DESCRIPTION
Title of Invention: PHARMACEUTICAL COMPOSITION FOR TREATMENT AND/OR
PREVENTION OF PANCREATIC CANCER
Technical Field
[0001]
The present invention relates to novel use of an antibody against CAPRTN-1 or a
fragment thereof in a medicament such as a therapeutic and/or preventive agent for pancreatic
cancer.
Background Art
[0002]
Cancer is the leading cause of death. This disease is currently treated principally by
surgical therapy in combination with radiation therapy and/or chemotherapy. In spite of
recent development of novel surgical techniques or discovery of novel anticancer agents, the
existing treatment of cancer has an insufficiently improved outcome, except for some cancer
types. With recent advances of molecular biology or cancer immunology, antibodies that
specifically react with cancer, cancer antigens that are recognized by cytotoxic T cells, genes
encoding such cancer antigens, and the like have been identified, raising expectations on
specific cancer therapy targeting the cancer antigens (Non Patent Literature 1).
[0003]
For reducing the adverse effect of cancer therapy, it is desired that peptides,
polypeptides, or proteins recognized as antigens of the cancer should rarely exist in normal
cells and specifically exist in cancer cells. In 1991, Boon et al. (Ludwig Institute for Cancer
Research, Belgium) isolated a human melanoma antigen MAGE1 recognized by CD8-
positive T cells by a cDNA expression cloning method using autologous cancer cell lines and
cancer-reactive T cells (Non Patent Literature 2). Then, a SEREX (serological identification
of antigens by recombinant expression cloning) method has been reported, which adopts a
gene expression cloning approach to identify tumor antigens recognized by antibodies
produced in response to autologous cancer in vivo in a cancer patient (Non Patent Literature 3
and Patent Literature 1). According to this method, some cancer antigens that are rarely
expressed in normal cells and are specifically expressed in cancer have been isolated (Non
Patent Literatures 4 to 9). In addition, cell therapy using immunocytes that specifically react

with cancer antigens or cancer-specific immunotherapy using vaccines or the like comprising
cancer antigens is under clinical trial targeting some of the isolated cancer antigens.
[0004]
In recent years, various antibody drugs for cancer treatment targeting antigenic
proteins on cancer cells have emerged in the world. These drugs have received attention
because of their certain efficacy as cancer-specific therapeutic agents. A large majority of
antigenic proteins targeted by the drugs, however, are also expressed in normal cells. As a
result of administering the antibodies, cancer cells as well as normal cells expressing the
antigens are damaged, disadvantageous^ resulting in adverse effects. Thus, if cancer
antigens specifically expressed on the surface of cancer cells can be identified and antibodies
targeting the antigens can be used as drugs, these antibody drugs can be expected to achieve
treatment with less adverse effects. In terms of the technical common sense of those skilled
in the art, pancreatic cancer is known to be difficult to treat. An effective drug having
sufficient effects on pancreatic cancer has not yet been developed.
[0005]
Cytoplasmic- and proliferation-associated protein 1 (CAPRIN-1) has been known as
an intracellular protein that is expressed upon activation or cell division of resting normal
cells and forms cytoplasmic stress granules with intracellular RNAs to participate in the
regulation of transport and translation of mRNAs. This protein has been found to be
specifically expressed on the surface of cancer cells such as breast cancer cells and is
therefore under study as a target of antibody drugs for cancer treatment (Patent Literature 2).
Patent Literature 2, however, did not confirm that CAPRTN-1 is expressed on pancreatic
cancer cells, and neither disclosed nor suggested that CAPRTN-1 can serve as an antigenic
protein for pancreatic cancer.
Prior Art Literatures
Patent Literature
[0006]
Patent Literature 1: U.S. Patent No. 5698396
Patent Literature 2: International Publication No. WO2010/016526
Non Patent Literature
[0007]
Non Patent Literature 1: Tsuyoshi Akiyoshi, "Japanese Journal of Cancer and Chemotherapy",
1997, Vol. 24, p. 551-519 (Japanese Journal of Cancer and Chemotherapy Publishers Inc.,
Japan)

Non Patent Literature 2: Bruggen P. et al., Science, 254: 1643-1647 (1991)
Non Patent Literature 3: Proc. Natl. Acad. Sci. USA, 92: 11810-11813 (1995)
Non Patent Literature 4: Int. J. Cancer, 72: 965-971 (1997)
Non Patent Literature 5: Cancer Res., 58: 1034-1041 (1998)
Non Patent Literature 6: Int. J. Cancer, 29: 652-658 (1998)
Non Patent Literature 7: Int. J. Oncol., 14: 703-708 (1999)
Non Patent Literature 8: Cancer Res., 56: 4766-4772 (1996)
Non Patent Literature 9: Hum. Mol. Genet., 6: 33-39 (1997)
Summary of Invention
Problem to be Solved by the Invention
[0008]
An object of the present invention is to identify a cancer antigenic protein specifically
expressed on the surface of pancreatic cancer cells and provide use of an antibody targeting
the protein as a therapeutic and/or preventive agent for pancreatic cancer.
Means for Solving the Problem
[0009]
As a result of conducting diligent studies, the present inventors have obtained a cDNA
encoding a protein specifically binding to an antibody present in serum derived from a cancer-
bearing organism, by the SEREX method using a canine testicular tissue-derived cDNA
library and the serum of a breast cancer-affected dog, and then prepared CAPRIN-1 having an
amino acid sequence represented by any of even-numbered SEQ ID NOs: 2 to 30, and a large
number of antibodies against these CAPRTN-1 proteins, on the basis of the obtained gene and
human, bovine, horse, mouse, and chicken homologous genes thereof. Then, the present
inventors have now found that CAPRIN-1 protein segments are specifically expressed on the
surface of pancreatic cancer cells, and also found that an antibody against CAPRIN-1
damages pancreatic cancer cells expressing CAPRIN-1. On the basis of these findings, the
present invention has been completed.
[0010]
Thus, the present invention has the following aspects:
The present invention provides a pharmaceutical composition for treatment and/or
prevention of pancreatic cancer, comprising, as an active ingredient, an antibody or a
fragment thereof which specifically has immunological reactivity with a CAPRIN-1 protein
or a fragment thereof comprising 7 to 12 or more consecutive amino acid residues.

[0011]
In an embodiment, the CAPRIN-1 protein has an amino acid sequence represented by
any of even-numbered SEQ ID NOs: 2 to 30, or an amino acid sequence having 80% or
higher, preferably 85% or higher, more preferably 90% or higher, further preferably 95% or
higher, far more preferably 97 to 99% or higher sequence identity to the amino acid sequence.
[0012]
In another embodiment, the antibody is a monoclonal antibody or a polyclonal
antibody.
[0013]
In a further embodiment, the antibody is a human antibody, a humanized antibody, a
chimeric antibody, a single-chain antibody, or a multispecific antibody.
[0014]
In a further embodiment, the antibody is an antibody having immunological reactivity
with a polypeptide having an amino acid sequence represented by SEQ ID NO: 273, 266, 270,
272, or 269, or an amino acid sequence having 80% or higher, preferably 85% or higher, more
preferably 90% or higher, further preferably 95% or higher, far more preferably 97 to 99% or
higher sequence identity to the amino acid sequence, or a fragment thereof.
[0015]
In a further embodiment, the antibody is any of the following antibodies (a) to (y) and
has immunological reactivity with the CAPRIN-1 protein, or the pharmaceutical composition
is for treatment and/or prevention of pancreatic cancer and is characterized by comprising the
antibody as an effective ingredient:
(a) an antibody comprising a heavy chain variable region comprising complementarity
determining regions (CDRs) of SEQ ID NOs: 37, 38, and 39 and a light chain variable region
comprising CDRs of SEQ ID NOs: 41, 42, and 43;
(b) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 47, 48, and 49 and a light chain variable region comprising CDRs of SEQ ID NOs: 51,
52, and 53;
(c) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 57, 58, and 59 and a light chain variable region comprising CDRs of SEQ ID NOs: 61,
62, and 63;
(d) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 67, 68, and 69 and a light chain variable region comprising CDRs of SEQ ID NOs: 71,
72, and 73;

(e) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 77, 78, and 79 and a light chain variable region comprising CDRs of SEQ ID NOs: 81,
82, and 83;
(f) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 87, 88, and 89 and a light chain variable region comprising CDRs of SEQ ID NOs: 91,
92, and 93;
(g) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 97, 98, and 99 and a light chain variable region comprising CDRs of SEQ ID NOs: 101,
102, and 103;
(h) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 107, 108, and 109 and a light chain variable region comprising CDRs of SEQ ID NOs:
111, 112, and 113;
(i) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 117, 118, and 119 and a light chain variable region comprising CDRs of SEQ ID NOs:
121, 122, and 123;
(j) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 127, 128, and 129 and a light chain variable region comprising CDRs of SEQ ID NOs:
121, 122, and 123;
(k) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 132, 133, and 134 and a light chain variable region comprising CDRs of SEQ ID NOs:
136, 137, and 138;
(1) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 142, 143, and 144 and a light chain variable region comprising CDRs of SEQ ID NOs:
146, 147, and 148;
(m) an antibody comprising a heavy chain variable region comprising CDRs of SEQ
ID NOs: 142, 143, and 144 and a light chain variable region comprising CDRs of SEQ ID
NOs: 152, 153, and 154;
(n) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 157, 158, and 159 and a light chain variable region comprising CDRs of SEQ ID NOs:
161, 162, and 163;
(o) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs:
171,172, and 173;

(p) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs:
177, 178, and 179;
(q) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs:
182, 183, and 184;
(r) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs:
187, 188, and 189;
(s) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs:
192, 193, and 194;
(t) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 197, 198, and 199 and a light chain variable region comprising CDRs of SEQ ID NOs:
201, 202, and 203;
(u) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 207, 208, and 209 and a light chain variable region comprising CDRs of SEQ ID NOs:
211,212, and 213;
(v) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 217, 218, and 219 and a light chain variable region comprising CDRs of SEQ ID NOs:
221, 222, and 223;
(w) an antibody comprising a heavy chain variable region comprising CDRs of SEQ
ID NOs: 227, 228, and 229 and a light chain variable region comprising CDRs of SEQ ID
NOs: 231,232, and 233;
(x) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 237, 238, and 239 and a light chain variable region comprising CDRs of SEQ ID NOs:
241, 242, and 243; and
(y) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID
NOs: 247, 248, and 249 and a light chain variable region comprising CDRs of SEQ ID NOs:
251, 252, and 253.
[0016]
In a further embodiment of the present invention, the antibody or the fragment thereof
is conjugated with an antitumor agent.
[0017]

The present invention further provides a pharmaceutical combination comprising the
pharmaceutical composition of the present invention and a pharmaceutical composition
comprising an antitumor agent.
[0018]
The present invention further provides a method for treating and/or preventing
pancreatic cancer, comprising administering the pharmaceutical composition or the
pharmaceutical combination of the present invention to a subject.
[0019]
The antibody against CAPRIN-1 used in the present invention damages pancreatic
cancer cells. Thus, the antibody against CAPRTN-1 is useful in the treatment and/or
prevention of pancreatic cancer.
Modes for Carrying Out the Invention
[0020]
The antibody against a CAPRTN-1 protein, specifically, a polypeptide having an amino
acid sequence represented by any of even-numbered SEQ ID NOs: 2 to 30, used in the present
invention can be evaluated for its antitumor activity, as described later, by examining in vivo
the inhibition of tumor growth in a cancer-bearing animal or by examining ex vivo the
presence or absence of immunocyte- or complement-mediated cytotoxic activity exhibited by
the antibody against tumor cells expressing the polypeptide.
[0021]
The nucleotide sequences of polynucleotides encoding proteins consisting of amino
acid sequences represented by even-numbered SEQ ID NOs (i.e., SEQ ID NOs: 2, 4, 6,..., 28,
and 30) of SEQ ID NOs: 2 to 30 are shown in odd-numbered SEQ ID NOs (i.e., SEQ ID NOs:
1, 3, 5,..., 27, and 29) of SEQ ID NOs: 1 to 29, respectively.
[0022]
The amino acid sequences represented by SEQ ID NOs: 6, 8, 10, 12, and 14 in the
Sequence Listing are amino acid sequences of CAPRIN-1 isolated as polypeptides
specifically binding to antibodies present in serum derived from a cancer-bearing dog; the
amino acid sequences represented by SEQ ID NOs: 2 and 4 are amino acid sequences of
CAPRTN-1 isolated as human homologous factors (homologs or orthologs) thereof; the amino
acid sequence represented by SEQ ID NO: 16 is an amino acid sequence of CAPRTN-1
isolated as a bovine homologous factor thereof; the amino acid sequence represented by SEQ
ID NO: 18 is an amino acid sequence of CAPRTN-1 isolated as a horse homologous factor
thereof; the amino acid sequences represented by SEQ ID NOs: 20 to 28 are amino acid

sequences of CAPRIN-1 isolated as mouse homologous factors thereof; and the amino acid
sequence represented by SEQ ID NO: 30 is an amino acid sequence of CAPRIN-1 isolated as
a chicken homologous factor thereof (see Example 1 described later). CAPRIN-1 is known
to be expressed upon activation or cell division of resting normal cells.
[0023]
The study of the present invention has revealed that CAPRIN-1 protein is expressed on
the surface of pancreatic cancer cells. According to the present invention, an antibody
binding to a portion expressed on the surface of pancreatic cancer cells in each CAPRIN-1
protein molecule is preferably used. Examples of the partial peptide of the CAPRIN-1
protein expressed on the surface of pancreatic cancer cells include polypeptides each
consisting of 7 to 12 or more, for example, 8 to 11 or more, consecutive amino acid residues
in a region of amino acid residue numbers (aa) 50 to 98, amino acid residue numbers (aa) 233
to 343, or amino acid residue number (aa) 527 to the C terminus of an amino acid sequence
represented by any even number (except for SEQ ID NOs: 6 and 18) of SEQ ID NOs: 2 to 30
in the Sequence Listing, and specifically include: an amino acid sequence represented by SEQ
ID NO: 271 or 273 (preferably, for example, a region of an amino acid sequence represented
by SEQ ID NO: 274 or 275 in the amino acid sequence represented by SEQ ID NO: 273); an
amino acid sequence represented by SEQ ID NO: 266 (preferably, for example, a region of an
amino acid sequence represented by SEQ ID NO: 267 or 268 in the amino acid sequence
represented by SEQ ID NO: 266), 270, 272, or 269 as a partial peptide of the CAPRIN-1
protein expressed on the surface of cancer cells; and an amino acid sequence having 80% or
higher, preferably 85% or higher, more preferably 90% or higher, further preferably 95% or
higher, for example, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence
identity to any of the above amino acid sequences. The antibody used in the present
invention includes all antibodies that bind to these peptides and exhibit antitumor activity.
[0024]
The antibody against CAPRIN-1 used in the present invention may be any type of
antibody that can exert antitumor activity and includes, for example, monoclonal antibodies,
polyclonal antibodies, recombinant antibodies, for example, synthetic antibodies,
multispecific antibodies (e.g., diabody and triabody), humanized antibodies, chimeric
antibodies, and single-chain antibodies (scFv), human antibodies, and their antibody
fragments, for example, Fab, F(ab')2, and Fv. These antibodies and fragments thereof can be
prepared by methods generally known to those skilled in the art. The antibody according to
the present invention is desirably an antibody capable of specifically binding to the CAPRIN-
1 protein and is preferably a monoclonal antibody. A polyclonal antibody may be used as

long as homogeneous antibodies can be stably produced. In the case of a human subject, a
human antibody or a humanized antibody is desirable for avoiding or suppressing rejection.
[0025]
As used herein, the phrase "specifically binding to the CAPRIN-1 protein" means that
the antibody specifically binds to the CAPRIN-1 protein without substantially binding to
other proteins.
[0026]
The antibody that can be used in the present invention can be examined for its
antitumor activity, as described later, by examining in vivo the inhibition of tumor growth in a
cancer-bearing animal or by examining in vitro the presence or absence of immunocyte- or
complement-mediated cytotoxic activity exhibited by the antibody against tumor cells
expressing the polypeptide.
[0027]
The subject to receive the treatment and/or prevention of pancreatic cancer according
to the present invention is a mammal such as a human, a pet animal, livestock, or a sport
animal, preferably a human.
[0028]
Hereinafter, antigen preparation, antibody preparation, and a pharmaceutical
composition according to the present invention will be described.
[0029]

Proteins or fragments thereof used as sensitizing antigens for obtaining the antibody
against CAPRIN-1 used in the present invention are not limited by animal species serving as
their origins, including humans, dogs, cattle, horses, mice, rats, and chickens. The proteins
or the fragments thereof, however, are preferably selected in view of compatibility with
parent cells for use in cell fusion. In general, mammal-derived proteins are preferred.
Particularly, human-derived proteins are preferred. For example, when CAPRIN-1 is human
CAPRIN-1, human CAPRIN-1 proteins, partial peptides thereof, or cells expressing human
CAPRIN-1 can be used.
[0030]
The nucleotide sequences and amino acid sequences of human CAPRIN-1 and
homologs thereof can be obtained, for example, by accessing to GenBank (NCBI, USA) to
use BLAST or FASTA algorithm (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-
5877, 1993; and Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997).
[0031]

In the present invention, with reference to the nucleotide sequence (SEQ ID NO: 1 or
3) or amino acid sequence (SEQ ID NO: 2 or 4) of human CAPRJN-1, targets are nucleic
acids or proteins consisting of sequences having 70% to 100%, preferably 80% to 100%,
more preferably 90% to 100%, further preferably 95% to 100%, for example, 97% to 100%,
98% to 100%, 99% to 100%, or 99.5% to 100% sequence identity to the nucleotide sequence
or amino acid sequence of the ORF or mature portion of the reference. As used herein, the
term "% sequence identity" means a percentage (%) of the number of identical amino acids
(or bases) to the total number of amino acids (or bases) when two sequences are aligned such
that the maximum degree of similarity or identity can be achieved with or without introduced
gaps.
[0032]
The fragments of each CAPRJN-1 protein have lengths ranging from the amino acid
length of an epitope (or an antigenic determinant), which is the smallest unit recognized by
the antibody, to less than the full-length of the protein. The epitope refers to a polypeptide
fragment having antigenicity or immunogenicity in mammals, preferably humans. Its
smallest unit consists of approximately 7 to 12 amino acid residues, for example, 8 to 11
amino acid residues. Specific examples thereof include an amino acid sequence represented
by SEQ ID NO: 273, 266, 270, 272, or 269 and an amino acid sequence having 80% or higher,
preferably 85% or higher, more preferably 90% or higher, further preferably 95% or higher,
far more preferably 97 to 99% or higher sequence identity to the amino acid sequence.
[0033]
Polypeptides comprising the above human CAPRTN-1 proteins and partial peptides
thereof can be synthesized according to chemical synthesis methods, for example, Fmoc
(fluorenylmethyloxycarbonyl) and tBoc (t-butyloxycarbonyl) methods (Seikagaku Jikken
Koza (Biochemical Experimentation Course in English) 1, the Japanese Biochemical Society
ed., Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Tokyo Kagaku
Dojin Co., Ltd. (Japan), 1981). Also, these polypeptides can be synthesized by routine
methods using various commercially available peptide synthesizers. Alternatively,
polynucleotides encoding the polypeptides may be prepared using genetic engineering
approaches known in the art (Sambrook et al., Molecular Cloning, the 2nd edition, Current
Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al.,
Short Protocols in Molecular Biology, the 3rd edition, A compendium of Methods from
Current Protocols in Molecular Biology (1995), John Wiley & Sons; etc.) and incorporated
into expression vectors, which are then introduced into host cells so that the host cells produce
the polypeptides. In this way, the polypeptides of interest can be obtained.

[0034]
The polynucleotides encoding the polypeptides can be readily prepared by genetic
engineering approaches known in the art or routine methods using commercially available
nucleic acid synthesizers. For example, a DNA comprising the nucleotide sequence of SEQ
ID NO: 1 can be prepared by PCR using a human chromosomal DNA or cDNA library as a
template and a pair of primers designed so as to be capable of amplifying the nucleotide
sequence represented by SEQ ID NO: 1. Reaction conditions for this PCR can be
appropriately determined. Examples of the conditions can include, but not limited to, 30
cycles each involving reaction steps consisting of 94°C for 30 seconds (denaturation), 55°C
for 30 seconds to 1 minute (annealing), and 72°C for 2 minutes (elongation) using
thermostable DNA polymerase (e.g., Taq polymerase) and a Mg2+-containing PCR buffer,
followed by reaction at 72°C for 7 minutes. The PCR approach, conditions, etc. are
described in, for example, Ausubel et al., Short Protocols in Molecular Biology, the 3rd
edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995),
John Wiley & Sons (particularly, Chapter 15).
[0035]
Also, appropriate probes or primers can be prepared on the basis of information about
the nucleotide sequences and the amino acid sequences represented by SEQ ID NOs: 1 to 30
in the Sequence Listing described herein, and used in the screening of, for example, a human
cDNA library, to isolate the desired DNA. Preferably, such a cDNA library is produced
from cells, organs, or tissues expressing proteins shown in even-numbered SEQ ID NOs: 2 to
30. Examples of such cells or tissues include cells or tissues derived from the testis or from
cancers or tumors such as leukemia, breast cancer, lymphoma, brain tumor, lung cancer,
colorectal cancer, and pancreatic cancer. These manipulations, including the preparation of
probes or primers, the construction of a cDNA library, the screening of the cDNA library, and
the cloning of the gene of interest, are known to those skilled in the art and can be performed
according to methods described in, for example, Sambrook et al., Molecular Cloning, the 2nd
edition, Current Protocols in Molecular Biology (1989), and Ausubel et al. (ibid). DNAs
encoding the human CAPRIN-1 proteins and the partial peptides thereof can be obtained from
the DNAs thus obtained.
[0036]
The host cells may be any cell capable of expressing the above polypeptides.
Examples of prokaryotic cells include, but not limited to, E. coli. Examples of eukaryotic
cells include, but not limited to: mammalian cells such as monkey kidney cells COS1 and
Chinese hamster ovary cells CHO; a human embryonic kidney cell line HEK293; mouse

embryonic skin cell line NIH3T3; yeast cells such as budding yeast and fission yeast cells;
silkworm cells; and Xenopus egg cells.
[0037]
In the case of using prokaryotic cells as the host cells, the expression vectors used have
an origin that permits replication in the prokaryotic cells, a promoter, a ribosomal binding site,
a multicloning site, a terminator, a drug resistance gene, an auxotrophic complementary gene,
etc. Examples of expression vectors for E. coli can include pUC series, pBluescript II, pET
expression systems, and pGEX expression systems. The DNAs encoding the above
polypeptides can be incorporated into such expression vectors, with which prokaryotic host
cells are then transformed, followed by culture of the obtained transformants so that the
polypeptides encoded by the DNAs are expressed in the prokaryotic host cells. In this
respect, the polypeptides may be expressed as fusion proteins with other proteins.
[0038]
In the case of using eukaryotic cells as the host cells, expression vectors for eukaryotic
cells having a promoter, a splicing region, a poly(A) addition site, etc. are used as the
expression vectors. Examples of such expression vectors can include pKAl, pCDM8,
pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV, pRS, pcDNA3, and pYES2 vectors.
In the same way as above, the DNAs encoding the above polypeptides can be incorporated
into such expression vectors, with which eukaryotic host cells are then transformed, followed
by culture of the obtained transformants so that the polypeptides encoded by the DNAs are
expressed in the eukaryotic host cells. In the case of using expression vectors such as
pIND/V5-His, pFLAG-CMV-2, pEGFP-Nl, or pEGFP-Cl, the polypeptides may be
expressed as various fusion proteins tagged with His tag (e.g., (His)6 to (His)io), FLAG tag,
myc tag, HA tag, GFP, or the like.
[0039]
The expression vectors can be introduced into the host cells using well known methods
such as electroporation, a calcium phosphate method, a liposome method, a DEAE dextran
method, microinjection, viral infection, lipofection, and binding with cell-penetrating peptides.
[0040]
The polypeptide of interest can be isolated and purified from the host cells by a
combination of separation procedures known in the art. Examples thereof include, but not
limited to, treatment with a denaturant (e.g., 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.
[0041]

Antibodies are usually heteromultimeric glycoproteins each comprising at least two
heavy chains and two light chains. The antibodies, except for IgM, are heterotetrameric
glycoproteins of approximately 150 kDa each composed of 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, though the number of disulfide bonds between heavy chains
varies among different immunoglobulin isotypes. Each of the heavy and light chains also
has an intrachain disulfide bond. Each heavy chain has a variable domain (VH region) at
one end, followed by a series of constant regions. Each light chain has a variable domain
(VL region) at one end and has a single constant region at the other end. The light chain
constant region is aligned with the first heavy chain constant region, while the light chain
variable domain is aligned with the heavy chain variable domain. Particular regions called
complementarity determining regions (CDRs) in the antibody variable domains exhibit
specific variability and impart binding specificity to the antibody. Portions relatively
conserved in the variable regions are called framework regions (FRs). The complete heavy
and light chain variable domains each comprise four FRs connected via three CDRs. These
three CDRs are called CDRH1, CDRH2, and CDRH3 in this order from the N-terminus of the
heavy chain. Likewise, the CDRs are called CDRL1, CDRL2, and CDRL3 in the light chain.
CDRH3 is most important for the binding specificity of the antibody for its antigen. In
addition, CDRs in each chain are kept close to each other by the FR regions and contribute to
the formation of an antigen-binding site in the antibody, together with CDRs in the other
chain. The constant regions do not directly contribute to antibody-antigen binding, but
exhibit various effector functions, for example, involvement in antibody-dependent cellular
cytotoxicity (ADCC), phagocytosis mediated by binding to an Fey receptor, half-
life/clearance rate mediated by a neonatal Fc receptor (FcRn), and complement-dependent
cytotoxicity (CDC) mediated by a Clq component in the complement cascade.
[0042]
Preparation of antibody>
The anti-CAPRIN-1 antibody according to the present invention means an antibody
having immunological reactivity with a full-length CAPRTN-1 protein or a fragment thereof.
[0043]

As used herein, the "immunological reactivity" means the property of the antibody
binding to the CAPRTN-1 antigen in vivo. Via such binding, the antibody exerts the function
of damaging (e.g., killing, suppressing, or regressing) tumor. Specifically, the antibody used
in the present invention is not limited by its type as long as the antibody can damage
pancreatic cancer as a result of binding to the CAPRIN-1 protein.
[0044]
Examples of the antibody include monoclonal antibodies, polyclonal antibodies,
synthetic antibodies, multispecific antibodies, human antibodies, humanized antibodies,
chimeric antibodies, single-chain antibodies, and antibody fragments (e.g., Fab, F(ab')2, and
Fv). Also, the antibody is any class of immunoglobulin molecule, for example, IgG, IgE,
IgM, IgA, IgD, or IgY, or any subclass, for example, IgGi, IgG2, IgG3, IgG4, IgAi, or IgA2.
[0045]
The antibody may be further modified by acetylation, formylation, amidation,
phosphorylation, PEGylation, or the like, in addition to glycosylation.
[0046]
Hereinafter, preparation examples of various antibodies will be described.
When the antibody of the present invention is a monoclonal antibody, for example,
CAPRTN-1 proteins, pancreatic cancer cells expressing CAPRIN-1, or a cell line thereof (e.g.,
Capan-2) is administered to each mouse for immunization. The spleen is extracted from this
mouse. After separation of spleen cells, the cells are fused with mouse myeloma cells.
Clones producing antibodies having a cancer cell growth inhibitory effect are selected from
among the obtained fusion cells (hybridomas). The hybridomas producing monoclonal
antibodies having a cancer cell growth inhibitory effect are isolated and cultured. The
antibody of interest can be prepared by purification from the culture supernatant according to
a general affinity purification method.
[0047]
The monoclonal antibody-producing hybridomas may be prepared, for example, as
follows: first, animals are immunized with sensitizing antigens according to a method known
in the art. This immunization method generally comprises intraperitoneally or
subcutaneously injecting the sensitizing antigens to mammals. Specifically, the sensitizing
antigens are diluted with or suspended in PBS (phosphate-buffered saline), physiological
saline, or the like into an appropriate amount and then mixed, if desired, with an appropriate
amount of a conventional adjuvant, for example, a complete Freund's adjuvant. After
emulsification, the resulting emulsion is administered to each mammal several times every 4

to 21 days. Alternatively, an appropriate carrier may be used for the immunization with
sensitizing antigens.
[0048]
After confirmation of a rise in the level of the desired antibody in the serum of the
mammal thus immunized, immunocytes are collected from the mammal and subjected to cell
fusion. Preferred examples of the immunocytes particularly include spleen cells.
[0049]
Mammalian myeloma cells are used as partner parent cells to be fused with the
immunocytes. Various cell lines known in the art, for example, P3U1 (P3-X63Ag8Ul), P3
(P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.l (Current Topics in
Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J.
Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D.H. et al., Cell (1976) 8, 405-415),
SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (deSt. Groth, S.F. et al., J.
Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S. J. Exp. Med. (1978) 148, 313-
323), and R210 (Galfre, G. et al., Nature (1979) 277, 131-133), are preferably used as the
myeloma cells.
[0050]
The cell fusion between the immunocytes and the myeloma cells can be performed
basically according to a method known in the art, for example, the method of Kohler and
Milstein (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).
[0051]
More specifically, the cell fusion is carried out, for example, in the presence of a cell
fusion promoter in a conventional nutrient medium. For example, polyethylene glycol
(PEG) or Sendai virus (hemagglutinating virus of Japan (HVJ)) is used as the fusion promoter.
If desired, an auxiliary such as dimethyl sulfoxide may be further added in order to enhance
fusion efficiency.
[0052]
The ratio between the immunocytes and the myeloma cells used can be arbitrarily set.
For example, the amount of the immunocytes is preferably set to 1 to 10 times the amount of
the myeloma cells. Examples of the medium that can be used in the cell fusion include
RPMI1640 and MEM media suitable for the growth of the myeloma cell lines as well as
conventional media for use in this type of cell culture. In addition, a serum supplement such
as fetal calf serum (FCS) may be used in combination with these cells.
[0053]

For the cell fusion, the immunocytes and the myeloma cells are well mixed in a
predetermined amount of the medium. A PEG solution (average molecular weight: for
example, approximately 1000 to 6000) preheated to approximately 37°C is usually added to
the mixture at a concentration of 30 to 60% (w/v) and mixed therewith to form the
hybridomas of interest. Subsequently, procedures of sequentially adding an appropriate
medium and removing the supernatant by centrifugation are repeated to remove cell fusion
agents or the like unfavorable for the growth of the hybridomas.
[0054]
The hybridomas thus obtained are cultured in a conventional selective medium, for
example, a HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine)
for selection. Culture in the HAT medium is continued for a period (usually, several days to
several weeks) sufficient for the death of cells (non-fused cells) other than the hybridomas of
interest. Subsequently, hybridomas producing the antibody of interest are screened for and
cloned as single clones by a conventional limiting dilution method.
[0055]
In addition to such obtainment of the hybridomas by the immunization of non-human
animals with antigens, hybridomas producing human antibodies having the desired activity
(e.g., cell growth inhibitory activity) may be obtained by sensitizing human lymphocytes, for
example, EB virus-infected human lymphocytes, with proteins, protein-expressing cells, or
lysates thereof in vitro and fusing the sensitized lymphocytes with human-derived myeloma
cells capable of dividing permanently, for example, U266 (Accession No. TIB 196).
[0056]
The monoclonal antibody-producing hybridomas thus prepared can be subcultured in a
conventional medium and can also be stored for a long period in liquid nitrogen.
[0057]
Specifically, the desired antigens or cells expressing the desired antigens are used as
sensitizing antigens in immunization according to a conventional immunization method.
The obtained immunocytes are fused with parent cells known in the art according to a
conventional cell fusion method. Monoclonal antibody-producing cells (hybridomas) can be
screened for by a conventional screening method to prepare the antibody of interest.
[0058]
Another example of the antibody that may be used in the present invention is a
polyclonal antibody. The polyclonal antibody can be obtained, for example, as follows:
[0059]

Serum is obtained from small animals such as mice, human antibody-producing mice,
or rabbits immunized with natural CAPRIN-1 proteins or recombinant CAPRIN-1 proteins
expressed as fusion proteins with GST or the like in microorganisms such as E. coli, or partial
peptides thereof. This serum is purified using, for example, ammonium sulfate precipitation,
protein A or protein G columns, DEAE ion-exchange chromatography, or affinity columns
coupled with CAPRIN-1 proteins or synthetic peptides to prepare the polyclonal antibody of
interest. In Examples described later, rabbit polyclonal antibodies against CAPRIN-1
proteins were prepared and confirmed to have an antitumor effect.
[0060]
In this context, for example, KM mice (Kirin Pharma Co., Ltd./Medarex) and Xeno
mice (Amgen Inc.) are known as the human antibody-producing mice (e.g., International
Publication Nos. WO02/43478 and WO02/092812). Complete human polyclonal antibodies
can be obtained from the blood of such mice immunized with CAPRIN-1 proteins or
fragments thereof. Alternatively, spleen cells may be isolated from the mice thus
immunized and fused with myeloma cells. In this way, human monoclonal antibodies can be
obtained.
[0061]
The antigens can be prepared according to, for example, a method using animal cells
(JP Patent Publication (Kohyo) No. 2007-530068 A (2007)) or a method using baculovirus
(e.g., International Publication No. W098/46777). Antigens having low immunogenicity
may be bound to immunogenic macromolecules such as albumin for immunization.
[0062]
Alternatively, recombinant antibodies may be used, which are produced using a gene
recombination technique which comprises: cloning antibody genes from hybridomas;
incorporating the antibody genes into appropriate vectors; and introducing the vectors into
hosts (see, e.g., Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC
MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN
PUBLISHERS LTD, 1990). Specifically, antibody variable region (V region) cDNAs are
synthesized from the mRNAs of hybridomas using reverse transcriptase. After obtainment
of DNAs encoding the antibody V regions of interest, the DNAs are ligated with DNAs
encoding the desired antibody constant regions (C regions). The resulting ligation products
are incorporated into expression vectors. Alternatively, the antibody V region-encoding
DNAs may be incorporated into expression vectors containing antibody C region DNAs.
These DNAs are incorporated into the expression vectors so as to be expressed under the
control of expression control regions, for example, an enhancer and a promoter. Next, host

cells can be transformed with the resulting expression vectors and allowed to express
antibodies.
[0063]
The anti-CAPRIN-1 antibody used in the present invention is preferably a monoclonal
antibody. Alternatively, the anti-CAPRIN-1 antibody of the present invention may be a
polyclonal antibody, a genetically engineered antibody (chimeric antibody, humanized
antibody, etc.), or the like.
[0064]
The monoclonal antibody includes human monoclonal antibodies, non-human animal
monoclonal antibodies (e.g., mouse, rat, rabbit, and chicken monoclonal antibodies), and the
like. The monoclonal antibody may be prepared by the culture of hybridomas obtained by
the fusion between spleen cells from non-human mammals (e.g., mice or human antibody-
producing mice) immunized with CAPRTN-1 proteins and myeloma cells. In Examples
described later, monoclonal antibodies were prepared and confirmed to have an antitumor
effect on pancreatic cancer. These monoclonal antibodies each comprise a heavy chain
variable (VH) region having an amino acid sequence of SEQ ID NO: 40, 50, 60, 70, 80, 90,
100, 110, 120, 130, 135, 145, 160, 170, 200, 210, 220, 230, 240, or 250 and a light chain
variable (VL) region having an amino acid sequence of SEQ ID NO: 44, 54, 64, 74, 84, 94,
104, 114, 124, 139, 149, 155, 164, 174, 180, 185, 190, 195, 204, 214, 224, 234, 244, or 254,
wherein the VH region comprises CDR1 represented by an amino acid sequence of SEQ ID
NO: 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 132, 142, 157, 167, 197, 207, 217, 227, 237, or
247, CDR2 represented by an amino acid sequence of SEQ ID NO: 38, 48, 58, 68, 78, 88, 98,
108, 118, 128, 133, 143, 158, 168, 198, 208, 218, 228, 238, or 248, and CDR3 represented by
an amino acid sequence of SEQ ID NO: 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 134, 144,
159, 169, 199, 209, 219, 229, 239, or 249, and the VL region comprises CDR1 represented by
an amino acid sequence of SEQ ID NO: 41, 51, 61, 71, 81, 91, 101, 111, 121, 136, 146, 152,
161, 171, 177, 182, 187, 192, 201, 211, 221, 231, 241, or 251, CDR2 represented by an amino
acid sequence of SEQ ID NO: 42, 52, 62, 72, 82, 92, 102, 112, 122, 137, 147, 153, 162, 172,
178, 183, 188, 193, 202, 212, 222, 232, 242, or 252, and CDR3 represented by an amino acid
sequence of SEQ ID NO: 43, 53, 63, 73, 83, 93, 103, 113, 123, 138, 148, 154, 163, 173, 179,
184, 189, 194, 203, 213, 223, 233, 243, or 253.
[0065]
The chimeric antibody is an antibody prepared from a combination of sequences
derived from different animals and is, for example, an antibody composed of mouse antibody
heavy and light chain variable regions and human antibody heavy and light chain constant

regions. The chimeric antibody can be prepared using a method known in the art which
involves, for example: ligating DNAs encoding antibody V regions with DNAs encoding
human antibody C regions; incorporating the resulting ligation products into expression
vectors; and introducing the vectors into hosts so that antibodies are produced.
[0066]
The polyclonal antibody includes antibodies obtained from human antibody-producing
animals (e.g., mice) immunized with CAPRIN-1 proteins.
[0067]
The humanized antibody, also called reshaped human antibody, is an engineered
antibody. The humanized antibody is constructed by grafting antibody CDRs derived from
an immunized animal into human antibody complementarity determining regions of a human
antibody. A general gene recombination approach therefor is also known.
[0068]
Specifically, DNA sequences designed so as to link mouse antibody CDRs and human
antibody framework regions (FRs) are synthesized by PCR using several prepared
oligonucleotides having terminal portions overlapping with each other. The obtained DNAs
are ligated with DNAs encoding human antibody constant regions. Subsequently, the
resulting ligation products are incorporated into expression vectors, which are then introduced
into hosts for antibody production to obtain the antibody of interest (see European Patent
Application Publication No. EP239400 and International Publication No. WO96/02576).
The human antibody FRs connected via CDRs are selected such that the complementarity
determining regions form a favorable antigen-binding site. If necessary, amino acids in the
framework regions of antibody variable regions may be substituted such that the
complementarity determining regions of the resulting reshaped human antibody form an
appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). In
addition, these framework regions may be replaced with framework regions derived from
various human antibodies (see International Publication No. W099/51743).
[0069]
The human antibody framework regions connected via CDRs are selected such that the
complementarity determining regions form a favorable antigen-binding site. If necessary,
amino acids in the framework regions of antibody variable regions may be substituted such
that the complementarity determining regions of the resulting reshaped human antibody form
an appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53: 851-856).
[0070]

Amino acids in variable regions (e.g., FRs) or constant regions of the chimeric
antibody or the humanized antibody thus prepared may be substituted, for example, by other
amino acids.
[0071]
The amino acid substitution is the substitution of, for example, less than 15, less than
10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acids,
preferably 1 to 5 amino acids, more preferably 1 or 2 amino acids. The substituted antibody
should be functionally equivalent to an unsubstituted antibody. The substitution is desirably
conservative amino acid substitution, which is the substitution between amino acids similar in
properties such as charge, side chains, polarity, and aromaticity. The amino acids can be
classified in terms of similar properties into, for example: 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 amino acids (threonine, valine, and isoleucine); and aromatic amino
acids (phenylalanine, tyrosine, tryptophan, and histidine).
[0072]
Examples of modified antibodies can include antibodies bound with various molecules
such as polyethylene glycol (PEG). In the modified antibody used in the present invention,
the substance to be bound is not limited. In order to obtain such a modified antibody, the
obtained antibody can be chemically modified. A method therefor has already been
established in the art.
[0073]
In this context, the phrase "functionally equivalent" means that an antibody concerned
has biological or biochemical activity similar to that of the antibody used in the present
invention, specifically, the antibody concerned has the function of damaging tumor and
essentially causes no rejection when applied to humans, for example. Examples of such
activity can include cell growth inhibitory activity and binding activity.
[0074]
A method for preparing a polypeptide functionally equivalent to a certain polypeptide,
which comprises introducing a mutation into a polypeptide, is well known to those skilled in
the art. For example, those skilled in the art can appropriately introduce a mutation into the
antibody used in the present invention using site-directed mutagenesis (Hashimoto-Gotoh, T.
et al., (1995) Gene 152, 271-275; Zoller, MJ., and Smith, M. (1983) Methods Enzymol. 100,
468-500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz,

HJ., (1987) Methods Enzymol. 154, 350-367; Kunkel, TA., (1985) Proc. Natl. Acad. Sci.
USA. 82, 488-492; and Kunkel (1988) Methods Enzymol. 85, 2763-2766) or the like, thereby
prepare an antibody functionally equivalent to the antibody of the present invention.
[0075]
An antibody that recognizes an epitope of a CAPRIN-1 protein recognized by each
anti-CAPRIN-1 antibody described above can be obtained by a method generally known to
those skilled in the art. For example, the antibody can be obtained by a method which
comprises determining the epitope of the CAPRIN-1 protein recognized by the anti-CAPRIN-
1 antibody by a conventional method (e.g., epitope mapping) and preparing an antibody using
a polypeptide having an amino acid sequence contained in the epitope as an immunogen, or a
method which involves determining an epitope for an antibody prepared by a conventional
method and selecting an antibody that recognizes the same epitope as that for the anti-
CAPRIN-1 antibody. As used herein, the "epitope" refers to a polypeptide fragment having
antigenicity or immunogenicity in mammals, preferably humans. Its smallest unit consists
of approximately 7 to 12 amino acids, preferably 8 to 11 amino acids.
[0076]
The antibody used in the present invention has an affinity constant Ka (kon/koff) of
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
lO^M^oratleastlO^M"1.
[0077]
The antibody used in the present invention can be conjugated with an antitumor agent.
The conjugation of the antibody with the antitumor agent can be performed via a spacer
having a group (e.g., a succinimidyl group, a formyl group, a 2-pyridyldithio group, a
maleimidyl group, an alkoxycarbonyl group, or a hydroxy group) reactive with an amino
group, a carboxyl group, a hydroxy group, a thiol group, or the like.
[0078]
Examples of the antitumor agent include the following antitumor agents publicly
known in literatures, etc.: paclitaxel, doxorubicin, daunorubicin, cyclophosphamide,
methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa,
carboquone, meturedopa, uredopa, altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin,
bullatacinone, camptothecin, bryostatin, 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, chlorozotocin, fotemustine, lomustine, nimustine,
ranimustine, calicheamicin, dynemicin, clodronate, esperamicin, aclacinomycin, actinomycin,
authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine, Adriamycin, epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin,
olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate,
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens (e.g.,
calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone),
aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside,
aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,
demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, lentinan,
lonidamine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine,
pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,
procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid,
triaziquone, roridin 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 inhibitors,
difluoromethylornithine (DMFO), retinoic acid, capecitabine, and pharmaceutically
acceptable salts (known in the art) and derivatives (known in the art) thereof.
[0079]
Alternatively, the antibody used in the present invention can be administered in
combination with an antitumor agent to produce a higher therapeutic effect. This approach
is adaptable to a patient with cancer expressing CAPRTN-1 either before or after surgical
operation. This approach can be applied, particularly after surgery, to CAPRIN-1-
expressing cancer, which has been treated conventionally with an antitumor agent alone, to
produce higher prevention of cancer recurrence or prolongation of survival time.
[0080]
Examples of the antitumor agent used in the combined administration include the
following antitumor agents publicly known in literatures, etc.: paclitaxel, doxorubicin,
daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan,

improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine,
triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide,
trimethylolomelamine, bullatacin, bullatacinone, camptothecin, bryostatin, 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,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, dynemicin,
clodronate, esperamicin, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin,
cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin,
detorbicin, 6-diazo-5-oxo-L-norleucine, Adriamycin, epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-
mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone,
dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide,
mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic
acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine,
diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, lentinan, lonidamine,
maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,
phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine,
razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid, triaziquone, roridin 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 inhibitors,
difluoromethylornithine (DMFO), retinoic acid, capecitabine, and pharmaceutically
acceptable salts (known in the art) and derivatives (known in the art) thereof. Of these
antitumor agents, cyclophosphamide, paclitaxel, docetaxel, vinorelbine, or the like is
particularly preferably used.
[0081]
Alternatively, the antibody used in the present invention may be bound to a
radioisotope publicly known in literatures, etc., such as 211At, 131I, 125I, 90Y, l86Re, 188Re,

153Sm, 212Bi, 32P, I75Lu, or 176Lu. Desirably, a radioisotope effective for the treatment or
diagnosis of tumor is used.
[0082]
The antibody used in the present invention is an antibody having immunological
reactivity with CAPRTN-1 or an antibody specifically binding to CAPRJN-1 and exhibits
cytotoxic activity or tumor growth inhibitory effect on pancreatic cancer. The antibody
should have a structure that causes little or no rejection in recipient animals. Examples of
such antibodies include human antibodies, humanized antibodies, chimeric antibodies (e.g.,
human-mouse chimeric antibodies), single-chain antibodies, and multispecific antibodies (e.g.,
diabody and triabody) when the recipient animals are humans. These antibodies have heavy
and light chain variable regions derived from a human antibody or have heavy and light chain
variable regions with complementarity determining regions (CDR1, CDR2, and CDR3)
derived from a non-human animal antibody and framework regions derived from a human
antibody. Alternatively, these antibodies are recombinant antibodies having heavy and light
chain variable regions derived from a non-human animal antibody and heavy and light chain
constant regions derived from a human antibody. The antibody of the present invention is
preferably the former two antibodies.
[0083]
Such recombinant antibodies can be prepared as follows: DNAs encoding monoclonal
antibodies (e.g., human, mouse, rat, rabbit, and chicken monoclonal antibodies) against
human CAPRIN-1 are cloned from antibody-producing cells such as hybridomas and used as
templates in RT-PCR or the like to prepare DNAs encoding the light and heavy chain variable
regions of the antibodies. The respective sequences of the light and heavy chain variable
regions and the respective sequences of CDR1, CDR2, and CDR3 in each region are
determined on the basis of 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)).
[0084]
Such a DNA encoding each variable region or a DNA encoding each CDR is prepared
using a gene recombination technique (Sambrook et al., Molecular Cloning A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. In this context,
the human monoclonal antibody-producing hybridomas can be prepared by immunizing
human antibody-producing animals (e.g., mice) with human CAPRIN-1 and then fusing
spleen cells excised from the immunized animals with myeloma cells. Aside from this,

DNAs encoding human antibody-derived light or heavy chain variable and constant regions
are prepared, if necessary, using a gene recombination technique or a DNA synthesizer.
[0085]
For the humanized antibody, DNAs in which the CDR coding sequences in DNAs
encoding a human antibody-derived light or heavy chain variable regions are substituted by
corresponding CDR coding sequences of a non-human animal (e.g., mouse, rat, or chicken)-
derived antibody can be prepared and ligated with the DNAs encoding human antibody-
derived light or heavy chain constant regions to prepare a DNA encoding the humanized
antibody.
[0086]
For the chimeric antibody, DNAs encoding light or heavy chain variable regions of a
non-human animal (e.g., mouse, rat, or chicken)-derived antibody can be ligated with DNAs
encoding human antibody-derived light or heavy chain constant regions to prepare a DNA
encoding the chimeric antibody.
[0087]
The single-chain antibody refers to an antibody comprising heavy and light chain
variable regions linearly linked to each other via a linker. A DNA encoding the single-chain
antibody can be prepared by ligating a DNA encoding the heavy chain variable region, a
DNA encoding the linker, and a DNA encoding the light chain variable region. In this
context, the heavy and light chain variable regions are both derived from a human antibody or
derived from a human antibody having CDRs substituted by CDRs of a non-human animal
(e.g., mouse, rat, or chicken)-derived antibody. The linker consists of 12 to 19 amino acids.
Examples thereof include (G4S)3 consisting of 15 amino acids (G.B. Kim et al., Protein
Engineering Design and Selection 2007, 20 (9): 425-432).
[0088]
The bispecific antibody (diabody) refers to an antibody capable of specifically binding
to two different epitopes. A DNA encoding the bispecific antibody can be prepared by
ligating, for example, a DNA encoding a heavy chain variable region A, a DNA encoding a
light chain variable region B, a DNA encoding a heavy chain variable region B, and a DNA
encoding a light chain variable region A in this order (provided that the DNA encoding a light
chain variable region B and the DNA encoding a heavy chain variable region B are ligated via
a DNA encoding a linker as described above). In this context, the heavy and light chain
variable regions are all derived from a human antibody or derived from a human antibody
having CDRs substituted by CDRs of a non-human animal (e.g., mouse, rat, or chicken-
derived antibody.

[0089]
The recombinant DNAs thus prepared can be incorporated into one or more
appropriate vectors, which are then introduced into host cells (e.g., mammalian cells, yeast
cells, and insect cells) so that the DNAs are (co)expressed to produce recombinant antibodies
(P.J. Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P.
Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS; and
J.W. Goding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS).
[0090]
Examples of the antibody of the present invention prepared by any of the methods
described above include the following antibodies (a) to (y):
(a) an antibody comprising a heavy chain variable region comprising complementarity
determining regions (CDRs) of SEQ ID NOs: 37, 38, and 39 and a light chain variable region
comprising CDRs of SEQ ID NOs: 41, 42, and 43 (e.g., an antibody comprising a heavy chain
variable region of SEQ ID NO: 40 and a light chain variable region of SEQ ID NO: 44);
(b) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
47, 48, and 49 and a light chain variable region comprising CDRs of SEQ ID NOs: 51, 52,
and 53 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 50 and a
light chain variable region of SEQ ID NO: 54);
(c) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
57, 58, and 59 and a light chain variable region comprising CDRs of SEQ ID NOs: 61, 62,
and 63 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 60 and a
light chain variable region of SEQ ID NO: 64);
(d) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
67, 68, and 69 and a light chain variable region comprising CDRs of SEQ ID NOs: 71, 72,
and 73 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 70 and a
light chain variable region of SEQ ID NO: 74);
(e) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
77, 78, and 79 and a light chain variable region comprising CDRs of SEQ ID NOs: 81, 82,
and 83 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 80 and a
light chain variable region of SEQ ID NO: 84);
(f) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
87, 88, and 89 and a light chain variable region comprising CDRs of SEQ ID NOs: 91, 92,
and 93 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 90 and a
light chain variable region of SEQ ID NO: 94);

(g) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
97, 98, and 99 and a light chain variable region comprising CDRs of SEQ ID NOs: 101, 102,
and 103 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 100 and
a light chain variable region of SEQ ID NO: 104);
(h) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
107, 108, and 109 and a light chain variable region comprising CDRs of SEQ ID NOs: 111,
112, and 113 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 110
and a light chain variable region of SEQ ID NO: 114);
(i) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
117, 118, and 119 and a light chain variable region comprising CDRs of SEQ ID NOs: 121,
122, and 123 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 120
and a light chain variable region of SEQ ID NO: 124);
(j) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
127, 128, and 129 and a light chain variable region comprising CDRs of SEQ ID NOs: 121,
122, and 123 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 130
and a light chain variable region of SEQ ID NO: 124);
(k) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
132, 133, and 134 and a light chain variable region comprising CDRs of SEQ ID NOs: 136,
137, and 138 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 135
and a light chain variable region of SEQ ID NO: 139);
(1) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
142, 143, and 144 and a light chain variable region comprising CDRs of SEQ ID NOs: 146,
147, and 148 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 145
and a light chain variable region of SEQ ID NO: 149);
(m) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
142, 143, and 144 and a light chain variable region comprising CDRs of SEQ ID NOs: 152,
153, and 154 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 145
and a light chain variable region of SEQ ID NO: 155);
(n) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
157, 158, and 159 and a light chain variable region comprising CDRs of SEQ ID NOs: 161,
162, and 163 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 160
and a light chain variable region of SEQ ID NO: 164);
(o) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs: 171,

172, and 173 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 170
and a light chain variable region of SEQ ID NO: 174);
(p) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs: 177,
178, and 179 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 170
and a light chain variable region of SEQ ID NO: 180);
(q) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs: 182,
183, and 184 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 170
and a light chain variable region of SEQ ID NO: 185);
(r) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs: 187,
188, and 189 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 170
and a light chain variable region of SEQ ID NO: 190);
(s) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
167, 168, and 169 and a light chain variable region comprising CDRs of SEQ ID NOs: 192,
193, and 194 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 170
and a light chain variable region of SEQ ID NO: 195);
(t) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
197, 198, and 199 and a light chain variable region comprising CDRs of SEQ ID NOs: 201,
202, and 203 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 200
and a light chain variable region of SEQ ID NO: 204);
(u) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
207, 208, and 209 and a light chain variable region comprising CDRs of SEQ ID NOs: 211,
212, and 213 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 210
and a light chain variable region of SEQ ID NO: 214);
(v) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
217, 218, and 219 and a light chain variable region comprising CDRs of SEQ ID NOs: 221,
222, and 223 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 220
and a light chain variable region of SEQ ID NO: 224);
(w) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
227, 228, and 229 and a light chain variable region comprising CDRs of SEQ ID NOs: 231,
232, and 233 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 230
and a light chain variable region of SEQ ID NO: 234);

(x) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
237, 238, and 239 and a light chain variable region comprising CDRs of SEQ ID NOs: 241,
242, and 243 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 240
and a light chain variable region of SEQ ID NO: 244);
(y) an antibody comprising a heavy chain variable region comprising CDRs of SEQ ID NOs:
247, 248, and 249 and a light chain variable region comprising CDRs of SEQ ID NOs: 251,
252, and 253 (e.g., an antibody comprising a heavy chain variable region of SEQ ID NO: 250
and a light chain variable region of SEQ ID NO: 254).
[0091]
In this context, the amino acid sequences represented by SEQ ID NOs: 67, 68, and 69,
SEQ ID NOs: 77, 78, and 79, SEQ ID NOs: 87, 88, and 89, SEQ ID NOs: 97, 98, and 99,
SEQ ID NOs: 107, 108, and 109, SEQ ID NOs: 117, 118, and 119, SEQ ID NOs: 127, 128,
and 129, SEQ ID NOs: 132, 133, and 134, SEQ ID NOs: 142, 143, and 144, SEQ ID NOs:
157, 158, and 159, SEQ ID NOs: 167, 168, and 169, SEQ ID NOs: 167, 168, and 169, SEQ
ID NOs: 197, 198, and 199, SEQ ID NOs: 207, 208, and 209, SEQ ID NOs: 217, 218, and
219, SEQ ID NOs: 227, 228, and 229, SEQ ID NOs: 237, 238, and 239, SEQ ID NOs: 247,
248, and 249 correspond to CDR1, CDR2, and CDR3, respectively, of a mouse antibody
heavy chain variable region. The amino acid sequences represented by SEQ ID NOs: 71, 72,
and 73, SEQ ID NOs: 81, 82, and 83, SEQ ID NOs: 91, 92, and 93, SEQ ID NOs: 101, 102,
and 103, SEQ ID NOs: 111, 112, and 113, SEQ ID NOs: 121, 122, and 123, SEQ ID NOs:
136, 137, and 138, SEQ ID NOs: 146, 147, and 148, SEQ ID NOs: 152, 153, and 154, SEQ
ID NOs: 161, 162, and 163, SEQ ID NOs: 171, 172, and 173, SEQ ID NOs: 177, 178, and
179, SEQ ID NOs: 182, 183, and 184, SEQ ID NOs: 187, 188, and 189, SEQ ID NOs: 192,
193, and 194, SEQ ID NOs: 201, 202, and 203, SEQ ID NOs: 211, 212, and 213, SEQ ID
NOs: 221, 222, and 223, SEQ ID NOs: 231, 232, and 233, SEQ ID NOs: 241, 242, and 243,
SEQ ID NOs: 251, 252 and 253 correspond to CDR1, CDR2, and CDR3, respectively, of a
mouse antibody light chain variable region.
[0092]
Also, the amino acid sequences represented by SEQ ID NOs: 37, 38, and 39, SEQ ID
NOs: 47, 48, and 49, or SEQ ID NOs: 57, 58, and 59 correspond to CDR1, CDR2, and CDR3,
respectively, of a chicken antibody heavy chain variable region. The amino acid sequences
represented by SEQ ID NOs: 41, 42, and 43, SEQ ID NOs: 51, 52, and 53, or SEQ ID NOs:
61, 62, and 63 correspond to CDR1, CDR2, and CDR3, respectively, of a chicken antibody
light chain variable region.
[0093]

Examples of the humanized antibody, the chimeric antibody, the single-chain antibody,
or the multispecific antibody used in the present invention include the following antibodies,
wherein the antibody (a) above is used as an example:
(i) an antibody comprising a heavy chain variable region comprising the amino acid
sequences of SEQ ID NOs: 37, 38, and 39 and the amino acid sequences of human antibody-
derived framework regions and a light chain variable region comprising the amino acid
sequences of SEQ ID NOs: 41, 42, and 43 and the amino acid sequences of human antibody-
derived framework regions;
(ii) an antibody comprising a heavy chain variable region comprising the amino acid
sequences of SEQ ID NOs: 37, 38, and 39 and the 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: 41, 42, and 43 and the amino acid sequences of human antibody-
derived framework regions, and a light chain constant region comprising a human antibody-
derived amino acid sequence; and
(iii) an antibody comprising a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 40, 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: 44, and a light chain constant region comprising a human antibody-
derived amino acid sequence.
[0094]
The sequences of the constant and variable regions of human antibody heavy and light
chains are available from, for example, NCBI (USA; GenBank, UniGene, etc.). For
example, the following sequences can be referred to: Accession No. J00228 for a human IgGi
heavy chain constant region; Accession No. J00230 for a human IgG2 heavy chain constant
region; Accession No. X03604 for a human IgG3 heavy chain constant region; Accession No.
K01316 for a human IgG4 heavy chain constant region; Accession Nos. V00557, X64135, and
X64133 for a human light chain K constant region; and Accession Nos. X64132 and X64134
for a human light chain X constant region.
[0095]
Preferably, these antibodies have cytotoxic activity and can thereby exert an antitumor
effect.
[0096]
The above particular sequences of the heavy and light chain variable regions and
CDRs in each antibody are provided merely for illustrative purposes. The antibody of the

present invention should not be limited by the particular sequences. Hybridomas capable of
producing anti-human CAPRIN-1 human antibodies or non-human animal antibodies (e.g.,
mouse antibodies) different from those described above are prepared, and monoclonal
antibodies produced by the hybridomas are recovered and assessed as being (or being not) the
antibodies of interest with immunological binding activity against human CAPRIN-1 and
cytotoxic activity as indicators. The monoclonal antibody-producing hybridomas of interest
are thereby identified. Then, DNAs encoding heavy and light chain variable regions of the
antibodies of interest are produced from the hybridomas and sequenced, as described above.
The DNAs are used for the preparation of the different antibodies.
[0097]
The antibody used in the present invention may be any of the antibodies (i) to (iii), etc.
having a substitution(s), deletion(s), or addition(s) of one or several (preferably 1 or 2) amino
acids, particularly in a framework region sequence and/or a constant region sequence, as long
as the antibody has such specificity that it can specifically recognize CAPRIN-1. In this
context, the term "several" means 2 to 5, preferably 2 or 3.
[0098]
The antitumor effect of the anti-CAPRIN-1 antibody used in the present invention on
CAPRIN-1-expressing pancreatic cancer cells seems to be brought about by the following
mechanism:
[0099]
effector cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC) against the CAPRIN-1-expressing cells.
[0100]
Thus, the anti-CAPRIN-1 antibody used in the present invention can be evaluated for
its activity by determining in vitro the ADCC activity or the CDC activity against CAPRIN-1 -
expressing pancreatic cancer cells as specifically shown below in Examples.
[0101]
The anti-CAPRIN-1 antibody used in the present invention binds to CAPRIN-1
proteins on pancreatic cancer cells and exhibits an antitumor effect through the above activity.
Thus, the anti-CAPRIN-1 antibody of the present invention is presumably useful in the
treatment or prevention of pancreatic cancer. Specifically, the present invention provides a
pharmaceutical composition for treatment and/or prevention of pancreatic cancer, comprising
the anti-CAPRIN-1 antibody as an active ingredient. The anti-CAPRIN-1 antibody used for
the purpose of administration to human bodies (antibody therapy) is preferably a human
antibody or a humanized antibody for reducing immunogenicity.

[0102]
The anti-CAPRIN-1 antibody with higher binding affinity for a CAPRIN-1 protein on
pancreatic cancer cell surface exerts stronger antitumor activity. Thus, a stronger antitumor
effect can be expected if an anti-CAPPJN-1 antibody having high binding affinity for the
CAPRIN-1 protein can be obtained. Such an antibody is adaptable to a pharmaceutical
composition intended for the treatment and/or prevention of pancreatic cancer. Desirably,
such high binding affinity 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, in terms of an association constant
(affinity constant) Ka (kon/koff), as described above.
[0103]

The ability of the antibody to bind to CAPRIN-1 can be determined by use of binding
assay using, for example, ELISA, Western blot, immunofluorescence, and flow cytometry
analysis, as described in Examples.
[0104]

The antibody that recognizes CAPRIN-1 can be tested for its reactivity with CAPRIN-
1 by an immunohistochemical method well known to those skilled in the art using a
paraformaldehyde- or acetone-fixed frozen section or paraformaldehyde-fixed paraffin-
embedded section of a tissue obtained from a patient during surgical operation or from an
animal carrying a xenograft tissue inoculated with a cell line expressing CAPRIN-1 either
spontaneously or after transfection.
[0105]
For immunohistochemical staining, the antibody reactive with CAPRIN-1 can be
stained by various methods. For example, the antibody can be visualized through reaction
with a horseradish peroxidase-conjugated goat anti-mouse antibody or goat anti-rabbit
antibody.
[0106]
Pharmaceutical composition>
A target of the pharmaceutical composition for treatment and/or prevention of
pancreatic cancer of the present invention is not particularly limited as long as the target is
pancreatic cancer (cells) expressing a CAPRIN-1 gene.
[0107]

The terms "tumor" and "cancer" used herein mean malignant neoplasm and are used
interchangeably with each other.
[0108]
Pancreatic cancer targeted in the present invention is pancreatic cancer expressing a
gene encoding a polypeptide comprising an amino acid sequence of any of even-numbered
SEQ ID NOs: 2 to 30 or a partial sequence thereof consisting of 7 to 12 or more consecutive
amino acids.
[0109]
Examples of the pancreatic cancer include, but not limited to, pancreatic ductal
carcinoma, invasive pancreatic ductal carcinoma, pancreatic adenocarcinoma, acinar cell
carcinoma, adenosquamous carcinoma, giant cell tumor, intraductal papillary-mucinous
neoplasm (IPMN), mucinous cystic neoplasm (MCN), pancreatoblastoma, serous
cystadenocarcinoma, solid-pseudopapillary tumor (SPT), gastrinomas (Zollinger-Ellison
syndrome), glucagonomas, insulinoma, multiple endocrine neoplasia type-1 (MEN1)
(Wermer's syndrome), nonfunctional islet cell tumor, somatostatinomas, and VIPomas.
[0110]
The recipient animals are mammals, for example, mammals including primates, pet
animals, livestock, and sport animals and are particularly preferably humans, dogs, and cats.
[0111]
In the case of using the antibody of the present invention as a pharmaceutical
composition, the pharmaceutical composition can be formulated by a method generally
known to those skilled in the art. For example, the pharmaceutical composition can be used
in the form of a parenteral injection of an aseptic solution or suspension with water or any
other pharmaceutically acceptable liquid. For example, the pharmaceutical composition may
be formulated with the antibody mixed in a unit dosage form required for generally accepted
pharmaceutical practice, in appropriate combination with pharmacologically acceptable
carriers or media, specifically, sterilized water, physiological saline, plant oil, an emulsifier, a
suspending agent, a surfactant, a stabilizer, a flavoring agent, an excipient, a vehicle, a
preservative, a binder, etc. The amount of the active ingredient in such a preparation is
determined such that an appropriate dose within the prescribed range can be achieved.
[0112]
An aseptic composition for injection can be formulated according to conventional
pharmaceutical practice using a vehicle such as injectable distilled water.
[0113]

Examples of aqueous solutions for injection include physiological saline, isotonic
solutions containing glucose and other adjuvants, for example, D-sorbitol, D-mannose, D-
mannitol, and sodium chloride. These solutions may be used in combination with an
appropriate solubilizer, for example, an alcohol (specifically, ethanol) or a polyalcohol (e.g.,
propylene glycol and polyethylene glycol), or a nonionic surfactant, for example, polysorbate
80 (TM) or HCO-60.
[0114]
Examples of oily solutions include sesame oil and soybean oil. These solutions may
be used in combination with a solubilizer such as benzyl benzoate or benzyl alcohol. The
solutions may be further mixed with a buffer (e.g., a phosphate buffer solution and a sodium
acetate buffer solution), a soothing agent (e.g., procaine hydrochloride), a stabilizer (e.g.,
benzyl alcohol and phenol), or an antioxidant. The injection solutions thus prepared are
usually charged into appropriate ampules.
[0115]
The pharmaceutical composition of the present invention is administered orally or
parenterally, preferably parenterally. Specific examples of its dosage forms include
injections, intranasal administration agents, transpulmonary administration agents, and
percutaneous administration agents. Examples of the injections include intravenous
injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection,
through which the pharmaceutical composition can be administered systemically or locally.
[0116]
Also, the administration method can be appropriately selected depending on the age,
weight, sex, symptoms, etc. of a patient. The dose of a pharmaceutical composition
containing the antibody or a polynucleotide encoding the antibody can be selected within a
range of, for example, 0.0001 to 1000 mg/kg of body weight per dose. Alternatively, the
dose can be selected within a range of, for example, 0.001 to 100000 mg/body of a patient,
though the dose is not necessarily limited to these numeric values. Although the dose and
the administration method vary depending on the weight, age, sex, symptoms, etc. of a patient,
those skilled in the art can appropriately select the dose and the method.
[0117]
The pharmaceutical composition of the present invention can be administered to a
subject to treat and/or prevent pancreatic cancer.
[0118]
The present invention further encompasses a method for treating and/or preventing
pancreatic cancer, comprising administering the pharmaceutical composition of the present

invention in combination with the antitumor agent as exemplified above or a pharmaceutical
composition comprising the antitumor agent to a subject. The antibody of the present
invention or the fragment thereof may be administered simultaneously with or separately from
the antitumor agent to the subject. In the case of separately administering these
pharmaceutical compositions, either one may be administered first or later. Their dosing
intervals, doses, administration routes, and the number of doses can be appropriately selected
by a specialist. The dosage forms of separate drugs to be administered simultaneously also
include, for example, pharmaceutical compositions each formulated by mixing the antibody
of the present invention or fragment thereof and the antitumor agent in a pharmacologically
acceptable carrier (or medium). The above descriptions about prescription, formulation,
administration routes, doses, cancer, etc. as to the pharmaceutical compositions and dosage
forms containing the antibody of the present invention are also applicable to any of the above-
described pharmaceutical compositions and dosage forms containing the antitumor agent.
Thus, the present invention also provides a pharmaceutical combination (also referred to as a
"pharmaceutical kit") for treatment and/or prevention of pancreatic cancer, comprising the
pharmaceutical composition of the present invention and a pharmaceutical composition
comprising the antitumor agent as exemplified above.
[0119]
The present invention also provides a pharmaceutical composition for treatment and/or
prevention of pancreatic cancer, comprising the antibody of the present invention or fragment
thereof and the antitumor agent together with a pharmacologically acceptable carrier.
[0120]
Alternatively, the antitumor agent may be conjugated with the antibody of the present
invention or fragment thereof. The resulting conjugate can be mixed with a
pharmacologically acceptable carrier (or medium) as described above and formulated into a
pharmaceutical composition.
Examples
[0121]
Hereinafter, the present invention will be described specifically with reference to
Examples. However, the scope of the present invention is not intended to be limited by
these specific examples.
[0122]
[Example 1] Identification of pancreatic cancer antigenic protein by SEREX method
(1) Preparation of cDNA library

Total RNAs were extracted from the testicular tissue of a healthy dog by the acid
guanidium-phenol-chloroform method. Poly-A RNAs were purified using Oligotex-dT30
mRNA purification Kit (manufactured by Takara Shuzo Co., Ltd.) according to the protocol
attached to the kit.
[0123]
The mRNAs (5 fag) thus obtained were used to synthesize a canine testicular cDNA
phage library. The cDNA phage library was prepared using cDNA Synthesis Kit, ZAP-
cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by
Stratagene Corp.) according to the protocols attached to the kits. The prepared cDNA phage
library had a size of 7.73 x 105 pfu/ml.
[0124]
(2) Screening of cDNA library using serum
The canine testicular cDNA phage library thus prepared was used in immunoscreening.
Specifically, host E. coli (XL 1-Blue MRF) was infected by the phages on a NZY agarose
plate (<|)90 x 15 mm) so as to give 2210 clones. The host was cultured at 42°C for 3 to 4
hours to form plaques. The plate was covered at 37°C for 4 hours with a nitrocellulose
membrane (Hybond C Extra: manufactured by GE Healthcare Bio-Sciences Ltd.) infiltrated
with IPTG (isopropyl-P-D- thiogalactopyranoside) for protein induction and expression to
transfer the proteins to the membrane. Then, the membrane was recovered, dipped in TBS
(10 mM tris-HCl, 150 mM NaCl, pH 7.5) containing 0.5% skimmed milk, and shaken
overnight at 4°C to suppress nonspecific reaction. This filter was reacted with 500-fold
diluted serum of an affected dog at room temperature for 2 to 3 hours.
[0125]
The above serum of an affected dog used was serum collected from a breast cancer-
affected dog. The serum was stored at -80°C and pretreated immediately before use. The
serum pretreatment was performed by the following method: host E. coli (XL 1-Blue MRF')
was infected by X ZAP Express phages having no foreign gene insert and then cultured
overnight on a NZY plate medium at 37°C. Subsequently, a 0.2 M NaHC03 (pH 8.3) buffer
containing 0.5 M NaCl was added to the plate. The plate was left standing at 4°C for 15
hours. Then, the supernatant was recovered as an E. co/j/phage extract. Next, the
recovered E. co/z'/phage extract was applied to a NHS-column (manufactured by GE
Healthcare Bio-Sciences Ltd.) to immobilize E. co/z'/phage-derived proteins thereon. The
serum of an affected dog was applied to this protein-immobilized column and reacted
therewith. Antibodies adsorbed on the E. coli and the phages were removed from the serum.

A serum fraction that had passed through the column was diluted 500-fold with TBS
containing 0.5% skimmed milk. This dilution was used as an immunoscreening material.
[0126]
The membrane blotted with the serum thus treated and the fusion proteins was washed
four times with TBS-T (0.05% Tween 20/TBS) and then reacted at room temperature for 1
hour with secondary antibodies goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated;
manufactured by BETHYL Laboratories, Inc.) diluted 5000-fold with TBS containing 0.5%
skimmed milk, followed by detection through enzymatic color reaction using NBT/BCIP
Stock Solution (manufactured by Roche Diagnostics K.K.). Colonies that matched with
color reaction-positive sites were collected from the NZY agarose plate (<|)90 x 15 mm) and
lysed in 500 ul of an SM buffer solution (100 mM NaCl, 10 mM MgClS04, 50 mM tris-HCl,
0.01% gelatin, pH 7.5). Secondary screening and tertiary screening were subsequently
performed in the same way as above until single color reaction-positive colonies were
obtained. In this way, 30940 phage clones reactive with IgG in the serum were screened for.
Then, 5 positive clones were isolated.
[0127]
(3) Homology search for isolated antigen gene
In order to subject these five positive clones thus isolated to nucleotide sequence
analysis, procedures of converting the phage vectors to plasmid vectors were performed.
Specifically, 200 ul of a host E. coli (XL 1-Blue MRF) solution prepared such that absorbance
OD600 became 1.0 was mixed with 250 ul of a purified phage solution and further with 1 ul
of ExAssist helper phage (manufactured by Stratagene Corp.), followed by reaction at 37°C
for 15 minutes. Then, 3 ml of an LB medium was added to the reaction mixture. The host
was cultured at 37°C for 2.5 to 3 hours, immediately thereafter incubated for 20 minutes in a
water bath of 70°C, and then centrifuged at 1000 x g at 4°C for 15 minutes to recover the
supernatant as a phagemid solution. Subsequently, 200 ul of a phagemid host E. coli
(SOLR) solution prepared such that absorbance OD600 became 1.0 was mixed with 10 ul of a
purified phage solution, followed by reaction at 37°C for 15 minutes. 50 ul of the reaction
mixture was inoculated to an LB agar medium containing ampicillin (final concentration: 50
ug/ml) and cultured overnight at 37°C. A single colony of transformed SOLR was collected
and cultured at 37°C in an LB medium containing ampicillin (final concentration: 50 |J.g/ml).
Then, plasmid DNAs having the inserts of interest were purified using QIAGEN plasmid
Miniprep Kit (manufactured by Qiagen N.V.).
[0128]

The full-length sequences of the inserts in the purified plasmids were analyzed by the
primer walking method using a T3 primer represented by SEQ ID NO: 31 and a T7 primer
represented by SEQ ID NO: 32. This sequencing analysis yielded gene sequences
represented by SEQ ID NOs: 5, 7, 9, 11, and 13. As a result of conducting homology search
with known genes using the nucleotide sequences of these genes and amino acid sequences
(SEQ ID NOs: 6, 8, 10, 12, and 14) encoded thereby and the homology search program
BLAST Search (http://www.ncbi.nlm.nih.gov/BLAST/), the obtained five genes were all
found to be genes encoding CAPRJN-1. The sequence identity among these five genes was
100% for their nucleotide sequences in regions to be translated into proteins and 99% for their
amino acid sequences. The sequence identity of these genes to genes encoding human
homologous factors was 94% for their nucleotide sequences in regions to be translated into
proteins and 98% for their amino acid sequences. The nucleotide sequences of the human
homologous factors are represented by SEQ ID NOs: 1 and 3, and their amino acid sequences
are represented by SEQ ID NOs: 2 and 4. Also, the sequence identity of the obtained canine
genes to a gene encoding a bovine homologous factor was 94% for their nucleotide sequences
in regions to be translated into proteins and 97% for their amino acid sequences. The
nucleotide sequence of the bovine homologous factor is represented by SEQ ID NO: 15, and
its amino acid sequence is represented by SEQ ID NO: 16. In this context, the sequence
identity between the genes encoding the human homologous factors and the gene encoding
the bovine homologous factor was 94% for their nucleotide sequences in regions to be
translated into proteins and 93 to 97% for their amino acid sequences. The sequence identity
of the obtained canine genes to a gene encoding a horse homologous factor was 93% for their
nucleotide sequences in regions to be translated into proteins and 97% for their amino acid
sequences. The nucleotide sequence of the horse homologous factor is represented by SEQ
ID NO: 17, and its amino acid sequence is represented by SEQ ID NO: 18. In this context,
the sequence identity between the genes encoding the human homologous factors and the
gene encoding the horse homologous factor was 93% for their nucleotide sequences in
regions to be translated into proteins and 96% for their amino acid sequences. The sequence
identity of the obtained canine genes to genes encoding mouse homologous factors was 87 to
89% for their nucleotide sequences in regions to be translated into proteins and 95 to 97% for
their amino acid sequences. The nucleotide sequences of the mouse homologous factors are
represented by SEQ ID NOs: 19, 21, 23, 25, and 27, and their amino acid sequences are
represented by SEQ ID NOs: 20, 22, 24, 26, and 28. In this context, the sequence identity
between the genes encoding the human homologous factors and the genes encoding the
mouse homologous factors was 89 to 91% for their nucleotide sequences in regions to be

translated into proteins and 95 to 96% for their amino acid sequences. The sequence identity
of the obtained canine genes to a gene encoding a chicken homologous factor was 82% for
their nucleotide sequences in regions to be translated into proteins and 87% for their amino
acid sequences. The nucleotide sequence of the chicken homologous factor is represented
by SEQ ID NO: 29, and its amino acid sequence is represented by SEQ ID NO: 30. In this
context, the sequence identity between the genes encoding the human homologous factors and
the gene encoding the chicken homologous factor was 81 to 82% for their nucleotide
sequences in regions to be translated into proteins and 86% for their amino acid sequences.
[0129]
(4) Analysis of CAPRIN-1 gene expression in human pancreatic cancer cell
The genes thus obtained were examined for their expression in four different human
pancreatic cancer cell lines (Capan-2, MIAPaCa-2, PANC-1, and BxPC-3) by RT-PCR.
Reverse transcription reaction was performed as follows: total RNAs were extracted from 50
to 100 mg of each tissue and 5 to 10 x 106 cells of each cell line using TRIZOL reagent
(manufactured by Invitrogen Corp.) according to the protocol attached thereto. From the
total RNAs, cDNAs were synthesized using Superscript First-Strand Synthesis System for
RT-PCR (manufactured by Invitrogen Corp.) according to the protocol attached thereto.
PCR reaction was performed as follows using primers (SEQ ID NOs: 33 and 34) specific for
the obtained genes: PCR was performed by 30 repetitive cycles each involving 94°C for 30
seconds, 60°C for 30 seconds, and 72°C for 30 seconds using Thermal Cycler (manufactured
by Bio-Rad Laboratories, Inc.) and a reaction solution with the total amount adjusted to 25 ul
by the addition of 0.25 ul of the sample prepared by the reverse transcription reaction and
each reagent and attached buffer (2 uM each of the primers, 0.2 mM each of dNTPs, and 0.65
U of ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.)). The above gene-
specific primers were designed to amplify a region of nucleotide numbers 698 to 1124 in the
nucleotide sequence (human CAPRIN-1 gene) of SEQ ID NO: 1. GAPDH-specific primers
(SEQ ID NOs: 35 and 36) were also used for a comparative control. As a result, the gene
expression was confirmed in all the human pancreatic cancer cell lines.
[0130]
[Example 2] Preparation of polyclonal antibody against human CAPRIN-1
1 mg of human CAPRIN-1 recombinant proteins prepared according to Example 3 of
WO2010/016526 was mixed with an equal volume of an incomplete Freund's adjuvant (IFA)
solution. This mixture was subcutaneously administered to each rabbit four times every two
weeks. Then, blood was collected to obtain antiserum containing polyclonal antibodies.
This antiserum was further purified using a protein G carrier (manufactured by GE Healthcare

Bio-Sciences Ltd.) to obtain polyclonal antibodies against CAPRIN-1. In addition, the
serum of a rabbit that received no antigen was purified using a protein G carrier in the same
way as above and used as control antibodies.
[0131]
[Example 3] Analysis of CAPRTN-1 protein expression in human pancreatic cancer
(1) Analysis of CAPRTN-1 protein expression on human pancreatic cancer cell
The four human pancreatic cancer cell lines (Capan-2, MIAPaCa-2, PANC-1, and
BxPC-3) confirmed to have CAPRIN-1 gene expression were examined for their expression
of CAPRTN-1 proteins on the cell surface. 106 cells of each human pancreatic cancer cell
line thus confirmed to have gene expression were centrifuged in a 1.5-ml microcentrifuge
tube. 2 ug (5 ul) of the anti-CAPRTN-1 polyclonal antibodies prepared in Example 2 was
added thereto. The mixture was suspended in PBS containing 95 ul of 0.1% fetal bovine
serum and then left standing for 1 hour on ice. After washing with PBS, the resulting
suspension was suspended in PBS containing 5 ul of FITC-labeled goat anti-rabbit IgG
antibodies (manufactured by Santa Cruz Biotechnology, Inc.) and 95 ul of 0.1% fetal bovine
serum (FBS) and left standing for 1 hour on ice. After washing with PBS, the fluorescence
intensity was measured using FACSCalibur (Becton, Dickinson and Company). On the
other hand, the same operation as above was performed using the control antibodies prepared
in Example 2 instead of the polyclonal antibodies against CAPRTN-1 to prepare a control.
As a result, the pancreatic cancer cells supplemented with the anti-human CAPRTN-1
polyclonal antibodies all exhibited fluorescence intensity at least 20% stronger than that of the
control. This demonstrated that CAPRTN-1 proteins are expressed on the cell membrane
surface of the human pancreatic cancer cell lines. The above rate of enhancement in
fluorescence intensity was indicated by the rate of increase in mean fluorescence intensity
(MFI) in each cell line and calculated according to the following expression:
Rate of increase in mean fluorescence intensity (Rate of enhancement in fluorescence
intensity) (%) = ((MFI of cells reacted with the anti-human CAPRTN-1 antibody) - (Control
MFI)) / (Control MFI) x 100
[0132]
(2) Expression of CAPRTN-1 protein in human pancreatic cancer tissue
40 pancreatic cancer tissue samples of a paraffin-embedded human pancreatic cancer
tissue array (manufactured by US Biomax, Inc.) were used in immunohistochemical staining.
The human pancreatic cancer tissue array was treated at 60°C for 3 hours and then placed in a
staining bottle filled with xylene, and procedures of replacing xylene with a fresh one every 5
minutes were performed three times. Next, similar operation was performed using ethanol

and PBS-T instead of xylene. The human pancreatic cancer tissue array was placed in a
staining bottle filled with a 10 mM citrate buffer solution (pH 6.0) containing 0.05% Tween
20, treated at 125°C for 5 minutes, and then left standing at room temperature for 40 minutes
or longer. Redundant water around each section was wiped off with Kimwipe. The section
on a glass slide was encircled with a Dako pen, and an appropriate amount of Peroxidase
Block (manufactured by Dako Japan Inc.) was added dropwise thereto. The glass slide was
left standing at room temperature for 5 minutes and then placed in a staining bottle filled with
PBS-T, and procedures of replacing PBS-T with a fresh one every 5 minutes were performed
three times. A PBS-T solution containing 10% FBS was applied thereto as a blocking
solution, and the glass slide was left standing at room temperature for 1 hour in a moist
chamber. The anti-CAPRJN-1 polyclonal antibodies prepared in Example 2 were prepared
into 10 [ig/ml solution with a PBS-T solution containing 5% FBS, and this solution was
applied thereto. The glass slide was left standing overnight at 4°C in a moist chamber and
washed with PBS-T for 10 minutes three times. Then, an appropriate amount of Peroxidase
Labelled Polymer Conjugated (manufactured by Dako Japan Inc.) was added dropwise thereto,
and the glass slide was left standing at room temperature for 30 minutes in a moist chamber.
After washing with PBS-T for 10 minutes three times, a DAB staining solution (manufactured
by Dako Japan Inc.) was applied thereto, and the glass slide was left standing at room
temperature for approximately 10 minutes. Then, the staining solution was discarded, and
the glass slide was washed with PBS-T for 10 minutes three times, then rinsed with distilled
water, placed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in this order for 1 minute
per solution, and then left standing overnight in xylene. The glass slide was taken out and
enclosed in Glycergel Mounting Medium (manufactured by Dako Japan Inc.), followed by
observation. As a result, the strong expression of CAPRJN-1 was confirmed in 36 (90%) out
of a total of 40 pancreatic cancer tissue samples.
[0133]
[Example 4] Antitumor effect (ADCC activity) of anti-CAPRIN-1 polyclonal antibody
on pancreatic cancer cell
The antibody against CAPRIN-1 was studied for its ability to damage pancreatic
cancer cells expressing CAPRJN-1. The polyclonal antibodies against human CAPRIN-1
obtained in Example 2 were used in this evaluation. 106 cells each of the human pancreatic
cancer cell lines Capan-2 and MIAPaCa-2 confirmed to have CAPRIN-1 expression were
collected into a 50-ml centrifuge tube, to which 100 uCi of chromium 51 was then added,
followed by incubation at 37°C for 2 hours. Then, the cells were washed three times with an
RPMI1640 medium containing 10% fetal calf serum and added at a density of 10 cells/well

to a 96-well V-bottom plate. The polyclonal antibodies against human CAPRIN-1 were
added thereto at a concentration of 1 |ag/well. Lymphocytes separated from human
peripheral blood were further added thereto at a density of 2 x 105 cells/well and cultured at
37°C for 4 hours under conditions of 5% CO2. After the culture, the amount of chromium
(Cr) 51 released from damaged tumor cells was measured in the culture supernatant to
calculate the ADCC activity of the anti-human CAPRIN-1 polyclonal antibodies against each
pancreatic cancer cell line. As a result, the addition of the polyclonal antibodies against
human CAPRIN-1 was confirmed to produce ADCC activity of 14% and 11% against Capan-
2 and MIAPaCa-2, respectively, whereas similar operations produced ADCC activity less
than 0.7% against both Capan-2 and MIAPaCa-2 using control antibodies prepared from
antigen-unimmunized rabbit peripheral blood and produced ADCC activity less than 0.5%>
even in the absence of antibodies. These results demonstrated that the antibody against
CAPRIN-1 can damage CAPRIN-1-expressing pancreatic cancer cells through its ADCC
activity. These results about cytotoxic activity were obtained by: mixing the antibody
against CAPRIN-1 used in the present invention, lymphocytes, and 103 tumor cells with
incorporated chromium 51, as described above: culturing the cells for 4 hours; after the
culture, measuring the amount of chromium 51 released into the medium; and calculating the
cytotoxic activity against the tumor cells according to the following equation for calculation :
*
Equation: Cytotoxic activity (%>) = Amount of chromium 51 released from the tumor
cells supplemented with the antibody against CAPRIN-1 and lymphocytes / Amount of
chromium 51 released from tumor cells supplemented with 1 N hydrochloric acid x 100.
[0134]
[Example 5] Preparation of mouse and chicken monoclonal antibodies against
CAPRIN-1
100 \ig of the human CAPRIN-1 recombinant proteins prepared in Example 2 was
mixed with an equal amount of MPL+TDM adjuvant (manufactured by Sigma-Aldrich Corp.).
This mixture was used as an antigen solution per mouse. The antigen solution was
intraperitoneally administered to each 6-week-old Balb/c mouse (manufactured by Japan SLC,
Inc.). Then, 3 and 24 boosters were performed every 1 week to complete immunization.
Three days after the final shot, the spleen of each mouse was excised and ground between two
sterilized glass slides. Procedures of washing with PBS(-) (manufactured by Nissui
Pharmaceutical Co., Ltd.) and removing the supernatant by centrifugation at 1500 rpm for 10
minutes were repeated three times to obtain spleen cells. The obtained spleen cells were
mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 10:1. 200 ul
of an RPMI1640 medium containing 10% FBS was heated to 37°C and mixed with 800 ul of

PEG1500 (manufactured by Boehringer Ingelheim GmbH), and the PEG solution thus
prepared was added to the cell mixture, which was then left standing for 5 minutes for cell
fusion. After removal of the supernatant by centrifugation at 1700 rpm for 5 minutes, the
cells were suspended in 150 ml of an RPMI1640 medium containing 15% FBS supplemented
with 2% equivalent of a HAT solution (manufactured by Life Technologies, Inc./Gibco)
(HAT selective medium). This suspension was inoculated to fifteen 96-well plates
(manufactured by Thermo Fisher Scientific Inc./Nunc) at a concentration of 100 ul/well.
The spleen cells and the myeloma cells were fused by culture at 37°C for 7 days under
conditions of 5% CO2 to obtain hybridomas.
[0135]
The prepared hybridomas were screened with the binding affinity of antibodies
produced by the hybridomas against CAPRIN-1 proteins as an indicator. A 1 |ag/ml solution
of the CAPRTN-1 proteins prepared in Example 2 was added to a 96-well plate at a
concentration of 100 ul/well and left standing at 4°C for 18 hours. Each well was washed
three times with PBS-T. Then, a 0.5% bovine serum albumin (BSA) solution (manufactured
by Sigma-Aldrich Corp.) was added thereto at a concentration of 400 ul/well and left standing
at room temperature for 3 hours. The solution in each well was discarded, and each well
was washed three times with 400 ul of PBS-T. Then, the culture supernatant of each
hybridoma obtained above was added to the well at a concentration of 100 ul/well and left
standing at room temperature for 2 hours. Each well was washed three times with PBS-T.
Then, HRP-labeled anti-mouse IgG (H+L) antibodies (manufactured by Invitrogen Corp.)
diluted 5000-fold with PBS were added to the well at a concentration of 100 ul/well and left
standing at room temperature for 1 hour. Each well was washed three times with PBS-T.
Then, a TMB substrate solution (manufactured by Thermo Fisher Scientific Inc.) was added
thereto at a concentration of 100 ul/well and left standing for 15 to 30 minutes to cause color
reaction. After the color development, the reaction was terminated by the addition of 1 N
sulfuric acid at a concentration of 100 ul/well. The absorbance was measured at 450 nm and
595 nm using an absorption spectrometer. As a result, several hybridomas producing
antibodies having high absorbance were selected.
[0136]
The selected hybridomas were added to a 96-well plate at a density of 0.5 cells/well
and cultured in the plate. One week later, hybridomas forming single colonies in the wells
were observed. The cells in these wells were further cultured, and the cloned hybridomas
were screened with the binding affinity of antibodies produced by the hybridomas against
CAPRIN-1 proteins as an indicator. A 1 |J.g/ml solution of the CAPPJN-1 proteins prepared

in Example 2 was added to a 96-well plate at a concentration of 100 ul/well and left standing
at 4°C for 18 hours. Each well was washed three times with PBS-T. Then, a 0.5% BSA
solution was added to the well at a concentration of 400 ul/well and left standing at room
temperature for 3 hours. The solution in each well was discarded, and each well was washed
three times with 400 ul of PBS-T. Then, the culture supernatant of each hybridoma obtained
above was added to the well at a concentration of 100 ul/well and left standing at room
temperature for 2 hours. Each well was washed three times with PBS-T. Then, HRP-
labeled anti-mouse IgG (H+L) antibodies (manufactured by Invitrogen Corp.) diluted 5000-
fold with PBS were added to the well at a concentration of 100 ul/well and left standing at
room temperature for 1 hour. Each well was washed three times with PBS-T. Then, a
TMB substrate solution (manufactured by Thermo Fisher Scientific Inc.) was added to the
well at a concentration of 100 ul/well and left standing for 15 to 30 minutes to cause color
reaction. After the color development, the reaction was terminated by the addition of 1 N
sulfuric acid at a concentration of 100 ul/well. The absorbance was measured at 450 nm and
595 nm using an absorption spectrometer. As a result, 150 hybridoma lines producing
mouse monoclonal antibodies reactive with CAPRIN-1 proteins were obtained.
[0137]
Next, these mouse monoclonal antibodies were screened for antibodies reactive with
the surface of cancer cells expressing CAPRTN-1. Specifically, 106 cells of a human breast
cancer cell line MDA-MB-231V were centrifuged in a 1.5-ml microcentrifuge tube. 100 ul
of the culture supernatant of each hybridoma obtained above was added thereto and left
standing for 1 hour on ice. After washing with PBS, FITC-labeled goat anti-mouse IgG
antibodies (manufactured by Invitrogen Corp.) diluted 500-fold with PBS containing 0.1%
FBS were added thereto and left standing for 1 hour on ice. After washing with PBS, the
fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company).
On the other hand, the same operation as above was performed using the serum of each
untreated 6-week-old Balb/c mouse diluted 500-fold with a medium for hybridoma culture,
instead of the antibodies, to prepare a control. As a result, 22 mouse monoclonal antibodies
(mouse monoclonal antibodies #1 to #22) having stronger fluorescence intensity than that of
the control, i.e., reactive with the surface of breast cancer cells, were selected.
[0138]
In order to prepare chicken monoclonal antibodies, 300 \xg of the antigenic proteins
(human CAPRTN-1) (SEQ ID NO: 2) prepared in Example 2 was mixed with an equal amount
of a complete Freund's adjuvant. This mixture was used as an antigen solution per chicken.
The antigen solution was intraperitoneally administered to each 7-week-old chicken. Then,

7 boosters were performed every 4 weeks to complete immunization. Four days after the
final shot, the spleen of each chicken was excised and ground between two sterilized glass
slides. Procedures of washing with PBS(-) (manufactured by Nissui Pharmaceutical Co.,
Ltd.) and removing the supernatant by centrifugation at 1500 rpm for 10 minutes were
repeated three times to obtain spleen cells. The obtained spleen cells were mixed with light
chain-deficient chicken myeloma cells established from chickens by transformation using
avian reticuloendotheliosis virus, at a ratio of 5:1. 200 ul of an IMDM medium containing
10% FBS was heated to 37°C and mixed with 800 ul of PEG1500 (manufactured by
Boehringer Ingelheim GmbH), and the PEG solution thus prepared was added to the cell
mixture, which was then left standing for 5 minutes for cell fusion. After removal of the
supernatant by centrifugation at 1700 rpm for 5 minutes, the cells were suspended in 300 ml
of an IMDM medium containing 10% FBS supplemented with 2% equivalent of a HAT
solution (manufactured by Life Technologies, Inc./Gibco) (HAT selective medium). This
suspension was inoculated to thirty 96-well plates (manufactured by Thermo Fisher Scientific
Inc./Nunc) at a concentration of 100 ul/well. The spleen cells and the chicken myeloma
cells were fused by culture at 37°C for 7 days under conditions of 5% CO2 to obtain
hybridomas.
[0139]
The prepared hybridomas were screened with the binding affinity of antibodies
produced by the hybridomas against CAPPJN-1 proteins as an indicator. A 1 (j.g/ml solution
of the CAPRIN-1 proteins prepared in Example 2 was added to a 96-well plate at a
concentration of 100 ul/well and left standing at 4°C for 18 hours. Each well was washed
three times with PBS-T. Then, a 0.5% bovine serum albumin (BSA) solution (manufactured
by Sigma-Aldrich Corp.) was added thereto at a concentration of 400 ul/well and left standing
at room temperature for 3 hours. The solution in each well was discarded, and each well
was washed three times with 400 ul of PBS-T. Then, the culture supernatant of each
hybridoma obtained above was added thereto at a concentration of 100 ul/well and left
standing at room temperature for 2 hours. Each well was washed three times with PBS-T.
Then, HRP-labeled anti-chicken IgY antibodies (manufactured by Sigma-Aldrich Corp.)
diluted 5000-fold with PBS were added to the well at a concentration of 100 ul/well and left
standing at room temperature for 1 hour. Each well was washed three times with PBS-T.
Then, a TMB substrate solution (manufactured by Thermo Fisher Scientific Inc.) was added
to the well at a concentration of 100 ul/well and left standing for 15 to 30 minutes to cause
color reaction. After the color development, the reaction was terminated by the addition of 1
N sulfuric acid at a concentration of 100 ul/well. The absorbance was measured at 450 nm

and 595 nm using an absorption spectrometer. As a result, several hybridomas producing
antibodies having high absorbance were selected.
[0140]
The selected hybridomas were added to a 96-well plate at a density of 0.5 cells/well
and cultured in the plate. One week later, hybridomas forming single colonies in the wells
were observed. The cells in these wells were further cultured, and the cloned hybridomas
were screened with the binding affinity of antibodies produced by the hybridomas against
CAPRTN-1 proteins as an indicator. A 1 ug/ml solution of the human CAPRJN-1 proteins
was added to a 96-well plate at a concentration of 100 ul/well and left standing at 4°C for 18
hours. Each well was washed three times with PBS-T. Then, a 0.5% BSA solution was
added to the well at a concentration of 400 ul/well and left standing at room temperature for 3
hours. The solution in each well was discarded, and each well was washed three times with
400 ul of PBS-T. Then, the culture supernatant of each hybridoma obtained above was
added thereto at a concentration of 100 ul/well and left standing at room temperature for 2
hours. Each well was washed three times with PBS-T. Then, HRP-labeled anti-chicken
IgY antibodies (manufactured by Sigma-Aldrich Corp.) diluted 5000-fold with PBS were
added to the well at a concentration of 100 ul/well and left standing at room temperature for 1
hour. Each well was washed three times with PBS-T. Then, a TMB substrate solution
(manufactured by Thermo Fisher Scientific Inc.) was added to the well at a concentration of
100 ul/well and left standing for 15 to 30 minutes to cause color reaction. After the color
development, the reaction was terminated by the addition of 1 N sulfuric acid at a
concentration of 100 ul/well. The absorbance was measured at 450 nm and 595 nm using an
absorption spectrometer. As a result, several hybridoma lines producing monoclonal
antibodies reactive with CAPRIN-1 proteins were obtained.
[0141]
Next, these monoclonal antibodies were screened for antibodies reactive with the
surface of cancer cells expressing CAPRTN-1. Specifically, 5 x 105 cells of a human breast
cancer cell line MDA-MB-231V were centrifuged in a 1.5-ml microcentrifuge tube. 100 ul
of the culture supernatant of each hybridoma obtained above was added thereto and left
standing for 1 hour on ice. After washing with PBS, FITC-labeled goat anti-chicken IgG
(H+L) antibodies (manufactured by SouthernBiotech) diluted 30-fold with PBS containing
0.1% FBS were added thereto and left standing for 1 hour on ice. After washing with PBS,
the fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and
Company). On the other hand, the same operation as above was performed using a medium
for hybridoma culture to prepare a control sample. As a result, 3 monoclonal antibodies

(chicken monoclonal antibodies #1, #2, and #3) having stronger fluorescence intensity than
that of the control, i.e., reactive with the surface of breast cancer cells expressing CAPRIN-1,
were selected.
[0142]
[Example 6] Characterization of selected antibody
(1) Cloning of variable region gene of anti-CAPRIN-1 monoclonal antibody
mRNAs were extracted from the hybridoma line producing each of the 22 mouse
monoclonal antibodies and 3 chicken monoclonal antibodies selected in Example 5. Genes
of heavy chain variable (VH) and light chain variable (VL) regions of all the anti-CAPRIN-1
monoclonal antibodies were obtained by RT-PCR using primers specific for mouse FR1- and
FR4-derived sequences for the mouse monoclonal antibody-producing hybridomas and
primers specific for chicken FR1- and FR4-derived sequences for the chicken monoclonal
antibody-producing hybridomas. For sequencing, these genes were cloned into pCR2.1
vectors (manufactured by Invitrogen Corp.).
[0143]
(1)-1 RT-PCR
mRNAs were prepared from 106 cells of each mouse monoclonal antibody-producing
hybridoma line using mRNA micro purification kit (manufactured by GE Healthcare Bio-
Sciences Ltd.) and reverse-transcribed using Superscript II 1st strand synthesis kit
(manufactured by Invitrogen Corp.) to synthesis cDNAs. These procedures were performed
according to the protocol attached to each kit. The antibody genes were amplified by PCR
using the obtained cDNAs. A mouse heavy chain FR1 sequence-specific primer (SEQ ID
NO: 257) and a mouse heavy chain FR4 sequence-specific primer (SEQ ID NO: 258) were
used for obtaining the VH region genes. Also, a mouse light chain FR1 sequence-specific
primer (SEQ ID NO: 259) and a mouse light chain FR4-specific primer (SEQ ID NO: 260)
were used for obtaining the VL region genes. These primers were designed with reference
to Jones, S.T. and Bending, M.M. Bio/Technology 9, 88-89 (1991). PCR employed Ex Taq
(manufactured by Takara Bio Inc.). The cDNA sample was added to 5 ul of 10 x EX Taq
Buffer, 4 ul of dNTP Mixture (2.5 mM), 2 ul each of the primers (1.0 uM), and 0.25 ul of Ex
Taq (5 U/ul), and the total amount of the solution was adjusted to 50 ul with sterilized water.
After treatment at 94°C for 2 minutes, PCR was performed under conditions of 30 cycles each
involving 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.
[0144]

Also, total RNA was extracted from 106 cells of each chicken monoclonal antibody-
producing hybridoma line using High Pure RNA Isolation Kit (manufactured by Roche
Diagnostics K.K.). Then, cDNAs were synthesized using PrimeScript II 1st strand cDNA
Synthesis Kit (manufactured by Takara Bio Inc.). These procedures were performed
according to the protocol attached to each kit. The chicken antibody heavy and light chain
variable region genes were separately amplified by PCR according to a routine method with
the synthesized cDNAs as templates using KOD-Plus-DNA Polymerase (manufactured by
Toyobo Co., Ltd.). A chicken heavy chain FR1 sequence-specific primer and a chicken
heavy chain FR4 sequence-specific primer were used for obtaining the chicken antibody VH
region genes. Also, a chicken light chain FR1 sequence-specific primer and a chicken light
chain FR4-specific primer were used for obtaining the VL region genes.
[0145]
(l)-2 Cloning
Each PCR product obtained above was electrophoresed on an agarose gel. DNA
bands excised for each of the VH and VL regions. Each DNA fragment was purified using
QIAquick Gel purification kit (manufactured by Qiagen N.V.) according to the protocol
attached thereto. Each DNA thus purified was cloned into pCR2.1 vector using TA cloning
kit (manufactured by Invitrogen Corp.). DH5a competent cells (manufactured by Toyobo
Co., Ltd.) were transformed with the ligated vector according to a standard method. Ten
clones of each transformant were cultured overnight at 37°C in a medium containing 100
(j,g/ml ampicillin. Then, each plasmid DNA was purified using Qiaspin Miniprep kit
(manufactured by Qiagen N.V.).
[0146]
(l)-3 Sequencing
The VH and VL region genes in each plasmid obtained above were sequenced using an
M13 forward primer (SEQ ID NO: 261) and an M13 reverse primer (SEQ ID NO: 262), a
fluorescence sequencer (DNA sequencer 3130XL manufactured by Applied Biosystems, Inc.),
and BigDye Terminator Ver 3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems,
Inc.) according to the protocols attached thereto. As a result, the sequence of each gene and
an amino acid sequence encoded thereby were determined.
[0147]
Specifically, these monoclonal antibodies each comprise a heavy chain variable (VH)
region having an amino acid sequence of SEQ ID NO: 40 (SEQ ID NO: 45), SEQ ID NO: 50
(SEQ ID NO: 55), SEQ ID NO: 60 (SEQ ID NO: 65), SEQ ID NO: 70 (SEQ ID NO: 75),
SEQ ID NO: 80 (SEQ ID NO: 85), SEQ ID NO: 90 (SEQ ID NO: 95), SEQ ID NO: 100

(SEQ ID NO: 105), SEQ ID NO: 110 (SEQ ID NO: 115), SEQ ID NO: 120 (SEQ ID NO:
125), SEQ ID NO: 130 (SEQ ID NO: 131), SEQ ID NO: 135 (SEQ ID NO: 140), SEQ ID
NO: 145 (SEQ ID NO: 150), SEQ ID NO: 160 (SEQ ID NO: 165), SEQ ID NO: 170 (SEQ ID
NO: 175), SEQ ID NO: 200 (SEQ ID NO: 205), SEQ ID NO: 210 (SEQ ID NO: 215), SEQ
ID NO: 220 (SEQ ID NO: 225), SEQ ID NO: 230 (SEQ ID NO: 235), SEQ ID NO: 240 (SEQ
ID NO: 245), or SEQ ID NO: 250 (SEQ ID NO: 255) (SEQ ID NO in the parentheses
represents a gene sequence) and a light chain variable (VL) region having an amino acid
sequence of SEQ ID NO: 44 (SEQ ID NO: 46), SEQ ID NO: 54 (SEQ ID NO: 56), SEQ ID
NO: 64 (SEQ ID NO: 66), SEQ ID NO: 74 (SEQ ID NO: 76), SEQ ID NO: 84 (SEQ ID NO:
86), SEQ ID NO: 94 (SEQ ID NO: 96), SEQ ID NO: 104 (SEQ ID NO: 106), SEQ ID NO:
114 (SEQ ID NO: 116), SEQ ID NO: 124 (SEQ ID NO: 126), SEQ ID NO: 139 (SEQ ID NO:
141), SEQ ID NO: 149 (SEQ ID NO: 151), SEQ ID NO: 155 (SEQ ID NO: 156), SEQ ID
NO: 164 (SEQ ID NO: 166), SEQ ID NO: 174 (SEQ ID NO: 176), SEQ ID NO: 180 (SEQ ID
NO: 181), SEQ ID NO: 185 (SEQ ID NO: 186), SEQ ID NO: 190 (SEQ ID NO: 191), SEQ
ID NO: 195 (SEQ ID NO: 196), SEQ ID NO: 204 (SEQ ID NO: 206), SEQ ID NO: 214 (SEQ
ID NO: 216), SEQ ID NO: 224 (SEQ ID NO: 226), SEQ ID NO: 234 (SEQ ID NO: 236),
SEQ ID NO: 244 (SEQ ID NO: 246), or SEQ ID NO: 254 (SEQ ID NO: 256) (SEQ ID NO in
the parentheses represents a gene sequence), wherein the VH region comprises CDR1
represented by an amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57,
SEQ ID NO: 67, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO: 107, SEQ
ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 132, SEQ ID NO: 142, SEQ ID NO: 157, SEQ
ID NO: 167, SEQ ID NO: 197, SEQ ID NO: 207, SEQ ID NO: 217, SEQ ID NO: 227, SEQ
ID NO: 237, or SEQ ID NO: 247, CDR2 represented by an amino acid sequence of SEQ ID
NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, SEQ ID NO: 88,
SEQ ID NO: 98, SEQ ID NO: 108, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 133,
SEQ ID NO: 143, SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO: 198, SEQ ID NO: 208,
SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 238, or SEQ ID NO: 248, and CDR3
represented by an amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59,
SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ
ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 144, SEQ ID NO: 159, SEQ
ID NO: 169, SEQ ID NO: 199, SEQ ID NO: 209, SEQ ID NO: 219, SEQ ID NO: 229, SEQ
ID NO: 239, or SEQ ID NO: 249, and the VL region comprises CDR1 represented by an
amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71,
SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 121,
SEQ ID NO: 136, SEQ ID NO: 146, SEQ ID NO: 152, SEQ ID NO: 161, SEQ ID NO: 171,

SEQ ID NO: 177, SEQ ID NO: 182, SEQ ID NO: 187, SEQ ID NO: 192, SEQ ID NO: 201,
SEQ ID NO: 211, SEQ ID NO: 221, SEQ ID NO: 231, SEQ ID NO: 241, or SEQ ID NO: 251,
CDR2 represented by an amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID
NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 102, SEQ ID NO:
112, SEQ ID NO: 122, SEQ ID NO: 137, SEQ ID NO: 147, SEQ ID NO: 153, SEQ ID NO:
162, SEQ ID NO: 172, SEQ ID NO: 178, SEQ ID NO: 183, SEQ ID NO: 188, SEQ ID NO:
193, SEQ ID NO: 202, SEQ ID NO: 212, SEQ ID NO: 222, SEQ ID NO: 232, SEQ ID NO:
242, or SEQ ID NO: 252, and CDR3 represented by an amino acid sequence of SEQ ID NO:
43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93,
SEQ ID NO: 103, SEQ ID NO: 113, SEQ ID NO: 123, SEQ ID NO: 138, SEQ ID NO: 148,
SEQ ID NO: 154, SEQ ID NO: 163, SEQ ID NO: 173, SEQ ID NO: 179, SEQ ID NO: 184,
SEQ ID NO: 189, SEQ ID NO: 194, SEQ ID NO: 203, SEQ ID NO: 213, SEQ ID NO: 223,
SEQ ID NO: 233, SEQ ID NO: 243, or SEQ ID NO: 253.
[0148]
The amino acid sequences of the heavy chain variable regions of the obtained
monoclonal antibodies are represented by SEQ ID NOs: 40, 50, SEQ ID NO: 60, SEQ ID
NO: 70, SEQ ID NO: 80, SEQ ID NO: 90, SEQ ID NO: 100, SEQ ID NO: 110, SEQ ID NO:
120, SEQ ID NO: 130, SEQ ID NO: 135, SEQ ID NO: 145, SEQ ID NO: 160, SEQ ID NO:
170, SEQ ID NO: 200, SEQ ID NO: 210, SEQ ID NO: 220, SEQ ID NO: 230, SEQ ID NO:
240, and SEQ ID NO: 250. The amino acid sequences of their light chain variable regions
are represented by SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ
ID NO: 84, SEQ ID NO: 94, SEQ ID NO: 104, SEQ ID NO: 114, SEQ ID NO: 124, SEQ ID
NO: 139, SEQ ID NO: 149, SEQ ID NO: 155, SEQ ID NO: 164, SEQ ID NO: 174, SEQ ID
NO: 180, SEQ ID NO: 185, SEQ ID NO: 190, SEQ ID NO: 195, SEQ ID NO: 204, SEQ ID
NO: 214, SEQ ID NO: 224, SEQ ID NO: 234, SEQ ID NO: 244, and SEQ ID NO: 254.
[0149]
Specifically, the mouse monoclonal antibody #1 comprises the heavy chain variable
region of SEQ ID NO: 70 and the light chain variable region of SEQ ID NO: 74; #2
comprises the heavy chain variable region of SEQ ID NO: 80 and the light chain variable
region of SEQ ID NO: 84; #3 comprises the heavy chain variable region of SEQ ID NO: 90
and the light chain variable region of SEQ ID NO: 94; #4 comprises the heavy chain variable
region of SEQ ID NO: 100 and the light chain variable region of SEQ ID NO: 104; #5
comprises the heavy chain variable region of SEQ ID NO: 110 and the light chain variable
region of SEQ ID NO: 114; #6 comprises the heavy chain variable region of SEQ ID NO: 120
and the light chain variable region of SEQ ID NO: 124; #7 comprises the heavy chain variable

region of SEQ ID NO: 130 and the light chain variable region of SEQ ID NO: 124; #8
comprises the heavy chain variable region of SEQ ID NO: 135 and the light chain variable
region of SEQ ID NO: 139; #9 comprises the heavy chain variable region of SEQ ID NO: 145
and the light chain variable region of SEQ ID NO: 149; #10 comprises the heavy chain
variable region of SEQ ID NO: 145 and the light chain variable region of SEQ ID NO: 155;
#11 comprises the heavy chain variable region of SEQ ID NO: 160 and the light chain
variable region of SEQ ID NO: 164; #12 comprises the heavy chain variable region of SEQ
ID NO: 170 and the light chain variable region of SEQ ID NO: 174; #13 comprises the heavy
chain variable region of SEQ ID NO: 170 and the light chain variable region of SEQ ID NO:
180; #14 comprises the heavy chain variable region of SEQ ID NO: 170 and the light chain
variable region of SEQ ID NO: 185; #15 comprises the heavy chain variable region of SEQ
ID NO: 170 and the light chain variable region of SEQ ID NO: 190; #16 comprises the heavy
chain variable region of SEQ ID NO: 170 and the light chain variable region of SEQ ID NO:
195; #17 comprises the heavy chain variable region of SEQ ID NO: 200 and the light chain
variable region of SEQ ID NO: 204; #18 comprises the heavy chain variable region of SEQ
ID NO: 210 and the light chain variable region of SEQ ID NO: 214; #19 comprises the heavy
chain variable region of SEQ ID NO: 220 and the light chain variable region of SEQ ID NO:
224; #20 comprises the heavy chain variable region of SEQ ID NO: 230 and the light chain
variable region of SEQ ID NO: 234; #21 comprises the heavy chain variable region of SEQ
ID NO: 240 and the light chain variable region of SEQ ID NO: 244; #22 comprises the heavy
chain variable region of SEQ ID NO: 250 and the light chain variable region of SEQ ID NO:
254.
[0150]
The amino acid sequences of the heavy chain variable regions of the obtained chicken
monoclonal antibodies are represented by SEQ ID NOs: 40, 50, and 60. The amino acid
sequences of their light chain variable regions are represented by SEQ ID NOs: 44, 54, and 64.
[0151]
Specifically, the chicken monoclonal antibody #1 comprises the heavy chain variable
region of SEQ ID NO: 40 and the light chain variable region of SEQ ID NO: 44, wherein the
heavy chain variable region has CDR1, CDR2, and CDR3 consisting of the amino acid
sequences of SEQ ID NOs: 37, 38, and 39, respectively, and the light chain variable region
has CDR1, CDR2, and CDR3 consisting of the amino acid sequences of SEQ ID NOs: 41, 42,
and 43, respectively; the chicken monoclonal antibody #2 comprises the heavy chain variable
region of SEQ ID NO: 50 and the light chain variable region of SEQ ID NO: 54, wherein the
heavy chain variable region has CDR1, CDR2, and CDR3 consisting of the amino acid

sequences of SEQ ID NOs: 47, 48, and 49, respectively, and the light chain variable region
has CDR1, CDR2, and CDR3 consisting of the amino acid sequences of SEQ ID NOs: 51, 52,
and 53, respectively; and the chicken monoclonal antibody #3 comprises the heavy chain
variable region of SEQ ID NO: 60 and the light chain variable region of SEQ ID NO: 64,
wherein the heavy chain variable region has CDR1, CDR2, and CDR3 consisting of the
amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively, and the light chain
variable region has CDR1, CDR2, and CDR3 consisting of the amino acid sequences of SEQ
ID NOs: 61, 62, and 63, respectively.
[0152]
(2) Preparation of human-chicken chimeric recombinant antibody and mouse-chicken
chimeric antibody
The gene amplification fragment of the heavy chain variable region (SEQ ID NO: 40)
of the chicken monoclonal antibody #1 obtained in the preceding paragraph (1) was treated at
both ends with restriction enzymes, then purified, and inserted according to a routine method
into a pcDNA4/myc-His (manufactured by Invitrogen Corp.) vector already having gene
inserts of a chicken antibody-derived leader sequence comprising SEQ ID NO: 263 and a
human IgGi H chain constant region comprising SEQ ID NO: 264. Also, the gene
amplification fragment of the light chain variable region (SEQ ID NO: 44) of the chicken
monoclonal antibody #1 was treated at both ends with restriction enzymes, then purified, and
inserted according to a routine method into a pcDNA3.1/myc-His (manufactured by
Invitrogen Corp.) vector already having gene inserts of a chicken antibody-derived leader
sequence comprising SEQ ID NO: 263 and a human IgGi L chain constant region comprising
SEQ ID NO: 265.
[0153]
Next, the recombinant vector having the gene insert of the heavy chain variable region
(SEQ ID NO: 40) of the chicken monoclonal antibody #1 and the recombinant vector having
the gene insert of the light chain variable region (SEQ ID NO: 44) of the chicken monoclonal
antibody #1 were introduced into CHO-K1 cells (obtained from Riken Cell Bank).
Specifically, 2 x 105 CHO-K1 cells were cultured in a Ham's F12 medium (manufactured by
Invitrogen Corp.) containing 1 ml of 10% FBS per well of a 12-well culture plate, and washed
with PBS(-). Then, a fresh Ham's F12 medium containing 1 ml of 10% FBS per well was
added to the well. 250 ng each of the vectors lysed in 30 ju.1 of OptiMEM (manufactured by
Invitrogen Corp.) was mixed with 30 ul of Polyfect transfection reagent (manufactured by
Qiagen N.V.), and this mixture was added to each well. The CHO-K1 cells cotransfected
with the recombinant vectors were cultured in a Ham's F12 medium containing 10% FBS

supplemented with 200 |ug/ml Zeocin (manufactured by Invitrogen Corp.) and 200 ug/ml
Geneticin (manufactured by Roche Diagnostics) and then inoculated to a 96-well plate at a
density of 0.5 cells/well to prepare a cell line stably producing a human-chicken chimeric
antibody #1 (#1) having the variable regions of the chicken monoclonal antibody #1. Cell
lines stably producing a human-chicken chimeric antibody #2 (#2) or a human-chicken
chimeric antibody #3 (#3) were also prepared in the same way as above as to the chicken
monoclonal antibodies #2 and #3.
[0154]
Each prepared cell line was cultured for 5 days in a 150-cm2 flask at a density of 5 x
105 cells/ml using 30 ml of a serum-free OptiCHO medium (manufactured by Invitrogen
Corp.) to obtain culture supernatants containing #1, #2, or #3.
[0155]
Likewise, the gene amplification fragment of the heavy chain variable region (SEQ ID
NO: 40) of the chicken monoclonal antibody #1 was treated at both ends with restriction
enzymes, then purified, and inserted according to a routine method into a pcDNA4/myc-His
(manufactured by Invitrogen Corp.) vector already having gene inserts of a chicken antibody-
derived leader sequence and a mouse IgGi H chain constant region. Also, the gene
amplification fragment of the light chain variable region (SEQ ID NO: 44) of the chicken
monoclonal antibody #1 was treated at both ends with restriction enzymes, then purified, and
inserted according to a routine method into a pcDNA3.1/myc-His (manufactured by
Invitrogen Corp.) vector already having gene inserts of a chicken antibody-derived leader
sequence and a mouse IgGi L chain constant region. These recombinant vectors were
introduced into CHO-K1 cells in the same way as above to prepare a cell line stably
producing a mouse-chicken chimeric antibody #1 having the variable regions of the chicken
monoclonal antibody #1. Cell lines stably producing a mouse-chicken chimeric antibody #2
(#2) or a mouse-chicken chimeric antibody #3 (#3) were also prepared in the same way as
above as to the chicken monoclonal antibodies #2 and #3.
[0156]
Each prepared cell line was cultured for 5 days in a 150-cm2 flask at a density of 5 x
105 cells/ml using 30 ml of a serum-free OptiCHO medium (manufactured by Invitrogen
Corp.) to obtain culture supernatants containing the mouse-chicken chimeric antibody #1, the
mouse-chicken chimeric antibody #2, or the mouse-chicken chimeric antibody #3.
[0157]
(3) Expression of CAPRIN-1 on pancreatic cancer cell surface using obtained
monoclonal antibody

Next, four pancreatic cancer cell lines (Capan-2, MIAPaCa-2, PANC-1, and BxPC-3)
confirmed to have CAPRIN-1 gene expression were examined for their expression of
CAPRIN-1 protein on the cell surface. 106 cells of each cell line were centrifuged in each
1.5-ml microcentrifuge tube. The cancer cell surface-reactive anti-CAPRIN-1 mouse
monoclonal antibodies #1 to #22 prepared in Example 4 and the culture supernatant (100 ul)
containing the anti-CAPRIN-1 mouse-chicken chimeric antibody #1, #2, or #3 prepared in the
preceding paragraph (2) were separately added to the tubes and left standing for 1 hour on ice.
After washing with PBS, the cells were suspended in FITC-labeled goat anti-mouse IgG
antibodies (manufactured by Invitrogen Corp.) diluted 500-fold with PBS containing 0.1%
FBS and left standing for 1 hour on ice. After washing with PBS, the fluorescence intensity
was measured using FACSCalibur (Becton, Dickinson and Company). On the other hand,
the same operation as above was performed using isotype control antibodies, instead of the
anti-CAPRIN-1 mouse monoclonal antibodies #1 to #22 and the culture supernatant
containing the mouse-chicken chimeric antibody #1, #2, or #3, to prepare a control. As a
result, all the cells supplemented with any of the monoclonal antibodies #1 to #22 and the
mouse-chicken chimeric antibodies #1, #2, and #3 had fluorescence intensity at least 20%
stronger than that of the control. As a specific example, Capan-2, MIAPaCa-2, PANC-1,
and BxPC-3 supplemented with the mouse-chicken chimeric antibody #1 all exhibited 200%
or higher enhancement in fluorescence intensity. This demonstrated that CAPRIN-1
proteins are expressed on the cell membrane surface of the human pancreatic cancer cell lines.
The above rate of enhancement in fluorescence intensity was indicated by the rate of increase
in mean fluorescence intensity (MFI) in each cell line and calculated according to the
following expression:
Rate of increase in mean fluorescence intensity (Rate of enhancement in fluorescence
intensity) (%) = ((MFI of cells reacted with the anti-human CAPRIN-1 antibody) - (Control
MFI)) / (Control MFI) x 100
[0158]
(4) Antitumor effect (ADCC activity) of anti-CAPRIN-1 antibody on human
pancreatic cancer cell
Of the antibodies obtained above, the human-chicken chimeric antibody #1 was used
to evaluate its cytotoxic activity (ADCC activity) against human pancreatic cancer cells.
The culture supernatant containing the human-chicken chimeric antibody #1 obtained in the
paragraph (2) was purified using Hitrap Protein A Sepharose FF (manufactured by GE
Healthcare Bio-Sciences Ltd.). After replacement with PBS(-), the solution was filtered
through a 0.22-um filter (manufactured by Millipore Corp.). The resulting antibody was

used for activity assay. 106 cells each of human pancreatic cancer cell lines MIAPaCa-2 and
Capan-2 were collected into a 50-ml centrifuge tube, to which 100 uCi of chromium 51 was
then added, followed by incubation at 37°C for 2 hours. Then, the cells were washed three
•a
times with an RPMI1640 medium containing 10% FBS and added at a density of 2 x 10
cells/well to a 96-well V-bottom plate to prepare target cells. The purified antibody was
added thereto at a concentration of 1.2 (o,g/well. A cell population containing human NK
cells was separated from human peripheral blood lymphocytes using the following approach:
human peripheral blood mononuclear cells were reacted with FITC fluorescent dye-labeled
antibodies (anti-human CD3 antibody, anti-human CD20 antibody, anti-human CD 19
antibody, anti-human CD1 lc antibody, or anti-HLA-DR antibody (Becton, and Dickinson and
Company)). A cell population containing NK cells unstained with the antibodies was
separated using a cell sorter (FACS Vantage SE (Becton, and Dickinson and Company)) or
human NK cell separation kit (NK Cell Isolation Kit (manufactured by Miltenyi Biotec K.K.)).
The obtained cell population containing NK cells was added to the plate at a density of 2 x
105 cells/well and cultured at 37°C for 4 hours under conditions of 5% CO2. After the
culture, the amount of chromium (Cr) 51 released from damaged tumor cells was measured in
the culture supernatant to calculate the ADCC activity of the anti-CAPRIN-1 antibody against
the pancreatic cancer cells. As a result, the human-chicken chimeric antibody #1 exhibited
32% cytotoxic activity against MIAPaCa-2, whereas a cytotoxic activity less than 5% was
obtained by using monoclonal antibodies reactive with the CAPRIN-1 protein itself but
unreactive with the surface of cancer cells or in the absence of antibodies. Also, the human-
chicken chimeric antibody #1 exhibited 20% or higher cytotoxic activity against Capan-2,
whereas a cytotoxic activity less than 5% was obtained by using monoclonal antibodies
reactive with the CAPRIN-1 protein itself but unreactive with the surface of cancer cells or in
the absence of antibodies. The anti-CAPRJN-1 mouse monoclonal antibodies #1 to #22, the
human-chicken chimeric antibody #2, and the human-chicken chimeric antibody #3 were also
examined for their cytotoxic activity against MIAPaCa-2 and Capan-2 in the same way as
above. As a result, these antibodies exhibited 10% or higher cytotoxic activity against both
pancreatic cancer cell lines, whereas a cytotoxic activity less than 5% was obtained by using
monoclonal antibodies reactive with the CAPRIN-1 protein itself but unreactive with the
surface of cancer cells or in the absence of antibodies. These results demonstrated that the
obtained anti-CAPRIN-1 monoclonal antibodies damage CAPRIN-1-expressing cancer cells
through their ADCC activity. These results about cytotoxic activity were obtained by:
mixing the antibody against CAPRIN-1 used in the present invention, a cell population
containing human NK cells, and 2 x 103 tumor cells with incorporated chromium 51, as

described above: culturing the cells for 4 hours; after the culture, measuring the amount of
chromium 51 released into the medium; and calculating the cytotoxic activity against the
tumor cells according to the following equation for calculation :
Equation: Cytotoxic activity (%) = Amount of chromium 51 released from the tumor
cells supplemented with the antibody against CAPRIN-1 and a cell population containing
human NK cells / Amount of chromium 51 released from tumor cells supplemented with 1 N
hydrochloric acid x 100.
[0159]
[Example 7] Antitumor effect of anti-CAPRIN-1 monoclonal antibody on mouse in
vivo
Next, the obtained anti-CAPRIN-1 monoclonal antibodies (human-chicken chimeric
antibody #1) were evaluated for their antitumor effects on cancer-bearing mice in vivo. Each
antibody used was column-purified from the culture supernatant in the same way as above.
[0160]
The monoclonal antibodies against CAPRIN-1 were studied for their antitumor effects
using cancer-bearing Balb/c nude mice in which a CAPRIN-1-expressing human pancreatic
cancer cell line Capan-2 was transplanted. 5 x 106 Capan-2 cells (purchased from ATCC)
per mouse were subcutaneously transplanted into the abdomens of 6 Balb/c nude mice
(manufactured by Japan SLC, Inc.) and grown until the size of tumor became approximately 5
mm in diameter. Each monoclonal antibody against CAPRIN-1 was intraperitoneally
administered at a dose of 200 |j.g (200 u!)/mouse to 3 of these cancer-bearing mice. Then,
the antibody was intraperitoneally administered to the cancer-bearing mice at the same dose
as above twice a week. The size of tumor was measured every day, and the antitumor effect
was observed. On the other hand, PBS(-) was administered instead of the antibody to the
remaining 3 cancer-bearing mice, which were in turn used as a control group. As a result, in
the groups that received the anti-CAPRIN-1 mouse monoclonal antibodies #1 to #22, tumor
was regressed to 84% (with the tumor volume in the control group defined as 100%) on day
27 after the start of antibody administration. In addition, tumor growth was reduced to 75%
at day 35. The human-chicken chimeric antibodies #1, #2, and #3 were also evaluated in the
same way as above. As a result, tumor growth was reduced to 80% in all the cases on day
27 after the start of antibody administration. These results demonstrated that the obtained
antibodies against CAPRIN-1 exert an in vivo antitumor effect on CAPRIN-1-expressing
human pancreatic cancer cells. The size of tumor was calculated in terms of volume
according to the equation: 0.5 x (Major axis x Minor axis x Minor axis).
[0161]

[Example 8] Identification of peptide in CAPRIN-1 protein to which anti-CAPRIN-1
antibody reactive with cancer cell surface binds
The cancer cell surface-reactive anti-CAPRIN-1 monoclonal antibodies #12 to #22
obtained above were used to identify partial sequences in CAPRIN-1 proteins recognized
thereby.
[0162]
First, DTT (manufactured by Sigma-Aldrich Corp./Fluka) was added at a final
concentration of 10 mM to 100 ul of a 1 ug/ul solution containing recombinant CAPRIN-1
proteins dissolved in PBS, and reacted at 95°C for 5 minutes to reduce disulfide bonds in the
CAPRIN-1 proteins. Next, 20 mM (final concentration) iodoacetamide (manufactured by
Wako Pure Chemical Industries, Ltd.) was added thereto, followed by the alkylation reaction
of thiol groups at 37°C for 30 minutes under shading conditions. 50 \ig each of the anti-
CAPRIN-1 monoclonal antibodies #12 to #22 was added to 40 u.g of the obtained reduced
alkylated CAPRIN-1 proteins. The total amount of each mixture was adjusted to 1 ml with a
20 mM phosphate buffer solution (pH 7.0). The resulting mixture was reacted overnight at
4°C while mixed by stirring.
[0163]
Next, trypsin (manufactured by Promega K.K.) was added at a final concentration of
0.2 |ag to each reaction mixture and reacted at 37°C for 1 hour, 2 hours, 4 hours, and 12 hours.
Then, the reaction mixture was mixed with protein A-glass beads (manufactured by GE
Healthcare Bio-Sciences Ltd.) blocked with PBS containing 1% BSA (manufactured by
Sigma-Aldrich Corp.) and washed with PBS in advance, 1 mM calcium carbonate, and NP-40
buffer solution (20 mM phosphate buffer solution (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1%
NP-40) and reacted for 30 minutes.
[0164]
Each reaction solution was washed with a 25 mM ammonium carbonate buffer
solution (pH 8.0), followed by the elution of antigen-antibody complexes using 100 ul of
0.1% formic acid. The eluate was analyzed by LC-MS using Q-TOF Premier (manufactured
by Waters-MicroMass). This analysis followed the protocol attached to the instrument.
[0165]
As a result, a polypeptide of SEQ ID NO: 273 was identified as a partial CAPRIN-1
sequence recognized by all of the anti-CAPRIN-1 monoclonal antibodies #12 to #22. In the
polypeptide of SEQ ID NO: 273, a peptide of SEQ ID NO: 274 was identified as a partial
sequence recognized by the monoclonal antibodies #13 to #16, #17 to #19, and #21. As its

partial sequence peptide, a peptide of SEQ ID NO: 275 was further found to be recognized by
the monoclonal antibodies #13 to #16.
[0166]
Also, the human-chicken chimeric monoclonal antibody #1, the human-chicken
chimeric monoclonal antibody #3, and the mouse monoclonal antibodies #1, #2, #3, #4, #5,
#6, #7, #8, #9, #10, and #11 were used to identify epitope peptides in CAPRIN-1 proteins
recognized thereby. 93 candidate peptides consisting of 12 to 16 amino acids in the amino
acid sequence of the human CAPRIN-1 protein were synthesized and each dissolved at a
concentration of 1 mg/ml in DMSO.
[0167]
Each peptide was dissolved at a concentration of 30 |J.g/ml in a 0.1 M sodium
carbonate buffer solution (pH 9.6). The solution was added at a concentration of 100 ul/well
to a 96-well plate (manufactured by Thermo Fisher Scientific Inc./Nunc, product No.:
436006) and left standing overnight at 4°C. The solution in each well was discarded, and 10
mM ethanolamine/0.1 M sodium carbonate buffer solution (PH 9.6) was added thereto at a
concentration of 200 ul/well and left standing at room temperature for 1 hour. Then, the
solution in each well was discarded, and each well was washed twice with PBS containing
0.5% Tween 20 (PBST) to prepare a peptide-immobilized plate.
[0168]
The cell culture supernatant containing the human-chicken chimeric monoclonal
antibody #1 (#1), the human-chicken chimeric monoclonal antibody #3 (#3), or the mouse
monoclonal antibody (#1, #2, #3, #4, #5, #6, #7, #8, #9, #10, or #11) was added at a
concentration of 50 ul/well to each plate thus obtained. After shaking at room temperature
for 1 hour, the solution in each well was discarded, and each well was washed three times
with PBST. Next, a secondary antibody solution containing HRP-labeled anti-human IgG
(manufactured by Invitrogen Corp.) antibodies diluted 3000- to 4000-fold with PBST was
added at a concentration of 50 ul/well to the human-chicken chimeric monoclonal antibody
wells, while a secondary antibody solution containing HRP-labeled anti-mouse IgG
(manufactured by Invitrogen Corp.) antibodies diluted 3000- to 4000-fold with PBST was
added at a concentration of 50 ul/well to the mouse monoclonal antibody wells. Then, the
solution in each well was discarded, and each well was washed six times with PBST.
[0169]
A TMB substrate solution (manufactured by Thermo Fisher Scientific Inc.) was added
thereto at a concentration of 100 ul/well and left standing for 15 to 30 minutes to cause color
reaction. After the color development, the reaction was terminated by the addition of 1 N

sulfuric acid at a concentration of 100 ul/well. The absorbance was measured at 450 nm and
595 nm using an absorption spectrometer. As a result, a polypeptide of SEQ ID NO: 266
was identified as a partial CAPRIN-1 sequence recognized by all of the anti-CAPRIN-1
antibodies human-chicken chimeric monoclonal antibody #1 and anti-CAPRIN-1 monoclonal
antibodies #1 to #5. In the polypeptide of SEQ ID NO: 266, a peptide of SEQ ID NO: 267
was identified as a partial sequence recognized by the human-chicken chimeric monoclonal
antibody #1 and the mouse monoclonal antibodies #3 and #4. In the polypeptide of SEQ ID
NO: 266, a peptide of SEQ ID NO: 268 was identified as a partial sequence recognized by the
mouse monoclonal antibodies #1, #2, and #5. Thus, the polypeptide of SEQ ID NO: 266
was found to contain an epitope region for the anti-CAPRIN-1 antibodies human-chicken
chimeric monoclonal antibody #1 and mouse monoclonal antibodies #1, #2, #3, #4, and #5.
Also, a polypeptide comprising the amino acid sequence of SEQ ID NO: 270 was identified
as a partial CAPRIN-1 sequence recognized by all of the anti-CAPRIN-1 monoclonal
antibodies #6, #7, and #8. Thus, the polypeptide of SEQ ID NO: 270 was found to contain
an epitope region for the anti-CAPRIN-1 antibodies #6, #7, and #8. In addition, a
polypeptide comprising the amino acid sequence of SEQ ID NO: 272 was identified as a
partial CAPRIN-1 sequence recognized by all of the anti-CAPRIN-1 monoclonal antibodies
#9, #10, and #11. Thus, the polypeptide of SEQ ID NO: 272 was found to contain an
epitope region for the anti-CAPRIN-1 antibodies #9, #10, and #11. In addition, a
polypeptide comprising the amino acid sequence of SEQ ID NO: 269 was identified as a
partial CAPRIN-1 sequence recognized by the human-chicken chimeric monoclonal antibody
#3. Thus, the polypeptide of SEQ ID NO: 269 was found to contain an epitope region for
the human-chicken chimeric monoclonal antibody #3.
Industrial Applicability
[0170]
The antibody of the present invention is useful in the treatment and/or prevention of
pancreatic cancer.
[0171]
All publications, patents, and patent applications cited herein are incorporated herein
by reference in their entirety.
Free Text for Sequence Listing
[0172]
SEQ ID NOs: 31 to 36, 130, and 257 to 262: Primers

We Claim:
1. A pharmaceutical composition for treatment and/or prevention of pancreatic cancer,
comprising, as an active ingredient, an antibody or a fragment thereof which specifically has
immunological reactivity with a CAPRIN-1 protein or a fragment thereof comprising 7 to 12
or more consecutive amino acid residues.
2. The pharmaceutical composition according to claim 1, wherein the CAPRIN-1
protein has an amino acid sequence represented by any of even-numbered SEQ ID NOs: 2 to
30, or an amino acid sequence having 80% or higher sequence identity to the amino acid
sequence.
3. The pharmaceutical composition according to claim 1 or 2, wherein the
pharmaceutical composition comprises, as an active ingredient, an antibody or a fragment
thereof which has immunological reactivity with the fragment of the CAPRIN-1 protein, the
fragment being a polypeptide consisting of 7 or more consecutive amino acid residues in a
region of amino acid residue numbers 50 to 98, a region of amino acid residue numbers 233 to
343, or a region of amino acid residue number 527 to the C terminus, of an amino acid
sequence represented by any of even-numbered SEQ ID NOs: 2 to 30 except for SEQ ID
NOs: 6 and 18, or a polypeptide comprising the polypeptide as a partial sequence.
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the
antibody or the fragment thereof is an antibody or a fragment thereof which has
immunological reactivity with a partial CAPRIN-1 polypeptide having an amino acid
sequence represented by SEQ ID NO: 273, 266, 270, 272, or 269, or an amino acid sequence
having 80% or higher sequence identity to the amino acid sequence, or a fragment thereof
comprising 7 to 12 or more consecutive amino acid residues.
5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the
antibody is a monoclonal antibody or a polyclonal antibody.
6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the
antibody is a human antibody, a humanized antibody, a chimeric antibody, a single-chain
antibody, or a multispecific antibody.
7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the
antibody or the fragment thereof is any of the following (a) to (y):
(a) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising complementarity determining regions (CDRs) of SEQ ID NOs: 37, 38, and 39 and
a light chain variable region comprising CDRs of SEQ ID NOs: 41, 42, and 43 and has
immunological reactivity with the CAPRIN-1 protein;

(b) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 47, 48, and 49 and a light chain variable region
comprising CDRs of SEQ ID NOs: 51, 52, and 53 and has immunological reactivity with the
CAPRIN-1 protein;
(c) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 57, 58, and 59 and a light chain variable region
comprising CDRs of SEQ ID NOs: 61, 62, and 63 and has immunological reactivity with the
CAPRIN-1 protein;
(d) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 67, 68, and 69 and a light chain variable region
comprising CDRs of SEQ ID NOs: 71, 72, and 73 and has immunological reactivity with the
CAPRIN-1 protein;
(e) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 77, 78, and 79 and a light chain variable region
comprising CDRs of SEQ ID NOs: 81, 82, and 83 and has immunological reactivity with the
CAPRIN-1 protein;
(f) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 87, 88, and 89 and a light chain variable region
comprising CDRs of SEQ ID NOs: 91, 92, and 93 and has immunological reactivity with the
CAPRIN-1 protein;
(g) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 97, 98, and 99 and a light chain variable region
comprising CDRs of SEQ ID NOs: 101, 102, and 103 and has immunological reactivity with
the CAPRIN-1 protein;
(h) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 107, 108, and 109 and a light chain variable region
comprising CDRs of SEQ ID NOs: 111, 112, and 113 and has immunological reactivity with
the CAPRIN-1 protein;
(i) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 117, 118, and 119 and a light chain variable region
comprising CDRs of SEQ ID NOs: 121, 122, and 123 and has immunological reactivity with
the CAPRIN-1 protein;
(j) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 127, 128, and 129 and a light chain variable region

comprising CDRs of SEQ ID NOs: 121, 122, and 123 and has immunological reactivity with
the CAPRIN-1 protein;
(k) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 132, 133, and 134 and a light chain variable region
comprising CDRs of SEQ ID NOs: 136, 137, and 138 and has immunological reactivity with
the CAPRIN-1 protein;
(1) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 142, 143, and 144 and a light chain variable region
comprising CDRs of SEQ ID NOs: 146, 147, and 148 and has immunological reactivity with
the CAPRIN-1 protein;
(m) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 142, 143, and 144 and a light chain variable region
comprising CDRs of SEQ ID NOs: 152, 153, and 154 and has immunological reactivity with
the CAPRIN-1 protein;
(n) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 157, 158, and 159 and a light chain variable region
comprising CDRs of SEQ ID NOs: 161, 162, and 163 and has immunological reactivity with
the CAPRIN-1 protein;
(o) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 167, 168, and 169 and a light chain variable region
comprising CDRs of SEQ ID NOs: 171, 172, and 173 and has immunological reactivity with
the CAPRIN-1 protein;
(p) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 167, 168, and 169 and a light chain variable region
comprising CDRs of SEQ ID NOs: 177, 178, and 179 and has immunological reactivity with
the CAPRIN-1 protein;
(q) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 167, 168, and 169 and a light chain variable region
comprising CDRs of SEQ ID NOs: 182, 183, and 184 and has immunological reactivity with
the CAPRIN-1 protein;
(r) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 167, 168, and 169 and a light chain variable region
comprising CDRs of SEQ ID NOs: 187, 188, and 189 and has immunological reactivity with
the CAPRIN-1 protein;

(s) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 167, 168, and 169 and a light chain variable region
comprising CDRs of SEQ ID NOs: 192, 193, and 194 and has immunological reactivity with
the CAPRIN-1 protein;
(t) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 197, 198, and 199 and a light chain variable region
comprising CDRs of SEQ ID NOs: 201, 202, and 203 and has immunological reactivity with
the CAPRIN-1 protein;
(u) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 207, 208, and 209 and a light chain variable region
comprising CDRs of SEQ ID NOs: 211, 212, and 213 and has immunological reactivity with
the CAPRIN-1 protein;
(v) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 217, 218, and 219 and a light chain variable region
comprising CDRs of SEQ ID NOs: 221, 222, and 223 and has immunological reactivity with
the CAPRIN-1 protein;
(w) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 227, 228, and 229 and a light chain variable region
comprising CDRs of SEQ ID NOs: 231, 232, and 233 and has immunological reactivity with
the CAPRIN-1 protein;
(x) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 237, 238, and 239 and a light chain variable region
comprising CDRs of SEQ ID NOs: 241, 242, and 243 and has immunological reactivity with
the CAPRIN-1 protein; and
(y) an antibody or a fragment thereof, which comprises a heavy chain variable region
comprising CDRs of SEQ ID NOs: 247, 248, and 249 and a light chain variable region
comprising CDRs of SEQ ID NOs: 251, 252, and 253 and has immunological reactivity with
the CAPRIN-1 protein.
8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the
antibody or the fragment thereof is conjugated with an antitumor agent.
9. A pharmaceutical combination comprising the pharmaceutical composition
according to any one of claims 1 to 8 and a pharmaceutical composition comprising an
antitumor agent.

10. A method for treating and/or preventing pancreatic cancer, comprising
administering the pharmaceutical composition according to any one of claims 1 to 8 or the
pharmaceutical combination according to claim 9, to a subject.