DESCRIPTION
Title of Invention: Medicament for Treating and/or Preventing Cancer
Technical Field
[0001]
The present invention relates to a medicament for treating and/or preventing a
cancer, characterized by combining an antibody or a fragment thereof having immunological
reactivity with a CAPRIN-1 protein, with an antitumor agent, and to the use of the same.
Background Art
[0002]
Cancer is the leading cause of death. Current therapies for cancer comprise
combinations of principal surgical therapy with radiation therapy and chemotherapy.
Furthermore, the current therapies comprise applying a similar therapy to all patients having
the same type and the same stage of a cancer . At least 40% of the patients fail a primary
therapy and thus are subjected to a series of further therapies. If the patients again fail the
therapies, cancer metastasis takes place, finally resulting in an increased possibility of death.
Thus, the current radiation therapy and the chemotherapy are unable to compatible with
various types of cancers or individual cancer patients, and the surgical therapy itself is also
currently insufficient for complete cure of cancers in almost all cases.
[0003]
Various antibody drugs targeting antigen proteins on cancer cells for treatment of
cancers have appeared throughout the world as a technique for overcoming the
above-described problems of cancer therapies. Specific examples are as follows. It has
been demonstrated that HERCEPTIN (registered trademark) comprising as an active
ingredient a monoclonal antibody specifically binding to Her2, the sales of which were
approved in 1998 as a therapeutic agent for patients with metastatic breast cancer, has such a
clinical effect that HERCEPTIN can decrease the number of death of recurrent and metastatic
breast cancer patients among Her2-overexpressing metastatic breast cancer patients. It has
also been demonstrated that HERCEPTIN does not cause any severe side effects other than
cardiac toxicity compared with conventional chemotherapeutics. As another noteworthy
feature, the therapeutic effects of a combined use of HERCEPTIN with chemotherapeutics
against breast cancer have been demonstrated (Patent Literatures 1-3) . However, most
antigenic proteins on cancer cells to be targeted by antibody drugs such as Her2 are also
expressed in normal cells, so that not only cancer cells but also normal cells expressing
antigens are also cytotoxically impaired by administration of antibodies. The resulting side
effects may cause for concern.
[0004]
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 (M11S1), 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 1). 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 2). 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 2).
[0005]
In addition, on the basis of the report of Non-patent Literature 1 that CAPRIN-1 is a
cell membrane protein, Patent Literatures 4 and 5 describe that CAPRIN-1 (as a cell
membrane protein) under the name of M11S1 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 2, it has been commonly
believed from the time of the filing of Patent Literature 4 to date that CAPRIN-1 is not
expressed on the surface of a cell. The contents of Patent Literatures 4 and 5 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
[0006]
Patent Literature 1 Japanese Patent Publication (Kokai) No. 2006-316040A
Patent Literature 2 U.S. Patent No. 7485302
Patent Literature 3 U.S. Patent No. 7449184
Patent Literature 4 U.S. Patent Publication No. 2008/0075722
Patent Literature 5 International Publication WO2005/100998
Non-patent Literature
[0007]
Non-patent Literature 1 J. Biol. Chem., 270: 20717-20723, 1995
Non-patent Literature 2 J. Immunol., 172: 2389-2400, 2004
Summary of the Invention
Problem to be Solved by the Invention
[0008]
Objects of the present invention are to identify a cancer antigen protein specifically
expressed on the surface of a cancer cell, to combine an antibody targeting the cancer antigen
protein with an antitumor agent, and thus to provide use as a medicament for treating and/or
preventing a cancer.
Means for Solving the Problem
[0009]
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 proteins are specifically expressed in the
cells of cancers, such as breast cancer, brain tumor, leukemia, lymphoma, lung cancer, uterine
cervix cancer, bladder cancer, esophageal cancer, colorectal cancer, gastric cancer, and renal
cancer cells; 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 are
combined with a specific antitumor agent, so that significant cancer therapeutic effects can be
obtained. On the basis of these findings, the present invention as described below was
completed.
[0010]
The term "cancer" as used herein is used interchangeably with tumor or carcinoma.
[0011]
The present invention has the following characteristics.
[0012]
(1) A medicament for treating and/or preventing a cancer, comprising a combination of an
antibody or a fragment thereof having immunological reactivity with a CAPRIN-1 protein and
one or two or more types of antitumor agents, wherein the antibody or fragment and the
antitumor agent or antitumor agents are combined together or separately.
[0013]
(2) The medicament according to (1) above, wherein the antibody or a fragment thereof
having immunological reactivity with the above CAPRIN-1 protein is an antibody or a
fragment thereof, which binds specifically to the extracellular region of a CAPRIN-1 protein
existing on the surface of a cancer cell.
[0014]
(3) The medicament according to (1) or (2) above, wherein the antibody or a fragment thereof
having immunological reactivity with the above CAPRIN-1 protein is an antibody or a
fragment thereof, which binds specifically to a polypeptide having the amino acid sequence
represented by SEQ ID NO: 37 in the extracellular region of the CAPRIN-1 protein existing
on the surface of a cancer cell, or an amino acid sequence having 80% or more sequence
identity with the amino acid sequence represented by SEQ ID NO: 37.
[0015]
(4) The medicament according to any one of (1) to (3) above, wherein the above CAPRIN-1
protein is from a human.
[0016]
(5) The medicament according to any one of (1) to (4) above, wherein the above antitumor
agent is any of antitumor agents as described herein.
[0017]
(6) The medicament according to (5) above, wherein the antitumor agent is selected from the
group consisting of cyclophosphamide, paclitaxel, docetaxel, vinorelbine, and
pharmaceutically acceptable salts and derivatives thereof.
[0018]
(7) The medicament according to any one of (1) to (6) 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.
[0019]
(8) The medicament according to any one of (1) to (7) above, wherein the antibody is a
monoclonal antibody, a polyclonal antibody, or a recombinant antibody.
[0020]
(9) The medicament according to any one of (1) to (8) above, wherein the antibody is a
human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, or a
bispecific antibody.
[0021]
(10) A method for treating and/or preventing a cancer, comprising administering the
medicament of any one of (1) to (9) above to a subject suspected of having a cancer.
[0022]
(11) The method according to (10) above, comprising administering to a subject the antibody
or a fragment thereof and an antitumor agent, which are contained in the above medicament,
simultaneously or separately.
[0023]
This description includes all or part of the contents as disclosed in the description
and/or drawings of Japanese Patent Application No. 2010-023455, from which the present
application claims the priority.
Advantageous Effect of the Invention
[0024]
According to the present invention, surprising synergistic effects of massive cancer
reduction and regression can be obtained without detection of significant side effects.
Brief Description of the Drawings
[0025]
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 cytotoxicity exhibited by anti-CAPRIN-1 monoclonal antibodies (#1
to #11) that are reactive with the cell surface of the MDA-MB-157 breast cancer cell line
which expresses CAPRIN-1. Reference No. 3 indicates a cytotoxic activity exhibited when
the anti-CAPRIN-1 monoclonal antibody #1 was added. Reference No. 4 indicates a
cytotoxic activity exhibited when the anti- CAPRIN-1 monoclonal antibody #2 was added.
Reference No. 5 indicates a cytotoxic activity exhibited when the anti-CAPRIN-1 monoclonal
antibody #5 was added. Reference No. 6 indicates a cytotoxic activity exhibited when the
anti-CAPRIN-1 monoclonal antibody #4 was added. Reference No. 7 indicates a cytotoxic
activity exhibited when the anti-CAPRIN-1 monoclonal antibody #5 was added. Reference
No. 8 indicates a cytotoxic activity exhibited when the anti-CAPRIN-1 monoclonal antibody
#6 was added. Reference No. 9 indicates a cytotoxic activity exhibited when the
anti-CAPRIN-1 monoclonal antibody #7 was added. Reference No. 10 indicates a cytotoxic
activity exhibited when the anti-CAPRIN-1 monoclonal antibody #8 was added. Reference
No. 11 indicates a cytotoxic activity exhibited when the anti-CAPRIN-1 monoclonal antibody
#9 was added. Reference No. 12 indicates a cytotoxic activity exhibited when the
anti-CAPRIN-1 monoclonal antibody #10 was added. Reference No. 13 indicates a
cytotoxic activity exhibited when the anti-CAPRIN-1 monoclonal antibody #11 was added.
Reference No. 14 indicates a cytotoxic activity exhibited when a monoclonal antibody that is
reactive with a CAPRIN-1 protein itself but not reactive with the surface of the cancer cell
was added. Reference No. 15 indicates a cytotoxic activity exhibited when PBS was added
instead of the antibodies.
Fig. 3 shows the anti-tumor effect obtained when cyclophosphamide, an antitumor
agent, was used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with
the surface of cancer cells in nude mice, into which the breast cancer cell line MCF-7
expressing CAPRIN-1 has been transplanted. Reference No. 16 indicates the tumor size of the
mouse when PBS was added instead of the antibody. Reference No. 17 indicates the tumor
size of the mouse when cyclophosphamide was administered. Reference No. 18 indicates
the tumor size of the mouse when the anti-CAPRIN-1 monoclonal antibody #2 was
administered. Reference No. 19 indicates the tumor size of the mouse when
cyclophosphamide and anti-Her2 antibody were administered. Reference No. 20 indicates the
tumor size of the mouse when cyclophosphamide and the anti-CAPRIN-1 monoclonal
antibody #2 were administered.
Fig. 4 shows the anti-tumor effect obtained when paclitaxel, an antitumor agent,
was used in combination with an anti-CAPRIN-1 monoclonal antibody that is reactive with
the surface of cancer cells in nude mice into which the breast cancer cell line MCF-7
expressing CAPRIN-1 has been transplanted. Reference No. 21 indicates the tumor size of
the mouse when PBS was administered instead of the antibody. Reference No. 22 indicates
the tumor size of the mouse when paclitaxel was administered. Reference No. 23 indicates
the tumor size of the mouse when the anti-CAPRIN-1 monoclonal antibody #2 was
administered. Reference No. 24 indicates the tumor size of the mouse when paclitaxel and
anti-Her2 antibody were administered. Reference No. 25 indicates the tumor size of the
mouse when paclitaxel and the anti-CAPRIN-1 monoclonal antibody #2 were administered.
Fig. 5 shows the anti-tumor effect obtained when docetaxel, an antitumor agent, was
used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with the surface of
cancer cells in nude mice, into which the breast cancer cell line MCF-7 expressing CAPRIN-1
has been transplanted. Reference No. 26 indicates the tumor size of the mouse when PBS was
administered instead of the antibody. Reference No. 27 indicates the tumor size of the
mouse when docetaxel was administered. Reference No. 28 indicates the tumor size of the
mouse when the anti-CAPRIN-1 monoclonal antibody #2 was administered. Reference No.
29 indicates the tumor size of the mouse when docetaxel and anti-Her2 antibody were
administered. Reference No. 30 indicates the tumor size of the mouse when docetaxel and the
anti-CAPRIN-1 monoclonal antibody #2 were administered.
Fig. 6 shows the anti-tumor effect obtained when vinorelbine, an antitumor agent,
was used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with the
surface of cancer cells in nude mice, into which the breast cancer cell line MCF-7 expressing
CAPRIN-1 has been transplanted. Reference No. 31 indicates the tumor size of the mouse
when PBS was added instead of an antibody. Reference No. 32 indicates the tumor size of
the mouse when vinorelbine was administered. Reference No. 33 indicates the tumor size of
the mouse when the anti-CAPRIN-1 monoclonal antibody #2 was administered. Reference
No. 34 indicates the tumor size of the mouse when vinorelbine and anti-Her2 antibody were
administered. Reference No. 35 indicates the tumor size of the mouse when vinorelbine and
the anti-CAPRIN-1 monoclonal antibody #2 were administered.
Fig. 7 shows the anti-tumor effect obtained when cyclophosphamide, an antitumor
agent, was used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with
the surface of cancer cells in nude mice, into which the breast cancer cell line MCF-7
expressing CAPRIN-1 has been transplanted. Reference No. 36 indicates the tumor size the
mouse when PBS was administered instead of an antibody. Reference No. 37 indicates the
tumor size the mouse when cyclophosphamide was administered. Reference No. 38
indicates the tumor size the mouse when the anti-CAPRIN-1 monoclonal antibody #9 was
administered. Reference No. 39 indicates the tumor size the mouse when cyclophosphamide
and anti-Her2 antibody were administered. Reference No. 40 indicates the tumor size the
mouse when cyclophosphamide and the anti-CAPRIN-1 monoclonal antibody #9 were
administered.
Fig. 8 shows the anti-tumor effect obtained when paclitaxel, an antitumor agent, was
used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with the surface of
cancer cells in nude mice, into which the breast cancer cell line MCF-7 expressing CAPRIN-1
has been transplanted. Reference No. 41 indicates the tumor size of the mouse when PBS was
administered instead of an antibody. Reference No. 42 indicates the tumor size of the mouse
when paclitaxel was administered. Reference No. 43 indicates the tumor size of the mouse
when the anti-CAPRIN-1 monoclonal antibody #9 was administered. Reference No. 44
indicates the tumor size of the mouse when paclitaxel and anti-Her2 antibody were
administered. Reference No. 45 indicates the tumor size of the mouse when paclitaxel and the
anti-CAPRIN-1 monoclonal antibody #9 were administered.
Fig. 9 shows the anti-tumor effect obtained when docetaxel, an antitumor agent, was
used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with the surface of
cancer cells in nude mice, into which the breast cancer cell line MCF-7 expressing CAPRIN-1
has been transplanted. Reference No. 46 indicates the tumor size of the mouse when PBS was
administered instead of the antibody. Reference No. 47 indicates the tumor size of the
mouse when docetaxel was administered. Reference No. 48 indicates the tumor size of the
mouse when the anti-CAPRIN-1 monoclonal antibody #9 was administered. Reference No.
49 indicates the tumor size of the mouse when docetaxel and anti-Her2 antibody were
administered. Reference No. 50 indicates the tumor size of the mouse when docetaxel and the
anti-CAPRIN-1 monoclonal antibody #9 were administered.
Fig. 10 shows the anti-tumor effect obtained when vinorelbine, an antitumor agent,
was used in combination with an anti-CAPRIN-1 monoclonal antibody reactive with the
surface of cancer cells in nude mice, into which the MCF-7 breast cancer cell line MCF-7
expressing CAPRIN-1 has been transplanted. Reference No. 51 indicates the tumor size of the
mouse when PBS was administered instead of the antibody. Reference No. 52 indicates the
tumor size of the mouse when vinorelbine was administered. Reference No. 53 indicates the
tumor size of the mouse when the anti-CAPRIN-1 monoclonal antibody #9 was administered.
Reference No. 54 indicates the tumor size of the mouse when vinorelbine and anti-Her2
antibody were administered. Reference No. 55 indicates the tumor size of the mouse when
vinorelbine and the anti-CAPRIN-1 monoclonal antibody #9 were administered.
Mode for Carrying Out the Invention
[0026]
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 immunocytes or complements
to tumor cells expressing the polypeptide in vitro, as described later.
[0027]
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.
[0028]
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 homologue. 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.
[0029]
It was known that CAPRIN-1 is not expressed on the surface of cells; however, the
inventors' study has now revealed that a portion of the CAPRIN-1 protein is expressed on the
surface of various cancer cells. In the present invention, antibodies which bind to the
CAPRIN-1 protein portion to be expressed on the surface of cancer cells are preferably used.
An example of a partial peptide of the CAPRIN-1 protein, which is expressed on the surface
of cancer cells, is a polypeptide consisting of a sequence of 7 or more continuous amino acid
residues within the region of amino acid residue Nos. (aa) 50-98 or 233-305 in the amino acid
sequences represented by even-numbered sequences of SEQ ID NOS: 2 to 30 in the sequence
listing excluding SEQ ID NOS: 6 and 18. Specific examples include the amino acid sequence
represented by SEQ ID NO: 37 and an amino acid sequence having 80% or more, preferably
85% or more, more preferably 90% or more, and further preferably 95% or more sequence
identity with the amino acid sequence. Examples of an antibody to be used in the present
invention include all antibodies (specifically) binding to these peptides (or (specifically)
recognizing these peptides or having immunological reactivity with these peptides) and
exhibiting anti-tumor activity.
[0030]
The above-described anti-CAPRJN-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 capable of
specifically binding to a CAPRIN-1 protein 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.
[0031]
The term "specifically binding to CAPRIN-1 protein" as used herein refers to
specifically bind to a CAPRIN-1 protein, but does not substantially bind to proteins other than
the CAPRIN-1 protein.
[0032]
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.
[0033]
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.
[0034]
Preparation of antigens and antibodies, medicaments, and the like relating to the
present invention are described below.
[0035]
Preparation of antigens for antibody preparation>
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.
[0036]
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).
[0037]
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.
[0038]
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 (continuous) amino acids, for example
8 to 11 (continuous) amino acids. Examples of a partial sequence of CAPRIN-1 protein
specifically binding to an antibody include a partial sequence comprising at least about 7 to
12 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.
[0039]
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.
[0040]
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 the like) 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).
[0041]
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, 2nd 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.
[0042]
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.
[0043]
When prokaryotic cells are used as host cells, an expression vector used herein
contains an origin replicable within prokaryotic cells, a promoter, a ribo some-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, pBIuescript 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.
[0044]
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.
[0045]
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.
[0046]
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.
[0047]

An antibody is a heteromultimeric glycoprotein that generally contains at least two
heavy chains and two light chains. Antibodies other than IgM, an antibody are 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 contain 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 in cooperation 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.
[0048]

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.
[0049]
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 cytotoxically impair a tumor, such as
leukemia, lymphoma, breast cancer, brain tumor, lung cancer, esophageal cancer, gastric
cancer, renal cancer, or colorectal cancer.
[0050]
Examples of an antibody include a monoclonal antibody, a polyclonal antibody, a
recombinant antibody (e.g., a synthetic antibody, a multispecific antibody, a humanized
antibody, a chimeric antibody, or a single chain antibody), a human antibody, and an antibody
fragment thereof (e.g., Fab, F(ab') or Fv). 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. Any of these antibodies or fragments thereof has
immunological reactivity with a CAPRIN-1 protein existing on the surface of cancer cells and
preferably to a polypeptide of the extracellular region thereof (preferably, specifically binds to
the protein or the polypeptide) and exhibits a cytotoxic activity against cancer.
[0051]
The antibody may further be modified by, in addition to glycosylation, acetylation,
formylation, amidation, phosphorylation, pegylation (PEG), or the like.
[0052]
Various antibody preparation examples are as described below.
[0053]
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.
[0054]
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.
[0055]
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.
[0056]
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).
[0057]
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.
[0058]
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,
polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used. If desired, an auxiliary
agent such as dimethyl sulfoxide may be added and used in order to enhance fusion efficiency.
[0059]
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.
[0060]
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.
[0061]
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.
[0062]
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).
[0063]
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.
[0064]
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.
[0065]
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.
[0066]
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. In Examples
described later, a rabbit polyclonal antibody against the peptide (represented by SEQ ID NO:
37) of a partial region (in the amino acid sequence of a CAPRIN-1 protein) that is expressed
on the surface of cancer cells was prepared and the anti-tumor effect was confirmed.
[0067]
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.
[0068]
An antigen can be prepared according to a method using animal cells (Japanese
Patent Publication (Kohyo) No. 2007-530068) or baculovirus (e.g., International 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.
[0069]
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.
[0070]
The anti-CAPRIN-1 antibody used in the present invention is preferably a
monoclonal antibody. However, the anti-CAPRIN-1 antibody of the present invention may
also be a polyclonal antibody, or a recombinant antibody or a genetically-modified antibody
(e.g., a chimeric antibody, a humanized antibody, a single chain antibody, or a bispecific
antibody), for example.
[0071]
Examples of the monoclonal antibody include human monoclonal antibodies and
non-human animal monoclonal antibodies (e.g., a mouse monoclonal antibody, a rat
monoclonal antibody, a rabbit monoclonal antibody, and a chicken monoclonal antibody).
The 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 or a human antibody-producing
mouse) immunized with a CAPRIN-1 protein, with a myeloma cell. In Examples described
later, mouse monoclonal antibodies were prepared and the anti-tumor effects were confirmed.
[0072]
These monoclonal antibodies comprise a heavy chain variable (VH) region
comprising the amino acid sequence of SEQ ID NO: 43, 73, 83, 93, 103, 113, or SEQ ID NO:
123 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID
NO: 47, 53, 58, 63, 68, 77, 87, 97, 107, 117, or 127, wherein: the VH region comprises CDR1
represented by the amino acid sequence of SEQ ID NO: 40, 70, 80, 90, 100, 110, or 120,
CDR2 represented by the amino acid sequence of SEQ ID NO: 41, 71, 81, 91, 101, 111, or
121, and CDR3 represented by the amino acid sequence of SEQ ID NO: 42, 72, 82, 92, 102,
112, or 122; and the VL region comprises CDR1 represented by the amino acid sequence of
SEQ ID NO: 44, 50, 55, 60, 65, 74, 84, 94, 104, 114, or 124, CDR2 represented by the amino
acid sequence of SEQ ID NO: 45, 51, 56, 61, 66, 75, 85, 95, 105, 115, or 125, and CDR3
represented by the amino acid sequence of SEQ ID NO: 46, 52, 57, 62, 67, 76, 86, 96, 106,
116, or 126.
[0073]
The chimeric antibody is prepared by combining sequences from different animals.
For example, the chimeric antibody comprises mouse antibody heavy chain and light chain
variable regions and human antibody heavy chain and light chain constant regions. Such a
chimeric antibody can be prepared by known methods. For example, the chimeric antibody
can be obtained by ligating DNA encoding an antibody V region to DNA encoding a human
antibody C region, incorporating the resultant ligate into an expression vector, introducing the
vector into a host, and then causing the host to produce the antibody.
[0074]
Examples of the polyclonal antibody include an antibody obtained by immunizing a
human antibody-producing animal (e.g., a mouse) with a CAPRIN-1 protein.
[0075]
The 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.
[0076]
Specifically, DNA sequences designed to have each of the CDRs of a mouse
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 EP239400 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).
[0077]
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).
[0078]
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.
[0079]
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.
[0080]
Antibodies may be chemically modified. Examples of such a modified antibody
include antibodies bound to various molecules such as polyethylene glycol (PEG) and
antitumor compounds (e.g., antitumor agents as exemplified later). 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.
[0081]
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.
[0082]
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.
[0083]
An antibody mat recognizes an epitope of a CAPRIN-1 protein to be recognized by
the above anti-CAPRIN-1 antibody, i.e. an antibody that specifically binds to the epitope, can
be obtained by methods 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.
[0084]
The affinity constant Ka(kon/k0ff) of the antibody to be used in the present invention
is preferably at least 107M_1, at least 108M"\ at least 5 x 108M_1, at least 109M_1, at least 5 x
109M"\ at least lO'V, at least 5 x 1010M"], at least 10nM"\ at least 5 x lt^M"1, at least
lO^M^oratleastlO^M-1.
[0085]
The antibody used in 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 with 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).
[0086]
Examples of an antitumor agent usable in the present invention include the following
known antitumor agents as in literatures and 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,
cryptophy cin 1, 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 (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, defofamine,
demecolcine, diaziquone, elfornithine, elliptinium acetate (elliptinium), epothilone, etoglucid,
lenthinan, lonidamine, maytansine, ansamitocine, mitoguazone, mitoxantrone, mopidanmol,
nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethyl
hydrazide, procarbazine, razoxane, rhizoxin, schizophyllan, spiro germanium, 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.
[0087]
Alternatively, a known radio isotope as in literatures and the like, such as 2uAt, 133I,
]25I, 90Y, 186Re, mRe, 153SM, 212Bi, 32P, 175Lu, or 176Lu can be bound to the antibody used in
the present invention. A desired radio isotope is effective for treatment or diagnosis of
tumor.
[0088]
The antibody used in the present invention is an antibody having immunological
reactivity with CAPRIN-1, an antibody specifically recognizing CAPRJN-1, or an antibody
specifically binding to CAPRIN-1, which exhibits cellular cytotoxic activity against cancers,
e.g., 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 a human, a
human antibody, a humanized antibody, a chimeric antibody (e.g., a human-mouse chimeric
antibody), a single chain antibody, and a bispecific antibody. These antibodies are:
recombinant antibodies in which heavy chain and light chain variable regions are from a
human antibody; recombinant antibodies in which heavy chain and light chain variable
regions comprise complementarity determining regions (CDR1, CDR2, and CDR3) from a
non-human animal antibody, and, framework regions from a human antibody; or recombinant
antibodies in which heavy chain and light chain variable regions are from a non-human
animal antibody, and, heavy chain and light chain constant regions are from a human antibody.
Preferable antibodies are the former two antibodies.
[0089]
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
the Kabat EU numbering system (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).
[0090]
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.
[0091]
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.
[0092]
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.
[0093]
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).
[0094]
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, only CDRs are substituted with CDRs of an antibody from a non-human
animal (e.g., a mouse, a rat, or a chicken) although the other regions are from a human
antibody.
[0095]
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).
[0096]
Examples of the antibody of the present invention prepared by the above method
include the following antibodies (a) to (k):
[0097]
(a) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 40, 41, and 42 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 44, 45, and 46 (preferably, an antibody comprising
the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ
ID NO: 47);
[0098]
(b) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 40, 41, and 42 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 50, 51, and 52 (preferably, an antibody comprising
the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ
ID NO: 53);
[0099]
(c) an antibody comprisingCDRl, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 40, 41, and 42 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 55, 56, and 57 (preferably, an antibody comprising
the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ
ID NO: 58);
[0100]
(d) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 40, 41, and 42 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 60, 61, and 62 (preferably, an antibody comprising
the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ
ID NO: 63);
[0101]
(e) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 40, 41, and 42 as CDR1, CDR2, and CDR3 of a heavy chain
variable region and CDR1, CDR2, and CDR3 of a light chain variable region comprising SEQ
ID NOS: 65, 66, and 67 (preferably, an antibody comprising the heavy chain variable region
of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 68);
[0102]
(f) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprisingSEQ ID NOS: 70, 71, and 72 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 74, 75, and 76 (preferably, an antibody comprising
the heavy chain variable region of SEQ ID NO: 73 and the light chain variable region of SEQ
ID NO: 77);
[0103]
(g) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 80, 81, and 82 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 84, 85, and 86 (preferably, an antibody comprising
the heavy chain variable region of SEQ ID NO: 83 and the light chain variable region of SEQ
ID NO: 87);
[0104]
(h) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 90, 91, and 92 and CDR1, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 94, 95, and 96 (for example, an antibody
comprising the heavy chain variable region of SEQ ID NO: 93 and the light chain variable
regionofSEQIDNO:97);
[0105]
(i) an antibody comprising CDR1, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 100, 101, and 102 and CDRl, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 104, 105, and 106 (for example, an antibody
comprising the heavy chain variable region of SEQ ID NO: 103 and the light chain variable
region of SEQ ID NO: 107);
(j) an antibody comprising CDRl, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 110, 111, and 112 and CDRl, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 114, 115, and 116 (for example, an antibody
comprising the heavy chain variable region of SEQ ID NO: 113 and the light chain variable
region of SEQ ID NO: 117); and
[0106]
(k) an antibody comprising CDRl, CDR2, and CDR3 of a heavy chain variable region
comprising SEQ ID NOS: 120, 121, and 122 and CDRl, CDR2, and CDR3 of a light chain
variable region comprising SEQ ID NOS: 124, 125, and 126 (for example, an antibody
comprising the heavy chain variable region of SEQ ID NO: 123 and the light chain variable
region of SEQ ID NO: 127).
[0107]
Here, the amino acid sequences represented by SEQ ID NOS: 40, 41, and 42, SEQ
ID NOS: 70, 71, and 72, SEQ ID NO: 80, 81, and 82, SEQ ID NO: 90, 91, and 92, SEQ ID
NO: 100, 101, and 102, SEQ ID NO: 110, 111, and 112, or SEQ ID NO: 120, 121, and 122
are CDR1, CDR2, and CDR3, respectively, of a mouse antibody heavy chain variable region.
The amino acid sequences represented by SEQ ID NO: 44, 45, and 46, SEQ ID NO: 50, 51,
and 52, SEQ ID NO: 55, 56, and 57, SEQ ID NO: 60, 61, and 62, SEQ ID NO: 65, 66, and 67,
SEQ ID NO: 74, 75, and 76, SEQ ID NO: 84, 85, and 86, SEQ ID NO: 94, 95, and 96, SEQ
ID NO: 104, 105, and 106, SEQ ID NO: 114, 115, and 116, or SEQ ID NO: 124, 125, and 126
are CDR1, CDR2, and CDR3, respectively, of a mouse antibody light chain variable region.
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:
[0108]
(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, the 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 (e.g., 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).
[0109]
(ii) 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, the heavy chain constant region comprises an amino acid sequence
from a human antibody, and, the 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 the light chain constant region comprises an amino acid
sequence from a human antibody (e.g., the antibody wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO: 43, and, the heavy chain constant region
comprises an amino acid sequence from a human antibody, as well as, the light chain variable
region comprises the amino acid sequence of SEQ ID NO: 47, and, the light chain constant
region comprises an amino acid sequence from a human antibody).
[0110]
In this context, 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 registration no. J00228 can be
referred to as a human IgGl heavy chain constant region, the sequence of registration no.
J00230 can be referred to as a human IgG2 heavy chain constant region, the sequence of
registration no. X03604 can be referred to as a human IgG3 heavy chain constant region, the
sequence of registration no. K01316 can be referred to as a human IgG4 heavy chain constant
region, the sequences of registration no. V00557, X64135, X64133, and the like can be
referred to as human light chain k constant regions, and the sequences of registration nos.
X64132, X64134, and the like can be referred to as human light chain X constant regions.
[0111]
The above antibodies preferably have cellular cytotoxic activity and thus can
exhibit anti-tumor effects.
[0112]
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 cellular 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.
[0113]
Furthermore, regarding the above antibody of the present invention, the sequence of
each of the above antibodies (i) to (iv), 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 (preferably, 1 or 2) amino acids, as long as it has
specificity for specific recognition of CAPRIN-1. Here the term "several" refers to 2 to 5, and
preferably 2 or 3.
[0114]
Antibodies used in the present invention can also be produced by gene
recombination techniques using 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.
[0115]
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.
[0116]
Further alternative examples of DNA encoding the antibody used in 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: 48. 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: 49. 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.
[0117]
The DNA of the present invention 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.
[0118]
Examples of DNA relating to the above antibodies (a) to (k) are as follows.
[0119]
(i) As DNA encoding a polypeptide that comprises the amino acid sequence of SEQ ID NO:
43, 73, 83, 93, 103, 113, 123, 133, 143, or 153, DNA comprising the nucleotide sequence of
SEQ ID NO: 48, 78, 88, 98, 108, 118, or 128.
[0120]
(ii) As DNA encoding a polypeptide that comprises the amino acid sequence of SEQ ID NO:
47, 53, 58, 63, 68, 77, 87, 97, 107, 117, 127, 137, 147, or 157, DNA comprising the
nucleotide sequence of SEQ ID NO: 49, 54, 59, 64, 69, 79, 89, 99, 109, 119, or 129.
[0121]
The anti-CAPRIN-1 antibodies used in the present invention are considered to
exhibit the anti-tumor effect against CAPRIN-1-expressing cancer cells through the following
mechanism:
[122]
the effector-cell-antibody-dependent cytotoxicity (ADC-C) of CAPRIN-1-expressing cells;
and the complement-dependent cytotoxicity (CDC) of CAPRIN-1-expressing cells.
[0123]
Therefore, the activity of an anti-CAPRIN-1 antibody to be used in the present
invention can be evaluated by measuring ex vivo the above ADCC activity or CDC activity
against CAPRIN-1 protein-expressing cancer cells, as specifically described in Examples
below.
[0124]
ADCC activity can be measured using a commercially available kit for measuring
cytotoxic activity such as a Cytotoxicity Detection Kit (Roche). According to the method,
ADCC activity can be measured by procedures comprising reacting a target cancer cell with
an anti-CAPRIN-1 antibody on ice, culturing the cell with an effector cell (e.g., PBMC) for 4
hours, and then measuring the enzyme activity of lactate dehydrogenase (LDH) or Cr51
radioactivity released in the medium in the culture supernatant. Also, CDC activity can be
measured by procedures comprising reacting a target cancer cell with an anti-CAPRIN-1
antibody on ice, culturing the cell with a solution containing complements (e.g., serum) for 4
hours, and then measuring enzyme activity or radioactivity similar to the above in the culture
supernatant.
[0125]
An anti-CAPRIN-1 antibody 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.
[0126]
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
(Wkoff) is preferably at least I07 M"1, at least 10s M"1, at least 5 x 108 M"1, at least 109 M"1, at
least 5 x 109 W\ at least 1010 M"1, at least 5 x 1010 M"1, at least 1011 M"1, at least 5 x 10H M"1,
at least 1032 M*1, or, at least 1013 M"1.
[0127]

The capacity of an antibody to bind to a CAPRIN-1 protein can be determined by
binding assay using ELISA, a Western blot method, immuno-fluorescence and flow
cytometric analysis, or the like as described in Examples.
[0128]

An antibody that recognizes a CAPRIN-1 protein can be tested for reactivity with
CAPRIN-1 by a method known by persons skilled in the art on the basis of
immunohistochernistry using paraformaldehyde- or acetone-fixed frozen sections or
paraformaldehyde-fixed paraffin-embedded tissue sections (prepared from tissue samples
obtained from a patient during surgery, or tissue samples obtained from an animal having
heterotransplant tissue inoculated with a cell system expressing CAPRIN-1, naturally or after
transfection).
[0129]
An antibody reactive with CAPRIN-1 can be stained by various methods for
immunohistochemical staining. For example, a horseradish peroxidase-conjugated goat
anti-mouse antibody or goat anti-rabbit antibody is caused to undergo reaction, a target
antibody can be visualized.
[0130]

The present invention is characterized by combining an anti-CAPRIN-1 antibody
with an antitumor agent as exemplified above. An anti-CAPRIN-i antibody and an
antitumor agent each having antitumor activity are administered in combination to a cancer
patient, so that synergistically significant anti-tumor effect, specifically the effect of causing
almost complete tumor regression in a cancer-bearing animal model, can be obtained as
described in Examples. Such special anti-tumor effect is observed when an anti-CAPRIN-1
antibody and an antitumor agent are used in combination, even when tumor growth gradually
increases over time. This effect is completely extraordinary.
[0131]
In the present invention, examples of an antitumor agent to be used in combination
with an anti-CAPRIN-1 antibody include all chemotherapeutics that are used, were used, or
will be used for treating various types of caners or tumors. Examples of such an antitumor
agent include an antimetabolic drug, an antibiotic anticancer agent, a plant alkaloid-based
anticancer agent, a topoisomerase inhibitor, and an antitumor alkylating agent. For example,
all antitumor agents (as exemplified above) known in literatures and the like are included
herein. Examples thereof include, but are not limited to, antitumor agents that are used in
Examples described below, such as anticancer agents (e.g., cyclophosphamide, paclitaxel,
docetaxel, and vinorelbine), the significant anti-tumor effects of which have been confirmed.
Therefore, in the present invention, one or two or more drugs selected from
cyclophosphamide, paclitaxel, docetaxel, vinorelbine, and pharmacologically acceptable salts
or derivatives thereof can be used as antitumor agents.
[0132]
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.
[0133]
The term "tumor" and "cancer" as used herein refers to malignant neoplasm and is
used interchangeably.
[0134]
The medicament of the present invention is characterized by comprising a
combination of an antibody or a fragment thereof having immunological reactivity with a
CAPRIN-1 protein and one or two or more types of antitumor agents, wherein the antibody or
fragment and the antitumor agents are combined together or separately. Specifically, when
these active ingredients are combined together, the above antibody or a fragment thereof and
the above antitumor agent(s) can be mixed together in a carrier (or an excipient) to be
formulated in the form of a pharmaceutical composition. On the other hand, when these
active ingredients are separately combined, a pharmaceutical composition containing the
above antibody or a fragment thereof as an active ingredient and a pharmaceutical
composition containing the above antitumor agent(s) as an active ingredient are separately
formulated so that a medicament can be produced in the form of a pharmaceutical kit. Such
a pharmaceutical composition and pharmaceutical kit are more specifically described below.
[0135]
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, and colorectal cancer.
[0136]
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, and rectal cancer.
[0137]
Moreover, preferable subjects are mammals including primates, pet animals,
domestic animals, animals for competition, and the like and are particularly preferably
humans, dogs, and cats.
[0138]
Pharmaceutical composition>
The active ingredients contained in the medicament for treating and/or preventing a
cancer of the present invention, i.e. the above antibody or a fragment thereof and the above
antitumor agent, can be formulated by a method known by persons skilled in the art in the
from of a pharmaceutical composition prepared by mixing them or in the form of individual
pharmaceutical compositions thereof.
[0139]
For example, the pharmaceutical composition can be used parenterally in the form of
an injection preparation such as an aseptic solution prepared with water or a
pharmacologically acceptable solution other than water or a suspension. 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.
Also, the pharmaceutical composition of the present invention can contain a
pharmacologically acceptable salt. As a pharmacologically acceptable salt, for example,
inorganic acid such as hydrochloric acid or phosphoric acid, or organic acid such as acetic
acid, tartaric acid, or mandelic acid can be used. Furthermore, a salt formed with a free
carboxyl group can be used. For example, such a salt can also be induced from an inorganic
base such as sodium, potassium, ammonium, calcium, hydroxide of iron (I), or the like, or an
organic base such as isopropylamine, trimethylamine, 2-ethyl aminoethanol, histidine, or
procaine.
[0140]
An aseptic composition for injection can be prescribed according to general
pharmaceutical practice using a vehicle such as distilled water for injection.
[0141]
Examples of an aqueous solution for injection include saline, an isotonic solution
containing dextrose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, 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).
[0142]
Examples of an oily fluid 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.
[0143]
Administration is oral or perenteral administration. Examples of perenteral
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. Also, in the case of perenteral administration, infusion
administration that is gradually or slowly performed taking much time is possible.
Administration methods can be appropriately selected depending on a patient's age, body
weight, sex, symptom, and the like. The pharmaceutical composition comprising an
antibody is preferably parenteraly administered. On the other hand, in the case of a
pharmaceutical composition comprising an antitumor agent, either oral administration or
parenteral administration is selected depending on types of antitumor agents and indications.
[0144]
In the case of the pharmaceutical composition for treating and/or preventing a cancer
of the present invention, the dosage of the above antibody can be selected from the range
between 0.0001 mg and 1000 mg per kg body weight, for example. Alternatively, for example,
the dosage can be selected from the range between 0.001 mg/body of a patient and 100000
mg/body of a patient; however, the dosage range is not always limited to these numerical
values. Furthermore, the dosage of the above antitumor agent can be selected from the range
between 1 and 1000 mg/body of a patient and preferably between 10 and 500 mg/body of a
patient, for example; however, the dosage range is not always limited to these numerical
values. In addition, the dosage and administration method are varied depending on a
patient's body weight, age, sex, symptom, and the like, but can be appropriately selected by
persons skilled in the art.
[0145]

Cancer treatment and/or prevention using the agent for treating and/or preventing a
cancer of the present invention involve various forms in addition to administration of the
agent as the above pharmaceutical composition. For example, the active ingredients of the
agent for treating and/or preventing a cancer of the present invention can be administered
simultaneously or can be separately administered in order. In a specific example, the active
ingredients can be administered at intervals of up to about 3 weeks, that is, during about 3
weeks after administration of the first active ingredient, the second active ingredient can be
administered. At this time, this administration may be performed subsequent to surgical
treatment, or surgical treatment may also be performed between the administration of the first
agent and the administration of the second agent. Also, the agent for treating and/or
preventing a cancer of the present invention may be administered according to a plurality of
administration cycles. For example, when the simultaneous administration of the active
ingredients of the agent for treating and/or preventing a cancer of the present invention is
performed, the pharmaceutical composition comprising both active ingredients is
administered with a cycle of about 2 days to about 3 weeks. Subsequently, where needed,
the therapeutic cycle may also be repeated according to the physician's judgment. Similarly,
when the formulation for administering the active ingredients in order is planned, the
administration periods of the individual agents are adjusted to be the same period. An
interval between cycles may vary from 0 to 2 months. The dosage of each active ingredient
of the agent for treating and/or preventing a cancer of the present invention can be set
similarly to the dosage used for administration of each active ingredient of the pharmaceutical
composition.
[0146]
Pharmaceutical kit>
The medicament for treating and/or preventing a cancer of the present invention
may be in the form of a pharmaceutical kit. The term "pharmaceutical kit" as used herein
refers to, in a method for treating or preventing a cancer, a package for using the above
anti-CAPRIN-1 antibody or a fragment thereof and the above antitumor agent that are active
ingredients in the form of individual pharmaceutical compositions. The package includes
instructions for administration of each active ingredient. Each active ingredient of the above
pharmaceutical composition for treating and/or preventing a cancer, which is contained in a
pharmaceutical kit can be in the form of a pharmaceutical composition that has been
formulated as described above so that active ingredients can be administered together or
separately. Also, a pharmaceutical kit contains the amounts of active ingredients sufficient
for a single dose or multiple doses so that each active ingredient can be administered
according to the above administration method.
[0147]
Based on the content described specifically above, the present invention further
provides a method for treating and/or preventing a cancer, comprising administering the
above medicament of the present invention to a subject suspected of having cancer (including
a subject with cancer). In an embodiment, an antibody or a fragment thereof and an
antitumor agent, which are contained in the above medicament, are administered
simultaneously or separately to the above subject.
Examples
[0148]
The present invention is described more specifically based on Examples, but the
scope of the present invention is not limited by these specific examples.
[0149]
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.
[0150]
A dog testis cDNA phage library was synthesized using the thus obtained mRNA (5
fig). 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 7.73xl05pfu/ml.
[0151]
(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.
[0152]
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 NaCl
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 co/z'/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.
[0153]
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 mMNaCl, 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.
[0154]
(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 OD60o of 1.0, 250 p,l 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
ul of a solution prepared from phagemid host Escherichia coli SOLR to give an absorbance
OD60o of 1.0 and 10 ul of the purified phage solution were mixed, followed by 15 minutes of
reaction at 37°C. 50 ul of the resultant was plated on LB agar medium containing ampicillin
(at final concentration of 50 ug/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 ug/ml) at 37°C. After culture, plasmid DNA carrying an insert of
interest was purified using a QIAGEN plasmid Miniprep Kit (QIAGEN).
[0155]
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.
[0156]
(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 5 mg of
each 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.
[0157]
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.
[0158]
(5) Immunohistochemical staining
(5)-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 intestine, esophagus, heart,
kidney, salivary gland, large intestine, 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 jam 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 anti-CAPRIN-1 monoclonal
antibody (monoclonal antibody #6; prepared in Example 3) of 10 ug/ml adjusted with a
blocking solution, which reacts with cancer cell surfaces and comprises the heavy chain
variable region of SEQ ID NO: 73 and the light chain variable region of SEQ ID NO: 77, 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 three times, and then an MOM biotin-Iabeled 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 three 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 10 minutes of washing with PBS-T was performed 3 times, a DAB
color-development solution (DAB 10 mg + 30% H202 10 ul/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 #9) comprising the heavy chain
variable region of SEQ ID NO: 103 and the light chain variable region of SEQ ID NO: 107.
[0159]
(5)-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 monoclonal
antibody #6 prepared in Example 3. 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 #9 prepared in Example 3.
[0160]
(5)-3 CAPRIN-1 expression in human breast cancer tissue
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 mM 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 monoclonal antibody #6 (prepared in Example 4) of 10 fig/ml adjusted
with a PBS-T solution containing 5% FBS, which reacts with cancer cell surfaces, 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 #2 or #9 prepared in Example 3.
[0161]
(5)-4 CAPRIN-1 expression in human malignant brain tumor
Immunohistochemical staining was performed according to a method similar to that
used in (5)-3 above with 247 malignant brain tumor tissue specimens on a paraffin-embedded
human malignant brain tumor tissue array (BIOMAX), using the monoclonal antibody #6
prepared in Example 3. 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 #2 or #9 prepared in
Example 3.
[0162]
(5)-5 CAPRIN-1 expression in human breast cancer metastasized lymph node
Immunohistochemical staining was performed according to a method similar to that
in (5)-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 monoclonal antibody #6 prepared in Example 3. 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 #2 or #9 prepared
in Example 3.
[0163]
(5)-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 monoclonal antibody #6 prepared in Example 3. 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 #2 or #9.
[0164]
Example 2 Preparation of novel human cancer antigen protein
(1) Preparation of recombinant protein
Based on the gene of SEQ ID NO: 1, a recombinant protein from the human
homologous gene was prepared by the following method. PCR was performed in a total
volume of 50 jil with 1 ul of cDNA (the expression of which had been confirmed by an
RT-PCR method for the cDNA used herein from among breast cancer tissue- or cell-derived
cDNAs), two types of primer (SEQ ID NOS: 38 and 39 comprising Sac I and Xho I restriction
enzyme cleavage sequences) of 0.4 (iM each, 0.2 mM dNTP, and 1.25 U PrimeSTAR HS
polymerase (Takara Shuzo Co., Ltd.), prepared by adding the reagents and an accompanying
buffer. 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).
[0165]
The thus purified DNA fragment was li gated 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.
(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 ug/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 mM, 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.
[0166]
The cells were suspended in phosphate buffered saline and then disrupted by
ultrasoni cation 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.
[0167]
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 raM 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 mM sodium chloride, and thus the purified fraction of the protein having the
amino acid sequence represented by SEQ ID NO: 2 was obtained.
[0168]
200 ul of each purified sample obtained by the above method was dispensed into 1
ml of reaction buffer (20 mM Tris-Hcl, 50 mM, NaCI, 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 urn (PALL) and then
the resultant was used for the following experiment.
[0169]
Example 3 Preparation of chicken monoclonal antibody against CAPRIN-1
100 ug of the antigen protein (human CAPRIN-1) represented by 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/c mice (Japan SLC Inc.), and then the
administration was performed 3 times every week (24 times of administration in total), and
thus immunization was completed. Each spleen was excised on day 3 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 ul 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 seeded on fifteen 96-well plates (Nunc) at 100 ul per well. Cells were
cultured under conditions of 7 days, at 37°C, in the presence of 5% CO2, so that hybridomas
prepared by fusion of splenocytes and myeloma cells were obtained.
[0170]
Hybridomas were selected using as a marker the binding affinity of the antibody
produced by the prepared hybridomas to the CAPRIN-1 protein. The CAPRIN-1 protein
solution (1 (-ig/ml) prepared in Example 1 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 jxl 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 ul 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, the HRP-labeled
anti-mouse IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was added at 100 ul
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 ul of a TMB substrate solution (Thermo) was
added per well and then left to stand for 15 to 30 minutes for color development reaction.
After color development, 100 jal 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.
[0171]
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
CAPRIN-1 protein. The CAPRIN-1 protein solution (1 |ag/ml) prepared in Example 1 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 ul of PBS-T per well. 100 ul
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 ul of a TMB substrate solution
(Thermo) was added per well, and then left to stand for 15 to 30 minutes for color
development 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, 150 hybridoma cell lines producing monoclonal
antibodies reactive with the CAPRIN-1 protein were obtained.
[0172]
Next, of those monoclonal antibodies, antibodies reactive to the cell surface of
breast cancer cells expressing CAPRIN-1 were selected. Specifically, 106 cells of the human
breast cancer cell line MDA-MB-231V were subjected to centrifugation with 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 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 serum (of a 6-week-old Balb/c mouse not treated with the antibodies)
diluted 500-fold with medium for culturing hybridomas, so that a control sample was
prepared. As a result, 11 monoclonal antibodies (#1 to #11) that had exhibited a
fluorescence intensity stronger than that of the control and that is, that reacted with the cell
surface of breast cancer cells, were selected.
[0173]
Example 4 Characterization of selected antibody
(1) Cloning of anti-CAPRIN-1 monoclonal antibody variable region gene
mRNA was extracted from each hybridoma cell line producing each of the 11
mouse monoclonal antibodies selected in Example 3. The genes of the heavy chain variable
(VH) and light chain valuable (VL) regions of all anti-CAPRIN-1 monoclonal antibodies were
obtained by an RT-PCR method using primers specific to a mouse FRl-derived sequence and
a mouse FR4-derived sequence. For sequence determination, these genes were cloned into a
pCR2.1 vector (Invitrogen). Furthermore, mRNA was extracted from two mouse-derived
hybridoma cell lines producing monoclonal antibodies reactive with the surface of
CAPRIN-1 -expressing breast cancer cells. The genes of the heavy chain variable (VH) region
and light chain variable (VL) region of each antibody were obtained by an RT-PCR method
using primers specific to the mouse FRl-derived sequence and the mouse FR4-derived
sequence. For sequence determination, these genes were cloned into a pCR2.1 vector
(Invitrogen).
[0174]
(1)-1 RT-PCR
After extraction of total RNA from 106 cells of each hybridoma cell line using a
High Pure RNA Isolation Kit (Roche), cDNA was synthesized using a PrimeScriptll 1st
strand cDNA Synthesis Kit (Takara). These procedures were performed according to
protocols attached to each kit.
[0175]
The gene of the mouse antibody heavy chain variable region and the gene of the
mouse antibody light chain variable region were separately amplified by a PCR method
according to a conventional method using the thus synthesized cDNA as a template and
KOD-Plus-DNA Polymerase (TOYOBO).
[0176]
To obtain the genes of the mouse antibody VH and VL regions, a primer (SEQ ID
NO: 130) specific to the mouse heavy chain FR1 sequence, a primer (SEQ ID NO: 131)
specific to the mouse heavy chain FR4 sequence, a primer (SEQ ID NO: 132) specific to the
mouse light chain FR1 sequence, a primer (SEQ ID NO: 133) specific to the mouse light
chain FR4 sequence were used.
[0177]
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 DH5 competent cells (TOYOBO)
according to a conventional method. 10 clones of each transformant were cultured overnight
in medium (100 (ig/ml ampicillin) at 37°C, and then plasmid DNA was purified using a
Qiaspin Miniprep kit (QIAGEN).
[0178]
(l)-2 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: 134) and an M13 reverse
primer (SEQ ID NO: 135) 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.
[0179]
The gene sequences of the heavy chain variable regions of the thus obtained
monoclonal antibodies are each represented by SEQ ID NOS: 48, 78, 88, 98, 108, 118, and
128, and the amino acid sequences of the heavy chain variable regions are each represented
by SEQ ID NOS: 43, 73, 83, 93, 103, 113, and 123. The gene sequences of the light chain
variable regions of the same monoclonal antibodies are each represented by SEQ ID NOS: 49,
54, 59, 64, 69, 79, 89, 99, 109, 119, and 129, and the amino acid sequences of the light chain
variable regions are each represented by SEQ ID NOS: 47, 53, 58, 63, 68, 77, 87, 97, 107,
117, and 127.
[0180]
Specifically, 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, #2 comprises the
heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID
NO: 53, #3 comprises the heavy chain variable region of SEQ ID NO: 43 and the light chain
variable region of SEQ ID NO: 58, #4 comprises the heavy chain variable region of SEQ ID
NO: 43 and the light chain variable region of SEQ ID NO: 63, #5 comprises the heavy chain
variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 68, #6
comprises the heavy chain variable region of SEQ ID NO: 73 and the light chain variable
region of SEQ ID NO: 77, #7 comprise the heavy chain variable region of SEQ ID NO: 83
and the light chain variable region of SEQ ID NO: 87, #8 comprises the heavy chain variable
region of SEQ ID NO: 93 and the light chain variable region of SEQ ID NO: 97, #9
comprises the heavy chain variable region of SEQ ID NO: 103 and the light chain variable
region of SEQ ID NO: 107, #10 comprises the heavy chain variable region of SEQ ID NO:
113 and the light chain variable region of SEQ ID NO: 117, and #11 comprises the heavy
chain variable region of SEQ ID NO: 123 and the light chain variable region of SEQ ID NO;
127.
(2) Expression of CAPRIN-1 on the surface of various cancer cells using anti-CAPRIN-1
antibodies #2 and #9
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, 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 (MNK28 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), one (1) lymphoma cell line (Ramos), one (1) uterine cervix cancer cell line
(SW756), one (1) bladder cancer cell line (T24), and one (1) esophageal cancer cell line
(KYSE180) were examined for expression of CAPRIN-1 protein on the cell surface of each
cell line using the culture supernatants of hybridomas producing #2 and #9 obtained in
Example 3. 106 cells of each cell line were centrifuged using a 1.5 ml microcentrifuge tube.
Each culture supernatant (100 ul) of hybridomas producing #2 and #9 obtained in Example 3
was added and then left to stand on ice for 1 hour. After washing with PBS, a FITC-labeled
goat-anti human IgG (H+L) antibody (SouthernBiotech) diluted 500-fold with PBS containing
0.1% FBS and a FITC-labeled anti-mouse IgG (H+L) antibody (Invitrogen) were 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 procedure similar to the above was performed using a medium for culturing
hybridomas, and it was used as a negative control. As a result, cells to which the antibodies
#2 and #9 had been added exhibited fluorescence intensity stronger by 20% or more than that
of the 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.
[0181]
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 the control)) / (MFI level of control) x 100.
[0182]
(3) Anti-tumor effect of anti-CAPRIN-1 antibodies on cancer cells (ADCC activity)
The above-selected anti-CAPRIN-1 monoclonal antibodies #1 to #11 were
evaluated for their cytotoxic activities against cancer cells (ADCC activity). Hybridomas
producing the monoclonal antibodies #1 to #11 were each cultured using hybridoma SFM
medium (Invitrogen). The thus obtained supernatant was purified using Hitrap ProteinA
Sepharose FF (GE HealthCare), replaced with PBS(-), and then filtered through 0.22 jam filter
(Millipore). The obtained filtrates were used as antibodies for assay. The human breast
cancer cell line MDA-MB-157 (106 cells) was collected in a 50-ml centrifuge tube, to which
100 uCi chromium(Cr)-51 was then added, and incubated at 37°C for 2 hours. Subsequently,
the cells were washed 3 times with RPMI1640 medium containing 10% FBS, and then
dispensed into each well of a 96-well V-bottom plate at 103 cells per well. The thus obtained
cells are used as target cell. The above purified antibodies (1 \i% each) were added to the
cells. Separately, mouse lymphocytes separated from the mouse spleen were further added (2
x 105 cells) and then cultured under conditions of 37°C and 5% CO for 4 hours. After
culture, the amount of chromium(Cr)-51 released from cytotoxically impaired tumor cells in a
culture supernatant was measured, so that the ADCC activity of each anti-CAPRIN-1
antibody against cancer cells was calculated. As a result, all the monoclonal antibodies #1 to
#11 exhibited 20% or more ADCC activity against MDA-MB-157. Specifically, for
example #1 exhibited 22.1% cytotoxic activity, #2 exhibited 29.1% cytotoxic activity, #6
exhibited 30.2% cytotoxic activity, and #9 exhibited 32.4% cytotoxic activity (see Fig. 1).
On the other hand, similar procedures were performed using the monoclonal antibodies
(which were prepared in Example 2) reactive with the CAPRIN-1 protein itself, but not
reactive with the surface of cancer cells, no cytotoxic activity was observed (see Fig. 1).
From the above results, it was demonstrated that the thus obtained anti-CAPRIN-1
monoclonal antibodies (#1 to #11) cytotoxically impaired CAPRIN-1-expressing cancer cells
by the ADCC activity. Similarly, the anti-CAPRIN-1 antibodies were examined for ADCC
activity against other human cancer cell lines 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 (MNK28 and MNK45), a colorectal cancer cell line
(SW480), a leukemia cell line (AML5), and a lymphoma cell line (Ramos). As a result, all
the monoclonal antibodies #1 to #11 exhibited ADCC activities higher than those of isotype
controls. Specifically, for example, #9 exhibited 12% or more (1.3% in the case of the isotype
control) activity against T98G, #9 exhibited 16% or more (3% in the case of isotype control)
against U373, #9 exhibited 24% or more (2.6% in the case of isotype control) activity against
A549, #9 exhibited 20% or more (0. 2% in the case of isotype control) activity against QG56,
#9 exhibited 23% or more (3.0% in the case of isotype control) against Caki-1, #9 exhibited
14% or more (1.5% in the case of isotype control) against ACHN, #9 exhibited 16% or more
activity (2.5% in the case of isotype control) against SW756, #9 exhibited 18% or more
activity (2.1% in the case of isotype control) against T24, #9 exhibited 22% or more activity
(3.0% in the case of isotype control) against KYSE180, #9 exhibited 15% or more activity
(1.7% in the case of isotype control) against MNK28, #9 exhibited 10% or more activity
(2.3% in the case of isotype control) against MNK45, #9 exhibited 17% or more activity
(1.3% in the case of isotype control) against SW480, #9 exhibited 10% or more activity (3.0%
in the case of isotype control) against AML5, and #9 exhibited 10% or more activity (4.1% in
the case of isotype control) against Ramos. It was demonstrated by the above results that the
obtained anti-CAPRIN-1 antibodies (#1 to #11) cytotoxically impaired various human cancer
cells expressing CAPR1N-1.
[0183]
(4) Anti-tumor effect of anti-CAPRIN-1 antibodies on cancer cells (CDC activity)
The above-selected anti-CAPRIN-1 monoclonal antibodies #1 to #11 were
evaluated for cytotoxic activity against cancer cells (CDC activity). Blood taken from a
rabbit was added to an Eppendorf tube, left to stand at room temperature for 60 minutes, and
then centrifuged at 3000 rpm for 5 minutes. Thus, the serum for assay of CDC activity was
prepared. 105 cells of MDA-MB-231V, a human breast cancer cell, were collected in a
50-ml centrifuge tube, to which 100 u.Ci chromium-51 was then added, and incubated at 37°C
for 2 hours. The cells were washed 3 times with RPMI medium containing 10% FBS,
suspended in RPMI medium containing the above-prepared rabbit serum at a concentration of
50%, and then dispensed into each well of a 96-well V-bottom plate at 103 cells per well.
The monoclonal antibodies #1 to #13 obtained in Example 3 were each added (1 ug each) to
the cells, which were then cultured under conditions of 37°C and 5% CO? for 4 hours. After
culture, the amount of chromium-51 released from cytotoxically impaired tumor cells in a
culture supernatant was measured, and the CDC activity of each anti-CAPRIN-1 monoclonal
antibody against MDA-MB-231V in a hybridoma supernatant was calculated. As a result,
all the monoclonal antibodies #1 to #11 exhibited 30% or more CDC activity. On the other
hand, no cytotoxic activity was observed when similar procedures were performed using the
monoclonal antibodies (which were prepared in Example 2) reactive with the CAPRIN-1
protein itself, but not reactive with the surface of cancer cells. Therefore, it was revealed
that the anti-CAPRIN-1 monoclonal antibodies (#1 to #11) can cytotoxically impair tumor
cells expressing CAPRIN-1, as also seen from the results of CDC activity.
[0184]
Example 5 Identification of a peptide in a CAPRIN-1 protein to which anti-CAPRIN-1
antibodies reactive with the surface of cancer cells bind
A partial sequence of a CAPRIN-1 protein to be recognized by the above-obtained
anti-CAPRIN-1 monoclonal antibodies #1 to #11 reactive with the surface of cancer cells was
identified using each antibody.
[0185]
First, DTT (Fluka) was added to 100 ui of a recombinant CAPRIN-1 protein
solution prepared by dissolving the protein to a concentration of 1 |ig/ul in PBS, so that the
final concentration was 10 mM, followed by 5 minutes of reaction at 95°C. Disulfide bonds
within the CAPRIN-1 protein were reduced, iodacetamide (Wako Pure Chemical Industries,
Ltd.) at a final concentration of 20 mM was added, and then the alkylation reaction of a thiol
group was performed at 37°C under light shielding conditions for 30 minutes. The
anti-CAPRIN-1 monoclonal antibodies #1 to #11 were each added (50 \xg each) to 40 ug of
the thus reduced and alkylated CAPRIN-1 protein. Each solution was diluted to a volume of
1 ml with 20 mM phosphate buffer (pH 7.0), followed by reacting at 4°C overnight while
agitating and mixing the solution.
[0186]
Next, trypsin (Promega) was added to a final concentration of 0.2 jj.g. After
reaction at 37°C for 1 hour, 2 hours, 4 hours, or 12 hours, each resultant was blocked with
PBS containing 1% BSA (Sigma) in advance, and then mixed with protein A-glass beads
(GE), which were previously washed with PBS, and 1 mM calcium carbonate in NP-40 buffer
(20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% NP-40), followed by 30
minutes of reaction for each solution.
[0187]
The reaction mixture was washed with 25mM ammonium carbonate buffer (pH 8.0),
and then an antigen-antibody complex was eluted using 100 ul of 0.1% formic acid. The
eluate was subjected to LC-MS analysis using Q-TOF Premier (Waters-MicroMass).
LC-MS analysis was conducted according to protocols attached to the apparatus.
[0188]
As a result, the polypeptide of SEQ ID NO: 37 was identified as a partial
CAPRIN-1 sequence, which was recognized by any of the anti-CAPRIN-1 monoclonal
antibodies #1 to #11.
[0189]
Example 6 Effect of antitumor agents on expression of CAPRIN-1 on the surface of cancer
cells
(1) Calculation of 50% inhibitory concentration of antitumor agents against cancer cell
To evaluate the effect of antitumor agents on the expression of CAPRIN-1 on the
surface of a cancer cell, the 50% inhibitory concentration of each antitumor agent was
calculated using the MCF-7 cancer cell. Using the MCF-7 human breast cancer cell line,
50% inhibitory concentrations of 4 types of antitumor agents that are currently used as
remedies for breast cancer (i.e., cyclophosphamide: "Endoxan" (registered trademark,
Shionogi & Co., Ltd.), paclitaxel: "Taxol" (registered trademark, Bristol-Myers), docetaxel:
"Taxotere" (registered trademark, Sanofi-aventis K.K.), vinorelbine: "Navelbine" (registered
trademark, Kyowa Hakko Kirin Co., Ltd.)), were examined. The cell line was prepared to 1
x 103 cells/ml and then cultured on a 6-well plate under conditions of 37°C and 5% C02 for
one day. Then, the cell was treated with each antitumor agent at final concentrations of
0.001 uM, 0.01 uM, 0.1 uM, 1.0 uM, and 10 uM, followed by 2-days culture under
conditions of 37°C and 5% CO2. After removal of the culture medium, the cell was washed
twice with PBS(-), to which 0.25% Trypsin-EDTA was then added in order to detach the cell
from the plate. The thus detached cell was suspended with PBS(-) to a volume of 100 ul, to
which 10 ul of 0.4% trypan blue stock solution was then added, and the mixture was
measured for counts of living cells using a hemocytometer. The rate of living cells in case
that the cell was treated with each antitumor agent (i.e., a chemotherapeutic) was calculated,
wherein the number of living cells in case that the cell was not treated with each antitumor
agent was designated at 100%. 50% inhibitory concentration was roughly estimated based
on the obtained values, and each antitumor agent was prepared to have a concentration around
the determined 50% inhibitory concentration, and thereafter procedures similar to the above
were further performed in order to calculate a more specific 50% inhibitory concentration.
[0190]
As an example, the results of examination for cyclophosphamide (an antitumor
agent) are described below. MCF-7 cell line was prepared to 1 x 10' cells/mL, and then
cultured on a 6-well plate under conditions of 37°C and 5% CO: for one day. Then, the cell
was treated with cyclophosphamide at final concentrations of 1 x 10" uM, 5x10" uM, 2 x
10"2 uM, and 1 x 10"2 jaM, followed by 2-days culture under conditions of 37°C and 5% CtX
After removal of the culture medium, the cell was washed with PBS(-)twice, to which 0.25%
Trypsin-EDTA was added in order to detach the cell from the plate. The detached cell was
suspended in PBS(-) to a volume of 100 ul, to which 10 ul of 0.4% trypan blue stock solution
was further added. The mixure was measured for counts of living ceils using a
hemocytometer. As a result, the 50% inhibitory concentration was determined to be 3 x 10"2
uM. The IC50 values of respective antitumor agents in each type of cancer cells were
calculated by using the same procedures. The results are shown in Table 1.
Table 1
50%> inhibitory concentration of antitumor agents
against MCF-7

[0191]
(2) Effect of antitumor agents on expression of CAPRIN-1 upon treatment of cancer cell with
them
The cancer cell line MCF-7 was treated with each antitumor agent at a 50%
inhibitory concentration which was calculated in (1) above, and the expression behavior of a
CAPRIN-1 protein on the cell surface was examined.
[0192]
The expression behavior of the CAPRIN-1 protein on the surface of the
above-treated MCF-7 human breast cancer cell was examined. The cell was prepared to a
concentration of 1 x lO^cells/ml, and then cultured on a 6-well plate under conditions of 37°C
and 5% CO2 for 1 day. Next, the cell was treated with antitumor agents at the 50%
inhibitory concentration calculated in (1) above, or with PBS(-) as a control, and then cultured
under conditions of 37°C and 5% CO2 for 2 days. After removal of the culture medium, the
cell was washed with PBS(-)twice and then detached from the plate using a cell scraper.
Thereafter, the cell was centrifuged with a 1.5-ml microcentrifuge tube. One (1) ug (5 ul) of
the mouse anti-CAPRIN-1 monoclonal antibody #9 was added to the separated cell, which
was further suspended in 95 yd of PBS containing 0.1% fetal calf serum and then left to stand
on ice for 1 hour. After washing with PBS, the cell was suspended in PBS containing 5 pi of
a FITC-labeled goat anti-rabbit IgG antibody (SantaCruz) and 95 pi of 0.1% fetal bovine
serum (FBS) 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, the same procedure as the above was performed using a control antibody instead
of the mouse anti-CAPRIN-1 monoclonal antibody, and it was used as a control. As a result,
no significant difference in fluorescence intensity was observed regardless of treatment with
an antitumor agent. Specifically, the fluorescence intensity obtained in the case of adding
the anti-CAPRIN-1 antibody to MCF-7 not treated with any antitumor agent indicated 32%
higher enhancement when compared with that in the case of adding the control antibody. On
the other hand, when MCF-7 was treated with the antitumor agent using the same procedure,
the 32% enhancement in fluorescence intensity was observed, and thus this result was the
same as that in the case of no treatment with the antitumor agent. It was revealed by these
results that the treatment of breast cancer cells with an antitumor agent has no effect on
expression of CAPRIN-1 on the surface of breast cancer cells. Here, the percentage of
enhancement in fluorescence intensity was represented by the percentage of increase in mean
fluorescence intensity (MFI level) in each cell and calculated by the following formula.
[0193]
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) - (control MFI level)) ¦*¦ (control MFI level) x 100.
[0194]
Example 7 In vivo combination therapy using anti-CAPRJN-1 antibody and antitumor agent
(1) With the use of tumor-bearing mice into which the CAPRIN-1 -expressing
MCF-7 human breast cancer cell line had been transplanted, the anti-tumor effect of the
combined use of an anti-CAPRJN-1 monoclonal antibody and antitumor agents was examined.
A method for examining the anti-tumor effect using mice into which MCF-7 had been
transplanted and the results thereof are as described below. 106 MCF-7 cells (purchased
from ATCC) were transplanted subcutaneously to the dorsal region of each of 280 nude mice
(Japan SLC Inc.). Mice were grown until each tumor reached a size of about 7 mm in
diameter.
[0195]
Next, as described specifically in the following experimental section 1 and
experimental section 2, tumor-bearing mice into which MCF-7 had been transplanted were
divided into 5 groups, a group to which only the anti-CAPRJN-1 antibody was administered, a
group to which only an antitumor agent (of 4 types) was administered, a group to which an
antitumor agent and an anti-Her2 antibody (mouse anti-human ErbB2 monoclonal antibody,
isotype: IgG2b (R&D systems, catalog No. MAB11291)) were administered in combination, a
group to which an antitumor agent and anti-CAPRJN-1 monoclonal antibody were
administered in combination, and a group to which control (PBS(-) was administered. In
addition, mouse PBMC was administered to all the administration groups.
[0196]
Experimental section 1>
(Group to which only anti-CAPRIN-1 antibody was administered)
The anti-CAPRIN-1 monoclonal antibody #2 was intraperitoneally administered to
each of 5 tumor-bearing mice at 5 mg/kg/shot on days 0, 4,8,11,15, and 17 after the start of
the experiment. PBMC separated from Balb/c mouse spleen was intravenously administered
to each mouse at 1 x 107cells/0.2 mL (RPMI1640) on days 0, 8, and 15 after the start of the
experiment.
[0197]
(Group to which cyclophosphamide was administered)
Cyclophosphamide was intraperitoneally administered to each of 5 tumor-bearing
mice at 80 mg/kg/shot on days 0 and 4 after the start of the experiment. PBMC separated from
Balb/c mouse spleen was intravenously administered to each mouse at 1 x 107 cells/0.2 mL
(RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0198]
(Group to which paclitaxel was administered)
Paclitaxel was intraperitoneally administered to each of 5 tumor-bearing mice at 15
mg/kg/shot on days 0 and 3 after the start of the experiment. PBMC separated from Baib/c
mouse spleen was intravenously administered to each mouse at 1 x 10 cells/0.2 mL
(RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0199]
(Group to which docetaxel was administered)
Docetaxel was intraperitoneally administered to each of 5 tumor-bearing mice at 10
mg/kg/shot on days 0 and 3 after the start of the experiment. PBMC separated from Balb/c
mouse spleen was intravenously administered to each mouse at 1 x 107 cells/0.2 mL
(RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0200]
(Group to which vinorelbine was administered)
Vinorelbine was intraperitoneally administered to each of 5 tumor-bearing mice at 1
mg/kg/shot on day 0 after the start of the experiment. PBMC separated from Balb/c mouse
spleen was intravenously administered to each mouse at 1 x 10 cells/0.2 mL (RPMI1640) on
days 0, 8, and 15 after the start of the experiment.
[0201]
(Group to which cyclophosphamide and anti-Her2 antibody were administered)
Cyclophosphamide was intraperitoneally administered to each of 5 tumor-bearing
mice at 80 mg/kg/shot on days 0 and 4 after the start of the experiment, and at the same time
the anti-Her2 antibody was intraperitoneally administered to each mouse at 5 mg/kg/shot on
days 0, 4, 8, 11, 15, and 17 after the start of the experiment. PBMC separated from Balb/c
mouse spleen was intravenously administered to each mouse at 1 x 107 cells/0.2 mL
(RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0202]
(Group to which paclitaxel and anti-Her2 antibody were administered)
Paclitaxel was intraperitoneally administered to each of 5 tumor-bearing mice at 15
mg/kg/shot on days 0 and 3 after the start of the experiment, and at the same time, the
anti-Her2 antibody was intraperitoneally administered to each mouse at 5 mg/kg/shot on days
0, 4, 8, 11, 15, and 17 after the start of the experiment. PBMC separated from Balb/c mouse
spleen was intravenously administered to each mouse at 1 x 10 cells/0.2 mL (RPMI1640) on
days 0, 8, and 15 after the start of the experiment.
[0203]
(Group to which docetaxel and anti-Her2 antibody were administered)
Docetaxel was intraperitoneally administered to each of 5 tumor-bearing mice at 10
mg/kg/shot on days 0 and 3 after the start of the experiment, and at the same time, the
anti-Her2 antibody was intraperitoneally administered to each mouse at 5 mg/kg/shot on days
0, 4, 8, 11. 15, and 17 after the start of the experiment. PBMC separated from Balb/c mouse
spleen was intravenously administered to each mouse at 1 x 107 cells/0.2 mL (RPMI1640) on
days 0, 8, and 15 after the start of the experiment.
[0204]
(Group to which vinorelbine and anti-Her2 were administered)
Vinorelbine was intraperitoneally administered to each of 5 tumor-bearing mice at 1
mg/kg/shot on day 0 after the start of the experiment, and at the same time, the anti-Her2
antibody was intraperitoneally administered to each mouse at 5 mg/kg/shot on days 0, 4, 8, 11,
15, and 17 after the start of the experiment. PBMC separated from Balb/c mouse spleen was
intravenously administered to each mouse at 1 x 107 cells/0.2 mL (RPMI1640) on days 0, 8,
and 15 after the start of the experiment.
[0205]
(Group to which cyclophosphamide and anti-CAPRIN-1 antibody were administered)
Cyclophosphamide was intraperitoneally administered to each of 5 tumor-bearing
mice at 80 mg/kg/shot on days 0 and 4 after the start of the experiment, and at the same time,
the anti-CAPRIN-1 monoclonal antibody #2 was intraperitoneally administered to each
mouse at 5 mg/kg/shot on days 0, 4, 8, 11, 15, and 17 after the start of the experiment.
PBMC separated from Balb/c mouse spleen was intravenously administered to each mouse at
1 x 107 cells/0.2 mL (RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0206]
(Group to which paclitaxel and anti-CAPRIN-1 antibody were administered)
Paclitaxel was intraperitoneally administered to each of 5 tumor-bearing mice at 15
mg/kg/shot on days 0 and 3 after the start of the experiment, and at the same time, the
anti-CAPRIN-1 monoclonal antibody #2 was intraperitoneally administered to each mouse at
5 mg/kg/shot on days 0, 4, 8, 11, 15, and 17 after the start of the experiment. PBMC
separated from Balb/c mouse spleen was intravenously administered to each mouse at 1 x 10
cells/0.2 mL (RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0207]
(Group to which docetaxel and anti-CAPRIN-1 antibody were administered)
Docetaxel was intraperitoneally administered to each of 5 tumor-bearing mice at 10
mg/kg/shot on days 0 and 3 after the start of the experiment, and at the same time, the
anti-CAPRIN-1 monoclonal antibody #2 was intraperitoneally administered to each mouse at
5 mg/kg/shot on days 0, 4, 8, 11, 15, and 17 after the start of the experiment. PBMC
separated from Balb/c mouse spleen was intravenously administered to each mouse at 1 x 107
cells/0.2 mL(RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0208]
(Group to which vinorelbine and anti-CAPRIN-1 antibody were administered)
Vinorelbine was intraperitoneally administered to each of 5 tumor-bearing mice at 1
mg/kg/shot on day 0 after the start of the experiment, and at the same time, the
anti-CAPRIN-1 monoclonal antibody #2 was intraperitoneally administered to each mouse at
5 mg/kg/shot on days 0, 4, 8, 11, 15, and 17 after the start of the experiment. PBMC
separated from Balb/c mouse spleen was intravenously administered to each mouse at 1 x 107
cells/0.2 mL (RPMI1640) on days 0, 8, and 15 after the start of the experiment.
[0209]

Experimental conditions similar to those for the experimental section 1 were used
for a group to which only an anti-CAPRIN-1 antibody was administered, a group to which
cyclophosphamide and the anti-CAPRIN-1 antibody were administered, a group to which
paclitaxel and the anti-CAPRIN-1 antibody were administered, a group to which docetaxel
and the anti-CAPRIN-1 antibody were administered, and a group to which vinorelbine and the
anti-CAPRIN-1 antibody were administered, except that the anti-CAPRIN-1 monoclonal
antibody #9 was administered as the anti-CAPRIN-1 antibody.
[0210]
Experimental conditions similar to those for the experimental section 1 were used
for a group to which cyclophosphamide was administered, a group to which paclitaxel was
administered, a group to which docetaxel was administered, a group to which vinorelbine was
administered, a group to which cyclophosphamide and an anti-Her2 antibody were
administered, a group to which paclitaxel and the anti-Her2 antibody were administered, a
group to which docetaxel and the anti-Her2 antibody were administered, and a group to which
vinorelbine and the anti-Her2 antibody were administered.
[0211]
Tumor sizes were measured every day and anti-tumor effect was observed for each
administration group of each of the above experimental sections. A group of 5 tumor-bearing
mice to which PBS(-) was administered instead of an antibody was used as a control group. In
addition, the tumor size was determined by calculating the volume using the formula of major
axis x minor axis x minor axis x 0.5.
[0212]
As a result of observation of the anti-tumor effect, in the experimental section 1, the
tumor was found to have regressed to about 79% in the group to which each antitumor agent
had been administered, about 56% in the group to which only the anti-CAPRIN-1 antibody
had been administered, and about 74% in the group to which each antitumor agent and the
anti-Her2 antibody had been administered, when the tumor volume in the control group, to
which PBS(-) had been administered on day 26 after the start of the experiment, was
designated at 100%. On the other hand, in the group to which each antitumor agent and the
anti-CAPRIN-1 antibody had been administered, the tumor was found to have regressed to
about several tens of % on day 14 and found to have almost completely regressed on and after
day 22 (see Fig. 3 to Fig. 6).
Also, in the experimental section 2, the tumor was found to have regressed to about
68% in the group to which each antitumor agent had been administered, about 45% in the
group to which only the anti-CAPRIN-1 antibody had been administered, and about 55% in
the group to which each antitumor agent and the anti-Her2 antibody had been administered,
when the tumor volume in the control group, to which PBS(-) had been administered, on day
26 after the start of the experiment was designated at 100%. On the other hand, in the group
to which each antitumor agent and the anti-CAPRIN-1 antibody had been administered,
tumors were found to have regressed to several tens of % on day 14 and also found to have
almost completely regressed on and after day 22 (see Fig. 7 to Fig. 10).
Industrial Applicability
[0213]
The antibodies of the present invention are useful for treating and/or preventing
cancers.
[0214]
All publications, patents, and patent applications cited herein are incorporated
herein by reference in their entirety.
Sequence Listing Free Text
[0215]
SEQ ID NO: 31: T3 primer
SEQ ID NO: 32: T7 primer
SEQ ID NOS: 33 and 34: primer
SEQ ID NOS: 35 and 36: GAPDH primer
SEQ ID NOS: 38 and 39: primer
SEQ ID NOS: 130 to 135: primer
We Claim:
1. A medicament for treating and/or preventing a cancer, comprising a combination of an
antibody or a fragment thereof having immunological reactivity with a CAPRIN-1 protein,
and one or two or more types of antitumor agent, wherein the antibody or fragment and the
antitumor agent or antitumor agents are combined together or separately.
2. The medicament according to claim 1, wherein the antibody or a fragment thereof having
immunological reactivity with a CAPRIN-1 protein is an antibody or a fragment thereof,
which binds specifically to the extracellular region of a CAPRIN-1 protein existing on the
surface of a cancer cell.
3. The medicament according to claim 1 or 2, wherein the antibody or a fragment thereof
having immunological reactivity with a CAPRIN-1 protein is an antibody or a fragment
thereof, which binds specifically to a polypeptide having the amino acid sequence represented
by SEQ ID NO: 37 in the extracellular region of the CAPRIN-1 protein existing on the
surface of a cancer cell, or an amino acid sequence having 80% or more sequence identity
with the amino acid sequence represented by SEQ ID NO: 37.
4. The medicament according to any one of claims 1 to 3, wherein the CAPRIN-1 protein is
from a human.
5. The medicament according to any one of claims 1 to 4, wherein the above antitumor agent
is any of the antitumor agents as described in the description.
6. The medicament according to claim 5, wherein the antitumor agent is selected from the
group consisting of cyclophosphamide, paclitaxel, docetaxel, vinorelbine, and
pharmaceutically acceptable salts and derivatives thereof.
7. The medicament according to any one of claims 1 to 6, 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.
8. The medicament according to any one of claims 1 to 7, wherein the antibody is a
monoclonal antibody, a polyclonal antibody, or a recombinant antibody.
9. The medicament according to any one of claims 1 to 8, wherein the antibody is a human
antibody, humanized antibody, chimeric antibody, single chain antibody, or bispecific
antibody.
10. A method for treating and/or preventing a cancer, comprising administering the
medicament according to any one of claims 1 to 9 to a subject suspected of having a cancer.
11. The method according to claim 10, comprising administering to a subject the antibody or
a fragment thereof and an antitumor agent, which are contained in the above medicament,
simultaneously or separately.