DESCRIPTION
PHARMACEUTICAL COMPOSITION FOR TREATMENT AND PREVENTION
OF
CANCERS
TECHNICAL FIELD
[0001] The present invention relates to a novel medical use of antibodies to CAPRIN-1 or
fragments thereof as, for example, therapeutic and/or preventive agents for cancer.
BACKGROUND OF INVENTION
[0002] Cancer is the leading cause of death. Treatment currently performed for cancer is
mainly surgical therapy, which can be combined with radiation therapy or chemotherapy. In
spite of development of new surgical methods and discovery of new anti-cancer agents in
recent years, treatment results of cancers are not greatly improved at present except for some
cancers. Through a recent progress of molecular biology and cancer immunology,
antibodies that are specifically reactive with cancers, cancer antigens recognized by cytotoxic
T cells, as well as the genes encoding the cancer antigens, have been identified, and
expectations for specific immunotherapies targeting cancer antigens have been raised
(Tsuyoshi AKIYOSHI, "Gan To Kagaku-Ryoho (Cancer and Chemotherapy)," 1997, vol. 24,
pp. 551-519 (Jp) (Cancer and Chemotherapy Publishers, Inc., Japan)).
[0003] In cancer treatment methods, in order to reduce side effects, it is desirable for
peptides, polypeptides, or proteins recognized as cancer antigens to be absent in almost all
normal cells but specifically present in cancer cells. In 1991, Boon et al of the Ludwig
Institute in Belgium isolated the human melanoma antigen MAGE 1 recognized by
CD8-positive T cells by the cDNA-expression cloning method using an autologous cancer
cell line and cancer-reactive T cells (Bruggen P. et al, Science, 254:1643-1647 (1991)).
Thereafter, the SEREX (serological identification of antigens by recombinant expression
cloning) method was reported, wherein tumor antigens recognized by antibodies produced
through response to an autologous cancer in the body of a patient with cancer can be
identified using the gene-expression cloning technique (Proc. Natl. Acad. Sci. USA,
92:11810-11813 (1995); and US Patent No. 5,698,396). By the SEREX method, some

cancer antigens, which are not substantially expressed in normal cells but are specifically
expressed in cancer cells, were isolated (Int. J. Cancer, 72: 965-971 (1997); Cancer Res., 58:
1034-1041 (1998); Int. J. Cancer, 29: 652-658 (1998); Int. J. Oncol, 14: 703-708 (1999);
Cancer Res., 56: 4766-4772 (1996); and Hum. Mol. Genet 6: 33-39, 1997). Further, clinical
trials of cell therapies using immunocytes that specifically react with cancer antigens, which
are some of the isolated cancer antigens, and cancer-specific immunotherapies using vaccines
comprising cancer antigens or the like have been conducted.
[0004] Meanwhile, in recent years, a variety of antibody medicines for cancer treatment
that target antigen proteins on cancer cells have come into existence. Such medicines used
as cancer-specific therapeutic agents exhibit drug efficacy to a certain extent, and thus they
have been gaining attention. However, most of target antigen proteins are also expressed on
normal cells. As a result of antibody administration, not only cancer cells, but also normal
cells, on which a target antigen has been expressed can be damaged, thereby causing a side
(or adverse) effect, which becomes problematic. Hence, it is expected that, if it becomes
possible to identify cancer antigens that are specifically expressed on the surface of a cancer
cell and to use antibodies targeting such antigens as medicaments, then treatment with
antibody medicines that cause fewer side effects could be realized.
[0005] Cytoplasmic-and proliferation-associated protein 1 (CAPRTN-1) is an intracellular
protein that is expressed when normal cells in resting phase are activated or undergo cell
division. CAPRTN-1 is also known to be involved in the regulation of the transport and
translation of mRNAs through formation of ctytoplasmic stress granules with RNA in a cell.
CAPRIN-1 has different names, such as GPI-anchored membrane protein 1 and membrane
component surface marker 1 protein (MUSI), as if this protein is known to be a membrane
protein. These different names are derived from the report (J. Biol. Chem., 270:
20717-20723, 1995) that the gene sequence of CAPRIN-1 originally has a GPI-binding region
and CAPRTN-1 is a membrane protein expressed in colon cancer cells. It was later reported
that the CAPRTN-1 gene sequence described in said report was not correct; i.e., a frame shift
took place by deletion of a single nucleotide from the CAPRTN-1 gene sequence currently
registered with GenBank or the like, so that 80 amino acids were deleted from the C-terminus
and the resulting artifact (74 amino acids) was the GPI binding portion in the report; and
another error was also present on the 5' side of the gene sequence, thereby resulting in
deletion of 53 amino acids from the N-terminus (J. Immunol, 172: 2389-2400, 2004).

Further, it has been reported that the protein encoded by the CAPRIN-1 gene sequence
currently registered with GenBank or the like was not a cell membrane protein (J. Immunol.,
172: 2389-2400, 2004).
[0006] In addition, based on the report of J. Biol. Chem., 270: 20717-20723, 1995 that
CAPRIN-1 is a cell membrane protein, US2008/0075722 and WO2005/100998 disclose that
CAPRIN-1 under the name of Ml 1S1 can be used for cancer therapy as a target of antibody
medicines for cancer therapy and as one of cell membrane proteins; however, the Examples
contain no description of the cancer therapy using an antibody against the protein. However,
as reported in J. Immunol., 172: 2389-2400, 2004, it was a common belief, from the time of
filing US2008/0075722 up to now, that CAPRIN-1 is not expressed on the surface of a cell,
and thus, it is obvious that the contents of US2008/0075722 and WO2005/100998 based only
on misinformation that CAPRIN-1 is a cell membrane protein should not be understood as
common technical knowledge of persons skilled in the art.
SUMMARY OF THE INVENTION
PROBLEM TO BE SOLVED BY THE INVENTION
[0009] An object of the present invention is to identify cancer antigen proteins
specifically expressed on the surface of cancer cells and to provide a use of antibodies
targeting such proteins as therapeutic and/orpreventive (or prophylactic) agents for cancer.
MEANS FOR SOLVING PROBLEM
[0010] As a result of intensive studies, the present inventors have now obtained cDNA
encoding a protein that binds to an antibody present in the serum from a tumor-bearing
organism by the SEREX method using testis tissue-derived cDNA libraries and sera from
dogs with breast cancer. With the use of the obtained canine genes and genes homologous
thereto from human, bovine, horse, mouse, and chicken, CAPRIN-1 proteins having amino
acid sequences shown in the even numbers of SEQ ID NOS: 2 to 30 (i.e., even-numbered
SEQ ID NOS: 2 to 30) and antibodies against the CAPRIN-1 proteins have now been
prepared. In addition, the present inventors have now found that CAPRIN-1 is specifically
expressed in the cells of breast cancer, brain tumor, leukemia, lymphoma, lung cancer,
esophageal cancer, colon cancer, gastric cancer, and kidney cancer, and that portions of the
CAPRIN-1 proteins are specifically expressed on the surface of such cancer cells. Further,

the present inventors have now found that antibodies against the CAPRIN-1 portions
expressed on cancer cell surfaces can damage (or impair) cancer cells expressing CAPRIN-1.
These findings have led to the completion of the present invention.
[0011] Therefore, the present invention has characteristics as described below.
[0012] The present invention provides a pharmaceutical composition for treatment and/or
prevention of a cancer, which comprises, as an active ingredient, an antibody or a fragment
thereof having an immunological reactivity with a CAPRIN-1 protein having an amino acid
sequence shown in any one of the even numbered SEQ ID NOS: 2 to 30 or 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 of any of the
even-numbered SEQ ID NOS: 2 to 30, or with a fragment of the CAPRIN-1 protein
comprising 7 or more consecutive amino acids.
[0013] In one embodiment of the present invention, the cancer is breast cancer, brain
tumor, leukemia, lymphoma, lung cancer, esophageal cancer, colon cancer, gastric (or
stomach) cancer, or kidney cancer.
[0014] In another embodiment of the present invention, the antibody is a monoclonal or
polyclonal antibody.
[0015] In another embodiment of the present invention, the antibody is a human antibody,
a humanized antibody, a chimeric antibody, a single-chain antibody, or a bispecific antibody.
[0016] In another embodiment of the present invention, the antibody is an antibody
having an immunological reactivity with a polypeptide having the amino acid sequence
shown in SEQ ID NO: 37 or SEQ ID NO: 136 or an amino acid sequence having 80% or
more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or
more sequence identigy with the amino acid sequence, or with a fragment of the polypeptide.
[0017] In another embodiment of the present invention, in the pharmaceutical
composition for treatment and/or prevention of a cancer comprising the antibody as an active
ingredient, the above antibody is any one of the antibodies (a) to (k) described below and has
an immunological reactivity with a CAPRIN-1 protein.
(a) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 44, 45, and 46.
[0018] (b) An antibody comprising a heavy-chain variable region comprising the

sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 50, 51, and 52.
[0019] (c) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 55, 56, and 57.
[0020] (d) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 60, 61, and 62.
[0021] (e) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 65, 66, and 67.
[0022] (f) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 70, 71, and 72 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 74, 75, and 76.
[0023] (g) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 80, 81, and 82 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 84, 85, and 86.
[0024] (h) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 90, 91, and 92 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 94, 95, and 96.
[0025] (i) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 100, 101, and 102 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 104, 105, and 106.
[0026] (j) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 110, 111, and 112 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 114, 115, and 116.
[0027] (k)
An antibody comprising a heavy-chain variable region comprising the sequences shown in
SEQ ID NOS: 120, 121, and 122 and a light-chain variable region comprising the sequences
shown in SEQ ID NOS: 124,125, and 126.
EFFECTS OF THE INVENTION

[0028] Antibodies against CAPRIN-1 used in the present invention damage (or impair)
cancer cells. Therefore, such antibodies against CAPRIN-1 are useful for treatment or
prevention of cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Fig. 1 shows expression patterns of genes encoding CAPRIN-1 proteins in normal
tissues and tumor cell lines. In this Fig., reference no. 1 shows the expression pattern of
each CAPRIN-1 coding gene, and reference no. 2 shows the expression pattern of GAPDH
gene.
Fig. 2 shows the cytotoxic activity of an antibody to CAPRIN-1 (or
anti-CAPRIN-1 antibody) against the breast cancer cell line expressing CAPRIN-1 gene
(T47D). In this Fig., reference no. 3 shows the activity after addition of the
anti-CAPRTN-l antibody, reference no. 4 shows the activity after addition of control antibody,
and reference no. 5 shows the activity in the absence of any antibody.
Fig. 3 shows the cytotoxic activity of an antibody to CAPRIN-1 (or
anti-CAPRIN-1 antibody ) against the breast cancer cell line expressing CAPRIN-1 gene
(MDA-MB-157). In this Fig., reference no. 6 show the activity after addition of the
anti-CAPRIN-1 antibody, reference no. 7 shows the activity after addition of control antibody
and reference no. 8 shows the activity in the absence of any antibody.
Fig. 4 shows the cytotoxicity against the breast cancer MDA-MB-157 cell line
expressing CAPRIN-1, wherein the cytotoxicity is exhibited by the monoclonal antibodies to
CAPRIN-1 (i.e., the monoclonal antibodies #1 to #11), which are reactive with the surface of
the cancer cell. Specifically, this Fig. shows the activity levels after addition of the #1
monoclonal antibody to CAPRIN-1 (reference no. 9), the #2 monoclonal antibody to
CAPRIN-1 (reference no. 10), the #3 monoclonal antibody to CAPRIN-1 (reference no. 11),
the #4 monoclonal antibody to CAPRIN-1 (reference no. 12), the #5 monoclonal antibody to
CAPRIN-1 (reference no. 13), the #6 monoclonal antibody to CAPRIN-1 (reference no. 14),
the #7 monoclonal antibody to CAPRIN-1 (reference no. 15), the #8 monoclonal antibody to
CAPRIN-1 (reference no. 16), the #9 monoclonal antibody to CAPRIN-1 (reference no. 17),
the #10 monoclonal antibody to CAPRIN-1 (reference no. 18), and the #11 monoclonal
antibody to CAPRIN-1 (reference no. 19), the activity level after addition of a monoclonal
antibody reactive with the CAPRIN-1 protein itself but not with the surface of the cancer cell

(reference no. 20), and the activity level after addition of PBS instead of each antibody
(reference no. 21).
Figs. 5a to 5c show the antitumor effect of the monoclonal antibodies to
CAPRIN-1 (i.e., the monoclonal antibodies #1 to #11) reactive with the surface of a cancer
cell, on Balb/c mice into which the mouse carcinoma CT26 cell line expressing CAPPJN-l
was transplanted. These Figs, show the mouse tumor sizes after administration of the #1
monoclonal antibody to CAPPJN-l (reference no. 22), the #2 monoclonal antibody to
CAPPJN-l (reference no. 23), the #3 monoclonal antibody to CAPPJN-l (reference no. 24),
the #4 monoclonal antibody to CAPPJN-l (reference no. 25), the #5 monoclonal antibody to
CAPPJN-l (reference no. 26), the #6 monoclonal antibody to CAPPJN-l (reference no. 27),
the #7 monoclonal antibody to CAPPJN-l (reference no. 28), the #8 monoclonal antibody to
CAPPJN-l (reference no. 29), the #9 monoclonal antibody to CAPPJN-l (reference no. 30),
the #10 monoclonal antibody to CAPPJN-l (reference no. 31), and the #11 monoclonal
antibody to CAPRIN-1 (reference no. 32), the mouse tumor size after administration of a
monoclonal antibody reactive with a CAPRIN-1 protein itself but not with the surface of the
cancer cell (reference no. 33), and the mouse tumor size after administration of PBS instead
of each antibody (reference no. 34).
Figs. 6a to 6c show the antitumor effect of monoclonal antibodies to CAPRIN-1
(i.e., the monoclonal antibodies #1 to #11) reactive with the surface of a cancer cell, on Balb/c
mice into which the mouse carcinoma N1E cell line expressing CAPRIN-1 was transplanted.
These Figs, show the mouse tumor sizes after administration of the #1 monoclonal antibody
to CAPRIN-1 (reference no. 35), the #2 monoclonal antibody to CAPRIN-1 (reference no. 36),
the #3 monoclonal antibody to CAPRIN-1 (reference no. 37), the #4 monoclonal antibody to
CAPRIN-1 (reference no. 38), the #5 monoclonal antibody to CAPPJN-l (reference no. 39),
the #6 monoclonal antibody to CAPPJN-l (reference no. 40), the #7 monoclonal antibody
against CAPPJN-l (reference no. 41), the #8 monoclonal antibody against CAPRIN-1
(reference no. 42), the #9 monoclonal antibody against CAPPJN-l (reference no. 43), the #10
monoclonal antibody to CAPPJN-l (reference no. 44), and the #11 monoclonal antibody
against CAPPJN-l (reference no. 45), the mouse tumor size after administration of a
monoclonal antibody reactive with a CAPRIN-1 protein itself but not with the surface of the
cancer cell (reference no. 46), and the mouse tumor size after administration of PBS instead
of each antiboddy (reference no. 47).

MODE FOR CARRYING OUT THE INVENTION
[0030] As described below, the antitumor activity of antibodies to the polypeptide shown
in any one of the even-numbered SEQ ID NOS: 2 to 30 used in the present invention can be
evaluated by examining in vivo the inhibition of tumor growth in a tumor-bearing animal, or
by examing in vitro whether or not immunocyte- or complement-mediated cytotoxic activity
against tumor cells expressing the polypeptide is exhibited.
[0031] In addition, the nucleotide sequences of polynucleotides encoding the proteins
consisting of the amino acid sequences shown in the even-numbered SEQ ID NOS: 2 to 30
(i.e., SEQ ID NOS: 2, 4, 6...28, and 30) are shown in the odd-numbered SEQ ID NOS: 1 to 29
(i.e., SEQ ID NOS: 1, 3, 5...27, and 29), respectively.
[0032] The amino acid sequences shown in SEQ ID NOS: 6, 8, 10, 12 and 14 in the
Sequence Listing disclosed according to the present invention are the amino acid sequences of
the CPRIN-1 protains, which were isolated, by the SEREX method using canine testis
tissue-derived cDNA libraries and sera from dogs with breast cancer, as polypeptides capable
of binding to antibodies specifically existing in the sera from tumor-bearing dogs; the amino
acid sequences shown in SEQ ID NOS: 2 and 4 are the amino acid sequences of the CPRIN-1
protains isolated as human homologs of said dog polypeptides; the amino acid sequence
shown in SEQ ID NO: 16 is the amino acid sequence of the CPRIN-1 protain isolated as a
bovine homolog of said dog polypeptide; the amino acid sequence shown in SEQ ID NO: 18
is the amino acid sequence of the CPRIN-1 protain isolated as an equine homolog of said dog
polypeptide; the amino acid sequences shown in (even-numbered) SEQ ID NOS: 20 to 28 are
the amino acid sequences of the CPRIN-1 protains isolated as murine homologs of said dog
polypeptides; and the amino acid sequence shown in SEQ ID NO: 30 is the amino acid
sequence of the CPRIN-1 protain isolated as a chicken homolog of said dog polypeptide (see
Example 1 described below). CAPRIN-1 is known to be expressed when activation or cell
division of normal cells in resting phase takes place.
[0033] It was known that CAPRIN-1 was not expressed on the surface of cells.
However, as a result of examination in connection with the present invention, it has been now
revealed that certain portions of CAPRIN-1 protein are expressed on the surfaces of various
cancer cells. According to the present invention, an antibody that binds to a portion within
CAPRIN-1 protein expressed on cancer cell surfaces is preferably used. Examples of the

partial peptides within CAPRIN-1 protein expressed on cancer cell surfaces include
polypeptides consisting of a sequence of 7 or more consecutive amino acids in the region of
the amino acid residue Nos. (or the amino acids (aa)) 50-98 or the amino acid residue Nos.
(aa) 233-305 in an amino acid sequence shown in any one of the even-numbered SEQ ID
NOS: 2 to 30, excluding SEQ ID NOS: 6 and 18, in the Sequence Listing. Specific
examples thereof include the amino acid sequence shown in SEQ ID NO: 37 or 136
(preferably, the region of the amino acid seqeunce shown in SEQ ID NO: 137 or 138 in the
amino acid sequence shown in SEQ ID NO: 136), or 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 said amino acid sequences. Antibodies of the present invention
include all antibodies capable of binding to the above peptides and having antitumor activity.
[0034] The antibodies to CAPRIN-1 usable in the present invention as described above
may be any types thereof, as long as they can exhibit antitumor activity. Examples thereof
include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific
antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain
antibodies (scFV), and fragments thereof such as Fab and F(ab')2. These antibodies and
fragments thereof can be prepared by methods known to persons skilled in the art. In the
present invention, antibodies capable of specifically binding to a CAPRIN-1 protein are
desirable. Such antibodies are preferably monoclonal antibodies; however, as long as
homogenous antibodies can be stably produced, polyclonal antibodies may also be used. In
addition, if the subject is a human, a human antibody or a humanized antibody is desirable in
order to avoid or inhibit the immunorejection.
[0035] The word "specifically binding to a CAPRIN-1 protein" as used herein means that
an antibody of interest specifically binds to the CAPRIN-1 protein and does not substantially
bind to other proteins.
[0036] As described below, the antitumor activity of an antibody used in the present
invention can be evaluated by examining in vivo the inhibition of tumor growth in a
tumor-bearing animal, or examing in vitro whether or not the immunocyte- or
complement-mediated cytotoxic activity against tumor cells expressing the polypeptide is
exhibited.
[0037] Moreover, the subjects in need of treatment and/or prevention of cancer accordnig
to the the present invention are mammals such as human, pet animals, livestock animals, or

sport animals. The preferred subject is a human.
[0038] Production of antigens, production of antibodies, and pharmaceutical compositions,
related to the present invention, will be explained below.
[0039]

Proteins or fragments thereof used as sensitizing antigens for obtaining antibodies
to CAPRIN-1 used in the present invention are not limited in terms of their origins such as
animals including, for example, humans, canines, bovines, horses, mice, rats, and chickens.
However, such proteins or fragments thereof are preferably selected in view of compatibility
with parent cells used for cell fusion. Mammal-derived proteins are generally preferable and
human-derived proteins are particularly preferable. For instance, if the CAPRIN-1 is human
CAPRIN-1, a human CAPRIN-1 protein, a partial peptide thereof, or cells capable of
expressing human CAPRTN-1 can be used.
[0040] Nucleotide sequences and amino acid sequences of human CAPRIN-1 and
homologs thereof can be obtained by, for example, accessing GenBank (NCBI, USA) and
using the BLAST or FASTA algorithm (Karlin and Altschul, Proc. Natl. Acad. Sci. USA,
90:5873-5877,1993; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997).
[0041] According to the present invention, when the nucleotide sequence (SEQ ID NO: 1
or 3) or the amino acid sequence (SEQ ID NO: 2 or 4) of human CAPRIN-1 is used as a base
sequence, targets are nucleic acids or proteins each consisting of a sequence having 70% to
100%, preferably 80% to 100%, more preferably 90% to 100%, and further 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 amino acid sequence of the ORF or mature portion of the
base nucleotide sequence or amino acid sequence. The term "% sequence identity" as used
herein means a percentage (%) of the number of identical amino acids (or nucleotides) to the
total number of amino acids (or nucleotides) in the case that two sequences are aligned such
that maximum similarity can be achieved with or without introduction of gaps.
[0042] Fragments of a CAPRIN-1 protein have lengths ranging from the amino acid
length of an epitope (or an antigenic determinant), which is the smallest unit of an antigen
recognized by an antibody, to less than the full-length of the protein. The epitope refers to a
polypeptide fragment having antigenicity or immunogenicity in mammals and preferably in
humans. The smallest unit of polypeptide fragment consists of approximately 7 to 12 amino

acids, and for example, 8 to 11 amino acids. A specific example thereof is the amino acid
sequence shown in SEQ ID NO: 37, SEQ ID NO: 137, or SEQ ID NO: 138, or 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 said amino acid sequence.
[0043] Polypeptides comprising the aforementioned human CAPRIN-1 protein and partial
peptides thereof can be synthesized according to chemical synthesis methods such as the
Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl
method) (the Japanese Biochemical Society (ed.), "Biochemical Experimentation Course
(Seikagaku Jikken Koza) 1," Protein Chemistry IV, Chemical Modification and Peptide
Synthesis, Kagaku-dojin Publishing Company, Inc. (Japan), 1981). Also, they can be
synthesized by general methods using a variety of commercially available peptide
synthesizers. In addition, polipeptides of interest can be obtained by preparing
polynucleotides encoding the above polypetides using known gene engineering methods
(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, etc.), incorporating each of the polynucleotides into an
expression vector and introducing the vector into a host cell, thereby allowing the host cell to
produce the polypeptide. By such a way, the dseired polypeptides can be obtained.
[0044] Polynucleotides encoding the aforementioned polypeptides can be readily
prepared by known gene engineering techniques or general methods using commercially
available nucleic acid synthesizers. For example, DNA comprising the nucleotide sequence
shown in SEQ ID NO: 1 can be prepared by PCR using a human chromosome DNA or cDNA
library as a template and a pair of primers designed to enable the amplification of the
nucleotide sequence shown in SEQ ID NO: 1. PCR conditions can be appropriately
determined. For example, such conditions may comprise conducting 30 cycles of the
reaction steps (as one cycle) consisitng of: 94°C, 30 seconds (denaturation); 55°C, 30 seconds
to 1 minute (annealing); and 72°C,r 2 minutes (elongation) using a thermostable DNA
polymerase (e.g., Taq polymerase) and a Mg -containing PCR buffer, followed by reaction at
72°C for 7 minutes after completion of the 30 cyles. However, the present invention is not
limited to the above-exemplified PCR conditions. PCR techniques and conditions are
described in, for example, Ausubel et al., Short Protocols in Molecular Biology, 3rd edition,

A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley
& Sons (Chapter 15, in particular).
[0045] In addition, desired DNA can be isolated by preparing appropriate probes and
primers based on information about the nucleotide and amino acid sequences shown in SEQ
ID NOS: 1 to 30 in the Sequence Listing described herein, and screening a human cDNA
library or the like with the use of such probes and primers. Preferably, such cDNA library is
produced from a cell, organ, or tissue in which the protein with any one of the even-numbered
SEQ ID NOS: 2 to 30 is expressed. Examples of the cell or tissue include cells or tissues
from testis and cancers or tumors, such as leukemia, breast cancer, lymphoma, brain tumor,
lung cancer, and colon cancer. Operations such as preparation of probes or primers,
construction of cDNA libraries, screening of cDNA libraries, and cloning of genes of interest,
as described above, are known to persons skilled in the art, and they can be carried out
according to, for example, the methods described in Sambrook et al., Molecular Cloning, the
2nd edition, Current Protocols in Molecular Biology (1989) and Ausbel et al. (ibid). DNAs
encoding human CAPRIN-1 protein and partial peptides thereof can be obtained from the thus
obtained DNAs.
[0046] The above-described host cells may be any cells, as long as they can express the
above-described polypeptides. An example of prokaryotic host cell includes, but is not
limited to, Escherichia coli. Examples of eukaryotic host cells include, but are not limited to,
mammalian cells such as monkey kidney cell (COS1), Chinese hamster ovary cell (CHO),
human embryonic kidney cell line (HEK293), and mouse embryonic skin cell line (NIH3T3),
yeast cells such as budding yeast and dividing yeast cells, silkworm cells, and Xenopus egg
cells.
[0047] When prokaryotic cells are used as host cells, an expression vector having an
origin replicable in prokaryotic cells, a promoter, a ribosome-binding site, a multicloning site,
a terminator, a drug resistance gene, an auxotrophic complementary gene, or the like can be
used. As expression vectors for Escherichia coli, pUC vectors, pBluescriptll, pET
expression systems, pGEX expression systems, and the like can be exemplified. A DNA
encoding the above polypeptide is incorporated into such an expression vector, a prokaryotic
host cell is transformed with the vector, and then the thus obtained transformed cell is
cultured, so that the polypeptide encoded by the DNA can be expressed in the prokaryotic
host cell. At this time, the polypeptide can also be expressed as a fusion protein with

another protein.
[0048] When eukaryotic cells are used as host cells, expression vectors for eukaryotic
cells having a promoter, a splicing region, a poly(A) addition site, or the like can be used.
Examples of such expression vectors include pKAl, pCDM8, pSVK3, pMSG, pSVL,
pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2. By similar procesures to
those mentioned above, a DNA encoding the aforementioned polypeptide is incorporated into
such an expression vector, an eukaryotic host cell is transformed with the vector, and then the
thus obtained transformed cell is cultured, so that the polypeptide encoded by the above DNA
can be expressed in the eukaryotic host cell. When pIND/V5-His, pFLAG-CMV-2,
pEGFP-Nl, pEGFP-Cl, or the like is used as an expression vector, the above polypeptide
may be expressed as a fusion protein with a tag, such as His tag (e.g., (His) to (His) ),
FLAG tag, myc tag, HA tag, or GFP.
[0049] For introduction of an expression vector into a host cell, well known methods can
be employed, such as electroporation, a calcium phosphate method, a liposome method, a
DEAE dextran method, microinjection, viral infection, lipofection, and binding with a
cell-membrane-permeable peptide.
[0050] Isolation and purification of a polypeptide of interest from host cells can be
performed using known isolation techniques in combination. Examples of such known
techniques include, but are not limited to, treatment using a denaturing agent such as urea or a
surfactant, ultrasonication, enzymatic digestion, salting-out, solvent fractionation and
precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric
focusing electrophoresis, ion exchange chromatography, hydrophobic chromatography,
affinity chromatography, and reverse phase chromatography.
[0051]

In general, antibodies are heteromultimeric glycoproteins each comprising at least
two heavy chains and two light chains. Meanwhile, antibodies except for IgM are
heterotetrameric glycoproteins (approximately 150 kDa) each comprising two identical light
(L) chains and two identical heavy (H) chains. Typically, each light chain is connected to a
heavy chain via a single covalent disulfide bond. However, the number of disulfide bonds
between heavy chains varies among different immunogloblin isotypes. Each of heavy chain
and light chain also has an intrachain disulfide bond(s). Each heavy chain has a variable

domain (VH region) at one end thereof, to which some constant regions are bound in series.
Each light chain has a variable domain (VL region) at one end thereof and has a single
constant region at the opposite end thereof. The constant region of a light chain is aligned
with the first constant region of a heavy chain and the light-chain variable domain is aligned
with the heavy-chain variable domain. A specific region of an antibody variable domain,
which is called "complementarity determining region (CDR)," exhibits specific variability so
as to impart binding specificity to an antibody. A relatively conserved portion in a variable
region is called a "framework region (FR)." A complete heavy-chain or light-chain viariable
domain comprises 4 FRs connected to each other via 3 CDRs. Such CDRs are called
"CDRH1," "CDRH2," and "CDRH3," respectively, in such order from the N-terminus in a
heavy chain. Similarly, for a light chain, they are called "CDRL1," "CDRL2," and
"CDRL3," respectively. CDRH3 plays the most important role in terms of antibody-antigen
binding specificity. In addition, CDRs in each chain are retained by FR regions in the state
that they are close to each other, and they contribute to the formation of antibody-antigen
binding sites with CDRs in a corresponding chain. Constant regions do not directly
contribute to antibody-antigen binding. However, they exhibit various effector functions
such as association with antibody-dependent cell-mediated cytotoxicity (ADCC),
phagocytosis through binding to an Fey receptor, half-life/clearance rate via an neonatal Fc
receptor (FcRn), and complement-dependent cytotoxicity (CDC) via a Clq component in the
complement cascade.
[0052]

The term "anti-CAPRIN-1 antibody" used in the present invention refers to an
antibody having an immunological reactivity with a full-length CAPRIN-1 protein or a
fragment thereof.
[0053] The term "immunological reactivity" used herein indicates the characteristics of an
anbibody binding in vivo to a CAPRIN-1 antigen. The tumor-damaging function (e.g., death,
inhibition, or regression) can be expressed as a result of such binding. Specifically, any type
of antibody may be used in the present invention as long as the antibody can bind to a
CAPRIN-1 protein to damage a tumor or a cancer such as leukemia, lymphoma, breast cancer,
brain tumor, lung cancer, esophageal cancer, gastric cancer, kidney cancer, or colon cancer.
[0054] Examples of such antibodies include monoclonal antibodies, polyclonal antibodies,

synthetic antibodies, multispecific antibodies, human antibodies, humanized antibodies,
chimeric antibodies, single-chain antibodies, and antibody fragments (e.g., Fab and F(ab')2).
In addition, examples of arbitrary immunoglobulin classes of such antibodies include IgG,
IgE, IgM, IgA, IgD, and IgY, and examples of arbitrary immunoglobulin subclasses include
IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
[0055] Antibodies may be further modified via acetylation, formylation, amidation,
phosphorylation, or pegylation (PEG), in addition to glycosylation.
[0056] Production examples for a variety of antibodies are described below.
[0057] In a case in which an antibody of interest is a monoclonal antibody, a breast cancer
SK-BR-3 cell line expressing CAPRIN-1 or the like is administered to mice for immunization,
followed by extraction of spleens from the mice. Cells are separated from each spleen and
then are fused with mouse myeloma cells. Clones capable of producing an antibody having
cancer cell growth inhibition action are selected from the obtained fusion cells (hybridomas).
A monoclonal antibody-producing hybridoma having cancer cell growth inhibition action is
isolated and cultured. An antibody of interest can be prepared via purification from the
culture supernatant by a general affinity purification method.
[0058] Also, a monoclonal antibody-producing hybridoma can be produced in a manner
described below, for example. First, an animal is immunized with a sensitizing antigen by a
known method. In a general method, immunization is carried out by intraperitoneally or
subcutaneously injecting a sensitizing antigen into a mammal. Specifically, a sensitizing
antigen is diluted with or suspended in PBS (Phosphate-Buffered Saline), physiological saline,
or the like to an appropriate resultant amount. If desired, an appropriate amount of a
conventional adjuvant (e.g., Freund's complete adjuvant) is mixed therewith. After
emulsification takes place, the resultant is administered to a mammal several times every 4 to
21 days. In addition, an adequate carrier can be used for immunization with a sensitizing
antigen.
[0059] As described above, after immunization of a mammal and confirmation of an
increase to a desired antibody level in serum, immunocytes are collected from the mammal
and subjected to cell fusion. Particularly preferable examples of immunocytes are
splenocytes.
[0060] Mammalian myeloma cells are used as relevant parent cells subjected to fusion
with the above immunocytes. For such myeloma cells, the following various examples of

known cell lines are preferably used: 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 (de St. Groth, S. F. et al, J. Immunol. Methods (1980)
35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), and R210 (Galfre, G. et
al, Nature (1979) 277, 131-133).
[0061] Basically, cell fusion of immunocytes and myeloma cells described above can be
carried out according to a known method such as the method of Kohler and Milstein et al.
(Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).
[0062] More specifically, cell fusion described above is carried out in the presence of a
cell fusion promoter in a conventional nutrients-containing culture solution, for example.
Examples of a fusion promoter to be used include polyethylene glycol (PEG) and Sendai virus
(HVJ: hemagglutinating virus of Japan). If desired, an adjuvant such as dimethylsulfoxide
may be further added for improvement of fusion efficiency.
[0063] The proportion of immunocytes used to that of myeloma cells used can be
arbitrarily determined. For example, the ratio of immunocytes to myeloma cells is
preferably 1:1 to 10:1. Examples of a culture solution that can be used for cell fusion
described above include an RPMI1640 culture solution and an MEM culture solution
adequate for growth of the above myeloma cell lines as well as other conventional culture
solutions used for this kind of cell culture. Further, a serum replacement such as fetal calf
serum (FCS) can be used in combination therewith.
[0064] For cell fusion, the above immunocytes and myeloma cells are sufficiently mixed
at predetermined amounts in the culture solution. A PEG solution (e.g., average molecular
weight: approximately 1000 to 6000) that has been previously heated to approximately 37°C
is added thereto at a concentration of generally 30% to 60% (w/v), followed by mixing. This
results in formation of hybridomas of interest. Subsequently, operational steps of sequential
addition of an appropriate culture solution and removal of the supernatant via centrifugation
are repeatedly carried out to remove cell fusion agent(s) and the like that are not preferable for
the growth of hybridomas.
[0065] The thus obtained hybridomas are cultured in a conventional selection culture
solution such as an HAT culture solution (a culture solution comprising hypoxanthine,

aminopterin, and thymidine) for selection. Culture in such an HAT culture solution is
continuously carried out for a sufficient time period (generally several days to several weeks)
for death of cells (non-fused cells) other than hybridomas of interest. Next, a conventional
limiting dilution method is employed to screen for hybridomas producing antibodies of
interest and to carry out single cloning.
[0066] Further, it is also possible to obtain human antibody-producing hybridomas having
desired activity (e.g., cell growth inhibition activity) in the following manner, as well as to
obtain the above hybridomas via immunization of non-human animals with antigens.
Human lymphocytes (e.g., human lymphocytes infected with EB virus) are sensitized in vitro
with a protein, protein-expressing cells, or a lysate thereof and sensitized lymphocytes are
fused with human-derived myeloma cells having the ability to parmanently divide (e.g.,
U266) (registration no. TIB 196).
[0067] Monoclonal antibody-producing hybridomas produced as above can be
subcultured in a conventional culture solution. In addition, they can be preserved in liquid
nitrogen for a long period of time.
[0068] Specifically, immunization is carried out using a desired antigen or cells
expressing a desired antigen as sensitizing antigen(s) according to a conventional
immunization method. The obtained immunocytes are fused with known parent cells by a
conventional cell fusion method. Then, monoclonal antibody-producing cells (hybridomas)
are screened for by a conventional screening method. Thus, antibody production can be
carried out.
[0069] Other examples of antibodies that can be used in the present invention include
polyclonal antibodies. For example, polyclonal antibodies can be used in a manner
described below.
[0070] Serum is obtained by immunizing small animals such as mice, human
antibody-producing mice, or rabbits with a naturally occurring CAPRIN-1 protein, a
recombinant CAPRIN-1 protein that has been expressed as a protein fused with GST or the
like in a microorganism such as Escherichia coli, or a partial peptide thereof. The serum is
purified via ammonium sulfate precipitation, protein A/protein G column chromatography,
DEAE ion-exchange chromatography, affinity column chromatography with a coulumn to
which a CAPRIN-1 protein or a synthetic peptide is coupled, or a similar technique for
preparation of polyclonal antibodies. In the Examples described below, a rabbit polyclonal

antibody was produced, and antitumor effects thereof were confirmed, such antibody being
against a partial peptide (with the sequence shown in SEQ ID NO: 37) of a domain in a
CAPRIN-1 protein amino acid sequence that is expressed on cancer cell surfaces.
[0071] A known human antibody-producing mouse used herein is, for example, a KM
Mouse (Kirin Pharma/Medarex) or a XenoMouse (Amgen) (e.g., WO02/43478 and
WO02/092812). When such mice are immunized with CAPRTN-1 proteins or fragments
thereof, complete human polyclonal antibodies can be obtained from blood. In addition,
human monoclonal antibodies can be produced by a method of fusing splenocytes collected
from immunized mice with myeloma cells.
[0072] Antigen preparation can be carried out in accordance with a method such as a
method using animal cells (JP Patent Publication (Kohyo) No. 2007-530068) or a method
using a baculovirus (e.g., W098/46777). If the immunogenicity of an antigen is low, an
antigen is bound to a macromolecule having immunogenicity, such as albumin. Then, the
antigen can be used for immunization.
[0073] Further, it is possible to use a gene recombinant antibody produced by cloning an
antibody gene from a hybridoma, incorporating the clone into an adequate vector, introducing
the vector into a host, and using a gene recombinant technique. (See, for example, Carl, A. K.
Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,
Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990.)
Specifically, cDNA of a variable region (V region) of an antibody is synthesized from mRNA
of a hybridoma with the use of a reverse transcriptase. After DNA encoding a V region of
an antibody of interest is obtained, such DNA is ligated to desired DNA encoding an antibody
constant region (C region). The resultant is incoporated into an expression vector.
Alternatively, DNA encoding an antibody V region may be incorporated into an expression
vector comprising DNA of an antibody C region. Such DNA is incorporated into an
expression vector in a manner such that it is expressed under control of an expression control
region such as an enhancer or a promoter. Next, host cells are transformed with such
expression vector, thereby allowing the antibody to be expressed.
[0074] Anti-CAPRIN-1 antibodies of the present invention are preferably monoclonal
antibodies. However, they may be polyclonal antibodies, gene-modified antibodies (such as
chimeric antibodies and humanized antibodies), and the like.
[0075] Monoclonal antibodies include human monoclonal antibodies and non-human

animal monoclonal antibodies (e.g., mouse monoclonal antibodies, rat monoclonal antibodies,
rabbit monoclonal antibodies, and chicken monoclonal antibodies). Monoclonal antibodies
can be produced by culturing hybridomas obtained via fusion of myeloma cells and
splenocytes from non-human mammals (e.g., mice or human antibody-producing mice)
immunized with CAPRIN-1 proteins. In the Examples described below, mouse monoclonal
antibodies were produced and antitumor effects thereof were confirmed. Such a monoclonal
antibody comprises a heavy-chain variable (VH) region having the amino acid sequence
shown in SEQ ID NO: 43, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO:
103, SEQ ID NO: 113, or SEQ ID NO: 123 and a light-chain variable (VL) region having the
amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID
NO: 63, SEQ ID NO: 68, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO:
107, SEQ ID NO: 117, or SEQ ID NO: 127. Here, the VH region comprises: CDR1
represented by the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 70, SEQ ID NO: 80,
SEQ ID NO: 90, SEQ ID NO: 100, SEQ ID NO: 110, or SEQ ID NO: 120; CDR2 represented
by the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 71, SEQ ID NO: 81, SEQ ID
NO: 91, SEQ ID NO: 101, SEQ ID NO: 111, or SEQ ID NO: 121; and CDR3 represented by
the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 72, SEQ ID NO: 82, SEQ ID NO:
92, SEQ ID NO: 102, SEQ ID NO: 112, or SEQ ID NO: 122. The VL region comprises:
CDR1 represented by the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID
NO: 55, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 74, SEQ ID NO: 84, SEQ ID NO: 94,
SEQ ID NO: 104, SEQ ID NO: 114, or SEQ ID NO: 124; CDR2 represented by the amino
acid sequence of SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID
NO: 66, SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 95, SEQ ID NO: 105, SEQ ID NO:
115, or SEQ ID NO: 125; and CDR3 represented by the amino acid sequence of SEQ ID NO:
46, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 76,
SEQ ID NO: 86, SEQ ID NO: 96, SEQ ID NO: 106, SEQ ID NO: 116, or SEQ ID NO: 126.
[0076] A chimeric antibody is an antibody produced by combining sequences from
different animals. An example thereof is an antibody consisting of 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 produced by a known method. For
example, it can be obtained by ligating DNA encoding an antibody V region to DNA
encoding a human antibody C region, incorporating the resultant into an expression vector,

and introducing the vector into a host for antibody production.
[0077] Polyclonal antibodies include antibodies obtained by immunizing human
antibody-producing animals (e.g., mice) with CAPRIN-1 proteins.
[0078] A humanized antibody is a modified antibody, and it is sometimes referred to as a
"reshaped human antibody." It is known that a humanized antibody is constructed by
transplanting CDRs of an immunized animal-derived antibody into complementarity
determining regions of a human antibody. Also, a general gene recombinant technique
therefor is known.
[0079] Specifically, a DNA sequence designed in a manner that allows mouse antibody
CDRs to be ligated to human antibody framework regions (FRs) is synthesized by the PCR
method using several oligonucleotides prepared in such a manner that the oligonucleotides
have portions overlapping each other at one end of each thereof. A humanized antibody can
be obtained by ligating the above obtained DNA to DNA encoding a human antibody constant
region, incorporating the resultant into an expression vector, and introducing the vector into a
host for antibody production (see EP-A-239400 and WO96/02576). Human antibody FRs
ligated to each other via CDRs are selected on the assmption that complementarity
determining regions can form a good antigen binding site. If necessary, amino acids in
framework regions of an antibody variable region may be substituted in such a manner that
complementarity determining regions in a reshaped human antibody form an appropriate
antigen binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). In addition, the
framework regions may be substituted with framework regions from a different human
antibody (see W099/51743).
[0080] Human antibody framework regions ligated to each other via CDRs are selected
on the assmption that complementarity determining regions can form good antigen binding
sites. If necessary, amino acids in framework regions of an antibody variable region may be
substituted in such a manner that complementarity determining regions in reshaped human
antibody form an appropriate antigen binding sites (Sato K. et al., Cancer Research 1993, 53:
851-856).
[0081] After a chimeric antibody or a humanized antibody is produced, amino acids in a
variable region (e.g., FR) or a constant region may be substituted, for example, with different
amino acids.
[0082] Here, the amino acid substitution is a substitution of, for example, less than 15,

less than 10, not more than 8, not more than 7, not more than 6, not more than 5, not more
than 4, not more than 3, or not more than 2 amino acids, preferably 1 to 5 amino acids, and
more preferably 1 or 2 amino acids. A substituted antibody should be functionally
equivelent to an unsubstituted antibody. The substitution is preferably a conservative amino
acid substitution, which is a substitution between amino acids having similar characteristics in
terms of charge, side chains, polarity, aromaticity, and the like. For example,
characteristically similar amino acids can be classified into the following types: 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, isoleucine); and
aromatic amino acids (phenylalanine, tyrosine, tryptophan, and histidine).
[0083] An example of an antibody modifier is an antibody bound to a molecule such as
polyethylene glycol (PEG). Regarding antibody modifiers of the present invention,
substances that bind to an antibody are not limited. Such an antibody modifier can be
obtained by chemically modifying an obtained antibody. A method of such modification has
been already established in the field related to the present invention.
[0084] The expression "functionally equivalent" used herein indicates a situation in which
an antibody of interest has biological or biochemical activity similar to that of an antibody of
the present invention. Specifically, such antibody has a function of damaging tumors and
causes essentially no rejection reaction when applied to humans. An example of such
activity is cell growth inhibition activity or binding activity.
[0085] A known method for preparing a polypeptide functionally equivalent to a given
polypeptide that is well known to persons skilled in the art is a method comprising
introducing a mutation into a polypeptide. For instance, a person skilled in the art can
adequately introduce a mutation into an antibody of the present invention using a site-specific
mutagenesis method (Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275; Zoller, MJ., and
Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al., (1984) Nucleic Acids
Res. 12, 9441-9456; Kramer, W. and Fritz, HJ., (1987) Methods Enzymol. 154, 350-367;
Kunkel, TA., (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492; or Kunkel (1988) Methods
Enzymol. 85, 2763-2766) or a similar method. Thus, an antibody funcitonally equivalent to
the antibody of the present invention can be prepared.

[0086] An aforemenioned antibody capable of recognizing an epitope of a CAPRIN-1
protein recognized by an anti-CAPRIN-1 antibody can be obtained by a method known to
persons skilled in the art. For example, it can be obtained by: a method comprising
determining an epitope of a CAPRIN-1 protein recognized by an anti-CAPRIN-1 antibody by
a general method (e.g., epitope mapping) and producing an antibody using a polypeptide
having an amino acid sequence contained in the epitope as an immunogen; or a method
comprising determining an epitope of an antibody produced by a general method and
selecting an antibody having an epitope identical to an epitope of an anti-CAPRIN-1 antibody.
Here, the term "epitope" refers to a polypeptide fragment having antigenicity or
immunogenicity in mammals and preferably in humans. The smallest unit threof consists of
approximately 7 to 12 amino acids and preferably 8 to 11 amino acids.
[0087] The affinity constant Ka (kon/koff) of an antibody of the present invention is
preferably at least 107 M"1, at least 108 M"1, at least 5 x 108 M-1, at least 109 M-1, at least 5 x
109 M-1, at least 1010 M-1, at least 5 x 1010 M-1, at least 1011 M-1, at least 5 x 1011 M-1, at least
1012M-1,oratleast 1013M-1.
[0088] An antibody of the present invention can be conjugated with an antitumor agent.
Binding between an antibody and an antitumor agent can be carried out via a spacer having a
group reactive to an amino group, a carboxyl group, a hydroxy group, a thiol group, or the
like (e.g., an imidyl succinate group, a formyl group, a 2-pyridyldithio group, a maleimidyl
group, an alkoxycarbonyl group, or a hydroxy group).
[0089] Examples of antitumor agents include the following antitumor agents known in
references or the like: paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate,
5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carboquone,
meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide,
triethilenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin,
bryo statin, callystatin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin,
pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine,
chlorozotociri, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, dynemicin,
clodronate, esperamicin, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin,
cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin,

detorbicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN, epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycig, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorabicin, denopterin, pteropterin, trimetrexate, fludarabine,
6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone,
aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside,
aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,
demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, lentinan,
lonidamine, maytansine, ansamitocine, mitoguazone, mitoxantrone, mopidanmol, nitraerine,
pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,
procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid,
triaziquone, roridine A, anguidine, urethane, vindesine, dacarbazine, mannomustine,
mitobronitol, mitolactol, pipobroman, gacytosine, docetaxel, chlorambucil, gemcitabine,
6-thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide,
ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate,
daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor,
difluoromethylornithine (DMFO), retinoic acid, capecitabine, and pharmacologically
acceptable salts or derivatives thereof.
[0090] Alternatively, it is also possible to bind a radioactive isotope such as At, I,
125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 175Lu, or 176Lu known in references and the like to an
antibody of the present invention. It is desirable for such radioactive isotopes to be effective
for tumor treatment or diagnosis.
[0091] An antibody of the present invention is an antibody having an immunological
reactivity with CAPRIN-1 or an antibody capable of specifically recognizing CAPRIN-1.
Such an antibody should be an antibody having a structure that allows a subject animal to
which the antibody is administered to completely or almost completely avoid a rejection
reaction. If the subject animal is a human, examples of the above antibody include human
antibodies, humanized antibodies, chimeric antibodies (e.g., human-mouse chimeric
antibodies), single-chain antibodies, and bispecific antibodies. Such an antibody is a
recombinant antibody having human antibody-derived heavy-chain and light-chain variable

regions, a recombinant antibody having heavy-chain and light-chain variable regions each
consisting of non-human animal antibody-derived complementarity determining regions
(CDR1, CDR2, and CDR3) and human antibody-derived framework regions, or a
recombinant antibody having non-human animal antibody-derived heavy-chain and
light-chain variable regions and human antibody-derived heavy-chain and light-chain constant
regions. The first two antibodies are preferable.
[0092] The above recombinant antibody can be produced in the manner described below.
DNA encoding a monoclonal antibody against human CAPRTN-1 (e.g., a human monoclonal
antibody, a mouse monoclonal antibody, a rat monoclonal antibody, a rabbit monoclonal
antibody, or a chicken monoclonal antibody) is cloned from an antibody-producing cell such
as a hybridoma. DNAs encoding a light-chain variable region and a heavy-chain variable
region of the antibody are produced by an RT-PCR method or the like using the obtained
clone as a template. Then, the sequences of a light-chain variable region and a heavy-chain
variable region or the sequences of CDR1, CDR2, and CDR3 are determined by 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)).
[0093] Further, such DNAs encoding variable regions or DNAs encoding CDRs are
produced by a gene recombinant technique (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 produced by
immunizing a human antibody-producing animal (e.g., a mouse) with human CAPRIN-1 and
fusing splenocytes from the spleen removed from the animal with myeloma cells. In
addition to the above, if necessary, DNAs encoding human antibody-derived light-chain or
heavy-chain variable regions and constant regions are produced by a gene recombinant
technique or a DNA synthesizer.
[0094] In the case of a humanized antibody, DNA in which the CDR coding sequences in
a DNA encoding a human antibody-derived light-chain or heavy-chain variable region have
been substituted with corresponding CDR coding sequences of an antibody from a non-human
animal (e.g., a mouse, a rat, or a chicken) is produced. The DNA obtained as above is
ligated to the DNA encoding a constant region of a human antibody-derived light chain or
heavy chain. Thus, DNA encoidng a humanized antibody can be produced.
[0095] In the case of a chimeric antibody, DNA encoding an antibody light-chain or

heavy-dhain variable region from a non-human animal (e.g., a mouse, a rat, or a chicken) is
ligated to the DNA encoding a human antibody-derived light-chain or heavy-chain constant
region. Thus, DNA encoding a chimeric antibody can be produced.
[0096] A single-chain antibody is an antibody in which a heavy-chain variable region and
a light-chain variable region are linearly ligated to each other via a linker. DNA encoding a
single-chain antibody can be produced by binding DNA encoding a heavy-chain variable
region, DNA encoding a linker, and a DNA encoding a light-chain variable region. Here, a
heavy-chain variable region and a light-chain variable region are those from a human
antibody or those from a human antibody in which CDRs alone have been substituted with
CDRs of an antibody from a non-human animal (e.g., a mouse, a rat, or a chicken). In
addition, the linker consists of 12 to 19 amino acids. An example thereof is (G4S)3
consisting of 15 amino acids (G. B. Kim et al., Protein Engineering Design and Selection
2007, 20 (9): 425-432).
[0097] A bispecific antibody (diabody) is an antibody capable of specifically binding to
two different epitopes in which, for example, 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 are bound to each other in such order
(provided that DNA encoding a light-chain variable region B and DNA encoding a
heavy-chain variable region B are bound to each other via DNA encoding a linker described
above). Thus, DNA encoding a bispecific antibody can be produced. Here, both a
heavy-chain variable region and a light-chain variable region are those from a human
antibody or those from a human antibody in which CDRs alone have been substituted with
CDRs of an antibody from a non-human animal (e.g., a mouse, a rat, or a chicken).
[0098] Recombinant DNA produced as above is incorporated into one or a plurality of
appropriate vector(s). Each such vector is introduced into a host cell (e.g., a mammal cell, a
yeast cell, or an insect cell) for (co)expression. Thus, a recombinant antibody can be
produced (P. J. Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997
WILEY, P. Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY
PRESS; J. W. Goding, Monoclonal Antibodies: Principles and Practice., 1993 ACADEMIC
PRESS).
[0099] Examples of an antibody of the present invention produced by the above method
include the following antibodies (a) to (k).

[0100] (a) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 44, 45, and 46 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 43 and a light-chain variable
region of SEQ ID NO:47).
[0101] (b) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 50, 51, and 52 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 43 and a light-chain variable
region of SEQ ID NO: 53).
[0102] (c) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 55, 56, and 57 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 43 and a light-chain variable
region of SEQ ID NO: 58).
[0103] (d) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 60, 61, and 62 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 43 and a light-chain variable
region of SEQ ID NO: 63).
[0104] (e) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 65, 66, and 67 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 43 and a light-chain variable
region of SEQ ID NO: 68).
[0105] (f) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 70, 71, and 72 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 74, 75, and 76 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 73 and a light-chain variable
region of SEQ ID NO:77).
[0106] (g) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 80, 81, and 82 and a light-chain variable region

comprising the sequences shown in SEQ ID NOS: 84, 85, and 86 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 83 and a light-chain variable
region of SEQ ID NO:87).
[0107] (h) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 90, 91, and 92 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 94, 95, and 96 (and preferably an antibody
composed of a heavy-chain variable region of SEQ ID NO: 93 and a light-chain variable
region of SEQ ID NO: 97).
[0108] (i) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 100, 101, and 102 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 104, 105, and 106 (and preferably an
antibody composed of a heavy-chain variable region of SEQ ID NO: 103 and a light-chain
variable region of SEQ ID NO: 107).
(j) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 110, 111, and 112 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 114, 115, and 116 (and preferably an
antibody composed of a heavy-chain variable region of SEQ ID NO: 113 and a light-chain
variable region of SEQ ID NO: 117).
[0109] (k) An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 120, 121, and 122 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 124, 125, and 126 (and preferably an
antibody composed of a heavy-chain variable region of SEQ ID NO: 123 and a light-chain
variable region of SEQ ID NO: 127).
[0110] Here, amino acid sequences shown in SEQ ID NOS: 40, 41, and 42, amino acid
sequences shown in SEQ ID NOS: 70, 71, and 72, amino acid sequences shown in SEQ ID
NOS: 80, 81, and 82, amino acid sequences shown in SEQ ID NOS: 90, 91, and 92, amino
acid sequences shown in SEQ ID NOS: 100, 101, and 102, amino acid sequences shown in
SEQ ID NOS: 110, 111, and 112, or amino acid sequences shown in SEQ ID NOS: 120, 121,
and 122 correspond to CDR1, CDR2, and CDR3 of mouse antibody heavy-chain variable
regions, respectively. In addition, amino acid sequences shown in SEQ ID NOS: 44, 45, and
46, amino acid sequences shown in SEQ ID NOS: 50, 51, and 52, amino acid sequences
shown in SEQ ID NOS: 55, 56, and 57, amino acid sequences shown in SEQ ID NOS: 60, 61,

and 62, amino acid sequences shown in SEQ ID NOS: 65, 66, and 67, amino acid sequences
shown in SEQ ID NOS: 74, 75, and 76, amino acid sequences shown in SEQ ID NOS: 84, 85,
and 86, amino acid sequences shown in SEQ ID NOS: 94, 95, and 96, amino acid sequences
shown in SEQ ID NOS: 104, 105, and 106, amino acid sequences shown in SEQ ID NOS:
114, 115, and 116, or amino acid sequences shown in SEQ ID NOS: 124, 125, and 126
correspond to CDR1, CDR2, and CDR3 of mouse antibody light-chain variable regions,
respectively.
[0111] In addition, a humanized antibody, a chimeric antibody, a single-chain antibody,
or a bispecific antibody of the present invention is, for example, the folllowing antibody (i) or
(ii) (an example of antibody (a) is described below).
[0112] (i) An antibody comprising: a heavy-chain variable region comprising the amino
acid sequences of SEQ ID NOS: 40, 41, and 42 and an amino acid sequence of a human
antibody-derived framework region; and a light-chain variable region comprising the amino
acid sequences of SEQ ID NOS: 44, 45, and 46 and amino acid sequences of human
antibody-derived framework regions (and preferably an antibody comprising the amino acid
sequence of SEQ ID NO: 43 in a heavy-chain variable region and the amino acid sequence of
SEQ ID NO: 47 in a light-chain variable region).
[0113] (ii) An antibody comprising: a heavy-chain variable region comprising the amino
acid sequences of SEQ ID NOS: 40, 41, and 42 and amino acid sequences of human
antibody-derived framework regions; a heavy-chain constant region comprising a human
antibody-derived amino acid sequence; a light-chain variable region comprising the amino
acid sequences of SEQ ID NOS: 44, 45, and 46 and amino acid sequences of human
antibody-derived framework regions; and a light-chain constant region comprising a human
antibody-derived amino acid sequence (and preferably an antibody comprising: a heavy-chain
variable region comprising the amino acid sequence of SEQ ID NO: 43; a heavy-chain
constant region comprising a human antibody-derived amino acid sequence; a light-chain
variable region comprising the amino acid sequence of SEQ ID NO: 47; and a light-chain
constant region comprising a human antibody-derived amino acid sequence).
[0114] In addition, sequences of human antibody heavy-chain and light-chain constant
and variable regions can be obtained from, for example, NCBI (U.S.A: GenBank, UniGene,
etc.). For example, the following sequences can be used as reference sequences for the
corresponding regions: the sequence with registration no. J00228 for a human IgG1

heavy-chain constant region; the sequence with registration no. J00230 for a human IgG2
heavy-chain constant region; the sequence with registration no. X03604 for a human IgG3
heavy-chain constant region; the sequence with registration no. K01316 for a human IgG4
heavy-chain constant region; the sequence with registration no. V00557, X64135, or X64133
for a human light-chain K constant region; and the sequence with registration no. X64132 or
X64134 for a human light-chain X constant region.
[0115] The above antibodies preferably have cytotoxic activity, thereby exhibiting
antitumor effects.
[0116] In addition, the above specific sequences of heavy-chain and light-chain variable
regions and CDRs in an antibody are merely described for exemplification. . It is obvious that
the present invention is not limited to particular sequences. A hybridoma capable of
producing a different human antibody or a non-human animal antibody (e.g., a mouse
antibody) against human CAPRIN-1 is produced. A moloclonal antibody produced by the
hybridoma is collected. Then, it is determined whether or not the obtained antibody is an
antibody of interest using, as indicators, immunological binding activity and cytotoxic activity
with respect to human CAPRIN-1. Thus, a monoclonal antibody-producing hybridoma of
interest is identified. Thereafter, as described above, DNAs encoding heavy-chain and
light-chain variable regions of an antibody of interest are produced from the hybridoma for
sequence determination. The DNAs are used for production of different antibodies.
[0117] Further, the above antibody of the present invention may be any one of antibodies
(i) to (iv) above having a substitution, deletion, or addition of one or several (and preferably,
1 or 2) amino acid(s), particularly in a framework region sequence and/or a constant region
sequence, as long as it has the specific property of specifically recognizing CAPRIN-1.
Here, the term "several amino acids" indicates 2 to 5 and preferably 2 or 3 amino acids.
[0118] Furthermore, according to the present invention, DNA encoding the above
anbibody of the present invention, DNA encoding a heavy chain or light chain of the antibody,
or DNA encoding a heavy-chain or light-chain variable region of the antibody is also
provided. For instance, in the case of antibody (a), examples of such DNA include: DNA
encoding a heavy-chain variable region comprising nucleotide sequences encoding the amino
acid sequences of SEQ ID NOS: 40, 41, and, 42; and DNA encoding a light-chain variable
region comprising nucleotide sequences encoding the amino acid sequences of SEQ ID NOS:
44, 45, and 46.

[0119] Complementarity determining regions (CDRs) encoded by DNAs of the above
sequences are regions that determine antibody specificity. Therefore, sequences encoding
the other regions (i.e., constant regions and framework regions) in an antibody may be
sequences from a different antibody. Here, different antibodies include antibodies from
non-human organisms. However, in view of reduction of side effects, human-derived
antibodies are preferable. That is to say, in the above case, DNA regions encoding
framework regions and constant regions of heavy and light chains preferably comprise
nucleotide sequences encoding the relevant amino acid sequences from a human antibody.
[0120] Further, different examples of DNA encoding an antibody of the present invention,
such as antibody (a), include DNA encoding a heavy-chain variable region comprising a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 and DNA in which
a region encoding a light-chain variable region comprises a nucleotide sequence encoding the
amino acid sequence of SEQ ID NO: 47. Here, an example of a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 43 is the nucleotide sequence of SEQ ID
NO: 48. In addition, an example of a nucleotide sequence encoding the amino acid sequence
of SEQ ID NO: 47 is the nucleotide sequence of SEQ ID NO: 49. Also, the above DNAs
encoding heavy-chain and light-chain constant regions preferably comprise nucleotide
sequences encoding the corresponding human antibody-derived amino acid sequences.
[0121] DNA of the present invention can be obtained by, for example, the aforementioned
methods or the following methods. First, total RNA is prepared from a hybridoma for an
antibody of the present invention using a commercially available RNA extraction kit. Then,
cDNA is synthesized with a reverse transcriptase using random primers and the like. Next,
cDNA encoding an antibody is amplified by a PCR method using, as primers,
oligonucleotides having sequenses conserved in variable regions of known mouse antibody
heavy-chain and light-chain genes. Sequences encoding constant regions can be obtained by
amplifying known sequences by a PCR method. The nucleotide sequence of the DNA can
be determined by a general method involving, for example, incorporation into a plasmid or
phage for sequence determination.
[0122] It is thought that antitumor effects of an anti-CAPRIN-1 antibody used in the
present invention upon CAPRIN-1-expressing cancer cells are exhibited through mechanisms
of cytotoxicities described below.
[0123] The cytotoxicities are effector cell-mediated antibody-dependent cellular

cytotoxicity (ADCC) against CAPRIN-1 -expressing cells and complement-dependent
cytotoxicity (CDC) against CAPRIN-1-expressing cells.
[0124] Accordingly, the activity of an anti-CAPRIN-1 antibody used in the present
invention can be evaluated via ex vivo determination of ADCC activity or CDC activity to
CAPRIN-1-expressing cancer cells as specifically described in the Examples mentioned
below.
[0125] An anti-CAPRIN-1 antibody used in the present invention binds to a
CAPRIN-1-protein on a cancer cell and exhibits antitumor effects based on the above activity.
Therefore, such antibody is believed to be useful for cancer treatment or prevention.
Specifically, according to the present invention, the pharmaceutical composition for treatment
and/or prevention of cancer that comprises, as an active incredient, an anti-CAPRIN-1
antibody, is provided. When an anti-CAPRIN-1 antibody is used for the purpose of
administering an antibody to humans (antibody treatment), it is preferably used in the form of
a human antibody or a humanized antibody in order to reduce immunogenicity.
[0126] In addition, as the binding affinity between an anti-CAPRIN-1 antibody and a
CAPRIN-1 protein on a cancer cell surface becomes higher, stronger antitumor activity can be
exhibited by an anti-CAPRIN-1 antibody. Therefore, if an anti-CAPRIN-1 antibody having
high binding affinity to a CAPRIN-1 protein can be obtained, even stronger antitumor effects
can be expected to be exhibited. Accordingly, it becomes possible to use such antibody as a
parmaceutical composition for cancer treatment and/or prevention. As described above, for
high binding affinity, the affinity constant Ka (kon/koff) is preferably at least 107 M-1, at least
108 M-1, at least 5 x 108 M-1, at least 109 M"1, at least 5 x 109 M-1, at least 1010 M-1, at least 5 x
1010 M-1, at least 1011 M-1, at least 5 x 1011 M-1, at least 1012 M-1, or at least 1013 M-1.
[0127]

The capacity of an antibody to bind to CAPRIN-1 can be specified via binding
assay using, for example, ELISA, a Western blot method, immunofluorescence, or
flowcytometry analysis as described in the Examples.
[0128]

An antibody that recognizes CAPRIN-1 can be tested in terms of reactivity with
CAPRIN-1 by an immunohistochemical method known to persons skilled in the art using a

frozen tissue section fixed with paraformaldehyde or acetone or a paraffin-embedded tissue
section fixed with paraformaldehyde. Such section is prepared from a tissue obtained from a
patient during surgery or an animal carrying xenograft tissue that has been innoculated with a
natural cell or transfected cell line that expresses CAPRIN-1.
[0129] For immunohistochemical staining, an antibody reactive to CAPRIN-1 can be
stained by a variety of methods. For example, it can be visualized by reacting with a
horseradish peroxidase-conjugated goat anti-mouse antibody or goat anti-rabbit antibody.
[0130]
Pharmaceutical composition>
A target of the pharmaceutical composition for treatment and/or prevention of
cancer of the present invention is not particularly limited as long as the target is a cancer (cell)
expressing the CAPPJN-1 gene.
[0131] Both the terms "tumor" and "cancer" used herein refer to malignant neoplasm, and
thus they are used in an exchangeable manner.
[0132] A cancer that can be a target in the present invention is a cancer expressing a gene
encoding a polypeptide comprising an amino acid sequence of any one of the even-numbered
SEQ ID NOS: 2 to 30 or a partical sequence consisting of 7 or more consecutive amino acids
of said amino acid sequence. Preferable examples thereof include breast cancer, brain tumor,
leukemia, lung cancer, lymphoma, mastocytoma, esophageal cancer, and colon cancer.
[0133] Examples of these specific cancers include, but are not limited to, breast
adenocarcinoma, composite type breast adenocarcinoma, malignant mammary mixed tumor,
intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell cancer, small cell
cancer, large cell cancer, glioma that is a tumor of neuroepithelial tissue, ependymoma,
neuronal tumor, embryonal neuroectodermal tumor, schwannoma, neurofibroma, meningioma,
chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma,
small-cell-to-medium-cell lymphoma, cecal cancer, ascending colon cancer, descending colon
cancer, transverse colon cancer, sigmoid colon cancer, and rectal cancer.
[0134] In addition, the subject animal of the present invention is a mammal. Examples
thereof include mammals such as primates, pet animals, livestock animals, and sport animals.
Humans, dogs, and cats are particularly preferable.
[0135] When an antibody used in the present invention is used as a pharmaceutical
composition, it can be formulated by a method known to persons skilled in the art. For

instance, it can be parenterally used in the form of a parenteral injection of: an aseptic
solution comprising water or a pharmacologically acceptable non-water solution; or a
suspension liquid. For example, in one possible case, it can be formulated with the
combined use of a pharmacologically acceptable carrier or medium and specifically sterilized
water, physiological saline, plant oil, an emulsifier, a suspension, a surfactant, a stabilizer, a
flavoring agent, an excipient, a vehicle, a preservative, or a binder in an appropriate manner
by mixing in a unit dosage form reqruied for a generally acceptable pharmaceutical
formulation. The amount of an active ingredient in a formulation is determined such that an
appropriate dosage within the indicated range can be achieved.
[0136] . An aseptic composition for injection purposes can be formulated in accordance
with general formulation practice using a vehicle such as distilled water for injection
purposes.
[0137] Examples of an aqueous solution for injection purposes include physiological
saline and isotonic solutions comprising glucose and other adjuvants such as D-sorbitol,
D-mannose, D-mannitol, and sodium chloride. Such solution may be used with an
appropriate dissolution aid. Examples of such dissolution aid include alcohols such as
ethanol and polyalcohol, propylene glycol, polyethylene glycol, and nonion surfactants such
as polysorbate 80(TM) and HCO-60.
[0138] Examples of oily liquid include sesame oil and soybean oil. Such oily liquid may
be used in combination with a dissolution aid such as benzyl benzoate or benzyl alcohol. In
addition, it may be mixed with a buffering agent such as a phosphate buffer solution, a
sodium acetate buffer solution, a soothing agent such as procaine hydrochloride, a stabilizer
such as benzyl alcohol, phenol, or an antioxidant. In general, a formulated injeciton solution
is introduced into an adequate ample.
[0139] The above pharmaceutical composition is orally or parenterally administered.
Preferably, it is parenterally administered. Specific examples of dosage forms include
injectable agents, intranasally-administered agents, transpulmonarily-administered agents, and
percutaneously-administered agents. For example, injectable agents can be systemically or
locally administered via intravenous injection, intramuscular injection, intraperitoneal
injection, or subcutaneous injection.
[0140] In addition, the administration method can be appropriately determined depending
on patient age, weight, gender, and symptoms. A single dose of a pharmaceutical

composition comprising an antibody or a polynucleotide encoding an antibody can be selected
within a range of, for example, 0.0001 mg to 1000 mg per kg of body weight. Alternatively,
the dose can be selected within a range of, for example, 0.001 to 100000 mg per patient's
body; however, it is not necessarily limited thereto. The dose and the administration method
are changed depending on patient age, weight, gender, and symptoms. However, persons
skilled in the art can appropriately select the dose and the method.
[0141]
Polypeptide and DNA>
According to the present invention, the following polypeptides and DNAs for
antibodies (a) to (k) described above are further provided.
[0142] (i) A polypeptide comprising the amino acid sequences of SEQ ID NO: 43, SEQ
ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 113, and SEQ
ID NO: 123, and DNA encoding the polypeptide.
[0143] (ii) A polypeptide comprising the amino acid sequences of SEQ ID NO: 47, SEQ
ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 77, SEQ ID NO:
87, SEQ ID NO: 97, SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 127, and DNA
encoding the polypeptide.
[0144] (iii) DNA comprising the nucleotide sequences of SEQ ID NO: 48, SEQ ID NO:
78, SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO: 108, SEQ ID NO: 118, and SEQ ID NO:
128.
[0145] (iv) DNA comprising the nucleotide sequences of SEQ ID NO: 49, SEQ ID NO:
54, SEQ ID NO: 59, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89,
SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119, and SEQ ID NO: 129.
[0146] (v) A heavy-chain CDR polypeptide comprising amino acid sequences selected
from the group consisting of amino acid sequences of SEQ ID NOS: 40, 41, and 42, amino
acid sequences of SEQ ID NOS: 70, 71, and 72, amino acid sequences of SEQ ID NOS: 80,
81, and 82, amino acid sequences of SEQ ID NOS: 90, 91, and 92, amino acid sequences of
SEQ ID NOS: 100, 101, and 102, amino acid sequences of SEQ ID NOS: 110, 111, and 112,
and amino acid sequences of SEQ ID NOS: 120, 121, and 122, and DNA encoding the
polypeptide.
[0147] (vi) A light-chain CDR polypeptide comprising amino acid sequences selected
from the group consisting of amino acid sequences of SEQ ID NOS: 44, 45, and 46, amino

acid sequences of SEQ ID NOS: 50, 51, and 52, amino acid sequences of SEQ ID NOS: 55,
56, and 57, amino acid sequences of SEQ ID NOS: 60, 61, and 62, amino acid sequences of
SEQ ID NOS: 65, 66, and 67, amino acid sequences of SEQ ID NOS: 74, 75, and 76, amino
acid sequences of SEQ ID NOS: 84, 85, and 86, amino acid sequences of SEQ ID NOS: 94,
95, and 96, amino acid sequences of SEQ ID NOS: 104, 105, and 106, amino acid sequences
of SEQ ID NOS: 114, 115, and 116, and amino acid sequences of SEQ ID NOS: 124, 125,
and 126, and DNA encoding the polypeptide.
[0148] These polypeptides and DNAs can be produced by a gene recombinant technique
as described above.
EXAMPLES
[0149] The present invention is hereafter described in greater detail with reference to the
following examples, although the scope of the present invention is not limited thereto.
[0150]
Example 1: Identification of new cancer antigen protein by SEREX method
(1) Construction of cDNA library
Total RNA was extracted from a testis tissue of a healthy dog by an Acid
guanidium-Phenol-Chloroform method and then a polyA RNA was purified according to
protocols included with an Oligotex-dT30 mRNA purification Kit (Takara Shuzo Co., Ltd.).
[0151] A canine testis cDNA phage library was synthesized using the thus obtained
mRNA (5 \ig). The cDNA phage library was constructed using a cDNA Synthesis Kit, a
ZAP-cDNA Synthesis Kit, and a ZAP-cDNA Gigapacklll Gold Cloning Kit
(STRATAGENE) according to protocols included with the kits. The size of the thus
constructed cDNA phage library was 7.73x 105pfu/ml.
[0152]
(2) Screening of cDNA library using serum
Immunoscreening was performed using the above constructed canine testis cDNA
phage library. Specifically, host Escherichia coli (XLl-Blue MRF') was infected with the
phage on an NZY agarose plate (O90 x 15 mm) so as to obtain 2210 clones. E. coli cells
were cultured at 42°C for 3 to 4 hours to form plaques. The plate was covered with a
nitrocellulose membrane (Hybond C Extra: GE Healthcare Bio-Science) impregnated with
IPTG (isopropyl-p-D-thiogalactoside) at 37°C for 4 hours, so that the protein was induced,

expressed, and then transferred to the membrane. Subsequently, the membrane was
collected and then immersed in TBS (10 mM Tris-HCl, 150 mM NaCl, and pH 7.5)
containing 0.5% powdered skim milk, followed by overnight shaking at 4°C, thereby
suppressing nonspecific reaction. The filter was reacted with a 500-fold diluted serum of a
canine patient at room temperature for 2 to 3 hours.
[0153] As the above serum of a canine patient, a serum collected from a canine patient
with breast cancer was used. These sera were stored at -80°C and then subjected to
pre-treatment immediately before use. A method for pretreatment of serum is as follows.
Specifically, host Escherichia coli (XL 1-Blue MRF') was infected with a X ZAP Express
phage in which no foreign gene had been inserted and then cultured overnight on a NZY plate
medium at 37°C. Subsequently, buffer (0.2 M NaHCO and pH 8.3) containing 0.5 M NaCl
was added to the plate, the plate was left to stand at 4°C for 15 hours, and then a supernatant
was collected as an Escherichia co/z/phage extract. Next, the thus collected Escherichia
co/z'/phage extract was applied to an NHS-column (GE Healthcare Bio-Science), so that an
Escherichia co/z'-phage-derived protein was immobilized. The serum of a canine patient was
applied to the protein-immobilized column for reaction and then Escherichia coli and an
antibody adsorbed to the phage were removed from the serum. The serum fraction that had
passed through the column was diluted 500-fold with TBS containing 0.5% powdered skim
milk. The resultant was used as an immunoscreening material.
[0154] A membrane onto which the treated serum and the above fusion protein had been
blotted was washed 4 times with TBS-T (0.05% Tween20/TBS) and then caused to react with
goat anti-dog IgG (Goat anti-Dog IgG-h+I HRP conjugated (BETHYL Laboratories)) diluted
5000-fold with TBS containing 0.5% powdered skim milk as a secondary antibody for 1 hour
at room temperature. Detection was performed via an enzyme coloring reaction using an
NBT/BCIP reaction solution (Roche). Colonies that matched sites positive for a coloring
reaction were collected from the NZY agarose plate (<£90 x 15 mm) and then lysed in 500 pi
of an SM buffer (100 mM NaCl, 10 mM MgClS04, 50 mM Tris-HCl, 0.01% gelatin, and pH
7.5). Until colonies positive for coloring reaction were unified, secondary screening and
tertiary screening were repeated so that 30,940 phage clones reacting with serum IgG were
screened for by a method similar to the above. Thus, 5 positive clones were isolated.
[0155]
(3) Homology search for isolated antigen gene

For nucleotide sequence analysis of the 5 positive clones isolated by the above
method, a procedure for conversion from phage vectors to plasmid vectors was performed.
Specifically, 200 ul of a solution was prepared to contain host Escherichia coli (XL 1-Blue
MRF') so that absorbance ODeoo was 1.0. The solution was mixed with 250 ul of a purified
phage solution and then with 1 ul of an ExAssist helper phage (STRATAGENE), followed by
15 minutes of reaction at 37°C. Three (3) ml of LB medium was added and then culture was
performed at 37°C for 2.5 to 3 hours. Immediately after culture, the temperature of the
solution was kept at 70°C by water bath for 20 minutes, centrifugation was performed at 4°C
and 1000 x g for 15 minutes, and then the supernatant was collected as a phagemid solution.
Subsequently, 200 ul of a solution was prepared to contain phagemid host Escherichia coli
(SOLR) so that absorbance ODgoo was 1.0. The solution was mixed with 10 p.1 of a purified
phage solution, followed by 15 minutes of reaction at 37°C. The solution (50 ul) was seeded
on LB agar medium containing ampicillin (final concentration of 50 p.g/ml) and then cultured
overnight at 37°C. Transformed SOLR single colony was collected and then cultured in LB
medium containing ampicillin (final concentration: 50 u.g/ml) at 37°C. A plasmid DNA
containing the insert of interest was purified using a QIAGEN plasmid Miniprep Kit
(QIAGEN).
[0156] The purified plasmid was subjected to analysis of the full-length insert sequence
by a primer walking method using the T3 primer of SEQ ID NO: 31 and the T7 primer of
SEQ ID NO: 32. As a result of sequence analysis, the gene sequences of SEQ ID NOS: 5, 7,
9, 11, and 13 were obtained. A homology search program, BLAST search
(http://www.ncbi.nlm.nih.gov/BLAST/), was performed using the nucleotide sequences of the
genes and the corresponding amino acid sequences (SEQ ID NOS: 6, 8, 10, 12, and 14). As
a result of this homology search with known genes, it was revealed that all of the 5 obtained
genes encoded CAPRIN-1. Regarding regions to be translated to proteins, the sequence
identity among the 5 genes was 100% in terms of nucleotide sequence and 99% in terms of
amino acid sequence. Also, regarding regions to be translated to proteins, the sequence
identity between the genes and genes encoding human factors homologous thereto (human
homologs) was 94% in terms of nucleotide sequence and 98% in terms of amino acid
sequence. The nucleotide sequences of the human homologues are shown in SEQ ID NOS:
1 and 3 and the amino acid sequences of the same are shown in SEQ ID NOS: 2 and 4. Also,
regarding regions to be translated to proteins, the sequence identity between the thus obtained

canine genes and a gene encoding a cattle homologue was 94% in terms of nucleotide
sequence and 97% in terms of amino acid sequence. The nucleotide sequence of the cattle
homologue is shown in SEQ ID NO: 15 and the amino acid sequence of the same is shown in
SEQ ID NO: 16. In addition, regarding regions to be translated to proteins, the sequence
identity between the genes encoding the human homologues and the gene encoding the cattle
homologue was 94% in terms of nucleotide sequence and ranged from 93% to 97% in terms
of amino acid sequence. Also, regarding regions to be translated to proteins, the sequence
identity between the obtained canine genes and a gene encoding an equine homologue was
93% in terms of nucleotide sequence and 97% in terms of amino acid sequence. The
nucleotide sequence of the equine homologue is shown in SEQ ID NO: 17 and the amino acid
sequence of the same is shown in SEQ ID NO: 18. In addition, regarding regions to be
translated to proteins, the sequence identity between the genes encoding the human
homologues and the gene encoding the equine homologue was 93 % in terms of nucleotide
sequence and 96% in terms of amino acid sequence. Also, regarding regions to be translated
to proteins, the sequence identity between the obtained canine genes and genes encoding
mouse homologues ranged from 87% to 89% in terms of nucleotide sequence and ranged
from 95% to 97% in terms of amino acid sequence. The nucleotide sequences of the mouse
homologues are shown in SEQ ID NOS: 19, 21, 23, 25, and 27 and the amino acid sequences
of the same are shown in SEQ ID NOS: 20, 22, 24, 26, and 28. In addition, regarding
regions to be translated to proteins, the sequence identity between the genes encoding the
human homologues and the genes encoding the mouse homologues ranged from 89% to 91%
in terms of nucleotide sequence and ranged from 95% to 96% in terms of amino acid
sequence. Also, regarding regions to be translated to proteins, the sequence identity between
the obtained canine genes and a gene encoding a chicken homologue was 82% in terms of
nucleotide sequence and 87% in terms of amino acid sequence. The nucleotide sequence of
the chicken homologue is shown in SEQ ID NO: 29 and the amino acid sequence of the same
is shown in SEQ ID NO: 30. In addition, regarding regions to be translated to proteins, the
sequence identity between the genes encoding the human homologues and the gene encoding
the chicken homologue ranged from 81% to 82% in terms of nucleotide sequence and was
86%> in terms of amino acid sequence.
[0157]
(4) Gene expression analysis in each tissue

Expression of the genes obtained by the above method in canine and human normal
tissues and various cell lines was examined by an RT-PCR method. A reverse transcription
reaction was performed as follows. Specifically, total RNA was extracted from each tissue
(50 mg to 100 mg) and each cell line (5 to 10 x 10 cells) using a TRIZOL reagent
(Invitrogen) according to protocols included therewith. cDNA was synthesized using the
total RNA and Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) according
to protocols included therewith. PCR was performed as follows using primers specific to the
obtained genes (SEQ ID NOS: 33 and 34). Specifically, PCR was performed by repeating
30 times a cycle of 94°C/30 seconds, 60°C/30 seconds, and 72GC/30 seconds using a Thermal
Cycler (BIO RAD) and a reaction solution adjusted to a total amount of 25 ul through
addition of each reagent and an attached buffer (0.25 ul of a sample prepared by reverse
transcription reaction, the above primers (2 uM each), dNTP (0.2 mM each), and 0.65 U of
ExTaq polymerase (Takara Shuzo)). In addition, the gene-specific primers mentioned above
were used to amplify the region between nucleotide number 206 and nucleotide number 632
in the nucleotide sequence (canine CAPRIN-1 gene) of SEQ ID NO: 5 and the region
between nucleotide number 698 and nucleotide number 1124 in the nucleotide sequence
(human CAPPJN-1 gene) of SEQ ID NO: 1. For comparison control, GAPDH-specific
primers (SEQ ID NOS: 35 and 36) were used at the same time. As a result, as shown in Fig.
1, strong expression was observed in testis in the case of healthy canine tissues, while
expression was observed in canine breast cancer and adenocarcinoma tissues. Furthermore,
expression of the human homologs homologous to the obtained genes was also confirmed.
As a result, similarly to the case of canine CAPPJN-1 genes, expression could be confirmed
only in the testis in the case of normal tissues. However, in the case of cancer cells,
expression was detected in many types of cancer cell lines, such as cell lines of breast cancer,
brain tumor, leukemia, lung cancer, and esophageal cancer. Expression was confirmed in a
particularly large number of breast cancer cell lines. Based on the results, it was confirmed
that CAPRTN-1 expression was not observed in normal tissues other than those of the testis
while CAPRIN-1 was expressed in many cancer cells and particularly in breast cancer cell
lines.
[0158] In addition, in Fig. 1, Reference No. 1 along the longitudinal axis indicates the
expression pattern of each of the above-identified genes and Reference No. 2 along the same
indicates the expression pattern of the GAPDH gene for comparison control.

[0159]
(5) Preparation of polyclonal antibody against CAPRIN-1-derived peptide
To obtain an antibody binding to CAPRIN-1, CAPRIN-1-derived peptide
(Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln (SEQ ID NO: 37)) was
synthesized. The peptide (1 mg) as an antigen was mixed with an incomplete Freund's
adjuvant (IFA) solution in an amount equivalent to the peptide. The mixture was
subcutaneously administered to a rabbit 4 times every 2 weeks. Subsequently, blood was
collected, so that an antiserum containing a polyclonal antibody was obtained. Furthermore,
the antiserum was purified using a protein G support (GE Healthcare Bio-Sciences) and then a
polyclonal antibody against the CAPRIN-1 -derived peptide was obtained. In addition, an
antibody obtained by purifying serum of rabbits to which no antigen had been administered
with the use of a protein G support in the manner described above was designated as a control
antibody.
[0160]
(6) Analysis of antigen protein expression on cancer cells
Next, it was examined whether or not the CAPRIN-1 protein was expressed on
cell surfaces of 7 types of breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1,
MDA-MB-231V, BT20, SK-BR-3, and MDA-MB-231T) in which CAPRIN-1 gene
expression had been strongly confirmed. Each human breast cancer cell line in which gene
expression had been confirmed (10 cells) as described above was centrifuged in a 1.5-ml
microcentrifugal tube. The polyclonal antibody against the CAPRTN-1-derived peptide (2
u.g)(5ul) prepared in (5) above was added thereto. The resultant was further suspended in
PBS containing 0.1% fetal bovine serum (95 ul) and then left to stand on ice for 1 hour.
After washing with PBS, the resultant was suspended in PBS containing an FITC-labeled goat
anti-rabbit IgG antibody (Santa Cruz Biotechnology, Inc.)(5 ul) and 0.1% fetal bovine serum
(FBS)(95 ul) and then left to stand on ice for 1 hour. After washing with PBS, fluorescence
intensity was measured using FACSCalibur (Becton, Dickinson and Company). Meanwhile,
a procedure similar to the above was performed using the control antibody prepared in (5)
above instead of the polyclonal antibody against a CAPRIN-1-derived peptide, so that a
control was prepared. As a result, fluorescence intensity was found to be at least 30%
stronger in all cells to which the anti-human CAPRIN-1 antibody had been added than that in
control cells. Specifically, the following increases in fluorescence intensity were confirmed:

MDA-MB-157: 184%; T47D: 221%; MRK-nu-1: 115%; MDA-MB-231V: 82%; BT20: 32%;
SK-BR-3: 279%; and MDA-MB-231T: 80%. Based on the above, it was confirmed that the
CAPRJN-1 protein was expressed on the cell surfaces of the above human cancer cell lines.
In addition, the rate of increase in fluorescence intensity is represented by the rate of increase
in mean fluorescence intensity (MFI value) in cells. It was calculated by the following
equation.
[0161]
Rate of increase in mean fluorescence intensity (rate of increase in fluorescence intensity) (%)
= ((MFI value of cells reacted with an anti-human CAPRIN-1 antibody) - (control MFI
value)) / (control MFI value) x 100
(7) Immunohistochemical staining
(7)-l CAPRIN-1 expression in normal mouse and canine tissues
Mice (Balb/c, female) and dogs (beagle dogs, female) were exsanguinated under
ether anesthesia and ketamine/isoflurane anesthesia. After laparotomy, organs (stomach,
liver, eyeball, thymus gland, muscle, bone marrow, uterus, small intestine, esophagus, heart,
kidney, salivary gland, large intestine (colon), mammary gland, brain, lung, skin, adrenal
gland, ovary, pancreas, spleen, and bladder) were each transferred to a 10-cm dish containing
PBS. Each organ was cut open in PBS and then subjected to perfusion fixation overnight
with 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The
perfusate was discarded, the tissue surface of each organ was rinsed with PBS, and then a
PBS solution containing 10% sucrose was added to a 50-ml centrifugal tube. Each tissue
was then placed in each tube and then shaken using a rotor at 4°C for 2 hours. Each solution
was substituted with a PBS solution containing 20% sucrose and then left to stand at 4°C until
tissues precipitated. Each solution was substituted with a PBS solution containing 30%
sucrose and then left to stand at 4°C until tissues precipitated. Each tissue was removed and
a necessary portion was excised with a surgical scalpel. Next, an OCT compound (Tissue
Tek) was applied and spread over each tissue surface, and then the tissues were placed on
Cryomold. Cryomold was placed on dry ice for rapid freezing. Tissues were sliced into
sections of 10 to 20 jam thickness using a cryostat (LEICA) and then the sliced tissue sections
were air-dried on glass slides for 30 minutes using a hair dryer, so that glass slides on which
sliced tissue sections had been placed were prepared. Next, each glass slide was placed in a
staining bottle filled with PBS-T (saline containing 0.05%) Tween20), so that a procedure

involving exchange with PBS-T every 5 minutes was performed 3 times. Excess water
around each specimen was removed using Kimwipes and then each section was encircled
using DAKOPEN (DAKO). As blocking solutions, a MOM mouse Ig blocking reagent
(VECTASTAIN) was applied onto mouse tissue and a PBS-T solution containing 10% FBS
was applied onto canine tissue. The resultants were left to stand in a moist chamber at room
temperature for 1 hour. Next, a solution was prepared to contain a monoclonal antibody
(monoclonal antibody #6) against CAPRIN-1 having the heavy-chain variable region of SEQ
ID NO: 73 and the light-chain variable region of SEQ ID NO: 77 and reacting with the cancer
cell surfaces prepared in Example 4, which antibody was adusted at a concentration of 10
(j,g/ml in the blocking solution. The solution was applied onto each slide glass and then left
to stand within a moist chamber at 4°C overnight. After 3 times wash, each 10 minutes, with
PBS-T, a MOM biotin-labeled anti-IgG antibody (VECTASTAIN) diluted 250-fold with the
blocking solution was applied onto each glass slide|and then left to stand within a moist
chamber at room temperature for 1 hour. After 3 times wash, each 10 minutes, with PBS-T,
an avidin-biotin ABC reagent (VECTASTAIN) was applied and then left to stand within a
moist chamber at room temperature for 5 minutes. After 3 times wash, each 10 minutes,
with PBS-T, a DAB staining solution (DAB 10 mg + 30% H202 10 ui/0.05 M Tris-HCl (pH
7.6) 50 ml) was applied and then the glass slides were left to stand within a moist chamber at
room temperature for 30 minutes. Glass slides were rinsed with distilled water and then a
hematoxylin reagent (DAKO) was applied. After being left to stand at room temperature for
1 minute, the glass slides were rinsed with distilled water. The glass slides were immersed
in 70%, 80%, 90%, 95%, and 100% ethanol solutions in such order for 1 minute each and
then left to stand in xylene overnight. The glass slides were removed, coverslipped with
Glycergel Mounting Medium (DAKO), and then observed. As a result, CAPRIN-1
expression was observed to a slight degree within cells in all salivary gland, kidney, colon,
and stomach tissues, but CAPRIN-1 expression was never observed on cell surfaces. Also,
absolutely no CAPRIN-1 expression was observed in tissues from other organs. In addition,
similar results were obtained when the monoclonal antibody against CAPRIN-1 having the
heavy-chain variable region of SEQ ID NO: 103 and the light-chain variable region of SEQ
ID NO: 107 (monoclonal antibody #9) was used.
[0162]
(7)-2 CAPRIN-1 expression in canine breast cancer tissue

With the use of 108 frozen canine breast cancer tissue specimens from dogs
diagnosed by pathological diagnosis as having malignant breast cancer, frozen section slides
were prepared by a method similar to the above and immunohistochemical staining was
performed using the monoclonal antibody #6 prepared in Example 4. As a result,
CAPRIN-1 expression was confirmed in 100 out of the 108 specimens (92.5%). CAPRIN-1
was particularly strongly expressed on the surfaces of highly atypical cancer cells. In
addition, similar results were obtained when the monoclonal antibody #9 produced in
Example 4 was used.
[0163]
(7)-3 CAPRIN-1 expression in human breast cancer tissue
Immunohistochemical staining was performed using 188 breast cancer tissue
specimens of a paraffin-embedded human breast cancer tissue array (BIOMAX). After 3
hours of treatment at 60°C, the human breast cancer tissue array was added to a staining bottle
filled with xylene and then xylene replacement every 5 minutes was performed 3 times.
Next, a similar procedure was performed using ethanol and PBS-T instead of xylene. The
human breast cancer tissue array was added to a staining bottle filled with 10 mM citrate
buffer (pH 6.0) containing 0.05% Tween20, treated for 5 minutes at 125°C, and then left to
stand at room temperature for 40 minutes or more. Excess water around each specimen was
removed using Kimwipes, each section was encircled using DAKOPEN, and then an
appropriate amount of Peroxidase Block (DAKO) was added dropwise. The resultant was
left to stand at room temperature for 5 minutes and then added to a staining bottle filled with
PBS-T. PBS-T replacement every 5 minutes was performed 3 times. As a blocking
solution, a PBS-T solution containing 10% FBS was applied and then left to stand within a
moist chamber at room temperature for 1 hour. Next, a solution was prepared to contain the
monoclonal antibody #6 reacting with the cancer cell surfaces prepared in Example 4 at a
concentration of 10 |ug/ml adjusted using a PBS-T solution containing 5% FBS. The
solution was applied and then left to stand overnight within a moist chamber at 4°C. After 3
times wash, each 10 minutes, with PBS-T, an appropriate amount of Peroxidase Labeled
Polymer Conjugated (DAKO) was added dropwise, and then the glass slides were left to stand
at room temperature for 30 minutes within a moist chamber. After 3 times wash, each 10
minutes, with PBS-T, a DAB staining solution (DAKO) was applied and then left to stand at
room temperature for 10 minutes. The DAB staining solution was discarded and then 10

minutes of wash was performed with PBS-T for 3 times. The glass slides were rinsed with
distilled water and then immersed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in
such order for 1 minute each and then left to stand in xylene overnight. The glass slides
were removed, coverslipped with Glycergel Mounting Medium (DAKO), and then observed.
As a result, strong CAPRTN-1 expression was observed for 138 (73%) out of the total 188
breast cancer tissue specimens. In addition, similar results were obtained when the
monoclonal antibody #9 prepared in Example 4 was used.
[0164]
(7)-4 CAPRTN-1 expression in human malignant brain tumor
With the use of 247 malignant brain tumor tissue specimens of paraffin-embedded
human malignant brain tumor tissue arrays (BIOMAX), immunohistochemical staining was
performed by a method similar to that in (7)-3 above using the monoclonal antibody #6
prepared in Example 4. As a result, strong CAPRTN-1 expression was observed in 227
(92%) out of the total 247 malignant brain tumor tissue specimens. In addition, similar
results were obtained when the monoclonal antibody #9 prepared in Example 4 was used.
[0165]
(7)-5 CAPRTN-1 expression in human breast cancer metastatic lymph node
With the use of 150 tissue specimens of human breast cancer metastatic lymph
nodes of paraffin-embedded human breast cancer metastatic lymph node tissue arrays
(BIOMAX), immunohistochemical staining was performed by a method similar to that in
(7)-3 above using the monoclonal antibody #6 prepared in Example 4. As a result, strong
CAPRTN-1 expression was observed in 136 (90%) out of the total 150 tissue specimens of
human breast cancer metastatic lymph nodes. Specifically, it was revealed that CAPRTN-1
is also strongly expressed in a cancer tissue that has metastasized from breast cancer. In
addition, similar results were obtained when the monoclonal antibody #9 prepared in Example
4 was used.
[0166]
Example 2: Antitumor effects (ADCC activity) of antibody against CAPRTN-1 upon cancer
cells
Next, it was examined whether or not an antibody against CAPRTN-1 would be
able to damage CAPRTN-1-expressing tumor cells. Evaluation was carried out using the
polyclonal antibody against a human CAPRTN-1-derived peptide prepared in Example 1.

Two types of human breast cancer cell lines (T47D and MDA-MB-157) (106 cells each), in
which CAPRTN-1 expression had been confirmed, were separately collected into a 50-ml
centrifugal tube. Chromium 51 (100 u.Ci) was added thereto, followed by incubation at
37°C for 2 hours. Thereafter, cells were washed 3 times with an RPMI1640 medium
containig 10% fetal calf serum and added to wells (10 cells per well) in 96-well V-bottom
plates. The above polyclonal antibody against a human CAPRIN-1-derived peptide was
added thereto (1 (4.g per well). Further, lymphocytes separated from rabbit peripheral blood
were added thereto (2 x 105 cells per well), followed by culture under conditions of 37°C and
5% CO2 for 4 hours. After culture, the level of chromium (Cr) 51 released from damaged
tumor cells in each culture supernatant was determined. Then, the ADCC activity of the
polyclonal antibody against a human CAPRTN-1-derived peptide to cancer cells was
calculated. As a result, ADCC activities against T47D (15.4%) and MDA-MB-157 (17.3%)
were confirmed (see Figs. 2 and 3). Meanwhile, substantially no activity was observed in a
case in which a procedure similar to the above was performed using the control antibody
prepared from peripheral blood of a rabbit that had not been immunized with an antigen
(Example 1 (5)) or in a case in which no antibody was added (see Figs. 2 and 3).
Accordingly, it was revealed that CAPRTN-1-expressing tumor cells can be damaged by
inducing ADCC activity with the use of an antibody against CAPRTN-1.
[0167] In addition, for cytotoxic activity, an antibody against CAPRTN-1 used in the
present invention, mouse lymphocytes, and 103 cells incorporating chromium 51 from a
leukemia cell line were mixed together and cultured for 4 hours. Thereafter, the level of
chromium 51 released into the medium was determined. Then, the cytotoxic activity to the
leukemia cell line was calculated by the following equation*.
[0168]
*Equation: Cytotoxic activity (%) = [(the level of chromium 51 released from T47D or
MDA-MB-157 to which an antibody against CAPRTN-1 and mouse lymphocytes were added)
/ (the level of chromium 51 released from target cells to which IN hydrochloric acid was
added)] x 100
[0169]
Example 3: Preparation of new human cancer antigen proteins
(1) Preparation of recombinant protein
A recombinant protein of a human homolog gene was prepared by the following

method based on the gene of SEQ ID NO: 1 obtained in Example 1. PCR was performed by
repeating 30 times a cycle of 98°C/10 seconds and 68°C/2.5 minutes using a Thermal Cycler
(BIO RAD) and a reaction solution adjusted to a total amount of 50 ul through addition of
each reagent and an attached buffer (1 ul of cDNA (which was from a variety of
tissue/cell-derived cDNAs prepared in Example 1 and observed for their expression by
RT-PCR), 2 types of primers (0.4 uM each; SEQ ID NOS: 38 and 39) containing Sacl and
Xhol restriction enzyme cleavage sequences, 0.2 mM dNTP, 1.25 U PrimeSTAR HS
polymerase (Takara Shuzo)). The above 2 types of primers were used to amplify the region
encoding the full-length amino acid sequence of SEQ ID NO: 2. After PCR, the thus
amplified DNA was subjected to 1% agarose gel electrophoresis and then a DNA fragment of
about 2.1 kbp was purified using a QIAquick Gel Extraction Kit (QIAGEN).
[0170] The purified DNA fragment was ligated to a pCR-Blunt cloning vector
(Invitrogen). The vector was transformed into Escherichia coli and then the plasmid was
collected. It was confirmed based on the sequence that the amplified gene fragment matched
the target sequence. The plasmid that matched the sequence of interest was treated with Sacl
and Xhol restriction enzymes and then the resultant was purified using a QIAquick Gel
Extraction Kit. Then, the gene sequence of interest was inserted into a pET30a expression
vector (Novagen) for Escherichia coli treated with Sacl and Xhol restriction enzymes. A
His tag-fused recombinant protein can be produced using the vector. The plasmid was
transformed into Escherichia coli BL21 (DE3) for expression and then expression induction
was performed using 1 mM IPTG, so that the target protein was expressed within Escherichia
coli.
(2) Purification of recombinant protein
Each above-obtained recombinant Escherichia coli expressing SEQ ID NO: 1 was
cultured at 37°C in LB medium containing 30 ug/ml kanamycin until the absorbance at 600
nm reached around 0.7. Then isopropyl-(3-D-l-thiogalactopyranoside was added to a final
concentration of 1 mM, followed by 4 hours of culture at 37°C. Subsequently, cells were
collected by 10 minutes of centrifugation at 4800 rpm. The cell pellet was suspended in
phosphate buffered saline and then centrifuged at 4800 rpm for 10 minutes for washing cells.
[0171] The cells were suspended in phosphate buffered saline and then subjected to
ultrasonication on ice. The thus ultrasonicated Escherichia coli lysate was centrifuged at 6000
rpm for 20 minutes. The thus obtained supernatant was used as a soluble fraction and the

thus obtained precipitate was used as an insoluble fraction.
[0172] The soluble fraction was added to a nickel chelate column (carrier: Chelating
Sepharose (TradeMark) Fast Flow (GE Healthcare), column capacity: 5 mL, 50 mM
hydrochloric acid buffer (pH 8.0) as equilibrated buffer)) prepared according to a
conventional method. The unadsorbed fraction was washed with 50 mM hydrochloric acid
buffer (pH 8.0) in an amount 10 times the capacity of the column 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. An elution
fraction of 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole (for which the
elution of the protein of interest had been confirmed by Coomassie staining) was added to a
strong anion exchange column (carrier: Q Sepharose (TradeMark) Fast Flow (GE Healthcare),
column capacity: 5 mL, and 20 mM phosphate buffer (pH 8.0) as equilibrated buffer). The
unadsorbed fraction was washed with 20 mM phosphate buffer (pH 7.0) in an amount 10
times the column capacity and 20 mM phosphate buffer (pH 7.0) containing 200 mM sodium
chloride. Immediately after washing, 5 beds were eluted using 20 mM phosphate buffer (pH
7.0) containing 400 mM sodium chloride. Thus, purified fractions of proteins each having
the amino acid sequence shown in SEQ ID NO: 2 were obtained.
[0173] 200 ul of each purified preparation obtained by the above method was dispensed
into 1 ml of reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 pH 7.4) and then 2
\xl of enterokinase (Novagen) was added. The preparation was left to stand at room
temperature overnight for reaction, His tag was cleaved, and then purification was performed
according to the attached protocols using an Enterokinase Cleavage Capture Kit (Novagen).
Next, 1.2 ml of each purified preparation obtained by the above method was substituted with
physiological phosphate buffer (Nissui Pharmaceutical Co., Ltd.) using ultrafiltration
NANOSEP 10K OMEGA (PALL). Sterilized filtration was performed using 0.22-um HT
Tuffryn Acrodisc (PALL) and then the resultants were used for the following experiments.
[0174]
Example 4: Preparation of monoclonal antibody against CAPRTN-1
The antigen protein (human CAPPJN-1) (100 ug) shown in SEQ ID NO: 2
prepared in Example 3 was mixed with a MPL+TDM adjuvant (Sigma) in an amount
equivalent to that of the antigen protein. The mixture was used as an antigen solution per
mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb/c mice

(Japan SLC Inc.) and then further administered 3 times or 24 times every week for completion
of immunization. Spleen was removed on day 3 after the final immunization and then
ground in between two sterilized glass slides. Each resultant was washed with PBS (-)
(Nissui) and then centrifuged at 1500 rpm for 10 minutes, so that a procedure to remove
supernatants was repeated 3 times. Thus, spleen cells were obtained. The thus obtained
spleen cells were mixed with the mouse myeloma cell SP2/0 (purchased from ATCC) at a
ratio of 10 : 1. The PEG solution prepared by mixing 200 µl of RPMI1640 medium
containing 10% FBS heated at 37°C and 800 µl of PEG1500 (Boehringer) was added to the
cells. The solution was left to stand for 5 minutes for cell fusion. Centrifugation was
performed at 1700 rpm for 5 minutes to remove supernatants. Cells were suspended in 150
ml of RPMI1640 medium (HAT selective medium) containing 15% FBS, to which 2%
equivalent of HAT solution (Gibco) had been added and then seeded onto fifteen 96-well
plates (Nunc) at 100 µl per well. Cells were cultured for 7 days under conditions of 37°C
and 5% CO2, so that hybridomas resulting from fusion of spleen cells to myeloma cells were
obtained.
[0175] Hybridomas were selected using as an indicator the binding affinity of the
antibody produced by the thus prepared hybridomas for the CAPRIN-1 protein. The
CAPRTN-1 protein solution (1 µg/ml) prepared in Example 3 was added at 100 ul per well of
96-well plates and then left to stand at 4°C for 18 hours. Each well was washed 3 times with
PBS-T, 0.5% Bovine Serum Albumin (BSA) solution (Sigma) was added at 400 µl per well,
and then the plates were left to stand at room temperature for 3 hours. The solution was
removed and then each well was washed 3 times with 400 µl of PBS-T. Each culture
supernatant of the hybridomas obtained above was added at 100 µl per well and then left to
stand at room temperature for 2 hours. Each well was washed 3 times with PBS-T, an
HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was
added at 100 µl per well and then left to stand at room temperature for 1 hour. Each well
was washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added at 100 µl per
well and then left to stand for 15-30 minutes, so that a color reaction was performed. After
color development, IN sulfuric acid was added at 100 µl per well to stop the reaction.
Absorbance at 450 nm and absorbance at 595 nm were measured using an absorption
spectrometer. As a result, a plurality of hybridomas producing antibodies with high
absorbances were selected.
48

[0176] The thus selected hybridomas were added at 0.5 hybridomas per well of 96-well
plates and then cultured. After 1 week, hybridomas forming single colonies in wells were
observed. Cells in these wells were further cultured. Hybridomas were selected using as
an indicator the binding affinity (of the antibody produced by cloned hybridomas) for the
CAPRTN-1 protein. The CAPRIN-1 protein solution (1 ug/ml) prepared in Example 3 was
added at 100 ul per well of 96-well plates and then left to stand at 4°C for 18 hours. Each
well was washed 3 times with PBS-T, a 0.5% BSA solution was added at 400 ul per well, and
then left to stand at room temperature for 3 hours. The solution was removed and then each
well was washed 3 times with 400 ul of PBS-T. Each culture supernatant of the hybridomas
obtained above was added at 100 ul per well and then left to stand at room temperature for 2
hours. Each well was washed 3 times with PBS-T, an HRP-labeled anti-mouse IgG (H+L)
antibody (Invitrogen) diluted 5000-fold with PBS was added at 100 µl per well and then left
to stand at room temperature for 1 hour. Each well was washed 3 times with PBS-T, a TMB
substrate solution (Thermo) was added at 100 µl per well and then left to stand for 15-30
minutes, so that a color reaction was performed. After color development, IN sulfuric acid
was added at 100 µl per well to stop the reaction. Absorbance at 450 nm and absorbance at
595 nm were measured using an absorption spectrometer. As a result, 150 hybridoma cell
lines producing monoclonal antibodies exerting reactivity with the CAPPJN-1 protein were
obtained.
[0177] Next, from among these monoclonal antibodies, monoclonal antibodies exerting
reactivity with the surfaces of breast cancer cells expressing CAPRIN-1 were selected.
Specifically, 106 cells of the MDA-MB-231V human breast cancer cell line were subjected to
centrifugation with a 1.5-ml microcentrifugal tube. The supernatant (100 pi) of each
hybridoma above was added and then left to stand on ice for 1 hour. After washing with
PBS, an FITC-labeled goat anti-mouse IgG antibody (Invitrogen) diluted 500-fold with PBS
containing 0.1% FBS was added and then left to stand on ice for 1 hour. After washing with
PBS, fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and
Company). Meanwhile, a procedure similar to the above was performed using untreated
serum of 6-week-old Balb/c mice diluted 500-fold with a hybridoma culture medium instead
of the antibody so that a control was prepared. As a result, 11 monoclonal antibodies (#1 to
#11) having fluorescence intensity stronger than that of the control; that is, reacting with the
surfaces of breast cancer cells were selected.

[0178]
Example 5: Characterization of selected antibodies
(1) Cloning of an anti-CAPRIN-1 monoclonal antibody variable region gene
mRNAs were extracted from hybridoma cell lines producing the 11 monoclonal
antibodies selected in Example 4. The heavy-chain variable (VH) region gene and the
light-chain variable (VL) region gene for every anti-CAPRIN-1 monoclonal antibody were
obtained by RT-PCR using primers specific to a mouse FR1-derived sequence and a mouse
FR4-derived sequence. For sequencing, the genes were separately cloned into pCR2.1
vectors (Invitrogen).
[0179]
(1)-1 RT-PCR
mRNA was prepared from each hybridoma cell line (106 cells) using an mRNA
micro purification kit (GE Healthcare). Each obtained mRNA was subjected to reverse
transcription using a SuperScriptll 1st strand synthesis kit (Invitrogen) for cDNA synthesis.
The above procedures were carried out according to the protocols attached to the kits.
[0180] Each obtained cDNA was used for antibody gene amplification by PCR.
[0181] In order to obtain the VH region gene, a primer specific to a mouse heavy-chain
FR1 sequence (SEQ ID NO: 130) and a primer specific to a mouse heavy-chain FR4 sequence
(SEQ ID NO: 131) were used. In addition, in order to obtain the VL region gene, a primer
specific to a mouse light-chain FR1 sequence (SEQ ID NO: 132) and a primer specific to a
mouse light-chain FR4 sequence (SEQ ID NO: 133) were used. These primers were
designed with reference to Jones, S. T. and Bending, M. M. Bio/Technology 9, 88-89 (1991).
For PCR, Ex-taq (Takara Bio Inc.) was used. Each cDNA sample was mixed with a 10 x
EX Taq Buffer (5 ul), dNTPs Mixture (2.5 mM)(4 ul), primers (1.0 uM)(2 ul each), and Ex
Taq (5U/ul)(0.25 ul). The total volume was adjusted to 50 ul with sterilized water. PCR
was carried out under conditions comprising, after treatment at 94°C for 2 minutes, 30 cycles
of a combination of denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, and
elongation reaction at 72°C for 1 minute.
[0182]
(l)-2 Cloning
Each PCR product obtained above was subjected to agalose gel electrophoresis,
followed by excision of DNA bands of the VH region and the VL region. • DNA was purified

using a QIAquick Gel purification kit (QIAGEN) according to the protocols attached to the
kit. Each purified DNA was cloned into a pCR2.1 vector using a TA cloning kit (Invitrogen).
Each DNA-ligated vector was transformed into DH5a competent cells (TOYOBO) according
to a conventional method. Each transformant (10 clones) was cultured overnight in a
medium (100 µg/ml ampicillin) at 37°C. The obtained plasmid DNA was purified using a
Qiaspin Miniprep kit (QIAGEN).
[0183]
(l)-3 Sequencing
Gene sequence analysis of the VH region and the VL region in each plasmid
obtained above was carried out using an Ml3 forward primer (SEQ ID NO: 134) and an Ml3
reverse primer (SEQ ID NO: 135) with a fluorescent sequencer (ABI; DNA sequencer
3130XL) and a BigDye terminater Ver. 3.1 cycle sequencing kit (ABI) in accordance with the
protocols attached to the kit. As a result, each gene sequence (identical in 10 clones) was
determined.
[0184] The obtained amino acid sequences of monoclonal antibody heavy-chain variable
regions are shown in SEQ ID NO: 43, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93,
SEQ ID NO: 103, SEQ ID NO: 113, and SEQ ID NO: 123. The obtained amino acid
sequences of light-chain variable regions are shown in SEQ ID NO: 47, SEQ ID NO: 53, SEQ
ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO:
97, SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 127.
[0185] Specifically, a 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. A monoclonal
antibody #2 comprises the heavy-chain variable region of SEQ ID NO: 43 and the light-chain
variable region of SEQ ID NO: 53. A monoclonal antibody #3 comprises the heavy-chain
variable region of SEQ ID NO: 43 and the light-chain variable region of SEQ ID NO: 58. A
monoclonal antibody #4 comprises the heavy-chain variable region of SEQ ID NO: 43 and
the light-chain variable region of SEQ ID NO: 63. A monoclonal antibody #5 comprises the
heavy-chain variable region of SEQ ID NO: 43 and the light-chain variable region of SEQ ID
NO: 68. A monoclonal antibody #6 comprises the heavy-chain variable region of SEQ ID
NO: 73 and the light-chain variable region of SEQ ID NO: 77. A monoclonal antibody #7
comprises the heavy-chain variable region of SEQ ID NO: 83 and the light-chain variable
region of SEQ ID NO: 87. A monoclonal antibody #8 comprises the heavy-chain variable

region of SEQ ID NO: 93 and the light-chain variable region of SEQ ID NO: 97. A
monoclonal antibody #9 comprises the heavy-chain variable region of SEQ ID NO: 103 and
the light-chain variable region of SEQ ID NO: 107. A monoclonal antibody #10 comprises
the heavy-chain variable region of SEQ ID NO: 113 and the light-chain variable region of
SEQ ID NO: 117. A monoclonal antibody #11 comprises the heavy-chain variable region of
SEQ ID NO: 123 and the light-chain variable region of SEQ ID NO: 127.
[0186]
(2) Expression of CAPRIN-1 on the cell surfaces of differnt cells caused with the use of the
obtained monoclonal antibodies
Next, it was examined whether or not the CAPRIN-1 protein was expressed on
cell surfaces of 7 types of breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1,
MDA-MB-231V, BT20, SK-BR-3, and DA-MB-231T) in which CAPRIN-1 gene expression
had been confirmed, 3 types of other breast cancer cell lines (MDA-MB-231C, MCF-7, and
ZR75-1), | types of glioma cell lines (T98G, SNB19, U251, and U87G), 3 types of kidney
cancer cell lines (Caki-1, Caki2, and A498), 1 type of a gastric cancer cell line (MKN45), 1
type of a colon cancer cell line (Caco2), 3 types of lung cancer cell lines (A549, QG56, and
PC8), and 3 types of leukemia cell lines (Namalwa, BDCM, and RPI1788). Each cell line
(106 cells) was centrifuged in a 1.5-ml microcentrifugal tube. The hybridoma supernatants
(100 ul each) containing monoclonal antibodies #1 to #10 against CAPRIN-1 prepared in
Example 4 reacting to cancer cell surfaces were separately added thereto and then left to stand
on ice for 1 hour. After washing with PBS, each resultant was suspended in an FITC-labeled
goat anti-mouse IgG antibody (Invitrogen Corporation) diluted 500-fold with PBS containing
0.1% FBS and then left to stand on ice for 1 hour. After washing with PBS, fluorescence
intensity was measured using FACSCalibur (Becton, Dickinson and Company). Meanwhile,
a procedure similar to the above was performed using, as a control, the control antibody
prepared in (5) above instead of the hybridoma supernatants containing monoclonal
antibodies #1 to #11 against CAPRIN-1, so that a control was prepared. As a result,
fluorescence intensity was found to be at least 30% stronger in all cells to which the
monoclonal antibodies #1 to #11 had been added than that in control cells. Specifically, the
following increases in fluorescence intensity were confirmed when, for example, the
monoclonal antibody #9 was used: MDA-MB-157: 211%; T47D: 145%; MRK-nu-1: 123%;
MDA-MB-231V: 251%; BT20: 168%; and MDA-MB-231T: 94%. Based on the above, it

was confirmed that the CAPRIN-1 protein was expressed on the cell surfaces of the above
human cancer cell lines. In addition, the rate of increase in fluorescence intensity is
represented by the rate of increase in mean fluorescence intensity (MFI value) in cells. It
was calculated by the following equation.
[0187]
Rate of increase in mean fluorescence intensity (rate of increase in fluorescence intensity) (%)
= ((MFI value of cells reacted with an anti-human CAPRIN-1 antibody) - (control MFI
value)) / (control MFI value) x 100
(3) Antitumor effects (ADCC activity) of antibodies against CAPRIN-1 upon cancer cells
The above selected monoclonal antibodies #1 to #11 against CAPRIN-1 were
evaluated in terms of cytotoxic activity (ADCC activity) to cancer cells. The hybridomas
producing monoclonal antibodies #1 to #11 were cultured using a hybridoma SFM medium
(Invitrogen). Each obtained supernatant was purified using Hitrap ProteinA Sepharose FF
(GE Healthcare), followed by substitution with PBS (-) and purification with a 0.22-u.m filter
(Millipore). Each resultant was used as an antibody for activity determination. The human
breast cancer MDA-MB-157 cell line (106 cells) was collected into a 50-ml centrifugal tube.
Chromium 51 (100 u,Ci) was added thereto, followed by incubation at 37°C for 2 hours.
Thereafter, cells were washed 3 times with an RPMI1640 medium containing 10% FBS.
The cells were added to wells (10 cells per well) in 96-well V-bottom plates. Thus, target
cells were prepared. The above purified antibodies were added thereto (1 u.g per well).
Further, mouse lymphocytes separated from mouse spleen (2x105 cells) were added thereto,
followed by culture under conditions of 37°C and 5% CO2 for 4 hours. After culture, the
level of chromium (Cr) 51 released from damaged tumor cells in each culture supernatant was
determined. Then, ADCC activity of each polyclonal antibody against a human
CAPRIN-1-derived peptide to cancer cells was calculated. As a result, all monoclonal
antibodies #1 to #11 exhibited ADCC activity against MDA-MB-157 (20% or more).
Specifically, Specifically, for example, the following cytotoxic activity results were obtained:
#1: 22.1%; #2: 29.1%; #6: 30.2%; and #9: 32.4% (see Fig. 4). Meanwhile, no cytotoxic
activity was confirmed in a case in which a procedure similar to the above was performed
using the monoclonal antibody reactive to a CAPRIN-1 protein itself but not to cancer cell
surfaces prepared in Example 4 (see Fig. 4). The above results showed that the obtained
anti-CAPRTN-1 monoclonal antibodies (#1 to #11) damaged CAPRIN-1-expressing cancer

cells by exhibiting ADCC activity.
[0188]
(4) Antitumor effects (CDC activity) of antibodies against CAPRTN-1 upon cancer cells
The above selected monoclonal antibodies #1 to #11 against CAPRTN-1 were
evaluated in terms of cytotoxic activity (CDC activity) to cancer cells. Blood collected from
rabbits by blood sampling was added to an Eppendorf tube and then left to stand at room
temperature for 60 minutes, followed by centrifugation at 3000 rpm for 5 minutes. Thus,
serum for CDC activity determination was prepared. The human breast cancer
MDA-MB-231V cell line (105 cells) was collected into a 50-ml centrifugal tube. Chromium
51 (100 uCi) was added thereto, followed by incubation at 37°C for 2 hours. Thereafter,
cells were washed 3 times with an RPMI medium containing 10% FBS and then suspended in
an RPMI containing 50% rabbit serum prepared above. The cells were added to wells (10
cells per well) in 96-well V-bottom plates. The antibodies #1 to #11 obtained in (3) above
were separately added to wells (1 jag per well), followed by culture under conditions of 37°C
and 5% CO2 for 4 hours. After culture, the level of chromium (Cr) 51 released from
damaged tumor cells in each culture supernatant was determined. The CDC activity against
MDA-MB-231V exhibited by the anti-CAPRIN-1 monoclonal antibody in each hybridoma
supernatant was calculated. As a result, all monoclonal antibodies #1 to #11 exhibited CDC
activity (30% or more). Meanwhile, no cytotoxic activity was confirmed in a case in which
a procedure similar to the above was performed using the monoclonal antibody reactive to a
CAPRIN-1 protein itself but not to cancer cell surfaces prepared in Example 4 (see Fig. 4).
Accordingly, it has been revealed that the monoclonal antibodies against CAPRIN-1 (#1 to
#11) can damage CAPRJN-1-expressing tumor cells also by exhibiting CDC activity.
[0189]
Example 6: In vivo antitumor effects of anti-CAPRIN-1 monoclonal antibodies upon mice
Next, in vivo antitumor effects of the obtained monoclonal antibodies #1 to #11
against CAPRTN-l upon tumor-bearing mice were evaluated. Antibodies used in this
Example were obtained by subjecting the supernatant of each hybridoma to colum
purification in the manner described above.
[0190] Antitumor effects of the monoclonal antibodies #1 to #11 against CAPRIN-1 were
examined using tumor-bearing mice into which a mouse-derived cancer cell line expressing
CAPRTN-1 had been transplanted. CT26 cells (purchased from ATCC) were subcuatneously

transplanted into the dorsal portions of 70 Balb/c mice (Japan SLC, Inc.)(106 cells per mouse).
Each tumor was allowed to grow until the diameter thereof became approximately 7 mm.
The tumor-bearing mice (60 out of 70) were subjected to intraperitoneal administration of
monoclonal antibodies #1 to #11 against CAPRTN-1 and one type of the monoclonal antibody
(reactive to the CAPRTN-1 protein itself but not to cancer cell surfaces) prepared in Example
4 (5 mice per antibody) at a dose of 300 p,g (300 pi) per mouse. Thereafter, each antibody
was intraperitoneally administered in the same dose to the relevant tumor-bearing mice 3
times in total during 2 days. The tumor size was measured every day for observation of
antitumor effects. The 10 remaining tumor-bearing mice were subjected to administration of
PBS (-) instead of an antibody. The group of these mice was designated as a control group.
As a result of observation of antitumor effects, in the case of the test group to which
monoclonal antibodies #1 to #11 against CAPRTN-1 had been administered, tumor regression
occurred to such an extent that the tumor volume at the start of antibody administration
(100%) decreased to 50% by Day 4, approximately 10% by Day 6, and several percents by
Day 8. Substantially complete tumor regression took place from Days 11 to 14 (see Fig. 5).
On the other hand, in the control group, the tumor volume increased to approximately 260%,
350%, 550%, and 800% of the original volume by Days 4, 6, 8, and 11, respectively (see Fig.
5). In addition, in the group of mice to which the monoclonal antibody (reactive to the
CAPPJN-1 protein itself but not to cancer cell surfaces) had been administered, antitumor
effects could not be exhibited and tumor growth occurred as in the control group. The
results indicate that the obtained monoclonal antibodies #1 to #11 against CAPRTN-1 exhibit
strong in vivo antitumor effects upon cancer cells expressing CAPRTN-1. In addiiton, the
tumor size was obtained by calculating the tumor volume by the following formula: long
diameter x short diameter x short diameter x 0.5.
[0191] Further, monoclonal antibodies #1 to #11 against CAPRTN-1 were administered in
the manner described above to tumor-bearing mice (Balb/c) into which mouse N1E cancer
cells (purchased from ATCC) had been transplanted. This resulted in complete tumor
regression by Day 15 after antibody administration. On the other hand, in the control group,
the tumor volume increased to as high as approximately 950% of the original volume (see Fig.
6).
[0192]
Example 7: Identification of a peptide in CAPRTN-1 protein, to which an antibody against

CAPRIN-1 reacting to cancer cell surface binds
With the use of monoclonal antibodies #1 to #11 against CAPRIN-1, reacting with
the surfaces of cancer cells (obtained above), partial sequences in the CAPRIN-1 protein to be
recognized by these monoclonal antibodies were identified.
[0193] First, DTT (Fluka) was added to 100 µl of a solution prepared by dissolving a
recombinant CAPRIN-1 protein at a concentration of 1 µg/ul with PBS to a final
concentration of 10 mM, followed by 5 minutes of reaction at 95 °C, so that reduction of
disulfide bonds within the CAPRIN-1 protein was performed. Next, iodoacetamide (Wako
Pure Chemical Industries, Ltd.) with a final concentration of 20 mM was added and then an
alkylation reaction was performed for thiol groups at 37°C for 30 minutes under shading
conditions. Fifty (50) µg each of monoclonal antibodies #1 to #11 against CAPRIN-1 was
added to 40 ug of the thus obtained reduced-alkylated CAPRIN-1 protein, the volume of the
mixture was adjusted to 1 mL of 20 mM phosphate buffer (pH 7.0), and then the mixture was
left to react overnight at 4°C while stirring and mixing each mixture.
[0194] Next, trypsin (Promega) was added to a final concentration of 0.2 fig. After 1
hour, 2 hours, 4 hours, and then 12 hours of reaction at 37°C, the resultants were mixed with
protein A-glass beads (GE), which were subjected in advance to blocking with PBS
containing 1% BSA (Sigma) and then to washing with PBS, in 1 mM calcium carbonate and
NP-40 buffer (20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl, and 1%
NP-40), followed by 30 minutes of reaction.
[0195] The reaction mixtures were each washed with 25 mM ammonium carbonate buffer
(pH 8.0) and then antigen-antibody complexes were eluted using 100 jtxl of 0.1% formic acid.
LC-MS analysis was conducted for eluates using Q-TOF Premier (Waters-MicroMass)
according to the protocols attached to the instrument.
[0196] As a result, the polypeptide of SEQ ID NO: 136 was identified as a partial
sequence of CAPRIN-1, which was recognized by all of the monoclonal antibodies #1 to #11
against CAPRIN-l. Furthermore, the peptide of SEQ ID NO: 137 was identified as a partial
sequence in the polypeptide of SEQ ID NO: 136 above, which peptide was recognized by the
monoclonal antibodies #2 to #5, #6 to #8, and #10. It was further revealed that the
monoclonal antibodies #2 to #5 recognized the peptide of SEQ ID NO: 138 that was a partial
sequence peptide of the peptide of SEQ IS NO: 137.

INDUSTRIAL APPLICABILITY
[0197] The antibodies of the present invention are useful for treatment and/or prevention
of cancers.
[0198] This description includes all or part of the contents as disclosed in the description
and/or drawings of Japanese Patent Application No. 2009-087285, to which the present
application claims the priority. In addition, all publications, patents, and patent applications
cited herein are incorporated herein by reference in their entirety.
FREE TEXT OF SEQUENCE LISTING
[0199]
Primers: SEQ ID NOS: 31 to 39 and 130 to 135

WE CLAIM:
1. A pharmaceutical composition for treatment and/or prevention of a cancer,
which comprises, as an active ingredient, an antibody or fragment thereof having an
immunological reactivity with a CAPRIN-1 protein having an amino acid sequence
shown in any one of the even numbered SEQ ID NOS: 2 to 30 or an amino acid
sequence having 80% or more sequence identity with the amino acid sequence ,or with
a fragment of the CAPRIN-1 protein comprising 7 or more consecutive amino acids.
2. The pharmaceutical composition according to claim 1, which comprises, as an
active ingredient, an antibody or fragment thereof having immunological reactivity with
a fragment of the CAPRIN-1 protein that is a polypeptide consisting of a sequence of 7
or more consecutive amino acids in the region of amino acid residue Nos. 50-98 or
amino acid residue Nos. 233-305 in an amino acid sequence shown in any one of the
even numbered SEQ ID NOS: 2 to 30, excluding SEQ ID NOS: 6 and 18, or a
polypeptide comprising the polypeptide as a partial sequence.
3. The pharmaceutical composition according to claim 1, which comprises, as an
active ingredient, an antibody or fragment thereof having an immunological reactivity
with a partial polypeptide of CAPRIN-1 having an amino acid sequence shown in SEQ
ID NO: 37 or SEQ ID NO: 136 or an amino acid sequence having 80% or more
sequence identigy with the amino acid sequence, or with a fragment of the partial
polypetide comprising 7 or more consecutive amino acids.
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein
the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal
cancer, or colon cancer.
5. The pharmaceutical composition according to any one of claims 1 to 4, wherein
the antibody is a monoclonal or polyclonal antibody.
6. The pharmaceutical composition according to any one of claims 1 to 4, wherein
the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single
chain antibody, or a bispecific antibody.

7. An antibody having an immunological reactivity with a polypeptide having the
amino acid sequence shown in SEQ ID NO: 37 or SEQ ID NO: 136 or an amino acid
sequence having 80% or more sequence identity with the amino acid sequence.
8. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 44, 45, and 46, and having an
immunological reactivity with a CAPRIN-1 protein.
9. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 50, 51, and 52 and having an
immunological reactivity with a CAPRIN-1 protein.
10. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 55, 56, and 57, and having an
immunological reactivity with a CAPRIN-1 protein.
11. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 60, 61, and 62, and having an
immunological reactivity with a CAPRIN-1 protein.
12. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 40, 41, and 42 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 65, 66, and 67, and having an
immunological reactivity with a CAPRIN-1 protein.
13. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 70, 71, and 72 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 74, 75, and 76, and having an
immunological reactivity with a CAPRIN-1 protein.
14. An antibody comprising a heavy-chain variable region comprising the

sequences shown in SEQ ID NOS: 80, 81, and 82 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 84, 85, and 86, and having an
immunological reactivity with a CAPRIN-1 protein.
15. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 90, 91, and 92 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 94, 95, and 96, and having an
immunological reactivity with a CAPRIN-1 protein.
16. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 100, 101, and 102 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 104, 105, and 106, and having an
immunological reactivity with a CAPRIN-1 protein.
17. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 110, 111, and 112 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 114, 115, and 116, and having an
immunological reactivity with a CAPRIN-1 protein.
18. An antibody comprising a heavy-chain variable region comprising the
sequences shown in SEQ ID NOS: 120, 121, and 122 and a light-chain variable region
comprising the sequences shown in SEQ ID NOS: 124, 125, and 126, and having an
immunological reactivity with a CAPRIN-1 protein.
19. The antibody according to any one of claims 7 to 18, which is a human
antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a
bispecific antibody.
20. A pharmaceutical composition for treatment and/or prevention of cancer, which
comprises, as an active ingredient, the antibody according to any one of claims 7 to 19
or a fragment thereof.
21. A method for treatment and/or prevention of a cancer using an antibody or
fragment thereof having an immunological reactivity with a CAPRIN-1 protein which
has an amino acid sequence shown in any one of the even numbered SEQ ID NOS: 2 to
30 or an amino acid sequence having 80% or more sequence identity with the amino

acid sequence, or with a fragment of the CAPRIN-1 protein comprising 7 or more
consecutive amino acids.
22. A method for treating and/or preventing a cancer characterized by conducting
the method using the antibody according to any one of claims 7 to 19 or a fragment
thereof.

The present invention relates to a pharmaceutical composition for treatment and/or
prevention of cancer, which comprises, as an active ingredient, an antibody or fragment
thereof having an immunological reactivity with a CAPRTN-1 protein or a fragment thereof
comprising 7 or more consecutive amino acids.