. 50 |il of the resultant
was plated on LB agar medium containing ampicillin (at final concentration of 50
u.g/ml) and then cultured overnight at 37°C. A single colony of transformed
SOLR was collected and then cultured on LB medium containing ampicillin (at
final concentration of 50 u.g/ml) at 37°C. After culture, plasmid DNA carrying
an insert of interest was purified using a QIAGEN plasmid Miniprep Kit
(QIAGEN).
[0157]
The purified plasmid was subjected to the analysis of the entire sequence
of the insert by the primer walking method using the T3 primer of SEQ ID NO: 31
and the T7 primer of SEQ ID NO: 32. The gene sequences of SEQ ID NOS: 5, 7,
9, 11, and 13 were obtained by the sequence analysis. With the use of the
nucleotide sequences of the genes and the amino acid sequences thereof (SEQ ID
NOS: 6, 8, 10, 12, and 14), homology search program BLAST search
(http://www.ncbi.nlm.nih.gov/BLAST/) was conducted for searching homology
with known genes. As a result, it was revealed that all the five obtained genes
were genes encoding CAPRIN-1. The sequence identities among the five genes
were 100% at the nucleotide sequence level and 99% at the amino acid sequence
level in the regions to be translated into proteins. The sequence identities of
these genes and the human homologue-encoding gene were 94% at the nucleotide
sequence level and 98% at the amino acid sequence level in the regions to be
translated into proteins. The nucleotide sequences of the human homologues are
represented by SEQ ID NOS: 1 and 3 and the amino acid sequences of the same are
represented by SEQ ID NOS: 2 and 4. Also, the sequence identities of the
obtained dog genes and the cattle homologue-encoding gene were 94% at the
nucleotide sequence level and 97% at the amino acid sequence level in the regions
to be translated into proteins. The nucleotide sequence of the cattle homologue is
represented by SEQ ID NO: 15 and the amino acid sequence of the same is
represented by SEQ ID NO: 16. In addition, the sequence identities of the human
homologue-encoding genes and the cattle homologue-encoding gene were 94% at
the nucleotide sequence level and 93% to 97% at the amino acid sequence level in
the regions to be translated into proteins. Also, the sequence identities of the
obtained dog genes and the horse homologue-encoding gene were 93% at the
nucleotide sequence level and 97% at the amino acid sequence level in the regions
to be translated into proteins. The nucleotide sequence of the horse homologue is
represented by SEQ ID NO: 17 and the amino acid sequence of the same is
represented by SEQ ID NO: 18. In addition, the sequence identities of the human
homologue-encoding genes and the horse homologue-encoding gene were 93% at
the nucleotide sequence level and 96% at the amino acid sequence level in the
regions to be translated into proteins. Also, the sequence identities of the
obtained dog genes and the mouse homologue-encoding genes were 87% to 89% at
the nucleotide sequence level and 95% to 97% at the amino acid sequence level in
the regions to be translated into proteins. The nucleotide sequences of the mouse
homologues are represented by SEQ ID NOS: 19, 21, 23, 25, and 27 and the amino
acid sequences of the same are represented by SEQ ID NOS: 20, 22, 24, 26, and
28. In addition, the sequence identities of the human homologue-encoding genes
and the mouse homologue-encoding genes were 89% to 91% at the nucleotide
sequence level and were 95% to 96% at the amino acid sequence level in the
regions to be translated into proteins. Also, the sequence identities of the
obtained dog genes and the chicken homologue-encoding gene were 82% at the
nucleotide sequence level and 87% at the amino acid sequence level in the regions
to be translated into proteins. The nucleotide sequence of the chicken homologue
is represented by SEQ ID NO: 29 and the amino acid sequence of the same is
represented by SEQ ID NO: 30. In addition, the sequence identities of the human
homologue-encoding genes and the chicken homologue-encoding gene were 81%
to 82% at the nucleotide sequence level and 86% at the amino acid sequence level
in the regions to be translated into proteins.
[0158]
(4) Gene expression analysis in each tissue
The expression of genes obtained by the above method was examined in
dog and human normal tissues and various cell lines by an RT-PCR method.
Reverse transcription reaction was performed as follows. Specifically, total RNA
was extracted from 50 mg to 100 mg of the tissue or 5 to 10 x 106 cells of the cell
line using a TRIZOL reagent (Invitrogen) according to the accompanying
protocols. cDNA was synthesized with the total RNA using a Superscript
First-Strand Synthesis System for RT-PCR (Invitrogen) according to the
accompanying protocols. PCR was performed as follows using primers of SEQ
ID NOS: 33 and 34 specific to the obtained genes. Specifically, reagents and an
accompanying buffer were added to 0.25 ul of the sample prepared by the reverse
transcription reaction to a total volume of 25 ul, so that the resultant contained the
above primers of 2 uM each, dNTPs of 0.2 mM each, and 0.65 U ExTaq
polymerase (Takara Shuzo Co., Ltd.). PCR was carried out by repeating a cycle
of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds 30 times
using a Thermal Cycler (BIO RAD). The above gene-specific primers are
capable of amplifying the region ranging from nucleotides 206 to 632 in the
nucleotide sequence of SEQ ID NO: 5 (dog CAPRIN-1 gene) and the region
ranging from nucleotides 698 to 1124 in the nucleotide sequence of SEQ ID NO: 1
(human CAPRIN-1 gene). As a control for comparison, GAPDH-specific primers
of SEQ ID NOS: 35 and 36 were also used concurrently. As a result, as shown in
Fig. 1, strong expression was observed in testis among normal dog tissues, while
expression was observed in dog breast cancer and adenocarcinoma tissues.
Moreover, the observation of the expression of the human homologues from the
obtained genes was also carried out. As a result, similarly to the case of the dog
CAPRIN-1 gene, expression could be observed in only testis among normal
tissues. However, in the case of cancer cells, expression was detected in many
types of cancer cell lines, including breast cancer, brain tumor, leukemia, lung
cancer, and esophageal cancer cell lines. Expression was observed particularly in
many breast cancer cell lines. It was confirmed by the results that the expression
of CAPRIN-1 is not observed in normal tissues other than testis, while CAPRIN-1
was expressed in many cancer cells and particularly in breast cancer cell lines.
[0159]
In Fig. 1, reference number 1 on each vertical axis indicates the
expression patterns of genes identified above and reference number 2 indicates the
expression patterns of the GAPDH gene as a control.
[0160]
(5) Preparation of polyclonal antibody against CAPRIN-1-derived peptide
To obtain an antibody binding to CAPRIN-1, the CAPRIN-1-derived
peptide represented by SEQ ID NO: 37 was synthesized, lmg of the peptide as
an antigen was mixed with an equivalent volume of an incomplete Freund's
adjuvant (IFA) solution. The mixture was subcutaneously administered to rabbits
4 times every 2 weeks. Blood was then collected and antiserum containing
polyclonal antibodies was obtained. Furthermore, the antiserum was purified
using a protein G carrier (GE HealthCare Bio-Sciences), so that polyclonal
antibodies against the CAPRIN-1-derived peptide were obtained. Also, the
serum of a rabbit to which no antigen had been administered was purified using a
protein G carrier in a manner similar to the above, and the resultant was used as a
control antibody.
[0161]
(6) Analysis of antigen protein expression on cancer cells
Next, 7 breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1,
MDA-MB-231V, BT20, SK-BR-3, and MDA-MB-231T) for which CAPRIN-1
gene expression had been observed at high levels were examined for CAPRIN-1
protein expression on the cell surfaces. 10 cells of each human breast cancer
cell line for which gene expression had been observed above were centrifuged in a
1.5-mI microcentrifuge tube. 2 u.g (5 ul) of the polyclonal antibodies against the
CAPRIN-1-derived peptide prepared in (5) above was added thereto. After
suspension with 95 ul of PBS containing 0.1% fetal calf serum, it was left to stand
on ice for 1 hour. After washing with PBS, the resultant was suspended in PBS
containing 5 ul of FITC-labeled goat anti-rabbit IgG antibody (SantaCruz) and 95
ul of 0.1% fetal bovine serum (FBS), and then the resultant was left to stand on ice
for 1 hour. After washing with PBS, fluorescence intensity was measured using a
FACS Caliber (Becton, Dickinson and Company). Meanwhile, procedures
similar to the above were performed using the control antibody prepared in (5)
above instead of the polyclonal antibodies against the CAPRIN-1-derived peptide
so that a control sample was obtained. As a result, all cells to which the
anti-human CAPRIN-1 antibody had been added exhibited enhanced fluorescence
intensity compared with the control. Specifically, fluorescence intensity was
enhanced to 193% in the case of MDA-MB-231V and 169% in the case of
SK-BR-3. It was revealed by these results that the CAPRIN-1 protein was
expressed on the cell membrane surfaces of the above human cancer cell lines.
The percentage of enhancement in the above fluorescence intensity was expressed
as percentage of increase in mean fluorescence intensity (MFI level) in each type
of cell and calculated by the following formula.
[0162]
Percentage of increase in mean fluorescence intensity (percentage of
enhancement in fluorescence intensity) (%) = ((MFI level of cells having reacted
with anti-human CAPRIN-1 antibody) - (MFI level of control)) / (MFI level of the
control) x 100.
[0163]
With a technique similar to the above, CAPRIN-1 expression was also
analyzed for 3 renal cancer cell lines (Caki-1, Caki-2, and A498), ovary cancer
cell line (SKOV3), lung cancer cell line (QG56), prostate cancer cell line (PC3),
uterine cervix cancer cell line (Hela), fibrosarcoma cell line (HT1080), 2 brain
tumor cell lines (T98G and U87MG), 2 mouse colorectal cancer cell lines (CT26
and colon 26), mouse breast cancer cell line (4T1), mouse melanoma cell line
(B16), and 2 mouse neuroblastoma cell lines (N1E-115 and Neuro2a). As a result,
CAPRIN-1 expression was confirmed in all cell lines. In addition, similar results
were obtained in the case of using the anti-CAPRIN-1 monoclonal antibody
(monoclonal antibody #1) comprising the heavy chain variable region of SEQ ID
NO: 43 and the light chain variable region of SEQ ID NO: 47, or the
anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #2) comprising the
heavy chain variable region of SEQ ID NO: 43 and the light chain variable region
of SEQ ID NO: 51, or the anti-CAPRIN-1 monoclonal antibody (monoclonal
antibody #3) comprising the heavy chain variable region of SEQ ID NO: 63 and
the light chain variable region of SEQ ID NO: 67 (obtained in Example 3).
[0164]
(7) Immunohistochemical staining
(7)-l CAPRIN-1 expression in mouse and dog normal tissues
Mice (Balb/c, female) and dogs (beagles, female) were exsanguinated
under ether anesthesia and ketamine/isoflurane anesthesia. After laparotomy,
each organ (stomach, liver, eyeball, thymus gland, muscle, bone marrow, uterus,
small bowel, esophagus, heart, kidney, salivary gland, large bowel, mammary
gland, brain, lung, skin, adrenal gland, ovary, pancreas, spleen, and bladder) was
transferred to a 10-cm dish containing PBS. Each organ was cut open in PBS and
then subjected to perfusion fixation overnight in 0.1 M phosphate buffer (pH 7.4)
containing 4% paraformaldehyde (PFA). The perfusion solution was discarded,
the tissue surface of each organ was rinsed with PBS, a PBS solution containing
10% sucrose was added to a 50-ml centrifuge tube, each tissue was added to the
tube, and then the tube was shaken using a rotor at 4°C for 2 hours. The solution
was replaced by a PBS solution containing 20% sucrose, and then left to stand at
4°C until the tissue sank. The solution was replaced by a PBS solution
containing 30% sucrose and then left to stand at 4°C until the tissue sank. The
tissue was removed and then needed portions were excised with a surgical scalpel.
Next, an OCT compound (Tissue Tek) was added to the tissue so that it was
thoroughly applied to the tissue surface, and then the tissue was placed in a
cryomold. The cryomold was placed on dry ice for quick freezing. Thereafter,
the tissue was sliced to 10 urn to 20 jam using a cryostat (LEICA). Slices were
air-dried on slide glasses using a hair dryer for 30 minutes, to prepare the sliced
tissue mounted on a slide glass. Next, each sample was placed in a staining
bottle filled with PBS-T (saline containing 0.05% Tween20) and then subjected to
replacement with PBS-T being repeated three times every 5 minutes. Excess
water around the sections was removed with Kimwipes, and then the sections were
circled using a DAKOPEN (DAKO). As blocking solutions, an MOM mouse Ig
blocking reagent (VECTASTAIN) and a PBS-T solution containing 10% FBS were
overlaid on mouse tissue and dog tissue, respectively, and then left to stand in a
moist chamber at room temperature for 1 hour. Next, a solution of the polyclonal
antibodies (reactive with the surfaces of cancer cells, prepared in (5) above)
against the CAPRIN-1 -derived peptide (SEQ ID NO: 37) of 10 ng/ml adjusted
with a blocking solution was placed on and then left to stand overnight in a moist
chamber at 4°C. 10 minutes of washing with PBS-T was performed 3 times, and
then an MOM biotin-labeled anti-IgG antibody (VECTASTAIN) diluted 250-fold
with the blocking solution was placed and then left to stand at room temperature
for 1 hour in a moist chamber. After ten (10) minutes of washing with PBS-T
was performed 3 times, an avidin-biotin ABC reagent (VECTASTAIN) was placed
on, and then the sample was left to stand in a moist chamber at room temperature
for 5 minutes. After ten (10) minutes of washing with PBS-T was performed 3
times, a DAB coloring solution (DAB 10 mg + 30% H202 10 jil/0.05 M Tris-HCl
(pH 7.6) 50 ml) was placed on, and then the sample was left to stand in a moist
chamber at room temperature for 30 minutes. After rinsing with distilled water, a
hematoxylin reagent (DAKO) was placed on, the sample was left to stand at room
temperature for 1 minute, and then rinsed with distilled water. The slide glass
was immersed in 70%, 80%, 90%, 95%, and then 100% ethanol solutions in such
order for 1 minute each and then left to stand overnight in xylene. The slide
glass was removed, sealed in Glycergel Mounting Medium (DAKO), and then
observed. As a result, the expression of CAPRIN-1 was slightly observed within
cells of each tissue of salivary gland, kidney, colon, and stomach, but the
expression of the same was not observed on cell surfaces. Furthermore, no
expression was observed in tissues from other organs. In addition, similar results
were obtained in the case of using the anti-CAPRIN-1 monoclonal antibody
(monoclonal antibody #1) comprising the heavy chain variable region of SEQ ID
NO: 43 and the light chain variable region of SEQ ID NO: 47, the anti-CAPRIN-1
monoclonal antibody (monoclonal antibody #2) comprising the heavy chain
variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID
NO: 51, or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #3)
comprising the heavy chain variable region of SEQ ID NO: 63 and the light chain
variable region of SEQ ID NO: 67 (obtained in Example 3).
[0165]
(7)-2 CAPRIN-1 expression in dog breast cancer tissue
Frozen section slides were prepared by a method similar to the above
using 108 frozen breast cancer tissue specimens of dogs pathologically diagnosed
as having malignant breast cancer, and immunohistochemical staining was
performed using the polyclonal antibody against the CAPRIN-1-derived peptide
(SEQ ID NO: 37) prepared in (5) above. As a result, the expression of
CAPRIN-1 was observed in 100 out of 108 specimens (92.5%) and CAPRIN-1 was
strongly expressed on the surfaces of cancer cells with a particularly high grade of
atypism. In addition, similar results were obtained in the case of using the
monoclonal antibody #1, #2, or #3 obtained in Example 3.
[0166]
(7)-3 CAPRIN-1 expression in human breast cancer tissues
Immunohistochemical staining was performed using 188 breast cancer
tissue specimens on a paraffin-embedded human breast cancer tissue array
(BIOMAX). After 3 hours of treatment of the human breast cancer tissue array at
60°C, the array was placed in a staining bottle filled with xylene, followed by
xylene replacement being repeated three times every 5 minutes. Next, a similar
procedure was performed with ethanol and PBS-T instead of xylene. The human
breast cancer tissue array was placed in a staining bottle filled with 10 raM citrate
buffer (pH 6.0) containing 0.05% Tween20. After 5 minutes of treatment at
125°C, the array was left to stand at room temperature for 40 minutes or more.
Excess water around the sections was removed with Kimwipes, the sections were
circled with a DAKOPEN, and Peroxidase Block (DAKO) was added dropwise in
appropriate amounts. After left to stand at room temperature for 5 minutes, the
array was placed in a staining bottle filled with PBS-T, followed by PBS-T
replacement being repeated three times every 5 minutes. As a blocking solution,
a PBS-T solution containing 10% FBS was placed on the array, and then the array
was left to stand in a moist chamber at room temperature for 1 hour. Next, a
solution of the polyclonal antibody against the CAPRIN-1-derived peptide (SEQ
ID NO: 37) prepared in (5) above having a concentration of 10 u.g/ml adjusted
with a PBS-T solution containing 5% FBS was placed on, and the array was left to
stand overnight in a moist chamber at 4°C. After ten (10) minutes of washing
with PBS-T was performed 3 times, Peroxidase Labeled Polymer Conjugated
(DAKO) was added dropwise in appropriate amounts and then the array was left to
stand in a moist chamber at room temperature for 30 minutes. After ten (10)
minutes of washing with PBS-T was performed 3 times, a DAB coloring solution
(DAKO) was placed on and then it was left to stand at room temperature for about
10 minutes. The coloring solution was discarded, 10 minutes of washing with
PBS-T was performed 3 times, and then it was rinsed with distilled water. The
array was immersed in 70%, 80%, 90%, 95%, and then 100% ethanol solutions in
such order for 1 minute each, and then left to stand in xylene overnight. The
slide glass was removed, sealed in Glycergel Mounting Medium (DAKO), and then
observed. As a result, the strong expression of CAPRIN-1 was observed in 138
out of a total of 188 breast cancer tissue specimens (73%). In addition, similar
results were obtained in the case of using the monoclonal antibody #1, #2, or #3
obtained in Example 3.
[0167]
(7)-4 CAPRIN-1 expression in human malignant brain tumor
Immunohistochemical staining was performed according to a method
similar to that used in (7)-3 above with 247 malignant brain tumor tissue
specimens on a paraffin-embedded human malignant brain tumor tissue array
(BIOMAX), using the polyclonal antibodies against the CAPRIN-1-derived
peptide (SEQ ID NO: 37) prepared in (5) above. As a result, the strong
expression of CAPRIN-1 was observed in 227 out of a total of 247 malignant brain
tumor tissue specimens (92%). In addition, similar results were obtained in the
case of using the monoclonal antibody #1, #2, or #3 obtained in Example 3
[0168]
(7)-5 CAPRIN-1 expression in human breast cancer metastasized lymph node
Immunohistochemical staining was performed according to a method
similar to that in (7)-3 above with 150 breast cancer metastasized lymph node
tissue specimens on a paraffin-embedded human breast cancer metastasized lymph
node tissue array (BIOMAX), using the polyclonal antibody against the
CAPRIN-1-derived peptide (SEQ ID NO: 37) prepared in (5) above. As a result,
the strong expression of CAPRIN-1 was observed in 136 out of a total of 150
breast cancer metastasized lymph node tissue specimens (90%). Specifically, it
was revealed that CAPRIN-1 was strongly expressed also in cancer tissues that
had metastasized from breast cancer. In addition, similar results were obtained in
the case of using the monoclonal antibody #1, #2, or #3 obtained in Example 3.
[0169]
(7)-6 CAPRIN-1 expression in various human cancer tissues
Immunohistochemical staining was performed according to a method
similar to the above with specimens on various paraffin-embedded human cancer
tissue arrays (BIOMAX), using the polyclonal antibody against the
CAPRIN-1-derived peptide (SEQ ID NO: 37) prepared in (5) above. As a result,
the strong expression of CAPRIN-1 was observed in esophageal cancer, colon
cancer, rectal cancer, lung cancer, renal cancer, bladder cancer, and uterine cervix
cancer. In addition, similar results were obtained in the case of using the
monoclonal antibody #1, #2, or #3 obtained in Example 3.
[0170]
Example 2 Preparation of human CAPRIN-1
(1) Preparation of recombinant protein
Based on the gene of SEQ ID NO: 1 obtained in Example 1, a
recombinant human CAPRIN-1 protein was prepared by the following method.
PCR was performed in a total volume of 50 |_d with 1 jal of cDNA, two primers
(SEQ ID NOS: 38 and 39 comprising Sac I and Xho I restriction enzyme cleavage
sequences) of 0.4 \\M each, 0.2 mM dNTP, and 1.25U PrimeSTAR HS polymerase
(Takara Shuzo Co., Ltd.), prepared by adding the reagents and an accompanying
buffer. The expression had been confirmed by an RT-PCR method for the cDNA
used herein from among various human-derived tissue- or cell-derived cDNAs
prepared in Example 1. PCR was preformed by repeating a cycle of 98°C for 10
seconds and 68°C for 2.5 minutes 30 times using a Thermal Cycler (BIO RAD).
The above two primers are capable of amplifying a region encoding the entire
amino acid sequence of SEQ ID NO: 2. After PCR, the thus amplified DNA was
subjected to electrophoresis on 1% agarose gel, and then an about 2.1 kbp DNA
fragment was purified using a QIAquick Gel Extraction Kit (QIAGEN).
[0171]
The thus purified DNA fragment was ligated to a cloning vector
PCR-Blunt (Invitrogen). After transformation of Escherichia coli with it,
plasmid was collected. It was verified by sequencing that the thus amplified gene
fragment has the sequence of interest. The plasmid having a matched sequence
with the sequence of interest was treated with Sac I and Xho I restriction enzymes
and then purified with a QIAquick Gel Extraction Kit. The gene sequence of
interest was inserted into an Escherichia coli expression vector pET30a (Novagen)
treated with Sac I and Xho I restriction enzymes. A His-tag fused recombinant
protein can be produced using the vector. The plasmid was transformed into
Escherichia coli for recombinant expression, BL21(DE3), and then expression was
induced with 1 mM IPTG, so that the protein of interest was expressed in
Escherichia coli.
[0172]
(2) Purification of recombinant protein
The above-obtained recombinant Escherichia coli expressing the gene of
SEQ ID NO: 1 was cultured in LB medium containing 30 ng/ml kanamycin at
37°C until absorbance at 600 nm reached around 0.7,
isopropyl-p-D-1-thiogalactopyranoside was added at a final concentration of 1
raM, and then cells were cultured at 37°C for 4 hours. Subsequently,
centrifugation was performed at 4800 rpm for 10 minutes and then cells were
collected. The resulting cell pellet was suspended in phosphate buffered saline
and centrifuged at 4800 rpm for 10 minutes, and then cells were washed.
[0173]
The cells were suspended in phosphate buffered saline and then
disrupted by ultrasonication on ice. The resulting lysate of the ultrasonicated
Escherichia coli was subjected to centrifugation at 6000 rpm for 20 minutes, and
then the resulting supernatant was regarded as a soluble fraction and the
precipitate was regarded as an insoluble fraction.
[0174]
The soluble fraction was added to a nickel chelate column adjusted
according to a conventional method (carrier: Chelating Sepharose™ Fast Flow
(GE HealthCare); column capacity of 5 ml; and equilibration buffer: 50 mM
hydrochloride buffer (pH 8.0)). Unadsorbed fractions were washed off with 50
mM hydrochloride buffer (pH 8.0) in an amount 10 times the column capacity and
20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole. Immediately
after washing, 6 beds were eluted with 20 mM phosphate buffer (pH 8.0)
containing 100 mM imidazole. The elution of the protein of interest was
confirmed by Coomassie staining on the elution fraction with 20 mM phosphate
buffer (pH 8.0) containing 100 mM imidazole, and then the elution fraction was
added to a strong anion exchange column (carrier: Q Sepharose™ Fast Flow (GE
HealthCare); column capacity of5 ml; and 20 mM phosphate buffer (pH 8.0) as an
equilibration buffer). An unadsorbed fraction was washed off with 20 mM
phosphate buffer (pH 7.0) in an amount 10 times the column capacity and 20 mM
phosphate buffer (pH 7.0) containing 200 mM sodium chloride. Immediately
after washing, 5 beds were eluted with 20 mM phosphate buffer (pH 7.0)
containing 400 raM sodium chloride, and thus the purified fraction of the protein
having the amino acid sequence represented by SEQ ID NO: 2 was obtained.
[0175]
200 \i\ of each purified sample obtained by the above method was
dispensed into 1 ml of reaction buffer (20 mM Tris-Hcl, 50 mM, NaCl, 2 mM
CaCl2' pH 7.4), followed by addition of 2 ul of enterokinase (Novagen). After
that, the resultant was left to stand overnight at room temperature for reaction so
that His-tag was cleaved off, and then purification was performed using an
Enterokinase Cleavage Capture Kit (Novagen) according to the accompanying
protocols. Next, 1.2 ml of the purified sample obtained by the above method was
subjected to the buffer replacement with physiological phosphate buffer (Nissui
Pharmaceutical Co., Ltd.) using an ultrafiltration NANOSEP 10K OMEGA
(PALL). Further, sterile filtration was performed using HT Tuffryn Acrodisc
0.22 p,m (PALL) and then the resultant was used for the following experiment.
[0176]
Example 3 Preparation of mouse monoclonal antibody against CAPRIN-1
100 u-g of the antigen protein (human CAPRIN-1) comprising the amino
acid sequence of SEQ ID NO: 2 prepared in Example 2 was mixed with an
equivalent amount of MPL+TDM adjuvant (Sigma), and then this was used as an
antigen solution per one mouse. The antigen solution was intraperitoneally
administered to 6-week-old Balb/cc mice (Japan SLC Inc.), and then the
administration was performed 7 times every week, and thus immunization was
completed. Each spleen was excised 3 days after the final immunization, and
sandwiched between two sterilized slide glasses and then crushed. The resultant
was washed with PBS(-) (Nissui) and then centrifuged at 1500 rpm for 10 minutes
to remove the supernatant. This procedure was repeated 3 times, so that
splenocytes were obtained. The thus obtained splenocytes and mouse myeloma
cells SP2/0 (purchased from ATCC) were mixed at a ratio of 10 : 1. A PEG
solution prepared by mixing 200 \i\ of RPMI1640 medium containing 10% FBS
heated at 37°C and 800 ul of PEG1500 (Boehringer) was added to the mixture, left
to stand for 5 minutes for cell fusion, and then subjected to centrifugation at 1700
rpm for 5 minutes. After removal of the supernatant, cells were suspended in 150
ml of RPMI1640 medium containing 15% FBS, supplemented with a HAT solution
(Gibco) (2% equivalent) (HAT selective medium), and then the cell suspension
was plated on fifteen 96-well plates (Nunc) at 100 ul per well. Cells were
cultured for 7 days at 37°C under conditions of 5% C02, so that hybridomas
prepared by fusion of splenocytes and myeloma cells were obtained.
[0177]
Hybridomas were selected using as a marker the binding affinity of the
antibody produced by the prepared hybridomas to human CAPRIN-1. The
CAPRIN-1 protein solution (1 |ig/ml) prepared in Example 2 was added to a
96-well plate at 100 ul per well and then left to stand at 4°C for 18 hours. Each
well was washed 3 times with PBS-T, 400 ul of a 0.5% Bovine Serum Albumin
(BSA) solution (SIGMA) was added per well, and then the plate was left to stand
at room temperature for 3 hours. The solution was removed, and then the wells
were washed three times with 400 u.1 of PBS-T per well. The culture supernatant
of the above-obtained hybridomas was added at 100 ul per well, and then left to
stand at room temperature for 2 hours. After washing each well three times with
PBS-T, an HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen) diluted
5000-fold with PBS was added at 100 u.1 per well and the resultant was then left to
stand at room temperature for 1 hour. After washing the wells three times with
PBS-T, 100 p,l of a TMB substrate solution (Thermo) was added per well and then
left to stand for 15 to 30 minutes for coloring reaction. After color development,
100 ul of IN sulfuric acid was added per well to stop the reaction, and then
absorbances at 450 nm and 595 nm were measured using an absorption
spectrometer. As a result, several hybridomas producing antibodies with high
absorbance values were selected.
[0178]
The thus selected hybridomas were added to a 96-well plate at 0.5 cells
per well and then cultured. After 1 week, hybridomas that had formed single
colonies in wells were observed. These cells in the wells were further cultured,
and then hybridomas were selected using as a marker the binding affinity of
antibodies produced by the cloned hybridomas to the human CAPRIN-1. The
CAPRIN-1 protein solution (1 (ag/ml) prepared in Example 3 was added to a
96-well plate at 100 ul per well, and then left to stand at 4°C for 18 hours. Each
well was washed with PBS-T three times, 400 ul of a 0.5% BSA solution was
added per well, and then the resultant was left to stand at room temperature for 3
hours. The solution was removed, and then the wells were washed three times
with 400 u.1 of PBS-T per well. 100 u.1 of each culture supernatant of the
above-obtained hybridomas was added per well, and then the plate was left to
stand at room temperature for 2 hours. After washing each well three times with
PBS-T, 100 ul of an HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen)
diluted 5000-fold with PBS was added per well and then left to stand at room
temperature for 1 hour. After washing the wells three times with PBS-T, 100 \iof a TMB substrate solution (Thermo) was added per well, and then left to stand
for 15 to 30 minutes for coloring reaction. After color development, 100 ul of
IN sulfuric acid was added per well to stop the reaction and then absorbances at
450 nm and 595 nm were measured using an absorption spectrometer. As a
result, 50 hybridoma cell lines producing monoclonal antibodies immunoreactive
with the human CAPRIN-1 were obtained.
[0179]
Next, of those monoclonal antibodies, antibodies reactive with the cell
surfaces of breast cancer cells expressing human CAPRIN-1 were selected.
Specifically, 106 cells of the MDA-MB-231V human breast cancer cell line were
centrifuged in a 1.5-ml microcentrifuge tube, and 100 ul of the culture supernatant
of each of the above hybridomas was added to the tube, and then the tube was left
to stand on ice for 1 hour. After washing with PBS, an FITC-labeled goat
anti-mouse IgG antibody (Invitrogen) diluted 500-fold with PBS containing 0.1%
FBS was added, and then left to stand on ice for 1 hour. After washing with PBS,
fluorescence intensity was measured using a FACS caliber (Becton, Dickinson and
Company). Meanwhile, procedures similar to the above were performed using
the serum of a 6-week-old Balb/c mouse that had not been treated with antibodies
and had been diluted 500-fold with medium for culturing hybridomas, so that a
control sample was obtained. As a result, three monoclonal antibodies
(monoclonal antibodies #1, #2, and #3) that had exhibited fluorescence intensity
stronger than that of the control, and that is, that reacted with the cell surfaces of
breast cancer cells, were selected.
[0180]
Example 4 Characterization of selected antibodies
(1) Cloning of genes of anti-human CAPRIN-1 mouse monoclonal antibody
variable regions
mRNA was extracted from each hybridoma cell line producing one of the
three monoclonal antibodies selected in Example 3. An RT-PCR method using
primers specific to the mouse FR1 -derived sequence and the mouse FR4-derived
sequence was performed therefor, and the genes of the heavy chain variable (VH)
regions and the genes of the light chain variable (VL) regions of all
anti-CAPRlN-1 monoclonal antibodies were obtained. For sequence
determination, these genes were cloned into a pCR2.1 vector (Invitrogen).
[0181]
(2) RT-PCR
mRNA was prepared from 106 cells of each hybridoma cell line using a
mRNA micro purification kit (GE HealthCare). The thus obtained mRNA was
reverse-transcribed and then cDNA was synthesized using a SuperScriptll 1st
strand Synthesis Kit (Invitrogen). These procedures were performed according to
protocols attached to each kit.
[0182]
Antibody gene amplification was performed by a PCR method using the
thus obtained cDNA.
[0183]
To obtain the gene of the VH region, a primer (SEQ ID NO: 54) specific
to the mouse heavy chain FR1 sequence and a primer (SEQ ID NO: 55) specific to
the mouse heavy chain FR4 sequence were used. Furthermore, to obtain the gene
of the VL region, a primer (SEQ ID NO: 56) specific to the mouse light chain FR1
sequence and a primer (SEQ ID NO: 57) specific to the mouse light chain FR4
were used. These primers were designed in reference to Jones, S. T. and Bending,
M. M. Bio/Technology 9, 88-89 (1991). Ex-taq (Takara Bio Inc.) was used for
PCR. A cDNA sample was added to 5 pi of 10 x EX Taq Buffer, 4 pi of dNTP
Mixture (2.5 mM), primers (1.0 pM) (2 pi each), and 0.25 pi of Ex Taq (5 U/pl),
and then the total amount thereof was adjusted with sterile water to 50 pi. PCR
was performed under the conditions: 2 minutes of treatment at 94°C, followed by
30 cycles of 1 minute of denaturation at 94°C, 30 seconds of annealing at 58°C,
and 1 minute of extension reaction at 72°C.
[0184]
(3) Cloning
The thus obtained PCR products were each subjected to agarose gel
electrophoresis, and DNA bands of the VH region and the VL region were excised.
DNA fragments were purified using a QIAquick Gel purification kit (QIAGEN)
according to the accompanying protocols. The purified DNA was cloned into a
pCR2.1 vector using a TA cloning kit (Invitrogen). The ligated vector was
transformed into DH5a competent cells (TOYOBO) according to a conventional
method. 10 clones of each transformant were cultured overnight in medium (100
|^g/ml ampicillin) at 37°C, and then plasmid DNA was purified using a Qiaspin
Miniprep kit (QIAGEN).
[0185]
(4) Sequence determination
The gene sequences of the VH region and the VL region in each plasmid
obtained above were analyzed with an M13 forward primer (SEQ ID NO: 58) and
an M13 reverse primer (SEQ ID NO: 59) on a fluorescence sequencer (DNA
sequencer 3130XL; ABI), using a Big Dye Terminator Ver3.1 Cycle Sequencing
Kit (ABI) according to the accompanying protocols. As a result, each gene
sequence was determined. The sequences were identical among the 10 clones.
[0186]
The thus obtained gene sequences encoding the monoclonal antibody
heavy chain variable regions are shown by SEQ ID NOS: 52 and 68, and the amino
acid sequences thereof are shown by SEQ ID NOS: 43 and 63; and the gene
sequences encoding the light chain variable regions are shown by SEQ ID NOS: 70,
53, and 69 and the amino acid sequences thereof are shown by SEQ ID NOS: 47,
51, and 67. Specifically, it was revealed that the monoclonal antibody #1
comprises the heavy chain variable region of SEQ ID NO: 43 and the light chain
variable region of SEQ ID NO: 47, and #2 comprises the heavy chain variable
region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51,
and #3 comprises the heavy chain variable region of SEQ ID NO: 63 and the light
chain variable region of SEQ ID NO: 67.
[0187]
Example 5 Identification of CAPRIN-1 epitope to be recognized by
anti-CAPRIN-1 monoclonal antibodies #1, #2 and #3
CAPRIN-1 epitope regions recognized by the anti-CAPRIN-1
monoclonal antibodies #1, #2, and #3 (obtained in Example 3) reactive with cancer
cell surfaces were identified.
[0188]
93 candidate peptides, each comprising 12 to 16 amino acids in the
amino acid sequence of the human CAPRIN-1 protein, were synthesized, and then
each peptide was dissolved in DMSO at a concentration of 1 mg/ml. Each
peptide was dissolved in 0.1 M sodium carbonate buffer (pH 9.6) at a
concentration of 30 pg/ml, added to a 96-well plate (Nunc, Product No. 436006) at
100 ul per well, and then left to stand overnight at 4°C. The solution was
discarded, 10 mM ethanolamine/0.1 M sodium carbonate buffer (PH9.6) was added
at 200 ul per well, and then the resultant was left to stand at room temperature for
1 hour. The solution was discarded and then the plate was washed twice with
PBS containing 0.5% Tween 20 (PBST), so that a plate onto which each peptide
had been immobilized was prepared.
[0189]
The cell culture supernatant containing the mouse monoclonal antibodies
(#1, #2, and #3) obtained in Example 3 was added at 50 u.1 per well, and then
shaken at room temperature for 1 hour. The solution was removed, followed by
three times of washing with PBST. Next, a secondary antibody solution prepared
by diluting a HRP-labeled anti-mouse IgG (Invitrogen) antibody 3000- to
4000-fold with PBST was added (50 jutl each) to the mouse monoclonal antibodies.
The solution was removed, followed by six times of washing with PBST.
[0190]
A TMB substrate solution (Thermo) was added at 100.pl per well and
then left to stand for 15 to 30 minutes for coloring reaction. After color
development, 1 N sulfuric acid was added at 100 pi per well to stop the reaction,
and then absorbances at 450 nm and 595 nm were measured using an absorption
spectrometer. As a result, a polypeptide comprising the amino acid sequence of
SEQ ID NO: 37 was identified as a partial CAPRIN-1 sequence recognized by both
anti-CAPRIN-1 monoclonal antibodies #1 and #2.
[0191]
Therefore, it was revealed that the polypeptide of SEQ ID NO: 37, a
partial sequence of human CAPRIN-1, contains the epitope regions for the
anti-CAPRIN-1 monoclonal antibodies #1, #2, and #3.
[0192]
Example 6 CAPRIN-1 expression on various cancer cell surfaces using
anti-CAPRIN-1 antibodies #1, #2, and #3
Next, 7 breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1,
MDA-MB-231V, BT20, SK-BR-3, and DA-MB-231T) for which CAPRIN-1 gene
expression had been observed, and the other 3 breast cancer cell lines
(MDA-MB-231C, MCF-7, and ZR75-1), 5 glioma cell lines (T98G, SNB19,
U251, U87MG, and U373), 4 renal cancer cell lines (Caki-1, Caki-2, A498, and
ACHN), 2 gastric cancer cell lines (MKN28 and MKN45), 5 colorectal cancer cell
lines (HT29, LoVo, Caco2, SW480, and HCT116), 3 lung cancer cell lines (A549,
QG56, and PC8), 4 leukemia cell lines (AML5, Namalwa, BDCM, RPI1788), 1
uterine cervix cancer cell line (SW756), 1 bladder cancer cell line (T24), 1
esophageal cancer cell line (KYSE180), and 1 lymphoma cell line (Ramos) were
examined for CAPRIN-1 protein expression on the cell surfaces of each cell line
using the culture supernatants containing #1, #2, and #3 obtained in Example 3.
106 cells of each cell line were centrifuged in a 1.5 ml microcentrifuge tube.
Each cell culture supernatant (100 ul) containing #1, #2, or #3 was added and then
left to stand on ice for 1 hour. After washing with PBS, a FITC-labeled goat-anti
mouse IgG (H+L) antibody (SouthernBiotech) diluted 500-fold with PBS
containing 0.1% FBS was added and then the resultant was left to stand on ice for
1 hour. After washing with PBS, fluorescence intensity was measured using a
FACS Calibur (Becton, Dickinson and Company). Meanwhile, a sample
subjected to a reaction with only a secondary antibody was used as a negative
control. As a result, cells to which the antibodies #1, #2, and #3 had been added
exhibited fluorescence intensity stronger by 20% or more than that of the negative
control. It was revealed by these results that the CAPRIN-1 protein was
expressed on the cell membrane surfaces of the above human cancer cell lines.
The percentage of enhancement in the above fluorescence intensity was expressed
as percentage of increase in mean fluorescence intensity (MFI level) in each type
of cell and calculated by the following formula.
[0193]
Percentage of increase in mean fluorescence intensity (percentage of enhancement
in fluorescence intensity)(%) = ((MFI level in cells having reacted with
anti-human CAPRIN-1 antibody) - (MFI level of control)) / (MFI level of control)
x 100.
[0194]
Example 7 Anti-tumor effects (ADCC activity and CDC activity) of
anti-CAPRIN-1 antibodies on cancer cells
Whether or not anti-CAPRIN-1 antibodies could damage cancer cells
expressing CAPRIN-1 was examined by measuring ADCC activity first.
Evaluation was performed using a polyclonal antibody against the human
CAPRIN-1-derived peptide (SEQ ID NO: 37) prepared in Example 1. 106 cells of
the MDA-MB-157 human breast cancer cell line for which CAPRIN-1 expression
had been confirmed were collected in a 50-ml centrifugal tube, 100 (j,Ci
chromium-51 was added, and then incubation was performed at 37°C for 2 hours.
Subsequently, the resultant was washed three times with RPMI1640 medium
containing 10% fetal calf serum. Cells were added to a 96-well V-bottom plate at
103 cells per well. 1 fag of the polyclonal antibody against the above human
CAPRIN-1-derived peptide was added to the wells and then lymphocytes (2 x 105
each) separated from rabbit peripheral blood were added, followed by 4 hours of
culture at 37°C under conditions of 5% CO2. After culture, the amount of
chromium (Cr)-51 released from damaged cancer cells in a culture supernatant was
measured, so that the ADCC activity of the polyclonal antibodies against the
human CAPRIN-1-derived peptide against cancer cells was calculated. As a
result, 18.1% ADCC activity against MDA-MB-157 was observed (see Fig. 2).
On the other hand, when similar procedures were performed using a control
antibody (Example 1 (5)) prepared from peripheral blood of a rabbit not
immunized with the antigen, and when no antibody was added, almost no activity
was observed (see Fig. 2). Therefore, it was revealed that the anti-CAPRIN-1
antibody can damage cancer cells expressing CAPRIN-1 by ADCC activity.
[0195]
Herein, the cytotoxic activity was determined as a cytotoxic activity
against a cancer cell line. Specifically, as described above, the result was
obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention,
a rabbit lymphocyte, and 103 cells of each cancer cell line that had incorporated
chromium-51, culturing cells for 4 hours, measuring the amount of chromium-51
released in medium after culture, and then calculating the cytotoxic activity
against the cancer cell line by the following formula *.
[0196]
* Formula: cytotoxic activity (%) = (the amount of chromium-51 released from
cancer cells upon addition of an anti-CAPRIN-1 antibody and a rabbit
lymphocyte) / (the amount of chromium-51 released from target cells to which IN
hydrochloric acid had been added) x 100.
[0197]
Next, the anti-CAPRIN-1 mouse monoclonal antibodies #1, #2, and #3
(obtained in Example 3) were evaluated for their cytotoxic activity against cancer
cells. Each cell culture supernatant producing #1, #2, or #3 was purified using
Hitrap ProteinA Sepharose FF (GE HealthCare), subjected to buffer replacement
with PBS(-), and then filtered with a 0.22 \xm filter (Millipore). The resultants
were used as antibodies for activity measurement. 106 cells of the MDA-MB-157
human breast cancer cell line were collected in a 50-ml centrifuge tube, 100 jaCi
chromium-51 was added, and then incubation was performed at 37°C for 2 hours.
Subsequently, the resultant was washed three times with RPMI1640 medium
containing 10% FBS. Cells were added to a 96-well V-bottom plate at 103 cells
per well for use as target cells. The above purified antibodies (1 |ig each) were
added to the cells. 5x10 cells of mouse splenocytes isolated from the spleen of a
6-week-old BALB/C mouse (Japan SIX Inc.) according to a conventional method
were further added and then cultured for 4 hours at 37°C under conditions of 5%
CO2. After culture, the amount of chromium-51 released from damaged tumor
cells in a culture supernatant was measured, and the cytotoxic activity of each
anti-CAPRIN-1 antibody against cancer cells was calculated. As negative
control samples, a sample prepared by adding PBS instead of the antibodies and a
sample prepared by adding an isotype control antibody instead of the antibodies
were used. As a result, the antibodies #1, #2, and #3 exhibited more than 25%
cytotoxic activity against MDA-MB-157. In contrast, the activity in the sample
prepared by adding PBS as a negative control and the activity in the sample
prepared by adding the isotype control antibody as a negative control were 1.7%
and 3.0%, respectively. Similarly, the antibodies #1, #2, and #3 were examined
for their cytotoxic activity against other cancer cells including glioma cell lines
T98G and U373, lung cancer cell lines A549 and QG56, renal cancer cell lines
Caki-1 and ACHN, a uterine cervix cancer cell line SW756, a bladder cancer cell
line T24, an esophageal cancer cell line KYSE180, gastric cancer cell lines
MKN28 and MKN45, a colorectal cancer cell line SW480, a leukemia cell line
AML5, and a lymphoma cell line Ramos. As a result, the antibody #1 exhibited
13.2% against T98G (2.5% in the case of isotype control), 16.0% against U373
(4.3% in the case of isotype control), 12.0% against A549 (4.5% in the case of
isotype control), 12.6% against QG56 (5.3% in the case of isotype control), 10.3%
against Caki-1 (4.5% in the case of isotype control), 9.0% against ACHN (3.8% in
the case of isotype control), 8.6% against SW756 (5.1% in the case of isotype
control), 13.0%o against T24 (3.8% in the case of isotype control), 8.9% against
KYSE180 (5.7%o in the case of isotype control), 16.2% against MKN28 (4.2% in
the case of isotype control), 12.1% against MKN45 (4.6% in the case of isotype
control), 13.4%) against SW480 (6.4% in the case of isotype control), 8.9% against
AML5 (4.7% in the case of isotype control), and 8.1% against Ramos (2.5%o in the
case of isotype control). Also the antibodies #2 and #3 exhibited similar results.
It was demonstrated by the above results that the thus obtained anti-CAPRIN-1
antibodies #1, #2, and #3 damage cancer cells expressing CAPRIN-1 by ADCC
activity. It was demonstrated by the above results that the thus obtained
anti-CAPRIN-1 mouse monoclonal antibodies #1, #2, and #3 damage cancer cells
expressing CAPRIN-1 by ADCC activity.
[0198]
Herein, the cytotoxic activity was determined as a cytotoxic activity
against a cancer cell line. Specifically, as described above, the result was
obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention,
a mouse splenocyte, and 103 cells of each cancer cell line that had incorporated
chromium-51, culturing cells for 4 hours, measuring the amount of chromium-51
released in medium after culture, and then calculating the cytotoxic activity
against the cancer cell line by the following formula*.
[0199]
* Formula: cytotoxic activity (%) = (the amount of chromium-51 released from
cancer cells upon addition of an anti-CAPRIN-1 antibody and a mouse splenocyte)
/ (the amount of chromium-51 released from target cells to which IN hydrochloric
acid had been added) x 100.
[0200]
Next, the obtained anti-CAPRIN-1 mouse monoclonal antibodies #1 and
#2 were evaluated for their cytotoxic activity (CDC activity) against cancer cells.
Blood collected from a rabbit was added to an Eppendorf tube, left to stand at
room temperature for 60 minutes, and then subjected to 5 minutes of
centrifugation at 3000 rpm. Thus, serum for measurement of CDC activity was
prepared. 105 cells of the MDA-MB-231V human breast cancer cell line were
collected in a 50-ml centrifuge tube, 100 u.Ci chromium-51 was added, and then
incubation was performed at 37°C for 2 hours. The resultant was washed three
times with RPMI medium containing 10% FBS. Subsequently, the cells were
suspended in RPMI medium containing the above-prepared rabbit serum (50%),
and then added to a 96-well V-bottom plate at 103 cells per well. 1 u.g each of the
mouse monoclonal antibodies #1 and #2 was added to the cells and then cells were
cultured for 4 hours at 37°C under conditions of 5% CO2. After culture, the
amount of chromium-51 released from damaged tumor cells in a culture
supernatant was measured, and then the CDC activity of each antibody against
MDA-MB-231V was calculated. As a result, the antibodies #1 and #2 both
exhibited more than 20% CDC activity. Also, no cytotoxic activity was observed
in a negative control group to which no antibody had been added. Therefore, it
was revealed that the antibodies #1 and #2 can damage tumor cells expressing
CAPRIN-1 also by CDC activity.
[0201]
Herein, the cytotoxic activity was determined as a cytotoxic activity
against a cancer cell line. Specifically, as described above, the result was
obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention,
serum, and 103 cells of each cancer cell line that had incorporated chromium-51,
culturing cells for 4 hours, measuring the amount of chromium-51 released in
medium after culture, and then calculating the cytotoxic activity against the cancer
cell line by the following formula*.
[0202]
* Formula: cytotoxic activity (%) = (the amount of chromium-51 released from
cancer cells upon addition of an anti-CAPRIN-1 antibody and serum) / (the
amount of chromium-51 released from target cells to which IN hydrochloric acid
had been added) x 100.
[0203]
Next, the thus obtained anti-CAPRIN-1 mouse monoclonal antibodies #1
and #2 were evaluated for their in vivo anti-tumor effects in tumor-bearing mice.
Antibodies used herein were prepared by column purification of the culture
supernatant of each cell producing #1 or #2 in the same manner as described
above.
[0204]
The anti-tumor effects of the antibodies #1 and #2 were examined using
tumor-bearing mice into which a mouse-derived cancer cell line expressing
CAPRIN-1 had been transplanted. 4T1 cells (purchased from ATCC) were
transplanted subcutaneously to the dorsal region of 30 Balb/c mice (Japan SLC
Inc.) at 5x105 cells/mouse. Tumors were allowed to grow to reach a size of about
5 mm in diameter. The antibodies #1 and #2 were each administered
intraperitoneally to 20 tumor-bearing mice from among the 30 tumor-bearing mice
in an amount of 200 p.g (200 ul)/mouse (each antibody was administered to 10
mice). Subsequently, the same amount of the antibody was administered
intraperitoneally to each tumor-bearing mouse 3 times in total within 2 days.
Tumor sizes were measured every day and anti-tumor effects were examined by
observation. Meanwhile, as a control group, PBS (-) was administered instead of
the antibodies to the remaining 10 tumor-bearing mice. The tumor size was
calculated as a volume using the formula: length of major axis x length of minor
axis x length of minor axis x 0.5.
[0205]
As a result of observation of anti-tumor effects, in the examination
group to which the anti-CAPRIN-1 mouse monoclonal antibodies #1 and #2 had
been administered, tumors were found to have almost completely regressed up to
day 16 after the administration of the antibodies. On the other hand, in the
control group to which PBS(-) had been administered, tumors were found to have
increased to about 820% on day 12. It was demonstrated by these results that the
obtained anti-human CAPRIN-1 mouse monoclonal antibodies #1 and #2 exhibit
strong in vivo anti-tumor effects on cancer cells expressing CAPRIN-1.
Industrial Applicability
[0206]
The antibodies of the present invention are useful for treating and/or
preventing cancer.
[0207]
All publications, patents, and patent applications cited herein are
incorporated herein by reference in their entirety.
Sequence Listing Free Text
[0208]
SEQ ID NO: 31: T3 primer
SEQ ID NO: 32: T7 primer
SEQ ID NOS: 33, 34, 38, 39, and 54-59: primers
SEQ ID NOS: 35 and 36: GAPDH primers
We Claim:
1. A pharmaceutical composition for treating and/or preventing a cancer,
comprising an antibody or a fragment thereof as an active ingredient that has
immunological reactivity with a partial polypeptide of CAPRIN-1, wherein
CAPRIN-1 is represented by any of the even-numbered sequences of SEQ ID
NOS: 2 to 30, and wherein the partial polypeptide comprises the amino acid
sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or
more sequence identity with the amino acid sequence of SEQ ID NO: 37.
2. The pharmaceutical composition according to claim 1, wherein the cancer is
breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mastocytoma, renal
cancer, uterine cervix cancer, bladder cancer, esophageal cancer, gastric cancer, or
colorectal cancer.
3. The pharmaceutical composition according to claim 1 or 2, wherein the
antibody is a monoclonal antibody or a polyclonal antibody.
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein
the antibody is a human antibody, humanized antibody, chimeric antibody, single
chain antibody, or bispecific antibody.
5. An antibody having immunological reactivity with a polypeptide that comprises
the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence
having 80% or more sequence identity with the amino acid sequence of SEQ ID
NO: 37.
6. The antibody according to claim 5, which has a cytotoxic activity against a
cancer cell expressing a CAPRIN-1 protein.
7. An antibody, which comprises a heavy chain variable region comprising SEQ ID
NOS: 40, 41, and 42 and a light chain variable region comprising SEQ ID NOS: 44,
45, and 46, and has immunological reactivity with a CAPRIN-1 protein.
8. An antibody, which comprises a heavy chain variable region comprising SEQ ID
NOS: 40, 41, and 42 and a light chain variable region comprising SEQ ID NOS: 48,
49, and 50 and has immunological reactivity with a CAPRIN-1 protein.
9. An antibody, which comprises a heavy chain variable region comprising SEQ ID
NOS: 60, 61, and 62 and a light chain variable region comprising SEQ ID NOS: 64,
65, and 66, and has immunological reactivity with a CAPRIN-1 protein.
10. The antibody according to any one of claims 5 to 9, which is a human antibody,
humanized antibody, chimeric antibody, single chain antibody, or bispecific
antibody.
11. A pharmaceutical composition for treating and/or preventing a cancer,
comprising the antibody or a fragment thereof of any one of claims 5 to 10 as an
active ingredient.
12. The pharmaceutical composition according to claim 11, wherein the cancer is
breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mastocytoma, renal
cancer, uterine cervix cancer, bladder cancer, esophageal cancer, gastric cancer, or
colorectal cancer.
13. A pharmaceutical combination for treating and/or preventing a cancer,
comprising the pharmaceutical composition of any one of claims 1 to 4 or the
pharmaceutical composition of claim 11 or 12, and a pharmaceutical composition
containing an antitumor agent.
14. A method for treating and/or preventing a cancer, comprising administering to
a subject the antibody or a fragment thereof of any one of claims 5 to 10 or the
pharmaceutical composition of claim 11 or 12.
15. A method for treating and/or preventing a cancer, comprising using
pharmaceutical compositions of the pharmaceutical combination of claim 13 in
combination in a subject.