# Patent

## Title
Pharmaceutical composition for treatment and/or prevention of pancreatic cancer

## Abstract

This invention relates to a pharmaceutical composition for treatment and/or prevention of pancreatic cancer, comprising as an active ingredient an antibody or a fragment thereof which has immunological reactivity with a CAPRIN-1 protein or a fragment thereof comprising 7 to 12 or more consecutive amino acid residues.

## Background
TECHNICAL FIELD
The present invention relates to novel use of an antibody against CAPRIN-1 or a fragment thereof in a medicament such as a therapeutic and/or preventive agent for pancreatic cancer.
BACKGROUND ART
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).
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.
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, disadvantageously 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.
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 CAPRIN-1 is expressed on pancreatic cancer cells, and neither disclosed nor suggested that CAPRIN-1 can serve as an antigenic protein for pancreatic cancer.
PRIOR ART LITERATURES
Patent Literature


    Patent Literature 1: U.S. Pat. No. 5,698,396
    Patent Literature 2: International Publication No. WO2010/016526


Non Patent Literature


    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
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
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 CAPRIN-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.
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.
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.
In another embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.
In a further embodiment, the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a multispecific antibody.
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.
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;
(l) 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.
In a further embodiment of the present invention, the antibody or the fragment thereof is conjugated with an antitumor agent.
The present invention further provides a pharmaceutical combination comprising the pharmaceutical composition of the present invention and a pharmaceutical composition comprising an antitumor agent.
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.
The antibody against CAPRIN-1 used in the present invention damages pancreatic cancer cells. Thus, the antibody against CAPRIN-1 is useful in the treatment and/or prevention of pancreatic cancer.
MODES FOR CARRYING OUT THE INVENTION
The antibody against a CAPRIN-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.
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.
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 CAPRIN-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 CAPRIN-1 isolated as a bovine homologous factor thereof; the amino acid sequence represented by SEQ ID NO: 18 is an amino acid sequence of CAPRIN-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.
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.
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.
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.
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.
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.
Hereinafter, antigen preparation, antibody preparation, and a pharmaceutical composition according to the present invention will be described.
<Preparation of Antigen for Antibody Preparation>
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.
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).
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 CAPRIN-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.
The fragments of each CAPRIN-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.
Polypeptides comprising the above human CAPRIN-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.
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).
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.
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 COST 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.
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.
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 pKA1, 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-N1, or pEGFP-C1, the polypeptides may be expressed as various fusion proteins tagged with His tag (e.g., (His)6 to (His)10), FLAG tag, myc tag, HA tag, GFP, or the like.
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.
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.
<Structure of Antibody>
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 C1q component in the complement cascade.
<Preparation of Antibody>
The anti-CAPRIN-1 antibody according to the present invention means an antibody having immunological reactivity with a full-length CAPRIN-1 protein or a fragment thereof.
As used herein, the “immunological reactivity” means the property of the antibody binding to the CAPRIN-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.
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, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.
The antibody may be further modified by acetylation, formylation, amidation, phosphorylation, PEGylation, or the like, in addition to glycosylation.
Hereinafter, preparation examples of various antibodies will be described.
When the antibody of the present invention is a monoclonal antibody, for example, CAPRIN-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.
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.
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.
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-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (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), 5194 (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.
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).
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.
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.
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.
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.
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).
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.
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.
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:
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.
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.
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. WO98/46777). Antigens having low immunogenicity may be bound to immunogenic macromolecules such as albumin for immunization.
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.
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.
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 CAPRIN-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.
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.
The polyclonal antibody includes antibodies obtained from human antibody-producing animals (e.g., mice) immunized with CAPRIN-1 proteins.
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.
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. WO99/51743).
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).
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.
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).
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.
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.
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, M J., and Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, H J., (1987) Methods Enzymol. 154, 350-367; Kunkel, T A., (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.
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.
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 5×108 M−1, at least 109 M−1, at least 5×109 M−1, at least 1010 M−1, at least 5×1010 M−1, at least 1011 M−1, at least 5×1011 M−1, at least 1012 M−1, or at least 1013 M−1.
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.
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, caminomycin, 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, elformithine, 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.
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 CAPRIN-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.
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, caminomycin, 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, elformithine, 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.
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, 186Re, 188Re, 153Sm, 212Bi, 32P, 175Lu, or 176Lu. Desirably, a radioisotope effective for the treatment or diagnosis of tumor is used.
The antibody used in the present invention is an antibody having immunological reactivity with CAPRIN-1 or an antibody specifically binding to CAPRIN-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.
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)).
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.
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.
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.
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).
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.
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).
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);

(l) 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).

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.
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.
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.

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 IgG1 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 κ constant region; and Accession Nos. X64132 and X64134 for a human light chain λ constant region.
Preferably, these antibodies have cytotoxic activity and can thereby exert an antitumor effect.
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.
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.
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:
effector cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the CAPRIN-1-expressing cells.
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.
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.
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-CAPRIN-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 5×108 M−1, at least 109 M−1, at least 5×109 M−1, at least 1010 M−1, at least 5×1010 M−1, at least 1011 M−1, at least 5×1011 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.
<Binding to Antigen-Expressing Cells>
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.
<Immunohistochemical Staining>
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.
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.
<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.
The terms “tumor” and “cancer” used herein mean malignant neoplasm and are used interchangeably with each other.
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.
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.
The recipient animals are mammals, for example, mammals including primates, pet animals, livestock, and sport animals and are particularly preferably humans, dogs, and cats.
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.
An aseptic composition for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as injectable distilled water.
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.
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.
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.
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.
The pharmaceutical composition of the present invention can be administered to a subject to treat and/or prevent pancreatic cancer.
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.
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.
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
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.
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.
The mRNAs (5 μg) 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 7.73×105 pfu/ml.
(2) Screening of cDNA Library Using Serum
The canine testicular cDNA phage library thus prepared was used in immunoscreening. Specifically, host E. coli (XL1-Blue MRF′) was infected by the phages on a NZY agarose plate (φ90×15 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-β-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.
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 (XL1-Blue MRF′) was infected by λ 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 NaHCO3 (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. coli/phage extract. Next, the recovered E. coli/phage extract was applied to a NHS-column (manufactured by GE Healthcare Bio-Sciences Ltd.) to immobilize E. coli/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.
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 (φ90×15 mm) and lysed in 500 μl of an SM buffer solution (100 mM NaCl, 10 mM MgClSO4, 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.
(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 μl of a host E. coli (XL1-Blue MRF′) solution prepared such that absorbance OD600 became 1.0 was mixed with 250 μl of a purified phage solution and further with 1 μl 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×g at 4° C. for 15 minutes to recover the supernatant as a phagemid solution. Subsequently, 200 μl of a phagemid host E. coli (SOLR) solution prepared such that absorbance OD600 became 1.0 was mixed with 10 μl of a purified phage solution, followed by reaction at 37° C. for 15 minutes. 50 μl of the reaction mixture was inoculated to an LB agar medium containing ampicillin (final concentration: 50 μg/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 μg/ml). Then, plasmid DNAs having the inserts of interest were purified using QIAGEN plasmid Miniprep Kit (manufactured by Qiagen N.V.).
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 (www.ncbi.nlm.nih.gov/BLAST/), the obtained five genes were all found to be genes encoding CAPRIN-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.
(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, PANG-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 10×106 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 μl by the addition of 0.25 μl of the sample prepared by the reverse transcription reaction and each reagent and attached buffer (2 μM 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.
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.
Example 3
Analysis of CAPRIN-1 Protein Expression in Human Pancreatic Cancer
(1) Analysis of CAPRIN-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 CAPRIN-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 mg (5 μl) of the anti-CAPRIN-1 polyclonal antibodies prepared in Example 2 was added thereto. The mixture was suspended in PBS containing 95 μl 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 μl of FITC-labeled goat anti-rabbit IgG antibodies (manufactured by Santa Cruz Biotechnology, Inc.) and 95 μl 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 CAPRIN-1 to prepare a control. As a result, the pancreatic cancer cells supplemented with the anti-human CAPRIN-1 polyclonal antibodies all exhibited fluorescence intensity at least 20% stronger than that of the control. 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)×100

(2) Expression of CAPRIN-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-CAPRIN-1 polyclonal antibodies prepared in Example 2 were prepared into 10 μg/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 CAPRIN-1 was confirmed in 36 (90%) out of a total of 40 pancreatic cancer tissue samples.
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 CAPRIN-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 μCi 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 103 cells/well to a 96-well V-bottom plate. The polyclonal antibodies against human CAPRIN-1 were added thereto at a concentration of 1 μg/well. Lymphocytes separated from human peripheral blood were further added thereto at a density of 2×105 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×100.

Example 5
Preparation of Mouse and Chicken Monoclonal Antibodies Against CAPRIN-1
100 μg 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 μl of an RPMI1640 medium containing 10% FBS was heated to 37° C. and mixed with 800 μl 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 μl/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.
The prepared hybridomas were screened with the binding affinity of antibodies produced by the hybridomas against CAPRIN-1 proteins as an indicator. A 1 μg/ml solution of the CAPRIN-1 proteins prepared in Example 2 was added to a 96-well plate at a concentration of 100 μl/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 μl/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 μl of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added to the well at a concentration of 100 μl/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 μl/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 μl/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 μl/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.
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 μg/ml solution of the CAPRIN-1 proteins prepared in Example 2 was added to a 96-well plate at a concentration of 100 μl/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 μl/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 μl of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added to the well at a concentration of 100 μl/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 μl/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 μl/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 μl/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.
Next, these mouse monoclonal antibodies were screened for antibodies reactive with the surface of cancer cells expressing CAPRIN-1. Specifically, 106 cells of a human breast cancer cell line MDA-MB-231V were centrifuged in a 1.5-ml microcentrifuge tube. 100 μl 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.
In order to prepare chicken monoclonal antibodies, 300 μg of the antigenic proteins (human CAPRIN-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 μl of an IMDM medium containing 10% FBS was heated to 37° C. and mixed with 800 μl 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 μl/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.
The prepared hybridomas were screened with the binding affinity of antibodies produced by the hybridomas against CAPRIN-1 proteins as an indicator. A 1 μg/ml solution of the CAPRIN-1 proteins prepared in Example 2 was added to a 96-well plate at a concentration of 100 μl/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 μl/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 μl of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at a concentration of 100 μl/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 μl/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 μl/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 μl/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.
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 μg/ml solution of the human CAPRIN-1 proteins was added to a 96-well plate at a concentration of 100 μl/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 μl/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 μl of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at a concentration of 100 μl/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 μl/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 μl/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 μl/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.
Next, these monoclonal antibodies were screened for antibodies reactive with the surface of cancer cells expressing CAPRIN-1. Specifically, 5×105 cells of a human breast cancer cell line MDA-MB-231V were centrifuged in a 1.5-ml microcentrifuge tube. 100 μl 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.
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.).
(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 μl of 10×EX Taq Buffer, 4 μl of dNTP Mixture (2.5 mM), 2 μl each of the primers (1.0 μM), and 0.25 μl of Ex Taq (5 U/μl), and the total amount of the solution was adjusted to 50 μl 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.
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.
(1)-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 μg/ml ampicillin. Then, each plasmid DNA was purified using Qiaspin Miniprep kit (manufactured by Qiagen N.V.).
(1)-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.
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.
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.
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.
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.
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.
(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 IgG1 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 IgG1 L chain constant region comprising SEQ ID NO: 265.
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, 2×105 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 μl of OptiMEM (manufactured by Invitrogen Corp.) was mixed with 30 μl 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 μg/ml Zeocin (manufactured by Invitrogen Corp.) and 200 μg/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.
Each prepared cell line was cultured for 5 days in a 150-cm2 flask at a density of 5×105 cells/ml using 30 ml of a serum-free OptiCHO medium (manufactured by Invitrogen Corp.) to obtain culture supernatants containing #1, #2, or #3.
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 IgG1 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 IgG1 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.
Each prepared cell line was cultured for 5 days in a 150-cm2 flask at a density of 5×105 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.
(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 μl) 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)×100

(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-μm 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 μCi 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% FBS and added at a density of 2×103 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 μg/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 CD19 antibody, anti-human CD11c 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 2×105 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-CAPRIN-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 2×103 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×100.

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.
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. 5×106 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 μg (200 μl)/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×(Major axis×Minor axis×Minor axis).
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.
First, DTT (manufactured by Sigma-Aldrich Corp./Fluka) was added at a final concentration of 10 mM to 100 μl of a 1 μg/μl 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 μg each of the anti-CAPRIN-1 monoclonal antibodies #12 to #22 was added to 40 μg 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.
Next, trypsin (manufactured by Promega K.K.) was added at a final concentration of 0.2 μg 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.
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 μl 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.
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.
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.
Each peptide was dissolved at a concentration of 30 μg/ml in a 0.1 M sodium carbonate buffer solution (pH 9.6). The solution was added at a concentration of 100 μl/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 μl/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.
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 μl/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 μl/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 μl/well to the mouse monoclonal antibody wells. Then, the solution in each well was discarded, and each well was washed six times with PBST.
A TMB substrate solution (manufactured by Thermo Fisher Scientific Inc.) was added thereto at a concentration of 100 μl/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 μl/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
The antibody of the present invention is useful in the treatment and/or prevention of pancreatic cancer.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
FREE TEXT FOR SEQUENCE LISTING
SEQ ID NOs: 31 to 36, 130, and 257 to 262: Primers

## Claims

1. A method for treating a CAPRIN-1-expressing pancreatic cancer, comprising administering a pharmaceutical composition to a patient with the pancreatic cancer, wherein the pharmaceutical composition comprises, as an active ingredient, an antibody or a fragment thereof which specifically binds a CAPRIN-1 protein expressed on the cell surface of the pancreatic cancer.

2. A method for treating a CAPRIN-1-expressing pancreatic cancer, comprising administering a pharmaceutical combination to a patient with the pancreatic cancer, wherein the pharmaceutical combination comprises: (1) a pharmaceutical composition comprising, as an active ingredient, an antibody or a fragment thereof which specifically binds a CAPRIN-1 protein expressed on the cell surface of the pancreatic cancer; and (2) a pharmaceutical composition comprising an antitumor agent.